Eosinophilic airway inflammation in asthmatic patients is associated with an altered airway microbiome.

BACKGROUND: Asthmatic patients have higher microbiome diversity and an altered composition, with more Proteobacteria and less Bacteroidetes compared with healthy control subjects. Studies comparing airway inflammation and the airway microbiome are sparse, especially in subjects not receiving anti-inflammatory treatment. OBJECTIVE: We sought to describe the relationship between the airway microbiome and patterns of airway inflammation in steroid-free patients with asthma and healthy control subjects. METHODS: Bronchoalveolar lavage fluid was collected from 23 steroid-free nonsmoking patients with asthma and 10 healthy control subjects. Bacterial DNA was extracted from and subjected to Illumina MiSeq sequencing of the 16S rDNA V4 region. Eosinophils and neutrophils in the submucosa were quantified by means of immunohistochemical identification and computerized image analysis. Induced sputum was obtained, and airway hyperresponsiveness to mannitol and fraction of exhaled nitric oxide values were measured. Relationships between airway microbial diversity and composition and inflammatory profiles were analyzed. RESULTS: In asthmatic patients airway microbial composition was associated with airway eosinophilia and AHR to mannitol but not airway neutrophilia. The overall composition of the airway microbiome of asthmatic patients with the lowest levels of eosinophils but not asthmatic patients with the highest levels of eosinophils deviated significantly from that of healthy subjects. Asthmatic patients with the lowest levels of eosinophils had an altered bacterial abundance profile, with more Neisseria, Bacteroides, and Rothia species and less Sphingomonas, Halomonas, and Aeribacillus species compared with asthmatic patients with more eosinophils and healthy control subjects. CONCLUSION: The level of eosinophilic airway inflammation correlates with variations in the microbiome across asthmatic patients, whereas neutrophilic airway inflammation does not. This warrants further investigation on molecular pathways involved in both patients with eosinophilic and those with noneosinophilic asthma.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

BACKGROUND: Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). RESULTS: A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=-0.33, P<0.04), but not in the non-cloned control pigs. CONCLUSION: The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

Gross and Histologic Anatomy of the Pelvic Ureter: Clinical Applications to Pelvic Surgery.

OBJECTIVE: To further evaluate relationships of the pelvic ureter to clinically relevant structures and to characterize the anatomy, histology, and nerve density of the distal ureter. METHODS: In this observational cadaveric study, 35 female cadavers were examined, 30 by gross dissections and five microscopically. Ureter length and segments of pelvic ureter were measured. Closest distances between the ureter and clinically relevant points were recorded. The distal pelvic ureter and surrounding parametrium were evaluated microscopically. Nerve density was analyzed using automated quantification of peripheral nerve immunostaining. Average measurements of nerve density in the anterior and posterior quadrants surrounding the ureter were statistically compared using a two-tailed t test. Descriptive statistics were used for analyses with distances reported as mean±SD (range). RESULTS: Gross dissections revealed ureter length of 26.3±1.4 (range 24-29) cm (right), 27.6±1.6 (25-30.5) cm (left). Lengths of ureter from pelvic brim to uterine artery crossover were 8.2±1.9 (4.4-11.5) cm (right), 8.5±1.5 (4.5-11.5) cm (left) and from crossover to bladder wall 3.3±0.7 (2.4-5.8) cm (right), 3.2±0.4 (2.6-4.1) cm (left). Intramural ureter length was 1.5±0.3 (1-2.2) cm (right) and 1.7±1.2 (0.8-2.5) cm (left). Distances from the ureter to uterine isthmus: median 1.7 (range 1-3.0) cm (right) and 1.7 (1.0-2.9) cm (left); lateral anterior vaginal fornix 1.5 (1.0-3.1) cm (right) and 1.7 (0.8-3.2) cm (left); lateral vaginal apex 1.3 (1.0-2.6) cm (right) and 1.2 (1.1-2.2) cm (left) were recorded. Microscopy demonstrated denser fibrovascularity posteromedial to the ureter. Peripheral nerve immunostaining revealed greater nerve density posterior to the distal ureter. CONCLUSION: Proximity of the ureter to the uterine isthmus and lateral anterior vagina mandates careful surgical technique and identification. The intricacy of tissue surrounding the distal ureter within the parametrium and the increased nerve density along the posterior distal ureter emphasizes the importance of avoiding extensive ureterolysis in this region.

