Crossreactivity of an Antiserum Directed to the Gram-Negative Bacterium Neisseria gonorrhoeae with the SNARE-Complex Protein Snap23 Correlates to Impaired Exocytosis in SH-SY5Y Cells.

Early maternal infections with Neisseria gonorrhoeae (NG) correlate to an increased lifetime schizophrenia risk for the offspring, which might be due to an immune-mediated mechanism. Here, we investigated the interactions of polyclonal antisera to NG (α-NG) with a first trimester prenatal brain multiprotein array, revealing among others the SNARE-complex protein Snap23 as a target antigen for α-NG. This interaction was confirmed by Western blot analysis with a recombinant Snap23 protein, whereas the closely related Snap25 failed to interact with α-NG. Furthermore, a polyclonal antiserum to the closely related bacterium Neisseria meningitidis (α-NM) failed to interact with both proteins. Functionally, in SH-SY5Y cells, α-NG pretreatment interfered with both insulin-induced vesicle recycling, as revealed by uptake of the fluorescent endocytosis marker FM1-43, and insulin-dependent membrane translocation of the glucose transporter GluT4. Similar effects could be observed for an antiserum raised directly to Snap23, whereas a serum to Snap25 failed to do so. In conclusion, Snap23 seems to be a possible immune target for anti-gonococcal antibodies, the interactions of which seem at least in vitro to interfere with vesicle-associated exocytosis. Whether these changes contribute to the correlation between maternal gonococcal infections and psychosis in vivo remains still to be clarified.

Comparison of clinical and laboratory characteristics during two major paediatric meningitis outbreaks of echovirus 30 and other non-polio enteroviruses in Germany in 2008 and 2013.

Viral meningitis is mainly caused by non-polio enteroviruses (NPEV). Large-scale data on the clinical characteristics between different outbreaks within the same region are lacking. This study aimed to analyse a possible influence of the circulating NPEV genotype on the disease outcome of affected children. A retrospective cohort study analysing two major outbreaks of NPEV meningitis in Germany in 2008 and 2013 was conducted in cooperation with the National Reference Centre for Poliomyelitis and Enteroviruses (NRC PE) and five German children's hospitals. A total of 196 patients with laboratory-confirmed NPEV meningitis were enrolled. In 2008, children with NPEV meningitis had significantly higher fever and showed more behavioural changes and less back pain. To better define typical findings in echovirus 30 (E-30) meningitis, patients were split into the following three groups: E-30 positive patients, patients with "Non E-30" infection and patients with "Untyped" NPEV infection. E-30 positive patients were significantly older and their disease course was more acute, with early admission to but also early discharge from hospital. E-30 positive patients showed a significantly higher rate of headache and meningism, and a lower rate of diarrhoea and clinically defined septicaemia when compared to the others. Regarding laboratory testing, E-30 positive patients presented with significantly elevated peripheral blood neutrophil counts when compared to patients with "Non E-30" or "Untyped" NPEV infection. In conclusion, E-30 meningitis in children shows a characteristic pattern of clinical features. To further characterise NPEV strains worldwide, continuous surveillance and typing of NPEV strains causing central nervous system disease is warranted.

Cross-reactivity of Antibodies Directed to the Gram-Negative Bacterium Neisseria gonorrhoeae With Heat Shock Protein 60 and ATP-Binding Protein Correlates to Reduced Mitochondrial Activity in HIBCPP Choroid Plexus Papilloma Cells.

