Robust and inexpensive equipment design for polymerase chain reaction detection of sequence mutations Cystic fibrosis in a mother and 2 children analysed

Every polymerase chain reaction (PCR) requires use of a temperature cycler for about 3 hours. Since there are many diagnostic tests using this technology, it is important that robust but inexpensive machinery is available. Such a standalone machine has been designed and used to analyse an interesting family in which another and her 2 children were diagnosed as having cystic fibrosis

A cell-based assay for the detection of neutralizing antibodies against alemtuzumab.

Aim: The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis. During treatment, anti-alemtuzumab antibodies may develop and reduce effective lymphocyte depletion in future treatment cycles. Results: Alemtuzumab-Alexa Fluor 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies. Conclusion: In this proof-of-concept study, a CHO-CD52 cell line has been developed and used to detect the presence of anti-alemtuzumab neutralizing antibodies. This platform provides the basis of an assay for routine screening of serum for neutralizing antibodies from patients treated with alemtuzumab

The impact of COVID-19 vaccines on male semen parameters: a retrospective cohort study

The emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic necessitated the development of adequate vaccines. Despite vaccines being demonstrated to be safe and effective for preventing severe disease and death, vaccine hesitancy remains. Reasons include concerns over adverse effects on male fertility, which have not been widely investigated. Therefore, this study is aimed at determining the impact of COVID-19 vaccination on semen parameters in a retrospective cohort study of South African males undergoing fertility assessment. The patients for this study were adult men who have previously undergone routine semen analysis for fertility assessment at Androcryos Andrology Laboratory (Johannesburg, South Africa) between March 2021 and March 2022. They also received vaccination within 3 months following a semen analysis and underwent a second semen analysis any time post-COVID-19 vaccination. From 277 records analysed, 46 patients met the inclusion criteria, receiving the Pfizer-BioNTech (BNT162b1) (63%), Johnson and Johnson (JNJ-78436735/Ad26.COV2S) (34.8%), and the AstraZeneca (AZD1222) (2.2%) vaccines. Sperm concentration significantly increased postvaccination (), with no significant changes in semen pH, volume, total sperm count, progressive motility, normal sperm morphology, or chromatin condensation. Results were not influenced by age, type of vaccine received, and the number of days following vaccination, as depicted by multiple regression analysis. In conclusion, there is no evidence of a negative impact of COVID-19 vaccination on male semen parameters, which is consistent with the emerging literature on COVID-19 vaccination and male fertility. COVID-19 vaccinations should not be dismissed based on fear of adverse effects on male fertility parameters

Detecting and predicting neutralization of alemtuzumab responses in MS

Objective: To test the hypothesis that anti-drug antibodies against alemtuzumab could become relevant after repeated treatments for some individuals, possibly explaining occasional treatment resistance. Methods: Recombinant alemtuzumab single-chain variable fragment antibody with a dual tandem nanoluciferase reporter linker was made and used to detect binding anti-drug antibodies. Alemtuzumab IgG Alexa-Fluor 488 conjugate was used in a competitive-binding cell based assay to detect neutralizing anti-drug antibodies. The assays were used to retrospectively screen, blinded, banked-serum samples from people with multiple sclerosis (n=32) who had received three or more cycles of alemtuzumab. Lymphocyte depletion was measured between baseline and about 1 month post-infusion. Results: The number of individuals showing limited depletion of lymphocytes increased with the number of treatment cycles. Lack of depletion was also a poor prognostic feature for future disease activity. Anti-drug antibody responses were detected in 29/32 (90.6%) individuals. Neutralizing antibodies occurred prior to the development of limited depletion in 6/7 individuals (18.8% of the whole sample). Pre-infusion, anti-drug antibody levels predicted limited, post-infusion lymphocyte depletion. Conclusions: Although anti-drug antibodies to alemtuzumab have been portrayed as being of no clinical significance, alemtuzumab-specific antibodies appear to be clinically relevant for some individuals, although causation remains to be established. Monitoring of, lymphocyte depletion and the anti-drug response may be of practical value in patients requiring additional cycles of alemtuzumab. Anti-drug antibody detection may help to inform on re-treatment or switching to another treatment

Improving the evidence base of Markov models used to estimate the costs of scaling up antiretroviral programmes in resource-limited settings

<p>Abstract</p> <p>Background</p> <p>Despite concerns about affordability and sustainability, many models of the lifetime costs of antiretroviral therapy (ART) used in resource limited settings are based on data from small research cohorts, together with pragmatic assumptions about life-expectancy. This paper revisits these modelling assumptions in order to provide input to future attempts to model the lifetime costs and the costs of scaling up ART.</p> <p>Methods</p> <p>We analysed the determinants of costs and outcomes in patients receiving ART in line with standard World Health Organization (WHO) guidelines for resource poor settings in a private sector managed ART programme in South Africa. The cohort included over 5,000 patients with up to 4 years (median 19 months) on ART. Generalized linear and Cox proportional hazards regression models were used to establish cost and outcome determinants respectively.</p> <p>Results</p> <p>The key variables associated with changes in mean monthly costs were: being on the second line regimen; receiving ART from 4 months prior to 4 months post treatment initiation; having a recent or current CD4 count <50 cells/µL or 50-199 cells/µl; having mean ART adherence <75% as determined by monthly pharmacy refill data; and having a current or recent viral load >100,000 copies/mL. In terms of the likelihood of dying, the key variables were: baseline CD4 count<50 cells/µl (particularly during the first 4 months on treatment); current CD4 count <50 cells/µl and 50-199 cells/µl (particularly during later periods on treatment); and being on the second line regimen. Being poorly adherent and having an unsuppressed viral load was also associated with a higher likelihood of dying.</p> <p>Conclusions</p> <p>While there are many unknowns associated with modelling the resources needed to scale-up ART, our analysis has suggested a number of key variables which can be used to improve the state of the art of modelling ART. While the magnitude of the effects associated with these variables would be likely to differ in other settings, the variables influencing costs and survival are likely to be generalizable. This is of direct relevance to those concerned about assessing the long-term costs and sustainability of expanded access to ART.</p

