The Effect of the COVID-19 Pandemic on Suicide Trends in Pretoria, South Africa.

ABSTRACT: Suicide is one of the worldwide leading causes of unnatural death. Pretoria is a capital city of South Africa and is the fourth most populated city in South Africa. Between 16 and 22 suicide cases are recorded daily in South Africa.Case files from the Pretoria Medico-Legal Laboratory were reviewed between 1 January 2015 to 31 December 2021, which spanned the COVID-19 pandemic. All cases of possible and probable suicides were included. One-way analysis of variance and Pearson chi2 tests were conducted to determine statistical significance in the observed trends.A total of 1820 possible and probable suicide cases were identified. The year following the outbreak of the COVID-19 pandemic accounted for the largest number of suicides. White males were the most heavily represented population, followed by Black males. Hanging was the most common method for males, whereas poisoning via ingestion and/or overdose was more commonly used by females.Suicides in Pretoria have increased since previous studies from 9.74% and 10.2% to 13.32%. Methods used have changed among various population groups over the past two decades. This paper examined changes in suicide trends in Pretoria compared to previous studies and highlighted correlations between suicide trends and COVID-19 lockdown measures.

Pro-Inflammatory Interactions of Dolutegravir with Human Neutrophils in an In Vitro Study.

There is increasing awareness of an association between the uptake of the HIV integrase inhibitor, dolutegravir, in first-line antiretroviral regimens with unusual weight gain and development of the metabolic syndrome, particularly in African women. Although seemingly unexplored, the development of systemic inflammation linked to the putative pro-inflammatory activity of dolutegravir represents a plausible pathophysiological mechanism of this unusual weight gain. This possibility was explored in the current study undertaken to investigate the effects of dolutegravir (2.5−20 µg/mL) on several pro-inflammatory activities of neutrophils isolated from the blood of healthy, adult humans. These activities included the generation of reactive oxygen species (ROS), degranulation (elastase release) and alterations in the concentrations of cytosolic Ca2+ using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of neutrophils to dolutegravir alone resulted in the abrupt, dose-related, and significant (p < 0.0039−p < 0.0022) generation of ROS that was attenuated by the inclusion of the Ca2+-chelating agent, EGTA, or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI), phospholipase C (U733122), myeloperoxidase (sodium azide) and phosphoinositol-3-kinase (wortmannin). In addition, exposure to dolutegravir augmented the release of elastase by stimulus-activated neutrophils. These pro-inflammatory effects of dolutegravir on neutrophils were associated with significant, rapid, and sustained increases in the concentrations of cytosolic Ca2+ that appeared to originate from the extracellular compartment, seemingly consistent with an ionophore-like property of dolutegravir. These findings are preliminary and necessitate verification in the clinical setting of HIV infection. Nevertheless, given the complex link between inflammation and obesity, these pro-inflammatory interactions of dolutegravir with neutrophils may contribute to unexplained weight gain, possibly via the development of insulin resistance.

Interleukin-10 and interleukin-1 receptor antagonist distinguish between patients with sepsis and the systemic inflammatory response syndrome (SIRS).

The current study evaluated the potential of clinical parameters and circulating biomarkers to distinguish sepsis from SIRS in patients admitted with systemic inflammation. Clinical parameters, leukocyte counts and platelets were measured on admission. Circulating C-reactive protein (CRP), procalcitonin (PCT) and cytokine concentrations were quantified using laser immunonephelometry, immunoluminescence and a Bio-Plex suspension bead array system respectively. Blood, sputum, urine, peritoneal and cerebrospinal fluid were sent for microscopy and culture. Based on clinical information and the results of microbiological testing, 62 patients were classified retrospectively into 2 groups, those with sepsis (n = 37) or SIRS (n = 25). Mean body temperature was higher and blood pressure lower in the sepsis patients. Circulating concentrations of CRP, PCT, interleukin (IL)-10 and IL-1 receptor antagonist (IL-1Ra) were significantly higher in patients with sepsis, with IL-10 identified as the best biomarker in differentiating sepsis from SIRS. The biomarkers that best predicted overall mortality were platelet counts >PCT ≥ CRP > IL-6 > IL-1Ra. These findings demonstrate that patients with sepsis have significantly increased levels of the immunosuppressive/anti-inflammatory cytokines, IL-1Ra and IL-10, compared to those with SIRS, consistent with a more intense counteracting anti-inflammatory response, while a biomarker profile including platelets, PCT, CRP, IL-6 and IL-1Ra may be useful to predict mortality.

