Статті в журналах з теми "Transmissio Blocking Therapy"

The objective of this study was to investigate the efficacy and potential side-effects of nucleotide/nucleoside analogues and hepatitis B immunoglobulin injection of newborns in blocking mother-to-child transmission of hepatitis B virus in the middle and late pregnancy period. 238 cases of enrolled pregnant women were divided into the Telbivudine group, the Tenofovir group, the Lamivudine group, and the hepatitis B immunoglobulin (HBIG) group. Enrolled patients received corresponding therapies. Clinical and laboratory data were collected. Results showed that the levels of HBV DNA of the enrolled pregnant women in the Telbivudine, Tenofovir, and Lamivudine groups decreased rapidly after 12 weeks of drug intervention compared with those in the control. HBsAg positive rate in newborns and in children 24 weeks after birth was 0/60, 0/60, 0/60, 3/30, and 11/28 in the Telbivudine, Tenofovir, Lamivudine, HBIG, and control groups, respectively. No significant side-effects were identified after following up to 12 months after birth. Our results show that routine HBV vaccine plus HBIG injections is insufficient in blocking mother-to-child HBV transmission. Administration of nucleotide/nucleoside analogues or HBIG at pregnancy is suggested to maximize the blocking of vertical HBV transmission.

Abstract As a newly emerged infectious disease, the coronavirus disease 2019 (COVID-19) has caused millions of deaths, resulting in a global health challenge. Currently, several vaccines have been approved with significant benefits against disease transmission. However, effective therapies are still needed for the clinical management of infected COVID-19 patients. Available evidence has indicated elevated levels of proinflammatory cytokines, including interleukin-6 (IL-6), in COVID-19 patients, with cytokine storm involving excessive cytokine release being observed in some severe cases. Several clinical studies have shown the promising effects of IL-6-blocking strategy in treating severe COVID-19 patients, but some observational studies have reported that IL-6-blocking therapy has no effects in preventing disease progression or death among COVID-19 patients. Herein, we review recent findings on the immunopathogenesis of COVID-19, with specific emphasis on the proinflammatory function of IL-6 and discuss the therapeutic potential of IL-6-blocking therapy for the treatment of COVID-19 patients, especially those with rheumatic diseases.

In this paper, we investigate the global dynamics of a system of delay differential equations which describes the interaction of hepatitis B virus (HBV) with both liver and blood cells. The model has two distributed time delays describing the time needed for infection of cell and virus replication. We also include the efficiency of drug therapy in inhibiting viral production and the efficiency of drug therapy in blocking new infection. We compute the basic reproduction number and find that increasing delays will decrease the value of the basic reproduction number. We study the sensitivity analysis on the key parameters that drive the disease dynamics in order to determine their relative importance to disease transmission and prevalence. Our analysis reveals that the model exhibits the phenomenon of backward bifurcation (where a stable disease-free equilibrium (DFE) co-exists with a stable endemic equilibrium when the basic reproduction number is less than unity). Numerical simulations are presented to evaluate the impact of time-delays on the prevalence of the disease.

In this study, a time-periodic viral infection model incorporating cell-to-cell infection and antiretroviral therapy has been investigated. The basic reproduction number ℛ 0 has been defined as a threshold parameter which governs whether or not the disease dies out. Theoretical results indicate that the disease goes to extinction if ℛ 0 < 1 and otherwise the disease will uniformly persist. The global stabilities of the equilibria for the corresponding autonomous model have been investigated by constructing suitable Lyapunov functions. Moreover, numerical simulations have been carried out to validate the obtained results. The results show that cell-to-cell infection mode may be a barrier to curing the viral infection and increasing the efficacy of protease inhibitors for blocking cell-to-cell infection which will benefit to weaken the severity of the viral infection.

Here, we review the literature assessing the role of transient receptor potential ankyrin 1 (TRPA1), a calcium-permeable non-selective cation channel, in various types of pain conditions. In the nervous system, TRPA1 is expressed in a subpopulation of nociceptive primary sensory neurons, astroglia, oligodendrocytes and Schwann cells. In peripheral terminals of nociceptive primary sensory neurons, it is involved in the transduction of potentially harmful stimuli and in their central terminals it is involved in amplification of nociceptive transmission. TRPA1 is a final common pathway for a large number of chemically diverse pronociceptive agonists generated in various pathophysiological pain conditions. Thereby, pain therapy using TRPA1 antagonists can be expected to be a superior approach when compared with many other drugs targeting single nociceptive signaling pathways. In experimental animal studies, pharmacological or genetic blocking of TRPA1 has effectively attenuated mechanical and cold pain hypersensitivity in various experimental models of pathophysiological pain, with only minor side effects, if any. TRPA1 antagonists acting peripherally are likely to be optimal for attenuating primary hyperalgesia (such as inflammation-induced sensitization of peripheral nerve terminals), while centrally acting TRPA1 antagonists are expected to be optimal for attenuating pain conditions in which central amplification of transmission plays a role (such as secondary hyperalgesia and tactile allodynia caused by various types of peripheral injuries). In an experimental model of peripheral diabetic neuropathy, prolonged blocking of TRPA1 has delayed the loss of nociceptive nerve endings and their function, thereby promising to provide a disease-modifying treatment.

In recent years, numerous studies have highlighted the significant use of botulinum neurotoxins (BoNTs) in the human therapy of various motor and autonomic disorders. The therapeutic action is exerted with the selective cleavage of specific sites of the SNARE’s protein complex, which plays a key role in the vesicular neuroexocytosis which is responsible for neural transmission. The primary target of the BoNTs’ action is the peripheral neuromuscular junction (NMJ), where, by blocking cholinergic neurons releasing acetylcholine (ACh), they interfere with neural transmission. A great deal of experimental evidence has demonstrated that BoNTs are also effective in blocking the release of other neurotransmitters or neuromodulators, such as glutamate, substance-P, and CGRP, and they can interfere with the function of glial cells, both at the peripheral and central level. The purpose of this review is to provide an update on the available experimental data from animal models that suggest or confirm the direct interactions between BoNTs and glial cells. From the data collected, it appears evident that, through mechanisms that are not yet fully understood, BoNTs can block the activation of spinal glial cells and their subsequent release of pro-inflammatory factors. BoNTs are also able to promote peripheral regeneration processes after nerve injury by stimulating the proliferation of Schwann cells. The data will be discussed in consideration of the possible therapeutic implications of the use of BoNTs on those pathological conditions where the contribution of glial cell activation is fundamental, such as in peripheral and central neuropathies.

SUMMARYVaccines are the cornerstone of influenza control policy, but can suffer from several drawbacks. Seasonal influenza vaccines are prone to production problems and low efficacies, while pandemic vaccines are unlikely to be available in time to slow a rapidly spreading global outbreak. Antiviral therapy was found to be beneficial during the influenza A(H1N1)pdm09 pandemic even with limited use; however, antiviral use has decreased further since then. We sought to determine the role antiviral therapy can play in pandemic and seasonal influenza control using conservative estimates of antiviral efficacy, and to assess if conservative but targeted strategies could be employed to optimize the use of antivirals. Using an age-structured contact network model for an urban population, we compared the transmission-blocking ability of a conservative antiviral therapy strategy to the susceptibility-reducing effects of a robust influenza vaccine. Our results show that while antiviral therapy cannot replace a robust influenza vaccine, it can play a role in reducing attack rates and eliminating outbreaks, and could significantly reduce public health burden when vaccine is either unavailable or ineffective. We also found that antiviral therapy, by treating those who are infected, is naturally a highly optimized strategy, and need not be improved upon with expensive targeted campaigns.

Background Neuromuscular disorders associated with muscular weakness and prolonged paralysis are common in critically ill patients. Acute myopathy has been described in patients receiving a combination therapy of corticosteroids and nondepolarizing neuromuscular blocking drugs for treatment of acute bronchospasm. The cause of this myopathy is not fully established and may involve drug interactions that perturb neuromuscular transmission. To investigate the interaction of corticosteroids with neuromuscular blocking drugs, the authors determined the effects of methylprednisolone and hydrocortisone alone and in combination with vecuronium on fetal (gamma-subunit containing) and adult (epsilon-subunit containing) subtypes of the muscle-type nicotinic acetylcholine receptor. Methods Functional channels were expressed in Xenopus laevis oocytes and activated with 1 microM acetylcholine. The resulting currents were recorded using a whole cell two-electrode voltage clamp technique. Results Both forms of the muscle-type acetylcholine receptor were potently inhibited by methylprednisolone and hydrocortisone, with concentrations producing 50% inhibition in the range of 400-600 microM and 1-2 mM, respectively. The corticosteroids produced noncompetitive antagonism of the muscle-type nicotinic acetylcholine receptor at clinical concentrations. Both receptor forms were also inhibited, even more potently, by vecuronium, with a concentration producing 50% inhibition in the range of 1-2 nM. Combined application of vecuronium and methylprednisolone showed additive effects on both receptor forms, which were best described by a two-site model, with each site independent. Conclusions The enhanced neuromuscular blockade produced when corticosteroids are combined with vecuronium may augment pharmacologic denervation and contribute to the pathophysiology of prolonged weakness observed in some critically ill patients.

Repurposed drugs that are safe and immediately available constitute a first line of defense against new viral infections. Despite limited antiviral activity against SARS-CoV-2, several drugs are being tested as medication or as prophylaxis to prevent infection. Using a stochastic model of early phase infection, we evaluate the success of prophylactic treatment with different drug types to prevent viral infection. We find that there exists a critical efficacy that a treatment must reach in order to block viral establishment. Treatment by a combination of drugs reduces the critical efficacy, most effectively by the combination of a drug blocking viral entry into cells and a drug increasing viral clearance. Below the critical efficacy, the risk of infection can nonetheless be reduced. Drugs blocking viral entry into cells or enhancing viral clearance reduce the risk of infection more than drugs that reduce viral production in infected cells. The larger the initial inoculum of infectious virus, the less likely is prevention of an infection. In our model, we find that as long as the viral inoculum is smaller than 10 infectious virus particles, viral infection can be prevented almost certainly with drugs of 90% efficacy (or more). Even when a viral infection cannot be prevented, antivirals delay the time to detectable viral loads. The largest delay of viral infection is achieved by drugs reducing viral production in infected cells. A delay of virus infection flattens the within-host viral dynamic curve, possibly reducing transmission and symptom severity. Thus, antiviral prophylaxis, even with reduced efficacy, could be efficiently used to prevent or alleviate infection in people at high risk.

