Disturbances in the IgG Antibody Profile in HIV-Exposed Uninfected Infants Associated with Maternal Factors.

Over the last 20 years, the incidence of vertical HIV transmission has decreased from 25%-42% to less than 1%. Although there are no signs of infection, the health of HIV-exposed uninfected (HEU) infants is notoriously affected during the first months of life, with opportunistic infections being the most common disease. Some studies have reported effects on the vertical transfer of antibodies, but little is known about the subclass distribution of these antibodies. We proposed to evaluate the total IgG concentration and its subclasses in HIV+ mothers and HEU pairs and to determine which maternal factors condition their levels. In this study, plasma from 69 HEU newborns, their mothers, and 71 control pairs was quantified via immunoassays for each IgG isotype. Furthermore, we followed the antibody profile of HEUs throughout the first year of life. We showed that mothers present an antibody profile characterized by high concentrations of IgG1 and IgG3 but reduced IgG2, and HEU infants are born with an IgG subclass profile similar to that of their maternal pair. Interestingly, this passively transferred profile could remain influenced even during their own antibody production in HEU infants, depending on maternal conditions such as CD4+ T-cell counts and maternal antiretroviral treatment. Our findings indicate that HEU infants exhibit an altered IgG subclass profile influenced by maternal factors, potentially contributing to their increased susceptibility to infections.

Neonatal Antibiotic Treatment Can Affect Stool Pattern and Oral Tolerance in Preterm Infants.

Preterm neonates are at high risk of infectious and inflammatory diseases which require antibiotic treatment. Antibiotics influence neonatal gut microbiome development, and intestinal dysbiosis has been associated with delayed gastrointestinal transit. Neonates who take less time to pass meconium have a better tolerance to enteral feeding. We analyzed the effect of neonatal antibiotic treatment on the stool pattern and oral tolerance in 106 preterm infants < 33 weeks gestational age. Neonates were classified in 3 groups according to neonatal antibiotic (ABT) treatment days: no antibiotics, 3−7 d ABT, and ≥8 d ABT. Preterm infants from the ≥8 d ABT group took longer to pass meconium and to start green and yellow stools, took longer to reach 100 and 150 mL/kg/day, and reached reduced volumes in enteral feeds at day of life 14 and 28 than infants from no ABT and 3−7 d ABT groups. Multiple linear regression models showed that neonatal antibiotic treatment, birth weight, invasive mechanical ventilation, surfactant, enteral feeding start day, neonatal parenteral nutrition, and neonatal fasting days are associated with the stool pattern and oral tolerance in preterm infants.

Impaired T helper cell responses in human immunodeficiency virus-exposed uninfected newborns.

INTRODUCTION: HIV-exposed uninfected (HEU) newborns suffer from higher risks of opportunistic infections during the first months of life compared to HIV-unexposed uninfected (HUU) newborns. Alterations in thymic mass, amounts of T helper (Th) cells, T-cell receptor diversity, and activation markers have been found in HEU newborns, suggesting alterations in T cell ontogeny and differentiation. However, little is known about the ability of these cells to produce specialized Th responses from CD4+ T cells. METHOD: To characterize the Th cell profile, we evaluated the frequency of Th1 (CD183+ CD194- CD196- /CXCR3+ CCR4- CCR6- ), Th2 (CD183- CD194+ CD196- /CXCR3- CCR4+ CCR6- ), Th17 (CD183- CD194+ CD196+ /CXCR3- CCR4+ CCR6+ ), and CD4+ CD25++ blood T-cell phenotypes in 50 HEU and 25 HUU newborns. Early activation markers on CD4+ T cells and the Th cytokine profile produced from mononuclear cells under polyclonal T cell stimulation were also studied. Additionally, we probed the ability of CD4+ T cells to differentiate into interferon (IFN)-γ-producing Th1 CD4+ T cells in vitro. RESULTS: Lower percentages of differentiated Th1 , Th2 , Th17, and CD4+ CD25++ T cells were found in blood from HEU newborns than in blood from HUU newborns. However, polyclonally stimulated Th cells showed a similar ability to express CD69 and CD279 but produced less secreted interleukin (IL)-2 and IL-4. Interestingly, under Th1 differentiation conditions, the percentages of CD4+ IFN-γ+ T cells and soluble IFN-γ were higher in HEU newborns than in HUU newborns. CONCLUSION: HEU neonates are born with reduced proportions of differentiated Th1 /Th2 /Th17 and CD4+ CD25++ T cells, but the intrinsic abilities of CD4+ T cells to acquire a Th1 profile are not affected by the adverse maternal milieu during development.

