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Snake venom, a complex mixture of proteins, has attracted human attention for centuries due to its associated mortality, morbidity and other therapeutic properties. In sub-Saharan Africa (SSA), where snakebites pose a significant health risk, understanding the genetic variability of snake venoms is crucial for developing effective antivenoms. The wide geographic distribution of venomous snake species in SSA countries demonstrates the need to develop specific and broad antivenoms. However, the development of broad antivenoms has been hindered by different factors, such as antivenom cross-reactivity and polygenic paratopes. While specific antivenoms have been hindered by the numerous snake species across the SSA region, current antivenoms, such as SAIMR polyvalent and Premium Serums & Vaccines, exhibit varying degrees of cross-reactivity. Such ability to cross-react enables the antivenoms to target multiple components from the different snake species. The advent of biotechnological innovations, including recombinant antibodies, small-molecule drugs, monoclonal antibodies and synthetic antivenoms, presents options for eliminating limitations associated with traditional plasma-derived antivenoms. However, challenges still persist, especially in SSA, in addressing genetic variability, as evidenced by inadequate testing capacity and limited genomic research facilities. This comprehensive review explores the genetic variability of snake venoms in SSA, emphasizing the venom composition of various snake species and their interactions. This information is critical in developing multiple strategies during antivenom development. Finally, it offers information concerning the need for extensive collaborative engagements, technological advancements and comprehensive genomic evaluations to produce targeted and effective antivenoms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Background: Parasitic infectious agents rarely occur in isolation. Epidemiological evidence is mostly lacking, and little is known on how the two common parasites Plasmodium and soil transmitted helminths (STH) interact. There are contradictory findings in different studies. Synergism, antagonism and neutral effect have been documented between Plasmodium and STH. This study investigated the impact of STH on clinical malaria presentation and treatment outcome., Methods: A matched case control study with a semi longitudinal follow up according to World Health Organization (WHO) antimalarial surveillance guideline was done among children aged 2 months to 9 years inclusively living in western rural areas of Bagamoyo, coastal region of Tanzania. Cases were children with uncomplicated and severe malaria enrolled from the health facilities while controls were children with asymptomatic Plasmodium parasitemia enrolled from the same community., Results: In simple conditional regression analysis there was a tendency for a protective effect of STH on the development of clinical malaria [OR = 0.6, 95% CI of 0.3-1.3] which was more marked for Enterobius vermicularis species [OR = 0.2, 95% CI of 0.0-0.9]. On the contrary, hookworm species tended to be associated with increased risk of clinical malaria [OR = 3.0, 95% CI of 0.9-9.5]. In multiple conditional regression analysis, the overall protective effect was lower for all helminth infection [OR = 0.8, 95% CI of 0.3-1.9] but remained significantly protective for E. vermicularis species [OR = 0.1, 95% CI of 0.0-1.0] and borderline significant for hookworm species [OR = 3.6, 95% CI of 0.9-14.3]. Using ordinal logistic regression which better reflects the progression of asymptomatic Plasmodium parasitemia to severe malaria, there was a 50% significant protective effect with overall helminths [OR = 0.5, 95% CI of 0.3-0.9]. On the contrary, hookworm species was highly predictive of uncomplicated and severe malaria [OR = 7.8, 95% (CI of 1.8-33.9) and 49.7 (95% CI of 1.9-1298.9) respectively]. Generally, children infected with STH had higher geometric mean time to first clearance of parasitemia., Conclusion: The findings of a protective effect of E. vermicularis and an enhancing effect of hookworms may explain the contradictory results found in the literature about impact of helminths on clinical malaria. More insight should be gained on possible mechanisms for these opposite effects. These results should not deter at this stage deworming programs but rather foster implementation of integrated control program for these two common parasites., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Salim Masoud et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Our limited knowledge about the ecological drivers of global arthropod decline highlights the urgent need for more effective biodiversity monitoring approaches. Monitoring of arthropods is commonly performed using passive trapping devices, which reliably recover diverse communities, but provide little ecological information on the sampled taxa. Especially the manifold interactions of arthropods with plants are barely understood. A promising strategy to overcome this shortfall is environmental DNA (eDNA) metabarcoding from plant material on which arthropods leave DNA traces through direct or indirect interactions. However, the accuracy of this approach has not been sufficiently tested. In four experiments, we exhaustively test the comparative performance of plant-derived eDNA from surface washes of plants and homogenized plant material against traditional monitoring approaches. We show that the recovered communities of plant-derived eDNA and traditional approaches only partly overlap, with eDNA recovering various additional taxa. This suggests eDNA as a useful complementary tool to traditional monitoring. Despite the differences in recovered taxa, estimates of community α- and β-diversity between both approaches are well correlated, highlighting the utility of eDNA as a broad scale tool for community monitoring. Last, eDNA outperforms traditional approaches in the recovery of plant-specific arthropod communities. Unlike traditional monitoring, eDNA revealed fine-scale community differentiation between individual plants and even within plant compartments. Especially specialized herbivores are better recovered with eDNA. Our results highlight the value of plant-derived eDNA analysis for large-scale biodiversity assessments that include information about community-level interactions., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