The stumbling block for the continued, single-drug use of praziquantel (PZQ) against schistosomiasis is less justified by the risk of drug resistance than by the fact that this drug is inactive against juvenal parasites, which will mature and start egg production after chemotherapy. Artemisinin derivatives, currently used against malaria in the form of artemisinin-based combination therapy (ACT), provide an opportunity as these drugs are not only active against malaria plasmodia, but surprisingly also against juvenile schistosomes. An artemisinin/PZQ combination would be complimentary, and potentially additive, as it would kill two schistosome life cycle stages and thus confer a transmission-blocking modality to current chemotherapy. We focus here on single versus combined regimens in endemic settings. Although the risk of artemisinin resistance, already emerging with respect to malaria therapy in Southeast Asia, prevents use in countries where ACT is needed for malaria care, an artemisinin-enforced praziquantel treatment (APT) should be acceptable in North Africa (including Egypt), the Middle East, China, and Brazil, as these countries are not endemic for malaria. Thanks to recent progress with respect to high-resolution diagnostics, based on circulating schistosome antigens in humans and molecular approaches for snail surveys, it should be possible to keep areas scheduled for schistosomiasis elimination under surveillance, bringing rapid response to bear on problems arising. The next steps would be to investigate where and for how long APT should be applied to make a lasting impact. A large-scale field trial in an area with modest transmission should tell how apt this approach is.

ABSTRACTThe discovery of the human immunodeficiency virus type 1 (HIV-1) in 1982 soon led to the identification and development of antiviral compounds to be used in treatment strategies for infected patients. Early in the epidemic, drug monotherapies frequently led to treatment failures because the virus quickly developed resistance to the single drug. Following the advent of highly active antiretroviral therapy (HAART) in 1995, dramatic improvements in HIV-1-infected patient health and survival were realized as more refined combination therapies resulted in reductions in viral loads and increases in CD4+T-cell counts. In the absence of an effective vaccine, prevention of HIV-1 infection has also gained traction as an approach to curbing the pandemic. The development of compounds as safe and effective microbicides has intensified and has focused on blocking the transmission of HIV-1 during all forms of sexual intercourse. Initial preclinical investigations and clinical trials of microbicides focused on single compounds effective against HIV-1. However, the remarkable successes achieved using combination therapy to treat systemic HIV-1 infection have subsequently stimulated the study and development of combination microbicides that will simultaneously inhibit multiple aspects of the HIV-1 transmission process by targeting incoming viral particles, virus-infected cells, and cells susceptible to HIV-1 infection. This review focuses on existing and developing combination therapies, covering preclinical development,in vitroandin vivoefficacy studies, and subsequent clinical trials. The shift in focus within the microbicide development field from single compounds to combination approaches is also explored.

Background: Interventions to block malaria transmission from humans to mosquitoes are currently in development. To be successfully implemented, key populations need to be identified where the use of these transmission-blocking and/or reducing strategies will have greatest impact. Methods: We used data from a longitudinally monitored cohort of children from Kilifi county located along the Kenyan coast collected between 1998-2016 to describe the distribution and prevalence of gametocytaemia in relation to transmission intensity, time and age. Data from 2,223 children accounting for 9,134 person-years of follow-up assessed during cross-sectional surveys for asexual parasites and gametocytes were used in logistic regression models to identify factors predictive of gametocyte carriage in this cohort. Results: Our analysis showed that children 1-5 years of age were more likely to carry microscopically detectable gametocytes than their older counterparts. Carrying asexual parasites and recent episodes of clinical malaria were also strong predictors of gametocyte carriage. The prevalence of asexual parasites and of gametocyte carriage declined over time, and after 2006, when artemisinin combination therapy (ACT) was introduced, recent episodes of clinical malaria ceased to be a predictor of gametocyte carriage. Conclusions: Gametocyte carriage in children in Kilifi has fallen over time. Previous episodes of clinical malaria may contribute to the development of carriage, but this appears to be mitigated by the use of ACTs highlighting the impact that gametocidal antimalarials can have in reducing the overall prevalence of gametocytaemia when targeted on acute febrile illness.

Background:Interventions to block malaria transmission from humans to mosquitoes are currently in development. To be successfully implemented, key populations need to be identified where the use of these transmission-blocking and/or reducing strategies will have greatest impact.Methods:We used data from a longitudinally monitored cohort of children from Kilifi county located along the Kenyan coast collected between 1998-2016 to describe the distribution and prevalence of gametocytaemia in relation to transmission intensity, time and age. Data from 2,223 children accounting for 9,134 person-years of follow-up assessed during cross-sectional surveys for asexual parasites and gametocytes were used in logistic regression models to identify factors predictive of gametocyte carriage in this cohort.Results:Our analysis showed that children 1-5 years of age were more likely to carry microscopically detectable gametocytes than their older counterparts. Carrying asexual parasites and recent episodes of clinical malaria were also strong predictors of gametocyte carriage. The prevalence of asexual parasites and of gametocyte carriage declined over time, and after 2006, when artemisinin combination therapy (ACT) was introduced, recent episodes of clinical malaria ceased to be a predictor of gametocyte carriage. Conclusions:Gametocyte carriage in children in Kilifi has fallen over time. Previous episodes of clinical malaria may contribute to the development of carriage, but this appears to be mitigated by the use of ACTs highlighting the impact that gametocidal antimalarials can have in reducing the overall prevalence of gametocytaemia when targeted on acute febrile illness.

Purpose. To investigate the efficacy of telbivudine (LdT) in blocking mother-to-child transmission (MTCT) of hepatitis B virus (HBV) during late pregnancy. Methods. A total of 651 pregnant women aged 18-40 in Nantong Third People’s Hospital and Hospital affiliated to Nantong University with positive hepatitis B surface antigen (HBsAg) and HBV DNA were enrolled between January 2011 and December 2015. Patients with HBV DNA≥106 copies/mL (n=251) received LdT during late pregnancy according to the patients’ will, while 136 high viral patients with HBV DNA≥106 copies/mL who did not take LdT therapy and 268 low viral patients with HBV DNA<106 copies/mL served as the controls. Results. At 7 months and 1 year postpartum, the basal HBV DNA serum level of treated patients declined significantly (P<0.001), while no obvious decline was observed in the untreated high viraemic controls (P<0.05) and untreated low viraemic controls (P<0.05). Only 1 infant (0.4%) in LdT group was HBsAg positive at 7 months, while 14 (5.2%) were in the untreated low viraemic controls (P<0.001) and 15 (11.0%) were in untreated high viraemic controls (P<0.001). Conclusion. For pregnant women with HBV DNA≥106 copies/mL, the use of LdT during late pregnancy could effectively reduce the MTCT rate of HBV.

ABSTRACTThe design of new antimalarial combinations to treatPlasmodium falciparuminfections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinantP. falciparumline expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the early-stage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC50s) below 2.5 μM. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of ≤5%, an average signal-to-noise ratio (S:N) of >30, and a Z′ of ∼0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy.

ABSTRACTThe maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency againsttransinfection of T cells byin vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

Atherosclerosis is the key pathogenesis of cardiovascular diseases; oxidative stress, which is induced by the generated excess reactive oxygen species (ROS), has been a crucial mechanism underlying this pathology. Nanoparticles (NPs) represent a novel strategy for the development of potential therapies against atherosclerosis, and multifunctional NPs possessing antioxidative capacities hold promise for amelioration of vascular injury caused by ROS and for evading off-target effects; materials that are currently used for NP synthesis often serve as vehicles that do not possess intrinsic biological activities; however, they may affect the surrounding healthy environment due to decomposition of products. Herein, we used nontoxic fucoidan, a sulfated polysaccharide derived from a marine organism, to develop chitosan–fucoidan nanoparticles (CFNs). Then, by binding to P-selectin, an inflammatory adhesion exhibited molecule expression on the endothelial cells and activated platelets, blocking leukocyte recruitment and rolling on platelets and endothelium. CFNs exhibit antioxidant and anti-inflammatory properties. Nevertheless, by now, the application of CFNs for the target delivery regarding therapeutics specific to atherosclerotic plaques is not well investigated. The produced CFNs were physicochemically characterized using transmission electron microscopy (TEM), together with Fourier transform infrared spectroscopy (FTIR). Evaluations of the in vitro antioxidant as well as anti-inflammatory activities exhibited by CFNs were based on the measurement of their ROS scavenging abilities and investigating inflammatory mediator levels. The in vivo pharmacokinetics and binding efficiency of the CFNs to atherosclerotic plaques were also evaluated. The therapeutic effects indicated that CFNs effectively suppressed local oxidative stress and inflammation by targeting P-selectin in atheromatous plaques and thereby preventing the progression of atherosclerosis.

Background/Aims: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. Results: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. Conclusion: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

ABSTRACTAn innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected withLeishmania infantumwere treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 hper os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.

