Ecological succession in the vaginal microbiota during pregnancy and birth.

The mother's vaginal microbiota represents the first microbes to which a child is exposed when delivered vaginally. However, little is known about the composition and development of the vaginal microbiota during pregnancy and birth. Here, we analyzed the vaginal microbiota of 57 women in pregnancy week 24, 36 and at birth after rupture of membranes but before delivery, and further compared the composition with that of the gut and airways of the 1-week-old child. The vaginal community structure had dramatic changes in bacterial diversity and taxonomic distribution, yet carried an individual-specific signature. The relative abundance of most bacterial taxa increased stepwise from week 24 of pregnancy until birth, with a gradual decline of Lactobacillus. Mother-to-child vertical transfer, as suggested by sharing, was modest, with the strongest transfer being for Clostridiales followed by Lactobacillales and Enterobacteriales. In conclusion, late gestation is associated with an increase in maternal vaginal microbiota diversity, and vaginal bacteria at birth only modestly predict the composition of the neonatal microbiota.

Does software optimization influence the radiologists' perception in low dose paediatric pelvic examinations?

PURPOSE: To investigate whether software optimisation can improve an observers' perception of image quality in low dose paediatric pelvic examinations. METHODS: Twenty-five consecutive patients (3-7 years old) were referred for a pelvic digital radiography (DR) examination. They were prospectively enrolled in the study over a 3-month period. Images were taken at 80 kV and 2-4 mAs depending on pelvic thickness (9-15 cm). A small focal spot, 130 cm SID: 10 cm air gap and 1 mm Al and 0.2 mm Cu additional filtration were also utilised. Images were acquired on a Canon DR detector and optimised using five different combinations of the multi-frequency processing software (Canon DR system version NE, Version 7.1 with SPECTRA) to comply with the ALARA principle. Image quality was blindly evaluated using the subjective Visual Grading Analysis (VGA) by five experienced musculoskeletal radiologists, including the evaluation of six anatomical image quality criteria (scored from 1 to 5). RESULTS: Consistently, the VGA results indicated that by using software optimised parameters, image quality was suitable for diagnosis in 48-71% of all images. Based on a VGC analysis all software optimised images did have significant better image quality then the one with just the clinical settings. Noise reduction was the software setting which influenced the image quality the most, area under the curve (AUC) of 0.8172 95%CI 0.7953-0.8375. CONCLUSION: Software optimisation improve the radiologists' perception of image quality and should thus be thoroughly considered within clinical practise. Noise reduction is the software parameter which has the greatest influence.

The microbiome of tunnels in hidradenitis suppurativa patients.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease defined by recurrent nodules, tunnels and scarring involving the intertriginous regions. Recent next-generation sequencing (NGS) studies suggest genera such as Prevotella spp., Peptoniphilus spp. and Porphyromonas spp. are associated with chronic and early HS lesions. However, a systematic investigation of the bacterial microbiome in HS tunnels remains unexplored using NGS. OBJECTIVE: We aimed to investigate the bacterial composition of the luminal white gelatinous material found in HS tunnels using NGS. METHODS: An exploratory study of patients with diagnosis of HS (n = 32) with tunnels. The tunnels were present either in the groin (n = 17) or in the axilla (n = 15). During deroofing of the tunnels, a sterile E-swab was taken of the luminal gelatinous material. The samples were investigated using NGS targeting 16S ribosomal RNA. RESULTS: The skin microbiome was characterized in 32 HS patients. Overall, five microbiome types were identified: Porphyromonas spp. (type I), Corynebacterium spp., (type II), Staphylococcus spp. (type III), Prevotella spp. (type IV) and Acinetobacter spp (type V). Porphyromonas spp. (type I) and Prevotella spp. (IV) were the most frequent genera found the tunnels. CONCLUSION: This study points to a potential association between the presence of certain anaerobic bacteria (Porphyromonas spp., Prevotella spp.) and HS tunnels. It may be speculated that these two genera are associated with the pathogenesis in HS either as drivers or as biomarkers.

Neonates colonized with pathogenic bacteria in the airways have a low-grade systemic inflammation.

