Perturbations of the T-cell receptor repertoire in response to SARS-CoV-2 in immunocompetent and immunocompromised individuals.

BACKGROUND: Functional T-cell responses are essential for virus clearance and long-term protection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, whereas certain clinical factors, such as older age and immunocompromise, are associated with worse outcome. OBJECTIVE: We sought to study the breadth and magnitude of T-cell responses in patients with coronavirus disease 2019 (COVID-19) and in individuals with inborn errors of immunity (IEIs) who had received COVID-19 mRNA vaccine. METHODS: Using high-throughput sequencing and bioinformatics tools to characterize the T-cell receptor ß repertoire signatures in 540 individuals after SARS-CoV-2 infection, 31 IEI recipients of COVID-19 mRNA vaccine, and healthy controls, we quantified HLA class I- and class II-restricted SARS-CoV-2-specific responses and also identified several HLA allele-clonotype motif associations in patients with COVID-19, including a subcohort of anti-type 1 interferon (IFN-1)-positive patients. RESULTS: Our analysis revealed that elderly patients with COVID-19 with critical disease manifested lower SARS-CoV-2 T-cell clonotype diversity as well as T-cell responses with reduced magnitude, whereas the SARS-CoV-2-specific clonotypes targeted a broad range of HLA class I- and class II-restricted epitopes across the viral proteome. The presence of anti-IFN-I antibodies was associated with certain HLA alleles. Finally, COVID-19 mRNA immunization induced an increase in the breadth of SARS-CoV-2-specific clonotypes in patients with IEIs, including those who had failed to seroconvert. CONCLUSIONS: Elderly individuals have impaired capacity to develop broad and sustained T-cell responses after SARS-CoV-2 infection. Genetic factors may play a role in the production of anti-IFN-1 antibodies. COVID-19 mRNA vaccines are effective in inducing T-cell responses in patients with IEIs.

Patient and provider perceptions of the relationship between alcohol use and TB and readiness for treatment: a qualitative study in South Africa.

Background: Unhealthy alcohol use is widespread in South Africa and has been linked to tuberculosis (TB) disease and poor treatment outcomes. This study used qualitative methods to explore the relationship between TB and alcohol use during TB treatment. Methods: Focus groups (FGs) were conducted with 34 participants who had previous or current drugsusceptible TB and self-reported current alcohol use. Eight interviews were conducted with healthcare workers who provide TB services in Worcester, South Africa. Results: In this rural setting, heavy episodic drinking is normalized and perceived to be related to TB transmission and decreased adherence to TB medication. Both healthcare workers and FG participants recommended the introduction of universal screening, brief interventions, and referral to specialized care for unhealthy alcohol use. However, participants also discussed barriers to the provision of these services, such as limited awareness of the link between alcohol and TB. Healthcare workers also specified resource constraints while FG participants or patients mentioned widespread stigma towards people with alcohol concerns. Both FG participants and health providers would benefit from education on the relationship between TB and unhealthy alcohol use as well and had specific recommendations about interventions for alcohol use reduction. Healthcare workers also suggested that community health worker-delivered interventions could support access to and engagement in both TB and alcohol-related services. Conclusion: Findings support strengthening accessible, specialized services for the identification and provision of interventions and psychosocial services for unhealthy alcohol use among those with TB.

Use of machine learning methods to understand discussions of female genital mutilation/cutting on social media.

Female genital mutilation/cutting (FGM/C) describes several procedures that involve injury to the vulva or vagina for nontherapeutic reasons. Though at least 200 million women and girls living in 30 countries have undergone FGM/C, there is a paucity of studies focused on public perception of FGM/C. We used machine learning methods to characterize discussion of FGM/C on Twitter in English from 2015 to 2020. Twitter has emerged in recent years as a source for seeking and sharing health information and misinformation. We extracted text metadata from user profiles to characterize the individuals and locations involved in conversations about FGM/C. We extracted major discussion themes from posts using correlated topic modeling. Finally, we extracted features from posts and applied random forest models to predict user engagement. The volume of tweets addressing FGM/C remained fairly stable across years. Conversation was mostly concentrated among the United States and United Kingdom through 2017, but shifted to Nigeria and Kenya in 2020. Some of the discussion topics associated with FGM/C across years included Islam, International Day of Zero Tolerance, current news stories, education, activism, male circumcision, human rights, and feminism. Tweet length and follower count were consistently strong predictors of engagement. Our findings suggest that (1) discussion about FGM/C has not evolved significantly over time, (2) the majority of the conversation about FGM/C on English-speaking Twitter is advocating for an end to the practice, (3) supporters of Donald Trump make up a substantial voice in the conversation about FGM/C, and (4) understanding the nuances in how people across cultures refer to and discuss FGM/C could be important for the design of public health communication and intervention.