The prevalence of occult endometrial cancer in women undergoing hysterectomy for benign indications.

OBJECTIVE: To estimate the frequency of occult endometrial cancer in women undergoing hysterectomy for benign indications. STUDY DESIGN: We performed a retrospective review of all patients undergoing hysterectomies for benign indications at our institution from 2006 to 2014. A departmental database was used to identify all hysterectomies performed, and institutional tumor registry was used to identify cases of endometrial carcinoma. Occult carcinomas were defined as cases with no suspicion preoperatively and histopathologic diagnosis of endometrial cancer postoperatively. RESULTS: A total of 6981 hysterectomies were performed for benign indications. Among these, thirteen patients (0.19%) were found to have occult endometrial cancer, with an overall rate of 1 in 537 patients (95% confidence interval 1:314-1:1008). Twelve patients had stage IA and one had stage IB disease. Median age of women found to have endometrial cancer was 50 years (range 35-72 years). The median BMI was 29.8 kg/m2 (range 21.3-50.4 kg/m2). The most common indications for hysterectomy were abnormal bleeding (47%), postmenopausal bleeding (15%), adnexal mass (15%), prolapse (15%), and endometrial hyperplasia without atypia (8%). Of the postmenopausal women that had bleeding, all patients underwent evaluation of the endometrium, however 75% of samples did not have adequate amount of endometrium to be evaluated and 25% were found to have hyperplasia. CONCLUSION: This is one of the largest single institution cohorts to examine occult malignancy. Unexpected endometrial carcinomas were found to occur in 0.19% or 1:537 (95% confidence interval 1:314-1:1008) hysterectomies for benign indications in our population. PRéCIS: Occult endometrial carcinomas are found to occur in 1:537 (0.19%) hysterectomies for benign indications.

Cognitive reactivity to sad mood provocation and the prediction of depressive relapse.

CONTEXT: Episode remission in unipolar major depression, while distinguished by minimal symptom burden, can also be a period of marked sensitivity to emotional stress as well as an increased risk of relapse. OBJECTIVE: To examine whether mood-linked changes in dysfunctional thinking predict relapse in recovered patients who were depressed. DESIGN: In phase 1 of this study, patients with major depressive disorder were randomly assigned to receive either antidepressant medication or cognitive behavior therapy. In phase 2, patients who achieved clinical remission underwent sad mood provocation and were then observed with regular clinical assessments for 18 months. SETTING: Outpatient psychiatric clinics at the Centre for Addiction and Mental Health, Toronto, Ontario. PARTICIPANTS: A total of 301 outpatients with major depressive disorder, aged 18 to 65 years, participated in phase 1 of this study and 99 outpatients with major depressive disorder in remission, aged 18 to 65 years, participated in phase 2. MAIN OUTCOME MEASURE: Occurrence of a relapse meeting DSM-IV criteria for a major depressive episode as assessed by the longitudinal interval follow-up evaluation and a Hamilton Depression Rating Scale score of 16 or greater. RESULTS: Patients who recovered through antidepressant medication showed greater cognitive reactivity following the mood provocation than those who received cognitive behavior therapy. Regardless of type of prior treatment, the magnitude of mood-linked cognitive reactivity was a significant predictor of relapse over the subsequent 18 months. Patients whose mood-linked endorsement of dysfunctional attitudes increased by a minimum of 8 points had a significantly shorter time to relapse than those whose scores were not as elevated. CONCLUSIONS: The vulnerability of remitted depressed patients for illness relapse may be related to the (re)activation of depressive thinking styles triggered by temporary dysphoric states. This is the first study to link such differences to prognosis following successful treatment for depression. Further understanding of factors predisposing to relapse/recurrence in recovered patients may help to shorten the potentially lifelong course of depression.

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype.

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.

Longitudinal assessment of gait abnormalities following penetrating ballistic-like brain injury in rats.

