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Background: Gestational Zika virus (ZIKV) infection is associated with the development of congenital Zika syndrome (CZS), which includes microcephaly and fetal demise. The magnitude and quality of orthoflavivirus-specific humoral immunity have been previously linked to the development of CZS. However, the role of ZIKV NS1-specific humoral immunity in mothers and children with prenatal ZIKV exposure and CZS remains undefined. In addition, considering that most of the at-risk population lives in dengue virus (DENV)-endemic areas, it is not clear what is the association between pre-existing DENV NS1-specific humoral immunity and CZS., Methods: Here, we studied 328 mothers and children with a clinical diagnosis and seropositivity for ZIKV infection during pregnancy, included during the 2015-2016 ZIKV epidemic in Colombia. We also performed clinical evaluation and pediatric neurological follow-up. The relative levels of circulating NS1-specific IgM and IgG against ZIKV and DENV were evaluated in mothers and children, and the association with the development of microcephaly was analyzed., Results: DENV and ZIKV IgG-NS1 antibodies in pregnant women were placentally transferred, and this passage and its duration in children depended on the maternal levels of the antibodies. We reported that higher concentrations of pre-existing DENV, but not ZIKV IgG-NS1 antibodies, were associated with a reduced risk of CZS-related microcephaly. Also, we observed that the IgM-NS1 response in infants is long-term and has a minor association with poor outcomes., Conclusions: The development of microcephaly in children prenatally exposed to ZIKV is associated with low plasma levels of placentally transferred, pre-existing DENV IgG-NS1 antibodies. These data are compatible with a protective role of anti-NS1 IgG antibodies against ZIKV infection during pregnancy and highlight the promising role of NS1 as an orthoflavivirus vaccine target in high-risk populations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Castro-Trujillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dengue is a significant health problem due to the high burden of critical infections during outbreaks. In 1997, the World Health Organization (WHO) classified dengue as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). It was revised in 2009 (updated in 2015), and the new guidelines recommended classifying patients as dengue without warning signs (DNS), dengue with warning signs (DWS), and severe dengue (SD). Although the utility of the revised 2009 classification for clinical studies is accepted, for immunological studies it needs to be clarified. We determined the usefulness of the 2009 classification for pediatric studies that analyze the circulating interleukin (IL)-6 and IL-8, two inflammatory cytokines. Plasma levels of IL-6 and IL-8 were evaluated in the acute and convalescent phases by flow cytometry in children with dengue classified using the 1997 and 2009 WHO guidelines. The plasma levels of IL-6 and IL-8 were elevated during the acute and decreased during convalescence, and both cytokines served as a good marker of acute dengue illness compared to convalescence. There were no differences in the plasma level of the evaluated cytokines among children with different clinical severity with any classification, except for the IL-8, which was higher in DWS than DNS. Based on the levels of IL-8, the 2009 classification identified DWS plus SD (hospital-treated children) compared to the DNS group [area under the curve (AUC): 0.7, p = 0.028]. These results support the utility of the revised 2009 (updated in 2015) classification in studies of immune markers in pediatric dengue., (© The Author(s) [2024]. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Secondary bacterial infection (SBI) occurs in a proportion of individuals with dengue and results in longer hospitalization, higher mortality, and increased health-related costs. However, the frequency, risk factors and predictive biomarkers of this comorbidity in pediatric dengue is partially known., Methods: We conducted a retrospective multicenter study in a dengue hyperendemic region of Colombia, analyzing 1597 children from two pediatric cohorts. We included children with confirmed dengue (mild to severe disease) and evaluated the rate of SBI, their clinical characteristics, diagnostic predictors and attention costs. We also assessed the diagnostic performance of plasma interleukin (IL)-6 for detecting SBI in pediatric dengue., Results: The frequency of SBI in children with dengue with warning signs in cohorts 1 and 2 was 2.4% and 7.3%, respectively, and this rate reached 30.7% and 38.2% in children with severe disease. Staphylococcus aureus and Escherichia