Richard Helmuth Glashoff

HIV infection is accompanied by production of pro-inflammatory cytokines, which are regarded as critical in neuronal damage, leading to brain dysfunction. To develop diagnostic tools and therapeutic interventions, we need to measure CNS response to immune activation, hence the need to identify specific cytokine biomarkers that are associated with brain damage in HIV infection. This cross-sectional retrospective study applied Magnetic Resonance Imaging (MRI) for brain volumetric measurements and high-throughput Luminex-based immunoassays to quantify plasma cytokine and chemokine concentrations. We then used generalized linear models and Partial Least Square Regression models to evaluate the association between brain volume and plasma cytokines in predominantly treatment-naïve participants with HIV. After adjusting for clinical and demographic variables, we observed that higher MCP-1 (p = 0.013) and RANTES (p = 0.002) remained significantly associated with lower cortical white matter volume, whereas the anti-inflammatory cytokine IL-9 (p = 0.025) and the growth factors PDGFBB (p = 0.012) and VEGF (p = 0.001) were associated with higher cortical white matter volume. Only IL-6 (p = 0.010) was significantly associated with lower subcortical grey matter volume. Higher concentrations of five pro-inflammatory cytokines, IL-6 (p = 0.0001), IL-8 (p = 0.018), GCSF (p = 0.004), MCP-1 (p = 0.004), and RANTES (p = 0.015), were associated with lower total grey matter volume. Associations of pro-inflammatory cytokines with lower brain volume could imply a link to mechanisms of HIV-associated brain damage, which may lead to neurocognitive impairment. Therefore, the use of highly sensitive neuroimaging and high-throughput immunoassays in HIV-associated brain disorders has potential applications in clinical assessments and therapeutic monitoring.

Immune and inflammatory alterations in multisystem inflammatory syndrome in children (MIS-C) as compared to Kawasaki disease (KD) were investigated in South Africa, a region of unique genetic background and high infectious disease burden. The observational study included MIS-C and KD patients during 4 severe acute respiratory syndrome coronavirus 2 waves (June 1, 2020–March 31, 2023) plus 12 healthy controls. Clinical features, routine inflammatory markers, hematological parameters, lymphocyte subsets and plasma inflammatory cytokines/chemokines were compared between groups. We enrolled 68 MIS-C, 18 KD, and 12 healthy controls. MIS-C patients had higher rates of Intensive Care Unit admission compared to KD (46% vs 17%; P = .03) and longer hospital stay (8.5 vs 6 days; P < .001). 8 MIS-C but no KD patients had an ejection fraction of < 40% (P = .07). Median lymphocyte counts were decreased in MIS-C, 1.2 cells/μL (interquartile range 0.7–2.3) versus KD 2.5 cells/μL (interquartile range 1.2–3.7), P = .02. Median CD3 + T-cell counts were lower in MIS-C (P = .04). Children with MIS-C had a higher median N-terminal pro-B-type natriuretic peptide of 5836 ng/L (1784–25,698) versus 7 ng/L (88–3262), P < .001 and Troponin T 25 ng/L (9–73) versus 7 ng/L (4–24), P = .01. Majority of cytokines/chemokines were elevated in both MIS-C and KD. When MIS-C was stratified by severity, significant differences in C-reactive protein (P < .001), total lymphocytes (P = .01), and N-terminal pro-B-type natriuretic peptide (P = .01) were observed. Inflammatory cytokine and chemokine levels were markedly raised in both KD and MIS-C. 3 markers were highlighted as indicators of MIS-C severity. There is a strong overlap in inflammatory marker alterations between MIS-C and KD at presentation in the African setting.

