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.

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.

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.

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.

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.

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.

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.

Performance of the Abbott SARS-CoV-2 IgG serological assay in South African 2 patients.

In late December 2019, pneumonia cases of unknown origin were reported in Wuhan, China. This virus was named SARS-CoV2 and the clinical syndrome was named coronavirus disease 19 (COVID-19). South Africa, despite strict and early lockdown has the highest infection rate in Africa. A key component of South Africa's response to SARSCoV2 was the rapid scale-up of diagnostic testing. The Abbott SARS-CoV2 assay detects IgG antibodies against the Nucleocapsid (N) protein of the SARS-CoV2 virus. This study undertook to validate and evaluate performance criteria of the Abbott assay and to establish whether this assay would show clinical utility in our population. Positive patients (n = 391) and negative controls (n = 139) were included. The Architect-i and Alinity-i systems were analyzers that were used to perform the SARS-CoV-2 IgG assay. In-house ELISA was incorporated into the study as a confirmatory serology test. A total of number of 530 participants was tested, 87% were symptomatic with infection and 13% were asymptomatic. When compared to RT-qPCR, the sensitivity of Architect and Alinity SARS-CoV2 assays was 69.5% and 64.8%, respectively. Specificity for Architect and Alinity assays was 95% and 90.3%, respectively. The Abbott assay was also compared to in house ELISA assay, with sensitivity for the Architect and Alinity assays of 94.7% and 92.5%, respectively. Specificity for Abbott Alinity assays was 91.7% higher than Abbott Architect 88.1%. Based on the current findings testing of IgG after 14 days is recommended in South Africa and supports other studies performed around the world.

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.

Diagnosis of human immunodeficiency virus associated disseminated intravascular coagulation.

INTRODUCTION: Disseminated intravascular Coagulation (DIC) is a thrombotic microangiopathy which may complicate a number of severe disease processes including sepsis. Development of microvascular thromboses results in consumption of coagulation factors and platelets and ultimate bleeding. Patients with HIV infection (PWH) often present with baseline dysregulation of the coagulation system which may increase severity and derangement of DIC presentation. Previously, we have shown that HIV is a significant risk factor for development of DIC. METHODOLOGY: We conducted a retrospective record review of all DIC screens submitted to our tertiary coagulation laboratory in Johannesburg, South Africa, over a one year period and compared the laboratory presentation of DIC in PWH with presentation of DIC in patients without HIV infection. RESULTS: Over the year, 246 patients fulfilled the International Society of Thrombosis and Haemostasis (ISTH) diagnostic criteria for DIC- 108 were confirmed HIV-infected and 77 were confirmed uninfected. PWH and DIC presented at a significantly earlier age (41 vs 46 years respectively, p<0.02). The prothrombin time was significantly more prolonged (30.1s vs 26.s), the d-dimer levels were substantially higher (5.89mg/L vs 4.52mg/L) and the fibrinogen (3.92g/L vs 1.73g/L) and platelet levels (64.8 vs 114.8x109/l) were significantly lower in PWH. PWH also showed significant synthetic liver dysfunction and higher background inflammation. CONCLUSION: PWH who fulfil the diagnostic criteria for DIC show significantly more dysregulation of the haemostatic system. This may reflect baseline abnormalities including endothelial dysfunction in the context of inflammation and liver dysfunction.

Operational characteristics of 30 lateral flow immunoassays used to identify COVID-19 immune response.

Serology or antibody tests for COVID-19 are designed to detect antibodies (mainly Immunoglobulin M (IgM) and Immunoglobulin G (IgG) produced in response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) infection. In this study, 30 lateral flow immunoassays were tested using serum or plasma from patients with confirmed SARS CoV-2 infection. Negative serological controls were accessed from a well-characterised bank of sera which were stored prior to February 2020. Operational characteristics and ease of use of the assays are reported. 4/30 (13%) of kits (Zheihang Orient Gene COVID-19 IgG/IgM, Genrui Novel Coronavirus (2019-nCoV) IgG/IgM, Biosynex COVID-19 BSS IgG/IgM, Boson Biotech 2019-nCoV IgG/IgM) were recommended for SAHPRA approval based on kit sensitivity. Of these, only the Orientgene was recommended by SAHPRA in August 2020 for use within the approved national testing algorithm while the remaining three received limited authorization for evaluation. All kits evaluated work on the same basic principle of immunochromatography with minor differences noted in the shape and colour of cartridges, the amount of specimen volume required and the test duration. Performance of the lateral flow tests were similar to sensitivities and specificities reported in other studies. The cassettes of the majority of kits evaluated (90%) detected both IgG and IgM. Only 23% of kits evaluated contained all consumables required for point-of-care testing. The study highlights the need for thorough investigation of kits prior to implementation.

Performance of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies in South Africa.

Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) has been identified as the causative agent for causing the clinical syndrome 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. South Africa is the current epicenter of SARS-CoV-2 infection in Africa. To optimize the diagnostic algorithm for SARS-CoV-2 in the South African setting, the study aims to evaluate the diagnostic performance of the EUROIMMUN Anti-SARS-CoV-2 assays. This study reported the performance of EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples targeting the recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Samples were collected from 391 individuals who had tested positive for SARS-CoV-2 and 139 SARS CoV-2 negative controls. Samples were stratified by number of days' post-PCR diagnosis and symptoms. The sensitivity of EUROIMMUN IgG was 64.1% (95% CI: 59.1-69.0%) and 74.3% (95% CI: 69.6-78.6%) for IgA and the specificity was lower for IgA [84.2% (95% CI: 77-89.2%)] than IgG [95.2% (95% CI: 90.8-98.4%)]. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay sensitivity was higher for IgA but low for IgG and improved for both assays in symptomatic individuals and at later timepoints post PCR diagnosis.

SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusion: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort.

The COVID-19 pandemic has affected all individuals across the globe in some way. Despite large numbers of reported seroprevalence studies, there remains a limited understanding of how the magnitude and epitope utilization of the humoral immune response to SARS-CoV-2 viral anti-gens varies within populations following natural infection. Here, we designed a quantitative, multi-epitope protein microarray comprising various nucleocapsid protein structural motifs, including two structural domains and three intrinsically disordered regions. Quantitative data from the microarray provided complete differentiation between cases and pre-pandemic controls (100% sensitivity and specificity) in a case-control cohort (n = 100). We then assessed the influence of disease severity, age, and ethnicity on the strength and breadth of the humoral response in a multi-ethnic cohort (n = 138). As expected, patients with severe disease showed significantly higher antibody titers and interestingly also had significantly broader epitope coverage. A significant increase in antibody titer and epitope coverage was observed with increasing age, in both mild and severe disease, which is promising for vaccine efficacy in older individuals. Additionally, we observed significant differences in the breadth and strength of the humoral immune response in relation to ethnicity, which may reflect differences in genetic and lifestyle factors. Furthermore, our data enabled localization of the immuno-dominant epitope to the C-terminal structural domain of the viral nucleocapsid protein in two independent cohorts. Overall, we have designed, validated, and tested an advanced serological assay that enables accurate quantitation of the humoral response post natural infection and that has revealed unexpected differences in the magnitude and epitope utilization within a population.

The influence of haemodialysis on CD4+ T-cell counts in people living with human immunodeficiency virus with end-stage kidney disease.

BACKGROUND: In South Africa it is estimated that 7.9 million people are living with human immunodeficiency virus (HIV). HIV is associated with an increased risk of kidney disease. For people living with HIV (PLWH) who develop end-stage kidney disease (ESKD), access to renal replacement therapy can be difficult. Kidney transplantation is a cost-effective option, with improved overall survival and better quality of life. In Johannesburg, the eligibility criteria for kidney transplantation include a sustained CD4+ T-cell count of > 200 cells/µL and suppressed HIV replication. OBJECTIVE: To investigate the influence of haemodialysis on the lymphocyte subsets in PLWH with ESKD. In addition, all available %CD4+ T-cell counts, absolute CD4+ T-cell counts and viral load measurements were collected to assess the longitudinal trends of these measurements in PLWH with ESKD. METHODS: This was a cross-sectional study comparing two groups. The HIV-infected study participants (n = 17) and HIV-uninfected controls (n = 17) were recruited from renal dialysis centres in Johannesburg from 2017 to 2018. Demographic data and social data were collected from all the study participants (n = 17). Blood samples were collected from all the study participants (before and after a haemodialysis session), and the lymphocyte subsets were then measured. The available longitudinal data for the serial CD4+ T-cell counts and HIV viral loads were collected (n = 14). RESULTS: Our cohort showed a statistically significant increase in the post-dialysis percentage of CD4+ T cells (5%, p < 0.001) and the absolute CD4+ T-cell counts (21 cells/µL, p < 0.03). The longitudinal trend analysis for the percentage of CD4+ T cells revealed a significant increase in five participants (36%), and a single patient (7%) had a significant decrease in the longitudinal trend analysis for the absolute CD4+ T-cell counts. The longitudinal trend analysis for HIV viral load revealed the majority of our participants were not virologically suppressed. CONCLUSION: This study showed that haemodialysis does not have an immediate negative impact on CD4+ T-cell count, suggesting that immunologic recovery is not impeded by treatment of the underlying ESKD.

The Utility of the Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Assay in Detecting Abnormalities in Lipid Metabolism and Cardiovascular Risk in an HIV-Infected South African Cohort.

People with HIV (PWH) have an increased prevalence of cardiovascular disease (CVD) compared to uninfected patients. Lipoprotein-associated phospholipase A2 (Lp-PLA2) catalyzes the synthesis of pro-inflammatory lipids that recruit monocytes. Current guidelines for assessing cardiovascular risk in HIV-infected patients suggest that Lp-PLA2 may be a useful surrogate marker for CVD health in this patient population. Lipoprotein-associated phospholipase A2, lipids, glucose, physical parameters, and carotid intimal-medial thickness (CIMT) were measured in 98 participants (49 HIV-uninfected, 27 antiretroviral therapy [ART]-naive PWH, and 22 ART-treated PWH). HIV viral load (VL) and CD4+ T-cell count were measured in HIV-infected participants. Lipoprotein-associated phospholipase A2 was increased in participants on protease inhibitor (PI) ART (median 50.5 vs 127.0 nmol/mL, P = .05) and correlated with age, body mass index, and cholesterol. Lipoprotein-associated phospholipase A2 was not related to Framingham risk score or CIMT but correlated directly with VL (r = .323, P = .025) and inversely with CD4+ T-cell count (r = -.727, P < .001). Lipoprotein-associated phospholipase A2 was increased in HIV-infected participants on PIs and correlated strongly with VL and CD4+ T-cell count suggesting that HIV-associated inflammation is linked to increased Lp-PLA2, providing a mechanistic link between HIV and CVD.