Dataset of cell viability and analytes released by cancer cell lines exposed to low pH and conditioned medium.

Cancer cells influence their microenvironment by secreting factors that promote tumour growth and survival while evading immune-mediated destruction. We previously determined the expression of secreted factors in breast and oesophageal squamous cell carcinoma cell lines (MCF-7 and WHCO6, respectively) using Luminex assays. These cells were subsequently treated with low pH medium to mimic in vivo acid exposure, and the effects on cell viability, proliferation, and secretion of cytokines, chemokines and growth factors were described [1]. Here, we present the datasets from these experiments in addition to data obtained from treating cell lines with conditioned medium from apoptotic cell cultures.

Hematological Complications of Human Immunodeficiency Virus (HIV) Infection: An Update From an HIV-Endemic Setting.

Medical professionals, particularly in regions with a high burden of human immunodeficiency virus (HIV), should be alert to the hematological complications of HIV, which may include cytopenias, malignancy, and coagulation disturbances. Patients may present with these conditions as the first manifestation of HIV infection. Hematological abnormalities are often multifactorial with opportunistic infections, drugs, malignancy, and HIV infection itself contributing to the clinical presentation, and the diagnosis should consider all these factors. Life-threatening hematological complications requiring urgent diagnosis and management include thrombotic thrombocytopenic purpura, superior mediastinal syndrome, spinal cord compression, and tumor lysis syndrome due to aggressive lymphoma. Antiretroviral therapy is the therapeutic backbone, including for patients with advanced HIV, in addition to specific therapy for the complication. This article reviews the impact of HIV on the hematological system and provides a clinical and diagnostic approach, including the role of a bone marrow biopsy, focusing on perspectives from sub-Saharan Africa.

Building blocks for better biorepositories in Africa.

BACKGROUND: Biorepositories archive and distribute well-characterized biospecimens for research to support the development of medical diagnostics and therapeutics. Knowledge of biobanking and associated practices is incomplete in low- and middle-income countries where disease burden is disproportionately high. In 2011, the African Society of Human Genetics (AfSHG), the National Institutes of Health (NIH), and the Wellcome Trust founded the Human Heredity and Health in Africa (H3Africa) consortium to promote genomic research in Africa and established a network of three biorepositories regionally located in East, West, and Southern Africa to support biomedical research. This manuscript describes the processes established by H3Africa biorepositories to prepare research sites to collect high-quality biospecimens for deposit at H3Africa biorepositories. METHODS: The biorepositories harmonized practices between the biorepositories and the research sites. The biorepositories developed guidelines to establish best practices and define biospecimen requirements; standard operating procedures (SOPs) for common processes such as biospecimen collection, processing, storage, transportation, and documentation as references; requirements for minimal associated datasets and formats; and a template material transfer agreements (MTA) to govern biospecimen exchange. The biorepositories also trained and mentored collection sites in relevant biobanking processes and procedures and verified biospecimen deposit processes. Throughout these procedures, the biorepositories followed ethical and legal requirements. RESULTS: The 20 research projects deposited 107,982 biospecimens (76% DNA, 81,067), in accordance with the ethical and legal requirements and established best practices. The biorepositories developed and customized resources and human capacity building to support the projects. [The biorepositories developed 34 guidelines, SOPs, and documents; trained 176 clinicians and scientists in over 30 topics; sensitized ethical bodies; established MTAs and reviewed consent forms for all projects; attained import permits; and evaluated pilot exercises and provided feedback. CONCLUSIONS: Biobanking in low- and middle-income countries by local skilled staff is critical to advance biobanking and genomic research and requires human capacity and resources for global partnerships. Biorepositories can help build human capacity and resources to support biobanking by partnering with researchers. Partnerships can be structured and customized to incorporate document development, ethics, training, mentorship, and pilots to prepare sites to collect, process, store, and transport biospecimens of high quality for future research.

Storage of Mycobacterium tuberculosis culture isolates in MicrobankTM beads at a South African laboratory.

