Search

This review seeks to educate clinicians and advocate for patients having acute-onset pediatric autoimmune encephalopathy. Primary care providers caring for children are not fully aware of the debilitating illness that changes the life of a child and a family overnight. Our goal is to heighten awareness of a) the initial diagnosis, b) treatment and c) information about referral of affected children by health professionals in Missouri and surrounding states.

Objective: To evaluate the safety and explore the efficacy of recombinant human lactoferrin (talactoferrin [TLf]) to reduce infection., Study Design: We conducted a randomized, double blind, placebo-controlled trial in infants with birth weight of 750-1500 g. Infants received enteral TLf (n = 60) or placebo (n = 60) on days 1 through 28 of life; the TLf dose was 150 mg/kg every 12 hours. Primary outcomes were bacteremia, pneumonia, urinary tract infection, meningitis, and necrotizing enterocolitis (NEC). Secondary outcomes were sepsis syndrome and suspected NEC. We recorded clinical, laboratory, and radiologic findings, along with diseases and adverse events, in a database used for statistical analyses., Results: Demographic data were similar in the 2 groups of infants. We attributed no enteral or organ-specific adverse events to TLf. There were 2 deaths in the TLf group (1 each due to posterior fossa hemorrhage and postdischarge sudden infant death), and 1 death in the placebo group, due to NEC. The rate of hospital-acquired infections was 50% lower in the TLf group compared with the placebo group (P < .04), including fewer blood or line infections, urinary tract infections, and pneumonia. Fourteen infants in the TLf group weighing <1 kg at birth had no gram-negative infections, compared with only 3 of 14 such infants in the placebo group. Noninfectious outcomes were not statistically significantly different between the 2 groups, and there were no between-group differences in growth or neurodevelopment over a 1-year posthospitalization period., Conclusion: We found no clinical or laboratory toxicity and a trend toward less infectious morbidity in the infants treated with TLf., Trial Registration: ClinicalTrials.gov: NCT00854633., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: The purpose of this study is to evaluate the effects of enteral lactoferrin on the fecal microbiome and contrast those influences with the neonatal intensive care unit (NICU) environment. We theorized that lactoferrin and the NICU habitat shape the fecal microbial composition of very preterm infants. Although functions attributed to lactoferrin include intestinal immune system development and emergence of a healthy gut microbiota, evidence is limited. Twenty-one very low birth weight (VLBW <1500 g) infants received twice-daily talactoferrin (TLf, a drug designation for recombinant human lactoferrin) or its excipient by gastric gavage from day 1-28 of life. Twenty-four-hour fecal samples were collected on day 21 of life and compared with fecal operational taxonomy units (OTUs) in treated and control infants in 2 NICUs. Workflow included fecal DNA isolation, generation of amplicons for the V1-V3 region of bacterial 16S ribosomal RNA, and sequencing of a gel-purified multiplex amplicon library using a Roche 454 GS FLX Titanium (Roche, Branford, Connecticut) platform and protocols. Fecal OTUs per infant were higher in NICU 1 vs NICU 2 (P < .001), consistent with fewer antibiotic days (P < .02) and a shorter duration of parenteral nutrition (P < .007) in NICU 1. Proteobacteria and Firmicutes were the major phyla in infants treated with TLf and placebo. Among Enterobacteriaceae, TLf prophylaxis reduced Enterobacter and Klebsiella, but increased Citrobacter in feces of VLBW infants. Citrobacter caused no neonatal infections in the study population. OTUs for Clostridiaceae increased in NICU 1 among infants treated with TLf. Importantly, OTUs of staphylococci were barely detectable in both NICUs among infants fed TLf. Fewer hospital-acquired infections occurred in infants treated with TLf vs controls, although the reduction was seen mostly in coagulase-negative