T-cell subpopulations αβ and γδ in cord blood of very preterm infants : The influence of intrauterine infection

Open Access: This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedPreterm infants are very susceptible to infections. Immune response mechanisms in this group of patients and factors that influence cord blood mononuclear cell populations remain poorly understood and are considered insufficient. However, competent immune functions of the cord blood mononuclear cells are also described. The aim of this work was to evaluate the T-cell population (CD3+) with its subpopulations bearing T-cell receptor (TCR) αβ or TCR γδ in the cord blood of preterm infants born before 32 weeks of gestation by mothers with or without an intrauterine infection. Being a pilot study, it also aimed at feasibility check and assessment of an expected effect size. The cord blood samples of 46 infants age were subjected to direct immunofluorescent staining with monoclonal antibodies and then analyzed by flow cytometry. The percentage of CD3+ cells in neonates born by mothers with diagnosis of intrauterine infection was significantly lower than in neonates born by mothers without infection (p = 0.005; Mann-Whitney U test). The number of cells did not differ between groups. Infection present in the mother did not have an influence on the TCR αβ or TCR γδ subpopulations. Our study contributes to a better understanding of preterm infants' immune mechanisms, and sets the stage for further investigations.Peer reviewedFinal Published versio

Group B Streptococcus vaccine development: present status and future considerations, with emphasis on perspectives for low and middle income countries.

Globally, group B Streptococcus (GBS) remains the leading cause of sepsis and meningitis in young infants, with its greatest burden in the first 90 days of life. Intrapartum antibiotic prophylaxis (IAP) for women at risk of transmitting GBS to their newborns has been effective in reducing, but not eliminating, the young infant GBS disease burden in many high income countries. However, identification of women at risk and administration of IAP is very difficult in many low and middle income country (LMIC) settings, and is not possible for home deliveries. Immunization of pregnant women with a GBS vaccine represents an alternate pathway to protecting newborns from GBS disease, through the transplacental antibody transfer to the fetus in utero. This approach to prevent GBS disease in young infants is currently under development, and is approaching late stage clinical evaluation. This manuscript includes a review of the natural history of the disease, global disease burden estimates, diagnosis and existing control options in different settings, the biological rationale for a vaccine including previous supportive studies, analysis of current candidates in development, possible correlates of protection and current status of immunogenicity assays. Future potential vaccine development pathways to licensure and use in LMICs, trial design and implementation options are discussed, with the objective to provide a basis for reflection, rather than recommendations

MicroRNA Fingerprints Identify miR-150 as a Plasma Prognostic Marker in Patients with Sepsis

BACKGROUND: The physiopathology of sepsis continues to be poorly understood, and despite recent advances in its management, sepsis is still a life-threatening condition with a poor outcome. If new diagnostic markers related to sepsis pathogenesis will be identified, new specific therapies might be developed and mortality reduced. Small regulatory non-coding RNAs, microRNAs (miRNAs), were recently linked to various diseases; the aim of our prospective study was to identify miRNAs that can differentiate patients with early-stage sepsis from healthy controls and to determine if miRNA levels correlate with the severity assessed by the Sequential Organ Failure Assessment (SOFA) score. METHODOLOGY/PRINCIPAL FINDINGS: By using genome-wide miRNA profiling by microarray in peripheral blood leukocytes, we found that miR-150, miR-182, miR-342-5p, and miR-486 expression profiles differentiated sepsis patients from healthy controls. We also proved by quantitative reverse transcription-polymerase chain reaction that miR-150 levels were significantly reduced in plasma samples of sepsis patients and correlated with the level of disease severity measured by the SOFA score, but were independent of the white blood counts (WBC). We found that plasma levels of tumor necrosis factor alpha, interleukin-10, and interleukin-18, all genes with sequence complementarity to miR-150, were negatively correlated with the plasma levels of this miRNA. Furthermore, we identified that the plasma levels ratio for miR-150/interleukin-18 can be used for assessing the severity of the sepsis. CONCLUSIONS/SIGNIFICANCE: We propose that miR-150 levels in both leukocytes and plasma correlate with the aggressiveness of sepsis and can be used as a marker of early sepsis. Furthermore, we envision miR-150 restoration as a future therapeutic option in sepsis patients

Detection of Streptococcus agalactiae colonization in pregnant women by using combined swab cultures: cross-sectional prevalence study

