Jörn-Hendrik Weitkamp

Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles

Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.

Maternal influences on fetal microbial colonization and immune development

While critical for normal development, the exact timing of establishment of the intestinal microbiome is unknown. For example, although preterm labor and birth have been associated with bacterial colonization of the amniotic cavity and fetal membranes for many years, the prevailing dogma of a sterile intrauterine environment during normal term pregnancies has been challenged more recently. While found to be a key contributor of evolution in the animal kingdom, maternal transmission of commensal bacteria may also constitute a critical process during healthy pregnancies in humans with yet unclear developmental importance. Metagenomic sequencing has elucidated a rich placental microbiome in normal term pregnancies likely providing important metabolic and immune contributions to the growing fetus. Conversely, an altered microbial composition during pregnancy may produce aberrant metabolites impairing fetal brain development and life-long neurological outcomes. Here we review the current understanding of microbial colonization at the feto-maternal interface and explain how normal gut colonization drives a balanced neonatal mucosal immune system, while dysbiosis contributes to aberrant immune function early in life and beyond. We discuss how maternal genetics, diet, medications, and probiotics inform the fetal microbiome in preparation for perinatal and postnatal bacterial colonization.

Candida infection in very low birth-weight infants: outcome and nephrotoxicity of treatment with liposomal amphotericin B (AmBisome).

SummarySystemic fungal infection occurs in 2 to 4.5% of very low birth-weight (VLBW) infants (<1,500 g) and may be fatal in 25 to 54%.Candida sp. is the major pathogen and amphotericin B the treatment of choice. To reduce side effects and optimize drug action, a formulation of amphotericin B encapsulated in liposomes (AmBisome®) has been introduced. Data on 21 VLBW infants who received a full course of AmBisome® was collected and its toxic effects with emphasis on nephrotoxicity and hypokalemia assessed. The median gestational age was 25 weeks (range 23–31) with a median birth-weight of 730 g (range 450–1,370). Antifungal therapy was started at a median age of 13 days (range 1–49). The median dose given was 2.6 mg/kg/day (range 1–5), and the median duration of therapy was 28 days (range 11–79), corresponding to a median cumulative dose of 71 mg/kg (range 12–271). Hypokalemia (<3.0 mmol/l) was observed in 30% before, and 15% during AmBisome® treatment. Twenty-one days after the termination of therapy, hypokalemia was not present in any patient. Median maximum daily potassium supplementation did not exceed doses usually recommended for VLBW infants. The median of the maximum creatinine levels before treatment was 121 μmol/l (range 71–221) and fell to 68 μmol/l (range 31–171) during treatment and 46 μmol/l (range 26–62) 21 days after the termination of therapy. All patients treated with AmBisome® eradicated fungi and recovered clinically. AmBisome® showed no certain nephrotoxicity in VLBW infants in this study.

Necrotising enterocolitis is characterised by disrupted immune regulation and diminished mucosal regulatory (FOXP3)/effector (CD4, CD8) T cell ratios

Background Necrotising enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Immaturity of gastrointestinal immune regulation may predispose preterm infants to NEC as FOXP3 T regulatory cells (Treg) are critical for intestinal immune homoeostasis. Objective To investigate the hypothesis that abnormal developmental regulation of lamina propria Treg would define premature infants with NEC. Design Lamina propria mononuclear cell populations from surgically resected ileum from 18 patients with NEC and 30 gestational age-matched non-NEC surgical controls were prospectively isolated. Polychromatic flow cytometry was performed to phenotype and analyse lamina propria T cell populations. The cytokine gene expression profile in NEC tissue was compared with that of non-NEC controls. Results The total number of Treg, CD4, or CD8 T cells in each ileum section was independent of gestational age, age or postmenstrual age and similar between patients with NEC and controls. In contrast, the ratio of Treg to CD4 T cells or Treg to CD8 T cells was significantly lower in NEC ileum than in infants without NEC (medians 2.9% vs 6.6%, p=0.001 and medians 6.6% vs 25.9%, p<0.001, respectively). For any given number of CD4 or CD8 T cells, Treg were, on average, 60% lower in NEC ileum than in controls. NEC tissue cytokine gene expression profiles were characteristic of inhibited Treg development or function. Treg/CD4 and Treg/CD8 ratios recovered between initial resection for NEC and reanastomosis. Conclusion The proportion of lamina propria Treg is significantly reduced in the ileum of premature infants with NEC and may contribute to the excessive inflammatory state of this disease.

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Infant and Adult Human B Cell Responses to Rotavirus Share Common Immunodominant Variable Gene Repertoires

Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (VH and VL) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the VH1 and VH4 families, for example, VH1–46, VH4–31, and VH4–61. This gene segment bias differed markedly from the VH3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant VH segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2–11 mo and those of adults were highly related when compared by VH, D, JH, VL, and JL segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.

