Abha Chopra

The abundant skin commensal, Staphylococcus epidermidis, is the leading cause of late-onset sepsis (LOS) in preterm infants but rarely causes infections in term infants and adults. Staphylococcal virulence mechanisms and the role of the preterm immune responses in driving these life-threatening infections remain poorly understood. Using an ex vivo sepsis model, we challenged whole blood from very preterm infants (30-32 weeks gestational age, GA; n = 8), term infants (> 37 weeks GA; n = 8), and young adults (18-25 years; n = 8) with either live S. epidermidis or S. aureus (~ 10
colony-forming units, CFU/ml) for 90 min. Dual RNA-sequencing (RNA-seq) was performed to simultaneously assess host and pathogen gene expression profiles, identifying common and pathogen-specific responses across cohorts. We found shared immune processes induced in all age groups upon bacterial challenge, including cytokine (IL1A, IL1B, IL6, IFNB1) and chemokine (CCL20, CCL3, CCL7, CXCL2) signalling. Preterm infants also exhibited unique responses, such as increased platelet activation and fibrin clot formation, Wnt signalling, and hypoxia pathways in response to S. epidermidis challenge. Our findings suggest that bacterial gene co-expression, including iron acquisition and heme biosynthesis genes, are also influenced by the hosts developmental age, highlighting the complexity of host-bacterial interactions in the early stages of neonatal sepsis.

Purpose: Despite ART initiation in the acute phase of HIV infection (AHI), the viral reservoir persists. In the NOVA study, the viral reservoir declined between 24 and 156 weeks after ART initiation in individuals treated during AHI and correlated with the presence of HIV-specific CD8+ T-cell responses at 24 weeks. Now we further explore the effect of selective CD8+ T-cell pressure on viral integration sites.

Method: Two individuals participating in the Netherlands Cohort Study on Acute HIV Infection (NOVA study) (P1 and P2) were selected for an in-depth study (clinical characteristics shown in Fig. 1). HIV-specific CD8+ T-cell proliferative responses upon HIV peptide stimulation were determined by flow cytometry. Viral isolates were sequenced and analyzed for the presence of CD8+ T-cell epitopes using a HLA-I class binding prediction tool. HIV proviral structure and integration sites were characterized using a modified Integrated Proviral Sequencing Assay (IPSA).
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Results: In P1, strong, broad HIV-specific proliferative CD8+ T-cell responses were observed at 24 weeks, which corresponded with a substantial number of viral escape mutations in HLA-A*02:01:01, HLA-B*13:02:01, HLA-B*15:01:01 (Gag), HLA-B*13:02:01 and HLA-B*15:01:01 (Nef) restricted epitopes. In P2, low frequencies of proliferating CD8+ T-cells were observed at 24 weeks, which is reflected by a relatively low number of escape mutations at that time point. In P2 viral escape was mostly observed in HLA-B*40:01:02 (Gag) restricted epitopes and in HLA-A*01:01 and HLA-A*02:01:01 (Nef and Pol) restricted epitopes (Fig. 2). IPSA demonstrated a strong decrease in intact HIV DNA between 24 and 156 weeks in both participants. Interestingly, 65-77% of intact proviruses were located in a genic site, of which 33-66% in antisense region implying selection for transcriptionally latent proviruses.
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Conclusions: This study found that broad HIV-specific CD8+ T-cell responses drive viral escape mutations and associates with proviral loss and propensity of antisense (silent) integration.

Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B*13:01 and HLA-B*38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis.
To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States (US) and South Africa (SA).
We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced SCAR patients in the US (n=63) and SA (n=26) compared to population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2.
In a multiple logistic regression model, HLA-B*44:03 (Pc<0.001, OR: 4.08), HLA-B*38:01 (Pc<0.001, OR: 5.66), and HLA-C*04:01 (Pc=0.003, OR: 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the US. HLA-B*44:03 was also associated with co-trimoxazole-induced DRESS in SA (Pc=0.019, OR: 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C*04:01 identified 94% and 78% of co-trimoxazole-induced SJS/TEN and DRESS in the US, respectively. The SEA risk allele HLA-B*13:01, with SPBS to HLA-B*44:03, was identified in just 1/63 US SCAR patients.
HLA alleles with SPBS to SEA-related risk alleles including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01 predisposed to co-trimoxazole-induced SCAR in the US and SA. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR.
HLA alleles including HLA-B*13:01 and HLA-B*38:02 are risk factors for co-trimoxazole-induced SCAR in Asian populations. However, the generalizability of these associations to other global populations is unknown but critical for population-appropriate risk stratification and diagnosis.
HLA alleles with shared peptide binding specificities (SPBS) to Asian-related risk alleles including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01 predisposed to co-trimoxazole-induced SCAR in the US and South Africa.
HLA alleles previously associated with co-trimoxazole-induced SCAR do not identify risk across populations. However, HLA alleles with SPBS provide biological plausibility and strategies for global and population-appropriate clinical risk stratification and diagnosis of cotrimoxazole-induced SCAR.

