Prior Virus Infection Research Articles

Prior viral infection primes cross-reactive CD8+ T cells that respond to mouse heart allografts.

Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

Genetic risk factors for severe and fatigue dominant long COVID and commonalities with ME/CFS identified by combinatorial analysis

BackgroundLong COVID is a debilitating chronic condition that has affected over 100 million people globally. It is characterized by a diverse array of symptoms, including fatigue, cognitive dysfunction and respiratory problems. Studies have so far largely failed to identify genetic associations, the mechanisms behind the disease, or any common pathophysiology with other conditions such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) that present with similar symptoms.MethodsWe used a combinatorial analysis approach to identify combinations of genetic variants significantly associated with the development of long COVID and to examine the biological mechanisms underpinning its various symptoms. We compared two subpopulations of long COVID patients from Sano Genetics’ Long COVID GOLD study cohort, focusing on patients with severe or fatigue dominant phenotypes. We evaluated the genetic signatures previously identified in an ME/CFS population against this long COVID population to understand similarities with other fatigue disorders that may be triggered by a prior viral infection. Finally, we also compared the output of this long COVID analysis against known genetic associations in other chronic diseases, including a range of metabolic and neurological disorders, to understand the overlap of pathophysiological mechanisms.ResultsCombinatorial analysis identified 73 genes that were highly associated with at least one of the long COVID populations included in this analysis. Of these, 9 genes have prior associations with acute COVID-19, and 14 were differentially expressed in a transcriptomic analysis of long COVID patients. A pathway enrichment analysis revealed that the biological pathways most significantly associated with the 73 long COVID genes were mainly aligned with neurological and cardiometabolic diseases.Expanded genotype analysis suggests that specific SNX9 genotypes are a significant contributor to the risk of or protection against severe long COVID infection, but that the gene-disease relationship is context dependent and mediated by interactions with KLF15 and RYR3.Comparison of the genes uniquely associated with the Severe and Fatigue Dominant long COVID patients revealed significant differences between the pathways enriched in each subgroup. The genes unique to Severe long COVID patients were associated with immune pathways such as myeloid differentiation and macrophage foam cells. Genes unique to the Fatigue Dominant subgroup were enriched in metabolic pathways such as MAPK/JNK signaling. We also identified overlap in the genes associated with Fatigue Dominant long COVID and ME/CFS, including several involved in circadian rhythm regulation and insulin regulation. Overall, 39 SNPs associated in this study with long COVID can be linked to 9 genes identified in a recent combinatorial analysis of ME/CFS patient from UK Biobank.Among the 73 genes associated with long COVID, 42 are potentially tractable for novel drug discovery approaches, with 13 of these already targeted by drugs in clinical development pipelines. From this analysis for example, we identified TLR4 antagonists as repurposing candidates with potential to protect against long term cognitive impairment pathology caused by SARS-CoV-2. We are currently evaluating the repurposing potential of these drug targets for use in treating long COVID and/or ME/CFS.ConclusionThis study demonstrates the power of combinatorial analytics for stratifying heterogeneous populations in complex diseases that do not have simple monogenic etiologies. These results build upon the genetic findings from combinatorial analyses of severe acute COVID-19 patients and an ME/CFS population and we expect that access to additional independent, larger patient datasets will further improve the disease insights and validate potential treatment options in long COVID.

Real-life epidemiology and current outcomes of hospitalized adults with invasive fungal infections.

We aimed to describe the current epidemiology of both hosts with invasive fungal infections (IFIs) and causative fungi. And, detail outcomes of these infections at 12 weeks in a real-life cohort of hospitalized patients. The study was retrospective and observational to describe IFI diagnosed in a tertiary hospital (February 2017-December 2021). We included all consecutive patients meeting criteria for proven or probable IFI according to EORTC-MSG and other criteria. A total of 367 IFIs were diagnosed. 11.7% were breakthrough infections, and 56.4% were diagnosed in the intensive care unit. Corticosteroid use (41.4%) and prior viral infection (31.3%) were the most common risk factors for IFI. Lymphoma and pneumocystis pneumonia were the most common baseline and fungal diseases. Only 12% of IFI occurred in patients with neutropenia. Fungal cultures were the most important diagnostic tests (85.8%). The most frequent IFIs were candidemia (42.2%) and invasive aspergillosis (26.7%). Azole-resistant Candida strains and non-fumigatus Aspergillus infections represented 36.1% and 44.5% of the cases, respectively. Pneumocystosis (16.9%), cryptococcosis (4.6%), and mucormycosis (2.7%) were also frequent, as well as mixed infections (3.4%). Rare fungi accounted for 9.5% of infections. Overall, IFI mortality at 12 weeks was 32.2%; higher rates were observed for Mucorales (55.6%), Fusarium (50%), and mixed infections (60%). We documented emerging changes in both hosts and real-life IFI epidemiology. Physicians should be aware of these changes to suspect infections and be aggressive in diagnoses and treatments. Currently, outcomes for such clinical scenarios remain extremely poor.

Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of varicella-zoster virus immunoglobulin G antibodies in fingerstick blood

Varicella zoster virus (VZV) causes childhood chickenpox, becomes latent in sensory ganglia and reactivates years later to cause shingles (Zoster) and postherpetic neuralgia in the elderly and immunosuppressed individuals. Serologic IgG tests can be used to determine if a person has antibodies to VZV from past varicella infection or had received varicella or zoster (shingles) vaccination. Commercial enzyme-linked immunosorbent assays (ELISAs) are currently used for the detection of VZV IgG antibodies in patient serum samples. However, ELISA tests require collection and processing of blood samples in a CLIA laboratory to separate serum or plasma for further testing. In this paper, we describe the development and testing of an antibody based Lateral Flow Immunochromatographic assay (LFA) device for the detection of VZV IgG in fingerstick whole blood. Analytical and clinical analyses were performed to compare the performance characteristics of the Viro VZV IgG LFA (VZV LFA) and the Diamedix VZV IgG ELISA. Analytical studies demonstrated the higher sensitivity of the VZV LFA compared to the ELISA by testing dilutions of the WHO VZV IgG serum International Standard. Clinical performance characteristics of the VZV LFA fingerstick whole blood assay were assessed at three point of care (POC) facilities by untrained users testing samples from 300 prospectively enrolled study subjects. VZV LFA results were compared with results obtained by testing serum samples obtained from the same study participants by the Diamedix VZV IgG ELISA. Two specimens with invalid results by the LFA assay were not included in the LFA performance calculations and nine equivocal ELISA results were included as positive for IgG results. The results from all three POC clinical sites demonstrated the higher sensitivity/positive percent agreement (PPA) (99.26%, 95% CI: 97.34–99.80) of the VZV LFA compared to the Diamedix VZV IgG ELISA (94.08%, 95% CI: 90.72–96.27). The specificity/negative percent agreement (NPA) of the VZV LFA compared to the ELISA test was calculated initially to be 39.29% (95% CI: 23.57–57.59) with 19 discordant test results out of 298 test results between the two assays (17 LFA positive/ELISA negative and two LFA negative/ELISA positive). The PPA and true NPA of the VZV LFA were determined by testing all 298 samples, including the discordant (19) and all concordant negative and positive (279) study subject serum samples, before and after blocking VZV gE antibody sites in the samples by spiking with VZV LFA gE capture antigen. The NPA improved to 100% (95% CI: 74.12–100) after the procedure when compared to the ELISA test results. The comparator ELISA PPA based on the spiking/blocking study remained as 94.08%, (95% CI: 90.72–96.27), comparable to test results from untreated samples.The VZV LFA has been demonstrated to be simple and sufficiently robust for use in CLIA-waived POC facilities by untrained healthcare professionals and to detect VZV IgG in 20 min from fingerstick whole blood. The VZV LFA therefore provides a fast, reliable, and highly sensitive method of determining prior VZV viral infection or varicella and zoster vaccination status.

An understanding of the immune dysfunction in susceptible people who develop the post-viral fatigue syndromes Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Long COVID

