Serologic tests for brucellosis aim to detect antibodies produced against membranous lipopolysaccharide of bacteria. Diagnostic use of this method is limited due to false positiveness. This study evaluates an alternative antigen to lipopolysaccharides (LPS), outer membrane 28-precursor-protein, of Brucella melitensis Rev1 for its diagnostic value. Omp28 precursor of B. melitensis Rev1 was cloned, expressed, and purified. 6-His and sumo epitope tags were used to tag the protein at N-termini. Omp28 gene was amplified based on the ORF sequence and cloned into a pETSUMO vector. The recombinant construct was propagated in Escherichia coli One Shot® Mach1™ cells then transformed into E. coli BL21(D3) cells for protein expression. The purified protein was studied in an indirect ELISA for diagnosis of brucellosis. Sera samples from 60 patients were screened by ELISA and the results were compared to Rose Bengal plate test. Recombinant antigen-based iELISA has given a successful outcome with the sensitivity, specificity, positive predictive value, and negative predictive value of 87.8%, 96.2%, 96.6%, and 78.78%, respectively. In conclusion, recombinant production and purification of the immunodominant Omp28 precursor protein has been achieved successfully in a one-step process with efficient yield and can be used for diagnosis of brucellosis in humans.

Ayaz, Akif; Yalcintepe, Sinem; Ozalp Yuregir, Ozge; Sahin, Yavuz; Ozer, Ahmet; Eser, Metin; Celik, Umit Author Information

In lacrimal system lacerations the canaliculi are involved in many of cases. Canalicular lacerations need urgent primary microsurgical repair. During surgery, it is extremely difficult to find the medial ostium of the canaliculi in severely damaged canaliculi cuts. In this instance, pigtail probe helps surgeon locate the medial portion of the cut canaliculi. In this study, a new technique has been presented for the complicated canalicular injuries. With this new technique the knot will stay in the nasal cavity unexposed, therefore the patients will be more comfortable during their recovery period.

Diabetic patients have increased susceptibility to infections, and urinary tract infections ( UTI) are the most common type in women with diabetes mellitus. Knowledge of bacterial clearance effectiveness following UTI in diabetics is sparse. In this study, the effects of diabetes on bacterial clearance efficiency and components of the innate immune system in response to UTI in a murine model were investigated. Streptozotocin-induced diabetic and control female C57BL/6J mice were infected with uropathogenic Escherichia coli, and bacterial load, expression of chemokines, and neutrophil infiltration in the bladder over time were investigated. Expression levels of histone deacetylases were also measured to address a potential mechanism underlying the phenotype. Bacterial clearance during UTI was significantly prolonged in diabetic mice relative to controls. Neutrophil infiltration in bladder tissue and urine, and both mRNA and protein expression of chemokines MIP-2, KC, MCP-1 and IL-6 in bladder tissue were diminished at early time points after infection in diabetic mice relative to controls. In addition, mRNA levels of histone deacetylases 1-5 were increased in diabetic mice. This is the first study to show an association of impaired bacterial clearance in diabetic mice with suppression of UTI-induced chemokine expression and neutrophil infiltration in the bladder.

Sirtuin1 (SIRT1) is a member of the Sirtuin family of class III histone deacetylases (HDACs) and a longevity factor in mammals initiating the cell survival mechanisms, as well as preventing the ischemic injury in heart. In Heart Failure (HF), an impairment in cell survival mechanism of cardiomyocytes is a notable factor in disease etiopathogenesis. Among many underlining factors, it is well known that oxidative stress comprises a critical impact on cardiomyocyte survival in HF. To date, less scrutiny has been given to the role of SIRT1 in patients with HF. The aim of the present study was to investigate expression levels of SIRT1 gene in compensated and decompensated HF groups, and their correlation with oxidative stress.

Sirtuin-1 (SIRT1) is a longevity factor in mammals initiating the cell survival mechanisms, and preventing ischemic injury in heart. In the etiopathogenesis of heart failure (HF), impairment in cardiomyocyte survival is a notable factor. Oxidative stress comprises a critical impact on cardiomyocyte lifespan in HF. The aim of the present study was to investigate SIRT1 expression in patients with compensated (cHF) and decompensated HF (dHF), and its correlation with oxidative stress. SIRT1 expression in peripheral leukocytes was examined using quantitative RT-PCR in 163 HF patients and 84 controls. Serum total oxidant status (TOS) and total antioxidant status (TAS) were measured via colorimetric assays, and oxidative stress index (OSI) was calculated. Lipid parameters were also determined by routine laboratory methods. SIRT1 mRNA expression was significantly downregulated in HF with more robust decrease in dHF (p=0.002, control vs cHF; p<0.001, control vs dHF). Markedly increased oxidative stress defined as elevated TOS, OSI and low TAS levels were detected in HF patients comparing with the controls (TAS; p=0.010, control vs cHF, p=0.045 control vs dHF, TOS; p=0.004 control vs cHF; p<0.001 control vs dHF, OSI; p<0.001 for both comparisons, respectively). With SIRT1 expression levels, TAS, TOS, OSI, and high density lipoprotein levels in cHF and dHF were determined correlated. SIRT1 expression were significantly reduced in both HF subtypes, particularly in dHF. SIRT1 expression was correlated with the oxidant levels and antioxidant capacity. Data suggest that SIRT1 may have a significant contribution in regulation of oxidant/antioxidant balance in HF etiology and compensation status.

Heart Failure comprises huge medical attention causing distinctive mortality and morbidity especially in westernized societies with a great impact on health care envisioning. The present study aimed to evaluate total antioxidant and oxidant capacities and advanced oxidative protein product levels via well-accepted methods in serum samples of patients with chronic heart failure (CHF). We also aimed to determine apoptosis levels in peripheral blood lymphocytes acquired from adult CHF patients and their the relationships with oxidative stress.

Diabetic individuals have increased susceptibility to urinary tract infection (UTI), a common, painful condition. During diabetes mellitus, non-enzymatic reactions between reducing sugars and protein amine groups result in excessive production of advanced glycation end products (AGEs) that accumulate in tissues. Since bacteria adhere to cell surfaces by binding to carbohydrates, we hypothesized that adherence of bacteria to the bladder in diabetics may be enhanced by accumulation of AGEs on urothelial surface proteins. Using a murine model of UTI, we observed increased adherence of type 1 fimbriated uropathogenic Escherichia coli (UPEC) to the bladder in streptozotocin-induced diabetic female mice compared with age-matched controls, along with increased concentrations of two common AGEs in superficial urothelial cells from diabetic bladders. Several lectins with different specificities exhibited increased binding to urothelial homogenates from diabetic mice compared with controls, and two of those lectins also bound to AGEs. Furthermore, mannose-binding type 1 fimbriae isolated from UPEC bound to different AGEs, and UPEC adherence to the bladder in diabetic mice, were inhibited by pretreatment of mice with the AGE inhibitor pyridoxamine. These results strongly suggest a role for urothelial AGE accumulation in increased bacterial adherence during UTI in diabetes.

The cause of chronic pelvic pain in interstitial cystitis/painful bladder syndrome (IC/PBS) remains unclear; autoimmunity is a possible etiology. We have recently shown that injection of a single immunogenic peptide of uroplakin 3A (UPK3A 65-84) induces experimental autoimmune cystitis (EAC) in female BALB/cJ mice that is unique among experimental models in accurately reflecting both the urinary symptoms and pelvic pain of IC/PBS. The aim of this project was to identify the roles of mast cells and mast cell chemoattractant/activator monocyte chemoattractant protein-1 [chemokine (C-C motif) ligand 2 (CCL2)] in the allodynia in this model. We immunized 6- to 8-wk-old female BALB/cJ mice with UPK3A 65-84 peptide and, 5-40 days later, observed increased responses to stimulation of the suprapubic abdominal and hindpaw surfaces with von Frey monofilaments compared with mice injected with adjuvant alone. Suprapubic and hindpaw tactile allodynia responses by EAC mice were blocked by instillation of lidocaine into the bladder but not by lidocaine in the uterus, confirming the bladder as the source of the hypersensitivity. Markedly increased numbers of activated mast cells and expression of CCL2 were found in the bladder after immunization with UPK3A 65-84. Hypersensitive responses were inhibited by mast cell stabilizer cromolyn sodium and antagonists of histamine receptors 1 and 2. Furthermore, BALB/cJ mice with deletion of the Ccl2 or chemokine (C-C motif) receptor 2 gene exhibited markedly reduced allodynia and accumulation of mast cells after UPK3A 65-84 immunization. These results show that UPK3A 65-84 immunization causes chronic visceral allodynia and suggest that it is mediated by CCL2-driven mast cell accumulation in the bladder.

A case is presented of a 24-year-old woman with acute, painless visual loss that occurred after sneezing. The patient had no previous ocular disease history. Ophthalmic work-up revealed a cilioretinal artery occlusion in the right eye. Transesophageal echocardiography showed a secundum atrial septal defect with right-to-left shunt. Sudden visual loss requires thorough investigation to determine the cause. In this case, examination revealed a retinal artery occlusion, whose risk factors include older age, systemic hypertension, diabetes mellitus, atherosclerosis, and giant cell arteritis (temporal) in more than 75% of patients. Especially in patients younger than 40 years, an embolus of cardiac origin (atrial septal defect, patent foramen ovale, or valvular) should be suspected as a cause of retinal artery occlusion. [ Ophthalmic Surg Lasers Imaging Retina. 2014;45:e30–e31.]

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder (II)1 Apr 20131153 ADVANCED GLYCATION END PRODUCTS ARE ADHERENCE FACILITATORS IN URINARY TRACT INFECTIONS Ahmet Ozer, Cengiz Z. Altuntas, Kenan Izgi, Fuat Bicer, Michael Kavran, and Firouz Daneshgari Ahmet OzerAhmet Ozer Cleveland, OH More articles by this author , Cengiz Z. AltuntasCengiz Z. Altuntas Cleveland, OH More articles by this author , Kenan IzgiKenan Izgi Cleveland, OH More articles by this author , Fuat BicerFuat Bicer Cleveland, OH More articles by this author , Michael KavranMichael Kavran Cleveland, OH More articles by this author , and Firouz DaneshgariFirouz Daneshgari Cleveland, OH More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.788AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Despite well studied functional perturbations of advanced glycation end products (AGEs) in molecular signaling pathways within the cell, no information is available about their possible mechanical role in host-pathogen interactions on epithelial surfaces. Bacteria colonize on cell surfaces predominantly by binding to the carbohydrates, therefore changes in the cell surface carbohydrates due to AGE accumulation might alter susceptibility to infections. Accumulation of AGEs in tissues increases greatly through diabetes mellitus (DM). AGEs on uroepithelial surfaces might contribute to the adherence of uropathogenic E. coli (UPEC) in urinary tract infections (UTI) in DM. METHODS STZ-induced diabetic, female C57BL/6J mice and aged-matched controls served as the main experimental groups. Mice were instilled with UPEC in the bladder lumen to induce UTI, and bacterial adherence was measured by counting colony forming units (CFU) generated from plated bladder homogenates. Following enzymatic harvesting of the upper layers of urothelium, the AGE content of the urothelial surfaces was measured by ELISA. Type 1 pili were purified from UPEC, biotinylated,and assayed for binding to AGEs in vitro by ELISA, using specific AGE products N-ε(carboxymethyl) lysine (CML)-BSA, N-ε(carboxyethyl) lysine (CEL)-BSA, and glucose-AGE-BSA. In vivo bacterial binding assays were completed in diabetic and control mice via transurethral instillation. Study groups were established based on the content of the instilled compounds as follows: UPEC only, UPEC + AGEs, UPEC + D-Mannose, UPEC + BSA, and 1xPBS only. Bladder homogenates were used to quantify bacterial CFU. RESULTS Significant increases in bladders of diabetic mice compared to controls were detected in UPEC adherence and AGE content (p<0.01 and p<0.001, respectively). AGEs showed significantly higher binding affinity in vitro to isolated type 1 pili, a distinct virulence factor on UPEC (p=0.0006 for CML, p=0.005 for CEL, and p=0.001 for glucose-AGE). Pre-incubation of UPEC with AGEs inhibited in vivo binding of UPEC to bladder epithelium significantly and to a similar extent as pre-incubation with D-mannose, a known binding inhibitor for type 1 pili, suggesting that UPEC can bind to AGEs on the urothelial surface. CONCLUSIONS AGE accumulation on bladder urothelium increases through DM, and AGEs may serve as bacterial adherence facilitators in UPEC-UTI. This new functional concept of AGEs can be subject to an extended area of studies addressing host-pathogen interactions and new therapeutic targets in diabetics. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e471 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ahmet Ozer Cleveland, OH More articles by this author Cengiz Z. Altuntas Cleveland, OH More articles by this author Kenan Izgi Cleveland, OH More articles by this author Fuat Bicer Cleveland, OH More articles by this author Michael Kavran Cleveland, OH More articles by this author Firouz Daneshgari Cleveland, OH More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The pathophysiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is enigmatic. Autoimmunity and impaired urothelium might lead the underlying pathology. A major shortcoming in IC/PBS research has been the lack of an appropriate animal model. In this study, we show that the bladder specific uroplakin 3A-derived immunogenic peptide UPK3A 65-84, which contains the binding motif for IA(d) MHC class II molecules expressed in BALB/c mice, is capable of inducing experimental autoimmune cystitis in female mice of that strain. A highly antigen-specific recall proliferative response of lymph node cells to UPK3A 65-84 was observed, characterized by selectively activated CD4+ T cells with a proinflammatory Th1-like phenotype, including enhanced production of interferon γ and interleukin-2. T cell infiltration of the bladder and bladder-specific increased gene expression of inflammatory cytokines were observed. Either active immunization with UPK3A 65-84 or adoptive transfer of peptide-activated CD4+ T cells induced all of the predominant IC/PBS phenotypic characteristics, including increased micturition frequency, decreased urine output per micturition, and increased pelvic pain responses to stimulation with von Frey filaments. Our study demonstrates the creation of a more specific experimental autoimmune cystitis model that is the first inducible model for IC/PBS that manifests all of the major symptoms of this debilitating condition.

Objectives: Animal models of vaginal distention (VD) have demonstrated increased expression of chemokine (C-C motif) ligand 7 (CCL7) In this study, we investigated the expression of CCL7 in mice models of simulated birth trauma-induced urinary incontinence using VD and pudendal nerve transection (PNT). Methods: Forty-nine mice were divided into 6 groups: VD, sham VD, PNT, sham PNT, anesthesia, and age-matched controls. The urethra, vagina, and rectum were harvested for the expression of CCL7 immediately or 24 hours after assigned procedure. Venous sampling for quantification of serum CCL7 was also performed. An analysis of variance model was used to compare the relative expression of CCL7 in each group. Results: Urethral CCL7 expression in the VD group was significantly higher than control group after 24 hours (P < 0.01). There was no difference in the urethral CCL7 expression in PNT, sham PNT, sham VD, or anesthesia groups compared with the controls. No statistically significant difference was noted in the vaginal and rectal expression of CCL7 between any of the groups except for sham PNT. Statistically significant differences were noted in the serum CCL7 expression in the VD, PNT, and sham PNT (P < 0.01 in all) groups after 24 hours compared with the control group. Conclusions: This study demonstrates overexpression of urethral CCL7 after VD but not PNT. This suggests that nerve injury does not contribute to the CCL7 overexpression. The overexpression of CCL7 in the serum of mice after VD suggests a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery.

The pathophysiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is poorly understood. Inflammatory and autoimmune mechanisms may play a role. We developed a murine model of experimental autoimmune prostatitis (EAP) that mimics the human phenotype of CP/CPPS. Eight-week-old mice were immunized subcutaneously with prostate-specific peptides in an emulsion of complete Freund's adjuvant. Mice were euthanized 10 days after immunization, and lymph node cells were isolated and assessed for recall proliferation to each peptide. P25 99-118 was the most immunogenic peptide. T-cell and B-cell immunity and serum levels of C-reactive protein and nitrate/nitrite levels were evaluated over a 9-wk period. Morphometric studies of prostate, 24-h micturition frequencies, and urine volume per void were evaluated. Tactile referred hyperalgesia was measured using von Frey filaments to the pelvic region. The unpaired Student's t-test was used to analyze differences between EAP and control groups. Prostates from p25 99-118-immunized mice demonstrated elevated gene expression levels of TNF-α, IL-17A, IFN-γ, and IL-1β, not observed in control mice. Compared with controls, p25 99-118-immunized mice had significantly higher micturition frequency and decreased urine output per void, and they demonstrated elevated pelvic pain response. p25 99-118 immunization of male SWXJ mice induced prostate-specific autoimmunity characterized by prostate-confined inflammation, increased micturition frequency, and pelvic pain. This autoimmune prostatitis model provides a useful tool for exploring the pathophysiology and new treatments.

We previously reported that mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), develop profound urinary bladder dysfunction. Because neurogenic bladder in MS patients causes marked bladder remodeling, we next examined morphometric and molecular alterations of the bladder in EAE mice. EAE was created in female SJL/J mice by immunization with the p139-151 encephalitogenic peptide of myelin proteolipid protein in complete Freund's adjuvant, along with intraperitoneal injections of Bordetella pertussis toxin. Seventy days after immunization, mice were scored for the level of neurological impairment and then killed. Spinal cord sections were assessed for demyelination, inflammation, and T cell infiltration; the composition of the bladder tissue was measured quantitatively; and gene expression of markers of tissue remodeling and fibrosis was assessed. A significant increase in the bladder weight-to-body weight ratio was observed with increasing neurological impairment, and morphometric analysis showed marked bladder remodeling with increased luminal area and tissue hypertrophy. Despite increased amounts of all tissue components (urothelium, smooth muscle, and connective tissue), the ratio of connective tissue to muscle increased significantly in EAE mice compared with control mice. Marked increases in mRNA expression of collagen type I α(2), tropoelastin, transforming growth factor-β3, and connective tissue growth factor (CTGF) were observed in EAE mice, as were decreased levels of mRNAs for smooth muscle myosin heavy chain, nerve growth factors, and muscarinic and purinergic receptors. Our results suggest that bladder remodeling corresponding to EAE severity may be due to enhanced expression of CTGF and increased growth of connective tissue.