Background: Mycobacterium tuberculosis complex (MTBC) isolates are typically stored at -70 °C in cryovials containing 1 mL aliquots of a liquid medium, with or without 50% glycerol. Multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. Small culture aliquots may be more practical; however, storage capacity remains challenging. MicrobankTM beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing MTBC isolates is uninvestigated. Objective: The study aimed to determine the feasibility of using MicrobankTM beads for long-term storage of MTBC isolates at a laboratory in South Africa. Methods: In February 2020, 20 isolates in liquid culture were stored in MicrobankTM beads, following an in-house developed protocol, at -70 °C. At defined time points (16 months [15 June 2021] and 21 months [18 November 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. Results: Stored liquid isolates demonstrated MTBC growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. Contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. Conclusion: MicrobankTM beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. MicrobankTM beads represent a storage solution for the medium-term storage of MTBC isolates. What this study adds: This study evaluated the use of MicrobankTM beads as an alternate method for storing MTBC culture isolates at -70 °C and provided a suitable option for medium-term storage of MTBC.

Validation of the Xpert Breast Cancer STRAT 4 Assay on the GeneXpert instrument to Assess Hormone Receptor, Ki67, and HER2 Gene Expression Status in Breast Cancer Tissue Samples.

Breast cancer is the commonest cause of cancer-related mortality in African females where patients often present later and with advanced disease. Causes for delayed diagnosis include restricted diagnostic access and international controversy on interpretation of ancillary tests like immunohistochemistry (IHC). Fine needle aspirates (FNAC) are an attractive alternative although may have reduced sensitivity. The Xpert Breast Cancer STRAT4 (STRAT4) (CE-IVD*) assay (Cepheid, Sunnyvale) is a semi-quantitative reverse-transcription polymerase chain reaction assay which detects messenger RNA (mRNA) expression in breast samples for estrogen receptor ( ESR1 ), progesterone receptor ( PGR1 ), human epidermal growth factor receptor/Erb-B2 receptor tyrosine kinase 2 (HER2/ ERBB2 ) and the proliferation marker, MKi67 . We assessed the performance of this assay on both formalin-fixed paraffin-embedded (FFPE, n=31) and matched FNAC (n=20) samples from patients presenting with breast cancer to the Johannesburg academic hospitals. IHC and Fluorescent in situ hybridization analysis (performed on HER2-indeterminate samples) was compared with the mRNA expression of the corresponding target genes in FFPE samples, and mRNA expression on FNAC samples was compared with the FFPE results for both mRNA expression and IHC. Concordance between IHC/FISH and Xpert Breast Cancer STRAT4 in FFPE and FNAC samples using the Quick lysis (Q) method (a research-use-only modification of the validated FFPE-lysis method), showed an overall percentage agreement for ESR1 expression of 90.3% and 81.3%, and for PGR1 expression at 86.7% and 81.3% respectively in FFPE and FNAC samples. Concordance was lowest for Ki67 expression, using a binary IHC cutoff for Ki67 positivity at ≥20% staining) at 83.9% and 62.5%, for FFPE and FNAC samples, respectively. This suggests that the STRAT4 assay may be a useful ancillary test in determining HR and Ki67 status in FFPE samples and that use on FNAC samples may be feasible. Future studies should expand the sample numbers and establish locally relevant cutoffs.

Assessing Biomarkers in Viral Infection.

Current biomarkers to assess the risk of complications of both acute and chronic viral infection are suboptimal. Prevalent viral infections like human immunodeficiency virus (HIV), hepatitis B and C virus, herpes viruses, and, more recently, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may be associated with significant sequelae including the risk of cardiovascular disease, other end-organ diseases, and malignancies. This review considers some biomarkers which have been investigated in diagnosis and prognosis of key viral infections including inflammatory cytokines, markers of endothelial dysfunction and activation and coagulation, and the role that more conventional diagnostic markers, such as C-reactive protein and procalcitonin, can play in predicting these secondary complications, as markers of severity and to distinguish viral and bacterial infection. Although many of these are still only available in the research setting, these markers show promise for incorporation in diagnostic algorithms which may assist to predict adverse outcomes and to guide therapy.

Value of diagnostic vaccination in diagnosis of humoral inborn errors of immunity.

Inborn errors of immunity (IEIs) or primary immunodeficiency diseases, are disorders caused by genetic defects affecting immune function. Clinically, IEI presents mainly as recurrent or severe infections, immune dysregulation (autoimmunity or autoinflammatory disorders), and lymphoproliferation with or without dysmorphic features. Humoral IEIs are the largest subgroup of IEI, with a wide spectrum of quantitative and qualitative antibody defects. These disorders are normally diagnosed based on immunological evaluation; diagnostic vaccination is part of this evaluation. This review examines the importance and relevance of diagnostic vaccination in the diagnosis of humoral IEIs and different technologies which can be utilised in diagnosis.