staphylococci-related bloodstream and central line infections (P = .06). TLf modified the fecal microbiome in VLBW infants, but care practices in the NICU habitat also contributed. Future research must establish whether elimination vs enrichment of gut-related microbiota reduces clinically significant hospital-acquired infections and promotes a healthy commensal microflora in the intestines of VLBW infants., Trial Registration: ClinicalTrials.gov: NCT00854633., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Health policy precludes neonatal vaccination against influenza. Hence, morbidity and mortality are high under 6 months of age. Lactoferrin may activate diminished numbers of dysfunctional dendritic cells and reverse neonatal vaccine failures. Aluminum hydroxide/ALUM recruits neutrophils that secrete lactoferrin at deposition sites of antigen. We theorized lactoferrin + influenza antigen initiates an equivalent antibody response compared to ALUM. Three-day-old mice received subcutaneously 30 μg of H1N1 hemagglutinin + 200 μg of bovine lactoferrin versus hemagglutinin + ALUM. Controls received hemagglutinin, lactoferrin, or ALUM. After 21 days, sera measured anti-H1N1 (ELISA) and neutralizing antibody (plaque assays). ELISA detected equal antibody production with lactoferrin + hemagglutinin compared to hemagglutinin + ALUM; both sera also neutralized H1N1 virus at a 1:20 dilution (p < 0.01). Controls had no anti-H1N1 antibody. Neonates given lactoferrin had no anaphylaxis when challenged four weeks later. Lactoferrin is a safe and effective adjuvant for inducing antibody against influenza in neonates., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Neonatal immunity exhibits weak Th1 but excessive Th2 responses, and the underlying mechanisms remain elusive. In this article, we show that neonatal basophils readily produce IL-4, a cytokine that proved to be pivotal in shaping the programs of both lymphocyte subsets. Besides promoting Th2 programs, IL-4 is captured by the IL-4 heteroreceptor (IL-4Rα/IL-13Rα1) expressed on dendritic cells and instigates IL-12 downregulation. Under these circumstances, differentiating Th1 cells upregulate IL-13Rα1, leading to an unusual expression of the heteroreceptor, which will serve as a death marker for these Th1 cells during rechallenge with Ag. The resulting Th1/Th2 imbalance impacts childhood immunity culminating in sensitivity to allergic reactions, susceptibility to microbial infection and perhaps poor efficacy of pediatric vaccines., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Eosinophils infiltrate intestinal tissue during necrotizing enterocolitis (NEC) and adult bowel diseases. We theorized that epithelial damage causes eosinophilic activation and recruitment at NEC onset., Objective: We studied the relationship between persistent blood eosinophilia and medical or surgical complications during NEC., Methods: NEC cases and controls at MU Children's Hospital (2008-2013) underwent review. A Likert scale measured NEC severity. We utilized an SPSS database for statistical analyses., Results: Of 50 NEC cases, infants in group 1 (n = 15) had eosinophilia <2 days after onset and those in group 2 (n = 25) had NEC but no persistent eosinophilia. Group 3 (n = 46) consisted of controls, i.e. infants without NEC matched for birth weight and gestational age and group 4 (n = 4) of preterm infants with infection and ≤5 days of eosinophilia. Hematologic assessment defined persistent eosinophilia as ≥5% eosinophils for ≥5 days after NEC onset. Absolute eosinophil counts were 2 times higher in group 1 than in group 2 (p = 0.002). The mean duration of eosinophilia was 8 days in group 1 versus 1 day in group 2 (p < 0.001). A Likert score of NEC severity was 3-fold higher in group 1 than in group 2 (p < 0.001). Compared to group 2, group 1 infants were 8 times more likely to have hepatic fibrosis or intestinal strictures., Conclusions: Early persistent blood eosinophilia is not currently a predictor of complications after the onset of NEC. This biomarker identifies immature infants at a high risk for adverse outcomes during NEC convalescence., (© 2015 S. Karger AG, Basel.)

