Protocol for ultrarapid immunostaining of frozen sections

Rapid immunostaining of frozen sections within a tolerable time span would be very helpful for intraoperative diagnosis. A protocol was therefore established using the enhanced polymer one-step staining (EPOS) system (Dako) with antibodies against leucocyte common antigen (LCA), cytokeratin (CK), and anti-melanoma (MEL). Best results with reliable and specific immunostaining and a labelling intensity comparable to standard immunostaining protocols were achieved with fixation of samples in 100% acetone for 20 seconds (CK, LCA) or two minutes (MEL), followed by incubation of the primary antibody and development of the chromogen reaction with 3,3'diaminobenzidine (DAB) for three and five minutes at 37 degrees C, respectively. The total procedure takes only 12 minutes, thus enabling rapid immunostaining on intraoperative frozen sections. Apart from its use in tumour classification, this method is especially useful in detecting tumour cells in sentinel lymph nodes.     

Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates

The prevalence of recently described mutation V176F, located in the beginning of the rpoB gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of rpoB mutations among 80 rifampin (RIF)-resistant Mycobacterium tuberculosis strains isolated in Germany during 1997. The most frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of rpoB mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical rpoB mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in M. tuberculosis. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance.     

Fatal disseminated Trichoderma longibrachiatum infection in an adult bone marrow transplant patient: species identification and review of the literature

Trichoderma longibrachiatum was recovered from stool surveillance cultures and a perirectal ulcer biopsy specimen from a 29-year-old male who had received an allogeneic bone marrow transplant for acute lymphoblastic leukemia. The amphotericin B (2.0 microgram/ml) and itraconazole (1.0 microgram/ml) MICs for the organism were elevated. Therapy with these agents was unsuccessful, and the patient died on day 58 posttransplantation. At autopsy, histologic sections from the lungs, liver, brain, and intestinal wall showed infiltration by branching septate hyphae. Cultures were positive for Trichoderma longibrachiatum. While Trichoderma species have been recognized to be pathogenic in profoundly immunosuppressed hosts with increasing frequency, this is the first report of probable acquisition through the gastrointestinal tract. Salient features regarding the identification of molds in the Trichoderma longibrachiatum species aggregate are presented.     

Long-term adaptation to prism-induced inversion of the retinal images

For 1 week, healthy human participants ( n=7) were devoid of normal vision by exposure to prism lenses that optically rotated their perceived world around the line of sight by 180 degrees. Adaptation to such prisms involved sustained and vigorous practice of the ability to redirect the unadapted efferent motor command; because prior to all visually guided movements, the to-be-executed efferent command was based on incorrect (prismatically reversed) spatial information. The time course of this sort of adaptation was systematically explored in Cooper-Shepard mental rotation (MR) tests and in naturalistic motor-tasks for the purpose of investigating whether mental rotations of the direction of the intended movement share common aspects with the process of MR. A control group ( n=7) intermittently exposed to the distorted spatial organization of the central visual field was studied in parallel. The main results were as follows: (a) the MR reaction times (RTs) day 1 with prisms appeared to be very similar to the normal RTs (day 1, no-prisms) with the one exception that subjects now responded within a prism (rotated) frame of spatial reference rather than within the environmentally upright. The visuomotor performance became grossly irregular and dysmetric. (b) The majority of the visuomotor adaptation functions began to level off on the 3rd day. (c) The increases in natural motor proficiency were accompanied by a systematic and noticeable decrease in magnitude of the MR Y-intercept obtained from the linear regression line calculated between each subject's RT and the various stimulus angles. MR slopes were stable through days 1-7 for both the experimental and control group. An increased correlation between rotational stimulus angle and RT suggested that the MR function also became progressively more tightly coupled to the stimulus angles. (d) Postadaptation measures of performance indicated the occurrence of selective and minimal adaptation in the natural motor tasks only. It is suggested that these results reflect an improved attentional (strategic) ability to replace incorrect (error producing) control signals with correct (error reducing) control signals. As a result, perceptual-motor start-up processes directly related to spatial coding and to the planning, initiation and correction of the intended direction of motor-or-mental movement improved while the subprocess ("stage") concerned with transformations of such movements remained unchanged. Visuomotor adaptation to inverting prisms engages, and thereby stimulates, a cortical system also invoked in the preparatory process of MR.     

Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3

Progesterone stimulation of Xenopus oocyte maturation requires the cytoplasmic polyadenylation-induced translation of mos and cyclin B mRNAs. One cis element that drives polyadenylation is the CPE, which is bound by the protein CPEB. Polyadenylation is stimulated by Aurora A (Eg2)-catalyzed CPEB serine 174 phosphorylation, which occurs soon after oocytes are exposed to progesterone. Here, we show that insulin also stimulates Aurora A-catalyzed CPEB S174 phosphorylation, cytoplasmic polyadenylation, translation, and oocyte maturation. However, these insulin-induced events are uniquely controlled by PI3 kinase and PKC-zeta, which act upstream of Aurora A. The intersection of the progesterone and insulin signaling pathways occurs at glycogen synthase kinase 3 (GSK-3), which regulates the activity of Aurora A. GSK-3 and Aurora A interact in vivo, and overexpressed GSK-3 inhibits Aurora A-catalyzed CPEB phosphorylation. In vitro, GSK-3 phosphorylates Aurora A on S290/291, the result of which is an autophosphorylation of serine 349. GSK-3 phosphorylated Aurora A, or Aurora A proteins with S290/291D or S349D mutations, have reduced or no capacity to phosphorylate CPEB. Conversely, Aurora A proteins with S290/291A or S349A mutations are constitutively active. These results suggest that the progesterone and insulin stimulate maturation by inhibiting GSK-3, which allows Aurora A activation and CPEB-mediated translation.     

Decreased markers of atopy in children with presumed early exposure to allergens, unhygienic conditions, and infections.

BACKGROUND: Several risk factors for the development of asthma and atopic disease in children have been described. Furthermore, there is consistent evidence that the prevalence of atopy increases with higher socioeconomic status. The knowledge about risk factors and preventive factors for atopy needs to be improved. OBJECTIVE: To compare 2 child populations (foster care and reference children) with different risk and protective factors for the development of atopy. METHODS: The study group consisted of 415 children, living in all 10 community foster homes in Lodz, a large industrial city in Poland. The study was performed from April 2, 2004, to April 30, 2006. The reference group consisted of 500 children, living with their parents at home, recruited from primary care centers. The primary outcome measures were skin prick test results and specific IgE in serum. Secondary outcomes included symptoms of allergic diseases and family history, including life conditions in early childhood. RESULTS: The full analysis set included 408 study children and 402 reference children. Significant differences were observed in the prevalence of atopy between the study and reference groups (11.3% vs 25.9%). We observed more positive skin prick test results in children from the reference group than in study children. To explain this phenomenon, we selected 16 variables that differ in both groups in early life and relate these to atopy. We found that the more cumulative features characteristic of the foster home population (poor living conditions), the lower the risk of atopy. CONCLUSION: Extremely unfavorable environmental circumstances, which are characteristic of the foster home population during early childhood, might prevent from atopy.     

c-Fos-dependent induction of the small ras-related GTPase Rab11a in skin carcinogenesis

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.     

Calcitonin gene-related peptide (CGRP) activates human neutrophils--inhibition by chemotactic peptide antagonist BOC-MLP.