Selected inflammatory and coagulation biomarkers pre-viral suppression in people with human immunodeficiency virus (HIV) infection without overt cardiovascular disease: Is there a need to redefine reference indices?

BACKGROUND: Human immunodeficiency virus (HIV) infection is prevalent in Africa and causes morbidity and mortality despite antiretroviral therapy (ART). Non-communicable complications of HIV infection include cardiovascular disease (CVD) with thromboses throughout the vascular tree. Ongoing inflammation and endothelial dysfunction in people living with HIV (PLWH) probably contribute significantly to HIV-related CVD. OBJECTIVES: A systematic review was conducted to inform interpretation of 5 biomarkers commonly measured in PLWH namely interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α), D-dimers, and soluble intracellular and vascular adhesion molecules-1 (sICAM-1 and sVCAM-1) to attempt to define a range for these values in ART naïve PLWH without overt CVD or additional comorbid diseases. METHODS: A systematic search was conducted for all studies documenting the levels of the above biomarkers in ART naïve PLWH published on the PubMed database from 1994 to 2020. RESULTS: The number of publications that reported medians above the assay values was: 4/15 for D-dimer; 0/5 for TNF-α, 8/16 for IL-6, 3/6 for sVCAM-1, and 4/5 for sICAM-1. CONCLUSION: The clinical utility of biomarkers is reduced by the lack of standardisation of the measurement of these parameters, absence of normal reference indices and the lack of uniformity of study protocols in different research centres. This review supports the ongoing use of D-dimers to predict thrombotic and bleeding events in PLWH since the weighted averages across study assays suggest that the median levels do not exceed the reference range. The role of inflammatory cytokine monitoring and measurement of endothelial adhesion markers is less clear.

The effect of acute acid exposure on immunomodulatory protein secretion, cell survival, and cell cycle progression in tumour cell lines.

Cancer develops when multiple systems fail to suppress uncontrolled cell proliferation. Breast cancers and oesophageal squamous cell carcinoma (OSCC) are common cancers prone to genetic instability. They typically occur in acidic microenvironments which impacts on cell proliferation, apoptosis, and their influence on surrounding cells to support tumour growth and immune evasion. This study aimed to evaluate the impact of the acidic tumour microenvironment on the production of pro-tumorigenic and immunomodulatory factors in cancer cell lines. Multiple factors that may mediate immune evasion were secreted including IL-6, IL-8, G-CSF, IP-10, GDF-15, Lipocalin-2, sICAM-1, and myoglobin. Others, such as VEGF, FGF, and EGF that are essential for tumour cell survival were also detected. Treatment with moderate acidity did not significantly affect secretion of most proteins, whereas very low pH did. Distinct differences in apoptosis were noted between the cell lines, with WHCO6 being better adapted to survive at moderate acid levels. Conditioned medium from acid-treated cells stimulated increased cell viability and proliferation in WHCO6, but increased cell death in MCF-7. This study highlights the importance of acidic tumour microenvironment in controlling apoptosis, cell proliferation, and immune evasion which may be different at different anatomical sites. Immunomodulatory molecules and growth factors provide therapeutic targets to improve the prognosis of individuals with cancer.

Distinguishing and overlapping laboratory results of thrombotic microangiopathies in HIV infection: Can scoring systems assist?

BACKGROUND: Patients with Human Immunodeficiency Virus (HIV) infection are at risk of thrombotic microangiopathies (TMAs) notably thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC). Overlap between laboratory results exists resulting in diagnostic ambiguity. METHODS: Routine laboratory results of 71 patients with HIV-associated TTP (HIV-TTP) and 81 with DIC with concomitant HIV infection (HIV-DIC) admitted between 2015 and 2021 to academic hospitals in Johannesburg, South Africa were retrospectively reviewed. Both the PLASMIC and the International Society of Thrombosis and Haemostasis (ISTH) DIC scores were calculated. RESULTS: Patients with HIV-TTP had significantly (P < .001) increased schistocytes and features of hemolysis including elevated lactate dehydrogenase (LDH)/upper-limit-of-normal ratio (median of 9 (interquartile range [IQR] 5-12) vs 3 (IQR 2-5)) but unexpectedly lower fibrinogen (median 2.8 (IQR 2.2-3.4) vs 4 g/L (IQR 2.5-9.2)) and higher D-dimer (median 4.8 (IQR 2.4-8.1) vs 3.6 g/L (IQR 1.7-6.2)) levels vs the HIV-DIC cohort. Patients with HIV-DIC were more immunocompromised with frequent secondary infections, higher platelet and hemoglobin levels, more deranged coagulation parameters and less hemolysis. Overlap in scoring systems was however observed. CONCLUSION: The laboratory parameter overlap between HIV-DIC and HIV-TTP might reflect a shared pathogenesis including endothelial dysfunction and inflammation and further research is required. Fibrinogen in DIC may be elevated as an acute phase reactant and D-dimers may reflect the extensive hemostatic activation in HIV-TTP. Inclusion of additional parameters in TMA scoring systems such the LDH/upper-limit-of-normal ratio, schistocytes count and wider access to ADAMTS-13 testing may enhance diagnostic accuracy and ensure appropriate utilization of plasma.