coli were the more frequent infectious agents. Increased total leukocytes and C-reactive protein levels, as well as high IL-6 at hospital admission, in children <48 months of age were early indications of SBI in dengue. Higher rates of organ dysfunction, the requirement of a longer hospitalization and a 2.3-fold increase in attention costs were observed in SBI., Conclusions: An important proportion of children with dengue course with SBI and exhibit higher morbidity. Elevated leukocytes, C-reactive protein and IL-6 in young children are early markers of SBI. Physicians should identify children with dengue and risk factors for SBI, microbiologically confirm the bacterial infection, and rationally and timely provide antimicrobial therapy., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Infections by dengue virus (DENV) and Zika virus (ZIKV) have some similar symptoms and a cross-reactive immune response, although with different risk populations and outcomes. Here, we evaluated the virologic characteristics and the nonstructural protein 1 (NS1)-specific antibody responses to DENV and ZIKV in children suspected of dengue in different epidemiologic moments in Colombia., Methods: Viral RNA, circulating NS1 and IgM/IgG specific for DENV and ZIKV were performed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) in 301 children suspected of dengue enrolled in a hospital setting during the ZIKV epidemic and a primary healthcare setting during a DENV epidemic. For the detection of DENV and ZIKV-specific IgM, an NS1-based ELISA was validated using characterized pediatric samples. Clinical and laboratory parameters were also evaluated., Results: DENV RNA or NS1 antigen was detected in the plasma of 62% of children, and in none, the ZIKV RNA was found. NS1-based ELISA for DENV and ZIKV IgM showed a sensitivity/specificity of 90/84% and 73/98%, respectively. Of 114 children without detectable viremia or antigenemia, 30.7%, 17.5%, 22% and 30% were IgM-DENV + , IgM-ZIKV + , IgM-DENV + ZIKV + and IgM-DENV - ZIKV - , respectively. The ZIKV/DENV IgM-NS1 ratio allows the identification of the infecting orthoflavivirus in 88% of the children with IgM-DENV + ZIKV + , confirming a high predominance of DENV infections in the 2 pediatric settings., Conclusion: Overall, 88% of the children with clinical suspicion of dengue had an identifiable orthoflaviviral infection, with 80% caused by DENV, 7% by ZIKV and 0.7% classified as recent infections or coinfection, demonstrating active viral cocirculation in the pediatric population of southern Colombia. The IgM-NS1 detection improved the identification of orthoflaviviral infections in children without viremia or antigenemia, suggesting it is a helpful complementary tool for medical personnel in tropical regions with high viral cocirculation and different clinical scenes., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is beginning to be tested. We evaluated the efficiency of the automated extraction of viral RNA for studies of pediatric dengue and zika. Clinical samples of children with dengue that were well characterized through RNA isolation by silica columns and serotype-specific nested RT-PCR (DENV-1 n  = 7, DENV-2 n  = 5, and negatives n  = 8) in addition to 40 pediatric plasma samples spiked with ZIKV (strain PRVA BC59) and 209 from negative pre-epidemic children were analyzed. RNA from patients was extracted by two automated standard and high-throughput protocols on the KingFisher™ Flex instrument. The isolated RNA was evaluated for concentration and purity by spectrophotometry, for structural and functional integrity by electrophoresis and expression of the RNase P gene, and usefulness in serotype-specific DENV detection by conventional and real-time RT-PCR. For the evaluation of ZIKV RNA, the commercial TaqMan Triplex® assay was used, along with a well-tested in-house RT-qPCR assay. The concentration of RNA (5.2 vs. 7.5 ng/ μ L, P =0.03) and the number of integral bands (9 vs. 11) were higher with the high-throughput protocol. However, the number of specimens serotyped for DENV by RT-qPCR was comparable for both protocols. The cycle thresholds of the TaqMan Triplex® commercial kit and the in-house assay for the detection of plasma ZIKV RNA isolated with the standard protocol showed a strong association ( r  = 0.93, P < 0.0001) and a Cohen Kappa index of 0.98 when all 249 samples were analyzed. These preliminary results suggest that automated instruments could be used in studies of cocirculating flaviviruses that have represented a public health problem in recent decades in Colombia. They boast advantages such as efficiency, precision, time savings, and lower risk of cross-contamination., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Sandra L. Delgado et al.)

