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A cross-sectional study was performed with 137 mothers and their children aged 0 - 35.9 months attending day-care centers (DCC) of Parobé and Três Coroas, Rio Grande do Sul, Brazil. The study analyzed the duration of breastfeeding among the children according to DCC in the same mothers' working place (internal DCC) or far from mothers' working place (external DCC). The prevalence of breastfeeding at 3, 6 and 12 months was 60%, 26% and 7%, respectively. There was a high statistically significant association between children in internal DCC and breastfeeding over 3 months of life (X(2)= 18.65; p = 0.00001). Mothers with children in internal DCC breastfed more during their working period then mothers with children in external DCC. The closeness of internal DCC to the working place may give the mothers a sense of serenity and safety. There is also more availability of the mothers to breastfeed during working time,this way maintaining the stimulation to breastmilk production and avoiding the introduction of bottle complements.

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent progress in the definition of molecules involved in immune regulation has led to the discovery of a number of type I membrane glycoproteins with a distinctive, cysteine-rich, repetitive domain structure within their extracellular regions. Because the prototype members of this family are receptors for cytokines (tumor necrosis factor [TNF] and nerve growth factor [NGF]), it was expected that the ligands for the other receptors would possess cytokine-like activities. This prediction has been fulfilled by the cloning of cDNA encoding a series of type II membrane glycoproteins, with homology to TNF, that bind to, and signal through, their cognate receptors. While the biological role of some of these ligand-receptor pairs remains obscure, at least two members of the family, CD40 and Fas, have proven their importance. The human X-linked immunodeficiency, hyper IgM syndrome, is the result of mutations in the CD40 ligand gene, and the Fas and Fas ligand genes are mutated in two mouse strains, lpr and gld, that develop autoimmune disease. These findings, together with other evidence, point to key roles of CD40/CD40 ligand interactions in immune activation, particularly in T-dependent B cell responses, and of Fas/Fas ligand in apoptosis and peripheral tolerance. These molecules, as well as the other ligands of the family, share the property of costimulation of T cell proliferation and are all expressed by activated T cells. More detailed analysis of the expression patterns of ligands and receptors on lymphocyte subpopulations will be necessary to define their different roles in immune activation and suppression.

A key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids is the 3,2-trans-enoyl-CoA isomerase (DCI; EC 5.3.3.8). It catalyzes the transformation of 3-cis and 3-trans intermediates arising during the stepwise degradation of all cis-, mono-, and polyunsaturated fatty acids to the 2-trans-enoyl-CoA intermediates. A genomic clone encoding the human DCI was isolated and characterized by use of the previously cloned human DCI cDNA. The entire gene encompasses approximately 12.5 kb, and the coding sequence is distributed over seven exons. One major and three minor transcription start sites were determined by primer extension analysis. In common with promoters of other housekeeping genes encoding mitochondrial proteins, the GC-rich, immediate 5'-flanking region of the DCI transcription initiation site lacks typical TATA and CAAT boxes; instead, two GC box consensus sequences are present. Introns 2 and 6 contain several Alu repetetive sequences. The human DCI gene locus was assigned to chromosome 16 by use of human-rodent somatic cell hybrids and to chromosome 16p13.3 by chromosomal in situ suppression hybridization studies.

<h4>Unlabelled</h4>In this study, chemiluminescent enzyme immunoassay (CLEIA) has been developed for the measurement of estrogen receptor (ER) in 34 gynecological oncological tissues from 15 cases of endometrial carcinoma, 15 cases of ovarian tumors and 4 cases of other gynecological tumors. The accuracy, specificity and sensitivity of CLEIA for ER were compared with that of dextran-coated charcoal (DCC) and enzyme immunoassay (EIA).<h4>Results</h4>(1) the standard curve of ER-CLEIA showed a significant linear correlation between the luminosity and ER concentration (r = 0.9786, P < 0.001); (2) The results of CLEIA and that of the other two methods were compared; the correlation coefficients were > 0.8 and their corresponding rates were > 90%; (3) the positive rate of ER determined by these three methods showed no significant differences, however, the mean value of ER-CLEIA was the highest among them. It is concluded that the CLEIA has a higher sensitivity than that of the other two methods for the measurement of ER.

Pyogenic liver abscess, which may lead to devastating consequences, is an uncommon medical problem in pediatrics and has generally been reported in compromised hosts. This article describes two patients with beta-thalassemia major and hemochromatosis complicated by Klebsiella pneumoniae liver abscess. One of the patients had severe complications, including subphrenic abscess, pleural effusion and meningitis. To present knowledge, the occurrence of K. pneumoniae liver abscess in patients with beta-thalassemia major has never before been reported in the literature.