As one of the most common malignancies worldwide, Hepatocellular carcinoma (HCC) has been treated by Sorafenib, which is the first approved target drug by FDA for advanced HCC. However, drug resistance is one of the obstacles to its application. As a typical characteristic of most solid tumors, hypoxia has become a key cause of resistance to chemotherapy and radiotherapy. It is important to elucidate the underlying mechanisms of Sorafenib resistance under hypoxia. In this study, the morphological changes of hepatocellular carcinoma cells were observed by Live Cell Imaging System and Transmission Electron Microscope; Sorafenib was found to induce necroptosis in liver cancer. Under hypoxia, the distribution of necroptosis related proteins was changed, which contributed to Sorafenib resistance. HSP90α binds with the necrosome complex and promotes chaperone-mediated autophagy (CMA) degradation, which leads necroptosis blocking and results in Sorafenib resistance. The patient-derived tumor xenograft (PDX) model has been established to investigate the potential therapeutic strategies to overcome Sorafenib resistance. 17-AAG inhibited HSP90α and presented obvious reversal effects of Sorafenib resistance in vivo and in vitro. All the results emphasized that HSP90α plays a critical role in Sorafenib resistance under hypoxia and 17-AAG combined with Sorafenib is a promising therapy for hepatocellular carcinoma.

As one of the most common malignancies worldwide, Hepatocellular carcinoma (HCC) has been treated by Sorafenib, which is the first approved target drug by FDA for advanced HCC. However, drug resistance is one of the obstacles to its application. As a typical characteristic of most solid tumors, hypoxia has become a key cause of resistance to chemotherapy and radiotherapy. It is important to elucidate the underlying mechanisms of Sorafenib resistance under hypoxia. In this study, the morphological changes of hepatocellular carcinoma cells were observed by Live Cell Imaging System and Transmission Electron Microscope; Sorafenib was found to induce necroptosis in liver cancer. Under hypoxia, the distribution of necroptosis related proteins was changed, which contributed to Sorafenib resistance. HSP90α binds with the necrosome complex and promotes chaperone-mediated autophagy (CMA) degradation, which leads necroptosis blocking and results in Sorafenib resistance. The patient-derived tumor xenograft (PDX) model has been established to investigate the potential therapeutic strategies to overcome Sorafenib resistance. 17-AAG inhibited HSP90α and presented obvious reversal effects of Sorafenib resistance in vivo and in vitro. All the results emphasized that HSP90α plays a critical role in Sorafenib resistance under hypoxia and 17-AAG combined with Sorafenib is a promising therapy for hepatocellular carcinoma.

Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.

Abstract BACKGROUND: Acute leukemia comprises malignant diseases of clonal character, to which specific treatment remains limited. Apoptosis induced by death receptor activation (i.e. by tumor necrosis factor-related apoptosis inducing ligand, TRAIL/APO2L) represents a potential method for cancer therapy. A loss of sensitivity to apoptosis represents one of the key molecular mechanisms of cancer cell resistance to chemo/immuno/radiotherapy. TRAIL, a member of the TNF family of death ligands, appears to specifically and efficiently kill tumor cells of diverse origin while sparing normal tissues. The TRAIL receptor family consists of five receptors: two death receptors (DR4, DR5), two decoy receptors (DcR1, DcR2), and osteoprotegerin (OPG). AIMS: Analysis of the molecular basis of TRAIL resistance and functional analysis of individual TRAIL receptors in HL60 myeloid leukemia cells. MATERIALS AND METHODS: TRAIL-resistant cells were selected from the original HL60 population using pressure of recombinant TRAIL. The expression of TRAIL receptors and other apoptosis regulating molecules and CD14 was analyzed by flow cytometry and by real time RT-PCR. Percentage of apoptotic cells was measured by flow cytometry using Annexin-V-FITC/PI apoptosis detection kit. The contribution of individual TRAIL receptors on the transmission of apoptotic signal was measured using blocking antibodies to TRAIL receptors. The TRAIL resistance related genome aberrations were analyzed by comparative genomic hybridization (CGH). RESULTS: Blocking antibodies to DR4 receptors significantly reduced the number of apoptotic HL60 cells compared to untreated controls. The blockage of DR5 receptors did not significantly inhibited TRAIL-induced cell death. Combination of anti-DR4 and anti-DR5 antibodies almost completely abrogated TRAIL-induced HL60 cell death and significantly reduced apoptosis compared to control or anti-DR4 antibody alone (p&lt;0.01). Blocking of decoy receptors (DcR1, DcR2, and OPG) did not significantly affect the apoptotic signaling in HL60 TRAIL-sensitive and TRAIL-resistant cell lines. The TRAIL-resistant HL60 phenotypes were characterized by the decreased expression of TRAIL receptors DR4, DR5, DcR1, and DcR2, decreased or increased expression of CD14, and unchanged or undetectable expression of OPG as compared to control TRAIL-sensitive HL60 cells. CGH showed the loss of genomic material of long arm of chromosome 8, at 8q2, in all tested HL60 TRAIL-resistant lines (n=6). Further, a loss of genomic material in at least two TRAIL-resistant lines was detected at 1p3, 8p11–22, and chromosome 19. The gain of genomic material in at least two TRAIL-resistant lines was detected at 1q21–23, 6q2, and 15q11.1–21.1. Other identified chromosomal aberrations were unique for individual TRAIL-resistant leukemia lines derived from HL60 cell line. SUMMARY/CONCLUSIONS: HL60 cells transduced TRAIL-specific apoptotic signal predominantly through TRAIL receptor DR4. Decoy receptors, including OPG, did not play a significant role in TRAIL resistance. The identified TRAIL-resistant phenotypes are associated with distinct genomic changes.

Abstract Drug resistance remains a significant obstacle in the successful treatment of cancer, highlighting the critical need to understand how oncogenes and cancer drugs impact cell physiology and resistance development. EML4-ALK is a receptor tyrosine kinase (RTK) fusion oncogene that drives 3-7% of lung cancer. Despite potent ALK inhibitors, EML4-ALK+ cancers frequently develop resistance during therapy. Recently, it was discovered that EML4-ALK and other RTK fusions form cytoplasmic protein condensates, and that condensate formation was required for oncogenic signaling. However, whether oncogenic condensates play a role in drug responses is unclear. In this study, we applied an optogenetic technique called ‘functional profiling’ to understand how EML4-ALK condensates impact cell signal transmission and drug response. Using light-stimulated RTKs, we found that EML4-ALK condensates strongly suppress signaling through transmembrane RTKs, including through EGFR, a central receptor in resistance development. Strikingly, treatment with ALK inhibitors (ALKi) rapidly restored and hypersensitized RTK signaling. We found that EML4-ALK condensates suppress RTK signals through sequestration of the downstream adapter Grb2, which is essential for signaling through EGFR and other RTKs. The release of Grb2 from condensates resensitized RTKs within 10s of minutes of ALKi addition. Resensitized RTKs, in turn, caused sporadic RTK activation pulses throughout the cell population, and pulses originated from paracrine RTK signals released by apoptotic neighbors. We found that these paracrine signals counteracted ALK inhibitor therapy and promoted survival and drug tolerance. Blocking paracrine signals through co-treatment of ALKi with inhibitors of either EGFR or matrix metalloproteases enhanced cell killing and minimized long-term drug tolerance. Our study uncovers a role for oncogenic condensates in drug resistance signaling, reveals a novel mechanism for oncogene-induced suppression of RTK signaling, and suggests novel co-therapies to more effectively treat cancers driven by EML4-ALK and possibly other RTK fusions. Our work also demonstrates the potential of functional optogenetic profiling for drug discovery to promote cancer therapy. Citation Format: David Gonzalez-Martinez, Lee Roth, Thomas Mumford, Yael Mosse, Asmin Tulpule, Trever Bivona, Lukasz Jan Bugaj. Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 848.

Medical science technology has improved tremendously over the decades with the invention of robotic surgery, gene editing, immune therapy, etc. However, scientists are now recognizing the significance of ‘biological circuits’ i.e., bodily innate electrical systems for the healthy functioning of the body or for any disease conditions. Therefore, the current trend in the medical field is to understand the role of these biological circuits and exploit their advantages for therapeutic purposes. Bioelectronics, devised with these aims, work by resetting, stimulating, or blocking the electrical pathways. Bioelectronics are also used to monitor the biological cues to assess the homeostasis of the body. In a way, they bridge the gap between drug-based interventions and medical devices. With this in mind, scientists are now working towards developing flexible and stretchable miniaturized bioelectronics that can easily conform to the tissue topology, are non-toxic, elicit no immune reaction, and address the issues that drugs are unable to solve. Since the bioelectronic devices that come in contact with the body or body organs need to establish an unobstructed interface with the respective site, it is crucial that those bioelectronics are not only flexible but also stretchable for constant monitoring of the biological signals. Understanding the challenges of fabricating soft stretchable devices, we review several flexible and stretchable materials used as substrate, stretchable electrical conduits and encapsulation, design modifications for stretchability, fabrication techniques, methods of signal transmission and monitoring, and the power sources for these stretchable bioelectronics. Ultimately, these bioelectronic devices can be used for wide range of applications from skin bioelectronics and biosensing devices, to neural implants for diagnostic or therapeutic purposes.

Disorders in the functions of platelets – blood cells responsible for the blood clots formation and prevention – are observed as independent diseases, as a complication of cancer and hematological diseases or as a result of a therapy. Nowadays, a test of platelet aggregation by aggregometry is the only diagnostic method for assessing the platelets functions. There are several varieties of aggregometry, which differ both in the method of recording the formation of platelet aggregates and in the method of preparing platelets for the experiment. In most laboratories, it is customary to conduct aggregometry in platelet-rich plasma in the presence of citrate ions. In this case, the concentration of calcium ions in plasma decreases, it prevents the thrombin formation and the plasma coagulation. On the other hand, it has long been known that platelet aggregation in response to ADP in the presence of calcium ions (in blood plasma collected in heparin or hirudin tubes, also blocking plasma clotting) is reversible: after 1-5 minutes after the addition of the activator, the disaggregation begins until the light transmission of the solution (platelet concentration) returns to its original level. This phenomenon is called "reversible” platelet aggregation. Reversible aggregation (“disaggregation”) is sometimes observed in aggregometry of citrate plasma, especially in pediatric patients. However, it is usually not considered normal and is considered a sign of platelet dysfunction. This review considers the known mechanisms of disaggregation in the presence or absence of calcium ions in the medium. The role of secondary activation of platelets as a potential cause of irreversible aggregation is discussed, as well as possible versions for explaining the results of aggregometry, when reversible platelet aggregation is observed.