The conceptual framework for a gut-brain axis has existed for decades. The Human Microbiome Project is responsible for establishing intestinal dysbiosis as a mediator of inflammatory bowel disease, obesity, and neurodevelopmental disorders in adults. Recent advances in metagenomics implicate gut microbiota and diet as key modulators of the bidirectional signaling pathways between the gut and brain that underlie neurodevelopmental and psychiatric disorders in adults. Evidence linking intestinal dysbiosis to neurodevelopmental disease outcomes in preterm infants is emerging. Recent clinical studies show that intestinal dysbiosis precedes late-onset neonatal sepsis and necrotizing enterocolitis in intensive care nurseries. Moreover, strong epidemiologic evidence links late-onset neonatal sepsis and necrotizing enterocolitis in long-term psychomotor disabilities of very-low-birth-weight infants. The notion of the gut-brain axis thereby supports that intestinal microbiota can indirectly harm the brain of preterm infants. In this review, we highlight the anatomy and physiology of the gut-brain axis and describe transmission of stress signals caused by immune-microbial dysfunction in the gut. These messengers initiate neurologic disease in preterm infants. Understanding neural and humoral signaling through the gut-brain axis will offer insight into therapeutic and dietary approaches that may improve the outcomes of very-low-birth-weight infants.

Background: Preliminary studies suggest that fish-oil lipid emulsion given parenterally to very preterm infants reduces the severity of retinopathy (ROP) and cholestasis., Methods: Infants weighing <1250 g at birth were randomly allocated to 2 groups: an experimental group of 60 infants that received an intravenous (IV) soybean, olive oil, and fish oil emulsion, and a control group of 70 infants that was given a parenteral soybean and olive oil emulsion. Plasma and erythrocyte concentrations of docosahexaenoic acid (DHA) were determined using a high-performance liquid chromatography-mass spectrometry analysis., Results: Nine infants in the fish oil group required laser therapy for ROP compared with 22 infants in the standard intralipid group (risk ratio [RR], 0.48; 95% confidence interval [CI], 0.24-0.96). Three infants in the fish oil group developed cholestasis compared with 20 infants in the standard intralipid group (RR, 0.18; 95% CI, 0.055-0.56). The mean plasma DHA concentrations in treated infants were 2.9-fold higher in the fish oil group than in control infants on the 7th and 14th days of life. The mean DHA content in erythrocytes of treated infants was 4.5-fold and 2.7-fold higher compared with controls at 7 and 14 days of age., Conclusions: Premature infants receiving an IV fat emulsion containing fish oil had less ROP requiring laser treatment and less cholestasis than those receiving a standard lipid emulsion. These infants also had higher plasma and erythrocyte DHA levels at 7 and 14 days, suggesting potential long-term neurodevelopmental benefits., (© 2013 American Society for Parenteral and Enteral Nutrition.)

Purpose of Review: There is an intense interest among neonatal caregivers as to whether lactoferrin given enterally may reduce the incidence of necrotizing enterocolitis in preterm infants. This review presents scientific and clinical evidence that lactoferrin alleviates or prevents this life-threatening disease., Recent Findings: Preclinical studies in neonatal rats showed that lactoferrin given orally before enteral infection with pathogenic Escherichia coli reduced bacteremia and mortality. A multicentered clinical trial found that very low-birth weight preterm infants given bovine lactoferrin had a significant reduction in late-onset sepsis; there was also a trend towards a diminished incidence of necrotizing enterocolitis. Although multicentered trials of lactoferrin use in preterm infants are near completion, regulatory burdens required to bring lactoferrin to the bedside may limit its availability., Summary: Extremely preterm infants should receive colostrum, a natural lactoferrin concentrate, immediately after birth and, ideally, continue on breast milk throughout the hospital stay. This practice appears well tolerated, but additional experience will tell us whether this practice reduces the prevalence of necrotizing enterocolitis.