Tobacco-Derived Lipopolysaccharide, Not Microbial Translocation, as a Potential Contributor to the Pathogenesis of Rheumatoid Arthritis.

Microbial lipopolysaccharides (LPS) have been implicated in the pathogenesis of rheumatoid arthritis (RA), possibly driving a systemic inflammatory response that may trigger the development and/or exacerbation of the disease. To explore the existence of this mechanism in African RA patients, we have measured systemic levels of LPS and its surrogate, LPS-binding protein (LBP), as well as those of intestinal fatty acid-binding protein (I-FABP), pulmonary surfactant protein D (SP-D), and cotinine in serum to identify possible origins of LPS, as well as associations of these biomarkers with rheumatoid factor (RF) and anticitrullinated peptide (aCCP) autoantibodies and the DAS 28-3 clinical disease severity score. A cohort of 40 disease-modifying antirheumatic drug-naïve, black South African RA patients rated by compound disease scores and 20 healthy subjects and 10 patients with chronic obstructive pulmonary disease (COPD) as controls were included in this study. Levels of the various biomarkers and autoantibodies were measured using a combination of ELISA and immunofluorimetric and immunoturbidometric procedures. LPS levels were lowest in the RA group compared to the healthy controls (p = 0.026) and COPD patients (p = 0.017), while LBP levels were also significantly lower in RA compared to the healthy individuals (p = 0.036). Levels of I-FABP and SP-D were comparable between all three groups. Categorisation of RA patients according to tobacco usage revealed the following significant positive correlations: LBP with C-reactive protein (p = 0.0137); a trend (p = 0.073) towards an association of LBP with the DAS 28-3 disease severity score; RF-IgG antibodies with both LPS and LBP (p = 0.033 and p = 0.041, respectively); aCCP-IgG antibodies with LPS (p = 0.044); and aCCP-IgG with RF-IgM autoantibodies (p = 0.0016). The findings of this study, several of them novel, imply that tobacco products, as opposed to microbial translocation, represent a potential source of LPS in this study cohort of RA patients, again underscoring the risks posed by tobacco usage for the development and severity of RA.

Comparison of the diagnostic potential of three anti-citrullinated protein antibodies as adjuncts to rheumatoid factor and CCP in a cohort of South African rheumatoid arthritis patients.

PURPOSE: A retrospective comparison of the prevalence and diagnostic value of anti-Sa, anti-CEP-1, and anti-MCV autoantibodies relative to those of the established autoantibodies, composite RF and anti-CCP-IgG used routinely for RA diagnosis as a component of the ACR 2010 criteria, in a cohort of disease-modifying anti-rheumatic drug naïve African RA patients (n = 75). METHODS: Serum concentrations of anti-Sa, anti-CEP-1 and anti-MCV autoantibodies were measured using ELISA procedures, while anti-CCP-IgG antibodies were determined by fluorescence enzyme immunoassay, and composite RF by latex-enhanced laser nephelometry. RESULTS: The seropositivity frequencies of anti-Sa, anti-CEP-1 and anti-MCV antibodies for the RA patients were 82, 72, 85%, respectively, while that of anti-CCP-IgG and RF was 87% for both. Overall, anti-MCV demonstrated the best specificity, positive predictive value (PPV), odds ratio and positive likelihood ratio of all the types of autoantibody tested. CONCLUSION: These observations in this unique cohort of RA patients indicated novel associations of all three autoantibodies in regard to HLA-SE risk alleles, disease severity and tobacco use that were not reported before. Elevated anti-Sa titers designated a propensity of higher disease and high-risk alleles in our cohort. Anti-CEP-1 association with HLA-SE homozygosity and high-risk alleles is also novel in this group. Of note, measurement of anti-MCV antibodies on presentation, either as an adjunctive or even as a stand-alone test, surpassed all other biomarkers investigated here and, therefore, may add value to clinical management.

Smoking and Air Pollution as Pro-Inflammatory Triggers for the Development of Rheumatoid Arthritis.