The effect of the neuropeptides substance P, neurokinin A and alpha-calcitonin gene-related peptide (CGRP) on human neutrophil granulocytes was investigated. Substance P induced secondary granule secretion at a concentration of 100 microM. CGRP induced a significant secretory response at 10 microM and thus appeared to be about 10 times more potent than substance P. Calcitonin and a fragment of CGRP, CGRP(8-37), had no effect on neutrophil degranulation. The chemotactic peptide antagonist BOC-MLP (100 microM) inhibited lactoferrin secretion mediated both by CGRP and chemotactic peptide FMLP almost completely, while secretion in response to tumour necrosis factor (TNF) was unaffected. Results from receptor binding studies showed that CGRP and N-formyl-methionyl-leucyl-phenylalanine (FMLP) do not compete for binding. This indicates that CGRP does not exert its effects by binding to the chemotactic peptide receptor. CGRP induced a rapid increase in the cytosolic-free calcium concentration and this increase was not, unlike that induced by FMLP, abolished by preincubation of the cells with pertussis toxin (1000 ng/ml). Therefore CGRP signal transduction in neutrophils appears to involve rapid changes in the cytosolic-free calcium concentration but not a pertussis toxin-sensitive G-protein. In summary, this is the first report to show that CGRP can directly activate neutrophil granulocytes, and this probably occurs via a cell surface receptor which is distinct from that of FMLP although both the CGRP and FMLP-mediated effects can be blocked by BOC-MLP.     

Chromosome 8p deletions are associated with invasive tumor growth in urinary bladder cancer.

Alterations of chromosome 8, including deletions of 8p, occur frequently in many tumors. In this study, fluorescence in situ hybridization was used to study the relationship between 8p deletions, 8q gains, and phenotype in bladder cancer. Cells from 87 tumors were examined by dual-labeling fluorescence in situ hybridization with a centromere 8 probe (pJM12) and P1 probes for 8p22, 8p12, 8q12, and 8q24. Both 8p22 deletions and 8q24 gains were strongly associated with tumor phenotype. There was a marked difference in 8p22 deletions between noninvasive (pTa) tumors (3/33) and minimally invasive (pT1) tumors (8/19; P = 0.005) whereas there was no significant difference between pT1 and muscle-invasive (pT2-4) tumors (19/35; P = 0.3926). Six tumors with 8p22 deletion were examined at 8p12. Three of these tumors showed no 8p12 deletion, narrowing down the site of a putative tumor suppressor gene distal to 8p12. In one other case, there was a marked increase in 8p12 copy number (> 40 per cell; amplification), suggesting the presence of an oncogene involved in bladder cancer at 8p12. The marked difference in 8p22 deletions between noninvasive (pTa) and minimally invasive (pT1) tumors is consistent with a role of a putative tumor suppressor gene on 8p for development of invasive tumor phenotype.     

Cytokeratin subsets can reliably distinguish Barrett's esophagus from intestinal metaplasia of the stomach

The histological distinction between intestinal metaplasia involving the distal esophagus (Barrett's esophagus [BE]) and intestinal metaplasia of the stomach has important clinical implications and can be difficult even with the use of histochemical mucin stains. Cytokeratin (CK) 7 and 20 are cytoplasmic structural proteins that show restricted expression in normal and malignant epithelia of the gastrointestinal tract. The aim of this study was to determine the use of CK7 and 20 expression in the histological distinction of BE from gastric intestinal metaplasia. CK7 and 20 immunostaining was performed on randomly selected surgical resection (n = 31) and biopsy specimens (n = 34) from patients with long-segment BE and gastric resection specimens (n = 11) and gastric cardia biopsy specimens (n = 13) in patients with histological evidence of intestinal metaplasia. A unique pattern of immunoreactivity designated the Barrett's CK7/20 pattern showed superficial CK20 staining and strong CK7 staining of both superficial and deep glands in 29 of 31 (94%) esophageal resection specimens and 34 of 34 (100%) esophageal biopsy specimens form patients with long-segment BE. A Barrett's CK7/20 pattern was not observed in gastric cardia biopsy specimens (n = 13) or gastric resection specimens (n = 11) in patients with histological evidence of intestinal metaplasia. The sensitivity, specificity, and positive predictive value of a Barrett's CK7/20 pattern for a diagnosis of long-segment BE was 97%, 100%, and 100%, respectively. CK7 and 20 reactivity patterns can reliably identify the location of intestinal metaplasia in the esophagus and stomach using histological material from both routine endoscopic biopsy and surgical resection specimens.