Antibacterial antibodies can cause neurologic side-effects by cross-reactivity with cellular antigens. Here we investigated interactions of antibodies to Neisseria gonorrhoeae (α-NG) - maternal infections by which increases the offspring's risk for later psychosis-with HIBCPP cells, a cell culture model of choroid plexus epithelium. Immunocytochemistry and Western blotting with α-NG, revealed organelle-like intracellular staining in HIBCPP cells, and labelling of several immunoreactive bands in cellular protein. Two-dimensional Western blotting revealed several immunopositive spots, most prominent of which were identified by mass spectrometry as mitochondrially localized proteins heat shock protein 60 (Hsp60) and ATP-binding protein ß-subunit (ATPB). Similarly α-NG interacted with commercial samples of these proteins as revealed by Western blotting. Three alternative methods (JC-1, Janus green and MTT staining) revealed α-NG to cause in HIBCPP cells a significant decrease in mitochondrial activity, which could be reverted by neuroleptic drugs. Immunoreactivity of α-NG with choroid plexus epithelium in human post mortem samples suggests in vivo relevance of these findings. Finally, distinctly different staining patterns of antibodies against Neisseria meningitidis (α-NM), confirmed antibody specificity. To our knowledge this is the first report that α-NG cross-reactivity with Hsp60 and ATPB impairs mitochondrial activity in choroid plexus epithelial cells, pathogenetic relevance of which needs further clarification.

Prevalence of antibodies against influenza A and B viruses in children in Germany, 2008 to 2010.

The prevalence of influenza A and B virus-specific IgG was determined in sera taken between 2008 and 2010 from 1,665 children aged 0-17 years and 400 blood donors in Germany. ELISA on the basis of whole virus antigens was applied. Nearly all children aged nine years and older had antibodies against influenza A. In contrast, 40% of children aged 0-4 years did not have any influenza A virus-specific IgG antibodies. Eightysix percent of 0-6 year-olds, 47% of 7-12 year-olds and 20% of 13-17 year-olds were serologically naïve to influenza B viruses. By the age of 18 years, influenza B seroprevalence reached approximately 90%. There were obvious regional differences in the seroprevalence of influenza B in Germany. In conclusion, seroprevalences of influenza A and influenza B increase gradually during childhood. The majority of children older than eight years have basal immunity to influenza A, while comparable immunity against influenza B is only acquired at the age of 18 years. Children aged 0-6 years, showing an overall seroprevalence of 67% for influenza A and of 14% for influenza B, are especially at risk for primary infections during influenza B seasons.

Prevalence of antibodies against hepatitis A virus among children and adolescents in Germany.

Since hepatitis A virus (HAV) infection during childhood is mostly asymptomatic, only seroprevalence studies can provide reliable information on incidence of HAV infection in children. The prevalence of anti-HAV antibodies was determined in sera taken in 2008 to 2010 from 1,645 children aged 0-17 years and in sera taken in 2010-2011 from 400 adult blood donors in Germany. For examination of trend over time, 715 sera collected between 1999 and 2006 from children at the age of 0-17 years within the federal state Thuringia were included. Antibody testing was carried out using the test kits ETI-AB-HAVK PLUS and ETI-HA-IGMK PLUS from DiaSorin. In children, the overall prevalence of antibodies was 10.8 %. After the seroprevalence declined from 8.8 % among the 0-2 year-olds to 2.4 % among the 3-4 year-olds, there was a significant increase to 20.5 % in the group of the 15-17 year-olds. Boys had with 12.7 % a significantly higher seroprevalence of anti-HAV antibodies compared to 8.8 % among girls. In adult blood donors, there was a HAV seroprevalence of 19.3 %. The likelihood of past infection or immunization within the age groups of children from 0 to 12 years differed significantly from that of adults. In conclusion, in Germany, only a small number of HAV infections occur in children, especially up to the age of 12 years. The proportion of susceptible children is greater than the proportion of susceptible adults. Thus, during outbreaks, the rate of infection among children would usually be higher than the rate among adults.

Natalizumab treatment during pregnancy - effects on the neonatal immune system.