ABSTRACT Background The asexual blood stages of the Plasmodium berghei life cycle including merozoites are attractive targets for transmission blocking vaccines and drugs. Improved understanding of P. berghei life cycle stage growth and development would provide new opportunities to evaluate antimalarial vaccines and drugs. Methods Blood stage samples from C57BL/6 albino mice infected with P. berghei sporozoites were singly stained with a high binding affinity deoxyribonucleic acid dye, YOYO-1, and measured by flow cytometry (FCM). Duplicate slides were made from samples and stained with diluted Giemsa’s and YOYO-1, respectively. Correlated results were compared by FCM, light microscopy, and fluorescent microscopy. Results Complete life cycle stage determination and analysis by FCM is reported to include merozoites, ring forms, trophozoites, immature, and mature schizonts. FCM demonstrated a clear separation between each stage using their unique fluorescence distribution. When compared to light microscopy, a strong correlation (r 2 = 0.925 to 0.974) was observed in determining the ring forms, trophozoites, and schizonts phases, but only a moderate correlation (r 2 = 0.684 to 0.778) was observed for merozoites. The identification and measurement of merozoites suggest that FCM is a useful technique to monitor the entire life stage of the parasite. Initial stage-specific data demonstrated that mefloquine has a mode of action on mature parasite forms, and artesunic acid was rapidly effective against merozoites and other immature and mature parasite forms with higher killing. Conclusion Blood stage parasites in each individual life stage, including merozoites, are reliably identified and quantified quickly by FCM, making this technique an ideal alternative to microscopy. This integrated whole life stage model, particularly with confirmed determination of merozoite population, could widely be used for drug and vaccine research in malaria therapy and prophylaxis.

Botulinum toxin type A-induced changes in the chemical coding of dorsal root ganglion neurons supplying the porcine urinary bladder Botulinum toxin type A (BTX) is a potent neurotoxin, which in recent years has been effectively applied in experimental treatments of many neurogenic disorders of the urinary bladder. BTX is a selective, presynaptically-acting blocking agent of acetylcholine release from nerve terminals what, in turn, leads to the cessation of somatic motor and/or parasympathetic transmission. However, application of this toxin in urological practice is still in the developmental stages and the full mechanism of its action remain elusive. Thus, the present study was aimed at investigating the neurochemical characterization of dorsal root ganglion (DRG) neurons supplying the porcine urinary bladder after BTX treatment. Retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall in six juvenile female pigs and three weeks later, intramural bladder injections of BTX (100 IU per animal) were carried out in all the animals. After a week, DRG from L1 to Cq1 were harvested from the pigs and neurochemical characterization of FB+ neurons was performed using double-labeling immunofluorescence technique on 10-μm-thick cryostat sections. BTX injections led to a significant decrease in the number of FB+ neurons containing substance P (SP), calcitonin gene-related peptide (CGRP), calbindin (CB), somatostatin (SOM) and neuronal nitric oxide synthase (nNOS) when compared with that found in the healthy animals (19% vs. 45%, 18% vs. 36%, 0.6% vs. 3%, 0.4 vs. 4% and 0.1% vs. 6%, respectively) These data demonstrated that BTX changed the chemical coding of bladder sensory neurons, and therefore this drug should be taken into consideration when it planning experimental therapy of selected neurogenic bladder disorders.

Background The most sensitive diagnostic criterion of myasthenia gravis is a decrement in the muscular response to repetitive stimulation. The authors hypothesized that myasthenia gravis patients who show a train-of-four ratio (T4/T1) &lt; 0.9 in the preanesthetic period will have increased sensitivity to nondepolarizing neuromuscular blocking agents compared with myasthenia gravis patients with preanesthetic T4/T1 &gt; or = 0.9. Methods After institutional review board approval was obtained, 20 electrophysiologically documented myasthenia gravis patients were studied. Current pyridostigmine therapy was continued until the morning of surgery. Before induction of anesthesia, neuromuscular transmission was recorded from the hypothenar muscles using electromyography with train-of-four stimulation of the ulnar nerve. According to the T4/T1 ratio, patients were assigned to the "normal" group (T4/T1 &gt; or = 0.9) or the "decrement" group (T4/T1 &lt; 0.9). After induction of intravenous anesthesia, the effective dose to achieve a 95% neuromuscular blockade (ED95) for atracurium was assessed with a cumulative bolus technique. Postoperatively, pyridostigmine was titrated to obtain a T4/T1 &gt; 0.75 and to treat residual myasthenic symptoms. Results In 14 patients, preanesthetic T4/T1 was &gt; or = 0.9 (normal), whereas 6 patients presented with T4/T1 &lt; 0.9 (decrement). Decrement patients had a lower ED95 of 0.07 +/- 0.03 mg/kg atracurium (mean +/- SD) compared with normal patients with an ED95 of 0.24 +/- 0.11 mg/kg atracurium (P = 0.002). All patients were extubated within 30 min after surgery. Postoperative pyridostigmine infusion did not differ significantly between groups. Conclusions The requirement for atracurium is significantly reduced in myasthenia gravis patients with a T4/T1 ratio &lt; 0.9 before anesthesia. This study indicates that routine neuromuscular monitoring in myasthenia gravis patients should be extended into the preinduction period to identify patients who require less atracurium.

A major cause of cancer cell resistance to chemotherapeutics is the blocking of apoptosis and induction of autophagy in the context of cell adaptation and survival. Therefore, new compounds are being sought, also among drugs that are commonly used in other therapies. Due to the involvement of histamine in the regulation of processes occurring during the development of many types of cancer, antihistamines are now receiving special attention. Our study concerned the identification of new mechanisms of action of azelastine hydrochloride, used in antiallergic treatment. The study was performed on HeLa cells treated with different concentrations of azelastine (15–90 µM). Cell cycle, level of autophagy (LC3 protein activity) and apoptosis (annexin V assay), activity of caspase 3/7, anti-apoptotic protein of Bcl-2 family, ROS concentration, measurement of mitochondrial membrane potential (Δψm), and level of phosphorylated H2A.X in response to DSB were evaluated by cytometric method. Cellular changes were also demonstrated at the level of transmission electron microscopy and optical and fluorescence microscopy. Lysosomal enzyme activities-cathepsin D and L and cell viability (MTT assay) were assessed spectrophotometrically. Results: Azelastine in concentrations of 15–25 µM induced degradation processes, vacuolization, increase in cathepsin D and L activity, and LC3 protein activation. By increasing ROS, it also caused DNA damage and blocked cells in the S phase of the cell cycle. At the concentrations of 45–90 µM, azelastine clearly promoted apoptosis by activation of caspase 3/7 and inactivation of Bcl-2 protein. Fragmentation of cell nucleus was confirmed by DAPI staining. Changes were also found in the endoplasmic reticulum and mitochondria, whose damage was confirmed by staining with rhodamine 123 and in the MTT test. Azelastine decreased the mitotic index and induced mitotic catastrophe. Studies demonstrated the multidirectional effects of azelastine on HeLa cells, including anti-proliferative, cytotoxic, autophagic, and apoptotic properties, which were the predominant mechanism of death. The revealed novel properties of azelastine may be practically used in anti-cancer therapy in the future.

ABSTRACTThe initial phases of acute human immunodeficiency virus type 1 (HIV-1) infection may be critical for development of effective envelope (Env)-specific antibodies capable of impeding the establishment of the latent pool of HIV-1-infected CD4+T cells, preventing virus-induced immune hyperactivation to limit disease progression and blocking vertical virus transmission. However, the initial systemic HIV-1 Env-specific antibody response targets gp41 epitopes and fails to control acute-phase viremia. African-origin, natural simian immunodeficiency virus (SIV) hosts do not typically progress to AIDS and rarely postnatally transmit virus to their infants, despite high milk viral loads. Conversely, SIV-infected rhesus macaques (RMs), Asian-origin nonnatural SIV hosts, sustain pathogenic SIV infections and exhibit higher rates of postnatal virus transmission. In this study, of acute SIV infection, we compared the initial systemic Env-specific B cell responses of AGMs and RMs in order to probe potential factors influencing the lack of disease progression observed in AGMs. AGMs developed higher-magnitude plasma gp120-specific IgA and IgG responses than RMs, whereas RMs developed more robust gp140-directed IgG responses. These gp120-focused antibody responses were accompanied by rapid autologous neutralizing responses during acute SIV infection in AGMs compared to RMs. Moreover, acute SIV infection elicited a higher number of circulating Env-specific memory B cells in peripheral blood of AGMs than in the blood of RMs. These findings indicate that AGMs have initial systemic Env-specific B cell responses to SIV infection distinct from those of a nonnatural SIV host, resulting in more functional SIV-specific humoral responses, which may be involved in impairing pathogenic disease progression and minimizing postnatal transmission.IMPORTANCEDue to the worldwide prevalence of HIV-1 infections, development of a vaccine to prevent infection or limit the viral reservoir remains an important goal. HIV-1-infected humans, as well as SIV-infected nonnatural SIV hosts, develop pathogenic infections and readily transmit the virus to their infants. Conversely, natural SIV hosts do not develop pathogenic infections and rarely transmit the virus to their infants. The immunologic factors contributing to these favorable outcomes in natural SIV hosts could prove invaluable for directing HIV-1 vaccine and therapy design. This study identified distinctions in the specificity and function of the initial systemic SIV envelope-specific B cell response that developed during acute SIV infection in natural and nonnatural SIV host species. Identification of distinct acute B cell responses in natural SIV hosts may inform vaccine strategies seeking to elicit similar responses prior to or during the initial phases of acute HIV-1 infection.