Introduction: Better understanding of hepatitis B virus (HBV) biomarkers is needed. We evaluated Hepatitis B core related antigen (HBcrAg), in the United Kingdom (UK) and South Africa (SA). Methods: We undertook a cross-sectional retrospective observational study of adults with chronic HBV infection from the UK (n=142) and SA (n=211). We recorded clinical and laboratory parameters and quantified HBcrAg. We report HBcrAg distribution, relationship with other biomarkers, and performance in risk stratification based on point of care test (POCT) thresholds. Results: SA and UK cohorts were similar in sex and age (p>0.05), but significantly different in ethnicity, HIV coinfection, HBeAg-positivity and proportion with HBV viral load (VL) >200,000 IU/ml (all p5.3 log ₁₀ U/ml compared to the UK (33% vs 9% respectively, p200,000 IU/ml in the UK, but not SA. HBcrAg positively correlated with alanine transferase (ALT) (p<0.001 in UK, p<0.01 in SA), and fibrosis/cirrhosis by APRI score (p = 0.03 in UK and p=0.008 in SA), but not by elastography or FIB-4 scores. Discussion: HBcrAg distribution and relationship with other biomarkers differs between settings. HBcrAg (including POCT) may be a useful proxy for VL, but less so as a marker of disease progression, Its use needs tailoring to represent diverse populations.

Developing a broadly protective vaccine covering most ETEC variants has been elusive. The most clinically advanced candidate yet is an oral inactivated ETEC vaccine (ETVAX^®). We report on the use of a proteome microarray for the assessment of cross-reactivity of anti-ETVAX^® IgG antibodies against over 4000 ETEC antigens and proteins. We evaluated 40 (pre-and post-vaccination) plasma samples from 20 Zambian children aged 10–23 months that participated in a phase 1 trial investigating the safety, tolerability, and immunogenicity of ETVAX^® adjuvanted with dmLT. Pre-vaccination samples revealed high IgG responses to a variety of ETEC proteins including classical ETEC antigens (CFs and LT) and non-classical antigens. Post-vaccination reactivity to CFA/I, CS3, CS6, and LTB was stronger than baseline among the vaccinated compared to the placebo group. Interestingly, we noted significantly high post-vaccination responses to three non-vaccine ETEC proteins: CS4, CS14, and PCF071 (p = 0.043, p = 0.028, and p = 0.00039, respectively), suggestive of cross-reactive responses to CFA/I. However, similar responses were observed in the placebo group, indicating the need for larger studies. We conclude that the ETEC microarray is a useful tool for investigating antibody responses to numerous antigens, especially because it may not be practicable to include all antigens in a single vaccine.

Summary: Inborn errors of immunity (IEI) are inherited monogenic disorders resulting in defective immune response. Non‐infectious presentations are increasingly more apparent. Widely available, cost‐effective early indicators are needed. Peripheral‐blood cytopenia may be a presenting laboratory feature or an observed secondary phenomenon. This retrospective review of the South African Primary Immunodeficiency Registry (SAPIDR) aimed to assess the haematological indices at presentation and their association with the International Union of Immunological Societies (IUIS) 2019 IEI classification and mortality. Of 396 patients on the SAPIDR, 66% (n = 257) had available haematological results. Sixty percent were males and 85% under 18 years. A majority (53%) had predominantly antibody deficiency. At presentation, infection was prominent (86%) followed by cytopenia (62%). Neutropenia was associated with IUIS III [odds ratio (OR) 3.65, confidence interval (CI) 1.44–9.25], thrombocytopenia with IUIS II (OR 14.39, CI 2.89–71.57), lymphopenia with IUIS I (OR 12.16, CI 2.75–53.73) and pancytopenia with IUSI I (OR 12.24, CI 3.82–39.05) and IUIS II (OR 5.99, CI 2.80–12.76). Cytopenia showed shorter overall survival (OR 2.81, CI 1.288–4.16). Cytopenias that are severe, persistent, unusual and/or recurrent should prompt further investigation for IEI. The full blood count and leucocyte differential may facilitate earlier identification and serve as an adjunct to definitive molecular classification.

