Proteomic analysis of seminal plasma in men with different spermatogenic impairment.

Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two-dimensional differential gel electrophoresis (2-D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type-3, beta-2-microglobulin, galectin-3-binding protein, prolactin-inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis.

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.

Evaluation of probiotic administration on the immune response of coccidiosis-vaccinated broilers.

Probiotics are nonpathogenic bacteria that can promote bird health by reducing pathogen colonization. Researchers have previously demonstrated that the avian immune response can be modulated with probiotics, which may provide a mechanism for the reported reductions in pathogens. We examined phagocyte oxidative burst and cell proliferation of vaccinated broilers administered probiotics. We hypothesized that the combination of probiotic bacteria and a vaccine would affect immune function. Two studies were conducted to evaluate this interaction in broilers. Treatments consisted of a negative control, probiotic, vaccine, or a probiotic + vaccine. Peripheral blood was collected on d 7, 14, and 21 of age. Heterophils and monocytes were evaluated for oxidative burst and lymphocytes were assayed for proliferation. In study 1, heterophil oxidative burst was higher (P ≤ 0.05) in each treatment that received probiotic on d 14 when compared with the negative control. On d 21, an enhanced (P ≤ 0.05) heterophil oxidative burst was observed in the probiotic treatment when compared with the other treatments. On d 14, monocyte oxidative burst was greater (P ≤ 0.05) in the probiotic + vaccine treatment when compared with all other treatments. An increase (P ≤ 0.05) in lymphocyte proliferation was observed among all treatments on d 7 when compared with the negative control. Both vaccine treatments had significant lymphocyte proliferation on d 14 when compared with the negative control. In study 2, the probiotic treatment was associated with greater levels in heterophil oxidative burst on d 7 when compared with all other treatments. On d 21, an increase (P ≤ 0.05) in heterophil oxidative burst was seen in the vaccine treatment when compared with the negative control. On d 7, increased (P ≤ 0.05) monocyte oxidative burst was observed in the vaccine treatment when compared with the negative control. No significant differences were observed in lymphocyte proliferation in any of the treatment groups. These data suggest that probiotics can modulate the immune response and may play a role in vaccination.

Assessing the health perspectives of unique populations of adolescents: a focus group study.

Few articles report on youth's perceptions about health-related issues and needs and even fewer have given voice to youth with disabilities, chronic health conditions, or youth in therapeutic foster care. The purpose of this study was to explore perceptions of health issues from the perspectives of youth placed in therapeutic foster care, youth with chronic medical conditions, and youth with disabilities. Twelve focus groups with 67 youth aged 13-20 were conducted in rural, suburban, and urban communities in the Commonwealth of Virginia. Data analysis revealed both within unique group themes and themes that cut across unique groups. Youth in therapeutic foster care with chronic health conditions or with disabilities perceive themselves as being different from their peers. Participants in each group expressed concerns about the unique issues that they confront daily and how these challenges differentiate them from youth who do not share their life experiences. They expressed the desire for other youth to understand them better and to not treat them differently. Discussion and implications focus on the need to create programs to support the unique needs of these youth.

Tec family kinases modulate thresholds for thymocyte development and selection.

Tec family kinases are implicated in T cell receptor (TCR) signaling, and combined mutation of inducible T cell kinase (Itk) and resting lymphocyte kinase (Rlk)/Txk in mice dramatically impairs mature T cell function. Nonetheless, mutation of these kinases still permits T cell development. While itk(-)(/)- mice exhibit mild reductions in T cells with decreased CD4/CD8 cell ratios, rlk(-)(/)-itk(-)(/)- mice have improved total T cell numbers yet maintain decreased CD4/CD8 ratios. Using TCR transgenics and an in vitro thymocyte deletion model, we demonstrate that mutation of Tec kinases causes graded defects in thymocyte selection, leading to a switch from negative to positive selection in rlk(-)(/)-itk(-)(/)- animals. The reduction in both positive and negative selection and decreased CD4/CD8 ratios correlates with decreased biochemical parameters of TCR signaling, specifically defects in capacitive Ca(2+) influx and activation of the mitogen-activated kinases extracellular signal-regulated kinase 1 and 2. Thus, Tec kinases influence cell fate determination by modulating TCR signaling, leading to altered thresholds for thymocyte selection. These results provide support for a quantitative model for thymic development and provide evidence that defects in negative selection can substantially alter thymic cellularity.