BACKGROUND: Pregnancies in women with severe relapsing-remitting multiple sclerosis treated with natalizumab constitute a major challenge, because withdrawal of the drug may cause relapses but continuation might have unknown effects on the infantile immune system. AIMS OF THE STUDY: To identify the impact of maternal natalizumab treatment during pregnancy on basic immune functions of the newborn. METHODS: Basic immunological testing and assessment of the chemotaxis rate of freshly isolated T lymphocytes in the presence and absence of CXCL12 was performed in two neonates, whose mothers were treated with natalizumab until the 34th week of pregnancy (pw). RESULTS: Both children had an uneventful birth. However, a reduction in the CXCL12-induced T-cell chemotaxis was found in both children. In contrast, the chemotaxis rate of unstimulated T lymphocytes was not altered. The distribution of the lymphocyte subpopulations was investigated only in case 1 and was normal. CONCLUSIONS: Here, we present to our knowledge the first assessment of T lymphocytes chemotaxis rate in two natalizumab-exposed newborns. A significant reduction in the CXCL12-induced chemotaxis rate of T lymphocytes has been observed and may compromise host defence function in early life. More clinical and immunological data on natalizumab-exposed neonates are warranted.

Clinical characteristics of children with lower respiratory tract infections are dependent on the carriage of specific pathogens in the nasopharynx.

A prospective clinical study was performed to correlate nasopharyngeal carriage of bacteria with the type of lower respiratory tract infections (LRTI) in hospitalised children. To determine bacterial load in nasopharyngeal aspirates (NPA) we used semiquantitative culturing and quantitative TaqMan-PCR for those pathogens difficult to culture. Specimens and clinical data were obtained from 311 children between 0 and 16 years of age with LRTI during the period of 2006-2008. The most common detected potentially pathogenic colonisers were Haemophilus influenzae (32.1 %), Moraxella catharralis (26.7 %), Staphylococcus aureus (17.7 %) and Streptococcus pneumoniae (16.7 %). As expected S. aureus was the most common coloniser in children less than 4 months of age, whereas H. influenzae detection peaked in older children. Co-colonisation with other bacterial pathogens were more often observed in children with S. aureus (46 %) and S. pneumoniae (49 %) than in those with H. influenzae (30 %) or M. catharralis (27 %). Children with S. aureus co-colonisation had higher levels of C-reactive-protein, received antibiotics more frequently and stayed longer in hospital than those with S. aureus single colonisation. In contrast, children with H. influenzae, M. catharralis or S. pneumoniae colonisation suffered more often from pneumonia than children with S. aureus colonisation. Coloniser specific analysis of bacterial quantity revealed no significant reduction of the bacterial carriage from the first to the second NPA. No correlation of a high bacterial load and occurrence of pneumonia could be detected. In conclusion, clinical characteristics in children with LRTIs are associated with a specific bacterial set of colonisers detected in the nasopharynx rather than on their quantity.

Cat scratch disease--heterogeneous in clinical presentation: five unusual cases of an infection caused by Bartonella henselae.

BACKGROUND: Cat-scratch disease (CSD) is common in children, however the wide spectrum of the clinical presentation of CSD may lead to delayed diagnosis. An atypical presentation of CSD includes in its differential diagnosis diseases such as tuberculosis, other mycobacterioses, Epstein-Barr-Virus infection (EBV) or malignant disease. Since, in a small number of cases, these diseases may be present concurrently with an active CSD, it is important to consider CSD early in the differential diagnosis and order the appropriate tests. These tests include serology and, where possible, histology including molecular diagnostic methods on tissue specimens. PATIENTS AND METHOD: We performed a case series of five patients treated in our hospital with a clinical diagnosis of cat-scratch disease, confirmed by serology. An analysis of the history and clinical symptoms associated specifically with an atypical presentation of CSD was performed. RESULTS: The clinical presentation of CSD no longer encompasses the original typical description from 1950, but rather presents with a wide spectrum of signs and symptoms, including the absence of a documented cat scratch, fever, primary lesions or peripheral lymphadenopathy. Low density lesions in spleen, liver and lymph nodes are typical findings in ultrasound, MRI, or CT. Ignoring CSD as a possibility in investigating possible malignancy or tuberculosis could lead to unnecessary hospitalisation and delay in the proper treatment. CONCLUSION: CSD should also be considered in differential diagnosis of any patient with intraabdominal lymphadenopathy, abdominal pain and fever of unknown origin. A careful history is important, however, often patients with CSD have no history of contact with cats. Therefore in atypical cases of CSD the finding of other clinical symptoms and performance of specific diagnostic tests is important. Our experience suggests that early serological testing for Bartonella henselae should be performed and may avoid invasive diagnostic procedures.