Viral infection in most people results in a transient immune/inflammatory response resulting in elimination of the virus and recovery where the immune system returns to that of the pre-infectious state. In susceptible people by contrast there is a transition from an acute immune response to a chronic state that can lead to an ongoing lifelong complex post-viral illness, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. This susceptibility is proposed to be genetic or be primed by prior health history. Complex abnormalities occur in immune cell functions, immune cell metabolism and energy production, and in cytokine immune modulator regulation. The immune system of the brain/central nervous system becomes activated leading to dysfunction in regulation of body physiology and the onset of many neurological symptoms. A dysfunctional immune system is core to the development of the post-viral condition as shown with diverse strategies of immune profiling. Many studies have shown changes in numbers and activity of immune cells of different phenotypes and their metabolism. Immune regulating cytokines show complex altered patterns and vary with the stage of the disease, and there are elements of associated autoimmunity. These complex changes are accompanied by an altered molecular homeostasis with immune cell transcripts and proteins no longer produced in a tightly regulated manner, reflected in the instability of the epigenetic code that controls gene expression. Potential key elements of the altered immune function in this disease needing further exploration are changes to the gut-brain-immune axis as a result of changes in the microbiome of the gut, and viral reactivation from latent elements of the triggering virus or from a prior viral infection. Long COVID, an Myalgic Encephalomyelitis/Chronic Fatigue Syndrome-like illness, is the post-viral condition that has arisen in large numbers solely from the pandemic virus Severe Acute Respiratory Syndrome Coronovirus-2. With over 760 million cases worldwide, an estimated ~100 million cases of Long COVID have occurred within a short period. This now provides an unprecedented opportunity to understand the progression of these post-viral diseases, and to progress from a research phase mainly documenting the immune changes to considering potential immunotherapies that might improve the overall symptom profile of affected patients, and provide them with a better quality of life.

PSII-B-21 Does Prior Viral Infection Exacerbate the Response to Heat Stress?

Abstract In rodent models, prior immune activation appears to exacerbate the response to heat stress (HS), presumably by compounding an inflammatory response by both insults. Thus, study objectives were to investigate whether prior viral infection aggravates the physiological and feed intake (FI) responses to HS. Crossbred barrows (n=45; 75.5±4.8 kg BW) were enrolled in two experimental periods (P). During P1 (2 d), pigs were fed ad libitum and housed in thermoneutral (20ºC) conditions. During P2 (6 d), pigs were exposed to an increased heat load (28 to 31ºC). Rectal temperature (TR), skin temperature (TS), and respiration rate (RR) were obtained twice daily. FI was measured daily as feed disappearance. Unexpectedly, pigs became sick during the experiment and tested positive for influenza on d 6 of P2. Based on TR obtained during P1, animals were retrospectively categorized into 1 of 2 groups: 1) High-TR (HT; top third of pigs; TR&amp;gt;39.32ºC; x̅=39.38ºC; n=15), or 2) Low-TR (LT; bottom third of pigs; TR&amp;lt; 39.10ºC; x̅=38.95ºC; n=15). Pigs in the middle third tier were excluded from the analysis. This ostensibly categorized pigs into a “sick” and “healthy” status. Data were statistically analyzed using the MIXED procedure in SAS. As expected, HS increased TR in both groups; however, HT had elevated TR during P2 relative to LT pigs (+0.5ºC; P&amp;lt; 0.01). Skin temperature and RR increased during HS but remained similar between HT and LT pigs (P&amp;gt;0.41). During P1, FI was similar between groups (P&amp;gt;0.33). During P2, HS reduced FI in both groups; however, the decrease was more severe in HT than LT pigs (53 vs. 37%, respectively; P=0.01). Altogether, HT pigs, presumably infected before HS exposure, had increased TR and a more drastic reduction in FI during HS than LT pigs. A better understanding of how viral or bacterial infection influences the response to HS would have pragmatic implications for all segments of animal agriculture.

Abstract #1088797: Graves' Disease Induced Vasospastic Angina and NSTEMI: An Unusual Link Between Endocrinology and Cardiology