SARS-CoV-2 Host Immunogenetic Biomarkers.

SARS-CoV-2 causes generally mild symptoms, with approximately 10-20% of cases progressing to severe disease. The pathophysiologic mechanisms by which SARS-CoV-2 causes severe disease are largely unknown. Data have indicated the involvement of different immunogenetic markers such as HLA, T, and B cells, to be associated with disease outcome. This has led to interest in these genes as potential biomarkers of SARS-CoV-2 susceptibility and for predicting prognosis and response to vaccines and other therapeutic strategies. In this chapter, we discussed outline protocols for characterizing these potential biomarkers and methods for identifying SARS-CoV-2 biomarkers using the Luminex® 100/200 technology and next-generation sequencing.

Evaluation Protocol for SARS-CoV-2 Serological Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of COVID-19. Accurate detection of SARS-CoV-2 infection is not only important for management of infected individuals but also to break the chain of transmission. Although the polymerase chain reaction (PCR) is the gold standard for diagnosis of acute SARS-CoV-2 infection, there are a number of limitations of these assays, which include the inability to detect past infection and decline in sensitivity 14 days post-symptom onset. There are several serology tests developed for the detection of SARS-CoV-2 antibodies including high-throughput serology platforms and lateral flow immunoassays. These tests should be evaluated for their performance to meet local regulations acceptance criteria. To optimize the diagnostic algorithm for SARS-CoV-2, this protocol describes the evaluation of serological antibody testing using various automated serology platforms and lateral flow immunoassays. This protocol was evaluated in both serum and plasma samples. The sample preparation, procedure, and data analysis are described. The protocol can be adapted for any serological testing.

Intestinal fatty acid binding protein (I-FABP) and CXC3L1 evaluation as biomarkers for patients at high-risk for coeliac disease in Johannesburg, South Africa.

Coeliac disease (CD) is an autoimmune disorder and one of the few gastroenteropathies with accurate serological testing. CD serology has decreased accuracy for patients on a gluten-free diet and for monitoring mucosal healing. New ancillary tests would, therefore, be useful. Intestinal Fatty Acid Binding Protein (I-FABP) and CX3CL1 (Fractalkine) are two promising biomarkers for CD but haven't been examined in patients who are at a high-risk for CD such as patients with type one diabetes (TID). This study, therefore, aimed to investigate serum levels of I-FABP and CX3CL1 in a cohort of South African patients with TID at a high-risk of developing CD. The serum I-FABP levels were significantly higher in CD-positive patients compared to CD-negative individuals (p = 0.03). No significant differences in the serum CX3CL1 levels were detected although this may reflect the impact of the comorbid autoimmune diseases had on the serum CX3CL1 levels. In conclusion, this study is the first to assess the levels of these biomarkers in a multiethnic population with comorbid autoimmune disease and determined I-FABP to be the more promising biomarker in such clinical contexts. Future research should focus on a diverse biomarker panel and longitudinal follow-up of patients at a high-risk for CD.

Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay.

Cell death is important in physiology, and can happen as a result of structural damage, or as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies, and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V/PI apoptosis assay.

Antigen-Based Point of Care Testing (POCT) for Diagnosing SARS-CoV-2: Assessing Performance.