Abstract Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

Abstract The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep mechanism. The viral envelope glycoproteins (env) binds first to CD4 and subsequently interacts with the V3 loop with a chemokine receptor, CCR5 or CXCR4, triggering the fusion event. Several findings suggest that viruses using CCR5 for entry (R5-tropic HIV-1) is the predominant species transmitted among patients. Importantly, CCR5 expression levels determine disease progression. CCR5 does not seem to be necessary for normal cell function, since individuals with a homozygous mutation (Δ32) do not appear to have clinical, immune alterations. Furthermore, these individuals are highly protected against transmission of R5-tropic HIV-1. Therefore, intervention strategies aimed at altering or blocking CCR5 expression may be beneficial for cellular protection and provide a clinical benefit against HIV-1 infection. We have constructed an HIV-1 derived vector, CAD-R5, expressing an intracellular single-chain antibody (intrabody) specific for CCR5 coupled to a KDEL endoplasmic reticulum retention signal. Intrabody expressing primary T-cells efficiently disrupted CCR5 cell surface expression with a 4.3-fold reduction in CCR5 mean fluorescence intensity (MFI) as compared to the control vector without the intrabody gene (7.9 and 33.6 MFI, respectfully). CAD-R5 transduced primary CD4 T-cells expressing the intrabody gene were resistant to R5-tropic HIV infection as shown by a 50 to 60-fold reduction in HIV-1 p24 concentration. Moreover, intrabody gene expressing cells demonstrated a selective advantage and enriched by 6.4-fold in a population of infected cells as compared to uninfected or infected cells containing vector without the intrabody gene. The SCID-human mouse model, produced by conjoining fetal human thymus and liver under the renal capsule, has become a staple for in vivo testing of T cell anti-retroviral therapy. When CAD-R5 transduced, fetal liver derived CD34+ stem cells were used for reconstitution of the human implants in SCID-hu mice (n = 2, 2 groups), 6-weeks later the human thymi displayed an average of 25% reporter gene expressing T cells. Thymocyte development was not altered by vector integration or loss of CCR5 cell surface expression as determined by the CD4+/CD8+ staining profile. Importantly, CCR5 intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge with a 3-fold reduction in viral load. These results validate the efficacy of lentiviral delivered CCR5 intrabody mediated protection from R5-tropic HIV-1. The findings also underscore the potential advantage of intrabody gene delivery to CD34+ stem cells, which allows differentiation of protected T cell progeny.

Abstract Normal karyotype AML patients with FLT3 ITD have a higher risk of relapse than those lacking this mutation (Estey EH, Am J Hematol. 2013). Our comparative genomic hybridization study (77 patients) showed that AML cases with as compared to those without FLT3 ITD had significantly lower number of amplifications or deletions (number of total CNV aberrations: 18±4 vs 51±8, p=0.003). The latter observation in concert with that of others (Thiede, Blood 2002) suggest that FLT3 ITD mutation if present in normal karyotype patients is a key player in the pathogenesis of leukaemia and targeting this mutation may be used successfully in FLT3 ITD positive patients relapsing post HSCT. This was also the case in the two patients presented in this study. Patient UPN 952, male, 53 years old, AML M4 (FAB), normal karyotype, received alloHSCT in 2 CR after completion of two lines of remission induction therapies (first: DA3+7 (Daunorubicin, Ara-C), HAM, high dose Ara-C; second: ICE (IDA, Ara-C, VP-16)) and then promptly transplanted. He relapsed 56 days after transplantation. Patient UPN 938, female, 50 years old, AML, normal karyotype, received as an induction DA 3+7, and for consolidation HAM and high dose Ara-C and completed only one course of the maintenance therapy and transplanted in CR. Both cases were transplanted from unrelated donors (10/10 HLA A, B, C, DR and DQ alleles matched), they received myeloablative conditioning (i.v. Busulfan and Cyclophosphamide) and Cyclosporin A as GvHD prophylaxis. At relapse they received salvage chemotherapy tailored to their biological performance (UPN 938 having Age Adjusted Charlson Comorbidity Index 3 received FLAG and UPN 952 with the index 9, ECOG 3, DA2+5). Both cases received in addition Sorafenib (two times 400 mg per day). The response was prompt and the marrow was free from blasts beginning from 11 day post chemotherapy. UPN 952 received as a maintenance only one course of 6-TG with low dose of Ara-C. Due to the substantial comorbidity and liver toxicity WHO3 further chemotherapy treatment was terminated. The patient was left only on Sorafenib (2 times 200 mg per day). UPN 938 was receiving the maintenance therapy (AML protocol) in 6 – 8 weeks intervals based on low dose Ara-C with 6TG or DNR and also Sorafenib (2 times 200 mg per day). In both cases FLT3 signalling pathway (FLT3 Pathway Mutation PCR Array, SABiosciences, Qiagen) revealed a lack of any additional mutation at the check points in FLT3, KRAS, HRAS, NRAS, MEK1, PIK3CA, BRAF and PTEN genes. UPN 938 had in addition to FLT3 ITD mutation c.1807_1808insATGAATATGATCTCAAAT (p.K602_W603insYEYDLK) insertion which relevance was not so far describe. Sorafenib resistance mutations were not found. The patients were on Sorafenib for 8 (UPN 938) and 9 months (UPN 952). The course of UPN938 was uncomplicated. The second case showed mild aGvHD symptoms evolving into cGvHD (skin lesions and dry eye) slowed down with rapamicine. Up to now the patients are in CR and free from FLT3 ITD. The main observation points: - Sorafenib with chemotherapy tailored to the biologic performance of patients contributes to the efficient salvage in patients with relapsing FLT3 ITD positive AML post HSCT and is also effective given alone as a maintenance. - Side effects were seen in one out of two patients likely associated with the blocking of VEGFR signalling transmission (hand foot skin rash, von Willebrandt factor activity – 388%). Conclusion: The use of multikinase inhibitor (Sorafenib) contributes effectively to salvage therapy in FLT3+ AML patients relapsing post alloHSCTSorafenib given alone is able to maintain long-lasting remissionSide effects are individually dependent Supported by NBiR CellsTherpy grant and Byer Health Care Disclosures No relevant conflicts of interest to declare.

Background: Ketamine might be effective in blocking central sensitization of pain transmission neurons through its effect on NMDA receptors in refractory Complex Regional Pain Syndrome (CRPS) patients. At higher doses, ketamine infusions can be associated with significant risks; outpatient therapy requires return visits for a 10-day period with variable efficacy and duration. Objective: This study determined the efficacy of a 5-day moderate dose, continuous racemic ketamine infusion. The pharmacodynamic responses to racemic ketamine and norketamine were examined. Design: Observational study Methods: In this study, ketamine was titrated from 10-40 mg/hour in 16 CRPS patients, and maintained for 5 days. Pain was assessed daily. Ketamine and norketamine concentrations were obtained on Day 1 before starting the infusion; at 60 to 90 minutes, 120 to 150 minutes, 180 to 210 minutes, and 240 to 300 minutes after the initiation of the infusion on Days 2, 3, 4, and 5; and on Day 5 at 60 minutes after the conclusion of the infusion. The plasma concentrations of (R)-ketamine, (S)-ketamine, (R)-norketamine and (S)-norketamine were determined using an enantioselective liquid chromatography – mass spectrometry method. Results: Ketamine and norketamine infusion rates stabilized 5 hours after the start of the infusion. The subjects showed no evidence of significant tachycardia, arterial oxygen desaturation, or hallucinatory responses. Subjects generally experienced minimal pain relief on day one followed by significant relief by day 3. Mean pain scores decreased from the 8-9 to 3-5 ranges; however, the analgesic response to ketamine infusion was not uniform. On day 5, there was little or no change in the pain measure assessed as the worst pain experienced over the last 24 hours in 37% of the subjects. (R)- and (S)-ketamine concentrations peaked at 240-300 min. (R)- and (S)-norketamine concentrations were lower and peaked on Day 2 of the infusion, as opposed to Day 1 for (R)- and (S)-ketamine. Significant pain relief was achieved by the second day of infusion and correlated with the maximum plasma levels of ketamine and norketamine. Pain relief continued to significantly improve over the 5 day infusion at concentrations of 200-225 ng/mL for (R)- and (S)-ketamine, and 90-120 ng/mL for(R)- and (S)-norketamine. Conclusions: A 5-day ketamine infusion for the treatment of severe CRPS provided significant (P <0.05) pain relief by Day 3 compared to baseline. The pain relief experienced on Day 2 of the infusion continued to improve over the 5-day infusion period and correlated with the maximum plasma levels of ketamine and norketamine. We speculate that downstream metabolites of ketamine and norketamine might be playing a role in its therapeutic efficacy. Key words: ketamine, norketamine, CRPS, pharmacodynamics, chronic pain, enantiomers