Scaling up health policies and services in low- and middle-income settings

"Scaling up" effective health services is high on the policy agendas of many countries and international agencies. The current concern has been driven by growing recognition both of the challenges of achieving the health-related Millennium Development Goals (MDGs) in many countries, and of the need to ensure that the increased resources for health channelled through disease-specific health initiatives are able generate health gain at scale. Effective and cost-effective interventions exist to address many of the major causes of disease burden in the developing world, but coverage of many of these services remains low. There is a substantial gap between what could be achieved and what is actually being achieved in terms of health improvement in low- and middle-income countries

The in vitro modulation of steroidogenesis by inflammatory cytokines and insulin in TM3 Leydig cells

BACKGROUND: Cytokines and hormones, including insulin, are known to modulate the hypothalamic-pituitary-testes axis and steroidogenesis, both centrally and peripherally. In the context of chronic inflammation and hyperinsulinaemia mediating male hypogonadism associated with obesity, metabolic syndrome and type 2 diabetes mellitus, these mechanisms are poorly understood and the impact of cytokines and insulin on Leydig cell steroidogenesis has not been fully elicited. This study aimed to further investigate the in vitro impact of TNFα, IL1ß, IL6, IL8 and insulin on Leydig cell function and steroidogenesis. METHODS: hCG-stimulated TM3 Leydig cells were exposed to various concentrations of TNFα, IL1ß, IL6, IL8 (100 ng/ ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml) and insulin (10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0.01 ng/ml) in optimal cell culture conditions over 48 h. Cell viability (XTT) and testosterone and progesterone concentrations (ELISA) were assessed using standardised laboratory techniques. RESULTS: TNFα significantly decreased cell viability and progesterone and testosterone concentrations in a dosedependent relationship. IL1ß and IL6 had a subtle but significant negative effect on cell viability and testosterone concentrations, with a marked significant decrease in progesterone concentration at all concentrations investigated. IL8 showed an increase in cell viability, with no significant effect on testosterone concentrations alongside a significant decrease in progesterone concentrations. Insulin significantly increased cell viability and testosterone concentrations in a dose dependent relationship, but interestingly significantly decreased progesterone concentrations. CONCLUSIONS: The inflammatory cytokines TNFα, IL1β and IL6 cause a dose dependent decline in steroidogenesis in TM3 Leydig cells. These results suggest that chronic inflammation may downregulate steroidogenesis in males via direct modulation of Leydig cell function. However, IL8 may stimulate TM3 Leydig cell growth. Insulin is associated with a dose-dependent increase in testosterone synthesis, with a significant decline in progesterone synthesis. With the phenomenon of insulin resistance, the literature is unclear on the potential role of hyperinsulinaemia in steroidogenesis. Further studies are warranted in order to fully elicit the molecular mechanisms and interactions of these molecules on male steroidogenesis

Rapid single-cell identification of Epstein-Barr virus-specific T-cell receptors for cellular therapy

BACKGROUND AND AIMS: Epstein-Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals. The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. METHODS AND RESULTS: Mononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαβ-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. CONCLUSIONS: The authors' approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies

Early and Late Direct Costs in a Southern African Antiretroviral Treatment Programme: A Retrospective Cohort Analysis

Gary Maartens and colleagues describe the direct heath care costs and identify the drivers of cost over time in an HIV managed care program in Southern Africa

The clinical and molecular spectrum of galactosemia in patients from the Cape Town region of South Africa

BACKGROUND: The objective of this study was to document the clinical, laboratory and genetic features of galactosemia in patients from the Cape Town metropolitan region. METHODS: Diagnoses were based on thin layer chromatography for galactosuria/galactosemia and assays of erythrocyte galactose-1-phosphate uridyltransferase (GALT) and galactokinase activities. Patients were screened for the common S135L and Q188R transferase gene mutations, using PCR-based assays. Screening for the S135L mutation in black newborns was used to estimate the carrier rate for galactosemia in black South Africans. RESULTS: A positive diagnosis of galactosemia was made in 17 patients between the years 1980 to 2001. All had very low or absent galactose-1-phosphate uridyltransferase (GALT) activity, and normal galactokinase levels. The mean age at diagnosis was 5.1 months (range 4 days to 6.5 months). A review of 9 patients showed that hepatomegaly (9/9), and splenomegaly, failure to thrive, developmental delay, bilateral cataracts (6/9) were the most frequent features at diagnosis. Six had conjugated hyperbilirubinemia. Four experienced invasive E. coli infection before diagnosis. Ten patients were submitted to DNA analysis. All 4 black patients and 2 of mixed extraction were homozygous for the S135L allele, while all 3 white patients were homozygous for the Q188R allele. The remaining patient of mixed extraction was heterozygous for the Q188R allele. The estimated carrier frequency of the S135L mutation in 725 healthy black newborns was 1/60. CONCLUSIONS: In the absence of newborn screening the delay in diagnosis is most often unacceptably long. Also, carrier frequency data predict a galactosemia incidence of approximately 1/14 400 for black newborns in the Cape Metropole, which is much higher than the current detection rate. It is thus likely that many patients go undetected