Acute effects of cadmium on preovulatory serum FSH, LH, and prolactin levels and on ovulation and ovarian hormone secretion in estrous rats

On the day of diestrus II CFY rats were given 5, 10, or 15 mg/kg cadmium chloride (CdCl₂) or 1.0 mL/kg of 0.9% NaCl. On the next day a group of animals was anesthetized with pentobarbital and blood was collected from the aorta at 13:00, 15:00, 16:30, or 18:00 h. for FSH, LH, prolactin (PRL), progesterone (P), and estradiol-17β (E₂) determination. On the day of the expected estrus, the second group of animals was anesthetized with pentobarbital and cannulas were inserted in one of the femoral arteries and veins, and in one of the utero-ovarian veins. Five-minute blood fractions were collected from the ovary for 40 min, and following the first blood samples, 10 IU hCG was injected iv. Ovarian venous outflow and blood pressure were continuously recorded. From the blood fractions, P and E₂ were determined, and their secretion rates were calculated. In a third group of treated animals, the ovaries were excised for histological examination, and oviducts were flushed for counting oocytes. CdCl₂ in the dose of 10 or 15 mg/kg increased the PRL serum levels at 13:00 h; it diminished FSH serum levels in the dose of 10 mg/kg and LH serum levels in the doses of 10 and 15 mg/kg at 15:00 h. The decrease in LH levels continued until 16:30 h in the dose of 10 mg/kg CdCl₂. In estrous animals, CdCl₂ did not influence the blood pressure and ovarian blood flow. In animals receiving 10 or 15 mg/kg CdCl₂, a decrease in basal secretion of P occurred. The hCG induced a rapid rise in P secretion of controls; the 5 mg/kg CdCl₂ diminished the effect of hCG, and the 10 and 15 mg/kg completely abolished it, but failed to influence the basal secretion of E₂ and its response to hCG. CdCl₂ prevented ovulation in 40% to 50% of the animals; however, when ovulation occurred, normal oocyte numbers were found. No alteration could be observed in the light microscopic histology of ovary.     

Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.     

NO-induced migraine attack: strong increase in plasma calcitonin gene-related peptide (CGRP) concentration and negative correlation with platelet serotonin release

The aim of the present study was to investigate changes in the plasma calcitonin gene-related peptide (CGRP) concentration and platelet serotonin (5-hydroxytriptamine, 5-HT) content during the immediate headache and the delayed genuine migraine attack provoked by nitroglycerin. Fifteen female migraineurs (without aura) and eight controls participated in the study. Sublingual nitroglycerin (0.5 mg) was administered. Blood was collected from the antecubital vein four times: 60 min before and after the nitroglycerin application, and 60 and 120 min after the beginning of the migraine attack (mean 344 and 404 min; 12 subjects). In those subjects who had no migraine attack (11 subjects) a similar time schedule was used. Plasma CGRP concentration increased significantly (P<0.01) during the migraine attack and returned to baseline after the cessation of the migraine. In addition, both change and peak, showed significant positive correlations with migraine headache intensity (P<0.001). However, plasma CGRP concentrations failed to change during immediate headache and in the subjects with no migraine attack. Basal CGRP concentration was significantly higher and platelet 5-HT content tended to be lower in subjects who experienced a migraine attack. Platelet serotonin content decreased significantly (P<0.01) after nitroglycerin in subjects with no migraine attack but no consistent change was observed in patients with migraine attack. In conclusion, the fact that plasma CGRP concentration correlates with the timing and severity of a migraine headache suggests a direct relationship between CGRP and migraine. In contrast, serotonin release from platelets does not provoke migraine, it may even counteract the headache and the concomitant CGRP release in this model.     