Inclusion body myositis (IBM) is a late-onset, treatment-resistant inflammatory myopathy. Approximately half of IBM patients develop autoantibodies against cytosolic 5′-nucleotidase 1A (cN1A), but their role in disease pathogenesis remains unclear. This pilot study examined the effects of anti-cN1A-positive IBM serum on human primary myotubes’ transcriptome profile, using anti-cN1A-negative IBM and healthy sera as controls. Exposure to anti-cN1A-positive serum altered the expression of 1126 genes, with upregulation of adaptive immune response genes, notably CTSH and CTSZ, encoding cathepsins H and Z. These findings were validated using a publicly available independent dataset comprising transcriptomes from fresh muscle tissue samples. NT5C1A mRNA, which encodes cN1A, was not detected in cultured myotubes regardless of the presence of autoantibodies. The findings suggest distinct pathological mechanisms in anti-cN1A-positive IBM, independent of direct antibody-target interactions. The role of cathepsins in IBM pathogenesis warrants further investigation.

The success of cancer immunotherapies has highlighted the importance of monitoring the anti-tumour T cell response. Patients with mesothelioma frequently present with a malignant pleural effusion (MPE) that is commonly drained regularly to alleviate symptoms. As MPE contains tumour cells, T cells and cytokines, it provides a unique opportunity to sample immune events at the tumour site. However, there is minimal information on how MPE T cells are distinct from those in the blood, and whether T cell phenotypes unique to each compartment correlate with survival. We characterised T cell populations of matched MPE and blood from 31 mesothelioma patients using flow cytometry and bulk T cell receptor beta (TCRβ) sequencing. MPE CD8+ and CD4+ T cells displayed increased expression of PD-1, TIGIT, LAG-3 and TIM-3 compared to blood, with co-expression of inhibitory receptors greatest on MPE CD8+ T cells with a tissue resident memory T cell phenotype (CD69+CD103+). CD8+ TCRβ repertoires displayed clonal overlap between MPE and blood, suggesting that a majority of T cells traffic between these compartments. Finally, we show that high expression of PD-1 on circulating CD4+ T cells is an independent prognostic factor for poor survival in this patient group. This work suggests that MPE T cell phenotypes differ from those in circulation, with blood-based T cell subsets more sensitive predictors of outcome in this study.

Few HIV-specific epitopes restricted by non-classical HLA-E have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope KF11 (KAFSPEVIPMF) restricted by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from eight people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8 responses were analyzed using scRNA-seq and scTCR-seq. Supernatants were also assessed for soluble protein profiling. HLA-I multimers were developed to identify CD8s restricted by HLA-B57 and/or HLA-E ex vivo. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, whereas E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27, findings which were corroborated through scRNA sequencing. TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03 as demonstrated by in vitro T cell reporter assays and ex vivo multimer screening. Ex vivo CD8s were singly restricted by HLA-B57 and HLA-E, with dual restriction only observed in PWH with lower viral load. These findings demonstrate that certain HIV-specific CD8s in PWH exhibit dual restriction by HLA-B*57 and HLA-E*01/03, leading to functionally distinct immune responses depending upon the restricting allele(s).

Background
Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B*13:01 and HLA-B*38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis.

Objective
To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States (US) and South Africa (SA).

Methods
We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced SCAR patients in the US (n=63) and SA (n=26) compared to population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2.

Results
In a multiple logistic regression model, HLA-B*44:03 (Pc<0.001, OR: 4.08), HLA-B*38:01 (Pc<0.001, OR: 5.66), and HLA-C*04:01 (Pc=0.003, OR: 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the US. HLA-B* 44:03 was also associated with co-trimoxazole-induced DRESS in SA (Pc=0.019, OR: 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C*04:01 identified 94% and 78% of co-trimoxazole-induced SJS/TEN and DRESS in the US, respectively. The SEA risk allele HLA-B*13:01, with SPBS to HLA-B*44:03, was identified in just 1/63 US SCAR patient.