Background: Pediatric dengue and sepsis share clinical and pathophysiologic aspects. Multiple inflammatory and regulatory cytokines, decoy receptors and vascular permeability factors have been implicated in the pathogenesis of both diseases. The differential pattern and dynamic of these soluble factors, and the relationship with clinical severity between pediatric dengue and sepsis could offer new diagnosis and therapeutic strategies., Methods: We evaluated the concentration levels of 11 soluble factors with proinflammatory, regulatory and vascular permeability involvement, in plasma from children with dengue or sepsis, both clinically ranging from mild to severe, in the early, late and convalescence phases of the disease., Results: During early acute infection, children with sepsis exhibited specific higher concentration levels of IL-6, vascular endothelial growth factor (VEGF), and its soluble decoy receptor II (sVEGFR2) and lower concentration levels of IL-10 and the soluble tumor necrosis factor receptor 2 (sTNFR2), in comparison with children with severe dengue. In addition, the circulating amounts of soluble ST2, and VEGF/sVEGFR2 were widely associated with clinical and laboratory indicators of dengue severity, whereas secondary dengue virus infections were characterized by an enhanced cytokine response, relative to primary infections. In severe forms of dengue, or sepsis, the kinetics and the cytokines response during the late and convalescence phases of the disease also differentiate., Conclusions: Dengue virus infection and septic processes in children are characterized by cytokine responses of a specific magnitude, pattern and kinetics, which are implicated in the pathophysiology and clinical outcome of these diseases., Competing Interests: Supported by Ministerio de Ciencia Tecnología e Innovación, grant 112451929026 (D.M.S.) and the Sistema General de Regalías del Departamento del Huila (S.G.R.), project BPIN 2020000100145 (C.F.N.) and Vicerrectoría de Investigación y Proyección Social, Universidad Surcolombiana (C.F.N.). The other authors have no conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled activation of inflammatory cells and an exaggerated release of cytokines. It can be triggered by different factors, including viruses, such as dengue. The objective of this study was to characterize the clinical and laboratory profiles of children with severe dengue and HLH, and to identify the risk factors for this clinical complication., Methods: An analytical study was conducted in children with severe dengue who were treated in an intensive care unit between January 2019 and March 2020. Clinical and laboratory factors were compared between patients with and without HLH., Results: HLH represented 13.4% (15/112) of children with severe dengue. Patients with HLH had a long-lasting fever (10.1 vs. 5.8 days; P = 0.012), low hemoglobin levels (7.6 vs. 10.8 g/dL; P = 0.000) and high aspartate aminotransferase values (4443 vs. 1061 U/L; P = 0.002), alanine transaminase (1433 vs. 487 U/L; P = 0.004), partial thromboplastin time (80.6 vs. 51.8 seconds; P = 0.010), prothrombin time (23.5 vs. 19.6 seconds; P = 0.024), triglycerides (333.7 vs. 223.2 mg/dL; P = 0.005), lactate dehydrogenase (4209 vs. 1947 U/L; P = 0.006), soluble CD25 (3488 vs. 1026 pg/mL; P = 0.014), and presented with higher frequency of myocarditis (66.7% vs. 38.3%; P = 0.048), hepatitis (5.3% vs. 1.3%; P = 0.014), bacterial coinfection (73.3% vs. 26.7%; P = 0.010) and fatal outcome (26% vs. 5%; P = 0.037)., Conclusions: HLH is a serious life-threatening clinical complication of dengue virus infection that must be considered, particularly during outbreaks., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Increased systemic levels of inflammatory cytokines have been associated with the development of pathophysiologic events during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To further explore differences in the pattern and dynamics of plasma cytokines in individuals with coronavirus disease-19 (COVID-19), and the relationship with disease mortality, here we evaluated the plasma levels of proinflammatory and regulatory cytokines in Colombian patient survivors and nonsurvivors of SARS-CoV-2 infection. Individuals with confirmed COVID-19, with other respiratory diseases requiring hospitalization, and healthy controls, were included. Plasma levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon-γ, IL-10, soluble tumor necrosis factor receptor I (sTNFRI), and transforming growth factor-β1 were measured by a bead-based assay or enzyme-linked immunosorbent assay and clinical, laboratory, and tomographic parameters were registered during hospitalization. The levels of most of the evaluated cytokines were increased in COVID-19 individuals relative to healthy controls. The levels of IL-6, IL-10, and sTNFRI were directly associated with the development of respiratory failure, immune dysregulation, and coagulopathy, as well as with COVID-19 mortality. Particularly, the early, robust, and persistent increase of circulating IL-6 characterized COVID-19 nonsurvivors, while survivors were able to counteract the inflammatory cytokine response. In addition, IL-6 systemic levels positively correlated with the tomographic extension of lung damage in individuals with COVID-19. Thus, an exacerbated inflammatory cytokine response, particularly mediated by IL-6 added to the inefficiency of regulatory cytokines, distinguishes COVID-19-associated tissue disturbances, severity, and mortality in Colombian adults.