Investigation of the prevalence of tetQ, tetX and tetX1 genes in Bacteroides strains with elevated tigecycline minimum inhibitory concentrations

In this study, the antibiotic susceptibilities to tigecycline and tetracycline of 35 selected Bacteroides fragilis group strains were determined by Etest, and the presence of tetQ, tetX, tetX1 and ermF genes was investigated by polymerase chain reaction (PCR). tetQ was detected in all 12 B. fragilis group isolates (100%) exhibiting elevated tigecycline minimum inhibitory concentrations (MICs) (≥8 μg/mL) as well as the 8 strains (100%) with a tigecycline MIC of 4 μg/mL, whilst tetX and tetX1 were present in 15% and 75% of these strains, respectively. All of these strains were fully resistant to tetracycline (MIC ≥ 16 μg/mL). On the other hand, amongst the group of strains with tigecycline MICs < 4 μg/mL (15 isolates), tetQ, tetX and tetX1 were found less frequently (73.3%, 13.3% and 46.7%, respectively). All but two strains harbouring the tetQ gene in this group were non-susceptible to tetracycline, with a MIC > 4 μg/mL. These data suggest that in most cases tigecycline overcomes the tetracycline resistance mechanisms frequently observed in Bacteroides strains. However, the presence of tetX and tetX1 genes in some of the strains exhibiting elevated MICs for tigecycline draws attention to the possible development and spread of resistance to this antibiotic agent amongst Bacteroides strains. The common occurrence of ermF, tetX, tetX1 and tetQ genes together predicted the presence of the CTnDOT-like Bacteroides conjugative transposon in this collection of Bacteroides strains.     

Altered Peripheral Invariant Natural Killer T Cells in Atopic Dermatitis

Background  

Conflicting data exist on the number of invariant NKT (iNKT) cells in atopic dermatitis (AD); furthermore, no data have been published on their functional capacity.     

Peritubular capillary basement membrane changes in chronic renal allograft rejection: Comparison of light microscopic and ultrastructural observations

Marked peritubular capillary basement membrane (PTCBM) multilayering, the ultrastructural feature of chronic antibody-mediated rejection (ABMR) of kidney allografts, was found to correspond histologically to PTCs with thickened BMs; such PTCs have been suggested as a novel histological marker of chronic rejection. We investigated whether scoring of PTCBM thickening can substitute the ultrastructural search for PTCBM multilayering. The thickening was graded in PAS- and Jones-stained sections in 110 biopsies from recipients with a late dysfunction, all examined ultrastructurally for transplant capillaropathy (≥3 PTCs with ≥5 BM layers). Grade 0 indicated no thickening. Grade 1 and grade 2 were assigned when the PTCBMs were as thick as or thicker than those of the non-atrophic tubules, and duplication/chain-like lamination of the PTCBM was noted in ≤3 or ≥4 high-power fields, respectively. The series was enrolled in subgroups of those with and those without histopathological lesions of chronic rejection. Fifty-six biopsies displayed lesions of chronic ABMR. Transplant capillaropathy was demonstrated in 40 biopsies. Grade 2 thickening furnished a substantial interobserver concordance rate (κ = 0.803) and correlated with the transplant capillaropathy. Jones staining performed somewhat better in scoring than PAS staining. Grade 2 thickening was verified in 35 biopsies involving chronic ABMR, and in one control biopsy (sensitivity 61.4%, specificity 0.98). Grade 1 thickening was not suggestive of chronic ABMR at all. In conclusion, grade 2 thickening can be regarded as the histopathological lesion of chronic ABMR; however, electron microscopy remains the gold standard in the assessment of PTCBM changes.     

X-linked myotubular myopathy: report of a case with novel mutation

Myotubular myopathy is a well-defined entity within the centronuclear myopathy subgroup of congenital myopathies. The authors present a patient with the most severe X-linked recessive type (XLMTM). A baby boy presented at birth with severe hypotonia, weak spontaneous movements, arthrogryposis, and respiratory insufficiency. Muscle biopsy showed features of myotubular myopathy. The diagnosis was confirmed and further specified by genetic analysis, revealing a novel frameshift mutation (1314-1315insT) of the myotubularin-coding MTM1 gene. This case underlines the importance of interdisciplinary analysis of congenital muscle diseases, including histomorphological and genetic investigations.     

