Histologic and Immunohistochemical Analyses of Soft Tissue Sarcomas From brca2-Mutant/ tp53-Mutant Zebrafish Are Consistent With Neural Crest (Schwann Cell) Origin.

The zebrafish ( Danio rerio) provides a powerful model for analyzing genetic contributors to cancer. Multiple zebrafish lines with cancer-associated genetic mutations develop soft tissue sarcomas that are histologically consistent with malignant peripheral nerve sheath tumor (MPNST). The goal of this study was to determine the phenotype of soft tissue sarcomas in a brca2-mutant/ tp53-mutant zebrafish line using immunohistochemical markers that are commonly expressed in mammalian MPNST. We classified 70 soft tissue sarcomas from a brca2-mutant/ tp53-mutant zebrafish cohort as MPNST, undifferentiated sarcoma, or other tumor based on histologic features. The expression of S100, CD57, and glial fibrillary acidic protein (GFAP) was analyzed in nonneoplastic neural tissues and tumor specimens by immunohistochemistry. Each marker was expressed in nonneoplastic neural tissues. In MPNST, S100 and CD57 were widely expressed in neoplastic cells, with greater consistency observed for CD57 expression. In undifferentiated sarcomas, results were variable and correlated to anatomic location. Coelomic undifferentiated sarcomas often exhibited widespread CD57 expression but limited S100 expression. In comparison, ocular undifferentiated sarcomas exhibited limited expression of both CD57 and S100. Overall, CD57 and S100 expression was significantly higher in MPNST than in undifferentiated sarcomas. GFAP was not expressed in any of the tumors. This study identified commercially available antibodies that are useful for analyzing S100, CD57, and GFAP expression in zebrafish. This study further shows a correlation between degree of histologic differentiation and expression of these markers in soft tissue sarcomas from brca2-mutant/ tp53-mutant zebrafish and suggests that these cancers are derived from the neural crest with differentiation toward myelinating Schwann cells.

Genetic diversity of native populations of Croton tetradenius Baill. using ISSR markers.

Brazil has about 300 Croton species in different types of vegetation. Croton tetradenius Baill., which is endemic to the Northeast region and predominant in the Caatinga vegetation, stands out among the several species of this genus. Considering the importance of knowing the genetic variability of a species, the objective of this study was to analyze the genetic diversity of the genotypes of natural populations of C. tetradenius in the State of Sergipe, using ISSR molecular markers. Forty individuals were sampled in four natural populations of the State of Sergipe, Brazil. Thirteen primers were used for DNA amplification using ISSR-PCR, totaling 77 amplified fragments, of which 94.8% were polymorphic. Results of the cluster analysis obtained by the Jaccard's similarity index, using the UPGMA method, resulted in the formation of six distinct clusters. Analysis of molecular variance (AMOVA), used to estimate the genetic variability among populations, revealed significant genetic variance (P < 0.01) between and within the studied populations, and most of the genetic diversity was found (87%) within populations. According to the Jaccard's similarity index, none of the studied plants was genetically identical. CTE210 and CTE305 presented high similarity index (0.76), while CTE105 presented low similarity index (<0.16) with all related individuals. ISSR markers were efficient and allowed the formation of a molecular profile, and had sufficient polymorphism to estimate the genetic variability between the accessions of the studied populations.

Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is relatively rare, yet the economic and social burden is substantial. Having accurate incidence and prevalence estimates would facilitate efficient allocation of healthcare resources. OBJECTIVE: To provide a comprehensive and critical review of the epidemiological literature on ALS. METHODS: MEDLINE and EMBASE (1995-2011) databases of population-based studies on ALS incidence and prevalence reporting quantitative data were analyzed. Data extracted included study location and time, design and data sources, case ascertainment methods and incidence and/or prevalence rates. Medians and interquartile ranges (IQRs) were calculated, and ALS case estimates were derived using 2010 population estimates. RESULTS: In all, 37 articles met the inclusion criteria. In Europe, the median incidence rate (/100,000 population) was 2.08 (IQR 1.47-2.43), corresponding to an estimated 15,355 (10,852-17,938) cases. Median prevalence (/100,000 population) was 5.40 (IQR 4.06-7.89), or 39,863 (29,971-58,244) prevalent cases. CONCLUSIONS: Disparity in rates among ALS incidence and prevalence studies may be due to differences in study design or true variations in population demographics such as age and geography, including environmental factors and genetic predisposition. Additional large-scale studies that use standardized case ascertainment methods are needed to more accurately assess the true global burden of ALS.

Renal lesions of nondomestic felids.

To comprehensively evaluate the occurrence of renal lesions in a variety of nondomestic felids, necropsy cases from 1978 to 2008 were reviewed from a municipal zoo and a large cat sanctuary for those in which the kidneys were examined histologically. Seventy exotic felids were identified (25 tigers, 18 lions, 6 cougars, 5 leopards, 3 snow leopards, 3 clouded leopards, 3 Canadian lynx, 2 ocelots, 2 bobcats, 2 cheetahs, 1 jaguar), and their histologic renal lesions were evaluated and compared. The most common lesion was tubulointerstitial nephritis (TIN); 36 of 70 (51%) cats were affected to some degree. Lymphocytic interstitial nephritis was the most common lesion in the tigers (9 of 25, 36%) and was rarely seen in other species. Although the renal pelvis was not available for all cats, 28 of 47 (60%) had some degree of lymphocytic pyelitis. There was no significant association between the presence of pyelitis and that of TIN. Only 1 cat had pyelonephritis. Renal papillary necrosis was present in 13 of 70 (19%) cats and was significantly associated with historical nonsteroidal anti-inflammatory drug treatment (odds ratio, 7.1; 95% confidence interval, 1.9 to 26.8). Only 1 cat (lion) had amyloid accumulation, and it was restricted to the corticomedullary junction. Primary glomerular lesions were absent in all cats. Intraepithelial pigment was identified in many of the cats but was not correlated with severity of TIN. Despite several previous reports describing primary glomerular disease or renal amyloidosis in exotic felids, these lesions were rare to absent in this population.

Influenza A virus in swine breeding herds: Combination of vaccination and biosecurity practices can reduce likelihood of endemic piglet reservoir.

Recent modelling and empirical work on influenza A virus (IAV) suggests that piglets play an important role as an endemic reservoir. The objective of this study is to test intervention strategies aimed at reducing the incidence of IAV in piglets and ideally, preventing piglets from becoming exposed in the first place. These interventions include biosecurity measures, vaccination, and management options that swine producers may employ individually or jointly to control IAV in their herds. We have developed a stochastic Susceptible-Exposed-Infectious-Recovered-Vaccinated (SEIRV) model that reflects the spatial organization of a standard breeding herd and accounts for the different production classes of pigs therein. Notably, this model allows for loss of immunity for vaccinated and recovered animals, and for vaccinated animals to have different latency and infectious periods from unvaccinated animals as suggested by the literature. The interventions tested include: (1) varied timing of gilt introductions to the breeding herd, (2) gilt separation (no indirect transmission to or from the gilt development unit), (3) gilt vaccination upon arrival to the farm, (4) early weaning, and (5) vaccination strategies of sows with different timing (mass and pre-farrow) and efficacy (homologous vs. heterologous). We conducted a Latin Hypercube Sampling and Partial Rank Correlation Coefficient (LHS-PRCC) analysis combined with a random forest analysis to assess the relative importance of each epidemiological parameter in determining epidemic outcomes. In concert, mass vaccination, early weaning of piglets (removal 0-7days after birth), gilt separation, gilt vaccination, and longer periods between introductions of gilts (6 months) were the most effective at reducing prevalence. Endemic prevalence overall was reduced by 51% relative to the null case; endemic prevalence in piglets was reduced by 74%; and IAV was eliminated completely from the herd in 23% of all simulations. Importantly, elimination of IAV was most likely to occur within the first few days of an epidemic. The latency period, infectious period, duration of immunity, and transmission rate for piglets with maternal immunity had the highest correlation with three separate measures of IAV prevalence; therefore, these are parameters that warrant increased attention for obtaining empirical estimates. Our findings support other studies suggesting that piglets play a key role in maintaining IAV in breeding herds. We recommend biosecurity measures in combination with targeted homologous vaccination or vaccines that provide wider cross-protective immunity to prevent incursions of virus to the farm and subsequent establishment of an infected piglet reservoir.