When infecting humans, Andes orthohantavirus (ANDV) may cause a severe disease called hantavirus cardiopulmonary syndrome (HCPS). Following non-specific symptoms, the infection may progress to a syndrome of hemorrhagic fever combined with hyper-acute cardiopulmonary failure. The case fatality rate ranges between 25-40%, depending on the outbreak. In this study, we present the follow-up of a male patient who recovered from HCPS six years ago. We demonstrate that the ANDV genome persists within the reproductive tract for at least 71 months. Genome sequence analysis early and late after infection reveals a low number of mutations (two single nucleotide variants and one deletion), suggesting limited replication activity. We can exclude the integration of the viral genome into the host genome, since the treatment of the specimen with RNAse led to a loss of signal. We demonstrate a long-lasting, strong neutralizing antibody response using pseudovirions expressing the ANDV glycoprotein. Taken together, our results show that ANDV has the potential for sexual transmission.

Proper disinfection and inactivation of highly pathogenic viruses is an essential component of public health and prevention. Depending on environment, surfaces, and type of contaminant, various methods of disinfection must be both efficient and available. To test both established and novel chemical disinfectants against risk group 4 viruses in our maximum containment facility, we developed a standardized protocol and assessed the chemical inactivation of the two Ebola virus variants Mayinga and Makona suspended in two different biological soil loads. Standard chemical disinfectants ethanol and sodium hypochlorite completely inactivate both Ebola variants after 30 s in suspension at 70% and 0.5% v/v, respectively, concentrations recommended for disinfection by the World Health Organization. Additionally, peracetic acid is also inactivating at 0.2% v/v under the same conditions. Continued vigilance and optimization of current disinfection protocols is extremely important due to the continuous presence of Ebola virus on the African continent and increased zoonotic spillover of novel viral pathogens. Furthermore, to facilitate general pandemic preparedness, the establishment and sharing of standardized protocols is very important as it allows for rapid testing and evaluation of novel pathogens and chemical disinfectants., (© 2023. Springer Nature Limited.)

Background: Coronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but outbreaks of Middle East Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV-1, and the recently identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 4 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections. Therefore we investigated the virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) against human coronavirus (HCoV) 229E, highly pathogenic MERS- and SARS-CoVs, as well as the newly identified SARS-CoV-2, in vitro., Methods: To evaluate the antiviral potential of the extract, we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract after infection to further evaluate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce® in re-constituted nasal epithelium., Results: In the current study, we found that HCoV-229E was irreversibly inactivated when exposed to Echinaforce® at 3.2 μg/ml IC 50 . Pre-treatment of cell lines, however, did not inhibit infection with HCoV-229E and post-infection treatment had only a marginal effect on virus propagation at 50 μg/ml. However, we did observe a protective effect in an organotypic respiratory cell culture system by exposing pre-treated respiratory epithelium to droplets of HCoV-229E, imitating a natural infection. The observed virucidal activity of Echinaforce® was not restricted to common cold coronaviruses, as both SARS-CoV-1 and MERS-CoVs were inactivated at comparable concentrations. Finally, the causative agent of COVID-19, SARS-CoV-2 was also inactivated upon treatment with 50μg/ml Echinaforce®., Conclusions: These results show that Echinaforce® is virucidal against HCoV-229E, upon direct contact and in an organotypic cell culture model. Furthermore, MERS-CoV and both SARS-CoV-1 and SARS-CoV-2 were inactivated at similar concentrations of the extract. Therefore we hypothesize that Echinacea purpurea preparations, such as Echinaforce®, could be effective as prophylactic treatment for all CoVs due to their structural similarities.