The SARS-CoV-2 coronavirus (COVID-19) pandemic has precipitated an enormous collaborative global effort within the scientific and medical community in search of therapeutic and preventative solutions. The aim of this review is to collate the key developments regarding pharmacological treatments tested and vaccine candidates that have been approved to treat and arrest the spread of COVID-19. Introduction COVID-19 Transmission The COVID-19 outbreak has caused one of the most widespread and significant public health crises in decades. It has become one of the leading causes of death internationally. The primary route of transmission from person-to-person is from airborne aerosol spread through close physical contact, particularly in enclosed, poorly ventilated areas.(1) Transmission through contaminated objects was originally considered a major transmission contributor; however, it is no longer considered a significant driver of the spread. Wearing masks has shown to be effective at preventing or curtailing viral transmission, especially when combined with other measures like social distancing and depopulation of indoor communal spaces.(2) Mechanism of action: The mechanism of action and entry into human physiology at a cellular level has been described previously.(3) Briefly, the virus binds and enters the host cell through a spike protein expressed on its surface. The infection begins when the long protruding spike proteins that latches on to the angiotensin-converting enzyme 2 (ACE-2), a receptor involved in regulating blood pressure ACE-2 protein. From this point, the spike transforms, unfolding and refolding itself, using coiled spring-like parts that start out buried at the core of the spike. The reconfigured spike hooks and docks the virus particle to the host cell. This forms a channel allowing the viral genetic material into the unsuspecting cell, in the case of COVID-19, type II lung cells. From this point onwards, most of the damage caused by COVID-19 results from the immune system going into overdrive to stop the virus from spreading.(4) The influx of immune cells to the infected tissue causes severe damage in the process of cleaning out the virus, infected cells, and bacterial infections with potentially lethal consequences. Treatments Medical therapies to treat COVI-19 evolved rapidly. Treatments include drugs that were approved by the US Food and Drug Administration (FDA) and drugs made available under FDA emergency use authorizations (EUA). The Centers for Disease Control and Prevention (CDC) has strongly encouraged clinicians, patients, and their advocates to consult the treatment guidelines published by the National Institute of Health (NIH). These guidelines are based on scientific evidence and expert opinion.(5) Several therapeutic modalities have been tested and deployed to treat the disease, some of which are summarized here. Anti-virals: Antivirals are drugs that arrest the replication of the virus. They are generally considered most effective when administered in the early phase of infection. Remdesivir: To date, remdesivir is currently the only antiviral approved under EUA by the FDA to treat COVID-19. The approval was based on findings that hospitalized patients who receivedremdesivir recovered faster.(6)Remdesivir can be administered either alone or in combination with other medications. Molnupiravir: An antiviral drug, previously known as EIDD-2801, appears safe and effective. Viral levels reduce to undetectable levels in COVID-19 patients after five days of administration, according to data from a US-based Phase II clinical trial. While molnupiravir is proven to inhibit coronavirus replication in infected patients, more data is required to determine whether it can prevent severe illness.(7) Lopinavir/ritonavir: Lopinavir/ritonavir are anti-human immunodeficiency virus (HIV) drugs. Both have been investigated and neither drug showed any efficacy in large randomized controlled trials in hospitalized COVID-19 patients.(8) Anti-inflammatories: One reason for mortality in COVID-19 infected patients is an overactive response by the patient’s immune system. This response leads to several inflammatory disorders, not least of which is the much publicized “cytokine storm”. The following outlines agents have been tested to dampen the inflammatory response to COVID-19. Dexamethasone: Dexamethasone is an anti-inflammatory corticosteroid used for many years to treat autoimmune conditions and allergic reactions. It is cheap and widely available and has been shown to reduce mortality in the sickest hospitalized patients by dampening the immune response.(9) A meta-analysis study evaluating the results of seven trials shows the death rates were lower in hospitalized patients who took one of three different corticosteroids — dexamethasone, hydrocortisone, or methylprednisolone.(6) Baricitinib: Baricitinib is an anti-inflammatory drug used for the treatment of rheumatoid arthritis. In November 2020, the FDA issued an EUA to use baricitinib in combination with remdesivir in hospitalized adults and children two years and older requiring respiratory support. However, there is not enough evidence to support the use of this therapy with or without remdesivir.(10) Antibody Based Treatments: Antibodies are proteins generated by the immune system to help fight infections, such as viruses, by binding to and destroying them. Antibody-based treatments are likely most helpful soon after infection, rather than after the disease has progressed. Monoclonal antibodies: Monoclonal antibodies are synthesized in the laboratory. The FDA has approved two monoclonal antibody treatments, one single antibody from Eli Lilly, and a combination of two antibodies from Regeneron. The Eli Lilly antibody, Bamlanivimab (LY-CoV555), works by blocking COVID-19 from entering and infecting human cells. Preliminary results indicated that patients with mild-to-moderate COVID-19 who received bamlanivimab were less likely to be hospitalized. Studies are still underway, both as a monotherapy and combination therapy. Regeneron’s treatment utilizes a combination of two monoclonal antibodies, casirivimab and imdevimab (REGN-COV2), referred to as an antibody cocktail. Preliminary trial data reported that REGN-COV2 reduced viral load and relieved symptoms sooner in non-hospitalized patients. These treatments are available for patients under EUAs, but more data is required before becoming part of routine care.(6) Convalescent plasma: One of the first groups of antibody-based treatments used convalescent plasma (plasma from recovered COVID-19 patients). This treatment involves administering plasma from a recovered individual into someone infected with the virus. Theoretically, the antibodies fromthe recovered individual neutralize the virus in the infected individual. Although the FDA issued an EUA for convalescent plasma for hospitalized patients with COVID-19, the data to date has been conflicting and inconclusive.(6) Anti-coagulants: Because of the systemic nature of COVID-19 where the circulatory system supplies all parts of the body, some COVID-19 deaths are believed to be caused by blood clots forming in major arteries and veins. A recent study has reported that full-dose blood thinners decreased the need for life support and improved outcome in hospitalized COVID-19 patients. (11) This worldwide large clinical trial, where full dose treatments were administered to moderately ill patients hospitalized for COVID-19, reduced the requirement of vital organ support—such as the need for ventilators. In addition to some of the FDA approved drugs cited in the previous section, multiple treatments were investigated during the early phase of the COVID crisis, with varying results.(12) In contrast to the overall trials for COVID-19 treatments, the programs initiated for vaccine development have been incredibly successful, surpassing all expectations. Vaccines From the outset of the COVID-19 pandemic, vaccines ultimately offer the most appealing and robust therapeutic modality as they prevent the disease from taking hold. This has led to a global vaccine R&D effort that is unprecedented in terms of scale and delivery. The urgency to create a vaccine for COVID‑19 led to expedited schedules that compressed the standard vaccine development timeline from years to months. At the time of writing, three vaccines have been authorized for emergency use by the FDA in the US, with more likely to come onstream as they progress through the development pipeline. A fourth vaccine, from Oxford-AstraZeneca, has also been approved for distribution within the European Union (EU). The three vaccines approved in the US are highly effective at preventing hospitalization, death, and severe disease. Vaccines work by triggering an immune response that generate highly specific antibodies against an antigen, in the case of COVID-19, the virus spike protein expressed on the surface of the virus. Moreover, the immune system is taught to recognize the spike protein specific to the virus. If this spike protein is encountered in the future, an immune response is swiftly mounted, thus preventing escalation of the virus. Two of the authorized vaccines, developed by both Pfizer/BioNTech and Moderna, have revolutionized a technology referred to as messenger RNA (mRNA) technology. This technology acts as a delivery system to cells within our bodies with specific instructions to carry out a specific task.(13) Of importance: mRNA vaccines do not use live virus, but rather a portion of the message encoding for the spike protein. mRNA is produced by DNA, but does not enter the nucleus of the cell containing the DNA. Once the mRNA vaccine finishes producing the protein that is expressed on the cell surface, it is broken down and removed by normal cellular processes. The Johnson and Johnson (J&J) and Oxford-Astra Zeneca vaccines utilize a more conventional approach, referred to as a viral vector. This vaccine utilizes a harmless modified version of a common cold virus to deliver the gene encoding for the spike protein into the cell.(13) Vaccine comparison: Both the Pfizer/BioNTech and Moderna vaccines have been reported to confer over 94% protection rates against symptomatic COVID-19 infection.(14,15) The single shot J&J vaccine has shown to be 85% effective at protecting against severe disease, 66% against preventing moderate to severe disease, and also appears to be effective against the South African variant of the virus (B.1.351).(16) Although superficially the single shot J&J appears less effective, it is difficult to compare all three vaccines directly because of differences in trial design and outcomes. From a logistical point of view, the J&J vaccine is advantageous as it is a single-dose regimen that can be stored for up to three months in a refrigerator. The most recent data from the AstraZeneca phase three trial reports that the vaccine is 76% effective against symptomatic cases of the virus.(17) Several other trials are ongoing Several other trials are ongoing. The most important point from the information collected from 7 large efficacy trials is that all vaccines confer 100% protection against severe disease, hospitalizations, and death. Moreover, it is not just the vaccinated individual who benefits from vaccination. Most vaccines also reduce transmission of infection among people, and in so doing, help protect those who fail to mount an effective immune response to vaccines or who cannot receive the vaccinate because of their age or compromised immune system.(18) Vaccines and viral variants: Several variants of the virus have been reported, with the two properties causing the most concern being enhanced contagion and immune response evasion. The current vaccines were developed based on the spike protein before it contained the mutations identified in the variants. A recently published study investigated the effectiveness of the FDA approved Pfizer/BioNTech vaccine against the newly-emerged variants from the United Kingdom (UK) and South Africa (SA).(19) While both variants are deemed more transmissible, the levels of antibody generated in response to the vaccine are so high that it seems unlikely that it will impact the overall efficacy of the vaccine for these strains. This preliminary study also highlights the ongoing evolution of COVID-19, necessitating continuous monitoring of the significances of viral mutations for vaccine efficacy. While research suggests that COVID-19 vaccines have lower efficacy against the variants, and further research is needed, the vaccines appear to provide protection against severe COVID-19.(20) Vaccine manufacturing and distribution: The development of the vaccines, from basic R&D through human clinical trials, has been carried out within a very rapid time frame. Ramping up production, however, has been slow and cumbersome. After a slow start, Pfizer/BioNTech andModerna have raised output by gaining experience, scaling up production lines, and taking other steps like making certain raw materials on their own.(21) Of the three candidates, AstraZeneca has already struck a deal with COVAX, the global initiative to distribute COVID-19 vaccines equitably. Moderna has partnered with Lonza and Catalent Inc. to manufacture and distribute millions of doses.(22) Moreover, a recent agreement between J&J and Catalent has secured a US FDA emergency clearance that allows Catalent’s facility to manufacture and distribute, thus aiding the vaccination supply. Vaccination and reinfection: The first large scale study investigating whether reinfection can occur recently reported that the vast majority of people who had COVID-19 are protected from catching it again, for at least six months.(23) This Danish study found that protection against repeat COVID-19 infection is both robust and detectable in the majority of individuals, 80% or more of the naturally infected population younger than 65 years. However, individuals aged 65 years and older had less than 50% protection against repeat infection, since the older age group is more susceptible serious illness. Their finding highlights the need for continued vigilance to keep themselves and others safe. This also indicates that vaccination of previously infected individuals should be done because natural protection cannot be relied upon, consistent with the general consensus that vaccines confer a level of immune response that is higher than previous COVID-19 infection. Follow-up studies will give a better idea of the duration of vaccine protection. Conclusion As the COVID-19 pandemic has demonstrated, it is extremely difficult to eliminate a virus from the human population once it has entered. The pandemic spread has been compounded because the virus spreads asymptomatically. That said, the virus is manageable, similar to the manner in which vaccines have worked against other preventable communicable diseases. Monitoring the protective effects of the different vaccines will likely last for several years. For now, the outlook is positive as global cases decline, the vaccines roll out, and the momentum to investigate and repurpose drugs continues. Acknowledgments The author is grateful to Tara Finn for the careful reading of this manuscript. Conflict of interest There is no conflict of interest. References Coronavirus disease (COVID-19): How is it transmitted? Bai, N. Still Confused About Masks? Here’s the Science Behind How Face Masks PreventCoronavirus. Murphy, J.F. COVID-19: An Immunological Perspective. MOJ Immunol. 2020, 7(1), 10. Kupferschmidt K, Cohen J. Science. Race to find COVID-19 treatments accelerates. 2020. Information for Clinicians on Investigational Therapeutics for Patients with COVID-19Centers for Disease Control and Prevention. Tran, J. The Latest Research on COVID-19 Treatments and Medications in the Pipeline. Drug launched at Emory reduces virus that causes COVID-19 to undetectable levels. Group RC. Lopinavir-ritonavir in patients admitted to hospital with COVID-19(RECOVERY): a randomised, controlled, open-label, platform trial. Lancet. Baragona, S. Treating COVID-19 One Year In: Better, but No Breakthrough. Harvard Health Publishing. Treatments for COVID-19. NIH: National Institutes of Health. Full-dose blood thinners decreased need for life support and improved outcome in hospitalized COVID-19 patients. Lehrer, S., Rheinstein, P.H. Ivermectin Docks to the SARS-CoV-2 Spike Receptor-binding Domain Attached to ACE2. 2020. Murphy JF. COVID-19 mRNA vaccines. Baden, L.R. et al Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. 2021 Information about the Moderna COVID-19 Vaccine. 2021 Ledford, H. J&J’s single-dose COVID vaccine raises hopes for faster rollout. 2021 Cohen, J. AstraZeneca lowers efficacy claim for COVID-19 vaccine, a bit, after board's rebuke. 2021 Emanuel, E. Take whatever COVID vaccine you can get. All of them stop death and hospitalization. 2021 Xuping, X et al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera. 2021 COVID-19 vaccines: Get the facts 2021. Loftus P. Covid-19 Vaccine Manufacturing in U.S. Races Ahead. 2021. COVAX Announces additional deals to access promising COVID-19 vaccine candidates; plans global rollout starting Q1 2021. Hansen HH et al Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: a population-level observational study.2021.