Estimating Neonatal Necrotizing Enterocolitis Based on Feeding Practices.

(1) Background: The relationship between enteral nutrition and neonatal necrotizing enterocolitis (NEC) among premature neonates is still unclear. The present work was designed to assess the relationship between NEC and feeding strategies compared to control infants. (2) Methods: A retrospective case-control study of premature infants (<35 weeks' gestation) with or without NEC that examined feeding practices and clinical characteristics at birth and 3, 7, and 14-day hospitalization, with a longitudinal and cross-sectional analysis. (3) Results: A total of 100 newborns with NEC diagnosis and 92 neonates without the disease with similar demographic and clinical characteristics were included. The median day of NEC diagnosis was 15 days (Interquartile Range (IQR) 5-25 days). A significantly higher number of neonates that were fasting on days 7 and 14 developed NEC (p < 0.05). In the longitudinal analysis, generalized linear and mixed models were fit to evaluate NEC association with feeding strategies and showed that exclusive mother's own milk (MM) and fortified human milk (FHM) across time were significantly less likely associated with NEC (p < 0.001) and that enteral fasting was positively related with NEC. In the cross-sectional analysis, a binary logistic regression model was fit and predicted 80.7% of NEC cases. MM was also found to correlate with a reduced risk for NEC (OR 0.148, 95% CI 0.044-0.05, p = 0.02), and in particular, on day 14, several factors were related to a decreased odd for NEC, including birth weight, antenatal steroids, and the use of FHM (p < 0.001). (4) Conclusions: MM and FHM were associated with less NEC compared to fasting on days 7 and 14. Feeding practices in Neonatal Intensive Care Units (NICUs) should promote exclusive MM across the two-week critical period as a potential guideline to improve NEC outcome.

Cymerus™ iPSC-MSCs significantly prolong survival in a pre-clinical, humanized mouse model of Graft-vs-host disease.

The immune-mediated tissue destruction of graft-vs-host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM). Administering iPSC-MSCs altered phosphorylation and cellular localization of the T cell-specific kinase, Protein Kinase C theta (PKCθ), attenuated disease severity, and prolonged survival in a humanized mouse model of GvHD. Finally, we evaluated a constellation of pro-inflammatory molecules on circulating PBMCs that correlated closely with disease progression and which may serve as biomarkers to monitor therapeutic response. Altogether, our data suggest Cymerus iPSC-MSCs offer the potential for an off-the-shelf, cell-based therapy to treat GvHD.

Estimation of Neonatal Intestinal Perforation Associated with Necrotizing Enterocolitis by Machine Learning Reveals New Key Factors.

Intestinal perforation (IP) associated with necrotizing enterocolitis (NEC) is one of the leading causes of mortality in premature neonates; with major nutritional and neurodevelopmental sequelae. Since predicting which neonates will develop perforation is still challenging; clinicians might benefit considerably with an early diagnosis tool and the identification of critical factors. The aim of this study was to forecast IP related to NEC and to investigate the predictive quality of variables; based on a machine learning-based technique. The Back-propagation neural network was used to train and test the models with a dataset constructed from medical records of the NICU; with birth and hospitalization maternal and neonatal clinical; feeding and laboratory parameters; as input variables. The outcome of the models was diagnosis: (1) IP associated with NEC; (2) NEC or (3) control (neither IP nor NEC). Models accurately estimated IP with good performances; the regression coefficients between the experimental and predicted data were R² > 0.97. Critical variables for IP prediction were identified: neonatal platelets and neutrophils; orotracheal intubation; birth weight; sex; arterial blood gas parameters (pCO2 and HCO3); gestational age; use of fortifier; patent ductus arteriosus; maternal age and maternal morbidity. These models may allow quality improvement in medical practice.

Enhanced TAT-Cre Protein Transduction for Efficient Gene Recombination in T cells.

Genetic manipulation has increased our understanding of gene function and led to the discovery of new therapeutic targets. Cre/LoxP DNA recombination is widely used for genetic studies in mammalian cells. The direct delivery of Cre recombinase fused to protein transduction domains (PTDs), such as TAT, has been described as a valid alternative to the conditional, site-specific Cre expression in transgenic mice. However, efficiently conveying proteins into live cells, especially primary T cells, remains a major challenge. In this study, we show that one of our recently developed PTDs synthetic mimic greatly enhances the cellular uptake of the TAT-Cre fusion protein, enabling significantly smaller amounts of the protein to be used. We used this technique in primary mouse T cells to successfully delete, ex vivo, two essential genes involved in regulating T cell activation, Notch1 and Rbpjκ. Ex vivo gene deletion resulted in substantial protein reduction, comparable to that obtained in vivo when Cre-expressing Notch1-floxed (MxCre±Notch1fl/fl) mice were treated with polyinosinic-polycytidylic acid (polyl/C), but in considerably less time, and without altering normal cell physiology. These results highlight several key advantages that include the ability to use less expensive protein (TAT-Cre), a major reduction in total experimental time and labor, and fewer side effects on the treated cells. This method should offer new opportunities for immunological studies, especially in the context of identifying novel therapeutic targets.

Key Clinical Factors Predicting Adipokine and Oxidative Stress Marker Concentrations among Normal, Overweight and Obese Pregnant Women Using Artificial Neural Networks.

Maternal obesity has been related to adverse neonatal outcomes and fetal programming. Oxidative stress and adipokines are potential biomarkers in such pregnancies; thus, the measurement of these molecules has been considered critical. Therefore, we developed artificial neural network (ANN) models based on maternal weight status and clinical data to predict reliable maternal blood concentrations of these biomarkers at the end of pregnancy. Adipokines (adiponectin, leptin, and resistin), and DNA, lipid and protein oxidative markers (8-oxo-2'-deoxyguanosine, malondialdehyde and carbonylated proteins, respectively) were assessed in blood of normal weight, overweight and obese women in the third trimester of pregnancy. A Back-propagation algorithm was used to train ANN models with four input variables (age, pre-gestational body mass index (p-BMI), weight status and gestational age). ANN models were able to accurately predict all biomarkers with regression coefficients greater than R² = 0.945. P-BMI was the most significant variable for estimating adiponectin and carbonylated proteins concentrations (37%), while gestational age was the most relevant variable to predict resistin and malondialdehyde (34%). Age, gestational age and p-BMI had the same significance for leptin values. Finally, for 8-oxo-2'-deoxyguanosine prediction, the most significant variable was age (37%). These models become relevant to improve clinical and nutrition interventions in prenatal care.

Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling.

We recently reported that maternal antibiotic treatment (MAT) of mice in the last days of pregnancy and during lactation dramatically alters the density and composition of the gastrointestinal microbiota of their infants. MAT infants also exhibited enhanced susceptibility to a systemic viral infection and altered adaptive immune cell activation phenotype and function. CD8+ effector T cells from MAT infants consistently demonstrate an inability to sustain interferon gamma (IFN-γ) production in vivo following vaccinia virus infection and in vitro upon T cell receptor (TCR) stimulation. We hypothesize that T cells developing in infant mice with gastrointestinal microbiota dysbiosis and insufficient toll-like receptor (TLR) exposure alters immune responsiveness associated with intrinsic T cell defects in the TCR signaling pathway and compromised T cell effector function. To evaluate this, splenic T cells from day of life 15 MAT infant mice were stimulated in vitro with anti-CD3 and anti-CD28 antibodies prior to examining the expression of ZAP-70, phosphorylated ZAP-70, phospho-Erk-1/2, c-Rel, total protein tyrosine phosphorylation, and IFN-γ production. We determine that MAT infant CD8+ T cells fail to sustain total protein tyrosine phosphorylation and Erk1/2 activation. Lipopolysaccharide treatment in vitro and in vivo, partially restored IFN-γ production in MAT effector CD8+ T cells and reduced mortality typically observed in MAT mice following systemic viral infection. Our results demonstrate a surprising dependence on the gastrointestinal microbiome and TLR ligand stimulation toward shaping optimal CD8+ T cell function during infancy.

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

Maternal Antibiotic Treatment Impacts Development of the Neonatal Intestinal Microbiome and Antiviral Immunity.