A common link between Endocrinology and Cardiology is T3, the active form of thyroid hormone. Approximately 5-25% of patients with hyperthyroidism have atrial fibrillation, but a far less common cardiovascular disease (CVD) associated with thyrotoxicosis is myocardial infarction (MI). There are few reported cases of hyperthyroidism causing STEMI, NSTEMI, coronary vasospasm, and autoimmune myocarditis. We present a previously healthy 58-year-old female seen at the emergency department (ED) for nighttime/early morning chest pain, dyspnea and diaphoresis. She denied prior episodes or family history of CVD. EKG showed incomplete right bundle branch block and anterior T-wave inversions. Troponin peaked at 13 ng/mL. Echocardiogram was unremarkable. Left heart catherization demonstrated non-obstructive coronary artery disease concerning for vasospasm. Pertinent laboratories showed decreased TSH (< 0.005 μU/mL) and elevated total T3 and free T4 (2.19 ng/dL). Differentials for thyrotoxicosis included primary hyperthyroidism, spurious results (patient was taking supplement with biotin and iodine), and stress-induced thyroid function alteration. Further workup revealed negative Thyroid Stimulating Immunoglobulin (TSI) and positive TSH Receptor Antibodies (TRAb). Chest pain resolved and she was discharged, but returned days later with same complaints and elevated troponin. Cardiac differentials were myocarditis in the setting of autoimmune thyroiditis vs vasospastic angina. Serial EKGs had no signs of myocarditis. She denied prior viral infection, autoimmune conditions, recreational drugs, appetite suppressants, ephedrine, or migraine medications. Cardiac MRI was not consistent with myocarditis, instead suspicion was raised for subacute MI with nonobstructive coronary arteries (MINOCA). Based on Coronary Vasomotion Disorders International Study Group (COVADIS) criteria, she had nitro-sensitive vasospastic angina. In the setting of thyrotoxicosis and positive TRAb, she was diagnosed with coronary vasospasm NSTEMI triggered by Graves'. Autoimmune thyroid disease may be undiagnosed 50% of the time, unknowingly contributing to CVD. T3 has direct and indirect cardiovascular effects. It promotes catecholaminergic stimulation and acts as a positive ionotrope and chronotrope. Excess T3 creates a hypermetabolic state that can rarely trigger severe coronary vasospasm. MI with < 50 % stenosis is defined as MINOCA, accounting for 6% of acute infarctions that undergo angiography. Non-atherosclerotic etiologies of MINOCA include coronary vasospasm, embolism/thrombus, anemia, hypotension, and thyrotoxicosis. Our patient had two of these, and to the best of our knowledge, this is the first reported case of a troponin >0.5 ng/mL secondary to Graves’ disease induced MINOCA.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

Coagulopathy signature precedes and predicts severity of end‐organ heat stroke pathology in a mouse model

BackgroundImmune challenge is known to increase heat stroke risk, although the mechanism of this increased risk is unclear. ObjectivesWe sought to understand the effect of immune challenge on heat stroke pathology. Patients/MethodsUsing a mouse model of classic heat stroke, we examined the impact of prior viral or bacterial infection on hematological aspects of recovery. Mice were exposed to heat either 48 or 72 hours following polyinosinic:polycytidylic acid (poly I:C) or lipopolysaccharide injection, time points when symptoms of illness (fever, lethargy, anorexia) were minimized or completely absent. ResultsEmploying multivariate supervised machine learning to identify patterns of molecular and cellular markers associated with heat stroke, we found that prior viral infection simulated with poly I:C injection resulted in heat stroke presenting with high levels of factors indicating coagulopathy. Despite a decreased number of platelets in the blood, platelets are large and non‐uniform in size, suggesting younger, more active platelets. Levels of D‐dimer and soluble thrombomodulin were increased in more severe heat stroke, and in cases of the highest level of organ damage markers D‐dimer levels dropped, indicating potential fibrinolysis‐resistant thrombosis. Genes corresponding to immune response, coagulation, hypoxia, and vessel repair were up‐regulated in kidneys of heat‐challenged animals; these correlated with both viral treatment and distal organ damage while appearing before discernible tissue damage to the kidney itself. ConclusionsHeat stroke‐induced coagulopathy may be a driving mechanistic force in heat stroke pathology, especially when exacerbated by prior infection. Coagulation markers may serve as accessible biomarkers for heat stroke severity and therapeutic strategies.

Viral infection dampens human fetal membrane type I interferon responses triggered by bacterial LPS

The maternal-fetal interface possesses innate immune strategies to protect against infections. We previously reported that prior viral infection of human fetal membranes (FMs) in vitro and mouse FMs in vivo sensitized the tissue to low dose bacterial LPS leading to augmented inflammation. The objective of this study was to examine FM production of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in the context of this polymicrobial model. Human FM explants and pregnant C57BL/6 mice were treated with or without low dose LPS following exposure to media or the γ-herpes virus, MHV-68. FM RNA was analyzed by qRT-PCR for type I IFNs, ISGs, upstream signaling, and MHV-68 open reading frames (ORFs). Pre-exposure to MHV-68 followed by LPS treatment inhibited the ability of LPS to induce human FM type I IFNs (IFNA, IFNB); ISGs (OAS, MxA, APOBEC3G) and upstream signaling mediators (RIG-I, TBK-1). Signaling mediators IRF-3 and IRF-7 were also reduced. In mouse FMs, pre-exposure to MHV-68 followed by LPS treatment reduced the ability of LPS to upregulate Ifna, Ifnb, Mxa, Irf7, and also reduced Irf3. MHV-68 infection of FMs induced ORF45 which targets IRF-7, and this was further augmented in response to a combination of MHV-68 and LPS. Together, these findings indicate that a viral infection blunts FM type I IFN production and signaling in response to LPS leading to a suppressed ISG response. Our studies suggest that a viral infection inhibits this protective FM response by negatively regulating IRF-7 through ORF45, leaving the maternal-fetal interface vulnerable to further viral attack.