Currently, the most accurate way to diagnose an active SARS-CoV-2 (COVID-19) infection is through detection of viral RNA using reverse transcription polymerase chain reaction (RT-PCR) test. While RT-PCR tests are the most sensitive for identifying infection, there are significant limitations, such as global access to sufficient test kits, turnaround times (TAT) from specimen collection to test result is often greater than 24 h and the need for skilled operators in accredited laboratories requiring specialized equipment. A rapid test performed at the point of care (POC) could provide a result within an approximate time of 30 min post specimen collection, be performed by a health care worker and comprise a simple workflow, improving both turnaround time and potentially decreasing costs (e.g., transport, cold-chain, skilled laboratory staff, complex equipment). Determining the performance of SARS-CoV-2 RT-PCR tests is, however, easier to assess than antigen-based POCT, as residual clinical specimens (swabs in universal transport media [UTM]) are readily available in laboratory environments, and do not require patient informed consent. Evaluating the performance of POCT requires informed-consent driven studies, with patients required to provide a standard of care specimen as well as study evaluation specimens, which is often not acceptable as nasopharyngeal swabbing can be invasive, clinical field trials are costly and time consuming. Many institutions and regulatory bodies also require preliminary data prior to use in field settings. Therefore, we have developed a method to determine the performance of antigen based POCT that can be used by implementers in national healthcare programs, regulators and rapid test developers. The method investigates both quantitative and qualitative parameters, with the latter providing insights into the capability for implementation and national program uptake.

Thrombotic thrombocytopenic purpura in HIV-infected patients: new twists on an old disease.

OBJECTIVE: Investigate the presence of inflammation, endothelial dysfunction and complement activation in patients with HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP) to support the hypothesis that these processes probably contribute to the development of this thrombotic microangiopathy. DESIGN: A prospective, investigational cohort study of 35 consecutive patients diagnosed with HIV-associated TTP presenting to three academic, tertiary care hospitals in Johannesburg, South Africa over 2 years. METHODS: The patients with HIV-TTP received therapeutic plasma therapy and supportive treatment. Demographic data, the results of routine investigations and patient outcomes were recorded. Peripheral blood samples were collected prior to and on completion of plasma therapy and the following additional parameters were assessed at both time points: activity of the von Willebrand factor (VWF) cleaving protease, a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs 13 (ADAMTS-13) and the presence of ADAMTS-13 autoantibodies, levels of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-alpha, and two endothelial cell adhesion molecules. Complement activation was assessed by sequential measurement of C3 and C4 as well as levels of the complement inhibitor, factor H. RESULTS: The inflammatory and endothelial activation markers were significantly ( P  < 0.001) elevated in the cohort of patients prior to plasma therapy compared with levels on discharge. Complement was activated and normalized with therapy. The ADAMTS-13 levels were reduced with significant auto-antibodies to this protease at presentation. CONCLUSION: Inflammation in HIV mediates endothelial damage and complement activation. This study proposes that these processes are probably contributory to the development of HIV-TTP, which can therefore be characterized in part as a complementopathy, resembling TTP-like syndrome.

HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP): A practical guide and review of the literature.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a serious thrombotic microangiopathy (TMA), is prevalent in the South African HIV-infected population. The exact pathogenesis of HIV-associated TTP (HIV-TTP) is however still unclear with diagnostic and therapeutic inconsistancies. METHODS: A systematic review of the published literature regarding HIV-TTP was performed. RESULTS: HIV-TTP is still associated with significant morbidity and mortality in Africa despite the availability of anti-retroviral therpy (ART). Diagnosis of HIV-TTP requires the presence of a micro-angiopathic haemolytic anaemia with significant red blood cell schistocytes and thrombocytopenia in the absence of another TMA but background activation of the coagulation system and inflammation in HIV infected people can result in diagnostic anbiguity. Plasma therapy in the form of infusion or exchange is successful but expensive, associated with side-effects and not widely available. Adjuvant immunosuppression therapy may of benefit in patients with HIV-TTP and ART must always be optimised. Endothelial dysfunction caused by chronic inflammation and complement activation most likely contributes to the development of HIV-TTP. CONCLUSION: The role of adjuvant immunomodulating therpy, the therapeutic targets and pathogenic contribution from endothelial dysfunction in HIV-TTP requires further investigation.

The acidic tumour microenvironment: Manipulating the immune response to elicit escape.

The success of cancer treatment relies on the composition of the tumour microenvironment which is comprised of tumour cells, blood vessels, stromal cells, immune cells, and extracellular matrix components. Barriers to effective cancer treatment need to be overcome, and the acidic microenvironment of the tumour provides a key target for treatment. This review intends to provide an overview of the effects that low extracellular pH has on components of the tumour microenvironment and how they contribute to immune escape. Further, potential therapeutic targets will be discussed.