Diastolic function of the heart in mixed connective tissue disease

The authors examined the right and left ventricle functions in patients with mixed connective tissue disease (MCTD) by Doppler echocardiography. Of 51 patients, 20 had temporary pulmonary arterial hypertension in their case history. According to our knowledge, this is the first study examining the use of Doppler echocardiography and tissue Doppler technique in MCTD patients. Of 51 MCTD patients, 20 had pulmonary arterial hypertension (PAH) in the past 2 years. Diameters of the right and left ventricle, systolic and diastolic blood pressure were measured both in the 51 MCTD patients and in the 30 control subjects (mean age 54.8±6.2 years in the case of patients and 54.2±8.8 years in the case of control subjects). To estimate the global ventricle functions, the myocardial performance index—as described by Tei et al. (J Am Soc Echocardiogr 6:838–874, 1996)—was applied, which reflects the ratio of the sum of the isovolumetric contraction and relaxation time as compared to the ejection time. The 20 MCTD patients with PAH received cyclophosphamide therapy for 1 year beside the pulse corticosteroid (CS) therapy. In the case of MCTD patients without PAH, different treatments were used: 12 out of 31 patients were treated with sulfasalazine, 5 of whom received a combination of CS and methotrexate, and 14 took nonsteroid antiinflammatory drugs. In the case of the 51 MCTD patients (20 with PAH and 31 without PAH), diastolic function disorder of the left ventricle was detected; the diastolic E _e/A _a velocity quotient of the lateral mitral anulus was lower (p<0.01), and the mean deceleration time was longer (p<0.001) than that of the control group. The Tei index demonstrated the damage of the global ventricle function. The Tei index of the right ventricle indicated global failure of the right ventricle function in the case of MCTD patients complicated with PAH (Tei index 0.36±0.07 in MCTD with PAH and 0.28±0.04 in MCTD without PAH, p<0.001). The right ventricle function of MCTD patients without PAH was no different from that of the control group. In the case of patients with MCTD, signs of the disorder of the left ventricle diastolic function were observed. Our results suggest that the global impairment of the left ventricle function is the consequence of the disease itself and not the side effect of the treatment. In the case of MCTD patients complicated with PAH, the signs of the right ventricle function impairment proved to be permanent.     

Comparison of a Rapid Molecular Method,the BD GeneOhm Cdiff Assay,to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing Clostridium difficile in Diarrheal Feces

Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.Toxin-producing Clostridium difficile strains are important pathogens among patients who are treated with antibiotics or chemotherapeutic agents not only in the hospital environment but also in the community (3, 6, 10). Since the recognition of outbreaks of C. difficile infection (CDI) caused by C. difficile PCR ribotype 027 in Canada, the United States, and several European countries, rapid and accurate diagnosis of CDI is very important to stop the spread of these strains (7, 8, 19). In addition, the increasing morbidity and mortality rates associated with CDI and the increasing number of recurrences and therapeutic failures also highlight the need for the development of a rapid and reliable detection method for toxigenic C. difficile in diarrheal feces (12).Only a few laboratories routinely use the tissue culture cytotoxicity and toxin neutralization assays for the detection of toxigenic C. difficile in feces, because they are labor-intensive and time-consuming and standardization is very difficult. Due to their rapid turnaround time, enzyme immunoassays (EIAs) that detect toxin A and/or toxin B in stool are used in most laboratories (11, 16). To increase the sensitivity of these tests and in some instances to facilitate epidemiological investigations, culture of C. difficile has become essential. In spite of this, most laboratories use a single toxin detection test on feces for detection of toxigenic C. difficile (4). In the last 10 years, in-house PCR and real-time PCR assays have been developed to detect C. difficile toxin genes. These assays have shown very good sensitivity and specificity and short turnaround times (1, 17). However, widespread use of PCR methods in routine clinical microbiology is limited because these tests require special DNA extraction procedures to eliminate PCR inhibitors from fecal specimens and they cost more than do traditional testing methods.The BD GeneOhm Cdiff assay provides a rapid method for the qualitative detection of the C. difficile toxin B gene (tcdB) in diarrheal specimens from patients suspected of having CDI. This test is based on the amplification of the tcdB gene and the detection of the amplified DNA using fluorogen-labeled probes. Amplification, detection, and interpretation of the results are done automatically by the SmartCycler instrument (Cepheid, Sunnyvale, CA).Our aims were to compare the performance of the BD GeneOhm Cdiff assay to those of the tissue culture cytotoxicity assay and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux, Marcy-l''Etoile, France), for the direct detection of toxins A and B from fecal samples.