Factors at de novo donor-specific antibody initial detection associated with allograft loss: a multicenter study.

We aimed to evaluate patient factors including nonadherence and viral infection and de novo donor-specific antibody (dnDSA) characteristics [total immunoglobulin G (IgG), C1q, IgG3, and IgG4] as predictors of renal allograft failure in a multicenter cohort with dnDSA. We performed a retrospective observational study of 113 kidney transplant recipients with dnDSA and stored sera for analysis. Predictors of death-censored allograft loss were assessed by Cox proportional modeling. Death-censored allograft survival was 77.0% (87/113) during a median follow-up of 2.2 (IQR 1.2-3.7) years after dnDSA detection. Predictors of allograft failure included medication nonadherence [HR 6.5 (95% CI 2.6-15.9)], prior viral infection requiring immunosuppression reduction [HR 5.3 (95% CI 2.1-13.5)], IgG3 positivity [HR 3.8 (95% CI 1.5, 9.3)], and time post-transplant (years) until donor-specific antibody (DSA) detection [HR 1.2 (95% CI 1.0, 1.3)]. In the 67 patients who were biopsied at dnDSA detection, chronic antibody-mediated rejection [HR 11.4 (95% CI 2.3, 56.0)] and mixed rejection [HR 7.4 (95% CI 2.2, 24.8)] were associated with allograft failure. We conclude that patient factors, including a history of viral infection requiring immunosuppression reduction or medication nonadherence, combined with DSA and histologic parameters must be considered to understand the risk of allograft failure in patients with dnDSA.

Vigilância das síndromes neurológicas notificadas no HFSE em um contexto de epidemia de Zika, chikungunya e dengue

Justificativa e Objetivos: a ocorrência de manifestações neurológicas no contexto da circulação dos vírus da dengue(DENV), Zika(ZIKV) e chikungunya(CHIKV) no Brasil é um desafio para a vigilância epidemiológica. Objetivo: descrever o perfil clínico-epidemiológico dos pacientes notificados no HFSE com síndromes neurológicas potencialmente associadas ao DENV, CHIKV e ZIKV, em 2015 e 2016. &#x0D; Métodos: estudo observacional, com análise de bases secundárias e coleta de dados em prontuários e fichas de investigação epidemiológica do HFSE. Incluídos todos os casos notificados em 2015 e 2016, que atendessem aos critérios: a) síndrome neurológica de origem indeterminada e registro de infecção viral prévia até 60 dias antes do início do quadro neurológico, confirmados laboratorialmente ou não; b) síndrome neurológica sem registro de infecção viral prévia até 60 dias antes do início do quadro neurológico, com confirmação laboratorial para DENV, CHIKV e ZIKV em líquor, soro ou urina. Variáveis analisadas: sexo, idade, raça/cor, escolaridade, município de residência, sinais e sintomas, diagnóstico sindrômico e etiológico do quadro neurológico, local/tempo de internação e evolução. &#x0D; Resultados: 72 casos notificados: 61,1% homens, idade mediana 37 anos, quadro neurológico com síndrome de Guillain-Barré(SGB) 34,7% e meningoencefalite 31,9%. Foram classificados como associados a arboviroses 39 casos (54,1%), dos quais 14 pelo critério laboratorial (3 ZIKV, 6 CHIKV, 4 DENV, 1 DENV+ CHIKV) e 25 por critério clínico-epidemiológico. Nos laboratorialmente confirmados o percentual de meningoencefalite (57,1%) ultrapassou SGB (35,7%). &#x0D; Conclusão: o diagnóstico diferencial entre as arboviroses nesse contexto é um desafio para a vigilância epidemiológica, destacando-se o predomínio da SGB e das meningoencefalites.&#x0D; PALAVRAS-CHAVE: infecção por arbovírus; manifestações neurológicas; Epidemiologia.