Methods for handling missing data in serially sampled sputum specimens for mycobacterial culture conversion calculation.

BACKGROUND: The occurrence and timing of mycobacterial culture conversion is used as a proxy for tuberculosis treatment response. When researchers serially sample sputum during tuberculosis studies, contamination or missed visits leads to missing data points. Traditionally, this is managed by ignoring missing data or simple carry-forward techniques. Statistically advanced multiple imputation methods potentially decrease bias and retain sample size and statistical power. METHODS: We analyzed data from 261 participants who provided weekly sputa for the first 12 weeks of tuberculosis treatment. We compared methods for handling missing data points in a longitudinal study with a time-to-event outcome. Our primary outcome was time to culture conversion, defined as two consecutive weeks with no Mycobacterium tuberculosis growth. Methods used to address missing data included: 1) available case analysis, 2) last observation carried forward, and 3) multiple imputation by fully conditional specification. For each method, we calculated the proportion culture converted and used survival analysis to estimate Kaplan-Meier curves, hazard ratios, and restricted mean survival times. We compared methods based on point estimates, confidence intervals, and conclusions to specific research questions. RESULTS: The three missing data methods lead to differences in the number of participants achieving conversion; 78 (32.8%) participants converted with available case analysis, 154 (64.7%) converted with last observation carried forward, and 184 (77.1%) converted with multiple imputation. Multiple imputation resulted in smaller point estimates than simple approaches with narrower confidence intervals. The adjusted hazard ratio for smear negative participants was 3.4 (95% CI 2.3, 5.1) using multiple imputation compared to 5.2 (95% CI 3.1, 8.7) using last observation carried forward and 5.0 (95% CI 2.4, 10.6) using available case analysis. CONCLUSION: We showed that accounting for missing sputum data through multiple imputation, a statistically valid approach under certain conditions, can lead to different conclusions than naïve methods. Careful consideration for how to handle missing data must be taken and be pre-specified prior to analysis. We used data from a TB study to demonstrate these concepts, however, the methods we described are broadly applicable to longitudinal missing data. We provide valuable statistical guidance and code for researchers to appropriately handle missing data in longitudinal studies.

Serological responses to human virome define clinical outcomes of Italian patients infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic respiratory infectious disease COVID-19. However, clinical manifestations and outcomes differ significantly among COVID-19 patients, ranging from asymptomatic to extremely severe, and it remains unclear what drives these disparities. Here, we studied 159 sequentially enrolled hospitalized patients with COVID-19-associated pneumonia from Brescia, Italy using the VirScan phage-display method to characterize circulating antibodies binding to 96,179 viral peptides encoded by 1,276 strains of human viruses. SARS-CoV-2 infection was associated with a marked increase in immune antibody repertoires against many known pathogenic and non-pathogenic human viruses. This antiviral antibody response was linked to longitudinal trajectories of disease severity and was further confirmed in additional 125 COVID-19 patients from the same geographical region in Northern Italy. By applying a machine-learning-based strategy, a viral exposure signature predictive of COVID-19-related disease severity linked to patient survival was developed and validated. These results provide a basis for understanding the role of memory B-cell repertoire to viral epitopes in COVID-19-related symptoms and suggest that a unique anti-viral antibody repertoire signature may be useful to define COVID-19 clinical severity.

Deep immune profiling uncovers novel associations with clinical phenotypes of Multisystem Inflammatory Syndrome in Children (MIS-C).