Quantitative detection of norovirus excretion in pediatric patients with cancer and prolonged gastroenteritis and shedding of norovirus.

Although chronic courses of norovirus infection have been described in immunocompromised patients, little is known about noroviral shedding and correlation with clinical symptoms in these patients. In this report, the quantitative courses of norovirus excretion in nine pediatric patients with hematologic and oncologic disorders and prolonged gastroenteritis were investigated. In a retrospective study multiple fecal samples from nine pediatric cancer patients were examined by a one-step real-time PCR. Clinical data of the patients were reviewed and virological data were correlated with clinical symptoms. All nine patients presented with prolonged illness and prolonged noroviral shedding. Vomiting and diarrhea were associated with high norovirus concentrations and norovirus excretion declined slowly in the patients. Retrospectively, initial PCR-testing for norovirus was performed with a median of 7 days after onset of symptoms. This finding hints at the difficulty of obtaining early diagnosis of the infection in these children. The patients were shedding high norovirus concentration over a long period of time. Results of sequential quantitative PCR-testing for norovirus correlated with clinical symptoms. Both clinical symptoms and quantitative PCR-testings help to define the severity of norovirus infection and to estimate the risk for transmission. To prevent the spread of the disease, usage of virocidal disinfectants and isolation procedures should be maintained as long as patients are positive for noroviruses. Since vomiting is frequent in pediatric patients with oncological conditions, a screening program for rapid detection of norovirus infection in this group of patients should be considered.

Combined trimethoprim and caspofungin treatment for severe Pneumocystis jiroveci pneumonia in a five year old boy with acute lymphoblastic leukemia.

Caspofungin was used for the first time with trimethoprim/sulfamethoxazole (TMP/SMX) for treatment of high risk Pneumocystis jiroveci pneumonia (PCP) in a pediatric immunocompromised patient. Despite the need for mechanical ventilation, the pediatric patient with relapsed acute lymphoblastic leukemia improved within nine days of treatment and showed no major side effects. The apparent relative lack of toxicity and of pharmacokinetic drug interactions makes caspofungin an attractive agent.

[The vaccination coverage among children aged 24 - 30 months in pediatric offices November 1999 - May 2001. The influence of socio-demographic characteristics]. / Die Durchimpfung von 24 - 30 Monate alten Kindern in pädiatrischen Praxen im Zeitraum von November 1999 bis Mai 2001 - Der Einfluss soziodemografischer Faktoren.

BACKGROUND: The goal of this study was to investigate the influence of socio-demographic factors on vaccination coverage of 24-30 month old children in paediatric practices. METHODS: Vaccination coverage (VC) was documented for 15,682 children in 196 paediatric practices mainly in the region of Nordrhein. Data on socio-demographic factors were collected for 8,457 children and their influence on the vaccination status (VS) was investigated by means of logistic regression analysis. RESULTS: Complete age-appropriate VC for all vaccines recommended by the Standing Committee for Vaccination (STIKO) was 49.9 %. Complete VC for Diphtheria/Tetanus (DT) was 74.6 % and for first dose measles/mumps/rubella (MMR), 82.1 %. The following factors were associated with complete VS for DT and MMR: completion of last recommended well-child visit, parents living together, low number of siblings, short time to reach practice, parents stating they felt adequately informed about vaccinations. Complete vaccination for DT was inversely associated with being health insured through social security and for MMR with the mother having graduated from high school. CONCLUSIONS: A high proportion of 24-30 month old children did not have an age appropriate VS. Targeted parental education and timely vaccination strategies that take into account socio-demographic risk factors for low vaccination uptake are required.

Acute hemorrhagic pericarditis in a child with pneumonia due to Chlamydophila pneumoniae.