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48-72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%-25%) and 5% (3%-8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-β, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Frydman is the Chief Science Officer at Coagulo Medical Technologies, Inc. I. Bosch is cofounder of E25Bio Company, the source of the murine mAbs anti-NS1 used here., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1  μ g/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas., Competing Interests: I. Bosch is cofounder of E25Bio Company, the source of the murine mAbs used in the FFA showing here., (Copyright © 2022 Sara Bolívar-Marin et al.)

Dengue is one of the most important vector-borne viral illnesses found in tropical and subtropical regions. Colombia has one of the highest rates of dengue cases in the Americas. Severe dengue virus (DENV) infection presents with capillary leakage, hemorrhage, and organ compromise, eventually leading to death. Over the years, there have been many efforts to develop a vaccine that guarantees protective immunity, but they have been partially successful, as such immunity would need to guarantee protection against four distinct viral serotypes. Absolute platelet count is a laboratory parameter used to monitor the clinical progression of DENV, as infection is often accompanied by thrombocytopenia. Although this finding is well described with respect to the natural history of the disease, there are various hypotheses as to the cause of this rapid decrease, and several in vivo and ex vivo models have been used to explain the effect of DENV infection on platelets and their precursors. DENV infects and activates platelets, facilitating their elimination through recognition by phagocytic cells and peripheral margination. However, infection also affects the precursors in the bone marrow by modulating megakaryopoiesis. The objective of this article is to explore various proposed mechanisms of DENV-induced thrombocytopenia to better understand the pathophysiology and clinical presentations of this highly relevant viral infection.

Objective: The aim of this study is to determine the utility of C reactive protein (CRP) and interleukin (IL)-6 in the diagnosis of neonatal sepsis (NS) in a neonatal intensive care unit (NICU) in the south of Colombia., Study Design: A nonmatched case-control study was conducted. Convenience sampling was performed. Data were obtained from clinical records. IL-6 levels were determined using enzyme-linked immunosorbent assay. Receiver operator characteristic (ROC) curve analysis and Youden's index were used to determine the optimal cutoffs for CRP and IL-6 levels in diagnosing NS, early-onset NS (EONS), and late-onset NS (LONS)., Results: Data from 31 cases and 62 controls were included. History of chorioamnionitis (infinite odds ratio [OR] [3.07-infinity]), and the presence of meconium-stained amniotic fluid during birth (OR: 9.04 [1.35-112]) were identified as risk factors for NS. Differences in CRP ( p  < 0.0001) and IL-6 ( p  < 0.0485) levels were also found, more significantly for LONS and EONS patients, respectively. In the diagnosis of LONS using CRP levels, the area under the ROC curve (AUC) was 0.8371 ( p  < 0.0001). The optimal cutoff was 0.53 mg/dL. For EONS diagnosis using IL-6, the AUC was 0.6869 ( p  = 0.0315) and the optimal cutoff was 17.75 pg/mL., Conclusion: Differences between CRP and IL-6 levels were found between control and NS groups. Furthermore, CRP showed greater potential diagnostic utility in the LONS group, whereas IL-6 showed greater potential utility in the EONS group., Key Points: · NS is a major morbimortality cause worldwide. · CRP and IL-6 levels may be useful NS biomarkers. · No biomarker alone is enough for the diagnosis of NS., Competing Interests: None declared., (Thieme. All rights reserved.)

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.

Background: Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1-4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiating DENV serotypes and none are currently commercially available., Methodology/principle Findings: We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitivity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 serotyping were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07-100%., Significance: Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions., Competing Interests: I.B., A.R., L.M., B.B.H., N.S., & L.G. are affiliated or employed by E25Bio Inc. (www.e25bio.com), a company that develops point-of-care diagnostics for fever-causing infectious agents.

Pulmonary tuberculosis (PTB) has been identified as a substantial public health threat and diagnostic challenge. A large proportion of patients exhibit negative smear tests despite active infection. The role of cytokines in the pathophysiology and clinical severity of PTB remains a controversial question. We evaluated the pattern of cytokines presents locally in patients with smear-negative PTB. Levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17 in bronchoalveolar lavage fluid (BALf) from patients with smear-negative PTB, as well as in those with other pulmonary diseases and controls, were performed by flow cytometry. ROC curve and a radiological severity scale were used to establish the potential diagnosis use and the relationship of the cytokine levels with disease severity, respectively. The levels of IL-6 were higher in the PTB (P = 0.0249) and pneumonia (P = 0.0047) groups compared to controls. Low to undetectable levels of TNF-α, IFN-γ, IL-2, IL-4, IL-10, and IL-17 were found in BALf, even after sample concentration using filtration columns and centrifugation. IL-6 levels measured in BALf could distinguish PTB patients or pneumonia patients from controls (AUC: 0.91, P = 0.002 and AUC: 0.86, P = 0.001, respectively), but not patients with PTB from those with pneumonia (AUC: 0.51, P = 0.86). IL-6 levels were related with the severity of PTB, as levels were higher in patients with higher radiological severity. These results confirm the importance of IL-6 in the immunopathology of smear-negative PTB., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia., Methods: A pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up., Results: Twenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF., Conclusions: This study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Host genetics is recognized as an influential factor for the development of dengue disease., Objective: This study evaluated the association of dengue with the polymorphisms rs8192284 for gene IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN., Materials and Methods: Of the 292 surveyed subjects, 191 were confirmed for dengue fever and the remaining 101 were included as controls. The genotypes were resolved using polymerase chain reaction and restriction fragment length polymorphism (PCRRFLP). In an attempt to determine the risk (Odds Ratio) of suffering dengue fever, data were analyzed using chi-square for alleles and logistic regression for both genotypes and allelic combinations. Confidence intervals were set to 95% for all tests regardless of the adjustment by either self-identification or ancestry., Results: For Afro-Colombians, the allele rs8192284 C offered protection against dengue [OR=0.425,(0.204-0.887), p=0.020]. The alleles rs7248637 A and rs3775290 A posed, respectively, an increased risk of dengue for Afro-Colombians [OR=2.389, (1.170-4.879), p=0.015] and Mestizos [OR=2.329, (1.283-4.226), p=0.005]. The reproducibility for rs8192284 C/C [OR=2.45, (1.05-5.76), p=0.013] remained after adjustment by Amerindian ancestry [OR=2.52, (1.04-6.09), p=0.013]. The reproducibility for rs3775290 A/A [OR=2.48, (1.09-5.65), p=0.033] remained after adjustment by European [OR=2.34, (1.02-5.35), p=0.048], Amerindian [OR=2.49, (1.09-5.66), p=0.035], and African ancestry [OR=2.37, (1.04-5.41), p=0.046]. Finally, the association of dengue fever with the allelic combination CAG [OR=2.07, (1.06-4.05), p=0.033] remained after adjustment by Amerindian ancestry [OR=2.16, (1.09-4.28), p=0.028]., Conclusions: Polymorphisms rs8192284 for IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN were associated with the susceptibility to suffer dengue fever in the sampled Colombian population.