BACKGROUND AND OBJECTIVES: The development of childhood asthma is associated with neonatal colonization with pathogenic bacteria in hypopharynx. Furthermore, established asthma is associated with systemic low-grade inflammation. We here report on the association between neonatal colonization with pathogenic bacteria in hypopharynx and the development of systemic low-grade inflammation. METHODS: Bacterial colonization of the hypopharynx with Moraxella catharralis, Haemophilus influenzae, and/or Streptococcus pneumoniae was assessed in asymptomatic children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000 ) cohort at age 1 month by culturing technique (N = 238) and by quantitative polymerase chain reaction (qPCR) technique (N = 249) and in the COPSAC2010 cohort by culturing at age 1 month (N = 622) and again at age 3 months (N = 613). Systemic low-grade inflammation was determined in both cohorts at age 6 months by measuring plasma levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (lL-6). RESULTS: In both cohorts, bacterial colonization was associated with increased levels of hs-CRP: COPSAC2000 , 1 month culturing (geometric mean ratio of colonized/noncolonized [95% CI]), 1.39 [0.97-2.01], P = .08; 1 month qPCR, 1.55 [1.14-2.10], P < .01; COPSAC2010 , 1 month, 1.52 [1.23-1.87], P < .01; and 3 month, 1.57 [1.30-1.90], P < .01. A multiparametric principal component analysis incorporating hs-CRP, TNF-α, and IL-6 confirmed a systemic inflammatory profile in children colonized with M. catharralis, H. influenzae. and/or S. pneumoniae in the hypopharynx compared to noncolonized children (P-values < .05). CONCLUSION: The composition of the upper airway microbiome in early life may cause systemic low-grade inflammation.

Moderate to severe hidradenitis suppurativa patients do not have an altered bacterial composition in peripheral blood compared to healthy controls.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease defined by recurrent nodules, tunnels and scarring involving the intertriginous skin. Patients with HS often report an array of systemic symptoms such as fatigue and malaise. The aetiology of these symptoms remains unclear. Previously, various bacteria have been associated with mature HS lesions, and bacteraemia has been reported in patients with HS using traditional culturing methods. Thus, we hypothesized that a low-grade bacteraemia contributes to the symptomatology in patients with HS. OBJECTIVE: To explore the potential presence of bacteraemia in patients with HS and healthy controls. METHOD: A case-control study. Compositions of bacteria in the blood of 27 moderate to severe HS patients and 26 healthy controls were investigated using next-generation 16S ribosomal RNA gene sequencing (NGS) and routine anaerobic and aerobic blood culturing. None of the participants received any antibiotics (systemic or topical therapy) within 1 month prior to the study. HS patients with a recent flare were randomly selected by consecutive recruitment of eligible patients from the Department of Dermatology, Zealand University Hospital, Denmark. Healthy controls were recruited from the University of Copenhagen as well as from the healthcare staff. RESULTS: The different bacterial compositions were investigated using NGS and traditional anaerobic and aerobic blood culturing. Our NGS analysis provided a previously unreported characterization of the bacterial composition in peripheral blood from patients with HS and healthy controls. Overall, our data demonstrated that patients with HS do not have a different bacterial composition in their peripheral blood than healthy controls. CONCLUSION: The study suggests that the self-reported symptoms in HS such as malaise and fatigue may not be linked to bacteraemia.

Upregulated baseline plasma CCL19 and CCR7 cell-surface expression on monocytes in early rheumatoid arthritis normalized during treatment and CCL19 correlated with radiographic progression.

