Genotype, oxidase status, and preceding infection or autoinflammation do not affect allogeneic HCT outcomes for CGD.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections and inflammatory conditions. Hematopoietic cell transplantation (HCT) is the definitive treatment for CGD, but questions remain regarding patient selection and impact of active disease on transplant outcomes. We performed a multi-institutional retrospective and prospective study of 391 patients with CGD treated either conventionally (non-HCT) enrolled from 2004 to 2018 or with HCT from 1996 to 2018. Median follow-up after HCT was 3.7 years with a 3-year overall survival of 82% and event-free survival of 69%. In a multivariate analysis, a Lansky/Karnofsky score <90 and use of HLA-mismatched donors negatively affected survival. Age, genotype, and oxidase status did not affect outcomes. Before HCT, patients had higher infection density, higher frequency of noninfectious lung and liver diseases, and more steroid use than conventionally treated patients; however, these issues did not adversely affect HCT survival. Presence of pre-HCT inflammatory conditions was associated with chronic graft-versus-host disease. Graft failure or receipt of a second HCT occurred in 17.6% of the patients and was associated with melphalan-based conditioning and/or early mixed chimerism. At 3 to 5 years after HCT, patients had improved growth and nutrition, resolved infections and inflammatory disease, and lower rates of antimicrobial prophylaxis or corticosteroid use compared with both their baseline and those of conventionally treated patients. HCT leads to durable resolution of CGD symptoms and lowers the burden of the disease. Patients with active infection or inflammation are candidates for transplants; HCT should be considered before the development of comorbidities that could affect performance status. This trial was registered at www.clinicaltrials.gov as #NCT02082353.

Posttransplantation late complications increase over time for patients with SCID: A Primary Immune Deficiency Treatment Consortium (PIDTC) landmark study.

BACKGROUND: The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children in the United States and Canada onto a retrospective multicenter natural history study of hematopoietic cell transplantation (HCT). OBJECTIVE: We investigated outcomes of HCT for severe combined immunodeficiency (SCID). METHODS: We evaluated the chronic and late effects (CLE) after HCT for SCID in 399 patients transplanted from 1982 to 2012 at 32 PIDTC centers. Eligibility criteria included survival to at least 2 years after HCT without need for subsequent cellular therapy. CLE were defined as either conditions present at any time before 2 years from HCT that remained unresolved (chronic), or new conditions that developed beyond 2 years after HCT (late). RESULTS: The cumulative incidence of CLE was 25% in those alive at 2 years, increasing to 41% at 15 years after HCT. CLE were most prevalent in the neurologic (9%), neurodevelopmental (8%), and dental (8%) categories. Chemotherapy-based conditioning was associated with decreased-height z score at 2 to 5 years after HCT (P < .001), and with endocrine (P < .001) and dental (P = .05) CLE. CD4 count of ≤500 cells/µL and/or continued need for immunoglobulin replacement therapy >2 years after transplantation were associated with lower-height z scores. Continued survival from 2 to 15 years after HCT was 90%. The presence of any CLE was associated with increased risk of late death (hazard ratio, 7.21; 95% confidence interval, 2.71-19.18; P < .001). CONCLUSION: Late morbidity after HCT for SCID was substantial, with an adverse impact on overall survival. This study provides evidence for development of survivorship guidelines based on disease characteristics and treatment exposure for patients after HCT for SCID.

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia.

Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/µL, there was a higher proliferative response with the PHA flow assay compared to the 3H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID.

Measuring the effect of newborn screening on survival after haematopoietic cell transplantation for severe combined immunodeficiency: a 36-year longitudinal study from the Primary Immune Deficiency Treatment Consortium.