Multisystem Inflammatory Syndrome in Children (MIS-C) is a systemic inflammatory condition that follows SARS-CoV2 infection or exposure in children. Clinical presentations are highly variable and include fever, gastrointestinal (GI) disease, shock, and Kawasaki Disease-like illness (MIS-C/KD). Compared to patients with acute COVID, patients with MIS-C have a distinct immune signature and expansion of TRVB11 expressing T cells. However, the relationship between immunological and clinical phenotypes of MIS-C is unknown. Here, we measured serum biomarkers, TCR repertoire, and SARS-CoV2-specific T cell responses in a cohort of 76 MIS-C patients. Serum biomarkers associated with macrophage and Th1 activation were elevated in patients with shock, consistent with previous reports. Significantly increased SARS-CoV-2-induced IFN-γ, IL-2, and TNF-α production were seen in CD4 + T cells from patients with neurologic involvement and respiratory failure. Diarrhea was associated with a significant reduction in shock-associated serum biomarkers, suggesting a protective effect. TRVB11 usage was highly associated with MIS-C/KD and coronary aneurysms, suggesting a potential biomarker for these manifestations in MIS-C patients. By identifying novel immunologic associations with the different clinical phenotypes of MIS-C, this study provides insights into the clinical heterogeneity of MIS-C. These unique immunophenotypic associations could provide biomarkers to identify patients at risk for severe complications of MIS-C, including shock and MIS-C/KD.

Understanding Early Pandemic Severe Acute Respiratory Syndrome Coronavirus 2 Transmission in a Medical Center by Incorporating Public Sequencing Databases to Mitigate Bias.

BACKGROUND: Throughout the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, healthcare workers (HCWs) have faced risk of infection from within the workplace via patients and staff as well as from the outside community, complicating our ability to resolve transmission chains in order to inform hospital infection control policy. Here we show how the incorporation of sequences from public genomic databases aided genomic surveillance early in the pandemic when circulating viral diversity was limited. METHODS: We sequenced a subset of discarded, diagnostic SARS-CoV-2 isolates between March and May 2020 from Boston Medical Center HCWs and combined this data set with publicly available sequences from the surrounding community deposited in GISAID with the goal of inferring specific transmission routes. RESULTS: Contextualizing our data with publicly available sequences reveals that 73% (95% confidence interval, 63%-84%) of coronavirus disease 2019 cases in HCWs are likely novel introductions rather than nosocomial spread. CONCLUSIONS: We argue that introductions of SARS-CoV-2 into the hospital environment are frequent and that expanding public genomic surveillance can better aid infection control when determining routes of transmission.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as "COVID toes," remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including MIS-C and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients. Summary: NET formation and degradation are dysregulated in pediatric and symptomatic adult patients with various complications of COVID-19, in association with disease severity. NET degradation impairments are multifactorial and associated with natural inhibitors of DNase 1, G-actin and anti-DNase1L3 and anti-NET antibodies. Infection with the Omicron variant is associated with decreased levels of NETs when compared to other SARS-CoV-2 strains.

Coronavirus Disease 2019 Vaccine Impact on Rates of Severe Acute Respiratory Syndrome Coronavirus 2 Cases and Postvaccination Strain Sequences Among Health Care Workers at an Urban Academic Medical Center: A Prospective Cohort Study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) vaccine trials and post-implementation data suggest that vaccination decreases infections. We examine vaccination's impact on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case rates and viral diversity among health care workers (HCWs) during a high community prevalence period. METHODS: In this prospective cohort study, HCW received 2 doses of BNT162b2 or mRNA-1273. We included confirmed cases among HCWs from 9 December 2020 to 23 February 2021. Weekly SARS-CoV-2 rates per 100,000 person-days and by time from first injection (1-14 and ≥15 days) were compared with surrounding community rates. Viral genomes were sequenced. RESULTS: SARS-CoV-2 cases occurred in 1.4% (96/7109) of HCWs given at least a first dose and 0.3% (17/5913) of HCWs given both vaccine doses. Adjusted rate ratios (95% confidence intervals) were 0.73 (.53-1.00) 1-14 days and 0.18 (.10-.32) ≥15 days from first dose. HCW ≥15 days from initial dose compared to 1-14 days were more often older (46 vs 38 years, P = .007), Latinx (10% vs 8%, P = .03), and asymptomatic (48% vs 11%, P = .0002). SARS-CoV-2 rates among HCWs fell below the surrounding community, an 18% vs 11% weekly decrease, respectively (P = .14). Comparison of 50 genomes from post-first dose cases did not indicate selection pressure toward known spike antibody escape mutations. CONCLUSIONS: Our results indicate an early positive impact of vaccines on SARS-CoV-2 case rates. Post-vaccination isolates did not show unusual genetic diversity or selection for mutations of concern.