Background: Etiologic studies provide evidence that IL-4R and IL-6R receptors may play important roles in the regulatory mechanisms of the development of clinical dengue, especially in children which is a segment of the population with high severe dengue risk. Moreover, the allele frequencies and genetic associations may be influenced by the populational genetic background. Therefore, we performed a case-control study to evaluate possible associations between SNPs in IL4R and IL6R genes and clinical dengue in children from two Colombian populations., Methods: We genotyped the rs1805016 (IL4R) and rs8192284 (IL6R) by PCR-RFLP method, in 298 symptomatic children and 648 asymptomatic controls. Three individual genetic ancestral proportions (APs) (European, Amerindian, African) were inferred by genotyping 29 AIMs (Ancestry informative markers). The variables gender, APs, and the population of origin were used like confusion variables., Results: We found IL4R-rs1805016 GG genotype and G-allele carriers and IL6R-rs8192284 AA genotype associated with clinical dengue in the pooled and Huila samples. Nevertheless, we found no association of these polymorphisms in the sample of Antioquia., Conclusions: For the first time, we report SNPs in IL4R and IL6R genes associated with clinical dengue, which contributes to understanding the genetic susceptibility to dengue disease. Moreover, these results may be influenced by genetic background and must be evaluated through functional analysis., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Functional immunological evidence supports the impact that the host genetic variability has on the susceptibility to develop asymptomatic or symptomatic dengue infection. Children are more prone to develop severe dengue. Thus, we have evaluated possible associations between single-nucleotide polymorphisms (SNPs) located in immune genes and the development of symptomatic dengue in children from two Colombian populations with differences in genetic backgrounds and geographical features. We genotyped 15 SNPs (in 12 genes) in 298 symptomatic children and 648 healthy controls. Ancestry proportions (APs) were inferred by genotyping 29 ancestry informative markers. We observed four SNPs associated with susceptibility to develop dengue in NOD1, RIPK2, MICB, or PLCE1 genes. Conversely, we found one SNP in TNF gene and two haplotypes in the IKBKE gene associated with resistance to develop dengue. These associations were adjusted by gender, APs, and the population of origin because the association of polymorphisms may be different in admixed populations like Colombian. To our knowledge, this is the first reported association study with dengue in IKBKE, RIPK2, and NOD1 genes. We have also confirmed previously reported associations in MICB and PLCE1 genes with dengue. Overall, our results contribute to the understanding of the genetic susceptibility/resistance to develop symptomatic dengue. Nevertheless, these associations must be validated through functional analysis.

In this study, we identified, at the single-cell level, naturally induced cytokine-producing circulating cells (CPCCs) in children with dengue virus (DENV) infection ranging clinically from mild to severe disease. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) CPCCs were detected in children with primary or secondary acute dengue virus (DENV) infection, and the pattern of these cytokines was similar to that seen in the supernatant of cultured peripheral blood mononuclear cells and partially comparable to that found in plasma. Monocytes, B cells, and myeloid dendritic cells (mDCs) were the primary CPCCs detected, and the frequency of mDCs was significantly higher in severe disease. B cells isolated from children with dengue spontaneously secreted TNF-α, IL-6, and interleukin 10, and supernatants from cultures of purified B cells induced activation of allogeneic T cells, supporting an antibody-independent function of these cells during DENV infection. Thus, CPCCs could be a new immune parameter with potential use to evaluate pathogenesis in this infection.