BACKGROUND: Severe combined immunodeficiency (SCID) is fatal unless durable adaptive immunity is established, most commonly through allogeneic haematopoietic cell transplantation (HCT). The Primary Immune Deficiency Treatment Consortium (PIDTC) explored factors affecting the survival of individuals with SCID over almost four decades, focusing on the effects of population-based newborn screening for SCID that was initiated in 2008 and expanded during 2010-18. METHODS: We analysed transplantation-related data from children with SCID treated at 34 PIDTC sites in the USA and Canada, using the calendar time intervals 1982-89, 1990-99, 2000-09, and 2010-18. Categorical variables were compared by χ2 test and continuous outcomes by the Kruskal-Wallis test. Overall survival was estimated by the Kaplan-Meier method. A multivariable analysis using Cox proportional hazards regression models examined risk factors for HCT outcomes, including the variables of time interval of HCT, infection status and age at HCT, trigger for diagnosis, SCID type and genotype, race and ethnicity of the patient, non-HLA-matched sibling donor type, graft type, GVHD prophylaxis, and conditioning intensity. FINDINGS: For 902 children with confirmed SCID, 5-year overall survival remained unchanged at 72%-73% for 28 years until 2010-18, when it increased to 87% (95% CI 82·1-90·6; n=268; p=0·0005). For children identified as having SCID by newborn screening since 2010, 5-year overall survival was 92·5% (95% CI 85·8-96·1), better than that of children identified by clinical illness or family history in the same interval (79·9% [69·5-87·0] and 85·4% [71·8-92·8], respectively [p=0·043]). Multivariable analysis demonstrated that the factors of active infection (hazard ratio [HR] 2·41, 95% CI 1·56-3·72; p<0·0001), age 3·5 months or older at HCT (2·12, 1·38-3·24; p=0·001), Black or African-American race (2·33, 1·56-3·46; p<0·0001), and certain SCID genotypes to be associated with lower overall survival during all time intervals. Moreover, after adjusting for several factors in this multivariable analysis, HCT after 2010 no longer conveyed a survival advantage over earlier time intervals studied (HR 0·73, 95% CI 0·43-1·26; p=0·097). This indicated that younger age and freedom from infections at HCT, both directly driven by newborn screening, were the main drivers for recent improvement in overall survival. INTERPRETATION: Population-based newborn screening has facilitated the identification of infants with SCID early in life, in turn leading to prompt HCT while avoiding infections. Public health programmes worldwide can benefit from this definitive demonstration of the value of newborn screening for SCID. FUNDING: National Institute of Allergy and Infectious Diseases, Office of Rare Diseases Research, and National Center for Advancing Translational Sciences.

Congenital CMV infection in a Brazilian neonatal intensive care unit: high prevalence among twin newborns.

BACKGROUND: Cytomegalovirus (CMV) is one of the most important pathogens associated with congenital infection worldwide. Most congenital CMV-infected infants are asymptomatic at birth; however, some can develop delayed sequelae, especially hearing loss. METHODS: This study aimed to investigate the prevalence of congenital CMV infection in a neonatal intensive care unit in a low-income region of Brazil. The objectives extended to identifying associated factors, assessing the clinical status of infected newborns, and undertaking a two-year follow-up to discern potential long-term consequences in the affected infants. This cross-sectional prospective study enrolled newborns up to three weeks of life requiring intensive medical care. We employed a convenience sampling method to include 498 newborns and 477 mothers in the study. Categorical variables underwent analysis employing Fisher's exact test, whereas the examination of continuous variables involved the Mann‒Whitney test. RESULTS: CMV DNA was detected in saliva/urine samples from 6 newborns (1.21%), confirming congenital infection. We noted a significantly greater incidence (OR: 11.48; 95% CI: 2.519-52.33; p = 0.0094) of congenital infection among twins (7.14%) than among nontwins (0.66%). The twin patients exhibited discordant infection statuses, suggesting that only one of the babies tested positive for CMV. Most of the infected children were born to mothers who initiated sexual activity at a younger age (p = 0.0269). Only three out of the six newborns diagnosed with CMV infection underwent comprehensive clinical assessments and received continuous follow-up until they reached two years of age. Only one of the children had weight and height measurements below the norm for their age, coupled with developmental delays. CONCLUSIONS: The prevalence of congenital CMV infection among newborns admitted to the NICU was low and similar to that in the general population. However, we found a significantly greater incidence of congenital CMV infection in twins than in singletons. Interestingly, the twin-infected patients exhibited discordant infection statuses, suggesting that CMV was present in only one of the babies. We also found that most of the infected children were born to mothers who initiated sexual activity at a younger age. Diagnostic accessibility and comprehensive surveillance programs are imperative for effectively managing and preventing congenital CMV infections.