In this study, we examined the role IL-13 receptor alpha 1 (IL-13Rα1) plays in macrophage differentiation and function. The findings indicate that IL-13Rα1 is expressed on the M2 but not on the M1 subset of macrophages and specifically heterodimerizes with the IL-4Rα chain to form a type II receptor, which controls the differentiation and function of these cells. Indeed, BM cells from IL-13Rα1(+/+) and IL-13Rα1(-/-) mice yield equivalent numbers of macrophages when cultured under M2 polarizing conditions. However, IL-13Rα1(-/-) BM cells yield a much higher number of macrophages than IL-13Rα1(+/+) BM cells when the differentiation is carried out under M1-polarizing conditions. Further analyses indicated that macrophages that express IL-13Rα1 also display surface markers associated with an M2 phenotype. In addition, the IL-13Rα1(+) macrophages were highly efficient in phagocytizing zymosan bioparticles both in vitro and in vivo, and supported differentiation of naïve T cells to a Th2 phenotype. Finally, when stimulated by IL-13, a cytokine that uses the heteroreceptor, the cells were able to phosphorylate STAT6 efficiently. These previously unrecognized findings indicate that IL-13Rα1 serves as a marker for M2 macrophages and the resulting heteroreceptor influences both their differentiation and function., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this article is to educate neonatal caregivers about metagenomics. This scientific field uses novel and ever changing molecular methods to identify how infants become colonized with microbes after birth. Publications using metagenomics appear infrequently in the neonatal literature because clinicians are unaccustomed to the analytical techniques, data interpretation, and illustration of the results. This review covers those areas. After a brief introduction of neonatal citations forthcoming from metagenomic studies, the following topics are covered: (1) the history of metagenomics, (2) a description of current and emerging instruments used to define microbial populations in human organs, and (3) how extensive databases generated by genome analyzers are examined and presented to readers. Clinicians may feel like they are learning a new language; however, they will appreciate this task is essential to understanding and practicing neonatal medicine in the future., (© 2013 S. Karger AG, Basel.)

Lactoferrin (LF) is a multifunctional protein and a member of the transferrin family. LF and lysozyme in breast milk kill bacteria. In the stomach, pepsin digests and releases a potent peptide antibiotic called lactoferricin from native LF. The antimicrobial characteristics of LF may facilitate a healthy intestinal microbiome. LF is the major whey in human milk; its highest concentration is in colostrum. This fact highlights early feeding of colostrum and also fresh mature milk as a way to prevent necrotizing enterocolitis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Current models of necrotizing enterocolitis (NEC) propose that intraluminal microbes destroy intestinal mucosa and activate an inflammatory cascade that ends in necrosis. We suggest an alternate hypothesis wherein NEC is caused by injury to Paneth cells (PCs) in the intestinal crypts. PCs are specialized epithelia that protect intestinal stem cells from pathogens, stimulate stem cell differentiation, shape the intestinal microbiota, and assist in repairing the gut. Our novel model of NEC uses neonatal mice and ablates PCs followed by enteral infection. We contrast this model with other animal examples of NEC and the clinical disease. Selective destruction of PCs using dithizone likely releases tumor necrosis factor-α and other inflammatory mediators. We propose that this event produces inflammation in the submucosa, generates platelet-activating factor, and induces a coagulopathy. The role of PCs in NEC is consistent with the onset of disease in preterm infants after a period of PC-related maturation, the central role of PCs in crypt-related homeostasis, the anatomic location of pneumatosis intestinalis close to the crypts, and the proximity of PCs to occluded blood vessels that cause coagulation necrosis of the intestinal villi. We offer this hypothesis to promote new thoughts about how NEC occurs and its potential prevention., (Copyright © 2012 S. Karger AG, Basel.)

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Normally, Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and proinflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found that they were significantly decreased compared with age-matched controls. We were able to model this loss in the intestine of postnatal day (P)14-P16 (immature) mice by treating them with the zinc chelator dithizone. Intestines from dithizone-treated animals retained approximately half the number of Paneth cells compared with controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae, we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology that is consistent with other currently used animal models, but this technique is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.

Introduction: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear., Methods: To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF + BIF). All groups were exposed to asphyxia and cold stress., Results: Like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. The expression of lysozyme, secretory phospholipase A(2) (sPLA(2)), pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF group with a high incidence of NEC, as compared with the DF and FF + BIF groups where the disease was attenuated., Discussion: We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut.

Bacterial translocation from the gastrointestinal tract is an important pathway initiating late-onset sepsis and necrotizing enterocolitis in very low-birth-weight infants. The emerging intestinal microbiota, nascent intestinal epithelia, naive immunity, and suboptimal nutrition (lack of breast milk) have roles in facilitating bacterial translocation. Feeding lactoferrin, probiotics, or prebiotics has presented exciting possibilities to prevent bacterial translocation in preterm infants, and clinical trials will identify the most safe and efficacious prevention and treatment strategies., ((c) 2010 Elsevier Inc. All rights reserved.)