Transforming growth factor beta inhibitory element in the rabbit matrix metalloproteinase-1 (collagenase-1) gene functions as a repressor of constitutive transcription.

Transforming growth factor beta (TGF-beta) is a potent modulator of the extracellular matrix, enhancing collagen synthesis and regulating expression of several genes that encode the matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix. In this study, we explored the mechanisms whereby TGF-beta inhibits expression of the MMP-1 (collagenase 1) gene. We used transient transfection and gel mobility shift assays to characterize a TGF beta inhibitory element (TIE) at -249 bp in the rabbit MMP-1 promoter, which is also conserved at -246 bp in the human gene. This sequence shares homology to a previously identified TIE in the rat stromelysin-1 (MMP-3) promoter, where it is located at -709 bp. Mutational analyses and transient transfections indicate that MMP-1 TIE functions both as a constitutive repressor of MMP-1 gene expression and, in the presence of TGF-beta, as an antagonist of transcriptional induction by phorbol esters. c-Fos binds to the TIE in the rabbit MMP-1 promoter, along with other nuclear proteins, even in the absence of treatment with TGF-beta. However, the pattern of proteins binding to the TIE is altered in the presence of nuclear extracts from TGF-beta-treated cells, suggesting that TGF-beta leads to an alteration in protein/DNA interaction, with subsequent modulation of MMP-1 gene expression. We conclude that in the rabbit MMP-1 promoter, the TIE has dual functions as a repressor of basal transcription and as a mediator of the biologic effects of TGF-beta. Furthermore, these dual functions provide additional and subtle mechanisms for regulating MMP-1 gene expression under a variety of biological and pathological conditions.

Infusional CHOP chemotherapy (CVAD) with or without chemosensitizers offers no advantage over standard CHOP therapy in the treatment of lymphoma: a Southwest Oncology Group Study.

PURPOSE: Two phase II studies were conducted to evaluate infusional cyclophosphamide, doxorubicin, vincristine, and dexamethasone chemotherapy, termed the CVAD regimen, alone (Southwest Oncology Group [SWOG] 9240) and with the chemosensitizers verapamil and quinine (SWOG 9125) to assess effects on response, survival, and toxicity in intermediate- and high-grade advanced-stage non-Hodgkin's lymphoma (NHL). The results were compared with the historic group of patients randomized to CHOP chemotherapy on Intergroup (INT) 0067 (SWOG 8516). PATIENTS AND METHODS: All patients had biopsy-proven intermediate- or high-grade NHL (lymphoblastic histology excluded), were ambulatory and previously untreated, and had bulky stage II, III, or IV disease. One hundred twelve patients were registered on SWOG 9240 and received cyclophosphamide 750 mg/m(2) by intravenous bolus day 1, doxorubicin 12.5 mg/m(2)/d and vincristine 0.5 mg/d delivered as a continuous 96-hour infusion on days 1 through 4, and dexamethasone 40 mg/d orally on days 1 through 4 (CVAD). Cycles were repeated every 21 days for eight cycles. One hundred patients on SWOG 9125 received the same chemotherapy and the chemosensitizers verapamil 240 mg bid and quinine 40 mg tid. Chemosensitizers were begun 24 hours before chemotherapy and continued for a total of 6 days. RESULTS: Eighty-one patients were eligible for each study. The complete response (CR) rates were 39% on SWOG 9125 and 31% on SWOG 9240. With a median follow-up of 5.8 years on SWOG 9125 and 4.5 years on SWOG 9240, the 2-year failure-free survival (FFS) rate was 42% on SWOG 9125 and 41% on SWOG 9240. Two-year overall survival (OS) rate was 64% on SWOG 9125 and 58% on SWOG 9240. These results are comparable to a 44% CR rate, a 2-year FFS of 46%, and 2-year OS of 63% observed in 225 patients treated with CHOP on INT 0067 (SWOG 8516). CONCLUSION: CVAD combination chemotherapy alone or with the chemosensitizers verapamil and quinine is not promising therapy with respect to improved response or OS in intermediate- and high-grade advanced-stage NHL.