The diagnosis of severe combined immunodeficiency: Implementation of the PIDTC 2022 Definitions.

BACKGROUND: Shearer et al in 2014 articulated well-defined criteria for the diagnosis and classification of severe combined immunodeficiency (SCID) as part of the Primary Immune Deficiency Treatment Consortium's (PIDTC's) prospective and retrospective studies of SCID. OBJECTIVE: Because of the advent of newborn screening for SCID and expanded availability of genetic sequencing, revision of the PIDTC 2014 Criteria was needed. METHODS: We developed and tested updated PIDTC 2022 SCID Definitions by analyzing 379 patients proposed for prospective enrollment into Protocol 6901, focusing on the ability to distinguish patients with various SCID subtypes. RESULTS: According to PIDTC 2022 Definitions, 18 of 353 patients eligible per 2014 Criteria were considered not to have SCID, whereas 11 of 26 patients ineligible per 2014 Criteria were determined to have SCID. Of note, very low numbers of autologous T cells (<0.05 × 109/L) characterized typical SCID under the 2022 Definitions. Pathogenic variant(s) in SCID-associated genes was identified in 93% of patients, with 7 genes (IL2RG, RAG1, ADA, IL7R, DCLRE1C, JAK3, and RAG2) accounting for 89% of typical SCID. Three genotypes (RAG1, ADA, and RMRP) accounted for 57% of cases of leaky/atypical SCID; there were 13 other rare genotypes. Patients with leaky/atypical SCID were more likely to be diagnosed at more than age 1 year than those with typical SCID lacking maternal T cells: 20% versus 1% (P < .001). Although repeat testing proved important, an initial CD3 T-cell count of less than 0.05 × 109/L differentiated cases of typical SCID lacking maternal cells from leaky/atypical SCID: 97% versus 7% (P < .001). CONCLUSIONS: The PIDTC 2022 Definitions describe SCID and its subtypes more precisely than before, facilitating analyses of SCID characteristics and outcomes.

Comparison of hematopoietic cell transplant conditioning regimens for hemophagocytic lymphohistiocytosis disorders.

BACKGROUND: Allogeneic hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis (HLH) disorders is associated with substantial morbidity and mortality. OBJECTIVE: The effect of conditioning regimen groups of varying intensity on outcomes after transplantation was examined to identify an optimal regimen or regimens for HLH disorders. METHODS: We studied 261 patients with HLH disorders transplanted between 2005 and 2018. Risk factors for transplantation outcomes by conditioning regimen groups were studied by Cox regression models. RESULTS: Four regimen groups were studied: (1) fludarabine (Flu) and melphalan (Mel) in 123 subjects; (2) Flu, Mel, and thiotepa (TT) in 28 subjects; (3) Flu and busulfan (Bu) in 14 subjects; and (4) Bu and cyclophosphamide (Cy) in 96 subjects. The day 100 incidence of veno-occlusive disease was lower with Flu/Mel (4%) and Flu/Mel/TT (0%) compared to Flu/Bu (14%) and Bu/Cy (22%) (P < .001). The 6-month incidence of viral infections was highest after Flu/Mel (72%) and Flu/Mel/TT (64%) compared to Flu/Bu (39%) and Bu/Cy (38%) (P < .001). Five-year event-free survival (alive and engrafted without additional cell product administration) was lower with Flu/Mel (44%) compared to Flu/Mel/TT (70%), Flu/Bu (79%), and Bu/Cy (61%) (P = .002). The corresponding 5-year overall survival values were 68%, 75%, 86%, and 64%, and did not differ by conditioning regimen (P = .19). Low event-free survival with Flu/Mel is attributed to high graft failure (42%) compared to Flu/Mel/TT (15%), Flu/Bu (7%), and Bu/Cy (18%) (P < .001). CONCLUSIONS: Given the high rate of graft failure with Flu/Mel and the high rate of veno-occlusive disease with Bu/Cy and Flu/Bu, Flu/Mel/TT may be preferred for HLH disorders. Prospective studies are warranted.