CONTEXTO E OBJETIVO: Colonização materna por Streptococcus agalactiae e sepse neonatal de início precoce têm despertado interesse na literatura mundial. A doença estreptocócica neonatal está associada com significantes índices de morbidade e mortalidade perinatal, principalmente nos recém-nascidos prematuros. O objetivo deste estudo foi avaliar a prevalência de colonização estreptocócica materna por meio da coleta de swabs combinados, comparada com coleta de swab num único local. TIPO DE ESTUDO E LOCAL: Estudo transversal na Faculdade de Medicina de Botucatu, Universidade Estadual Paulista. MÉTODOS: Foram obtidas amostras de 405 pacientes com idade gestacional entre 35 e 37 semanas. Swabs da região perianal retal, introito vaginal e terço distal da parede vaginal foram cultivados em caldo seletivo de Todd-Hewitt. Colônias sugestivas de Streptococcus agalactiae foram submetidas aos testes de catalase e CAMP (Christie, Atkins, Munch-Petersen). Para avaliação da positividade de cultura de swabs combinados foi empregado o teste de Tukey para comparação de proporções. RESULTADOS: A prevalência de colonização estreptocócica foi de 25,4%. Dentre as pacientes com cultura positiva, 28,1% tiveram positividade em apenas um local de coleta, 24,2% em dois locais simultaneamente e 47,5% nos três locais avaliados. Associação de swabs de dois locais de coleta aumentou significativamente o isolamento estreptocócico, comparado a único swab (P < 0,05), exceto para coleta perianal. (retal) Utilização de swabs combinados de três locais de coleta mostrou taxas de isolamento estatisticamente superiores. CONCLUSÃO: Na cultura de swabs combinados de três locais de coleta houve maior prevalência de colonização materna por Streptococcus agalactiae, comparada com coleta de swabs em um único local.CONTEXT and OBJECTIVE: Maternal Streptococcus agalactiae colonization and early-onset neonatal sepsis have aroused interest in the worldwide literature. Streptococcal neonatal disease is associated with significant morbidity and mortality in the perinatal period, especially among premature neonates. The aim of this study was to assess the prevalence of maternal streptococcal colonization by using combined swab cultures, compared with swab collection from a single site. DESIGN and SETTING: Cross-sectional study at Faculdade de Medicina de Botucatu, Universidade Estadual Paulista. METHODS: Samples were obtained from 405 patients at gestational ages of 35 to 37 weeks. Swabs from the perianal (rectal) region, vaginal introitus and upper lateral vaginal vault were cultured in Todd-Hewitt selective broth. Colonies suggestive of Streptococcus agalactiae were subjected to the catalase and CAMP (Christie, Atkins, Munch-Petersen) tests. To evaluate the positivity of combined swab cultures, Tukey's test was used for comparison of proportions. RESULTS: The prevalence of streptococcal colonization was 25.4%. Among the patients with positive cultures, 28.1% had this at only one collection site, 24.2% simultaneously at two sites and 47.5% at all three sites. Associating the swabs from two collection sites significantly increased streptococcal isolation, compared with a single swab (P < 0.05), except for perianal (rectal) collection. Use of combined swabs from three collection sites showed statistically higher isolation rates. CONCLUSION: In combined swab cultures collected from three collection sites, the prevalence of maternal Streptococcus agalactiae colonization was higher than in swabs collected from a single site.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Estadual Paulista Faculdade de Medicina de Botucatu Department of PathologyUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Institute of BiosciencesUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Department of Gynecology and ObstetricsUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Department of PathologyUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Institute of BiosciencesUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Department of Gynecology and ObstetricsFAPESP: 07/51704-7FAPESP: 06/55307-

Nosocomial infection in a newborn intensive care unit (NICU), South Korea

BACKGROUND: This study aimed to determine the occurrence of nosocomial infections (NIs), including infection rates, main infection sites, and common microorganisms. Patients included in the study were taken from a newborn intensive care unit (NICU), in a hospital in South Korea. METHODS: A retrospective cohort study was performed by reviewing chart. The subjects were 489 neonates who were admitted to the NICU, survived longer than 72 hours, and not transferred to another unit, between Jan. 1. 1995 to Sep. 30, 1999. NIs were identified according to the NNIS definition. Data were analyzed with descriptive statistics. RESULTS: Cumulative incidence rate for NIs was 30.3 neonates out of 100 admissions, with a total of 44.6 infections. The incidence density was average 10.2 neonates and 15.1 infections per 1000 patient days. The most common infections were pneumonia (28%), bloodstream infection (26%), and conjunctivitis (22%). Major pathogens were Gram-positives such as Staphylococcus aureus and coagulase-negative staphylococci. The factors associated with NI was less than 1500 g of birth weight, less than 32 weeks of gestational age, and less than 8 of apgar score. There's no statistical difference in discharge status between two groups, but hospital stay was longer in subjects with nosocomial infection than those without infection. CONCLUSION: Although the distribution of pathogens was similar to previous reports, a high rate of nosocomial infection and in particular conjunctivitis was observed in this study that merits further evaluation