Seroprevalence of SARS-CoV-2 IgG antibodies and risk factors in health care workers at an academic medical center in Boston, Massachusetts.

Healthcare workers (HCWs) are at an increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus that causes Coronavirus Disease (COVID-19). We aim to assess the seroprevalence of SARS-CoV-2 IgG among healthcare workers and compare risk-factors between seropositive and seronegative HCWs. In this observational study, serum samples were collected from HCWs between July 13th to 26th, 2020 at Boston Medical Center (BMC). Samples were subsequently tested for SARS-CoV-2 IgG antibody using the Abbott SARS-CoV-2 IgG assay. Participants also answered a questionnaire capturing data on demographics, history of COVID-19 symptoms, occupation, infection prevention and control measures. Overall, 95 of 1743 (5.5%) participants tested positive for SARS-CoV-2 IgG. Of these, 1.8% of the participants had mild or no COVID-19 symptoms and did not require a diagnostic test. Seropositivity was not associated with gender, occupation, hand hygiene and personal protective equipment (PPE) practices amongst HCWs. However, lack of physical distancing among health care workers in work areas and break room was associated with seropositivity (p = 0.05, p = 0.003, respectively). The majority of the HCWs are negative for SARS-CoV-2 IgG. This data highlights the need to promote infection prevention measures, and the importance of distance amongst co-workers to help mitigate infection rates.

COVID-19 vaccine impact on rates of SARS-CoV-2 cases and post vaccination strain sequences among healthcare workers at an urban academic medical center: a prospective cohort study.

BACKGROUND: COVID-19 vaccine trials and post-implementation data suggest vaccination decreases SARS-CoV-2 infections. We examine COVID-19 vaccination's impact on SARS-CoV-2 case rates and viral diversity among healthcare workers (HCW) during a high community prevalence period. METHODS: A prospective cohort study from Boston Medical Center (BMC)'s HCW vaccination program, where staff received two doses of BNT162b2 or mRNA-1273. We included PCR-confirmed SARS-CoV-2 cases among HCWs from December 09, 2020 to February 23, 2021. Weekly SARS-CoV-2 rates per 100,000 person-day overall and by time from first injection (1-14 and >14 days) were compared with surrounding community rates. Viral genomes were sequenced from SARS CoV-2 positive samples. RESULTS: SARS-CoV-2 cases occurred in 1.4% (96/7109) of HCWs given at least a first dose and 0.3% (17/5913) of HCWs given both vaccine doses. Adjusted SARS-CoV-2 infection rate ratios were 0.73 (95% CI 0.53-1.00) 1-14 days and 0.18 (0.10-0.32) >14 days from first dose. HCW SARS-CoV-2 cases >14 days from initial dose compared to within 14 days were more often older (46 versus 38 years, p=0.007), Latinx (10% versus 8%, p=0.03), and asymptomatic (48% versus 11%, p=0.0002). SARS-CoV-2 rates among HCWs fell below those of the surrounding community, with a 18% versus 11% weekly decrease respectively (p=0.14). Comparison of 48 SARS-CoV-2 genomes sequenced from post-first dose cases did not indicate selection pressure towards known spike-antibody escape mutations. CONCLUSIONS: Our results indicate a positive impact of COVID-19 vaccines on SARS-CoV-2 case rates. Post-vaccination isolates did not show unusual genetic diversity or selection for mutations of concern. MAIN POINT: Cases of SARS-CoV-2 among health care workers dropped rapidly with COVID-19 vaccination. Sequencing 48 breakthrough infections (overwhelmingly in 14 days after 1st dose) showed no clear sign of any differences in spike protein compared with time-matched, unvaccinated control sequences.

Assessing abuse potential of new analgesic medications following market release: an evaluation of Internet discussion of tapentadol abuse.