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

SARS-CoV-2 infection associated with hepatitis in an infant with X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (X-SCID) is a disorder of adaptive immunity caused by mutations in the IL-2 receptor common gamma chain gene resulting in deficiencies of T and natural killer cells, coupled with severe dysfunction in B cells. X-SCID is lethal without allogeneic stem cell transplant or gene therapy due to opportunistic infections. An infant with X-SCID became infected with SARS-CoV-2 while awaiting transplant. The patient developed severe hepatitis without the respiratory symptoms typical of COVID-19. He was treated with convalescent plasma, and thereafter was confirmed to have SARS-CoV-2 specific antibodies, as detected with a microfluidic antigen array. After resolution of the hepatitis, he received a haploidentical CD34 selected stem cell transplant, without conditioning, from his father who had recovered from COVID-19. SARS CoV-2 was detected via RT-PCR on nasopharyngeal swabs until 61 days post transplantation. He successfully engrafted donor T and NK cells, and continues to do well clinically.

Infections in Infants with SCID: Isolation, Infection Screening, and Prophylaxis in PIDTC Centers.

PURPOSE: The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children with severe combined immunodeficiency (SCID) in a prospective natural history study of hematopoietic stem cell transplant (HSCT) outcomes over the last decade. Despite newborn screening (NBS) for SCID, infections occurred prior to HSCT. This study's objectives were to define the types and timing of infection prior to HSCT in patients diagnosed via NBS or by family history (FH) and to understand the breadth of strategies employed at PIDTC centers for infection prevention. METHODS: We analyzed retrospective data on infections and pre-transplant management in patients with SCID diagnosed by NBS and/or FH and treated with HSCT between 2010 and 2014. PIDTC centers were surveyed in 2018 to understand their practices and protocols for pre-HSCT management. RESULTS: Infections were more common in patients diagnosed via NBS (55%) versus those diagnosed via FH (19%) (p = 0.012). Outpatient versus inpatient management did not impact infections (47% vs 35%, respectively; p = 0.423). There was no consensus among PIDTC survey respondents as to the best setting (inpatient vs outpatient) for pre-HSCT management. While isolation practices varied, immunoglobulin replacement and antimicrobial prophylaxis were more uniformly implemented. CONCLUSION: Infants with SCID diagnosed due to FH had lower rates of infection and proceeded to HSCT more quickly than did those diagnosed via NBS. Pre-HSCT management practices were highly variable between centers, although uses of prophylaxis and immunoglobulin support were more consistent. This study demonstrates a critical need for development of evidence-based guidelines for the pre-HSCT management of infants with SCID following an abnormal NBS. TRIAL REGISTRATION: NCT01186913.

Microglial Cells Impact Gut Microbiota and Gut Pathology in Angiotensin II-Induced Hypertension.

RATIONALE: Increased microglial activation and neuroinflammation within autonomic brain regions have been implicated in sustained hypertension, and their inhibition by minocycline-an anti-inflammatory antibiotic-produces beneficial effects. These observations led us to propose a dysfunctional brain-gut communication hypothesis for hypertension. However, it has been difficult to reconcile whether an anti-inflammatory or antimicrobial action is the primary beneficial effect of minocycline in hypertension. Accordingly, we utilized chemically modified tetracycline-3 (CMT-3)-a derivative of tetracycline that has potent anti-inflammatory activity-to address this question. OBJECTIVE: Test the hypothesis that central administration of CMT-3 would inhibit microglial activation, attenuate neuroinflammation, alter selective gut microbial communities, protect the gut wall from developing hypertension-associated pathology, and attenuate hypertension. METHODS AND RESULTS: Rats were implanted with radiotelemetry devices for recording mean arterial pressure. Ang II (angiotensin II) was infused subcutaneously using osmotic mini-pumps to induce hypertension. Another osmotic mini-pump was surgically implanted to infuse CMT-3 intracerebroventricularly. Intracerebroventricular CMT- 3 infusion was also investigated in SHR (spontaneously hypertensive rats). Physiological, pathological, immunohistological parameters, and fecal microbiota were analyzed. Intracerebroventricular CMT-3 significantly inhibited Ang II-induced increases in number of microglia, their activation, and proinflammatory cytokines in the paraventricular nucleus of hypothalamus. Further, intracerebroventricular CMT-3 attenuated increased mean arterial pressure, normalized sympathetic activity, and left ventricular hypertrophy in Ang II rats, as well as in the SHR. Finally, CMT-3 beneficially restored certain gut microbial communities altered by Ang II and attenuated pathological alterations in gut wall. CONCLUSIONS: These observations demonstrate that inhibition of microglial activation alone was sufficient to induce significant antihypertensive effects. This was associated with unique changes in gut microbial communities and profound attenuation of gut pathology. They suggest, for the first time, a link between microglia and certain microbial communities that may have implications for treatment of hypertension.