T Cell Repertoire Maturation Induced by Persistent and Latent Viral Infection Is Insufficient to Induce Costimulation Blockade Resistant Organ Allograft Rejection in Mice.

CD28:CD80/86 pathway costimulation blockade (CoB) with the CD80/86-specific fusion protein CTLA4-Ig prevents T cell-mediated allograft rejection in mice. However, in humans, transplantation with CoB has been hampered by CoB-resistant rejection (CoBRR). CoBRR has been attributed in part to pathogen-driven T cell repertoire maturation and resultant heterologous alloreactive memory. This has been demonstrated experimentally in mice. However, prior murine models have used viral pathogens, CoB regimens, graft types, and/or antigen systems atypically encountered clinically. We therefore sought to explore whether CoBRR would emerge in a model of virus-induced memory differentiation designed to more closely mimic clinical conditions. Specifically, we examined mouse homologs of clinically prevalent viruses including murine polyomavirus, cytomegalovirus, and gammaherpesvirus 68 in the presence of clinically relevant maintenance CoB regimens using a fully MHC-mismatched, vascularized allograft model. Infected mice developed a significant, sustained increase in effector memory T cells consistent with that seen in humans, but neither developed heterologous alloreactivity nor rejected primarily vascularized heterotopic heart transplants at an increased rate compared with uninfected mice. These results indicate that memory acquisition alone is insufficient to provoke CoBRR and suggest that knowledge of prior latent or persistent viral infection may have limited utility in anticipating heterologous CoB-resistant alloimmunity.

Lymphopenia is associated with late onset Pneumocystis jirovecii pneumonia in solid organ transplantation.

Pneumocystis jirovecii pneumonia (PJP) affected 5%-15% of solid organ transplant (SOT) recipients prior to universal prophylaxis, classically with trimethoprim-sulfamethoxazole (TMP-SMX). Guidelines generally recommend 6-12months of prophylaxis post-SOT, yet optimal duration and robust PJP risk stratification have not been established. A retrospective, single-center, case-control study of PJP among SOT recipients from January 1998 to December 2013 was conducted. Cases had positive PJ direct fluorescent antibody assay of respiratory specimens. Controls were matched 4:1 by nearest date of SOT. Univariate testing and multivariate logistic regressions were performed. Fifteen cases were identified among 5505 SOT recipients (0.27% rate) and analyzed vs 60 controls. PJP occurred on average 6.1years (range 0.9-13.8) post-SOT; no case was receiving PJP prophylaxis at diagnosis. Most were treated with reduced immunosuppression and TMP-SMX plus steroids (80%). Six patients (40%) required critical care; 3 (20%) died. There were no significant demographic differences, though cases tended to be older at SOT (54 vs 48years, P=.1). In univariate analysis, prior viral infection was more common among cases (67% vs 37%, P=.08). Lower absolute lymphocyte count (ALC) at diagnosis date was strongly associated with PJP (400 vs 1230×106 cells/μL, P<.001); odds of infection were high with ALC ≤ 500×106 cells (OR 18.7, P<.01). Pneumocystis jirovecii pneumonia is a rare, late complication of SOT with significant morbidity and mortality. Severe lymphopenia may be useful in identifying SOT recipients who warrant continued or reinstated PJP prophylaxis.

The differentiated airway epithelium infected by influenza viruses maintains the barrier function despite a dramatic loss of ciliated cells.

Virus-host interactions in the respiratory epithelium during long term influenza virus infection are not well characterized. Therefore, we developed an air-liquid interface culture system for differentiated porcine respiratory epithelial cells to study the effect of virus-induced cellular damage. In our well-differentiated cells, α2,6-linked sialic acid is predominantly expressed on the apical surface and the basal cells mainly express α2,3-linked sialic acid. During the whole infection period, release of infectious virus was maintained at a high titre for more than seven days. The infected epithelial cells were subject to apoptosis resulting in the loss of ciliated cells together with a thinner thickness. Nevertheless, the airway epithelium maintained trans-epithelial electrical resistance and retained its barrier function. The loss of ciliated cells was compensated by the cells which contained the KRT5 basal cell marker but were not yet differentiated into ciliated cells. These specialized cells showed an increase of α2,3-linked sialic acid on the apical surface. In sum, our results help to explain the localized infection of the airway epithelium by influenza viruses. The impairment of mucociliary clearance in the epithelial cells provides an explanation why prior viral infection renders the host more susceptible to secondary co-infection by another pathogen.