Antifibroblast antibodies from systemic sclerosis patients bind to {alpha}-enolase and are associated with interstitial lung disease.

OBJECTIVE: To identify target antigens of antifibroblast antibodies (AFA) in systemic sclerosis (SSc) patients. PATIENTS AND METHODS: In the first part, sera from 24 SSc patients (12 with pulmonary arterial hypertension (PAH) and 12 without) and 36 idiopathic PAH patients, tested in pooled sera for groups of three, were compared with a sera pool from 14 healthy controls (HC). Serum IgG reactivity was analysed by the use of a two-dimensional electrophoresis and immunoblotting technique with normal human fibroblasts antigens. In the second part, serum IgG reactivity for two groups: 158 SSc, 67 idiopathic PAH and 100 HC; and 35 SSc and 50 HC was tested against alpha-enolase from Saccharomyces cerevisiae and recombinant human (rHu) alpha-enolase, respectively, on ELISA. RESULTS: In the first part, alpha-enolase was identified as a main target antigen of AFA from SSc patients. In the second part, 37/158 (23%) SSc patients, 6/67 (9%) idiopathic PAH patients and 4/100 (4%) HC (p<0.001) had anti-S cerevisiae alpha-enolase antibodies; 12/35 (34%) SSc patients and 3/50 (6%) HC had anti-rHu alpha-enolase antibodies (p = 0.001). In SSc, the presence of anti-S cerevisiae alpha-enolase antibodies was associated with interstitial lung disease (ILD), decreased total lung capacity (73.2% vs 89.7%; p<0.001) and diffusion capacity for carbon monoxide (47.4% vs 62.3%; p<0.001), and antitopoisomerase 1 antibodies (46% vs 21%; p = 0.005) but not anticentromere antibodies (11% vs 34%; p = 0.006). Results were similar with rHu alpha-enolase testing. CONCLUSION: In SSc, AFA recognise alpha-enolase and are associated with ILD and antitopoisomerase antibodies.

Tec kinases: modulators of lymphocyte signaling and development.

The Tec kinases are implicated as important components of the antigen receptor signaling required for proper lymphocyte activation and development. Recent data suggest that these kinases contribute to multiprotein complexes containing LAT and SLP-76 in T cells, and BLNK/SLP-65 in B cells, which are required for activation of PLC-gamma and downstream pathways.

Increased level of Eimeria sensitivity to diclazuril after using a live coccidial vaccine.

Anticoccidial vaccine and an anticoccidial drug rotation program were compared to determine which program was more effective in producing coccidia populations sensitive of 1 ppm diclazuril. The study used an anticoccidial drug-sensitivity battery test (AST) to determine the baseline level of diclazuril sensitivity to field isolates of Eimeria spp. from seven broiler complexes that had used diclazuril. Based on percentage reduction in weight gain and lesion scores, 25% or fewer of the isolates were effectively controlled by diclazuril. Following the baseline sampling, four of the complexes switched to a nondiclazuril in-feed anticoccidial drug program and three of the complexes switched to a vaccination program for two broiler grow-out cycles as the sole coccidiosis-control program. This study demonstrated that the vaccine used (Coccivac-B) contained anticoccidial drug-sensitive strains. Eimeria isolates were subsequently collected from the identical houses and diclazuril AST results were compared with the baseline AST results. Following the two grow-out cycles, sensitivity of the isolates to diclazuril from the four complexes that continued to use in-feed anticoccidial drugs remained essentially unchanged. The isolates from the three complexes that switched to the vaccination program demonstrated a marked increase in diclazuril sensitivity, with 60%-100% of the isolates from each complex effectively controlled by diclazuril. Vaccination with the anticoccidial drug-sensitive strains produced a measurable increase in the level of sensitivity to diclazuril.

Treating gastro-oesophageal reflux disease during pregnancy and lactation: what are the safest therapy options?