Revisiting the dengue epidemic of 2011 in Paraguay: molecular epidemiology of dengue virus in the Asuncion metropolitan area.

BACKGROUND: Dengue is one of the most important re-emerging viral diseases and the most common human arthropod-borne viral infection worldwide. Any of the four Dengue virus serotypes (DENV-1 to 4) can cause asymptomatic infections or clinical manifestations that range in severity from a mild, self-limited illness, to a severe disease characterized by a shock syndrome that can lead to death. Paraguay suffers periodic epidemic outbreaks of dengue since 1988 when the DENV-1 was introduced in the country. Epidemics caused by all four serotypes have been reported and the country. Although dengue is endemic in Paraguay, few studies have described the molecular epidemiology of DENV in the country, which is important to understand the local and global spread, as well as the evolution of this pathogen. METHODS: This was a cross-sectional study of a convenience sample. Suspected dengue patients of any age were recruited from the Emergency Laboratory of the Central Hospital of the Institute of Social Welfare, Asuncion, Paraguay, from February to June of 2011. A DENV antigen test was used to confirm the infection. The protein E gene sequences of isolated viruses were sequenced for phylogenetic analysis. RESULTS: Dengue was confirmed in 55.1% of the participants (n = 98/178). The most frequent clinical findings were fever, headache, and myalgia. Identity analyses of the protein E gene sequence of 56 viruses isolated showed the circulation of DENV-1 (n = 45) and DENV-2 (n = 11) in the Asuncion metropolitan area in 2011. Molecular epidemiology analyses suggest that DENV-1 was introduced into Paraguay from Argentina, while the DENV-2 from Brazil, replacing previous virus lineages. CONCLUSIONS: We have analyzed the molecular epidemiology of DENV-1 and DENV-2 isolated in Paraguay in 2011. We found strong evidence that DENV-1 was introduced into Paraguay from Argentina, while the DENV-2 from Brazil, replacing previous virus lineages. Molecular epidemiology studies are of great interest to analyze the dynamic of DENV spread, which are useful for early implementation of containment measures to reduce the risk of explosive epidemics caused by this virus.

SCID genotype and 6-month posttransplant CD4 count predict survival and immune recovery.

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4+ and CD4+CD45RA+ cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4+ cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

A phase I study of panobinostat in children with relapsed and refractory hematologic malignancies.

Background: Panobinostat demonstrates activity against pediatric cancers in vitro. A phase I trial in children with refractory hematologic malignancies was conducted. Study design: The trial evaluated two schedules of oral panobinostat using 3 + 3 dose escalations in 28-day cycles. For children with leukemia, panobinostat was given once daily three days a week each week at 24, 30 and 34 mg/m2/day. For children with lymphoma, panobinostat was given once daily three days a week every other week at 16, 20 and 24 mg/m2/day. Cerebrospinal fluid (CSF) from Day 29 of the first cycle, when available, was evaluated for PK. The study was registered on clinicaltrials.gov (NCT01321346) Results: Twenty-two subjects enrolled with leukemia. Five enrolled at dose level 1, 6 at dose level 2, and 11 at dose level 3. There was one dose limiting toxicity (DLT) in the leukemia arm at dose level 3 (Grade 4 hypertriglyceridemia), but no maximum tolerated dose (MTD) was identified. No subjects required removal from protocol therapy for QTc prolongation. PK studies were available in 11 subjects with similar exposure in children as in adults. Four Day 29 CSF specimens were found to have panobinostat levels below the lower limit of quantification. Five subjects with lymphoma were enrolled and received study drug, and 4 were evaluable for DLT. A DLT was reported (Grade 3 enteritis) on the lymphoma arm. Conclusions: Panobinostat was tolerated in heavily pretreated pediatric subjects. Gastrointestinal effects were observed on this study. There were no cardiac findings. There were no responses.