INTRODUCTION: Smoking is now well recognized not only as a risk factor for rheumatoid arthritis (RA), but also as a determinant of disease activity, severity, response to therapy, and possibly mortality. METHODS: Studies, mostly recent, which have provided significant insights into the molecular and cellular mechanisms which underpin the pathogenesis of smoking-related RA, as well as the possible involvement of other types of outdoor and indoor pollution form the basis of this review. RESULTS: Smoking initiates chronic inflammatory events in the lungs. These, in turn, promote the release of the enzymes, peptidylarginine deiminases 2 and 4 from smoke-activated, resident and infiltrating pulmonary phagocytes. Peptidylarginine deiminases mediate conversion of various endogenous proteins to putative citrullinated autoantigens. In genetically susceptible individuals, these autoantigens trigger the production of anti-citrullinated peptide, pathogenic autoantibodies, an event which precedes the development of RA. CONCLUSIONS: An increasing body of evidence has linked chronic inflammatory events in the lungs of smokers, to the production of anti-citrullinated peptide autoantibodies and development of RA. Creation of awareness of the associated risks, assessment of smoking status and implementation of compelling antismoking strategies must be included in the routine clinical management of patients presenting with suspected RA. IMPLICATIONS: Chronic inflammatory mechanisms operative in the lungs of smokers lead to the production of anti-citrullinated protein antibodies which, in turn, drive the development of RA. These mechanistic insights not only reinforce the association between smoking and risk for RA, but also the necessity to increase the level of awareness in those at highest risk.

Reliable and cost-effective serodiagnosis of rheumatoid arthritis.

Early diagnosis of patients with rheumatoid arthritis (RA) optimises therapeutic benefit and the probability of achieving disease remission. Notwithstanding clinical acumen, early diagnosis is dependent on access to reliable serodiagnostic procedures, as well as on the discerning application and interpretation of these. In the case of RA, however, no disease-specific serodiagnostic procedure is available due to the multi-factorial and polygenic nature of this autoimmune disorder. This has resulted in the development of an array of serodiagnostic procedures based on the detection of autoantibodies reactive with various putative autoantigens. Other procedures based on measurement of elevations in the concentrations of systemic biomarkers of inflammation, most commonly acute phase reactants and cytokines/chemokines, are used as objective indices of disease activity. Following a brief overview of RA research in African populations, the current review is focused on those autoantibodies/biomarkers, specifically rheumatoid factor, anti-citrullinated peptide antibodies and C-reactive protein, which are currently recognised as being the most reliable and cost-effective with respect to disease prediction and diagnosis, as well as in monitoring activity and outcome.

Pitfalls in the assessment of smoking status detected in a cohort of South African RA patients.

This study was conceived in an attempt to explain the unexpectedly high frequency of elevated levels of serum cotinine measured retrospectively in a cohort of predominantly black South African females with rheumatoid arthritis (RA), findings that were inconsistent with the smoking histories derived from health questionnaires. The discrepant findings suggested either a greater tendency towards underreporting of smoking status in the study cohort, or possible confounding effects of the use of smokeless tobacco products. In addition to the cohort of RA patients (n = 138, of whom 115 (83 %) were female), blood samples were also taken from a second cohort consisting of 29 declared smokers, 18 (62 %) of whom where females, 29 smokeless tobacco (SLT) users (all female), and 22 non-users of any tobacco products, 18 (82 %) of whom were females. Serum cotinine levels were determined using an ELISA procedure. Cotinine levels of >10.0 ng/ml were detected in serum specimens from 43 (31 %), RA patients of whom 35 (81 %) were female, with a median value of 50.1 ng/ml and interquartile range (iqr) of 68.6. Only 18 of the 35 females indicated that they smoked. The groups of declared smokers and SLT users had equivalent median serum cotinine levels of 88.0 ng/ml (iqr = 10.8 ng/ml) and 87.0 ng/ml (iqr = 15.6 ng/ml), respectively, while cotinine was undetectable in specimens from non-tobacco product users (<0.2 ng/ml). Users of SLT products in South Africa are predominantly female and have serum cotinine levels which are comparable with those of current smokers, raising concerns about the validity of measurement of cotinine as the sole objective marker of smoking status in populations with high usage of SLTs. This situation can be rectified by ensuring that usage of SLT products is accurately recorded in health questionnaires, while inclusion of measurement of one or more additional, objective biomarkers of smoking in combination with cotinine may enable reliable distinction between smoking and usage of SLTs which, given the associated risks, is a strategy of particular relevance in RA.