Mannheimia haemolytica biofilm formation on bovine respiratory epithelial cells

Mannheimia haemolytica is the most important bacterial agent associated with the bovine respiratory disease complex (BRDC), which causes worldwide economic losses to the cattle industry. M. haemolytica cells initially colonize the tonsillar crypts in the upper respiratory tract of cattle, from where they can subsequently descend into the lungs to cause disease. Many bacteria exist as biofilms inside their hosts. We hypothesize that M. haemolytica colonization of cattle during its commensal state may include biofilm formation. To begin to assess this possibility, we developed an in vitro system to study biofilm formation directly on bovine respiratory epithelial cells. Using fixed primary bovine bronchial epithelial cells, we observed M. haemolytica biofilm formation after a 48h incubation period at 37°C. Addition of mucin, the main component of mucus present in the upper respiratory tract, decreased M. haemolytica biofilm formation on bovine epithelial cells. We investigated the effects of prior viral infection of the epithelial cells on subsequent biofilm formation by M. haemolytica and found negligible effects. Utilization of this model system will provide new insights into the potential role of biofilm formation by M. haemolytica in the pathogenesis of BRDC.

82. Mapping the Humoral Immune Response to AAV by Molecular Docking and Cryo-Electron Microscopy for the Design of Next-Generation AAV Vectors

Despite constant improvements in gene therapy with AAV vectors, including use of new serotypes, routes of administration, and transgene cassette engineering, immune responses continue to be a major obstacle to therapeutic translation. Neutralizing antibodies (NAb) against the capsid generated by prior viral infection or AAV vector administration significantly reduce not only the effective patient population but also the overall efficacy of an AAV-based gene therapy. Here, by cloning out and evaluating anti-AAV antibodies from singly-sorted memory B cells from seropositive individuals, we have designed an approach that allows us to look at the humoral immune response globally, to hone in on the immunogenic regions of the capsid itself, and to compare responses between individuals in an unbiased and therapeutically-relevant setting. In this study, we screened a panel of 30 normal human donors, selected one with high pre-existing NAb titers (1:320 for AAV2, 1:40 for AAV3B), then sorted out switched memory B cells by negative selection, seeding on irradiated ms3T3-CD40L feeder cells and culturing for 2 weeks in the presence of IL-2 and IL-21. Following supernatant screening for anti-AAV antibody production, over 100 AAV-reactive clones were identified. After isolation and cloning using nested PCR, antibodies were evaluated for AAV capsid binding as well as neutralizing capacity. To date, all antibodies demonstrated binding to AAV2 and AAV3B as well as a panel of additional AAV serotypes (8, 9, rh10, rh32.33), suggesting that AAV-binding, yet non-neutralizing antibodies may possess broad serotype specificity.To identify the epitopes for these anti-AAV antibodies, we first took a high-throughout, predictive approach. Antibody variable region sequences were placed into a generic antibody scaffold and their three-dimensional structure modeled using Kotai Antibody Builder and RosettaAntibody followed by validation using COOT. The resulting structures were then iteratively docked onto the published structure of AAV3B using PIPER to identify the most energetically-favorable binding conformation. Thus far, the vast majority of footprint residues lie in variable regions of the capsid comprising and surrounding the 3-fold spikes. For a number of antibodies, initial prediction-directed capsid alanine scanning experiments have shown decreased antibody binding at the predicted residues, supporting the use of this approach. More comprehensive mutagenesis experiments are underway to further validate the approach and more completely map immunogenic epitopes of the AAV capsid proteins. In addition, cryo-EM analysis is currently underway for a number of these Fab-AAV complexes for additional, direct observation of the repertoire of binding footprints. These studies will provide information critical to understanding the antibody response to AAV and guide future attempts to rationally design next-generation capsids that are able to evade the anti-AAV antibody response.

Neuroenteric Stimulation for Gastroparesis.