Hematopoietic stem cell transplantation for CD40 ligand deficiency: Results from an EBMT/ESID-IEWP-SCETIDE-PIDTC study.

BACKGROUND: CD40 ligand (CD40L) deficiency, an X-linked primary immunodeficiency, causes recurrent sinopulmonary, Pneumocystis and Cryptosporidium species infections. Long-term survival with supportive therapy is poor. Currently, the only curative treatment is hematopoietic stem cell transplantation (HSCT). OBJECTIVE: We performed an international collaborative study to improve patients' management, aiming to individualize risk factors and determine optimal HSCT characteristics. METHODS: We retrospectively collected data on 130 patients who underwent HSCT for CD40L deficiency between 1993-2015. We analyzed outcome and variables' relevance with respect to survival and cure. RESULTS: Overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were 78.2%, 58.1%, and 72.3% 5 years after HSCT. Results were better in transplantations performed in 2000 or later and in children less than 10 years old at the time of HSCT. Pre-existing organ damage negatively influenced outcome. Sclerosing cholangitis was the most important risk factor. After 2000, superior OS was achieved with matched donors. Use of myeloablative regimens and HSCT at 2 years or less from diagnosis associated with higher OS and DFS. EFS was best with matched sibling donors, myeloablative conditioning (MAC), and bone marrow-derived stem cells. Most rejections occurred after reduced-intensity or nonmyeloablative conditioning, which associated with poor donor cell engraftment. Mortality occurred mainly early after HSCT, predominantly from infections. Among survivors who ceased immunoglobulin replacement, T-lymphocyte chimerism was 50% or greater donor in 85.2%. CONCLUSION: HSCT is curative in patients with CD40L deficiency, with improved outcome if performed before organ damage development. MAC is associated with better OS, EFS, and DFS. Prospective studies are required to compare the risks of HSCT with those of lifelong supportive therapy.

CCR2 Plays a Protective Role in Rocio Virus-Induced Encephalitis by Promoting Macrophage Infiltration Into the Brain.

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

Outcomes after bloodstream infection in hospitalized pediatric hematology/oncology and stem cell transplant patients.

BACKGROUND: Pediatric hematology/oncology (PHO) patients receiving therapy or undergoing hematopoietic stem cell transplantation (HSCT) often require a central line and are at risk for bloodstream infections (BSI). There are limited data describing outcomes of BSI in PHO and HSCT patients. METHODS: This is a multicenter (n = 17) retrospective analysis of outcomes of patients who developed a BSI. Centers involved participated in a quality improvement collaborative referred to as the Childhood Cancer and Blood Disorder Network within the Children's Hospital Association. The main outcome measures were all-cause mortality at 3, 10, and 30 days after positive culture date; transfer to the intensive care unit (ICU) within 48 hours of positive culture; and central line removal within seven days of the positive blood culture. RESULTS: Nine hundred fifty-seven BSI were included in the analysis. Three hundred fifty-four BSI (37%) were associated with at least one adverse outcome. All-cause mortality was 1% (n = 9), 3% (n = 26), and 6% (n = 57) at 3, 10, and 30 days after BSI, respectively. In the 165 BSI (17%) associated with admission to the ICU, the median ICU stay was four days (IQR 2-10). Twenty-one percent of all infections (n = 203) were associated with central line removal within seven days of positive blood culture. CONCLUSIONS: BSI in PHO and HSCT patients are associated with adverse outcomes. These data will assist in defining the impact of BSI in this population and demonstrate the need for quality improvement and research efforts to decrease them.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Full genome sequence of Rocio virus reveal substantial variations from the prototype Rocio virus SPH 34675 sequence.

Rocio virus (ROCV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae. We present an updated sequence of ROCV strain SPH 34675 (GenBank: AY632542.4), the only available full genome sequence prior to this study. Using next-generation sequencing of the entire genome, we reveal substantial sequence variation from the prototype sequence, with 30 nucleotide differences amounting to 14 amino acid changes, as well as significant changes to predicted 3'UTR RNA structures. Our results present an updated and corrected sequence of a potential emerging human-virulent flavivirus uniquely indigenous to Brazil (GenBank: MF461639).