Deaths from bee stings: a report of three cases from Pretoria, South Africa.

In South Africa bee stings are most commonly caused by either Apis mellifera capensis or A. mellifera scutellata, indigenous species which are notoriously aggressive when compared to European honey bees. According to Statistics South Africa, 109 deaths were documented for the period 2001-2011 as having been caused by hornets, wasps, and bees (ICD10-X26). This appears to be a small number but, as was reported in Australia, these statistics might be inaccurate due to either over- or underreporting of cases. We report 3 cases of fatalities due to bee stings, including one with postmortem features of diffuse intravascular coagulopathy. A brief overview of the venom of the honey bee, reactions following a bee sting and possible mechanisms of death are presented. Confirming the diagnosis in these cases may be very problematic for the forensic pathologist, as in many cases minimal history is available and both external and internal examination could fail to reveal any specific signs of bee sting or other obvious morphologic abnormalities. Thus, there is a need for reliable confirmatory or supportive diagnostic tests.

Circulating anti-citrullinated peptide antibodies, cytokines and genotype as biomarkers of response to disease-modifying antirheumatic drug therapy in early rheumatoid arthritis.

BACKGROUND: To measure circulating anti-citrullinated peptide antibodies (ACPA) and cytokines pre- and 6 months post-therapy as a strategy to predict and optimize responses to traditional disease-modifying antirheumatic drugs (DMARDs) in early RA, which is an unmet need in developing countries. PATIENTS AND METHODS: A cohort of 140 predominantly (88.5 %) black female South African patients with early RA was treated with synthetic DMARDs, mostly methotrexate (MTX) alone, or in combination with low-dose oral corticosteroids (CS). Circulating ACPA and a panel of circulating cytokines/chemokines/growth factors were measured at baseline and after 6 months of therapy in relation to disease activity and Shared Epitope (SE). RESULTS: Following 6 months of therapy, the median simplified disease activity index (SDAI) declined from a baseline of 41.4 to 16.0 (p = 0.0001) for the entire cohort, which was paralleled by significant falls in median serum ACPA levels (516.6 vs. 255.7 units/ml, p = <0.0001) and several of the circulating cytokines (IL-4, IL-7, IL-8, G-CSF, VEGF; p < 0.0010 - p < 0.0001) which were most evident in the subgroup of patients treated with a combination of MTX and CS. Although biomarker concentrations decreased most notably in the low-disease activity group post-therapy, no significant correlations between these biomarkers and disease activity were observed, Baseline ACPA levels, but not SDAI or cytokines, were significantly higher in the subgroup of risk allele-positive patients (561.1 vs. 331.9 units/ml, p < 0.05), while no associations with ACPA and a smoking history were evident. CONCLUSIONS: The use of DMARDs in RA is associated with significant decreases in ACPA and cytokines which did not correlate with changes in SDAI, precluding the utility of serial measurement of these biomarkers to monitor early responses to therapy, but may have prognostic value.

Transforming growth factor-ß1 and soluble co-inhibitory immune checkpoints as putative drivers of immune suppression in patients with basal cell carcinoma.

The current study compared the levels and possible associations between systemic soluble immune checkpoints (sICPs, n = 17) and a group of humoral modulators of immune suppressor cells (n = 7) in a cohort of patients with basal cell carcinoma (BCC, n = 40) and a group of healthy control subjects (n = 20). The seven humoral modulators of immunosuppressor cells were represented by the enzymes, arginase 1 and fibroblast activation protein (FAP), the chemokine, RANTES (CCL5) and the cytokines, interleukin-10 and transforming growth factor-ß1 (TGF-ß1), as well as the M2-type macrophage markers, soluble CD163 (sCD163) and sCD206. The plasma levels of six co-inhibitory sICPs, sCTLA-4, sLAG-3, sPD-1, sPD-L1, sTIM-3 and sPD-L2 were significantly elevated in the cohort of BCC patients (p<0.001-p<0.00001), while that of sBTLA was significantly decreased (p<0.006). Of the co-stimulatory sICPs, sCD27 and sGITR were significantly increased (p<0.0002 and p<0.0538) in the cohort of BCC patients, while the others were essentially comparable with those of the control participants; of the dual active sICPs, sHVEM was significantly elevated (p<0.00001) and TLR2 comparable with the control group. A correlation heat map revealed selective, strong associations of TGF-ß1 with seven co-stimulatory (z = 0.618468-0.768131) and four co-inhibitory (z = 0.674040-0.808365) sICPs, as well as with sTLR2 (z = 0.696431). Notwithstanding the association of BCC with selective elevations in the levels of a large group of co-inhibitory sICPs, our novel findings also imply the probable involvement of TGF-ß1 in driving immunosuppression in this malignancy, possibly via activation of regulatory T cells. Notably, these abnormalities were present in patients with either newly diagnosed or recurrent disease.

A radiologic-pathologic study of the histopathologic variants of ameloblastomas and their proliferation indices.

OBJECTIVES: This study aimed to analyze the clinicoradiologic features and Ki-67 proliferation indices between the histopathologic variants of ameloblastomas (ABs) for possible associations. STUDY DESIGN: The diagnosis and histopathologic variant were confirmed for all cases by experienced Oral and Maxillofacial Pathologists. Immunohistochemistry for Ki-67 was performed on the most representative formalin-fixed paraffin-embedded tissue block. Demographic, clinical data and radiologic features were analyzed from patient records and available radiographic examinations. The investigators were blinded to the histopathologic variant and proliferation index when the clinicoradiologic features were assessed. RESULTS: The current study included 116 cases of AB in the final sample. The indolent behavior of the unicystic variant was supported by their low proliferation index and slow growth paired with low frequencies of cortical destruction, loss of teeth, root resorption, and encroachment on anatomical structures. In contrast, the comparatively high proliferation index of the plexiform variant correlated with their fast growth and pain. Furthermore, high radiologic frequencies of cortical destruction, loss of teeth, and encroachment of surrounding anatomical structures supported their more aggressive clinical course. CONCLUSION: Statistically significant differences were noted between certain variants and Ki-67, location, borders, locularity, and cortical destruction, providing better insight into their biological behavior.

Serum matrix metalloproteinase-3 in comparison with acute phase proteins as a marker of disease activity and radiographic damage in early rheumatoid arthritis.

Matrix metalloproteinase-3 (MMP-3) is involved in the immunopathogenesis of rheumatoid arthritis (RA), but little is known about its relationship to genetic susceptibility and biomarkers of disease activity, especially acute phase reactants in early RA. MMP-3 was measured by ELISA in serum samples of 128 disease-modifying, drug-naïve patients and analysed in relation to shared epitope genotype, a range of circulating chemokines/cytokines, acute phase reactants, autoantibodies, cartilage oligomeric protein (COMP), and the simplified disease activity index (SDAI). MMP-3 was elevated >1.86 ng/ml in 56.25% of patients (P < 0.0001), correlated with several biomarkers, notably IL-8, IL-6, IFN γ , VEGF and COMP (r values = 0.22-0.33, P < 0.014-0.0001) and with CRP and SAA levels (r = 0.40 and 0.41, resp., P < 0.0000) and SDAI (r = 0.29, P < 0.0001), but not with erosions or nodulosis. However, the correlations of CRP and SAA with SDAI were stronger (respective values of 0.63 and 0.54, P < 0.001 for both). COMP correlated with smoking, RF, and MMP-3. MMP-3 is significantly associated with disease activity, inflammatory mediators and cartilage breakdown, making it a potential biomarker of disease severity, but seemingly less useful than CRP and SAA as a biomarker of disease activity in early RA.

Comparative upper-quarter posture analysis of female adolescent freestyle swimmers and non-swimmers.

ABSTRACTRepetitive upper-quarter limb movements imposed by freestyle swimming may lead to muscle length and tension changes, predisposing adolescent swimmers to postural malalignment. The study aimed to quantify the static upper-quarter postural alignment of competitive female adolescent freestyle swimmers, and compare their results to non-swimming peers and angles of spinal sagittal posture available in the literature. A cross-sectional study design was employed. The evaluation group (EVAL) consisted of 35 competitive swimmers (15 ± 3 y; 166.5 ± 9.9 cm; 65.5 ± 7.7 kg) and the control group (CON) of 36 peers (15 ± 3 y; 164.2 ± 6.7 cm; 62.1 ± 9.1 kg). Spinal sagittal posture was measured by photographic posture analysis. Median ± interquartile range and inter-group differences were calculated. Significant differences (p = 0.00) between groups for all variables, with a moderate - large effect, were observed. EVAL demonstrated restricted median scores for head-tilt angle (-8.7°), cervical angle (-13.3°) and shoulder protraction - retraction angle (-24°), and a greater score for thoracic angle (+7.4°), when compared to CON. EVAL and CON deviated from proposed criterion scores for cervical and thoracic angles, with EVAL also deviating from head-tilt angle and shoulder protraction - retraction angle criteria. In this group of adolescent participants, postural malalignment may have been exacerbated by years of freestyle swim training. HighlightsSignificant differences with medium to large effect sizes were noted in the angles of spinal sagittal posture between the competitive female adolescent freestyle swimmers and their non-swimming peers.Competitive female adolescent freestyle swimmers appeared to be more vulnerable to postural features in the upper quarter that were not within the desirable angles of spinal sagittal posture.The inherent nature of competitive freestyle swimming and the natural consequence of long-term training may explain the moderate to large effect sizes observed.

Dysregulation of systemic soluble immune checkpoints in early breast cancer is attenuated following administration of neoadjuvant chemotherapy and is associated with recovery of CD27, CD28, CD40, CD80, ICOS and GITR and substantially increased levels of PD-L1, LAG-3 and TIM-3.

Neoadjuvant chemotherapy (NAC) may alter the immune landscape of patients with early breast cancer (BC), potentially setting the scene for more effective implementation of checkpoint-targeted immunotherapy. This issue has been investigated in the current study in which alterations in the plasma concentrations of 16 soluble co-stimulatory and co-inhibitory, immune checkpoints were measured sequentially in a cohort of newly diagnosed, early BC patients (n=72), pre-treatment, post-NAC and post-surgery using a Multiplex® bead array platform. Relative to a group of healthy control subjects (n=45), the median pre-treatment levels of five co-stimulatory (CD27, CD40, GITRL, ICOS, GITR) and three co-inhibitory (TIM-3, CTLA-4, PD-L1) soluble checkpoints were significantly lower in the BC patients vs. controls (p<0.021-p<0.0001; and p<0.008-p<0.00001, respectively). Following NAC, the plasma levels of six soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in activation of CD8+ cytotoxic T cells, were significantly increased (p<0.04-p<0.00001), comparable with control values and remained at these levels post-surgery. Of the soluble co-inhibitory checkpoints, three (LAG-3, PD-L1, TIM-3) increased significantly post-NAC, reaching levels significantly greater than those of the control group. PD-1 remained unchanged, while BTLA and CTLA-4 decreased significantly (p<0.03 and p<0.00001, respectively). Normalization of soluble co-stimulatory immune checkpoints is seemingly indicative of reversal of systemic immune dysregulation following administration of NAC in early BC, while recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. Although a pathological complete response (pCR) was documented in 61% of patients (mostly triple-negative BC), surprisingly, none of the soluble immune checkpoints correlated with the pCR, either pre-treatment or post-NAC. Nevertheless, in the case of the co-stimulatory ICMs, these novel findings are indicative of the immune-restorative potential of NAC in early BC, while in the case of the co-inhibitory ICMs, elevated levels of soluble PD-L1, LAG-3 and TIM-3 post-NAC underscore the augmentative immunotherapeutic promise of targeting these molecules, either individually or in combination, as a strategy, which may contribute to the improved management of early BC.

Systemic Immune Dysregulation in Early Breast Cancer Is Associated With Decreased Plasma Levels of Both Soluble Co-Inhibitory and Co-Stimulatory Immune Checkpoint Molecules.

Breast cancer cells exploit the up-regulation or down-regulation of immune checkpoint proteins to evade anti-tumor immune responses. To explore the possible involvement of this mechanism in promoting systemic immunosuppression, the pre-treatment levels of soluble co-inhibitory and co-stimulatory immune checkpoint molecules, as well as those of cytokines, chemokines, and growth factors were measured in 98 newly diagnosed breast cancer patients and compared with those of 45 healthy controls using multiplex bead array and ELISA technologies. Plasma concentrations of the co-stimulatory immune checkpoints, GITR, GITRL, CD27, CD28, CD40, CD80, CD86 and ICOS, as well as the co-inhibitory molecules, PD-L1, CTLA-4 and TIM-3, were all significantly lower in early breast cancer patients compared to healthy controls, as were those of HVEM and sTLR-2, whereas the plasma concentrations of CX3CL1 (fractalkine), CCL5 (RANTES) and those of the growth factors, M-CSF, FGF-21 and GDF-15 were significantly increased. However, when analyzed according to the patients' breast cancer characteristics, these being triple negative breast cancer (TNBC) vs. non-TNBC, tumor size, stage, nodal status and age, no significant differences were detected between the plasma levels of the various immune checkpoint molecules, cytokines, chemokines and growth factors. Additionally, none of these biomarkers correlated with pathological complete response. This study has identified low plasma levels of soluble co-stimulatory and co-inhibitory immune checkpoint molecules in newly diagnosed, non-metastatic breast cancer patients compared to healthy controls, which is a novel finding seemingly consistent with a state of systemic immune dysregulation. Plausible mechanisms include an association with elevated levels of M-CSF and CCL5, implicating the involvement of immune suppressor cells of the M2-macrophage/monocyte phenotype as possible drivers of this state of systemic immune quiescence/dysregulation.

Elevated Levels of Soluble CTLA-4, PD-1, PD-L1, LAG-3 and TIM-3 and Systemic Inflammatory Stress as Potential Contributors to Immune Suppression and Generalized Tumorigenesis in a Cohort of South African Xeroderma Pigmentosum Patients.

Xeroderma Pigmentosum (XP), an autosomal recessive disorder characterized by ultraviolet radiation-induced abnormalities of DNA excision and repair pathways is associated with early development of cutaneous cancers. Intracellular oxidative stress has also been proposed as a contributor to the occurrence of skin cancers. However, little is known about the possible augmentative contributions of chronic inflammation, immune suppression and oxidative stress to the pathogenesis of malignancies associated with other subtypes of XP. This has been addressed in the current study, focused on the measurement of systemic biomarkers of inflammation, immune dysfunction and oxidative damage in XP patients, consisting of XP-C, XP-D and XP-E cases, including those XP-C cases who had already developed multiple skin malignancies. The inflammatory biomarker profile measured in XP patients and healthy control subjects included the cytokines, interleukins (ILs)-2, -4, -6, -10, interferon-γ (IFN- γ) and tumor-necrosis factor-α (TNF-α), the acute phase reactant, C-reactive protein (CRP), and cotinine (as an objective indicator of smoking status). Immune suppression was detected according to the levels of five soluble inhibitory immune checkpoint proteins (CTLA-4, PD-1, PD-L1, LAG-3 and TIM-3), as well as those of vitamin D, while oxidative stress was determined according to the circulating levels of the DNA adduct, 8-hydroxy-2-deoxyguanosine (8-OH-dG). These various biomarkers were measured in plasma using immunofluorimetric, nephelometric and ELISA procedures. Significant elevations in IL-6 (P<0.01) and TNF-α (P<0.0001), but none of the other cytokines, as well as increased levels of all five soluble inhibitory immune checkpoints (P=0.032-P=0.0001) were detected in the plasma of the XP patients. C-reactive protein and vitamin D were increased and decreased, respectively (both P<0.0001), while only one participant had an elevated level of plasma cotinine. Surprisingly, the levels of 8-OH-dG were significantly (P=0.0001) lower in the group of XP patients relative to a group of healthy control subjects. The findings of increased levels of pro-inflammatory cytokines and, in particular, those of the soluble immune checkpoints, in the setting of decreased vitamin D and moderately elevated levels of CRP in XP patients suggest a possible secondary role of ongoing, inflammatory stress and immune suppression in the pathogenesis of XP-associated malignancies.

Systemic levels of the soluble co-inhibitory immune checkpoints, CTLA-4, LAG-3, PD-1/PD-L1 and TIM-3 are markedly increased in basal cell carcinoma.

Although co-inhibitory immune checkpoint proteins are primarily involved in promoting cell-cell interactions that suppress adaptive immunity, especially tumor immunity, the soluble cell-free variants of these molecules are also detectable in the circulation of cancer patients where they retain immunosuppressive activity. Nevertheless, little is known about the systemic levels of these soluble co-inhibitory immune checkpoints in patients with various subtypes of basal cell carcinoma (BCC), which is the most invasive and treatment-resistant type of this most commonly-occurring malignancy. In the current study, we have measured the systemic concentrations of five prominent co-inhibitory immune checkpoints, namely CTLA-4, LAG-3, PD-1/PD-L1 and TIM-3, as well as those of C-reactive protein (CRP) and vitamin D (VD), in a cohort of patients (n = 40) with BCC, relative to those of a group of control participants, using the combination of multiplex bead array, laser nephelometry and ELISA technologies, respectively. The median systemic concentrations of CRP and VD were comparable between the two groups; however, those of all five immune checkpoints were significantly elevated (P = 0.0184 - P = < 0.00001), with those of CTLA-4 and PD-1 being highly correlated (r = 0.87; P < 0.00001). This seemingly novel finding not only identifies the existence of significant systemic immunosuppression in BCC, but also underscores the therapeutic promise of immune checkpoint targeted therapy, as well as the potential of these proteins to serve as prognostic/predictive biomarkers in BCC.

Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism.

Dolutegravir is a highly potent HIV integrase strand transfer inhibitor that is recommended for first-line anti-retroviral treatment in all major treatment guidelines. A recent study has shown that people taking this class of anti-retroviral treatment have a substantially higher risk of early-onset cardiovascular disease, a condition shown previously to be associated with increased platelet reactivity. To date, few studies have explored the effects of dolutegravir on platelet activation. Accordingly, the current study was undertaken with the primary objective of investigating the effects of dolutegravir on the reactivity of human platelets in vitro. Platelet-rich plasma, isolated platelets, or buffy coat cell suspensions prepared from the blood of healthy adults were treated with dolutegravir (2.5-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin, or a thromboxane A2 receptor agonist U46619. Expression of platelet CD62P (P-selectin), formation of heterotypic neutrophil:platelet aggregates, and calcium (Ca2+) fluxes were measured using flow cytometry and fluorescence spectrometry, respectively. Dolutegravir caused dose-related potentiation of ADP-, thrombin- and U46619-activated expression of CD62P by platelets, as well as a significant increases in formation of neutrophil:platelet aggregates. These effects were paralleled by a spontaneous, receptor-independent elevation in cytosolic Ca2+ that appears to underpin the mechanism by which the antiretroviral agent augments the responsiveness of these cells to ADP, thrombin and U46619. The most likely mechanism of dolutegravir-mediated increases in platelet cytosolic Ca2+ relates to a combination of lipophilicity and divalent/trivalent metal-binding and/or chelating properties of the anti-retroviral agent. These properties are likely to confer ionophore-type activities on dolutegravir that would promote movement of Ca2+ across the plasma membrane, delivering the cation to the cytosol where it would augment Ca2+-dependent intracellular signaling mechanisms. These effects of dolutegravir may lead to hyper-activation of platelets which, if operative in vivo, may contribute to an increased risk for cardiometabolic co-morbidities.

Virus Etiology, Diversity and Clinical Characteristics in South African Children Hospitalised with Gastroenteritis.

BACKGROUND: Viral gastroenteritis remains a major cause of hospitalisation in young children. This study aimed to determine the distribution and diversity of enteric viruses in children ≤5 years, hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital, Pretoria, South Africa, between July 2016 and December 2017. METHODS: Stool specimens (n = 205) were screened for norovirus GI and GII, rotavirus, sapovirus, astrovirus and adenovirus by multiplex RT-PCR. HIV exposure and FUT2 secretor status were evaluated. Secretor status was determined by FUT2 genotyping. RESULTS: At least one gastroenteritis virus was detected in 47% (96/205) of children. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). Norovirus genotypes GI.3, GII.2, GII.3, GII.4, GII.7, GII.12, GII.21, and rotavirus strains G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6], G9P[8] and sapovirus genotypes GI.1, GI.2, GII.1, GII.4, GII.8 were detected; norovirus GII.4[P31] and rotavirus G3P[4] predominated. Asymptomatic norovirus infection (GI.3, GI.7, GII.4, GII.6, GII.13) was detected in 22% of 46 six-week follow up stools. HIV exposure (30%) was not associated with more frequent or severe viral gastroenteritis hospitalisations compared to unexposed children. Rotavirus preferentially infected secretor children (p = 0.143) and norovirus infected 78% secretors and 22% non-secretors. CONCLUSION: Rotavirus was still the leading cause of gastroenteritis hospitalisations, but norovirus caused more severe symptoms.