Abstract Background Since the World Health Organization recommended single low-dose (0.25 mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant Plasmodium falciparum, several single-site studies have been conducted to assess efficacy. Methods An individual patient meta-analysis to assess gametocytocidal and transmission-blocking efficacy of PQ in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (1) gametocyte carriage in the first 2 weeks post treatment; and (2) the probability of infecting at least 1 mosquito or of a mosquito becoming infected. Results In 2574 participants from 14 studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytemia on day 0 (odds ratio [OR], 0.22; 95% confidence interval [CI], .17–.28 and OR, 0.12; 95% CI, .08–.16, respectively). Rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (P = .010 for day 7). Addition of 0.25 mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. Conclusions Transmission blocking is achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25–PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

ABSTRACT Since 2012, a single low dose of primaquine (SLDPQ; 0.25 mg/kg of body weight) with artemisinin-based combination therapies has been recommended as the first-line treatment of acute uncomplicated Plasmodium falciparum malaria to interrupt its transmission, especially in low-transmission settings of multidrug resistance, including artemisinin resistance. Policy makers in Cambodia have been reluctant to implement this recommendation due to primaquine safety concerns and a lack of data on its efficacy. In this randomized controlled trial, 109 Cambodians with acute uncomplicated P. falciparum malaria received dihydroartemisinin-piperaquine (DP) alone or combined with SLDPQ on the first treatment day. The transmission-blocking efficacy of SLDPQ was evaluated on days 0, 1, 2, 3, 7, 14, 21, and 28, and recrudescence by reverse transcriptase PCR (RT-PCR) (gametocyte prevalence) and membrane feeding assays with Anopheles minimus mosquitoes (gametocyte infectivity). Without the influence of recrudescent infections, DP-SLDPQ reduced gametocyte carriage 3-fold compared to that achieved with DP. Of 48 patients tested on day 0, only 3 patients were infectious to mosquitoes (∼6%). Posttreatment, three patients were infectious on day 14 (3.5%, 1/29) and on the 1st and 7th days of recrudescence (8.3%, 1/12 for each); this overall low infectivity precluded our ability to assess its transmission-blocking efficacy. Our study confirms the effective gametocyte clearance of SLDPQ when combined with DP in multidrug-resistant P. falciparum infections and the negative impact of recrudescent infections due to poor DP efficacy. Artesunate-mefloquine (ASMQ) has replaced DP, and ASMQ-SLDPQ has been deployed to treat all patients with symptomatic P. falciparum infections to further support the elimination of multidrug-resistant P. falciparum in Cambodia. (This study has been registered at ClinicalTrials.gov under identifier NCT02434952.)

Myasthenia gravis is an autoimmune disorder affecting the neuromuscular junction, which is characterized by the production of autoimmune antibodies to acetylcholine or muscle-specific kinase receptors, causing an error in transmission of nerve impulses to various muscles. The hallmark of myasthenia gravis is “grave or serious” fluctuating muscle weakness. Ocular, respiratory, bulbar, and skeletal muscles are most commonly affected; therefore, patients often present with fatigable ptosis, blurry vision, diplopia, change in facial expression, dysphagia, dysarthria, dyspnea, and limb weakness. Many medications, including fluroquinolone, aminoglycoside, magnesium sulfate, quinidine, and select beta blockers, are known to unmask or exacerbate symptoms of myasthenia gravis. Although the pathogenesis is not entirely understood, T lymphocytes are thought to play a role by blocking the acetylcholine receptors and causing antibody production. In the era of new immune-modulating therapies emerging for treatment of different cancers, their role in inducing a proinflammatory state has become apparent, thus highlighting a clear need to increase awareness about their role in inducing myasthenia gravis or myasthenia-like symptoms.

Auxiliary grafts have a high risk of Hepatitis B virus (HBV) infection in patients with chronic HBV-related diseases. Hepatitis B virus-related auxiliary partial orthotopic liver transplantation (APOLT) cases were reviewed to show the results of current methods to block native-to-graft HBV transmission. Three patients received APOLT for HBV-related liver cirrhosis and a recurrent upper gastrointestinal hemorrhage between April 2015 and January 2017 by the liver transplant team of Beijing Friendship Hospital affiliated with Capital Medical University. All three patients were positive for HBV surface antigen (HBsAg) and had a negative HBV DNA test result before transplantation. After auxiliary transplantations, HBsAg was found to be positive in two patients and negative in one patient. To avoid graft infection of HBV, entecavir-based therapy was employed and the remnant native livers of the recipients were removed 51–878 days after liver transplantation. Then, serum conversions of HBsAg were found in all three cases. For the first time, this case series shows the possibility of blocking the transmission of HBV from a native liver to a graft in auxiliary transplantation by entecavir-based therapy. Among the cases, a left lobe graft was successfully implanted as a replacement of the right lobe of the recipient, which is also discussed.

Abstract Background Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. Methods We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. Results We elucidated that keratin-18 (KRT18) and α/β-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. Conclusions As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

One of the most common routes of chronic hepatitis B virus (HBV) infection is mother-to-child transmission (MTCT). Approximately 6.4 million children under the age of five have chronic HBV infections worldwide. HBV DNA high level, HBeAg positivity, placental barrier failure, and immaturity of the fetal immune are the possible causes of chronic HBV infection. The passive-active immune program for children, which consists of the hepatitis B vaccine and hepatitis B immunoglobulin, and antiviral therapy for pregnant women who have a high HBV DNA load (greater than 2 × 105 IU/ml), are currently two of the most important ways to prevent the transmission of HBV from mother to child. Unfortunately, some infants still have chronic HBV infections. Some studies have also found that some supplementation during pregnancy can increase cytokine levels and then affect the level of HBsAb in infants. For example, IL-4 can mediate the beneficial effect on infants’ HBsAb levels when maternal folic acid supplementation. In addition, new research has indicated that HBV infection in the mother may also be linked to unfavorable outcomes such as gestational diabetes mellitus, intrahepatic cholestasis of pregnancy, and premature rupture of membranes. The changes in the immune environment during pregnancy and the hepatotropic nature of HBV may be the main reasons for the adverse maternal outcomes. It is interesting to note that after delivery, the women who had a chronic HBV infection may spontaneously achieve HBeAg seroconversion and HBsAg seroclearance. The maternal and fetal T-cell immunity in HBV infection is important because adaptive immune responses, especially virus-specific CD8 T-cell responses, are largely responsible for viral clearance and disease pathogenesis during HBV infection. Meanwhile, HBV humoral and T-cell responses are important for the durability of protection after fetal vaccination. This article reviews the literature on immunological characteristics of chronic HBV-infected patients during pregnancy and postpartum, blocking mother-to-child transmissions and related immune mechanisms, hoping to provide new insights for the prevention of HBV MTCT and antiviral intervention during pregnancy and postpartum.