Objective: To compare the effect of 2 prebiotic/probiotic products on weight gain, stool microbiota, and stool short-chain fatty acid (SCFA) content of premature infants., Patients and Methods: This randomized, blinded, placebo-controlled trial included 90 premature infants treated with either a dietary supplement containing 2 lactobacillus species plus fructooligosaccharides (CUL, Culturelle, ConAgra, Omaha, NE), a supplement containing several species of lactobacilli and bifidobacteria plus fructooligosaccharides (PBP, ProBioPlus DDS, UAS Laboratories, Eden Prairie, MN), or placebo (a dilute preparation of Pregestamil formula) twice daily for 28 days or until discharge if earlier. The primary outcome was weight gain. Secondary outcomes were stool bacterial analysis by culture and 16S rDNA quantitative polymerase chain reaction and stool SCFA content measured by high performance liquid chromatography., Results: Both prebiotic/probiotic combinations contained more bacterial species than noted on the label. No significant effect on infant growth of either prebiotic/probiotic supplement was observed. By cultures, 64% of infants receiving PBP became colonized with bifidobacteria, compared with 18% of infants receiving CUL and 27% of infants receiving placebo (chi-square, P = 0.064). No differences were noted between groups in colonization rates for lactobacilli, Gram-negative enteric bacteria, or staphylococci. By 16S rDNA polymerase chain reaction analysis, the bifidobacteria content in the stools of the infants receiving PBP was higher than in the infants receiving CUL or placebo (Kruskal-Wallis, P = 0.011). No significant differences in stool SCFA content were detected between groups. No adverse reactions were noted., Conclusions: Infants receiving PBP were more likely to become colonized with bifidobacteria. No significant differences in weight gain or stool SCFA content were detected.

Background: Patent ductus arteriosus (PDA) is frequent and potentially pathologic in preterm infants. A simple bedside tool to screen for ductal patency would assist in the care of extremely low birth weight (ELBW) infants., Objective: To investigate the utility of near-infrared spectroscopy (NIRS) in identifying ELBW infants who would benefit from early echocardiography., Methods: Tissue oxygen saturation (S(t)O(2)) was measured by NIRS in the lungs, brain, skeletal muscle and kidney of 20 ELBW infants. Comparisons were made between the S(t)O(2) in these organs and the need for intervention for a PDA. All studies were performed within the first 4 days of life. Similar measurements were performed following treatment with indomethacin in nine of the patients., Results: The S(t)O(2) of skeletal muscle (left deltoid) and kidney differed between the infants who were treated for PDA and those who were not (p = 0.01 for both). As a screen for a PDA requiring intervention, deltoid S(t)O(2) had sensitivity 77% and specificity 83%, and kidney S(t)O(2) had sensitivity 85% and specificity 83%. Following treatment with indomethacin, the low S(t)O(2) in the deltoid and kidney increased toward the range seen in patients who did not require treatment of a PDA. Inter- and intra-observer variability ranged from minimal to high., Conclusion: This pilot study of a portable NIRS device shows encouraging efficacy in identifying ELBW infants who were likely to benefit from early echocardiography and subsequent intervention to close a PDA. Further study is warranted.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.

Paneth cells are specialized epithelia in the small bowel that secrete antimicrobial proteins. Paneth cells are vital to the innate immunity of the small bowel in adult mammals, but their role during neonatal infection of the small bowel is not well established. Dithizone selectively damages Paneth cells, and when dithizone-treated newborn rats are infected enterally with Escherichia coli, the numbers of E. coli cells in their jejunal and ileal lavage fluid are significantly increased compared to controls. The data support that Paneth cells are necessary for neonatal antibacterial defense.

Amniotic fluid (AF) is a complex substance essential to fetal well-being. This article reviews recent discoveries and the current understanding of the origin and circulation of AF and its nutritive, protective, and diagnostic functions. Future directions for AF research are also discussed.