Two activator protein-1 elements in the matrix metalloproteinase-1 promoter have different effects on transcription and bind Jun D, c-Fos, and Fra-2.

Collagenase (matrix metalloproteinase-1, MMP-1) plays a central role in connective tissue metabolism as the only enzyme capable of degrading interstitial collagens at neutral pH. We used fragments of the rabbit collagenase promoter ranging from 1800 to 182 bp to measure transcriptional activity of the activator protein-1 (AP-1) site at -77. Mutation at -77 in this sequence greatly reduced basal transcription in all constructs. However, mutant constructs with at least 321 bp of promoter responded to phorbol myristate acetate, similar to their native counterparts, implicating upstream regions in mediating this response. Through mutagenesis and analysis of DNA-protein interactions, we also identified and characterized a novel AP-1 site at -186. Mutation at -186 in 321 bp of promoter modestly lowered basal activity but, in contrast to mutation at -77, reduced phorbol responsiveness by 50%. Mobility shift assays demonstrated specific inducible binding at both sites. DNA/protein complexes at both AP-1 sites contain c-Fos and Jun D proteins, while Fra-2 is present only at the -77 site. These studies (1) demonstrate cooperativity between these two AP-1 sites, (2) implicate the -186 site in phorbol inducibility and (3) identify specific members of the Fos and Jun families binding to these sites.

Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription, and mRNA stability.

Matrix metalloproteinase-1 (MMP-1) is one of three collagenases that can degrade the interstitial collagens, types I, II, and III at neutral pH. As these collagens are the most abundant proteins in the body, collagenase plays a critical role in modeling and remodeling the extracellular matrix. Therefore, it is not surprising that MMP-1 gene expression can be regulated at multiple points. Procollagenase can be activated by mechanisms that generate an active enzyme with differing specific activities, and the active enzyme can be inhibited by complexing with either the tissue inhibitor of metalloproteinases (TIMPs) or alpha 2 macroglobulin. The activator protein-1 (AP-1) site in the collagenase promoter plays a prominent role in the transcriptional control of the collagenase gene. It is essential for basal transcription, and contributes to induction by phorbol esters, although other sites in the proximal promoter are essential. In contrast, transactivation by cytokines such as Interleukin-1 depends on sequences in more distal regions of the promoter. Posttranscriptional mechanisms also regulate gene expression, and several cytokines and growth factors increase the stability of the collagenase transcript. Finally, glucocorticoid hormones repress transcription of the collagenase gene by the interaction of glucocorticoid receptors with the AP-1 proteins, Fos and Jun. Retinoids also suppress transcription by mechanisms that involve down-regulation of fos and jun mRNA, sequestration of Fos and Jun proteins, and the formation of complexes of retinoic acid receptors (RAR/RXR heterodimers) and AP-1 proteins on the DNA. These multiple points of regulation assure precise control of collagenolytic activity in a variety of physiologic and pathologic conditions.

Differentiation of an immortalized CNS neuronal cell line decreases their susceptibility to cytotoxic T cell lysis in vitro.