Introduction: Various studies from the past years examine the changing conditions and challenges in the veterinary sector. Secured access to public and private care services is a prerequisite for a holistically oriented health care system ("One Health"). In the present study, a multidimensional concept of accessibility to care services was used for the first time to determine and visualize the density of the animal health care system in Switzerland. Traditional indicators used to describe care structures focus either on availability or accessibility. In order to overcome the limitations of traditional indicators, the family of methods known as Floating-Catchment-Area-Methods (FCA) has been developed in care geographical research. The strength of FCA methods lies in the fact that they output accessibility independent of administrative boundaries and at the same time consider the spatial distance and available capacities. The study provides insight into the density of animal health care services using FCA methods and geographical information systems (GIS). Data on providers of veterinary services in the companion animal sector and, on the demand side, data on dogs and cats kept in Switzerland served as illustrative example. The result was interactive maps of the density of health care and the structure of spatial accessibility to veterinary providers and consumers. As expected, high spatial accessibility is found in the urban centers and the agglomerations of the Central Plateau. In contrast, spatial accessibility to medical services for dogs and cats is often lower in peripheral areas. Due to hitherto unavailable data, various analyses had to be postponed for the time being. For example, the model could of course be extended to all animal species and all types of medical services. In addition, it would also be possible to forecast the future density of health care, or to optimize the care system. Together with the relevant industry stakeholders, these gaps could be closed, and the model and the resulting findings could be further differentiated. The results should serve private actors in the concerned value chains, but also decision-makers in the public veterinary service, governmental authorities, agricultural bodies, universities, etc. as a basis for strategic decisions regarding the issue of medical supply density and care services in the animal sector.

Background: Caring for individuals with progressive, disabling forms of multiple sclerosis (MS) presents ongoing, complex challenges in health care delivery, especially access to care. Although mobility limitations represent a major hurdle to accessing comprehensive and coordinated care, fragmentation in current models of health care delivery magnify the problem. Importantly, individuals with disabling forms of MS are exceedingly likely to develop preventable secondary complications and to incur significant suffering and increased health care utilization and costs., Methods: A house call program, Multiple Sclerosis at Home Access (MAHA), was implemented. The program was designed to provide comprehensive services and prevent common complications. Key aspects included monthly house calls, continuity among providers, and a multidisciplinary team led by a comprehensivist, a provider bridging subspecialty and primary care. A total of 21 adult patients (Expanded Disability Status Scale score ≥7.5) completed 1 full year of the program., Results: During the 2-year preevaluation and postevaluation period, half of the hospital admissions were related to secondary and generally preventable complications. Aside from a single outlying individual important to the evaluation, in the year after program implementation, decreases were found in number of individuals hospitalized, hospitalizations/skilled facility admissions, and hospital days; the total number of overall emergency department (ED) visits decreased; and ED-only visits increased (ie, ED visits without hospital admission). Patient satisfaction reports and quality indicators were positive. Fifty percent of patients participated in supplementary televisits., Conclusions: This program evaluation suggests that a house call-based practice is a viable solution for improving care delivery for patients with advanced MS and disability., Competing Interests: Dr. Healey has received grants from the Multiple Sclerosis Foundation. Dr. Zabad has been a site investigator or principal investigator for clinical trials funded by Biogen, Genentech, Novartis, and Sun Pharma; in the past 2 years, has served as a consultant for Bayer, Genzyme, Teva Neuroscience, and TG Therapeutics and has given unbranded lectures sponsored by Teva; and is also a member of the adjudication committee for a clinical trial of biotin in primary and secondary progressive MS sponsored by PAREXEL and MedDay Pharmaceuticals. The other authors declare no conflicts of interest.