Hereditary angioedema (HAE) is a heterogeneous genetic disease caused by a deficit in C1 inhibitor (C1-INH) and clinically characterized by sudden events of edema, swelling, and pruritus. Here, we describe the first SERPING1 genotyping in 22 subjects from 4 non-related families, all from southern Colombia. The previously reported heterozygous gene mutations, c.1081C>T (p.Gln361*), c.1396C>G (p.Arg466Gly), c.1029+84G>A, or c.106&#95;107del (p.Ser36Phefs*21), were found in 12 patients. Of note, a single patient clinically characterized as severe HAE type 2 expressed mutations in exon 8 and intron 6, whereas all the others have type 1 HAE and expressed one pathogenic variant. One of the subjects, a 5-year-old girl was discovered to have a pathogenic variant, and she is still asymptomatic. This is the first report focused on HAE genetic analysis in a Colombian population.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During dengue virus (DENV) infection, a blockage of secretion of cytokines such as tumor necrosis factor (TNF)-α and members of the interferon (IFN) family has been described in vitro. We evaluated the functionality of monocytes as well as dendritic, B and T cells isolated from children with mild and severe dengue. Compared with those of healthy children, stimulated monocytes, CD4 + T cells and dendritic cells from children with dengue had lower production of proinflammatory cytokines. The interferon axis was dramatically modulated by infection as plasmacytoid dendritic cells (pDCs) and CD4 + T cells had low production of IFN-α and IFN-γ, respectively; plasma levels of IFN-α and IFN-γ were lower in severely ill children, suggesting a protective role. Patients with antigenemia had the highest levels of IFN-α in plasma but the lowest frequency of IFN-α-producing pDCs, suggesting that DENV infection stimulates a systemic type I IFN response but affects the pDCs function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dengue is a major public health problem in tropical regions around the world. Viral and immune host factors determine the clinical courses of the infection. We analyzed the dynamics of viremia (by real-time polymerase chain reactions), antigenemia (through detection of the viral non-structural protein [NS]-1 by enzyme-linked immunosorbent assays) and the frequency of virus-infected peripheral blood mononuclear cells (PBMCs) (by multiparametric flow cytometry) in children with primary or secondary dengue virus (DENV) infection in mild to severe cases. Additionally, we evaluated the association of these factors with clinical severity and laboratory parameters. The levels of viremia and antigenemia peaked during the early days of illness and these viral parameters were correlated (rho=0.37, P=0.003). Circulating monocytes were the most naturally infected subset within the PBMCs population, with kinetics similar to those of viremia and antigenemia. The levels of viremia and antigenemia were higher in children with primary infections than in those with secondary infections (P≤0.04). Although there were no associations between the three evaluated factors and clinical severity, the levels of plasma NS1 and the frequency of dengue virus-infected monocytes correlated with prolonged coagulation times. In short, the viremia, antigenemia and infected monocytes were detected early and were not related to clinical severity. The magnitude of antigenemia and infected circulating monocytes was associated with coagulation disorders., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tumor necrosis factor (TNF)-α is a key cytokine in the pathogenesis of dengue virus infection, and its accurate detection in several types of human samples is critical. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for the detection of TNF-α, but multiplexed bead-based assays such as cytometric bead array (CBA) are now frequently used. Here, using ELISA and two CBAs commercially available, we measured TNF-α concentrations in plasma and serum from children with acute dengue virus infection and healthy controls. To evaluate the detection efficiency and factors affecting it, spiked recovery and immune complex dissociation assays were also performed. The levels of TNF-α evaluated by ELISA in paired serum and plasma samples from children with dengue positively correlated (rho = 0.99, p < 0.0001). Children with dengue had higher levels of plasma TNF-α than those of healthy children (p = 0.004). The ELISA detected TNF-α in a higher number of plasma samples than the CBA (p < 0.0001), and both methods only correlated when TNF-α was evaluated in buffer-based solutions but not in plasma, indicating the presence of a factor interfering with the detection of TNF-α in plasma. The recovery of several types of human recombinant TNF-α was dramatically decreased in plasma but not in tissue culture media (p ≤ 0.01), and this effect was similar in the plasma obtained from the children with dengue or the healthy controls. The dissociation of immune complexes did not improve TNF-α recovery. Dilution of the plasma samples increased the recovery of TNF-α, but at high concentrations of the cytokine. In short, plasma affects the efficiency of TNF-α detection, and this effect should be considered in the measurement of this cytokine.