Gastro-oesophageal reflux and heartburn are reported by 45 to 85% of women during pregnancy. Typically, the heartburn of pregnancy is new onset and is precipitated by the hormonal effects of estrogen and progesterone on lower oesophageal sphincter function. In mild cases, the patient should be reassured that reflux is commonly encountered during a normal pregnancy: lifestyle and dietary modifications may be all that are required. In a pregnant woman with moderate to severe reflux symptoms, the physician must discuss with the patient the benefits versus the risks of using drug therapy. Medications used for treating gastro-oesophageal reflux are not routinely or vigorously tested in randomised, controlled trials in women who are pregnant because of ethical and medico-legal concerns. Safety data are based on animal studies, human case reports and cohort studies as offered by physicians, pharmaceutical companies and regulatory authorities. If drug therapy is required, first-line therapy should consist of nonsystemically absorbed medications, including antacids or sucralfate, which offer little, if any, risk to the fetus. Systemic therapy with histamine H2 receptor antagonists (avoiding nizatidine) or prokinetic drugs (metoclopramide, cisapride) should be reserved for patients with more severe symptoms. Proton pump inhibitors are not recommended during pregnancy except for severe intractable cases of gastrooesophageal reflux or possibly prior to anaesthesia during labour and delivery. In these rare situations, animal teratogenicity studies suggests that lansoprazole may be the best choice. Use of the least possible amount of systemic drug needed to ameliorate the patient's symptoms is clearly the best for therapy. If reflux symptoms are intractable or atypical, endoscopy can safely be performed with conscious sedation and careful monitoring the mother and fetus.

Necrotic enteritis in cage-reared commercial layer pullets.

Necropsy of five 12-week-old pullets from a flock of 99,300 suffering from an increased mortality rate revealed enlarged, gas-filled intestines, the mucosal surfaces of which had the "dirty turkish towel" appearance typical of necrotic enteritis. Although the pullets had been raised entirely in cages, intestinal scrapings revealed the presence of Eimeria maxima. Histopathological findings were compatible with necrotic enteritis. Clostridium perfringens was isolated by anaerobic culture from the intestines. Mortality returned to normal after bacitracin and amprolium were added to the feed.

Relationship of age at parabiosis and time of embryonic death on extent of mixing of embryonic blood.

Two studies were conducted to determine the extent of blood chimerism from parabionts joined at different ages and the mortality pattern after joining. Complete mixing of blood was found in 100% of the pairs joined at 13 days, 80% at 15 days, and 25% at 17 days of incubation. Therefore, interfusing of blood components could only be assured when the embryos were joined no later than 13 days of incubation. Mortality after parabiosis occurred in three peaks. The first and largest peak (16.4% mortality) occurred at 12 hr after parabiosis with substantial mortality through 36 hr. The second peak (4.6% mortality) occurred between 108 and 120 hr after joining of the eggs. The third and final peak (5.6% mortality) occurred between 192 hr and hatch. Surgical trauma, bacterial contamination, and the inability of the embryo to escape the shell once pipped are possible explanations since these events were associated with clusters of embryo deaths. The use of single surviving parabiont as a means of increasing the efficiency of the parabiotic technique is discussed.

Parabiosis between avian embryos resistant and susceptible to Rous sarcoma virus.

Parabiosis of chicken embryos was used to determine if resistance or susceptibility to Rous sarcoma virus (RSV) tumors could be transferred between RSV-resistant (R) and RSV-susceptible (S) embryos. Eggs from RSV-R and RSV-S lines were parabiosed at 12 days of incubation. One week after hatching the chicks were inoculated in the wing web with RSV. Birds were examined for tumor lesions through 9 weeks of age. Lesion scores of the nonparabiosed RSV-S line were significantly higher than nonparabiosed RSV-R lines. Lesion scores of RSV-R chicks that had been parabiosed to RSV-S chicks were higher than nonparabiosed RSV-R chicks. Lesion scores of RSV-S chicks parabiosed to RSV-R chicks were not different from nonparabiosed RSV-S chicks. These results suggest that the resistance to RSV was not transferred between parabionts but that there was significant transfer of susceptibility from the RSV-S embryos to the RSV-R embryos.

A preliminary comparison of reinforcer assessment methods for children with attention deficit hyperactivity disorder.

We evaluated the relative treatment utility of a verbal forced-choice questionnaire, child nomination, and direct observation for identifying the most potent reinforcers for children with attention deficit hyperactivity disorder. Results demonstrated that all three methods were more likely to disagree than to agree, that a forced-choice format may enhance verbal reinforcer assessment, and that further development and evaluation of verbal reinforcer-assessment methods are needed.

A comparison of reinforcer assessment methods: the utility of verbal and pictorial choice procedures.

We compared three methods of stimulus preference assessment for verbal children and specifically evaluated the utility of a verbal choice procedure for assessing relative reinforcer value. Using a token system, relative preference for five categories of reinforcers, representing 15 different stimuli, was assessed by three methods: a reinforcer survey, a verbal stimulus-choice questionnaire, and a pictorial stimulus-choice procedure. Results showed that the verbal and pictorial stimulus-choice assessments accurately identified high- and low-preference categories for 3 of 4 participants. Survey results alone often rated multiple categories as high preference, were less likely to identify low-preference categories, and were less likely to correspond with the results of a reinforcer assessment.

The differential effects of teacher and peer attention on the disruptive classroom behavior of three children with a diagnosis of attention deficit hyperactivity disorder.

We conducted functional analyses of classroom disruption during contingent teacher and peer attention conditions for 3 children with attention deficit hyperactivity disorder. Attention provided by peer confederates appeared to function as a distinct form of positive reinforcement for all 3 children.

A preliminary analysis of interactive effects between common classroom contingencies and methylphenidate.

To assess the drug-behavior interaction effects with an 8-year-old boy wih attention deficit hyperactivity disorder, common classroom antecedent (e.g., seating arrangement) and consequent (e.g., peer prompts) stimuli were alternated within a school day while drug conditions (methylphenidate vs. placebo) were alternated across days. The results suggested that peer attention maintained disruptive behavior when methylphenidate was absent but not when it was present.

A report of congenital indifference to pain as diagnosed in infancy.

As physicians and nurses, we have a fundamental obligation to consider patient welfare above all else. The case presented demonstrates the depth of this obligation. Our patient, who was diagnosed during infancy with congenital indifference to pain, can neither feel nor perceive the meaning of pain. Congenital indifference to pain is a rare disorder, which is characterized by normal intelligence, development, and sensation despite an absence of pain perception. There is no cure and treatment difficulties are plentiful. Educating and maintaining an open, consistent relationship with the family and caregivers is essential to reduce the morbidity of this condition.

Neurocognitive predictors of functional outcome two to 13 years after identification as ultra-high risk for psychosis.

BACKGROUND AND AIM: Little is known about the relationship between neurocognitive performance and functional outcome before the onset of frank psychosis. This longitudinal study aimed to investigate neurocognitive predictors of poor functional outcome in a group identified as ultra-high risk (UHR) for psychosis between two and 13 years prior. METHOD: Individuals (N=230) identified as UHR for psychosis at the PACE Clinic in Melbourne completed assessment of psychopathology, functioning and neurocognition at baseline and follow-up. The mean length of follow-up was 7.26 years (SD 3.05). RESULTS: Forty-one individuals with the poorest functional outcome were identified. Only 48.8% of this group had transitioned to psychosis. Poor functional outcome was associated with reduced performance at baseline in the specific neurocognitive domains of verbal learning and memory, processing speed and attention, and verbal fluency, but not global cognitive impairment. Reduced performance on a verbal story recall task, in combination with higher negative symptoms at baseline, was the best predictor of later poor outcome. Baseline positive psychotic symptoms and GAF scores were not associated with later poor outcome. DISCUSSION: To date, this is the longest follow-up study of an UHR sample. Poor functional outcome was associated with specific neurocognitive decrements, regardless of transition to psychosis. The detection of individuals with poor functioning at follow-up, against a background of previously identified risk factors for psychotic disorder, may yield a valid group in which to study biomarkers and treatment of schizophrenia.