Background: Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a primary immunodeficiency (PID) characterised by a predisposition to infection by weakly-pathogenic mycobacteria. In countries with a high prevalence of tuberculosis (TB), individuals with MSMD are also prone to infections by Mycobacterium tuberculosis. Several MSMD-associated genes have been described, all resulting in a disruption of IL-12 and IFN-γ cytokine axis, which is essential for control of mycobacterial infections. An accurate molecular diagnosis, confirmed by phenotypic and functional immune investigations, is essential to ensure that the patient receives optimal treatment and prophylaxis for infections. The aim of this study was to implement a set of functional assays to assess the integrity of the IL-12-IFN-γ cytokine pathways in patients presenting with severe, persistent, unusual and/or recurrent TB, mycobacterial infections or other clinical MSMD-defining infections such as Salmonella. Methods: Blood was collected for subsequent PBMC isolation from 16 participants with MSMD-like clinical phenotypes. A set of flow cytometry (phenotype and signalling integrity) and ELISA-based (cytokine production) functional assays were implemented to assess the integrity of the IL-12-IFN-γ pathway. Results: The combination of the three assays for the assessment of the integrity of the IL-12-IFN-γ pathway was successful in identifying immune deficits in the IL-12-IFN-γ pathway in all of the participants included in this study. Conclusions: The data presented here emphasise the importance of investigating PID and TB susceptibility in TB endemic regions such as South Africa as MSMD and other previously described PIDs relating to TB susceptibility may present differently in such regions. It is therefore important to have access to in vitro functional investigations to better understand the immune function of these individuals. Although functional assays alone are unlikely to always provide a clear diagnosis, they do give an overview of the integrity of the IL-12-IFN-γ pathway. It would be beneficial to apply these assays routinely to patients with suspected PID relating to mycobacterial susceptibility. A molecular diagnosis with confirmed functional impairment paves the way for targeted treatment and improved disease management options for these patients.

Immune activation, which is accompanied by the production of proinflammatory cytokines, is a strong predictor of disease progression in HIV infection. Inflammation is critical in neuronal damage linked to HIV-associated neurocognitive disorders. We examined the relationship between plasma cytokine levels and deficits in neurocognitive function. Multiplex profiling by Luminex^® technology was used to quantify 27 cytokines/chemokines from 139 plasma samples of people living with HIV (PLWH). The relationship of plasma cytokine markers, clinical parameters, and cognitive impairment, was assessed using Spearman correlations. Partial least squares regression and variable importance in projection scores were used for further evaluation of the association. Forty-nine (35.3%) participants exhibited neurocognitive impairment based on a global deficit score (GDS) of at least 0.5 and 90 (64.7%) were classified as nonimpaired. Twenty-three (16.5%) initiated on combination antiretroviral therapy for 4 weeks before cognitive assessment and 116 (83.5%) were not on treatment. We identified five proinflammatory cytokines that were significant predictors of GDS namely, IP-10 (β = 0.058; p = .007), RANTES (β = 0.049; p = .005), IL-2 (β = 0.047, p = .006), Eotaxin (β = 0.042, p = .003), and IL-7 (β = 0.039, p = .003). IP-10 and RANTES were the strongest predictors of GDS. Both cytokines correlated with plasma viral load and lymphocyte proviral load and were inversely correlated with CD4⁺ T cell counts. IP-10 and RANTES formed a separate cluster with highest proximity. Study findings describe novel associations among IP-10, RANTES, cognitive status, plasma viral load, and cell-associated viral load.

Background: The X-linked recessive primary immunodeficiency disease (PIDD) Wiskott-Aldrich syndrome (WAS) is identified by an extreme susceptibility to infections, eczema and thrombocytopenia with microplatelets. The syndrome, the result of mutations in theWASgene which encodes the Wiskott-Aldrich protein (WASp), has wide clinical phenotype variation, ranging from classical WAS to X-linked thrombocytopaenia and X-linked neutropaenia. In many cases, the diagnosis of WAS in first affected males is delayed, because patients may not present with the classic signs and symptoms, which may intersect with other thrombocytopenia causes.Case presentation: Here, we describe a three-year-old HIV negative boy presenting with recurrent infections, skin rashes, features of autoimmunity and atopy. However, platelets were initially reported as normal in numbers and morphology as were baseline immune investigations. An older male sibling had died in infancy from suspected immunodeficiency. Uncertainty of diagnosis and suspected severe PIDD prompted urgent further molecular investigation. Whole exome sequencing identifiedc. 397 G > Aas a novel hemizygous missense mutation located in exon 4 ofWAS.Conclusion: With definitive molecular diagnosis, we could target treatment and offer genetic counselling and prenatal diagnostic testing to the family. The identification of novel variants is important to confirm phenotype variations of a syndrome.