The CD27 and CD38 antigens are highly expressed on the plasmablast surface, and a massive plasmablast response has been described for dengue virus infection. Soluble CD27 and CD38 forms (sCD27 and sCD38, respectively) increase after immune activation. Here, we show increased sCD27 release in cultures of purified polyclonally stimulated B cells. T and B cells isolated from children with dengue spontaneously produced higher levels of sCD27 but not sCD38, compared with healthy children (P=0.03), and sCD27 levels positively correlated with plasmablast frequency in the cultures (rho=0.58, P=0.01). Children with dengue had higher plasma levels of sCD27 and sCD38 than healthy children, which decreased during convalescence. Plasma sCD27 was higher in severe than with mild dengue, but the opposite was observed for sCD38. These findings support a potential new role for B cells in dengue pathogenesis, and sCD27 and sCD38 are novel biomarkers associated with clinical outcome during dengue virus infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6-12 months old) and children (1-14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = -0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue., Competing Interests: The authors have declared that no competing interests exist.

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3 + CD8 + amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3 + cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = -0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Bacterial meningitis (BM) is a pyogenic infection present in the subarachnoid space, potentially fatal and frequently associated with neurological sequelae. During BM, cytokines (CTs) are locally produced. We sought to determine the CTs' clinical role as disease severity predictors in adults, which is not completely clear. Using a bead-based flow cytometric assay, levels of six CTs were determined in cerebrospinal fluid (CSF) and plasma from 18 adult BM patients and 19 uninfected controls. Long-term neurological sequelae were evaluated using the Glasgow Outcome Scale (GOS). All evaluated CTs were higher in CSF than in plasma, and the levels of CSF interleukin (IL)-6, IL-8, IL-10, IL-1β, and tumor necrosis factor-α and plasma IL-10 and IL-12p70 were significantly higher in patients with severe sepsis than with sepsis, suggesting an association with clinical severity. There was a strong negative correlation between CSF IL-6 and plasma IL-12p70 with GOS score, supporting the possible role of these CTs in the development of neurological long-term sequelae. These findings could be helpful to identify candidates to receive neuroprotective treatments and early physiotherapy schemes.

B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.

Identification of early determinants of dengue disease progression, which could potentially enable individualized patient care are needed at present times. Soluble ST2 (sST2) has been recently reported to be elevated in the serum of children older than 2 years old and adults with dengue infection and it was correlated with secondary infections as well as with severe presentations of the disease. The mechanism by which secreted ST2 is linked to severe dengue and plasma leakage remains unclear. One possibility is that IL-33 ligand may be elevated, contributing to membrane bound ST2 as part of the immune activation in dengue infection. We determined plasma levels of sST2 and the ligand IL-33 in 66 children with acute secondary dengue infections clinically classified using the guidelines of the World Health Organization, 2009. Dengue infection showed significant increases in cytokines IL-12p70, IL-10, IL-8, IL-6, IL-1β and TNFα measured by flow cytometry based assay compared to uninfected individuals. In contrast, IL-33 levels remained unchanged between infected and uninfected individuals. The levels of sST2 positively correlated with values of IL-6 and IL-8 and inversely correlated with number of median value of platelet levels. In addition to circulating cytokine positive correlations we found that sST2 and isoenzyme creatine kinase-MB (CK-MB), a marker of myocardial muscle damage present in severe dengue cases were associated. Our pediatric study concluded that in dengue infections sST2 elevation does not involve concomitant changes of IL-33 ligand. We propose a study to assess its value as a predictor factor of disease severity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

Objective: Dengue is the most important arthropod-borne viral disease in the world; it can be life-threatening because of liver involvement. Aim Determining liver involvement frequency and severity in dengue-infected children., Methods: This was a descriptive case series study which involved studying 108 dengue-infected children aged less than 13 years old whose infection had been confirmed by the detection of dengue-specific IgM and NS1 in plasma. Clinical and biochemical parameters were used for evaluating liver involvement, including transaminases and albumin. Hepatitis A and leptospira infection were also evaluated by using ELISA to detect pathogen-specific IgM in plasma during acute and convalescence phases. The study was carried out at a teaching hospital in Neiva from June 2009 to May 2010., Results: Ninety-eight of the aforementioned cases were clinically classified as dengue with warning signs (DWS) and 10 as severe dengue (SD). Two out of three DWS patients and all SD patients had some degree of liver involvement, shown clinically and biochemically. Regardless of the clinical classification, hepatomegaly was the main clinical sign of liver involvement and was present in 85% of all the children in the study. It is worth noting that 5 patients had probable dengue and leptospirosis co-infection, this being the first instance of this in Colombia. None of the cases analyzed here had acute hepatitis A., Conclusions: Liver compromise should be considered in confirmed cases of dengue as shown in this series of children. Leptospirosis must be considered as differential diagnosis and also as causing co-infection in a febrile child.