Gastroparesis (GP) is a syndrome characterized by delayed gastric emptying in association with symptoms of epigastric pain, nausea, and vomiting in the absence of mechanical obstruction. The prevalence of gastroparesis has been estimated at 24 per 100,000, with women more commonly affected than men. Diabetes appears to be the underlying cause in approximately 25% of patients, while connective tissue disorders, autoimmune disorders, prior gastric surgery, ischemia, and medications make up a smaller percentage of the remaining identifiable causes. However, the largest group of GP patients falls into the idiopathic category (~50%); many of these patients likely develop GP as a result of a prior viral infection. Symptoms of gastroparesis develop due to a number of different pathophysiologic processes, including disorders of fundic accommodation, antroduodenal dyscoordination, a weak antral pump, gastric dysrhythmias, abnormal duodenal feedback, and enhanced visceral sensation. Once the diagnosis of GP is made, the clinician has a number of different treatment options. For patients with mild to moderate symptoms, dietary modifications in conjunction with or without prokinetics and antiemetics are often all that is required. However, many patients with severe symptoms who fail to respond to standard therapy may benefit from neuroenteric stimulation (gastric electrical stimulation). This monograph will review the role of the neuroenteric stimulator therapy for gastroparesis, discuss possible mechanisms of action of neuroenteric stimulation, review data from recently published studies on its efficacy, and discuss patient selection and adverse events.

Prior Viral Infection Increases Heat Stroke Severity in Mice

Anecdotal evidence suggests that prior viral or bacterial infection increases heat stroke (HS) severity. We hypothesized that (1) Poly I:C (PIC; 100 mg, IP) or lipopolysaccharide (LPS; 50 mg/kg, IP) injection 48h or 72h prior to heat exposure (HE) would exacerbate core temperature (Tc; radiotelemetry; ±0.1°C) responses and HS severity of male C57BL/6J mice, and (2) these responses would occur in the absence of overt PIC or LPS sickness behaviors. PIC and LPS induced 2.0°C and 1.1°C fevers, respectively, and decreased activity (ACT) compared to saline (SAL; equivalent volume). PIC caused greater body weight (BW), food intake (FI) and water intake (WI) loss than LPS. Tc, ACT, BW, FI and WI of LPS mice were similar to SAL by 48h. FI and WI of PIC mice were similar to SAL by 72h; however, BW of PIC mice did not recover to pre‐injection levels by 72h. Heating time (190 min), thermal load (181°C·Min) and dehydration (9.6%) during HE were similar between PIC, LPS and SAL mice. PIC was associated with more severe HS recovery responses regardless of prior injection time. PIC injection 48h prior to HE decreased HS survival (60%) within ~4h of recovery and the PIC survivors showed a trend towards greater hypothermia depth (marker of HS severity; 30.4°C) compared to LPS (31.3°C) and SAL (31.5°C) mice. PIC injection 72h prior to HE had no effect on HS survival, but hypothermia depth was greater (29.2°C) compared to LPS (31.1°C) and SAL (31.2°C; ANOVA, P=0.022). Our data indicate that prior viral infection has no effect on Tc responses during HE, but exacerbates HS severity during recovery even after sickness behaviors are no longer evident. Future studies will delineate the inflammatory pathways mediating decreased HS survival during the early stages of HS recovery. Author views not official US Army or DOD policy

Ventricular septal defect repair in children during first year of life in a public hospital

Ventricular septal defect (VSD) is the most common congenital heart disease; primary surgical closure is the usual strategy for repairing it. Our objective is to describe results of surgical repair in children under 1 year of age and analyze risk factors for morbidity and mortality. Retrospective study; all patients with VSD repaired between 2004 and 2011 were included. Demographic, surgical procedure and postoperative variables were recorded: age, weight, genetic syndrome, type of VSD, length of stay, complications and outcome. Risk factors of mortality and morbidity: age < 6 m, weight < 3 kg, Down, malnutrition and respiratory infection prior syndrome were analyzed. 256 patients, age 5.3 months (21d-1y), weight 4.75 kg (2.2 to 13), 32% with Down syndrome and 17.5% with preoperative mechanical ventilation were operated. Perimembranous VSD was the most frequent type (62%). 28% experienced complications and 7% required reoperation for bleeding, infection or defect. The median hospital stay was 6 days (1-185). Postoperative 30 days mortality was 3%. Age < 6 m, weight < 3 kg, malnutrition and prior respiratory viral infection were associated with prolonged hospitalization, but no risk factors for mortality were identified. The primary surgical closure of the VSD is a procedure with satisfactory results at our institution.