Chlamydophila pneumoniae is mainly responsible for respiratory tract infections but has also been associated with endocarditis and myocarditis. We report a case of pneumonia in a child with hemorrhagic pericardial effusion with a positive result by a new C. pneumoniae TaqMan PCR, suggesting a pericardial inflammation directly induced by C. pneumoniae. C. pneumoniae should be suspected in patients with community-acquired pneumonia and concurrent pericarditis. Empirical treatment with azithromycin seems feasible.

Toxic epidermal necrolysis in a premature infant of 27 weeks' gestational age.

Toxic epidermal necrolysis (TEN) is very rare in the newborn period. So far, three cases of TEN in newborns have been reported worldwide. We report a premature infant of 27 weeks' gestational age with TEN at 4 weeks of age. Sepsis treated by an antibiotic combination regimen preceding the TEN was a common feature of all four cases. In our patient, coagulase-negative staphylococci could be identified by blood culture, whereas the previously reported patients suffered from Klebsiella pneumoniae sepsis or Escherichia coli sepsis. Possibly, the uniform association with septic infection in the cases of TEN in the neonatal period might hint at a causal association, thus differentiating it from TEN in older children or adults.

Blood culture sampling rates at a German pediatric university hospital and incidence of invasive pneumococcal disease.

BACKGROUND: Recent pediatric surveillance studies suggest the incidence of pneumococcal bacteremia, but not meningitis, is lower in Germany than in most developed countries. Suboptimal case assessment in routine clinical practice has been suspected of contributing to this apparent discrepancy. METHODS: We aimed to assess the blood culture sampling rate at a German pediatric university hospital and the disease burden associated with pneumococcal bacteremia in children under 5 years of age. The study design was retrospective, based on data-linkage and chart review. RESULTS: Blood cultures were frequently obtained in sepsis (96%; CI 78-99%) and meningitis (95%; CI 77-99%), but less commonly in pneumonia (49%; CI 43-54%) and fever without focus (48%; CI 38-59%). Pneumococci were the most common source of clinically significant bacteremia in previously healthy children. CONCLUSION: These blood culture sampling rates may be insufficient for the sensitive detection of pneumococcal bacteremia. Epidemiological surveillance based on poorly standardized diagnostic practices is prone to under-assessment.

A human flora-associated rat model of the breast-fed infant gut.

OBJECTIVES: Bacterial colonization of the infant gut may have important influences on the development of gastrointestinal, respiratory, and allergic disease. Early diet is a major determinant of the gut microflora. It is very difficult to carry out studies in human infants that can investigate the interaction of diet, flora, and mucosa. In this study we have developed an infant human flora-associated (IHFA) rat model to allow such investigation. METHODS: Germ-free infant rats were infected with fecal bacteria from exclusively breast-fed infants and were maintained on a modified infant formula for 8 weeks. The fecal and cecal contents were collected and compared with feces of breast-fed infants for bacterial populations, bacterial metabolites, and enzymes and for the ability to inhibit adhesion of pathogenic bacteria to human mucosal cells. RESULTS: The IHFA cecum and feces were dominated by lactic acid bacteria, Bifidobacterium, and lactobacilli, which were representative of the infant feces. The fecal short-chain fatty acid profile was dominated by acetic and lactic acid in a similar manner to human infant feces. Other bacterial metabolites were similar to those of the human infant. Rat intestinal samples were able to inhibit the adhesion of pathogens to mucosal cells, but to a lesser extent than the human samples. CONCLUSIONS: This IHFA infant model of the intestinal flora of the breast-fed infant is considered valid for studying the effect of diet on bacterial colonization and metabolism.

Systemic activation of the immune system during ganciclovir treatment following intratumoral herpes simplex virus type 1 thymidine kinase gene transfer in an adolescent ependymoma patient.

During ganciclovir treatment of an adolescent ependymoma patient two weeks after intracranial implantation of HSVtk retroviral vector producer cells, increasing numbers of peripheral T- and B-cells were found as well as enhanced T-cell activation and elevated serum levels of interleukin 12 and soluble Fas ligand. These findings suggest the systemic activation of the immune system during ganciclovir treatment in our patient. The induction of an immune response by HSVtk/ganciclovir supports the concept of an anti-tumor vaccination effect by prodrug activating gene therapy systems and may open new promising perspectives for enhancing therapeutic efficiency by combined prodrug activating and immunological gene therapy strategies.