Microbial colonization of the infant gastrointestinal tract (GIT) begins at birth, is shaped by the maternal microbiota, and is profoundly altered by antibiotic treatment. Antibiotic treatment of mothers during pregnancy influences colonization of the GIT microbiota of their infants. The role of the GIT microbiota in regulating adaptive immune function against systemic viral infections during infancy remains undefined. We used a mouse model of perinatal antibiotic exposure to examine the effect of GIT microbial dysbiosis on infant CD8(+) T cell-mediated antiviral immunity. Maternal antibiotic treatment/treated (MAT) during pregnancy and lactation resulted in profound alterations in the composition of the GIT microbiota in mothers and infants. Streptococcus spp. dominated the GIT microbiota of MAT mothers, whereas Enterococcus faecalis predominated within the MAT infant GIT. MAT infant mice subsequently exhibited increased and accelerated mortality following vaccinia virus infection. Ag-specific IFN-γ-producing CD8(+) T cells were reduced in sublethally infected MAT infant mice. MAT CD8(+) T cells from uninfected infant mice also demonstrated a reduced capacity to sustain IFN-γ production following in vitro activation. We additionally determined that control infant mice became more susceptible to infection if they were born in an animal facility using stricter standards of hygiene. These data indicate that undisturbed colonization and progression of the GIT microbiota during infancy are necessary to promote robust adaptive antiviral immune responses.

CXCR4 expression on pathogenic T cells facilitates their bone marrow infiltration in a mouse model of aplastic anemia.

Aplastic anemia (AA) is a disease characterized by T-cell-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells. Physiologically, T cells migrate to the BM in response to chemokines, such as SDF-1α, the ligand for CXCR4. However, how T cells traffic to the BM in AA is poorly understood. CXCR4 is aberrantly expressed in immune-mediated diseases and its regulation by nuclear factor-κB (NF-κB) in cancer models is well documented. In this study, we show that CXCR4 is highly expressed on BM-infiltrating CD4(+) and CD8(+) T cells in a mouse model of AA. Inhibiting CXCR4 in AA mice, using CXCR4(-/-) splenocytes or AMD3100, significantly reduced BM infiltration of T cells. We also report that NF-κB occupancy at the CXCR4 promoter is enhanced in BM-infiltrating CD8(+) T cells of AA mice. Moreover, inhibiting NF-κB signaling in AA mice using Bay11 or dehydroxymethylepoxyquinomicin, or transferring p50(-/-) splenocytes, decreased CXCR4 expression on CD8(+) T cells, significantly reduced BM infiltration of T cells, and strongly attenuated disease symptoms. Remarkably, therapeutic administration of Bay11 significantly extended survival of AA mice. Overall, we demonstrate that CXCR4 mediates migration of pathogenic T cells to the BM in AA mice, and inhibiting NF-κB signaling may represent a novel therapeutic approach to treating AA.

Therapeutic targeting of NOTCH signaling ameliorates immune-mediated bone marrow failure of aplastic anemia.

Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defined. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1(IC) and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infiltrating T cells during active disease. Conditionally deleting Notch1 or administering γ-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH1(IC) was significantly elevated and bound to the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH1(IC) levels, decreased NOTCH1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention.

Human CD4(+) effector T lymphocytes generated upon TCR engagement with self-peptides respond defectively to IL-7 in their transition to memory cells.

The peripheral repertoire of CD4(+) T lymphocytes contains autoreactive cells that remain tolerant through several mechanisms. However, nonspecific CD4(+) T cells can be activated in physiological conditions as in the course of an ongoing immune response, and their outcome is not yet fully understood. Here, we investigate the fate of human naive CD4(+) lymphocytes activated by dendritic cells (DCs) presenting endogenous self-peptides in comparison with lymphocytes involved in alloresponses. We generated memory cells (Tmem) from primary effectors activated with mature autologous DCs plus interleukin (IL)-2 (Tmauto), simulating the circumstances of an active immune response, or allogeneic DCs (Tmallo). Tmem were generated from effector cells that were rested in the absence of antigenic stimuli, with or without IL-7. Tmem were less activated than effectors (demonstrated by CD25 downregulation) particularly with IL-7, suggesting that this cytokine may favour the transition to quiescence. Tmauto and Tmallo showed an effector memory phenotype, and responded similarly to polyclonal and antigen-specific stimuli. Biochemically, IL-7-treated Tmallo were closely related to conventional memory lymphocytes based on Erk-1/2 activation, whereas Tmauto were more similar to effectors. Autologous effectors exhibited lower responses to IL-7 than allogeneic cells, which were reflected in their reduced proliferation and higher cell death. This was not related to IL-7 receptor expression but rather to signalling deficiencies, according to STAT5 activation These results suggest that ineffective responses to IL-7 could impair the transition to memory cells of naive CD4(+) T lymphocytes recognizing self-peptides in the setting of strong costimulation.