Proper ligament tension in knee flexion within cruciate-retaining (CR) total knee arthroplasty has long been associated with clinical success; however, traditional balancing principles have assumed that the distal femoral joint line (DFJL) affects only extension. The purpose of this study was to determine the effect DFJL may have on ligament strains and tibiofemoral kinematics of CR knee designs in flexion. A computational analysis was performed using a musculoskeletal modeling system for two different knee implants, the high-flex CR (HFCR) and guided-motion CR (GMCR). Tibiofemoral kinematics and ligament strain were measured at 90-degree knee flexion while the implants' DFJL was incrementally shifted proximally. Femoral implant position and kinematics were used to determine the femur's anteroposterior position relative to the tibia. The change in the femoral medial condyle position relative to the tibia was 0.33 mm and 0.53 mm more anterior per each 1-mm elevation of the DFJL for HFCR and GMCR, respectively. The change in the lateral condyle position was 0.20 mm more anterior and 0.06 mm more posterior for HFCR and GMCR, respectively. The strain in the lateral and medial collateral ligaments changed minimally with elevation of the DFJL. In both implants, strain increased in the anterior lateral and posterior medial bundles of the posterior collateral ligament with elevation of the DFJL, whereas strain decreased in the iliotibial band and iliotibial patellar band. Our findings suggest that DFJL affects ligament tension at 90-degree knee flexion and therefore flexion balance for CR implants. Elevating the DFJL to address tight extension space in a CR knee while flexion space is well balanced could result in increased flexion tension especially when the flexion-extension mismatch is large. To achieve balanced flexion and extension, the amount of DFJL elevation may need to be reduced., Competing Interests: M.B.C.: Acelity: paid consultant, Acelity Surgical Advisory Board: paid consultant, Bone and Joint Journal 360: editorial or governing board, Exactech, Inc: paid consultant, Intellijoint: paid consultant; Stock or stock Options, Journal of Orthopaedics and Traumatology: editorial or governing board, Link Orthopaedics: paid consultant, Smith & Nephew: paid consultant; Research support, Techniques in Orthopaedics: editorial or governing board, Theravance Biopharma: paid consultant, Zimmer: paid consultant. D.H.: Smith & Nephew: employee; Stock or stock Options, S.L.: Smith & Nephew: employee; Stock or stock Options, R.S.: AAOS: board or committee member, American Association of Hip and Knee Surgeons: Board or committee member, Arthroplasty Today: editorial or governing board, Gauss surgical: Stock or stock Options, Intelijoint: paid consultant; Stock or stock Options, Journal of Arthroplasty: editorial or governing board, Smith & Nephew: paid consultant; research support., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Despite the overall successful outcomes following primary total knee arthroplasty (TKA) and the concept that a well-balanced TKA yields a more successful result, concerns still remain in the reported literature regarding the patellofemoral joint. Diminished outcomes have been associated with poorly balanced or placed patella implants. The effect of different techniques to achieve flexion-extension balance and the use of posterior stabilized (PS) versus cruciate retaining (CR) implant designs on patellofemoral balancing has not been previously studied. The purpose of this study is to utilize a validated computational analysis software to simulate the effects of varying implant positions and sizes of femoral components. The patellofemoral retinaculum (PFR) load was significantly affected by some conditions, while others did not reach significance. The proximal-distal implant position with knee flexion angle ( p  < 0.001), the implant size ( p  < 0.001), and the implant bearing type (CR/PS) ( p  < 0.05) were significant. For the proximal-distal implant position and knee flexion angle, a more proximal implant position (elevating the joint line) increased the PFR load from 15 to 30°, and a more proximal implant position reduced retinaculum load from 60 to 135°. However, at 45°, implant position does not affect retinaculum load. Achieving the appropriate balance between the dynamic nature of both the tibiofemoral and the patellofemoral interaction in TKA has proven to be complex and challenging to manage. Balancing of a TKA is essential to the proper functioning and overall longevity of the implant. These results demonstrate that patellofemoral balance is affected by implant size and position during flexion-extension gap balancing., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Working in accordance with biosafety level four practices is highly complex and time-consuming. Therefore, the respective laboratory protocols should be as uniform as possible, simple to perform and straightforward in readout. Here we describe the successful application of a standardized 24-well plate focus assay protocol for the titration of Zaire ebolavirus (two isolates), Marburg virus (three isolates), Lassa virus (two isolates), Crimean Congo hemorrhagic fever virus (one isolate), and tick-borne encephalitis virus (two isolates). Viral titers are determined based on a simple visual readout. The protocol exhibits high precision, with coefficients of variation for interassay variability ranging between 0.05 and 0.21 and those for intraassay variability between 0.08 and 0.23. All reagents required for the test, including primary and secondary antibodies, are commercially available, facilitating the establishment of the protocol in other laboratories., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The etiologic cause of encephalitis, meningitis or meningo-encephalitis is unknown in up to 70% of cases. Clinical shotgun metagenomics combined with host depletion is a promising technique to identify infectious etiologies of central nervous system (CNS) infections. We developed a straightforward eukaryotic host nucleic acid depletion method that preserves intact viruses and bacteria for subsequent shotgun metagenomics screening of clinical samples, focusing on cerebrospinal fluid (CSF). A surrogate CSF sample for a CNS infection paradigm was used to evaluate the proposed depletion method consisting of selective host cell lysis, followed by enzymatic degradation of the liberated genomic DNA for final depletion with paramagnetic beads. Extractives were subjected to reverse transcription, followed by whole genome amplification and next generation sequencing. The effectiveness of the host depletion method was demonstrated in surrogate CSF samples spiked with three 1:100 dilutions of Influenza A H3N2 virus (qPCR Ct-values 20.7, 28.8, >42/negative). Compared to the native samples, host depletion increased the amount of the virus subtype reads by factor 7127 and 132, respectively, while in the qPCR negative sample zero vs. 31 (1.4E-4 %) virus subtype reads were detected (native vs. depleted). The workflow was applied to thirteen CSF samples of patients with meningo-/encephalitis (two bacterial, eleven viral etiologies), a serum of an Andes virus infection and a nose swab of a common cold patient. Unlike surrogate samples, host depletion of the thirteen human CSF samples and the nose swab did not result in more reads indicating presence of damaged pathogens due to, e.g., host immune response. Nevertheless, previously diagnosed pathogens in the human CSF samples (six viruses, two bacteria), the serum, and the nose swab (Human rhinovirus A31) were detected in the depleted and/or the native samples. Unbiased evaluation of the taxonomic profiles supported the diagnosed pathogen in two native CSF samples and the native and depleted serum and nose swab, while detecting various contaminations that interfered with pathogen identification at low concentration levels. In summary, damaged pathogens and contaminations complicated analysis and interpretation of clinical shotgun metagenomics data. Still, proper consideration of these issues may enable future application of metagenomics for clinical diagnostics.

