Characterization of the immune response in HIV-associated cryptococcal meningitis

Despite widespread roll out of anti-retroviral therapy (ART), cryptococcal meningitis (CM) remains a significant cause of morbidity and mortality among HIV-infected persons in sub-Saharan Africa. Host immune response is central to the pathogenesis, and not only influences susceptibility to infection, but clinical presentation and outcome. Animal studies suggest that macrophage activation is a crucial component of the host immune response but human studies are limited. This thesis aims to characterize the activation state of CSF macrophages and blood monocytes in persons with HIV-associated CM, assess the effect of ART on the immune response, and identify immune correlates of severe disease and poor outcome. A prospective cohort study was conducted in Cape Town, South Africa (Innate Immunity in Cryptococcal Disease (IICD)). Persons with HIV-associated CM were enrolled and the immune response in CSF and blood examined using flow cytometry and cytokine analysis. Soluble markers of macrophage activation (sCD14, sCD163) and the ex vivo responses of whole blood and monocytes to TLR ligands and cryptococcal preparations were also measured. Additional CSF and serum samples were obtained from the Cryptococcal Optimal ART timing (COAT) trial (South Africa and Uganda) to examine the immune consequences of earlier ART initiation in cryptococcal meningitis. In this trial, earlier ART was associated with increased mortality in CM compared with a deferred ART strategy (8 days vs. 5 weeks post CM diagnosis respectively). CSF flow cytometry showed that CD8 T cells were the most common cells in the CSF followed by neutrophils and CD4 T cells. Both CD14+ and CD14- CSF macrophages were identified and expressed a range of surface markers associated with different activation states. Cryptococci were identified using flow cytometry as CD45 negative cells; cryptococcal counts using flow cytometry showed a very strong correlation with quantitative culture. There was no suggestion that raised intracranial pressure occurred as a result of a pro-inflammatory response; instead, persons with high intracranial pressure had a greater CSF fungal burden, a significantly reduced CSF cellular infiltrate (particularly CD4 T cells, CD8 T cells and double negative T cells) and larger cryptococcal cells. Contrary to animal studies, there was no association between macrophage activation and fungal burden. ART appeared to have a more profound effect on the immune response at site of disease (CSF) compared to the blood. In the IICD study, ART was associated with an increased frequency of CD4 T cells and decreased frequency of CD8 T cells in the CSF, along with increased expression of CD206 and CD16 on the surface of CSF monocyte- macrophages, suggesting an alternatively activated (M2) phenotype. These changes were closely related to plasma HIV viral load, even in subjects not taking ART. In the COAT trial, participants who received earlier ART initiation were more likely to have a CSF cellular infiltrate by day 14 compared to those in the deferred ART arm (8 days vs. 5 weeks post CM diagnosis respectively). This primarily occurred among persons with an initial paucity of CSF cellular response, the same sub-group identified in the COAT trial to be at highest risk of mortality following early ART initiation, and the same group previous shown in cohort studies to be at increased risk of IRIS. Furthermore, participants in the Kampala earlier ART arm had increased sCD14 and sCD163 concentrations in their CSF at day 14 suggesting increased macrophage activation in the central nervous system (CNS). These findings are supportive of the hypothesis that the increased mortality associated with earlier ART initiation in the COAT trial was immunologically driven. Finally, immune correlates of fatal outcome were examined. Persons who died by week 12 in the IICD study were noted to have a lower proportion of double negative T cells and lower concentrations of IFN-γ in the CSF at baseline. Non-survivors also had evidence of increased immune activation in the blood, elevated IL-10, decreased monocyte HLA-DR expression, impaired monocyte TNF-α response to LPS, and decreased IFN-γ responses to LPS in whole blood. These features are consistent with those of monocyte anergy and the compensatory anti-inflammatory response syndrome. The cause for this immune signature among persons with HIV-associated cryptococcal meningitis was not clear and warrants further study; the downstream effects of HIV-1 induced immune activation or the immune modulatory effects of cryptococcal capsule are both possibilities

Flow cytometry to assess CSF fungal burden in cryptococcal meningitis

Fungal burden in the cerebrospinal fluid is an important determinant of mortality in cryptococcal meningitis but its use to aid clinical decision-making is hampered by the time involved to perform quantitative cultures. Here we demonstrate the potential of flow cytometry as a novel and rapid technique to address this

The CSF immune response in HIV-1-associated cryptococcal meningitis: macrophage activation, correlates of disease severity and effect of antiretroviral therapy.