Traumatic brain injury (TBI) results in enduring motor and cognitive dysfunction. Although gait disturbances have been documented among TBI patients, few studies have profiled gait abnormalities in animal models of TBI. We sought to obtain a comprehensive longitudinal analysis of gait function following severe penetrating ballistic-like brain injury (PBBI) in rats. Rats were subjected to either unilateral frontal PBBI, probe insertion alone, or sham surgery. Sensorimotor performance was assessed using the CatWalk automated gait analysis system. Baseline measurements were taken 3 days prior to injury and detailed analysis of gait was performed at 1, 3, 7, 14, and 28 days post-injury. Both PBBI and probe-inserted rats displayed altered static and dynamic gait parameters that were primarily evident during the early (<7 days) post-injury phase and were resolved by 1 month post-injury. PBBI produced more severe deficits compared to probe-alone which were reflected in the number, magnitude, and resolution time of abnormal gait parameters. While altered parameters were detected in all four paws, they were more apparent on the contralateral side. Gait parameters including paw pressure, print area, swing speed, and stride length were significantly decreased whereas stance, swing, and step cycle duration were increased compared to sham. Overall, altered gait patterns detected using the CatWalk system in the PBBI model were injury-severity dependent, resolved at later time points, and appeared similar to those reported in severe TBI patients. These results indicate that the CatWalk may be most useful for neuroprotection studies that focus on the acute/subacute recovery period after TBI.

Characterisation of gut microbiota in Ossabaw and Göttingen minipigs as models of obesity and metabolic syndrome.

BACKGROUND: Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs. METHODS AND FINDINGS: The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs' cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine. CONCLUSION: Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.

Novel TaqMan® PCR for detection of Leptospira species in urine and blood: pit-falls of in silico validation.

In the acute phase of leptospirosis, the diagnosis can be established with high sensitivity by testing blood and urine samples with polymerase chain reaction (PCR). However, only few real-time PCR assays have been validated for diagnostic use. The diagnostic accuracy of a novel TaqMan® PCR (LipL32 real-time PCR) targeting the lipl32 gene (or hap-1) and a previously described TaqMan® PCR (16S real-time PCR) targeting the rrs gene coding for 16S rRNA was evaluated when applied to both urine and blood specimens from humans suspected of leptospirosis. Applied to at least two blood cultures LipL32 real-time PCR had a sensitivity of 86%, and a specificity of 100%; and 16S real-time PCR had a sensitivity of 100%, and a specificity of 97%. Applied to urine samples, patients that were positive by the reference methods were also positive by both real-time PCR assays (n=4). For LipL32 real-time PCR the specificity was 100%, while for 16S real-time PCR it was only 91.5% due to unexpected cross-reactions with other bacteria. The analytical sensitivity was close to the theoretical limit-of-detection for both assays detecting all described human pathogenic species. We report a specific real-time PCR assay for detection of Leptospira, i.e., LipL32 real-time PCR that has been validated for diagnostic application in both urine and blood specimens from humans. We further show that a previously described 16S real-time PCR no longer can be recommended for diagnostic use due to a low specificity.

Severity profile of penetrating ballistic-like brain injury on neurofunctional outcome, blood-brain barrier permeability, and brain edema formation.

This study evaluated the injury severity profile of unilateral, frontal penetrating ballistic-like brain injury (PBBI) on neurofunctional outcome, blood-brain barrier (BBB) permeability, and brain edema formation. The degree of injury severity was determined by the delivery of a water-pressure pulse designed to produce a temporary cavity by rapid (<40 ms) expansion of the probe's elastic balloon calibrated to equal 5%, 10%, 12.5%, or 15% of total rat brain volume (control groups consisted of sham surgery or insertion of the probe only). Neurofunctional assessments revealed motor and cognitive deficits related to the degree of injury severity, with the most clear-cut profile of PBBI injury severity depicted by the Morris water maze (MWM) results. A biphasic pattern of BBB leakage was detected in the injured hemisphere at all injury severity levels at 4 h post-injury, and again at 48-72 h post-injury, which remained evident out to 7 days post-PBBI in the 10% and 12.5% PBBI groups. Likewise, significant brain edema was detected in the injured hemisphere by 4 h post-injury and remained elevated out to 7 days post-injury in the 10% and 12.5% PBBI groups. However, following 5% PBBI, significant levels of edema were only detected from 24 h to 48h post-injury. These results identify an injury severity profile of BBB permeability, brain edema, and neurofunctional impairment that provides sensitive and clinically relevant outcome metrics for studying potential therapeutics.

Expanding the diagnostic use of PCR in leptospirosis: improved method for DNA extraction from blood cultures.