Neurotropic tick borne encephalitis virus (TBEV) causes life-threatening disease, and accounts for most cases of tick-transmitted viral infections in Central and Eastern Europe and Russia. No specific treatment for TBEV infections exists, and vaccination is recommended for people at risk. So far, various nucleoside analogues have been investigated in vitro as potential candidates for treatment of TBEV infections. However, in vitro experiments with more complex cell culture systems, such as organotypic culture slices which model the sophisticated architecture of the target tissue are lacking. Using TBEV as a model, we investigated the suitability of rat organotypic cerebellum slices (OCS) to study the effectiveness of nucleoside analogues with a well-known anti-TBEV activity. In these OCS, 50 μM of the nucleoside analogues 2'-C-methyladenosine (2'-CMA) and especially 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) exhibited strong inhibitory effects on TBEV replication, reducing viral titers to an average of 103-fold and TBEV RNA content 60-90-fold. In contrast, the influence of 2'-C-methylcytidine (2'-CMC) on TBEV replication was very weak, reducing virus titers by 10-fold and TBEV RNA content by 3-fold. In agreement with other studies, there was no noticeable difference in TBEV titers between OCS treated with 50 μM of Ribavirin and the DMSO treated controls. All tested nucleoside analogues exhibited excellent cytotoxicity profiles at concentrations of 50 μM. Our findings in OCS were highly comparable to data obtained in cell line culture systems. Therefore, OCS represent an ideal in vitro approach to study antivirals against TBEV and possibly other neurotropic viruses., Competing Interests: The authors have declared that no competing interests exist.

Background: Throughout Europe, Ixodes ricinus transmits numerous pathogens. Its widespread distribution is not limited to rural but also includes urbanized areas. To date, comprehensive data on pathogen carrier rates of I. ricinus ticks in urban areas of Switzerland is lacking., Results: Ixodes ricinus ticks sampled at 18 (sub-) urban collection sites throughout Switzerland showed carrier rates of 0% for tick-borne encephalitis virus, 18.0% for Borrelia burgdorferi (sensu lato), 2.5% for Borrelia miyamotoi, 13.5% for Rickettsia spp., 1.4% for Anaplasma phagocytophilum, 6.2% for "Candidatus Neoehrlichia mikurensis", and 0.8% for Babesia venatorum (Babesia sp., EU1). Site-specific prevalence at collection sites with n > 45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples., Conclusions: Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite.