Fluconazole prophylaxis for prevention of invasive fungal infections in targeted highest risk preterm infants limits drug exposure

Objective:Previous reports suggest a benefit of fluconazole prophylaxis in extremely low birth weight (ELBW) infants <1000 g. Our aim was to evaluate if limiting fluconazole prophylaxis to targeted highest risk infants effectively prevents invasive fungal infections, has no undesired side effects and limits unnecessary drug exposure.Study Design:This nonrandomized retrospective pre-post intervention study compared two groups of infants: (1) Infants <26 weeks gestation and/or <750 g birth weight, requiring central vascular access and admitted to the Monroe Carell Jr Childrens Hospital at Vanderbilt neonatal intensive care unit (NICU) prior to 5 days of age, who received fluconazole prophylaxis and (2) a matched control group from the year prior to prophylaxis. This target population was selected for fluconazole prophylaxis based on prior infection control data from our institution and a number needed to treat of <15 to prevent one episode of fungemia. Following implementation and integration through the institutions computerized physician order entry (CPOE) system, provider adherence to the protocol was assessed during the prophylaxis period.Result:A total of 86 patients were included in the study, 44 in the no-prophylaxis group and 42 in the prophylaxis group. In the targeted prophylaxis group, no invasive fungal infections were observed as compared to nine infants with invasive infections in the no-prophylaxis group (P=0.004). No significant adverse effects were recorded. Targeting the highest risk infants reduced the number of infants <1000 g requiring prophylaxis from 80 to 42 (48% reduction) with no preventable infection missed. Provider compliance was 91% following implementation of this protocol through the CPOE system using a standardized order set.Conclusion:Targeting the highest risk infants for fluconazole prophylaxis through CPOE can effectively prevent invasive fungal infections and limit drug exposure with no unwanted side effects.

VH1–46 Is the Dominant Immunoglobulin Heavy Chain Gene Segment in Rotavirus-Specific Memory B Cells Expressing the Intestinal Homing Receptor α4β7

Memory B cells expressing the intestinal homing marker α4β7 are important for protective immunity against human rotavirus (RV). It is not known whether the B cell repertoire of intestinal homing B cells differs from B cells of the systemic compartment. In this study, we analyzed the RV-specific VH and VL repertoire in human IgD− B cells expressing the intestinal homing marker α4β7. The mean frequency of RV-specific B cells in the systemic compartment of healthy adult subjects was 0.6% (range, 0.2–1.2). The mean frequency of IgD− B cells that were both RV specific and α4β7 was 0.04% (range, 0.01–0.1), and a mean of 10% (range, 1–32) of RV-specific peripheral blood human B cells exhibited an intestinal homing phenotype. We previously demonstrated that VH1–46 is the dominant Ab H chain gene segment in RV-specific systemic B cells from adults and infants. RV-specific systemic IgD− or intestinal homing IgD−/α4β7+ B cells in the current study also used the gene segment VH1–46 at a high frequency, while randomly selected B cells with those phenotypes did not. These data show that VH1–46 is the immunodominant gene segment in human RV-specific effector B cells in both the systemic compartment and in intestinal homing lymphocytes. The mean replacement/silent mutation ratio of systemic compartment IgD− B cells was >2, consistent with a memory phenotype and antigenic selection. Interestingly, RV-specific intestinal homing IgD−/α4β7+ B cells using the VH1–46 gene segment were not mutated, in contrast to systemic RV-specific IgD− B cells.

Abnormal heart rate characteristics before clinical diagnosis of necrotizing enterocolitis

Objective:Earlier diagnosis and treatment of necrotizing enterocolitis (NEC) in preterm infants, before clinical deterioration, might improve outcomes. A monitor that measures abnormal heart rate characteristics (HRC) of decreased variability and transient decelerations was developed as an early warning system for sepsis. As NEC shares pathophysiologic features with sepsis, we tested the hypothesis that abnormal HRC occur before clinical diagnosis of NEC.Study Design:Retrospective review of Bells stage II to III NEC cases among infants <34 weeks gestation enrolled in a prospective randomized clinical trial of HRC monitoring at three neonatal intensive care units.Result:Of 97 infants with NEC and HRC data, 33 underwent surgical intervention within 1 week of diagnosis. The baseline HRC index from 1 to 3 days before diagnosis was higher in patients who developed surgical vs medical NEC (2.06±1.98 vs 1.22±1.10, P=0.009). The HRC index increased significantly 16 h before the clinical diagnosis of surgical NEC and 6 h before medical NEC. At the time of clinical diagnosis, the HRC index was higher in patients with surgical vs medical NEC (3.3±2.2 vs 1.9±1.7, P<0.001).Conclusion:Abnormal HRC occur before clinical diagnosis of NEC, suggesting that continuous HRC monitoring may facilitate earlier detection and treatment.

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.