RN33B cells are a temperature-sensitive neuronal cell line derived from rat E12 medullary raphe nucleus (Whittemore and White (1993) Brain Research 615, 27-40). Undifferentiated RN33B cells express class I but not class II antigens of the major histocompatibility complex (MHC), and intercellular adhesion molecule-1 (ICAM-1), a ligand for lymphocyte function associated antigen-1 (LFA-1), expressed on cytotoxic T lymphocytes (CTLs). Treatment of undifferentiated RN33B cells with interferon-gamma (IFN-gamma) upregulated both class I MHC and ICAM-1. After neuronal differentiation, expression of class I MHC antigens or ICAM-1 was undetected, even after IFN-gamma treatment. The neuronally differentiated RN33B cells were also markedly less susceptible to lysis by alloantigen-specific CTLs. These data suggest that intrinsic to the differentiation of CNS neurons is a mechanism to escape CTL-mediated cell lysis.

In vitro labeling strategies for identifying primary neural tissue and a neuronal cell line after transplantation in the CNS.

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS transplantation studies were tested by first characterizing the labels in vitro. The labels that were tested included 4',6-diamidino-2-phenylindole hydrochloride, 1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate-conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3'-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or tetanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in vivo after CNS transplantation. In vitro, 1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neurons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Most labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retained in cellular debris. In vivo, DiI was visualized in transplanted, DiI-labeled raphe neurons, but not in astrocytes up to 1 mo posttransplant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. beta-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No beta-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels for use in CNS transplantation studies should be optimized for the specific population of donor cells under study, with the initial step being characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be assessed.

Do HMOs care for the chronically ill?

This DataWatch refutes the notion that chronic illness is more prevalent among persons covered by indemnity insurance than by health maintenance organizations (HMOs). This is true even when health status and sociodemographic factors are accounted for in the analysis. The study analyzes the prevalence of chronic illness among privately insured nonelderly persons in HMOs and indemnity plans, using data from the 1992 National Health Interview Survey. More data are needed to examine this issue for Medicare and Medicaid populations.

Target regulation of neuronal differentiation in a temperature-sensitive cell line derived from medullary raphe.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen, a clonal cell line, RN33B, was isolated by serial dilution. At 33 degrees C, RN33B cells divide with a doubling time of 48 h and show T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. RN33B cells are immortal, but not transformed, as they will not grow in soft agar. At non-permissive temperature (38.5 degrees C), T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33 degrees C re-express T antigen, but do not de-differentiate or begin dividing. Co-culture with embryonic hippocampus and cerebral cortex, but not medullary raphe or spinal cord, resulted in significantly greater survival, more complex neuronal morphology, and enhanced expression of neuronal-specific antigens. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. These data suggest that the observed target regulation of RN33B cell neuronal differentiation in co-culture may be mediated by neurotrophin(s).

Acidic and basic fibroblast growth factor levels in spinal cord cultures are not regulated by alterations in heparan sulfate proteoglycan expression.

The present study was undertaken to assess both the levels of acidic and basic fibroblast growth factors in spinal cord cultures and to determine how they were presented to responsive cells. Western blots detected a single acidic fibroblast growth factor-like protein (17 kDa) and two (18 kDa, 24 kDa) basic fibroblast growth factor-immunoreactive proteins, the levels of which varied with the antibody used. Levels of all three proteins were unaltered in cultures grown in the presence of a mitotic inhibitor, which greatly reduced the number of astrocytes. Cell blots showed increased survival of spinal cord neurons at Mr that corresponded with the three proteins detected immunologically. Solubilized cultures separated on a P100 column showed mitogenic activity for NIH3T3 cells from 17-18 and 24 kDa fractions. Treatment of the cultures with heparitinase did not decrease the levels of acidic and basic fibroblast growth factors detected by Western blots, suggesting that these proteins were not associated with extracellular membrane heparan sulfate proteoglycans. The major fraction of both proteins appeared to be intracellular with a minor amount complexed with extracellular matrix proteins. An inhibitor of xylose-linked proteoglycan synthesis significantly altered heparan sulfate proteoglycan deposition into extracellular matrix, but did not alter the levels of acidic or basic fibroblast growth factors detected by Western blots, or the levels of choline acetyltransferase, glutamic acid decarboxylase, or aspartate aminotransferase activities. These results indicate that both acidic and basic fibroblast growth factors are stored predominantly intracellularly, with only a minor fraction complexed with extracellular proteins. We suggest that these intracellular proteins may be released following injury in the CNS and mediate a cascade of neuroprotective events.

Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin.

A patient with Haemophilus aphrophilus endocarditis was successfully treated with ciprofloxacin. The response to treatment with cefotaxime and netilmicin for 12 days was poor but was satisfactory to a 6 weeks' course of ciprofloxacin.

Genotyping of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease by Repetitive Elements-Based Polymerase Chain Reaction and DNA-DNA Hybridization.

ABSTRACT The bacterium that causes cucurbit yellow vine disease (CYVD) has been placed in the species Serratia marcescens based on 16S rDNA and groE sequence analysis. However, phenotypic comparison of the organism with S. marcescens strains isolated from a variety of ecological niches showed significant heterogeneity. In this study, we compared the genomic DNA of S. marcescens strains from different niches as well as type strains of other Serratia spp. through repetitive elements-based polymerase chain reaction (rep-PCR) and DNA-DNA hybridization. With the former, CYVD strains showed identical banding patterns despite the fact that they were from different cucurbit hosts, geographic locations, and years of isolation. In the phylogenetic trees generated from rep-PCR banding patterns, CYVD strains clearly were differentiated from other strains but formed a loosely related group with S. marcescens strains from other niches. The homogeneity of CYVD strains was supported further by the DNA relatedness study, in that labeled DNA from the cantaloupe isolate, C01-A, showed an average relative binding ratio (RBR) of 99%, and 0.33% divergence to other CYVD strains. Used as a representative strain of CYVD, the labeled C01-A had a RBR of 76%, and a 4.5% divergence to the S. marcescens type strain. These data confirm the previous placement of CYVD strains in S. marcescens. Our investigations, including rep-PCR, DNA-DNA hybridization, and previous phenotyping experiments, have demonstrated that CYVD-associated strains of S. marcescens cluster together in a group significantly different from other strains of the species.

Varicose veins as a source of adult human endothelial cells.

Endothelial cells can be harvested from segments of adult human saphenous vein in a varicose condition removed from patients having single or bilateral vein ligation and stripping. The cells are harvested by scraping with a scalpel, seeded on to gelatin coated or Primaria flasks and are passaged by removal with a rubber policeman. The cells cultured in this manner are maintained in a growth medium that is not supplemented with growth factors. The cells grow with a cobblestone monolayer morphology, possess angiotensin converting enzyme activity and react with antibodies to Factor VIII antigen. The cells fluoresce brightly after reaction with monoclonal antibodies specific for human endothelial cells. Thus, stripped varicose vein segments provide a readily available source of endothelial cells.

Use of microcarriers to isolate and culture pulmonary microvascular endothelium.