Conclusion
HLA alleles with SPBS to SEA-related risk alleles including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01 predisposed to co-trimoxazole-induced SCAR in the US and SA. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host–bacterial gene expression during sepsis. The DRIB protocol yielded 2.10–6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6–24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51–68% (8.4–10.9 million) reads, while 0.5–6.7% (79,496–789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host–bacterial gene co-expression dynamics in sepsis progression and outcomes.

Introduction
Cryptosporidium hominis is the dominant Cryptosporidium species infecting humans, but most advances in developing robust in vitro culturing platforms for Cryptosporidium have utilised C. parvum. Consequently, there is relatively little available information specific to the biology and life cycle of C. hominis. The present study utilised a pumpless and tubeless gut-on-chip to generate a physiologically relevant in vitro environment by applying a constant fluid shear stress of 0.02 dyn cm-2 to HCT-8 cells.

Methods
Gut-on-chips were fabricated using standard soft lithography. C. hominis oocysts isolated from human pathology samples were used to infect the human ileocecal colorectal adenocarcinoma (HCT-8) cell line under a constant fluid shear stress of 0.02 dyn cm-2. Parasite growth was assessed using a C. hominis-specific quantitative PCR, a Cryptosporidium genus-specific immunofluorescence assay, and scanning electron microscopy. Differences in the HCT-8 transcriptome with and without fluid shear stress, and the host-parasite interaction, were both assessed using bulk transcriptomics.

Results
Transcriptomic analysis of the HCT-8 cell line cultured within the gut-on-chip demonstrated a metabolic shift towards oxidative phosphorylation when compared to the same cell line cultured under static conditions. Extended C. hominis (subtype IdA15G1) cultures were sustained for up to 10 days within the gut-on-chip as shown by a C. hominis-specific qPCR and a Cryptosporidium genus-specific immunofluorescence assay, which demonstrated ~30-fold amplification in the gut-on-chip over the duration of the experiment. Scanning electron microscopy of infected monolayers identified trophozoites, meronts, merozoites, macrogamonts, microgamonts, and possible gamont-like stages at 48 h post-infection. The potential role of gamonts in the Cryptosporidium life cycle remains unclear and warrants further investigation. Transcriptomes of HCT-8 cells infected with C hominis revealed upregulation of biological processes associated with cell cycle regulation and cell signalling in C. hominis-infected cells under fluid shear stress compared to static culture.

Conclusions
These data demonstrate that bioengineered gut-on-chip models support extended C. hominis growth and can be used to interrogate responses of host cells to infection. Owing to its relative simplicity, the pumpless and tubeless gut-on-chip can be accessible to most laboratories with established HCT-8 infection models for Cryptosporidium culture.

Persistent systemic inflammation is associated with an elevated risk of cardiometabolic diseases. However, the characteristics of the innate and adaptive immune systems in individuals who develop these conditions remain poorly defined. Doublets, or cell-cell complexes, are routinely eliminated from flow cytometric and other immune phenotyping analyses, which limits our understanding of their relationship to disease states. Using well-characterized clinical cohorts, including participants with controlled human immunodeficiency virus (HIV) as a model for chronic inflammation and increased immune cell interactions, we show that circulating CD14+ monocytes complexed to CD3+ T cells are dynamic, biologically relevant, and increased in individuals with diabetes after adjusting for confounding factors. The complexes form functional immune synapses with increased expression of proinflammatory cytokines and greater glucose utilization. Furthermore, in persons with HIV, the CD3+ T cell: CD14+ monocyte complexes had more HIV copies compared to matched CD14+ monocytes or CD4+ T cells alone. Our results demonstrate that circulating CD3+ T-cell: CD14+ monocyte pairs represent dynamic cellular interactions that may contribute to inflammation and cardiometabolic disease pathogenesis. CD3+ T-cell: CD14+ monocyte complexes may originate or be maintained, in part, by chronic viral infections. These findings provide a foundation for future studies investigating mechanisms linking T cell-monocyte cell-cell complexes to developing immune-mediated diseases, including HIV and diabetes.