Although rollout of combined antiretroviral treatment (cART) has blunted human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) onset, there is increased development of cardiovascular diseases (CVDs) in HIV-infected individuals. While most HIV-infected individuals on cART achieve viral suppression, this may not necessarily result in complete immunological recovery. This study therefore evaluated T-cell-mediated changes and coagulation markers in HIV-positive individuals to ascertain their potential to increase CVD risk. Eighty participants were recruited (Worcester, South Africa), and fasted blood was collected to evaluate: 1) immune activation (CD38 expression on CD4⁺and CD8⁺T cells) and thrombus formation [tissue factor (CD142)] on CD4⁺and CD8⁺T cells; 2) monocyte subpopulations (nonclassical, intermediate, and classical); and 3) classical regulatory T (Treg) cells with activation markers [glycoprotein A repetitions predominant (GARP) and special AT-rich sequence-binding protein 1 (SATB-1)]. High- and low-density lipoprotein subclasses (Lipoprint) were also determined. This study revealed four key findings for HIV-positive patients: 1) coexpression of the CD142 coagulation marker together with immune activation on both CD4⁺and CD8⁺T cells during chronic infection stages; 2) Treg cell activation and upregulated GARP and SATB-1 contributing to Treg dysfunction in chronic HIV; 3) proatherogenic monocyte subset expansion with significant correlation between T-cell activation and macrophage activation (marker: CD163); and 4) significant correlation between immune activation and lipid subclasses, revealing crucial changes that can be missed by traditional lipid marker assessments (LDL and HDL). These data also implicate lipopolysaccharide-binding protein as a crucial link between immune activation, lipid alterations, and increased CVD risk.NEW & NOTEWORTHY With combined antiretroviral treatment rollout, HIV-AIDS patients are increasingly associated with cardiovascular diseases onset. This study demonstrated the significant interplay between adaptive immune cell activation and monocyte/macrophage markers in especially HIV-positive individuals with virological failure and on second line treatment. Our data also show a unique link between immune activation and lipid subclass alterations, revealing important changes that can be missed by traditional lipid marker assessments (e.g., LDL and HDL).

Background: Antiretroviral therapy (ART) started between 7 and 12 weeks of age improves neurodevelopmental outcomes in HIV-infected (HIV+) infants, but the impact of even earlier initiation is not yet described.Objective: We assessed the early neurodevelopment of HIV+ infants who started ART within 21 days of life. Methods: Participants were enrolled from the public sector birth HIV-diagnosis program. Inclusion criteria included: birth weight 2000g, infant commencing ART 6 weeks and no infant Cytomegalovirus disease. ART included Zidovudine/Lamivudine/Nevirapine for the first 2 weeks, the latter then replaced by Lopinavir/Ritonavir. Once body weight 3kg and gestational age 44 weeks, Abacavir replaced Zidovudine. The Griffiths Mental Development Scales (GMDS) were administered at 10-12 months.Results: Of 29 infants assessed, 23 (79%) were girls. Mean birth weight was 3002 ± 501g. Twenty-four mothers (83%) received ART during pregnancy. Seven (24%) infants were diagnosed HIV+ within 48 hours of birth. Median [IQR] viral load (VL) at diagnosis was 3904 [259;16922] copies/ml, age starting ART was 6.0 [3;10] days and age at VL suppression was 19.1 [15; 36] weeks. At the GMDS assessment nine (31%) participants had detectable VL and 26 (90%) had WHO clinical stage I disease. GMDS was performed at a mean age of 11.5±0.8 months. Mean quotients were within the average range: Global Griffiths score was 103.6±10.9 and mean quotients on the subscales ranged from lowest 95.9±13.4 for Locomotor to highest 112.8±11.3 for Hearing-and-language.Conclusion: Preliminary findings in this small group suggest that early neurodevelopmental scores are within the normal range in infants with perinatal HIV-infection who started ART at a median of 6 days.