We have previously shown that human myeloid dendritic cells treated with purified rotavirus induce an allogenic Th1 response. To determine if rotavirus in the context of an intestinal microenvironment modulates the function of dendritic cells, we treated these cells with supernatants from non-infected or infected Caco-2 cells and studied their capacity to promote Th1 or Th2 responses. Dendritic cells treated with supernatants from rotavirus-infected Caco-2 cells promoted a significantly lower Th1 response, in comparison with those treated with purified rotavirus. We wanted to establish if TGF-β1, induced, or TSLP, not induced, during rotavirus infection, could mediate this effect. Neutralization of TGF-β but not TSLP in the supernatant prior to treatment of dendritic cells increased their capacity to promote a Th1 response. The results suggest that the TGF-β1 induced by rotavirus could be an immune evasion mechanism, and may partially explain the poor rotavirus-specific T cell response we have previously evidenced., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have shown previously that rotavirus (RV) can infect murine intestinal B220(+) cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.

We quantified circulating total, rotavirus (RV) and Tetanus toxin (TT) memory B cells (mBc) in healthy adults using a limiting dilution assay (LDA) and a flow cytometry assay (FCA) that permit evaluation of both CD27+ and CD27- mBc. RV mBc were enriched in the CD27-, IgG+ and in the CD27+, IgM+ subsets. The numbers of RV mBc were higher by FCA than by LDA and results of the two assays did not correlate. TT IgGmBc and RV IgA mBc determined by FCA and by LDA correlated with TT plasma IgG and RV plasma IgA, respectively. The mean ratio of specific mBc/mug/ml of the corresponding plasma immunoglobulin was lower for TT IgG than for RV IgA mBc. Our studies contribute to understand the relationship between circulating mBc and serological memory, and enhance our capacity to develop better correlates of protection against RV disease.

Rotavirus (RV) infection of the intestine is the major cause of severe dehydrating diarrhea in infants around the world. Although protective immunity against RV, especially acquired B and T-cell responses, has been extensively studied, our understanding of RV immunity remains incomplete. In addition, the interaction between various protective immune mechanisms in the gut and specific enteric immune suppressor systems that normally exert a regulatory function on mucosal immunity has not been extensively investigated. Among the candidate suppressor systems, we hypothesized that CD4+ CD25+ Foxp3+ regulatory T (Treg) cells may play a role in modulating RV immunity since such cells are naturally present in large numbers in the intestine and function nonspecifically. Here we demonstrate that neonatal murine RV (EC) infection induces an expansion of the Treg cell population and the magnitude of the T cell mediated immune response is modulated by Treg cells. Accordingly, when natural Treg cells in neonatal mice were depleted before virus infection, both CD4+ and CD8+ T-cell responses to RV, such as proliferation and IFN-gamma secretion, were enhanced in mesenteric lymph nodes (MLNs) and the spleen. Interestingly, increased proliferation of CD19+ B cells from Treg cell depleted animals was also observed. Finally, we analyzed the in vivo effect of the Treg cell depletion on diarrheal disease, virus shedding and IgA RV-specific response. Treg cell depletion did not affect these functions. Our studies of immune modulatory Treg cells in the RV infection model may promote a better understanding of the basis for RV immunity as well as providing valuable clues for the development of more immunogenic RV vaccines.

In a double blind trial, 319 fully immunized children received two doses of either placebo or 10(6.7) focus-forming units of the attenuated RIX4414 human rotavirus (RV) vaccine ("all-in-one" formulation). Plasma RV-specific IgA (RV IgA), stool RV IgA, and circulating total and RV memory B cells (CD19+ IgD+/- CD27+) with an intestinal homing phenotype (alpha4beta7+ CCR9+/-) were measured, after the first and second doses, as potential correlates of protection. After the first and/or second dose, 54% of vaccinees and 13% of placebo recipients had plasma RV IgA. Before vaccination, most (95%) of the children (of both study groups) were breast-fed and had stool RV IgA (68.64%). Coproconversion (4-fold increase) after the first and/or second dose was observed in 32.7% of vaccinees and 17.4% of placebo recipients. No significant difference was seen when comparing the frequencies of any subset of memory B cells between vaccinees and placebo recipients. Statistically significant weak correlations were found between plasma RV IgA titers and coproconversion, and several subsets of memory B cells. The vaccine provided 74.8% protection (95% confidence interval, 30.93-92.62) against any RV gastroenteritis and 100% protection (95% confidence interval, 14.53-100) against severe RV gastroenteritis. When vaccinees and placebo recipients were considered together, a correlation was found between protection from disease and plasma RV IgA measured after dose 2 and RV memory (IgD- CD27+ alpha4beta7+ CCR9+) circulating B cells measured after dose 1. However, the correlation coefficients for both tests were low (<0.2), suggesting that other factors are important in explaining protection from disease.

We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1beta, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells.