Methodological aspects of Universal immuno-PCR on standard tubes.

One of the most used formats in inmuno-polymerase chain reaction (IPCR) is known as "Universal" IPCR (signal-generating complexes is based on conjugates of biotinylated DNA, biotinylated IgG and avidin). In the present study, we evaluated the utility of using mono- and bi-biotinylated DNA probes, pre-self-assembled DNA-neutravidin complex, blocking step and glutaraldehyde pretreatment of standard PCR tubes to improve the analytical performance of the hTSH-IPCR assay. The use of pre-self-assembled mono-biotinylated DNA-neutravidin complex enhances both the sensitivity and the reproducibility of the hTSH-IPCR assay, even without blocking step: hTSH-IPCR assay showed an improved limit of detection (LOD: 0.01 µIU/ml), calibration sensitivity (SEN: 2.4) and analytic sensitivity (γ: 9 µIU/ml-1) in comparison with both a self-made ELISA and a commercial one.

Development of a quantitative immuno-polymerase chain reaction assay to detect and quantify low levels of human thyroid stimulating hormone.

In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immuno-polymerase chain reaction (IPCR) assay specific for detection of human thyroid stimulating hormone (hTSH). Several anti-hTSH monoclonal antibodies (MAbs) were generated using hybridoma technology. Two pairs of MAbs (B-4 and B-9) were rationally selected and the optimal assay conditions of sandwich ELISAs were established. The ELISA prototypes were evaluated with standards calibrated with WHO 2nd International Reference Preparation for hTSH and in comparison with a commercial ELISA Kit. Although the limit of detection (LOD) was 0.1 µIU/ml in all cases, B-9-ELISA showed an analytical performance similar to commercial ELISA Kit. Therefore, we selected the B-9 ELISA to develop a hTSH-IPCR assay applying an "Universal-IPCR" format in standard PCR tubes without pretreatment. The signal amplification was achieved through the interaction between the biotinylated detection MAb and mono-biotinylated DNA probe pre-self-assembled with neutravidin. The hTSH-IPCR assay showed a significant increase in terms of the slope definition of sensitivity in low levels range. Our results support the potential of IPCR technique for being applied in clinical diagnosis of thyroid states.

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 µg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.

The eggshell features and clutch viability of the broad-snouted caiman (Caiman latirostris) are associated with the egg burden of organochlorine compounds.

Organochlorine compounds (OCCs) are toxic and have been identified as endocrine-disrupting chemicals (EDCs). The broad-snouted Caiman (Caiman latirostris) is an oviparous species widely distributed in South America with potential to accumulate OCCs. The eggshell is formed during passage of the eggs through the oviduct. Since the oviduct is a target of hormone actions, exposure to OCCs could modify eggshell quality, thus affecting clutch viability. Eight clutches were collected from wetlands of Parana River tributaries, in north-eastern Argentina. Two to four eggs per clutch were used to establish the burden of OCCs, eggshell thickness and eggshell porosity. The remaining eggs were incubated in controlled conditions. Ten days after hatching, hatchling survival was assessed. Organochlorine pesticide residues (OCPs) were found in all clutches, while polychlorinated biphenyls (PCBs) were present in all but one clutch. The principal contributors to the OCP burden were members of the DDT family and oxychlordane. Eggshell thickness was 400.9±6.0 µm and, unexpectedly, no association between eggshell thickness and the OCC burden was found. The number of pores in the outer surface was 25.3±4.3 pores/cm². A significant inverse correlation between porosity and OCC burden was found (Pearson r= -0.81, p= 0.01). Furthermore, a decrease in caiman survival with decreased pore density was observed (Pearson r= 0.73, p= 0.04). Our findings highlight another potential negative impact of current and past use of OCCs on wildlife species.

Laryngeal sequelae secondary to surgical treatment for recurrent respiratory papillomatosis in children.

OBJECTIVE: to describe the frequency and predictive factors associated with laryngeal scarring caused by surgical treatment of recurrent respiratory papillomatosis (RRP) in children. INTRODUCTION: RRP is an important cause of hoarseness and respiratory obstruction in children. The current standard of care for RRP is the systematic and repetitive surgical interventions. The repetitive surgeries may increase the risk of sequelae. A larger number of surgeries, the surgical technique used, and disease severity are related to an increased risk of scarring. MATERIAL AND METHODS: A retrospective, descriptive review of the medical charts of patients with RRP younger than 18 years was conducted. Between 2014 and 2017, 79 patients were identified; five patients were excluded. Demographic and clinical data were recorded and analyzed. The patients were divided into two groups, one with and the other without surgical sequelae, for comparison to identify sequela-associated factors. RESULTS: 75 patients, 40 (53.3%) male, were analyzed. Age at symptom onset ranged from 2 months to 13 years. Median age at the time of diagnosis was 42 months. Overall, 44% presented with disseminated disease. A median of nine (range, 1 to 86) surgeries were performed in each patient with a median of two (range, 0.6 to 10) of the average number of surgeries per year per patient. 29 patients (38.7%) had laryngeal sequelae. When comparing the patients with and without sequelae, statistically significant differences were found in the variables of dissemination during the course of the disease, overall number of surgeries and mean number of surgeries per year, history and number of previous surgeries at an outside institution, urgent surgeries, and CO2 laser use. Patients who underwent more than 10 surgeries or who had a history of previous surgeries at an outside institution had a higher frequency of laryngeal scarring in multivariate analysis. CONCLUSION: Scarring secondary to surgical treatment for RRP is common. Surgery-related variables seem to be predisposing factors. A greater number of surgeries and surgeries performed at less specialized centers are strongly related to this complication. Future studies with a larger sample size are necessary to determine whether other factors are involved.

Bilateral enucleation due to multi-bacterial fulminant endogenous panophthalmitis. / Enucleación bilateral por panoftalmitis endógena polimicrobiana fulminante.

The case is presented of a 62 year-old woman with a rapid, progressive bilateral decrease in visual acuity and panuveitis with orbital cellulitis. She was also in poor general condition, with emesis and fever. Septicaemia due to Klebsiella pneumoniae and bilateral endogenous panophthalmitis were diagnosed. The ocular infection quickly progressed to sclerokeratitis and bilateral perforation despite broad spectrum systemic antibiotic management, and eventually the patient required bilateral enucleation. Microbiological cultures of the surgical pieces identified Klebsiella pneumoniae and Candida magnoliae. To our knowledge, this is the third published case that required bilateral enucleation or evisceration due to endogenous panophthalmitis, and the first case of endogenous ocular infection caused by Candida magnoliae.

The UV filter benzophenone 3, alters early follicular assembly in rat whole ovary cultures.

Our goal was to study the effect of BP3 (benzophenone 3) in the follicular assembly and the potential involvement of Foxl2 pathway using whole ovary cultures. Ovaries were collected from Wistar rats at birth, treated in vitro with vehicle (0.01% DMSO), BP3 (5.8 nM, 276 nM, 576 nM and 876 nM) or ESR2 inhibitor (0.1 nM), and cultured for 7 days. Nest breakdown, follicular assembly and the expression of several regulators of these processes (p27, Foxl2, Sox9, Bmp2, Cyp19 and Fst) were evaluated. In vitro exposure to BP3 (5.8 nM) decreased the population of total oocytes, the number of nests per ovary and early primary follicles population. In addition, BP3 (5.8 nM) induced overexpression of Foxl2 mRNA levels through ESR2 but increased Fst mRNA levels independently from ESR2 or Foxl2. We also observed that the number of p27-positive oocytes was decreased after BP3 (5.8 nM). On the other hand, exposure to BP3-276 increased total oocytes, the number of nests per ovary and decreased primary follicles. In addition, BP3-276 induced no changes of Foxl2 mRNA levels through ESR2 but increased Fst mRNA levels independently from ESR2 or Foxl2. In conclusion, our study clearly shows that exposure to BP3 is to perturb the early events of germ cell development as showed here in whole ovary cultures.

High incidence of anti-cytokine autoantibodies in dogs with immune diseases suggests important immuno-regulatory functions.