Novel protein transduction domain mimics as nonviral delivery vectors for siRNA targeting NOTCH1 in primary human T cells.

RNA interference technology has recently been highlighted as a powerful research method as well as a potential therapeutic treatment for several diseases. However, the delivery of small interfering RNA (siRNA) into T cell lines and primary blood cells is exceedingly challenging, as they are resistant to transfection by conventional reagents. As a result, there is an unmet need for nonviral, efficient, and easily prepared carriers for siRNA delivery into hard-to-transfect cell types. Here, we report a novel system based on protein transduction domain mimics (PTDMs), generated by ring opening metathesis polymerization, for intracellular delivery of siRNA molecules. PTDM-based siRNA delivery induced efficient NOTCH1 knockdown in Jurkat T cells and human peripheral blood mononuclear cells without any measured toxicity. Furthermore, delivering siRNA to NOTCH1 in human peripheral blood cells modulated cell proliferation and differentiation of T cells into T(H)1 cells.

Human CD16+ and CD16- monocyte subsets display unique effector properties in inflammatory conditions in vivo.

Two major subsets of human Mo are identified based on CD14 and CD16 expression: the classical CD16(-) Mo and the minor CD14(+)CD16(+) Mo. In vitro studies suggested distinct function and differentiation potential for each cell population. However, the in vivo relevance of these findings remains unclear. To evaluate the development and function of human Mo in an in vivo model, we transferred both Mo subpopulations into the peritoneum of immunocompromised mice in homeostatic or inflammatory conditions. Inflammation was induced with soluble LPS or particulate zymosan. CD16(+) were more phagocytic and produced higher amounts of TNF and IL-6 than CD16(-) Mo early after transfer with zymosan. They also produced higher levels of ß2-defensin in any condition evaluated, which could represent a new marker for this subpopulation. In contrast, differentiating CD16(-) Mo (24 h after transfer) acquired greater APC capacity in LPS-induced peritonitis, whereas none of the Mo subsets attained this ability with zymosan. CX(3)CL1 supported the survival of both Mo subsets in vivo. Similar Mo subpopulations were present in human peritonitis. These results support the idea of specialized roles of the Mo subset, where CD16(+) might act in an immediate innate immune response, whereas CD16(-) could have a major role as APCs.

Tolerogenic dendritic cells generated with different immunosuppressive cytokines induce antigen-specific anergy and regulatory properties in memory CD4+ T cells.

Dendritic cells (DCs) are professional APCs involved in the initiation of both immunity and immunological tolerance. In autoimmune diseases or graft rejections, most reactive lymphocytes are effector/memory cells. It is believed that memory T cells are more resistant to tolerance induction than naive lymphocytes; however, studies on mechanisms for their efficient tolerization are still scarce. In this study, we generated human monocyte-derived DCs by culture with GM-CSF and IL-4 (control DCs), as well as tolerogenic DCs (tDCs) by adding IL-10, IL-10/TGF-beta1, or IL-10/IL-6. Cells were maturated with TNF-alpha/PGE(2). Compared with control DCs, tDCs had similar expression of HLA-DR, CD80, and CD86, lower expression of CD40, higher levels of macrophage markers, enhanced endocytic ability, increased secretion of IL-6, IL-10 (only tDCs generated with IL-10 and tDCs generated with IL-10/IL-6), and PGE(2), and lower secretion of IL-12 and IL-23. In vitro, tDCs had the capacity to induce anergy in tetanus toxoid-specific memory CD4(+) T cells, whereas the proliferative response to an unrelated Ag was intact. Anergy could be reverted upon exposure to IL-2. tDC-primed T cells have low suppressive ability. Nevertheless, the generation of both anergic and regulatory T cells was more efficient with tDCs generated with IL-10/TGF-beta1. Microarray-based gene expression profiling reflected modulated expression of several transcripts in tDCs. Surface CLIP-HLA-DR complexes and intracellular thrombospondin-1 were increased in the three tDCs. CD39 was highly expressed only in tDC-TGF, which correlated with increased adenosine production. We propose that these molecules, together with IL-10 and prostanoids, are key factors to induce Ag-specific tolerance in memory T cells.