Microcarriers of known diameter can be used to collect endothelial cells from microvessels of the same or slightly smaller internal diameter. The procedure is illustrated by collection of endothelial cells from rabbit pulmonary pre-capillary vessels. The lungs are perfused free of blood with physiological saline and then cold vessels. The lungs are perfused free of blood with physiological saline and then cold (4 degrees C) saline (containing EDTA, 0.02%, and microcarriers 600/ml; 40-60 micrometers diameter) is perfused via the pulmonary artery. The direction of flow is reversed periodically to collect the bead-cell harvest from the arterial side. Cold shock and EDTA cause the endothelial cells to detach from the vessels under conditions such that the cells remain attached to the microcarriers. The selective attachment to microcarriers is apparently aided by the tight fit of the beads within vessels of the same diameter. Beads do not emerge on the venous side, all being trapped at the pre-capillary level. Electron microscopic examination of lungs fixed during the perfusion shows that the beads lodge in terminal arterioles and pre-capillary vessels (approximately 40-60 micrometers in diameter, with one, sometimes incomplete, muscle layer). Endothelial cells recovered on microcarriers can be allowed to migrate on to flasks and back on to beads. The resultant cultures have an endothelial morphology and possess high levels of angiotensin coverting enzyme and carboxypeptidase N activity.

Brewers dried yeast as a source of mannan oligosaccharides for weanling pigs.

Brewers dried yeast, a source of mannan oligosaccharides (MOS), was assessed as an alternative to an antimicrobial agent (carbadox) for young pigs in two experiments. The yeast contained 5.2% MOS. Agglutination tests confirmed adsorption of several serovars of E. coli and Salmonella spp. onto the yeast product. In Exp. 1, seven replicates (five pigs per pen) of 22-d-old pigs were fed a nonmedicated basal diet or the basal diet with carbadox (55 mg/kg), yeast (3%), or a combination of 3% yeast and 2% citric acid for 28 d. Carbadox did not improve growth performance. Growth rate and feed intake were depressed (P < 0.05) in pigs fed yeast alone or in combination with acid. Log counts of total coliforms, Escherichia coli, and Clostridium perfringens in feces were not affected by diet, but Bifidobacteria spp. counts were lower (P < 0.05) in pigs fed the yeast + acid diet and lactobacilli counts were higher (P < 0.05) in pigs fed yeast. Fecal pH and VFA concentrations and intestinal morphological traits were not consistently affected by diet. Serum IgG levels were elevated in the yeast + acid (P < 0.01) group. In Exp. 2, the effects of yeast and carbadox additions to the diet on enteric microbial populations in young pigs housed in isolation units were evaluated. Pigs (n = 24) were weaned at 11 d of age (4.1 kg BW) and placed in isolation chambers (two pigs per chamber) equipped with individual air filtering systems and excrement containers. Treatments were a nonmedicated basal diet and the basal diet with 55 mg/kg of carbadox or with 3% yeast. Diets were fed for 29 d, then each pig was orally dosed with approximately 9.5 x 10(8) CFU of E. coli K88. Daily fecal E. coli K88 counts were not different (P > 0.05) among treatments, but fecal shedding of carbadox-resistant coliforms was higher (P < 0.01) during the 9-d period in pigs fed carbadox. Total fecal coliforms were consistently lower throughout the postinoculation period in pigs fed yeast (P < 0.05). Yeast reduced colonization oftotal coliforms in the duodenum,jejunum, cecum, and colon, but it did not have a consistent effect on colonization of E. coli K88. Pigs fed yeast tended (P < 0.10) to have higher serum IgG levels than controls. In these experiments, brewers dried yeast and carbadox had minimal effects on growth, microbial populations, and intestinal health traits of early-weaned pigs, but certain serum immunological traits were enhanced by feeding yeast.

Written information about epidural analgesia for women in labour: did it improve knowledge?

There is evidence that despite a distressed appearance, women in labour should be informed about the side effects and risks associated with epidural analgesia. An audit of 100 women who had used epidural analgesia for labour in our hospital and who had received a verbal explanation of the benefits, risks and side effects of epidural analgesia showed that the level of knowledge was low. An A5 laminated epidural information card was prepared summarising this information. The midwife and the anaesthetist used the card during labour as a focus for verbal discussion and as written reinforcement for the woman and her partner. A repeat audit of a further 100 women showed a statistically significant improvement in the level of knowledge about epidural analgesia. This audit suggests that the use of a written information card is beneficial. It improves and reinforces the process of giving information thus assisting the consent process.