Autoantibodies against cytokines have been associated with immunodeficiency, susceptibility to infectious diseases, autoimmunity and inflammation in humans, but have not yet been investigated in the Veterinary field so far. The aim of the current study was to determine the presence of anti-cytokine autoantibodies in canines suffering from various conditions including recurrent infections, autoimmune diseases and cancer in comparison to healthy controls. This is the first report of the presence of autoantibodies against cytokines in dogs. A total of 101 serum samples (51 patients and 50 clinically healthy dogs) from the state of Mexico and surroundings were analysed using a multiplex bead-based flow cytometry assay. Results show significant levels of various anti-cytokine autoantibodies in diseased dogs but not in healthy controls. In addition we show distinct associations of various disease types to the specificity of anti-cytokine autoantibodies and to response complexities. Apart from the direct functional/causal implication of anti-cytokine auto-antibodies on disease processes, this findings point to the possibility to use anti-cytokine response patterns as diagnostic tools.

Regional changes in the spatio-temporal pattern of progesterone receptor (PR) expression in the guinea-pig genital tract as parturition approaches.

Normal parturition in guinea-pig involves changes in responsiveness of the genital tract to estrogen and progesterone. To better characterize endocrine control of guinea-pig parturition, protein and mRNA expression of estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were quantitatively evaluated in lower (LUS) and upper (UUS) uterine segments, cervix (C) and pubic symphyseal ligament (PSL) at three stages of pregnancy (established based on interpubic distance, 0mm: non-relaxed group, 4-6mm: 5 days before parturition, 11-14 mm: 1-2 days prepartum) and immediately after parturition. Towards parturition, no changes in PR mRNA levels were recorded in the UUS, whereas the LUS displayed a gradual increase. PR transcripts exhibited decreased levels during parturition in C and PSL. Levels of PR mRNA were increased in the LUS compared with that of the UUS only at parturition. Regarding protein expression during parturition, PR levels decreased in the UUS whereas in the LUS increased. In C and PSL, PR protein expression decreased 1-2 days prepartum and remained low during parturition. None of the regions studied showed changes in mRNA or protein expression of ERalpha. Therefore, functional regionalization of the guinea-pig genital tract is associated with changes in the spatio-temporal pattern of PR expression as parturition approaches.

Laryngeal reconstruction in children with recurrent respiratory papillomatosis.

OBJETIVES: To describe our experience in reconstructive laryngeal surgery in patients with recurrent respiratory papillomatosis (RRP). INTRODUCTION: RRP is a rare laryngeal disease requiring multiple surgical endoscopic interventions during its course. These interventions may cause secondary lesions that may compromise airway patency. Open larynx reconstructive surgery, as tracheostomy, is a procedure considered to potentially favor extralaryngeal papilloma dissemination. In patients with RRP, the use of endoscopic posterior cricoid split and rib grafting has not been previously described. METHODS: The clinical charts of 230 patients with RRP seen between 1996 and 2017 were reviewed. All patients who underwent airway expansion procedures either by open or endoscopic approach were included in the study. RESULTS: Four patients with RRP underwent laryngeal surgery for laryngeal stenosis were included. A double-stage open approach was used in two patients and a single-stage endoscopic approach in the remaining two. The two tracheostomized patients were decannulated while tracheostomy was avoided in the two patients who underwent a single-stage endoscopic procedure. Two patients had active papillomatous lesions limited to the larynx at the time of surgery; no dissemination was observed during follow-up (cases 1 and 3). One patient had extralaryngeal disseminated papilomatosis; surgery did not lead to an increased lesion load compared to presurgical lesions (case 4). The patient who did not have active lesions did not have recurrence (case 2). CONCLUSIONS: Reconstructive laryngeal surgery is a safe and effective option in the management of stenotic sequelae resulting from the surgical treatment of RRP, allowing for decannulation or avoiding tracheostomy.

Impaired ovarian response to exogenous gonadotropins in female rat offspring born to mothers perinatally exposed to Bisphenol A.