Background  Lung transplantation is the only treatment option for many patients with end-stage pulmonary disease. Therefore, postthoracotomy pain therapy is of vital interest. Thoracic epidural analgesia (EPI) is the "gold standard" for postthoracotomy pain, but especially in lung transplantation contraindications, and potential infectious complications limit its advantages. Under these circumstances surgically placed postthoracotomy catheter-assisted continuous paravertebral intercostal nerve block (PVB) could be of advantage. Methods  We performed a retrospective cohort study of patients who underwent lung transplantation between 2005 and 2012. Groups were defined according to the type of postoperative pain therapy: PVB, EPI, and SYS (systemic analgesia). Total 44 patients were eligible. Results  Postoperative opioid requirement of the PVB and EPI group was comparable and less than that of the SYS group. Patients of the PVB group were weaned earlier from mechanical ventilation after lung transplantation. Conclusion  The potency of postoperative pain therapy of EPI and PVB seemed to be comparable and superior to SYS. Considering the risks and benefits, PVB could be a better choice than EPI for postthoracotomy pain therapy, especially in lung transplantation., Competing Interests: Disclosures: The authors declare no conflict of interest. The study has not been funded by any organization., (Georg Thieme Verlag KG Stuttgart · New York.)

Tuberculosis (TB) remains a global health problem, with vaccination being a necessary strategy for disease containment and elimination. A TB vaccine should be safe and immunogenic as well as efficacious in all affected populations, including HIV-infected individuals. We investigated the induction and maintenance of vaccine-induced memory CD4(+) T cells following vaccination with the subunit vaccine H1/IC31. H1/IC31 was inoculated twice on study days 0 and 56 among HIV-infected adults with CD4(+) lymphocyte counts of >350 cells/mm(3). Whole venous blood stimulation was conducted with the H1 protein, and memory CD4(+) T cells were analyzed using intracellular cytokine staining and polychromatic flow cytometry. We identified high responders, intermediate responders, and nonresponders based on detection of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) expressing central (TCM) and effector memory CD4(+) T cells (TEM) 182 days after the first immunization. Amplicon-based transcript quantification using next-generation sequencing was performed to identify differentially expressed genes that correlated with vaccine-induced immune responses. Genes implicated in resolution of inflammation discriminated the responders from the nonresponders 3 days after the first inoculation. The volunteers with higher expression levels of genes involved in antiviral innate immunity at baseline showed impaired H1-specific TCM and TEM maintenance 6 months after vaccination. Our study showed that in HIV-infected volunteers, expression levels of genes involved in the antiviral innate immune response affected long-term maintenance of H1/IC31 vaccine-induced cellular immunity. (The clinical trial was registered in the Pan African Clinical Trials Registry [PACTR] with the identifier PACTR201105000289276.)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults., Methods: HIV-infected adults with CD4+ T cell counts >350/mm3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5:1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay., Results: 47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015). Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells., Conclusion: H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response., Trial Registration: Pan African Clinical Trials Registry (PACTR) PACTR201105000289276.

Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR). Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property. However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure. Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures. Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible. The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene.

Two key players in adrenal steroid hormone biosynthesis are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2 that catalyze the final steps in the biosynthesis of cortisol and aldosterone, respectively. Overproduction of both hormones contributes to a number of severe diseases, as illustrated by the association of elevated aldosterone levels with hypertension and higher mortality in congestive heart failure, and by Cushing's syndrome as the clinical correlate of chronic hypercortisolism. Thus, CYP11B1 and CYP11B2 comprise new targets for drug treatment and selective inhibitors of both enzymes are of high pharmacological interest. To facilitate the search for such compounds, we have established novel test procedures using recombinant fission yeast strains that stably express these enzymes. The aim of this study was to compare the inhibition profiles displayed by these enzymes in established mammalian cell culture test systems to those obtained with the new fission yeast assays, and to evaluate the usefulness of the Schizosaccharomyces pombe strains as screening systems for the identification of novel lead compounds. Using these test systems, we were able to identify a new and very selective CYP11B2 inhibitor (SIAS-1) that displayed no detectable interference with CYP11B1 activity.

A measuring method for the detection of ultra-weak photon-radiation from living matter is described. Also the fluorescence-radiation is provable. The latter is of necessity for special-fluorescence investigations. A proof-sensibility of phi = 1.483 10(-16)W (respectively 7.63 10(-17)W for the distance of 65.5 mm-filter - slide replaced by trial-slide) is calculable for lambda = 554 nm with the device constant found out.

A theorem for Poisson convergence on realizations of two-dimensional Branching Random Walks with an underlying continuous time Markov Branching Process is proved. This result can be used to gain an approximation for the number of cells having sustained a certain deficiency after a long time in multistage carcinogenesis.

"The distribution of the maximum and the extinction probability for a Markovian population is derived. Asymptotic growth is described, using the sequence of sojourn times. A regularity criterion for the processes under consideration exists under certain assumptions. For a class of processes with specific population-dependent transition rates the asymptotic behaviour is given explicitly.", (excerpt)