OBJECTIVES: The aim of this study was to measure, in early rheumatoid arthritis (RA) patients, the concentration of CC-chemokine ligand 19 (CCL19) in plasma and the cell-surface expression of CC-chemokine receptor 7 (CCR7) on circulating monocytes and CD4+ T lymphocytes and to analyse correlations with disease activity and 5-year radiographic progression. METHOD: In disease-modifying anti-rheumatic drug (DMARD)-naïve RA patients (disease duration < 6 months), we measured plasma CCL19 by enzyme-linked immunosorbent assay (ELISA) (n = 160) and CCR7 cell-surface expression on monocytes and CD4+ T lymphocytes by flow cytometry (n = 40) at baseline and after 1 year of treatment with methotrexate (MTX) or methotrexate+cyclosporin A (MTX/CyA). Radiographic progression was scored by the van der Heijde-modified Total Sharp Score (TSS) from 0 to 5 years. RESULTS: Increased baseline CCL19 (median 85 pg/mL, range 31-1008 pg/mL, p = 0.01) decreased after 1 year (median 31 pg/mL, range 31-1030 pg/mL, p < 0.001) and 5 years (median 31 pg/mL, range 31-247 pg/mL, p < 0.001) to a level below the controls (n = 45) (median 60 pg/mL, range 31-152 pg/mL). Baseline plasma CCL19 levels [p = 0.011, 95% confidence interval (CI) 0.0030-0.0176], anti-cyclic citrullinated peptide (anti-CCP) antibody status (p = 0.002, 95% CI 0.61-2.38), and TSS > 0 at baseline (p < 0.001, 95% CI 1.21-3.16) were independent predictors of 5-year radiographic progression evaluated by multiple logistic regression in contrast to never smoked, C-reactive protein (CRP), gender, age, number of tender (NTJ) and swollen joints (NSJ), and 28-joint Disease Activity Score (DAS28). Increased CCR7 expression on monocytes (p = 0.008) correlated to CRP (p = 0.006, r = 0.52) and normalized (n = 15) after 1 year (p = 0.02). CONCLUSIONS: In DMARD-naïve RA patients, CCL19 plasma level and CCR7 surface expression on monocytes were upregulated and normalized after 1 year of treatment. Increased baseline plasma CCL19 level, anti-CCP antibody status, and TSS > 0 at baseline correlated independently with 5-year radiographic progression.

Up-regulated dipeptidyl-peptidase IV (CD26) on monocytes was unaffected by effective DMARD treatment in early steroid and DMARD-naive rheumatoid arthritis.

OBJECTIVES: To study the CD26 density on monocytes and CD4+ T-lymphocytes in steroid and DMARD-naïve, early rheumatoid arthritis (RA) patients and to analyse for correlations with disease activity, including long-term radiographic progression. METHODS: Forty patients with active, early steroid and DMARD naïve RA (<6 months' duration) were randomised to treatment with methotrexate (MTX) versus MTX and cyclosporine A (CYA). Controls were 15 healthy age and gender matched subjects. Peripheral blood mononuclear cells were analysed for CD26 density by flow cytometry at baseline and after 52 weeks. Radiographic progression was scored by delta total Sharp-van der Heijde score (TSS) from 0 to 5 years. RESULTS: The density of CD26 on monocytes (CD3-CD14+) in RA was up-regulated compared to healthy controls (p<0.0001) and remained unaffected by clinically effective DMARD treatment after 52 weeks. In anti-CCP positive RA patients (n=18) baseline CD26 density on monocytes correlated to 5-year radiographic progression (p=0.008, r=0.60). The density of CD26 did not correlate to DAS28, the swollen or tender joint count or CRP-level at baseline or at year one. The CD26 density on CD4+ T-lymphocytes at week 0 was comparable to healthy controls (p=0.34). CONCLUSIONS: The up-regulated density of CD26 on monocytes in steroid and DMARD naïve active early RA was unaffected by 52 weeks of effective DMARD treatment and correlated to 5-year radiographic progression.

Commonly used reference genes are actively regulated in in vitro stimulated lymphocytes.

Quantitative polymerase chain reaction (Q-PCR) studies of urine sediments have demonstrated an increased expression of cytotoxin genes during episodes of acute rejection of renal allografts. To compensate for differences in initial sample size and cDNA preparation, standard Q-PCR experiments involve normalization to a reference gene. Although stable expression of reference genes is a prerequisite for any Q-PCR analysis, commonly used reference genes have demonstrated a varying expression across tissues and various stimuli. In this study, cellular expression of several reference genes was investigated in a mixed lymphocyte reaction as a model of gene expression during alloreactive T-lymphocyte activation and acute rejection. Gene expression was quantified using Q-PCR, normalized to cell counts obtained by DNA quantification and corrected for cell polyploidy using flow cytometry. Examined reference genes were 18S rRNA, beta-actin (ACTB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1) and peptidylprolyle isomerase B (PPIB). This study also examined two novel T-lymphocyte-specific reference genes: CD3E and CD8B. HMBS and HPRT were 18.8- and 7.4-fold upregulated, respectively, ACTB was 5.3-fold upregulated, PPIB was 3.2-fold upregulated while 18S rRNA remained stably expressed. The T-lymphocyte-specific reference gene CD3E remained stable while CD8B was upregulated 2.3-fold. In conclusion, several commonly used reference genes were actively regulated during alloreactive T-lymphocyte activation. Additionally, we introduce two stable T-lymphocyte-specific reference genes that might be useful in a Q-PCR analysis of T-lymphocyte-specific cytotoxin genes in urine sediments, as they overcome the contribution of reference gene mRNA from cells irrelevant for diagnosis.

A case of anaphylactic shock possibly caused by intravesical Hexvix.

BACKGROUND: We report a case of a 69-year-old Caucasian male who experienced a severe drop in blood pressure and a skin rash after fluorescence cystoscopy and transurethral resection of the prostate. The diagnostic agent used for fluorescence cystoscopy and suspected of causing the anaphylaxis was hexaminolevulinate hydrochloride (Hexvix). INCIDENT: A profound fall in blood pressure associated with atrial fibrillation, the appearance of a rash, vomiting and transient loss of consciousness occurred immediately after surgery. The hypotension persisted for several hours despite intensive treatment. The reaction commenced approximately 5 h after exposure to Hexvix. There was an increase in serum tryptase (almost nine-fold) and a positive skin prick test to undiluted Hexvix. CONCLUSION: The mechanism of the anaphylaxis is uncertain, but is considered likely to be a non-immunoglobulin E-mediated allergic reaction possibly caused by Hexvix. This is the first reported case of a severe allergic reaction after intravesical instillation of Hexvix.

lacZ-neoR transfected glioma cells in syngeneic rats: growth pattern and characterization of the host immune response against cells transplanted inside and outside the CNS.

The rat glioma cell lines BT4C and BT4Cn were stably transfected with the bacterial lacZ-neomycin resistance (neoR) gene construct. Both transfected (BT4ClacZ and BT4CnlacZ) and untransfected cell lines were injected intracerebrally and subcutaneously into rats. Survival time, morphology, growth rate and immunological properties of the tumors were studied. Survival time was significantly prolonged after intracerebral implantation of the transfected cell lines. No similar response was found in nude rats, indicating an immunological response towards the lacZ-neoR-transfected cells in immunocompetent animals. Morphological observations showed that the lacZ-neoR-transfected gliomas were smaller and had a distinct boundary with the normal brain tissue, whereas the parental cell lines revealed a more diffuse growth pattern. Immunostaining showed a higher proportion of immunocompetent cells infiltrating the lacZ-neoR-transfected tumors. After s.c. injection, the lacZ-neoR-transfected BT4C cell line had a prolonged lag phase before assuming a growth rate similar to that of the parental cells. The BT4CnvlacZ tumors initially grew fastest, but then disappeared within 3 weeks. A similar response was observed with mock-transfected tumor cells. A (3)HTdR-incorporation assay on spleen cells from rats transplanted s.c. with BT4CnvlacZ cells showed a 10-fold increase in cell activation as compared with rats with BT4Cn tumors. A humoral response towards the transfected cells was verified by Western-blot analyses.

High-resolution integrated map encompassing the breast cancer loss of heterozygosity region on human chromosome 16q22.1.

Loss of heterozygosity (LOH) on the long arm of human chromosome 16 is a common genetic alteration observed in both invasive ductal and invasive lobular breast carcinomas. We have generated a high-resolution integrated map encompassing the smallest region of LOH overlap within chromosome 16q22.1 (SRO2). Southern hybridization experiments using more than 140 probes resulted in the assembly of 152 bacterial large-insert clones into a 2.8-Mb contig covering SRO2. The structure of the contig was verified by long-range mapping using total human genomic DNA, and the contig orientation was determined by fluorescence in situ hybridization. A total of 68 transcripts have been identified in the map. One of the genes residing within SRO2 is the E-cadherin gene, CDH1, which has previously been shown to be mutated in lobular breast carcinomas, resulting in loss of E-cadherin expression. In most cases of ductal carcinoma, which is the major mammary cancer type, E-cadherin is normally expressed, suggesting that other genes within 16q22.1 are involved in the development of this tumor subtype. The high-resolution map presented in this study provides a valuable resource for identification of tumor suppressor genes expected to be involved in the etiology of breast carcinomas.

Infectious agents in acute respiratory disease in horses in Ontario.

A study of acute respiratory disease in horses in Ontario was undertaken to determine the identity of current causative infectious agents. A nasopharyngeal swab was designed and utilized to maximize isolation of viruses, mycoplasma, and pathogenic bacteria. Serum samples were collected for parallel determination of antibody titers to equine influenza virus type A subtype 1 (H7N7) and subtype 2 (H3N8), equine rhinovirus types 1 and 2, equine herpesvirus type 1, Mycoplasma equirhinius, and Mycoplasma felis. Equine rhinovirus type 2 was recovered from 28/92 horses tested, and equine influenza virus type A, subtype 2, was recovered from 5. The mycoplasma and bacteria isolated were consistent with those commonly associated with nonspecific respiratory diseases in horses, except that Streptococcus pneumoniae capsular type 3 was isolated from 10 horses.

Serologic diagnosis of caprine arthritis-encephalitis by ELISA with two recombinant proteins in a parallel testing format.

A scheme for screening sera for antibodies to caprine arthritis-encephalitis virus (CAEV) was evaluated for its ability to identify positive and negative samples in a population with heterogeneous risk factors, using the criteria of sensitivity, specificity and positive predictive value. Five hundred caprine serum samples were tested using a transmembrane recombinant-based ELISA. Those that gave positive results were considered positive, while those with equivocal or negative results were retested with a core recombinant protein-based ELISA. Equivocal results after the second test were considered indeterminate and retesting is advised. Using this approach, a sensitivity, specificity and positive predictive value of 98.8%, 97.2% and 98.6% were obtained. These values are superior to those obtained by these tests used individually. The high sensitivity, specificity and predictive value of this new scheme of CAEV screening make it an attractive addition to any control or eradication program.

Migratory patterns of lac-z transfected human glioma cells in the rat brain.

Malignant brain tumors are characterized by extensive tumor-cell infiltration into the normal brain tissue. The present work describes the migratory behavior of human glioma cells transplanted into the adult rat brain with the aim of exploiting the extent of active cell migration and passive cell displacement within the central nervous system. To detect every transplanted tumor cell, a stably bacterial beta-galactosidase (lac-z) transfected human glioma cell line was used. To distinguish between an active cell migration process and passive cell displacement, rat brains were also implanted with inert fluorescent polystyrene microspheres and the distribution of tumor cells and microspheres was studied 1 hr and 3 days after implantation. One hour after implantation the tumor cells were strictly localized at the implantation site. However, 3 days after implantation, both tumor cells and microspheres showed an extensive distribution within the brain. Confirming earlier neuropathological and experimental studies, it is shown that the lac-z-transfected glioma cells had the capacity to move within the Virchow-Robin and subarachnoid spaces. However, since fluorescent microspheres were also found in these areas, this spread of tumor cells may be primarily mediated by the extensive cerebrospinal fluid flow that exists within the brain. Three days after implantation, the glioma cells also showed an active migration over the corpus callosum. In comparison, the fluorescent microspheres showed only limited spread along the callosal body. It is concluded that the bacterial lac-z gene can be stably transfected into human glioma cells and, since every tumor cell can be visualized within the brain, this model provides a tool for studying the mechanisms behind tumor-cell invasion of the brain.