The ovary is sensitive to disruption by the environmental estrogen Bisphenol A (BPA). Our aim was to investigate whether perinatal exposure to BPA (50µg/kgday), orally administered, affects ovarian response to exogenous gonadotrophins (PMSG or PMSG+hCG) in prepubertal female offspring. An altered response to gonadotrophins was observed in BPA-exposed rats. Increased proportion of antral follicles, altered levels of ovarian steroidogenic enzymes, gonadotropin receptors, AR and ERß were observed in PMSG group. Besides that, in response to PMSG+hCG, a persistent high Fshr mRNA expression and a decreased number of follicles with high expression of PR before ovulation were observed. After ovulation, there was an increase in antral atretic follicles, reduced Lhcgr mRNA expression and high serum levels of E2. Therefore, an early exposure to a low dose of BPA during perinatal period induces ovarian changes leading to an altered response to exogenous gonadotropin treatment later in life.

Nasopharyngeal angiofibroma in the elderly: report of a case.

Although nasopharyngeal angiofibromas usually occur during puberty and adolescence, thus being considered as juvenile, very rare documented reports describe this condition in old persons. The case of a 62-year-old man with a nasopharyngeal angiofibroma successfully resected is presented. It was the only lesion found in an old individual among 283 cases seen during a 20-year period in a large city hospital.

Renovasculopathies of hypertension in Mexico City.

OBJECTIVE: To examine the severities of renovasculopathies at autopsy in Mexico City, comparing birth cohorts, cause of death categories, and urban versus rural residence. DESIGN: Autopsies conducted in 1954, 1974, and 1991 were chosen from archived materials to represent men and women aged 15 to 84 years. Hematoxylin- and eosin-stained sections of kidney were retrieved and evaluated morphometrically. SETTING: Department of Pathology, Hospital General, Mexico City. PATIENTS: Patients of the Hospital General evaluated at autopsy. MAIN OUTCOME MEASURES: Renovasculopathies of hypertension include the defining features of benign nephrosclerosis, arterial intimal fibroplasia and arteriolar hyalinization. Fibroplasia is measured as average intimal thickness of renocortical arteries, expressed as percent of outer diameter. Hyalinization is measured by counting the number of affected arterioles in a unit area of cortical section. RESULTS: This study confirms a former study that shows much lower levels of renovasculopathy in Mexico City than in blacks and whites of New Orleans. The cohort born in 1894 showed slightly more involvement than those of 1914, 1934, 1954, and 1971, which did not differ from each other. No differences were found between urban and rural residence nor among the categories of tuberculosis, cirrhosis, cancer, and other conditions unrelated to atherosclerosis. CONCLUSIONS: The remarkable constancy of the renovasculopathies in most comparisons includes little change throughout the 20th century, keeping stable the substantial Mexico-US differences over much of this time frame.

Dissociation in the control of cervical eosinophilic infiltration and collagenolysis at the end of pregnancy or after pseudopregnancy in ovariectomized steroid-treated rats.

Previous studies have demonstrated that during parturition the physiological ripening that permits dilation of the cervical canal is due to a widespread collagenolysis that follows a heavy polymorphonuclear leukocyte invasion of the uterine cervix. The aim of this study was to investigate whether there is any association between the ovarian steroid hormones involved in rat parturition and this phenomenon. Pregnant or pseudopregnant rats were ovariectomized (OVX) at Day 9 and then given a hormonal treatment (sufficient to maintain fetal viability) until Day 23. Cervical biopsies, taken from animals killed intrapartum or 1 h before expected parturition, were studied for eosinophilic infiltration and collagen birefringence. Intrapartum or sham-OVX pregnant rats showed a massive eosinophilic infiltration and a widespread collagenolysis as indicated by a loss of collagen birefringence. Ovariectomized pregnant rats treated with estrogen plus progesterone or with progesterone alone showed neither infiltration nor collagenolysis. In rats OVX during pseudopregnancy, estrogen given alone induced a significant infiltration of eosinophils in the cervical stroma; however, treatment with the combination of estrogen and progesterone was not able to promote eosinophilic infiltration. Collagenolysis was absent in all pseudopregnant animals. These results show that estrogen induced a cervical eosinophilic infiltration in rats but that when progesterone was added to the estrogen treatment the infiltration was not present; in addition, none of the steroid hormones assessed were responsible for the collagenolysis found in the cervical tissue at term.