Immune modulation may improve outcome in HIV-associated cryptococcal meningitis. Animal studies suggest alternatively activated macrophages are detrimental but human studies are limited. We performed a detailed assessment of the cerebrospinal fluid (CSF) immune response and examined immune correlates of disease severity and poor outcome, and the effects of antiretroviral therapy (ART). We enrolled persons ≥18 years with first episode of HIV-associated cryptococcal meningitis. CSF immune response was assessed using flow cytometry and multiplex cytokine analysis. Principal component analysis was used to examine relationships between immune response, fungal burden, intracranial pressure and mortality, and the effects of recent ART initiation (<12 weeks). CSF was available from 57 persons (median CD4 34/μL). CD206 (alternatively activated macrophage marker) was expressed on 54% CD14+ and 35% CD14- monocyte-macrophages. High fungal burden was not associated with CD206 expression but with a paucity of CD4+, CD8+ and CD4-CD8- T cells and lower IL-6, G-CSF and IL-5 concentrations. High intracranial pressure (≥30cmH2O) was associated with fewer T cells, a higher fungal burden and larger Cryptococcus organisms. Mortality was associated with reduced interferon-gamma concentrations and CD4-CD8- T cells but lost statistical significance when adjusted for multiple comparisons. Recent ART was associated with increased CSF CD4/CD8 ratio and a significantly increased macrophage expression of CD206. Paucity of CSF T cell infiltrate rather than alternative macrophage activation was associated with severe disease in HIV-associated cryptococcosis. ART had a pronounced effect on the immune response at the site of disease

Patterns of size variation in bees at a continental scale: does Bergmann’s rule apply

Body size latitudinal clines have been widley explained by the Bergmann’s rule in homeothermic vertebrates. However, there is no general consensus in poikilotherms organisms in particular in insects that represent the large majority of wildlife. Among them, bees are a highly diverse pollinators group with high economic and ecological value. Nevertheless, no comprehensive studies of species assemblages at a phylogenetically larger scale have been carried out even if they could identify the traits and the ecological conditions that generate different patterns of latitudinal size variation. We aimed to test Bergmann’s rule for wild bees by assessing relationships between body size and latitude at continental and community levels. We tested our hypotheses for bees showing different life history traits (i.e. sociality and nesting behaviour). We used 142 008 distribution records of 615 bee species at 50 50 km (CGRS) grids across the West Palearctic. We then applied generalized least squares fitted linear model (GLS) to assess the relationship between latitude and mean body size of bees, taking into account spatial autocorrelation. For all bee species grouped, mean body size increased with higher latitudes, and so followed Bergmann’s rule. However, considering bee genera separately, four genera were consistent with Bergmann’s rule, while three showed a converse trend, and three showed no significant cline. All life history traits used here (i.e. solitary, social and parasitic behaviour; ground and stem nesting behaviour) displayed a Bergmann’s cline. In general there is a main trend for larger bees in colder habitats, which is likely to be related to their thermoregulatory abilities and partial endothermy, even if a ‘season length effect’ (i.e. shorter foraging season) is a potential driver of the converse Bergmann’s cline particularly in bumblebees

Numerical Analysis of Ca2+ Signaling in Rat Ventricular Myocytes with Realistic Transverse-Axial Tubular Geometry and Inhibited Sarcoplasmic Reticulum

The t-tubules of mammalian ventricular myocytes are invaginations of the cell membrane that occur at each Z-line. These invaginations branch within the cell to form a complex network that allows rapid propagation of the electrical signal, and hence synchronous rise of intracellular calcium (Ca2+). To investigate how the t-tubule microanatomy and the distribution of membrane Ca2+ flux affect cardiac excitation-contraction coupling we developed a 3-D continuum model of Ca2+ signaling, buffering and diffusion in rat ventricular myocytes. The transverse-axial t-tubule geometry was derived from light microscopy structural data. To solve the nonlinear reaction-diffusion system we extended SMOL software tool (http://mccammon.ucsd.edu/smol/). The analysis suggests that the quantitative understanding of the Ca2+ signaling requires more accurate knowledge of the t-tubule ultra-structure and Ca2+ flux distribution along the sarcolemma. The results reveal the important role for mobile and stationary Ca2+ buffers, including the Ca2+ indicator dye. In agreement with experiment, in the presence of fluorescence dye and inhibited sarcoplasmic reticulum, the lack of detectible differences in the depolarization-evoked Ca2+ transients was found when the Ca2+ flux was heterogeneously distributed along the sarcolemma. In the absence of fluorescence dye, strongly non-uniform Ca2+ signals are predicted. Even at modest elevation of Ca2+, reached during Ca2+ influx, large and steep Ca2+ gradients are found in the narrow sub-sarcolemmal space. The model predicts that the branched t-tubule structure and changes in the normal Ca2+ flux density along the cell membrane support initiation and propagation of Ca2+ waves in rat myocytes

Cultural diversity teaching and issues of uncertainty: the findings of a qualitative study

BACKGROUND: There is considerable ambiguity in the subjective dimensions that comprise much of the relational dynamic of the clinical encounter. Comfort with this ambiguity, and recognition of the potential uncertainty of particular domains of medicine (e.g.--cultural factors of illness expression, value bias in diagnoses, etc) is an important facet of medical education. This paper begins by defining ambiguity and uncertainty as relevant to clinical practice. Studies have shown differing patterns of students' tolerance for ambiguity and uncertainty that appear to reflect extant attitudinal predispositions toward technology, objectivity, culture, value- and theory-ladeness, and the need for self-examination. This paper reports on those findings specifically related to the theme of uncertainty as relevant to teaching about cultural diversity. Its focus is to identify how and where the theme of certainty arose in the teaching and learning of cultural diversity, what were the attitudes toward this theme and topic, and how these attitudes and responses reflect and inform this area of medical pedagogy. METHODS: A semi-structured interview was undertaken with 61 stakeholders (including policymakers, diversity teachers, students and users). The data were analysed and themes identified. RESULTS: There were diverse views about what the term cultural diversity means and what should constitute the cultural diversity curriculum. There was a need to provide certainty in teaching cultural diversity with diversity teachers feeling under considerable pressure to provide information. Students discomfort with uncertainty was felt to drive cultural diversity teaching towards factual emphasis rather than reflection or taking a patient centred approach. CONCLUSION: Students and faculty may feel that cultural diversity teaching is more about how to avoid professional, medico-legal pitfalls, rather than improving the patient experience or the patient-physician relationship. There may be pressure to imbue cultural diversity issues with levels of objectivity and certainty representative of other aspects of the medical curriculum (e.g.--biochemistry). This may reflect a particular selection bias for students with a technocentric orientation. Inadvertently, medical education may enhance this bias through training effects, and accommodate disregard for subjectivity, over-reliance upon technology and thereby foster incorrect assumptions of objective certainty. We opine that it is important to teach students that technology cannot guarantee certainty, and that dealing with subjectivity, diversity, ambiguity and uncertainty is inseparable from the personal dimension of medicine as moral enterprise. Uncertainty is inherent in cultural diversity so this part of the curriculum provides an opportunity to address the issue as it relates to patient care

Namilumab or infliximab compared with standard of care in hospitalised patients with COVID-19 (CATALYST): a randomised, multicentre, multi-arm, multistage, open-label, adaptive, phase 2, proof-of-concept trial

BACKGROUND: Dysregulated inflammation is associated with poor outcomes in COVID-19. We aimed to assess the efficacy of namilumab (a granulocyte-macrophage colony stimulating factor inhibitor) and infliximab (a tumour necrosis factor inhibitor) in hospitalised patients with COVID-19, to prioritise agents for phase 3 trials. METHODS: In this randomised, multicentre, multi-arm, multistage, parallel-group, open-label, adaptive, phase 2, proof-of-concept trial (CATALYST), we recruited patients (aged ≥16 years) admitted to hospital with COVID-19 pneumonia and C-reactive protein (CRP) concentrations of 40 mg/L or greater, at nine hospitals in the UK. Participants were randomly assigned with equal probability to usual care or usual care plus a single intravenous dose of namilumab (150 mg) or infliximab (5 mg/kg). Randomisation was stratified by care location within the hospital (ward vs intensive care unit [ICU]). Patients and investigators were not masked to treatment allocation. The primary endpoint was improvement in inflammation, measured by CRP concentration over time, analysed using Bayesian multilevel models. This trial is now complete and is registered with ISRCTN, 40580903. FINDINGS: Between June 15, 2020, and Feb 18, 2021, we screened 299 patients and 146 were enrolled and randomly assigned to usual care (n=54), namilumab (n=57), or infliximab (n=35). For the primary outcome, 45 patients in the usual care group were compared with 52 in the namilumab group, and 29 in the usual care group were compared with 28 in the infliximab group. The probabilities that the interventions were superior to usual care alone in reducing CRP concentration over time were 97% for namilumab and 15% for infliximab; the point estimates for treatment–time interactions were –0·09 (95% CI –0·19 to 0·00) for namilumab and 0·06 (–0·05 to 0·17) for infliximab. 134 adverse events occurred in 30 (55%) of 55 patients in the namilumab group compared with 145 in 29 (54%) of 54 in the usual care group. 102 adverse events occurred in 20 (69%) of 29 patients in the infliximab group compared with 112 in 17 (50%) of 34 in the usual care group. Death occurred in six (11%) patients in the namilumab group compared with ten (19%) in the usual care group, and in four (14%) in the infliximab group compared with five (15%) in the usual care group. INTERPRETATION: Namilumab, but not infliximab, showed proof-of-concept evidence for reduction in inflammation—as measured by CRP concentration—in hospitalised patients with COVID-19 pneumonia. Namilumab should be prioritised for further investigation in COVID-19. FUNDING: Medical Research Council