Group B Streptococcal β-Hemolysin/Cytolysin Directly Impairs Cardiomyocyte Viability and Function

BACKGROUND: Group B Streptococcus (GBS) is a leading cause of neonatal sepsis where myocardial dysfunction is an important contributor to poor outcome. Here we study the effects of the GBS pore-forming beta-hemolysin/cytolysin (Bh/c) exotoxin on cardiomyocyte viability, contractility, and calcium transients. METHODOLOGY/PRINCIPAL FINDINGS: HL-1 cardiomyocytes exposed to intact wild-type (WT) or isogenic Deltabeta h/c mutant GBS, or to cell-free extracts from either strain, were assessed for viability by trypan blue exclusion and for apoptosis by TUNEL staining. Functionality of exposed cardiomyocytes was analyzed by visual quantitation of the rate and extent of contractility. Mitochondrial membrane polarization was measured in TMRE-loaded cells exposed to GBS beta h/c. Effects of GBS beta h/c on calcium transients were studied in fura-2AM-loaded primary rat ventricular cardiomyocytes. Exposure of HL-1 cardiomyocytes to either WT GBS or beta h/c extracts significantly reduced both rate and extent of contractility and later induced necrotic and apoptotic cell death. No effects on cardiomyocyte viability or function were observed after treatment with Deltabeta h/c mutant bacteria or extracts. The beta h/c toxin was associated with complete and rapid loss of detectable calcium transients in primary neonatal rat ventricular cardiomyocytes and induced a loss of mitochondrial membrane polarization. These effects on viability and function were abrogated by the beta h/c inhibitor, dipalmitoyl phosphatidylcholine (DPPC). CONCLUSIONS/SIGNIFICANCE: Our data show a rapid loss of cardiomyocyte viability and function induced by GBS beta h/c, and these deleterious effects are inhibited by DPPC, a normal constituent of human pulmonary surfactant.. These findings have clinical implications for the cardiac dysfunction observed in neonatal GBS infections

Predominant constitutive CFTR conductance in small airways

BACKGROUND: The pathological hallmarks of chronic obstructive pulmonary disease (COPD) are inflammation of the small airways (bronchiolitis) and destruction of lung parenchyma (emphysema). These forms of disease arise from chronic prolonged infections, which are usually never present in the normal lung. Despite the fact that primary hygiene and defense of the airways presumably requires a well controlled fluid environment on the surface of the bronchiolar airway, very little is known of the fluid and electrolyte transport properties of airways of less than a few mm diameter. METHODS: We introduce a novel approach to examine some of these properties in a preparation of minimally traumatized porcine bronchioles of about 1 mm diameter by microperfusing the intact bronchiole. RESULTS: In bilateral isotonic NaCl Ringer solutions, the spontaneous transepithelial potential (TEP; lumen to bath) of the bronchiole was small (mean ± sem: -3 ± 1 mV; n = 25), but when gluconate replaced luminal Cl(-), the bionic Cl(- )diffusion potentials (-58 ± 3 mV; n = 25) were as large as -90 mV. TEP diffusion potentials from 2:1 NaCl dilution showed that epithelial Cl(- )permeability was at least 5 times greater than Na(+ )permeability. The anion selectivity sequence was similar to that of CFTR. The bionic TEP became more electronegative with stimulation by luminal forskolin (5 μM)+IBMX (100 μM), ATP (100 μM), or adenosine (100 μM), but not by ionomycin. The TEP was partially inhibited by NPPB (100 μM), GlyH-101* (5–50 μM), and CFTR(Inh)-172* (5 μM). RT-PCR gave identifying products for CFTR, α-, β-, and γ-ENaC and NKCC1. Antibodies to CFTR localized specifically to the epithelial cells lining the lumen of the small airways. CONCLUSION: These results indicate that the small airway of the pig is characterized by a constitutively active Cl(- )conductance that is most likely due to CFTR

Utility of In Vivo Transcription Profiling for Identifying Pseudomonas aeruginosa Genes Needed for Gastrointestinal Colonization and Dissemination

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia

Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation

SalK/SalR, a Two-Component Signal Transduction System, Is Essential for Full Virulence of Highly Invasive Streptococcus suis Serotype 2

BACKGROUND: Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: 89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant DeltasalKR restored its soaring pathogenicity. Colonization experiments showed that the DeltasalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the DeltasalKR mutant. CONCLUSIONS/SIGNIFICANCE: These findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI