Escherichia coli pode causar infecção intestinal e extra-intestinal, de origem comunitária ou hospitalar, prevalecendo como agente de infecção do trato urinário (ITU). O objetivo do presente estudo foi caracterizar a produção de <font face=\"Symbol\">b-lactamases de espectro estendido (ESBL), grupos filogenéticos, e a relação clonal em isolados clínicos de E. coli recuperados de pacientes ambulatoriais e internados atendidos em um Hospital Universitário de São Paulo, no período de 2005 a 2007. Seis por cento (34/562) dos isolados de E. coli estudados foram caracterizados como produtores de ESBL, sendo associados exclusivamente a infecções extra-intestinais, tanto nos pacientes ambulatoriais (10/28, 36%) como nos internados (18/28, 64%), dos quais 56% (19/34) foram recuperados de uroculturas. Os isolados produtores de ESBL exibiram um fenótipo multirresistente apresentando um perfil de resistência a ampicilina (100%), cefalotina (100%), cefotaxima (100%), ceftazidima (79%), sulfametoxazol-trimetoprim (62%), gentamicina (56%), ciprofloxacina (50%) e amicacina (6%) e permanecendo suscetíveis ao imipinem. A produção de ESBL esteve associada com a presença de genes do tipo blaCTX-M-2 (94%, 32/34), blaCTX-M-15 (3%, 01/34) e blaCTX-M-1 (3%, 01/34). Entre os isolados produtores de ESBL, os grupos filogenéticos B1 (53%, 18/34) e A (18%, 6/34), de baixa virulência, foram predominantes sobre os grupos filogenéticos, de alta virulência, B2 (12%, 4/34) e D (18%, 6/34). De fato, os genes de virulência pap, cnf1, sfa, hly, e iuc, associados com adesão, invasão e disseminação, não foram identificados. A tipagem genotípica por PFGE (utilizando a enzima Xbal) com posterior análise em dendrograma, identificou a presença de 31 clusters entre os 34 isolados produtores de ESBL. Em resumo, a alta incidência de isolados clonalmente não relacionados, pertencentes aos grupos filogenéticos A e B1, de baixa virulência, sugere que cepas comensais de E. coli podem adquirir genes de resistência do tipo blaCTX-M por transferência horizontal contribuindo no estabelecimento e no prognóstico de infecções extra-intestinais, principalmente do trato urinário. / Escherichia coli can produce both intestinal and extraintestinal community- or nosocomial-acquired infection, being the main agent of urinary tract infection (UTI). The aim of this study was to characterize the extended-spectrum beta-lactamase (ESBL) production, phylogenetic groups, and clonal relationship among E. coli clinical isolates recovered from inpatients and outpatients admmited at a university hospital in São Paulo. During 2005 to 2007, six percents (34/562) E. coli isolates were characterized as ESBL producers, being associated exclusively to extraintestinal infections in both inpatients (10/28, 36%) and outpatients (18/28, 64%), of which 56% (19/34) were recovered from urine cultures. ESBL-producing E. coli exhibited a multidrug-resistant phenotype with a resistance profile to ampicillin (100%), cephalotin (100%), cefotaxime (100%), ceftazidime (79%), sulphamethoxazole-trimethoprim (62%), gentamicin (56%), ciprofloxacin (50%), and amikacin (6%), and remaining susceptible to imipenem. In this regard, ESBL production was associated with the presence of blaCTX-M-2 (94%, 32/34), blaCTX-M-15 (3%,1/34) and blaCTX-M-1 (94%, 32/34) genes. On the other hand, low-virulence phylogenetic groups B1 (53%, 18/34) and A (18%, 6/34) were predominant over high-virulence phylogenetic groups B2 (12%, 4/34) and D (18%, 6/34), among ESBL-producing E. coli isolates studied. In fact, pap, cnf1, sfa, hly, and iuc virulence genes associated with adhesion, invasion and dissemination were not identified. Finally, genotyping by PFGE (using XbaI restriction) with subsequent cluster analysis (dendrogram) revealed the presence of 31 cluster among 34 ESBL-producing E. coli. In summary, the high prevalence of clonally unrelated ESBL-producing E. coli belonging to low-virulence phylogenetic groups A and B1 suggest that comensal E. coli can acquire blaCTX-M-like resistance genes through horizontal gene tranfer, contributing to the establishment and outcome of extraintestinal infections, mainly in the urinary tract.