Enteral nutrition with human milk lowers the incidence of necrotizing enterocolitis in preterm human infants. Lactoferrin, the major whey protein in human milk, has many functions related to host defense against bacterial infection. Here, we hypothesize that lactoferrin also helps terminate bacterial invasion of enterocytes via a detachment-induced apoptosis called anoikis. Death of infected epithelia by anoikis prevents local spread of bacterial pathogens because the bacteria are trapped within the cell. Such infected, apoptotic and sloughed epithelia also cannot infect the lower gastrointestinal tract, and the epithelia exit the body in the stool. Currently, anoikis is a phenomenon related to the renewal of enterocytes, and it is not recognized as an anti-bacterial host defense. We suggest that anoikis of infected enterocytes is a process in which lactoferrin plays an important role. In a pilot study in which neonatal rats were pre-treated with intra-gastric recombinant human lactoferrin, we found evidence of epithelia with anoikis in ileal fluid after enteric infection. This finding was rarely seen in infected neonatal rats without pre-treatment with lactoferrin. Quantitative analysis of intestinal lavage specimens and quantitative stereology of apoptotic epithelia in this model will be required to verify the theory. We propose that oral use of recombinant human lactoferrin might have these hypothesized and other anti-bacterial effects in preterm infants, and hence, this protein might prevent necrotizing enterocolitis in preterm infants who cannot take human milk.

Background and Aims: Extremely preterm human infants have increased susceptibility to small bowel infection. We hypothesized that early colonization of the immature small intestine with Lactobacillus GG (LGG), and use of a recombinant lactoferrin (rhLF) to promote growth of LGG, would enhance gut defenses against enteroinvasive Escherichia coli., Methods: Newborn rat pups were treated with nothing, intra-gastric LGG, or rhLF + LGG on days 3 and 4 of life. Gut colonization by LGG was quantified in lavaged jejunal and ileal fluid and gut wall homogenates on day 5 of life. Separate studies used similarly treated litters of newborn rats that were infected late on day 4 of life with E. coli [10(12) CFU/kg]. Sixteen hours later, the numbers of E. coli were measured in small bowel fluid and gut wall homogenates., Results: Control pups initially had lactic acid bacteria colonize the bowel, but these bacteria were not LGG. Pups treated with LGG or rhLF + LGG had significantly higher numbers of LGG in the ileum versus jejunum. Contrary to our hypothesis, rhLF did not augment LGG colonization. After E. coli-related gut infection, planktonic [lavage fluid] and epithelia-adherent growth [gut wall homogenates] of E. coli in the small bowel were most effectively reduced by pre-treatment with rhLF and LGG (P < .05)., Conclusion: Prophylactic therapy with recombinant human lactoferrin and the probiotic, Lactobacillus GG, act to enhance defenses against invasive E. coli in the nascent small intestine. We suggest that rhLF and LGG are therapeutic agents that may reduce necrotizing enterocolitis and gut-related sepsis in preterm human infants.

Objective: The purpose of this study was to develop a model of chronic intrauterine and fetal infection with Prevotella bivia, an anaerobe of the lower genital tract that is associated often with bacterial vaginosis., Study Design: Thirty timed pregnant New Zealand White rabbits on gestational day 21 were inoculated with P bivia or saline solution in a planned ratio of 4:1 (24 P bivia: 6 saline solution). Rabbits were inoculated 6 cm transcervically with 10(5) to 10(8) colony-forming units/uterine horn of P bivia or with saline solution. Necropsy was scheduled on days 4, 6, or 7 after inoculation. Cultures were collected from blood, uterus, amniotic fluid and fetal brain, lung, and heart. Tissues from placenta, uterus, fetal brain, and lung were evaluated with the histologic inflammation score, with a range of 0 to 13. Amniotic fluid was assayed for tumor necrosis factor-alpha by bioassay. Animals with contamination by other organisms were excluded. Categoric data were evaluated with the use of the Fisher exact test, and continuous data were evaluated with the use of the Wilcoxon rank sum., Results: After the exclusion of 8 animals because of contamination with other organisms, 22 animals were evaluated. Of 3 rabbits with an inoculum of 10(8) P bivia colony-forming units/horn, 2 animals (67%) had fever within 24 hours. These results were not compatible with chronic, subclinical infection. Therefore, 14 does had inocula of 10(5-6) P bivia colony-forming units/horn, with necropsy planned at day 4 (n=5 animals), day 6 (n=3 animals), and day 7 (n=6 animals), and 5 animals were inoculated with saline solution. Animals that had been inoculated with P bivia were significantly more likely to have a positive culture than were those animals that were inoculated with saline solution (64% vs 0%; P<.04). Preterm delivery without fever occurred in 21% of does (3/14 does) that were inoculated with P bivia overall and in 33% of the does (3/9 does) that were followed for 6 to 7 days. No saline-solution inoculated animal had preterm birth. There was an increase in amniotic fluid tumor necrosis factor-alpha levels over time in the P bivia group (P=.12). Histologic inflammation scores were not significantly different between P bivia and saline solution groups., Conclusion: Inoculation with P bivia at 10(5-6) colony-forming units/horn leads to chronic intrauterine and fetal infection that are accompanied by preterm birth in up to 33% of cases. This model may serve to explore the mechanism of preterm birth that is induced by chronic infection with genital tract anaerobes.

Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA-/- knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

Objective: To describe a novel center for perinatal medicine and law in a school of medicine and assess its inaugural presentation of the role of expert witnesses in a mock trial setting., Methods: The center's first program was an obstetrics and gynecology Grand Rounds that staged an abbreviated mock trial. The case summary was read. An attorney then conducted the direct examination of the plaintiff's obstetrics and gynecology and neonatal expert witnesses. The audience acted as the jury and anonymously voted electronically after the direct examination. The plaintiff's attorney then conducted the cross-examination of the defense experts on issues pertaining to possible bias and expert compensation with only a limited inquiry into substantive medical issues. A second vote was taken. A posttrial panel discussion and questionnaire evaluated the importance of this program to medical education., Results: The first vote indicated 86% of the jury decided that negligent obstetric management contributed substantially to the infant's injury, and 82% of the attendees felt that negligent neonatal care was a substantial factor in the infant's injury. After the cross-examination, 63% of the jurors now felt that negligent obstetric management contributed to the brain damage; whereas only 39% of the participants concluded that negligent newborn care was responsible for the injury. During posttrial discussion, the audience suggested that cross-examination of defense experts on issues of bias rather than medical care negatively affected their perception of the examining attorney's case., Conclusion: Analysis of the questionnaire showed that the attendees strongly appreciated experiencing this abbreviated mock trial and indicated that the center was a welcome addition to medical education.

Objective: To evaluate the effects of prenatal cigarette smoke exposure on newborn heart rate following the physiologic challenge of birth., Design: Nonexperimental, comparative., Participants: A convenience sample of 130 full-term, healthy newborns who were born at a suburban medical center., Main Outcome Measures: Cotinine is the major metabolite of nicotine and was measured in venous cord blood. The heart rate was monitored at 1 minute intervals during the first 4 hours of life. Infants were categorized into three groups based on the cotinine level: < 0.05 ng/ml (n = 68), 0.05-6.0 ng/ml (n = 39), and > 6.0 ng/ml (n = 23). These levels corresponded, respectively, to no exposure, passive, and active exposure of the mother to nicotine., Results: A one-way ANOVA was significant for maximum heart rate, F(2, 127) = 9.26, p = .001; range of heart rate, F(2, 127) = 5.4, p = .006; and variance of heart rate, F(2, 127) = 5.24, p = .007. Post hoc multiple comparisons found that newborns with cotinine levels > 6.0 ng/ml differed significantly from infants with cotinine levels < 0.05 ng/ml and 0.05-6.0 ng/ml in maximum heart rate, range of heart rate, and variance of heart rate., Conclusions: These findings suggest that newborns with cotinine levels > 6.0 ng/ml have a limited ability to maximize and vary their heart rate. Cardiac output in the newborn is primarily dependent on heart rate. If unable to maximize cardiac output during times of stress, the newborn is potentially at an increased risk for morbidity and possible mortality.