Restriction of Toxoplasma gondii growth in human brain microvascular endothelial cells by activation of indoleamine 2,3-dioxygenase.

One of the first steps in the development of cerebral toxoplasmosis is the penetration of the blood-brain barrier, which is comprised of microvascular endothelial cells. We examined the capacity of human brain microvascular endothelial cells (HBMEC) to interact with Toxoplasma gondii. We found that stimulation of HBMEC with gamma interferon (IFN-gamma) resulted in the induction of toxoplasmostasis. The capacity of HBMEC to restrict Toxoplasma growth after IFN-gamma stimulation was enhanced in the presence of tumor necrosis factor alpha (TNF-alpha). In addition, we found that IFN-gamma induced a strong induction of indoleamine 2,3-dioxygenase (IDO) activity in HBMEC, and this enzyme activity was enhanced by costimulation with TNF-alpha. The addition of excess amounts of tryptophan to the HBMEC cultures resulted in a complete abrogation of the IFN-gamma-TNF-alpha-mediated toxoplasmostasis. We therefore conclude that IDO induction contributed to the antiparasitic effector mechanism inducible in HBMEC by IFN-gamma and TNF-alpha.

Potential role of human brain microvascular endothelial cells in the pathogenesis of brain abscess: inhibition of Staphylococcus aureus by activation of indoleamine 2,3-dioxygenase.

Cerebral abscess is a rare complication of staphylococcal septicemia in infants associated with high mortality and morbidity. In the pathogenesis of abscess formation, S. aureus, one major causative agent, interacts with endothelial cells of the brain vessels before reaching the central nervous system. This study examined the growth of S. aureus in human brain microvascular endothelial cells (HBMEC) cultures stimulated with cytokines. IFN-gamma inhibited S. aureus replication by the induction of indoleamine 2,3-dioxygenase (IDO) in HBMEC. This activation of IDO in HBMEC could be shown by RT-PCR and by detection of kynurenine in culture supernatants of activated cells. Resupplementation of L-tryptophan abrogated the inhibitory effect of IFN-gamma on the growth of staphylococci, hence confirming the activation of indoleamine 2,3-dioxygenase as being responsible for the induced bacteriostasis. Addition of TNF-alpha enhanced the IFN-gamma mediated antibacterial effects, whereas TNF-alpha alone had no influence on staphylococcal growth. Stimulation of HBMEC with IFN-gamma failed to activate inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO). Thus, intra- and extracellular depletion of L-tryptophan seems to be an important process in the defense against staphylococcal brain abscesses by means of creating an unfavorable microenvironment.

Influence of glutamine and glycyl-glutamine on in vitro lymphocyte proliferation in children with solid tumors.

It is reported that the proliferative response of lymphocytes is lowered in patients with solid tumors. Glutamine is a major nutrient for rapidly proliferating cells. Unlike glutamine itself, the dipeptide glycyl-glutamine as a source for glutamine is stable in aqueous solutions ex vivo. In order to evaluate the possible therapeutic role of glutamine in lymphocyte proliferation, we investigated its influence on lymphocytes of children with solid tumors before and after chemotherapy. Lymphocytes were collected from 21 children and adolescents suffering from solid tumors (before and after chemotherapy) and from healthy controls. Glutamine and glycyl-glutamine, respectively, were added to cell cultures at concentrations between zero and 1.0 mmol/l. ConA or SAC served as T- or B-cell mitogens, respectively. The lymphocyte proliferation in the healthy control group was similar in degree to lymphocyte proliferation seen in the patients with solid tumors, regardless of the mitogen used. No difference in the degree of lymphocyte proliferation before or after chemotherapy was seen with either source of glutamine. Specific subgroups of malignancies showed trends that differed from the overall findings, but these differences were not found to be statistically significant. Routine supplementation with glutamine in children with solid tumors to enhance lymphocyte function is not supported by the data gained from in vitro proliferation tests.