OBJECTIVE: Research on substance abusers in treatment suggests that tapentadol, a prescription analgesic, may have relatively low abuse potential. Messages posted by recreational drug abusers on online forums were examined for amount of discussion and endorsement for abuse of tapentadol and comparator drugs. METHODS: Internet messages posted between January 1, 2011 and September 30, 2012 on seven drug-abuse web forums were evaluated. Proportions of posts and unique authors discussing tapentadol were compared with eight comparator compounds. Postcontent was coded to compare endorsement for abuse of tapentadol with two comparators, one drug with high desirability for abuse and one with low desirability for abuse. RESULTS: A total of 1,940,121 messages posted during the study period were copied from selected web forums. The proportion of all posts discussing tapentadol (proportion = 0.0003) was significantly lower than any of the comparator compounds (range of odds ratios from 16.6 to 104.3; P < 0.001). The proportion of unique authors was also lower. Posts coded for endorsement (N = 2,117) yielded an endorsement ratio (Ero) of 2.14 for tapentadol, which was significantly lower than the highly desirable for abuse oxymorphone (ERo = 5.08; P = 0.0011) and was as low as tramadol (ERo = 1.66), which has a long-established profile of low abuse and desirability for abuse. CONCLUSIONS: Recreational abusers posting on web forums appear to be less interested in abusing tapentadol when compared with other, selected prescription analgesics based on the amount of discussion (i.e., fewer posts and authors mentioning tapentadol). Endorsement of the product for abuse was also low.

Monitoring of internet forums to evaluate reactions to the introduction of reformulated OxyContin to deter abuse.

BACKGROUND: Reformulating opioid analgesics to deter abuse is one approach toward improving their benefit-risk balance. To assess sentiment and attempts to defeat these products among difficult-to-reach populations of prescription drug abusers, evaluation of posts on Internet forums regarding reformulated products may be useful. A reformulated version of OxyContin (extended-release oxycodone) with physicochemical properties to deter abuse presented an opportunity to evaluate posts about the reformulation in online discussions. OBJECTIVE: The objective of this study was to use messages on Internet forums to evaluate reactions to the introduction of reformulated OxyContin and to identify methods aimed to defeat the abuse-deterrent properties of the product. METHODS: Posts collected from 7 forums between January 1, 2008 and September 30, 2013 were evaluated before and after the introduction of reformulated OxyContin on August 9, 2010. A quantitative evaluation of discussion levels across the study period and a qualitative coding of post content for OxyContin and 2 comparators for the 26 month period before and after OxyContin reformulation were conducted. Product endorsement was estimated for each product before and after reformulation as the ratio of endorsing-to-discouraging posts (ERo). Post-to-preintroduction period changes in ERos (ie, ratio of ERos) for each product were also calculated. Additionally, post content related to recipes for defeating reformulated OxyContin were evaluated from August 9, 2010 through September 2013. RESULTS: Over the study period, 45,936 posts related to OxyContin, 18,685 to Vicodin (hydrocodone), and 23,863 to Dilaudid (hydromorphone) were identified. The proportion of OxyContin-related posts fluctuated between 6.35 and 8.25 posts per 1000 posts before the reformulation, increased to 10.76 in Q3 2010 when reformulated OxyContin was introduced, and decreased from 9.14 in Q4 2010 to 3.46 in Q3 2013 in the period following the reformulation. The sentiment profile for OxyContin changed following reformulation; the post-to-preintroduction change in the ERo indicated reformulated OxyContin was discouraged significantly more than the original formulation (ratio of ERos=0.43, P<.001). A total of 37 recipes for circumventing the abuse-deterrent characteristics of reformulated OxyContin were observed; 32 were deemed feasible (ie, able to abuse). The frequency of posts reporting abuse of reformulated OxyContin via these recipes was low and decreased over time. Among the 5677 posts mentioning reformulated OxyContin, 825 posts discussed recipes and 498 reported abuse of reformulated OxyContin by such recipes (41 reported injecting and 128 reported snorting). CONCLUSIONS: After introduction of physicochemical properties to deter abuse, changes in discussion of OxyContin on forums occurred reflected by a reduction in discussion levels and endorsing content. Despite discussion of recipes, there is a relatively small proportion of reported abuse of reformulated OxyContin via recipes, particularly by injecting or snorting routes. Analysis of Internet discussion is a valuable tool for monitoring the impact of abuse-deterrent formulations.

CD8+ cells enhance resistance to pulmonary serotype 3 Streptococcus pneumoniae infection in mice.

Despite the success of the pneumococcal conjugate vaccine, pneumococcal pneumonia remains a significant clinical problem, and there is still much to learn about natural resistance and cellular immunity to pneumococcus. We investigated the role of T lymphocytes in resistance to serotype (ST) 3 Streptococcus pneumoniae in an intranasal infection model in C57BL/6 (wild-type [Wt]) and CD8(+) (CD8(-/-))- and CD4(+) (MHC class II(-/-))-deficient mice. CD8(-/-) mice exhibited significantly more bacterial dissemination and lung inflammation and a significantly more lethal phenotype than Wt mice. However, there was no difference in the bacterial dissemination, lung inflammation, or survival of Wt and MHC class II(-/-) mice. Perforin (Pfn)(-/-) and IFN-γ(-/-) mice, which were used to dissect the role of CD8(+) T cells in our model, also exhibited a more lethal survival phenotype than Wt mice. Comparison of lung chemokine/cytokine levels by Luminex and cellular recruitment by FACS in Wt mice and knockout strains revealed that CD8(-/-) and IFN-γ(-/-) mice, which had the most lethal survival phenotype, had more CD4(+)IL-17(+) T (Th17) cells, IL-17, neutrophil chemoattractants, and lung neutrophils, and fewer regulatory T cells than Wt mice. CD4(+) T cell depletion improved the survival of ST-infected CD8(-/-) mice, and survival studies in Th17-deficient mice revealed that the Th17 response was dispensable for ST3 resistance in our model. Taken together, these findings demonstrate that CD8(+) cells are required, but CD4(+) T cells are dispensable for resistance to ST3 pneumonia in mice and suggest a previously unsuspected role for CD8(+) cells in modulating the inflammatory response to ST3.

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function.

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.

Cryptococcus neoformans laccase catalyses melanin synthesis from both D- and L-DOPA.

The human fungal pathogen Cryptococcus neoformans produces melanin in the presence of various substrates, including the L enantiomer of 3,4-dihydroxyphenylalanine (DOPA). The enzyme laccase catalyses the formation of melanin by oxidizing L-DOPA, initiating a series of presumably spontaneous reactions that ultimately leads to the polymerization of the pigment in the yeast cell wall. There, melanin protects the cell from a multitude of environmental and host assaults. Thus, the ability of C. neoformans to produce pigments from a variety of available substrates is likely to confer a survival advantage. A number of C. neoformans isolates of different serotypes produced pigments from D-DOPA, the stereoisomer of L-DOPA. Acid-resistant particles were isolated from pigmented C. neoformans cells grown in the presence of D-DOPA. Biophysical characterization showed the particles had a stably detectable free-radical signal by EPR, and negative zeta potential, similar to L-DOPA-derived particles. No major differences were found between L- and D-DOPA ghosts in terms of binding to anti-melanin antibodies, or in overall architecture when imaged by electron microscopy. C. neoformans cells utilized L- and D-DOPA at a similar rate. Overall, our results indicate that C. neoformans shows little stereoselectivity for utilizing DOPA in melanin synthesis. The ability of C. neoformans to use both L and D enantiomers for melanization implies that this organism has access to a greater potential pool of substrates for melanin synthesis, and this could potentially be exploited in the design of therapeutic inhibitors of laccase.

Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Th1 cells in vivo.

Adaptive regulatory T cells that develop from naive CD4 cells in response to exposure to Ag can act as immunotherapeutic agents to control immune responses. We show that effectors generated from murine islet-specific CD4 cells by TCR stimulation with IL-2 and TGF-beta1 have potent suppressive activity. They prevent spontaneous development of type 1 diabetes in NOD mice and inhibit development of pancreatic infiltrates and disease onset orchestrated by Th1 effectors. These regulatory T cells do not require innate CD25+ regulatory cells for generation or function, nor do they share some characteristics typically associated with them, including expression of CD25. However, the adaptive population does acquire the X-linked forkhead/winged helix transcription factor, FoxP3, which is associated with regulatory T cell function and maintains expression in vivo. One mechanism by which they may inhibit Th1 cells is via FasL-dependent cytotoxicity, which occurs in vitro. In vivo, they eliminate Th1 cells in lymphoid tissues, where Fas/FasL interactions potentially play a role because Th1 cells persist when this pathway is blocked. The results suggest that adaptive regulatory CD4 cells may control diabetes in part by impairing the survival of islet-specific Th1 cells, and thereby inhibiting the localization and response of autoaggressive T cells in the pancreatic islets.