Quantification of progesterone and 17-β estradiol in mouse serum by liquid chromatography-tandem mass spectrometry Authors: Benjamin Kennard, Allison Cobble, Amy Gravitte, Keleigh Galloway, Jen Kintner, Jennifer Hall, Stacy Brown Introduction: In the United States, Chlamydia trachomatis is a commonly appearing sexually transmitted infection1. It affects the U.S. healthcare system to a tune of about $500 million dollars annually2. In women, it generally appears asymptomatic and can lead to severe secondary complications such as pelvic inflammatory diseases or infertility1. Female sex hormones, estrogen and progesterone, are being identified to have a role in chlamydial infection. Specifically, this study aims to create quantification methods to detect levels of estrogen and progesterone in mice, infected with Chlamydia muridarum, plasma samples. Methods: Progesterone samples were prepared using solid-liquid extraction (SLE+) cartridges with ethyl acetate as the elution solvent. Estradiol samples were prepared using liquid-liquid extraction (LLE) with methyl tert-butyl ether and subsequent derivatization with DMIS. Following sample preparation, hormones were quantified in samples using LC-MS/MS with a gradient elution of 1 mM ammonium fluoride in water and acetonitrile. The separation was achieved using a UCT C18 column (100 x 21.mm, 1.8 μm particle size) maintained at 50oC. The mass spectrometer was set up to isolate molecular ions for progesterone (m/z 315.0910) and derivatized estradiol (m/z 431.1835). Quantification was facilitated by the use of deuterium-labeled internal standards and their corresponding molecular ions in the mass spectrometer (d9-progesterone; m/z 324.1230 and d5-estradiol; m/z 436.2922). Results: Several aspects of the assay presented have been optimized for maximum analyte recovery and analytical sensitivity, including column choice, mobile phase, derivatizing agents for estradiol, and extraction protocols for progesterone. The LC-MS/MS method was investigated for precision and accuracy over three separate days. The dynamic range of the progesterone assay was 5 – 100 ng/mL, with a limit of detection of 1 ng/mL. Likewise, the estradiol assay was linear in the range of 5 – 100 ng/mL, with a limit of detection of 0.5 ng/mL. The average precision, represented by % RSD was 0.74 – 8.5% and 6.3 – 13.4% for progesterone and estradiol, respectively. The accuracy of the method, represented by % error was 1.6 – 14.4% and 4.0 – 10.5% for progesterone and estradiol, respectively. Successful validation was defined as < 15% RSD and error (< 20% at the limit of quantification), per current FDA Guidelines. Conclusions: The developed LC-MS/MS method is specific for progesterone and estradiol, and the extraction is suitable for preparation of mouse serum samples. This assay could be successfully applied to hormone quantification in mouse samples to support the investigation of the link between chlamydia infection and hormone levels in female animals. References 1. Chlamydia - 2017 Sexually Transmitted Diseases Surveillance. https://www.cdc.gov/std/stats17/chlamydia.htm. Accessed October 23, 2018. 2. Owusu-Edusei K, Chesson HW, Gift TL, et al. The Estimated Direct Medical Cost of Selected Sexually Transmitted Infections in the United States, 2008. Sex Transm Dis. 2013;40(3):197-201. doi:10.1097/OLQ.0b013e318285c6d2

Chlamydia trachomatis is the most prevalent agent of bacterial sexually transmitted infections in the world. However, a profuse number of cases are unreported, as the infection is often asymptomatic. Sequelae such as pelvic inflammatory disease, an increased risk of cervical cancer, premature birth, and perinatal infections in pregnant women can occur. Inflammation occurs in the body in response to infection or injury. Although inflammation can lead to some unwanted secondary effects, such as pain, it serves to return the body to homeostasis by restoring injured tissues and eliminating pathogens. One recently identified connection between the central nervous system and the immune system that regulates inflammation is the cholinergic anti-inflammatory pathway (CAP). In the CAP, pathogen-associated molecular patterns stimulate the vagus nerve to activate the pathway, which ultimately results in acetylcholine (ACh) release, which down regulates inflammation. We hypothesized that genital chlamydial infection would increase the expression of choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, in the female murine genital tract, therefore down regulating inflammation and promoting chlamydial infection. Transgenic female mice carrying a ChAT-promoter driven GFP reporter gene were vaginally infected with C. muridarum. Mice were sacrificed on days 3, 9, 15, and 21 post infection; cervical, uterine horn, and ovarian tissues were removed and embedded in paraffin. Small sections of each tissue were cut and mounted onto slides. The tissue sections were then stained for the expression of ChAT using immunohistochemical techniques. Finally, tissue sections were viewed under a microscope for positive staining and the data was analyzed. The results indicated that there is a significant increase in the number of cells that express ChAT in genital tract of chlamydia-infected mice versus non-infected mice.

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...

Chronic kidney disease (CKD) progresses faster in males than females; however, the underlying mechanisms remain poorly understood. Sex differences in glomerular capillary morphology has been hypothesized to contribute, in part, to the increased susceptibility to hypertension-induced renal injury and CKD progression in males, but this has not been investigated. The goal of the present study was to assess glomerular capillary morphology in male vs. female rats with intact kidneys and after uninephrectomy (UNX). We hypothesized that glomerular capillary radii (RCAP) and length (LCAP) would be greater in male rats. Male (n=4) and female (n=4) with intact kidneys and UNX (n=4 males, n=4 females) provided a 0.4% NaCl diet and water ad libitum. Kidneys were perfusion-fixed, the left kidney was excised, and a 3 mm transverse section through the midline of the kidney was selected for further processing. Multiple 1 mm3 cubes were randomly excised from the left, middle, and right regions of the outer cortex, embedded in EPONTM, sectioned (1 μm), and stained with toluidine blue. Four glomeruli from each region were randomly selected for stereological analysis. Glomerular tuft volume (VG), RCAP, and LCAP were assessed. In rats with intact kidneys, no significant sex differences were observed in VG, RCAP, or LCAP. VG, RCAP, and LCAP were significant greater in both male and female rats with UNX vs. respective rats with intact kidneys. In rats with UNX, males exhibited a significantly greater VG and LCAP, but not RCAP, as compared to females despite no significant differences in relative kidney weight. These data indicate that males exhibit greater compensatory increases in LCAP following UNX. The greater capillary length may lead to reduced podocyte density, a well-known mechanism that increases the susceptibility to CKD progression.