Huanglongbing (HLB) is a destructive citrus bacterial disease caused by Candidatus Liberibacter asiaticus (Ca.Las) and cannot be cured by current pesticides. Root lesion and Tylenchulus semipenetrans juveniles were observed in HLB-affected citrus tree roots. We hypothesize that root treatment with fosthiazate (FOS) and Cupric-Ammonium Complex (CAC) will improve the root growth and inhibit HLB. CAC is a broad spectrum fungicide and can promote growth of crops. FOS kills Tylenchulus semipenetrans and protects roots from damage by harmful bacteria such as Ca.Las. After 90 days of combination treatment of FOS and CAC through root drenches, the citrus grew new roots and its leaves changed their color to green. The inhibition rate of Ca.Las reached more than 90%. During treatment process, the chlorophyll content and the root vitality increased 396 and 151%, respectively, and starch accumulation decreased by 88%. Transmission electron microscopy (TEM) and plant tissue dyeing experiments showed that more irregular swollen starch granules existed in the chloroplast thylakoid system of the HLB-infected leaves. This is due to the blocking of their secretory tissue by starch. TEM and flow cytometry experiments in vitro showed the synergistic effects of FOS and CAC. A transcriptome analysis revealed that the treatment induced the differential expression of the genes which involved 103 metabolic pathways. These results suggested that the cocktail treatment of FOS and CAC may effectively kill various pathogens including Ca.Las on citrus root and thus effectively control HLB.

ABSTRACT The development of an effective maternal HIV-1 vaccine that could synergize with antiretroviral therapy (ART) to eliminate pediatric HIV-1 infection will require the characterization of maternal immune responses capable of blocking transmission of autologous HIV to the infant. We previously determined that maternal plasma antibody binding to linear epitopes within the variable loop 3 (V3) region of HIV envelope (Env) and neutralizing responses against easy-to-neutralize tier 1 viruses were associated with reduced risk of peripartum HIV infection in the historic U.S. Woman and Infant Transmission Study (WITS) cohort. Here, we defined the fine specificity and function of the potentially protective maternal V3-specific IgG antibodies associated with reduced peripartum HIV transmission risk in this cohort. The V3-specific IgG binding that predicted low risk of mother-to-child-transmission (MTCT) was dependent on the C-terminal flank of the V3 crown and particularly on amino acid position 317, a residue that has also been associated with breakthrough transmission in the RV144 vaccine trial. Remarkably, the fine specificity of potentially protective maternal plasma V3-specific tier 1 virus-neutralizing responses was dependent on the same region in the V3 loop. Our findings suggest that MTCT risk is associated with neutralizing maternal IgG that targets amino acid residues in the C-terminal region of the V3 loop crown, suggesting the importance of the region in immunogen design for maternal vaccines to prevent MTCT. IMPORTANCE Efforts to curb HIV-1 transmission in pediatric populations by antiretroviral therapy (ART) have been highly successful in both developed and developing countries. However, more than 150,000 infants continue to be infected each year, likely due to a combination of late maternal HIV diagnosis, lack of ART access or adherence, and drug-resistant viral strains. Defining the fine specificity of maternal humoral responses that partially protect against MTCT of HIV is required to inform the development of a maternal HIV vaccine that will enhance these responses during pregnancy. In this study, we identified amino acid residues targeted by potentially protective maternal V3-specific IgG binding and neutralizing responses, localizing the potentially protective response in the C-terminal region of the V3 loop crown. Our findings have important implications for the design of maternal vaccination strategies that could synergize with ART during pregnancy to achieve the elimination of pediatric HIV infections.

ABSTRACT Low-dose primaquine is recommended to prevent Plasmodium falciparum malaria transmission in areas threatened by artemisinin resistance and areas aiming for malaria elimination. Community treatment campaigns with artemisinin-based combination therapy in combination with the gametocytocidal primaquine dose target all age groups, but no studies thus far have assessed the pharmacokinetics of this gametocytocidal drug in African children. We recruited 40 children participating in a primaquine efficacy trial in Burkina Faso to study primaquine pharmacokinetics. These children received artemether-lumefantrine and either a 0.25- or a 0.40-mg/kg primaquine dose. Seven blood samples were collected from each participant for primaquine and carboxy-primaquine plasma levels determinations: one sample was collected before primaquine administration and six after primaquine administration according to partially overlapping sampling schedules. Physiological population pharmacokinetic modeling was used to assess the impact of weight, age, and CYP2D6 genotype on primaquine and carboxy-primaquine pharmacokinetics. Despite linear weight normalized dosing, the areas under the plasma concentration-time curves and the peak concentrations for both primaquine and carboxy-primaquine increased with age and body weight. Children who were CYP2D6 poor metabolizers had higher levels of the parent compound, indicating a lower primaquine CYP2D6-mediated metabolism. Our data indicate that primaquine and carboxy-primaquine pharmacokinetics are influenced by age, weight, and CYP2D6 genotype and suggest that dosing strategies may have to be reconsidered to maximize the transmission-blocking properties of primaquine. (This study has been registered at ClinicalTrials.gov under registration no. NCT01935882.)

Abstract Background The efficacy and safety of artemether–lumefantrine (AL) and dihydroartemisinin–piperaquine (DP) against asexual parasites population has been documented. However, the effect of these anti-malarials on sexual parasites is still less clear. Gametocyte clearance following treatment is essential for malaria control and elimination efforts; therefore, the study sought to determine trends in gametocyte clearance after AL or DP treatment in children from a malaria-endemic site in Kenya. Methods Children aged between 0.5 and 12 years from Busia, western Kenya with uncomplicated Plasmodium falciparum malaria were assigned randomly to AL or DP treatment. A total of 334 children were enrolled, and dried blood spot samples were collected for up to 6 weeks after treatment during the peak malaria transmission season in 2016 and preserved. Plasmodium falciparum gametocytes were detected by qRT-PCR and gametocyte prevalence, density and mean duration of gametocyte carriage were determined. Results At baseline, all the 334 children had positive asexual parasites by microscopy, 12% (40/334) had detectable gametocyte by microscopy, and 83.7% (253/302) children had gametocytes by RT-qPCR. Gametocyte prevalence by RT-qPCR decreased from 85.1% (126/148) at day 0 to 7.04% (5/71) at day 42 in AL group and from 82.4% (127/154) at day 0 to 14.5% (11/74) at day 42 in DP group. The average duration of gametocyte carriage as estimated by qRT-PCR was slightly shorter in the AL group (4.5 days) than in the DP group (5.1 days) but not significantly different (p = 0.301). Conclusion The study identifies no significant difference between AL and DP in gametocyte clearance. Gametocytes persisted up to 42 days post treatment in minority of individuals in both treatment arms. A gametocytocidal drug, in combination with artemisinin-based combination therapy, will be useful in blocking malaria transmission more efficiently.

Abstract Background Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, although its cytotoxicity might be hampered by autophagy. In this study, we examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells. Methods The effects of PON in inducing autophagy were determined both in vitro, using SK-N-BE(2), SH-SY5Y, and IMR-32 human neuroblastoma cell lines, and in vivo, using zebrafish and mouse models. Single and combined treatments with chloroquine (CQ)—a blocking agent of lysosomal metabolism and autophagic flux—and PON were conducted, and the effects on cell viability were determined using metabolic and immunohistochemical assays. The activation of the autophagic flux was analyzed through immunoblot and protein arrays, immunofluorescence, and transmission electron microscopy. Combination therapy with PON and CQ was tested in a clinically relevant neuroblastoma mouse model. Results Our results confirm that, in neuroblastoma cells and wild-type zebrafish embryos, PON induces the accumulation of autophagy vesicles—a sign of autophagy activation. Inhibition of autophagic flux by CQ restores the cytotoxic potential of PON, thus attributing to autophagy a cytoprotective nature. In mice, the use of CQ as adjuvant therapy significantly improves the anti-tumor effects obtained by PON, leading to ulterior reduction of tumor masses. Conclusions Together, these findings support the importance of autophagy monitoring in the treatment protocols that foresee PON administration, as this may predict drug resistance acquisition. The findings also establish the potential for combined use of CQ and PON, paving the way for their consideration in upcoming treatment protocols against neuroblastoma.

Abstract Background Malaria remains a significant public health challenge in regions of the world where it is endemic. An unprecedented decline in malaria incidences was recorded during the last decade due to the availability of effective control interventions, such as the deployment of artemisinin-based combination therapy and insecticide-treated nets. However, according to the World Health Organization, malaria is staging a comeback, in part due to the development of drug resistance. Therefore, there is an urgent need to discover new anti-malarial drugs. This article reviews the literature on natural products with antiplasmodial activity that was reported between 2010 and 2017. Methods Relevant literature was sourced by searching the major scientific databases, including Web of Science, ScienceDirect, Scopus, SciFinder, Pubmed, and Google Scholar, using appropriate keyword combinations. Results and Discussion A total of 1524 compounds from 397 relevant references, assayed against at least one strain of Plasmodium, were reported in the period under review. Out of these, 39% were described as new natural products, and 29% of the compounds had IC50 ≤ 3.0 µM against at least one strain of Plasmodium. Several of these compounds have the potential to be developed into viable anti-malarial drugs. Also, some of these compounds could play a role in malaria eradication by targeting gametocytes. However, the research into natural products with potential for blocking the transmission of malaria is still in its infancy stage and needs to be vigorously pursued.