BACKGROUND: Leptospirosis is a neglected zoonosis of ubiquitous distribution. Symptoms are often non-specific and may range from flu-like symptoms to multi-organ failure. Diagnosis can only be made by specific diagnostic tests like serology and PCR. In non-endemic countries, leptospirosis is often not suspected before antibiotic treatment has been initiated and consequently, relevant samples for diagnostic PCR are difficult to obtain. Blood cultures are obtained from most hospitalized patients before antibiotic therapy and incubated for at least five days, thus providing an important source of blood for PCR diagnosis. However, blood cultures contain inhibitors of PCR that are not readily removed by most DNA-extraction methods, primarily sodium polyanetholesulfonate (SPS). METHODOLOGY/PRINCIPAL FINDINGS: In this study, two improved DNA extraction methods for use with blood cultures are presented and found to be superior in recovering DNA of Leptospira interrogans when compared with three previously described methods. The improved methods were easy and robust in use with all tested brands of blood culture media. Applied to 96 blood cultures obtained from 36 patients suspected of leptospirosis, all seven patients with positive convalescence serology were found positive by PCR if at least one anaerobic and one aerobic blood culture, sampled before antibiotic therapy were tested. CONCLUSIONS/SIGNIFICANCE: This study suggests that a specific and early diagnosis can be obtained in most cases of severe leptospirosis for up to five days after initiation of antimicrobial therapy, if PCR is applied to blood cultures already sampled as a routine procedure in most septic patients.

Longitudinal characterization of motor and cognitive deficits in a model of penetrating ballistic-like brain injury.

Traumatic brain injury (TBI) produces a wide range of motor and cognitive changes. While some neurological symptoms may respond to therapeutic intervention during the initial recovery period, others may persist for many years after the initial insult, and often have a devastating impact on quality of life for the TBI victim. The aim of the current study was to develop neurobehavioral testing parameters designed to provide a longitudinal assessment of neurofunctional deficits in a rodent model of penetrating ballistic-like brain injury (PBBI). We report here a series of experiments in which unilateral frontal PBBI was induced in rats, and motor/cognitive abilities were assessed using a battery of tests ranging from 30 min to 10 weeks post-injury. The results showed that PBBI produced consistent and significant (1) neurological deficits (neuroscore examination: 30 min to 10 weeks post-PBBI), (2) sensorimotor dysfunction in the contralateral forelimb (forelimb asymmetry task: 7 and 21 days), (3) motor dysfunction (balance beam task: 3-7 days; and fixed-speed rotarod task: 3-28 days), and (4) spatial learning deficits in the Morris water maze (MWM) task out to 10 weeks post-injury. Overall, the results of this study demonstrate that PBBI produces enduring motor and cognitive deficits, and identifies the optimal task and testing parameters for facilitating longitudinal screening of promising therapeutic interventions in this brain injury model.

A novel examination of atypical major depressive disorder based on attachment theory.

OBJECTIVE: While a large body of descriptive work has thoroughly investigated the clinical correlates of atypical depression, little is known about its fundamental origins. This study examined atypical depression from an attachment theory framework. Our hypothesis was that, compared to adults with melancholic depression, those with atypical depression would report more anxious-ambivalent attachment and less secure attachment. As gender has been an important consideration in prior work on atypical depression, this same hypothesis was further tested in female subjects only. METHOD: One hundred ninety-nine consecutive adults presenting to a tertiary mood disorders clinic with major depressive disorder with either atypical or melancholic features according to the Structured Clinical Interview for DSM-IV Axis-I Disorders were administered a self-report adult attachment questionnaire to assess the core dimensions of secure, anxious-ambivalent, and avoidant attachment. Attachment scores were compared across the 2 depressed groups defined by atypical and melancholic features using multivariate analysis of variance. The study was conducted between 1999 and 2004. RESULTS: When men and women were considered together, the multivariate test comparing attachment scores by depressive group was statistically significant at p < .05. Between-subjects testing indicated that atypical depression was associated with significantly lower secure attachment scores, with a trend toward higher anxious-ambivalent attachment scores, than was melancholia. When women were analyzed separately, the multivariate test was statistically significant at p < .01, with both secure and anxious-ambivalent attachment scores differing significantly across depressive groups. CONCLUSION: These preliminary findings suggest that attachment theory, and insecure and anxious-ambivalent attachment in particular, may be a useful framework from which to study the origins, clinical correlates, and treatment of atypical depression. Gender may be an important consideration when considering atypical depression from an attachment perspective.