The 14-kDa Vpr protein of human immunodeficiency virus type 1 (HIV-1) serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest in proliferating T lymphocytes, and the modulation of HIV-1-induced apoptosis. Extracellular Vpr has been detected in the sera and cerebral spinal fluid of HIV-infected patients. However, it is not known whether such forms of Vpr are biologically active. Vpr contains a carboxy-terminal basic amino acid rich segment stretch that is homologous to domains that mediate the energy- and receptor-independent cellular uptake of polypeptides by a process termed protein transduction. Similar functional protein-transducing domains are present in HIV-1 Tat, herpes simplex virus-1 DNA-binding protein VP22, and the Drosophila antennapedia homeotic transcription factor. We now demonstrate effective transduction of biologically active, synthetic Vpr (sVpr) as well as the Vpr-beta-galactosidase fusion protein. However, in contrast to other transducing proteins, Vpr transduction is not enhanced by protein denaturation, and Vpr's carboxy-terminal basic domain alone is not sufficient for its transduction across biological membranes. In contrast, the full-length Vpr protein effectively transduces a broad array of cells, leading to dose-dependent G2 cell-cycle arrest and apoptosis. Addition of Vpr into the extracellular medium also rescues the replication of Vpr-deficient strains of HIV-1 in human macrophage cultures. Native Vpr may thus be optimized for protein transduction, a feature that might enhance and extend the pathological effects of HIV infection.

HIV-1 viral protein R (Vpr) is a small, highly conserved accessory protein encoded by the HIV genome that serves many functions in the viral life cycle. Vpr induces G2 cell cycle arrest, which is thought to indirectly enhance viral replication by increasing transcription from the LTR. Vpr has also been implicated in facilitating infection of nondividing cells, most notably macrophages. Because Vpr is a nucleo-cytoplasmic shuttling protein, its role in enhancing viral replication in macrophages may be mediated through enhanced entry of the HIV preintegration complex through the limiting nuclear pore. Free Vpr is detectable in the serum of patients, and in vitro studies implicate extracellular forms of Vpr as an effector of cellular responses mediated through its ability to transduce through intact cytoplasmic membranes. We review the biologic properties of Vpr, focusing on its mechanism of action, role in HIV replication, and significance for host pathogenesis.

Objective: In a pregnant rabbit model using intracervical inoculation of Escherichia coli with delayed antibiotic therapy, we investigated the rate of positive cultures and histologic inflammation of maternal and fetal compartments and the concentration of tumor necrosis factor-alpha in the amniotic fluid for up to 5 days., Study Design: New Zealand White rabbits at 70% gestation were inoculated intracervically with 10(3) - 10(4) colony-forming units of E coli per uterine horn. At varying intervals after inoculation (0.5 - 4.0 hours), antibiotic therapy was initiated with ampicillin-sulbactam. Primary outcomes were positive cultures and histologic inflammation score. Tumor necrosis factor-alpha levels in the amniotic fluid were determined by bioassay., Results: A total of 60 animals were inoculated with E coli. At the endpoint, uterine cultures were positive more commonly than in the fetus or amniotic fluid (41.8% vs 27.5% vs 17.3%, respectively), which was consistent with an ascending pathway of infection. Inflammation scores were similar in uterus and placenta but lower in fetal lung and absent in fetal brain (2.8 vs 3.1 vs 0.84 vs 0.0, respectively). Comparing the durations of delay in antibiotic administration, we found a significant increase in positive uterine cultures and a significant increase in histologic inflammation score with increasing delay. The proportion of dead pups within a litter was significantly associated with the log of the tumor necrosis factor-alpha concentration in amniotic fluid and the degree of histologic inflammation in the uterus, but not with amniotic fluid or other culture positivity., Conclusion: The administration of therapeutic doses of antibiotic does not consistently eradicate bacteria from the rabbit uterus nor, more importantly, from the fetus and the amniotic fluid. Obtaining a negative amniotic fluid culture does not exclude either infection in the decidua or the fetus or histologic inflammation with tumor necrosis factor-alpha elaboration.

HIV infection of non-dividing cellular targets like macrophages requires successful passage of the viral preintegration complex (PIC) across an intact nuclear envelope. Unique but redundant nuclear import signals reside within the HIV integrase, matrix, and Vpr proteins as well as the 'DNA flap'; these signals appear to facilitate PIC transport through the limiting nuclear pores. We discuss recent studies that have advanced our understanding of this key step in the HIV life cycle.