<h4>Aims</h4>To investigate colonic metaplasia of goblet and columnar epithelial cells in ileal pouch mucosa; to correlate this with the degree of morphological and inflammatory change; and to assess whether such changes are related to the presence of faecal stasis.<h4>Methods</h4>Biopsy specimens of ileal pouch mucosa were taken from 31 patients (30 with ulcerative colitis, one with familial adenomatous polyposis) either before (eight patients) or after (23 patients) ileostomy closure. A simple morphological technique was used to assess changes in villous height. Inflammatory change was estimated using an established scoring system for pouchitis, and acquisition of colonic antigens was determined by immunohistochemistry using three monoclonal antibodies which recognise components of the two major epithelial cell types in the colorectum. The degree of staining with the monoclonal antibodies was graded and the grades correlated with an index of villous atrophy and with the inflammatory scores.<h4>Results</h4>Five of eight (63%) pre-closure and 15 of 23 (65%) post-closure biopsy specimens showed increased staining with an antibody against components of columnar epithelial cells. One of eight (12%) pre-closure and 15 of 23 (65%) post-closure biopsy specimens stained with an antibody for colonic mucin. Although both types of staining showed a positive correlation with the pouchitis score, they also occurred in the absence of inflammation.<h4>Conclusions</h4>Both goblet and columnar cells acquire colonic characteristics which are incomplete, but may represent a true adaptive response as they can develop in the absence of inflammation. As the change in goblet cells occurs after ileostomy closure, faecal stasis is likely to be a major contributory factor. Changes in columnar cells may occur before ileostomy closure in the absence of faecal stasis.

<h4>Aims</h4>To detect cytoplasmic and nuclear antigens using flow cytometry in acute leukaemia and to use this technique for double marker combinations.<h4>Methods</h4>Cytoplasmic staining was carried out in samples from 40 cases of acute leukaemia with monoclonal antibodies against the myeloid antigen CD13, the lymphoid antigens CD3, CD22, mu chain and the enzymes terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO). The cells were fixed with paraformaldehyde and permeabilised with Tween 20 and Becton Dickinson's FACS lysing solution. Flow cytometry results were compared in the same cases with immunocytochemistry results using the alkaline phosphatase anti-alkaline phosphatase method.<h4>Results</h4>The gentle permeabilisation induced by this method permitted preservation of the membrane antigens and the size and morphology of the cells. The results using flow cytometry were comparable with those obtained using immunocytochemistry, with nearly complete concordance in most cases.<h4>Conclusions</h4>This technique is simple, rapid, sensitive and reproducible and it is suitable for double staining procedures, such as nuclear and cytoplasmic, nuclear and membrane, or cytoplasmic and membrane. It therefore provides a powerful tool for extending the use of immunophenotyping for the diagnosis and follow up of acute leukaemia. It could also be used for the investigation of minimal residual disease.

We report the use of recombinant VIIa (rFVIIa) in the treatment of five ICHs in two factor VIII-deficient patients with inhibitors. In four of five ICHs, rFVIIa was the only factor replacement used at doses of 60-135 micrograms/kg every 2-4 hr for 12-14 days. Hemostasis at the primary site of bleeding was achieved in all cases, and all patients survived with no permanent neurologic deficits. However, the patient who received the highest dose of rFVIIa during the first 4 days of therapy developed clinical symptoms consistent with a cerebral vascular accident of the brainstem characterized by acute onset of truncal ataxia and upward-gaze nystagmus on day 8 of rFVIIa therapy. While receiving rFVIIa therapy for treatment of these five ICHs, four treatment courses were complicated by bleeding at sites other than the primary site, including two episodes of localized oozing at central line insertion sites, two episodes of hemarthrosis, and two episodes of epistaxis. Antifibrinolytic therapy with tranexamic acid was effective in two of these episodes. Laboratory evaluation revealed shortening of the PT, variable shortening without normalization of the APTT, peak factor VII activity levels 7-30-fold higher than normal baseline values, and normal antithrombin III (ATIII) and alpha 2-antiplasmin levels. In four of five ICHs, there was a 20-40% decrease in fibrinogen levels from baseline. The decrease in fibrinogen was accompanied by an increase in fibrin degradation products in 3/5 episodes and a 15-35% decrease in plasminogen activity levels in 2/5 episodes. Tissue factor pathway inhibitor (TFPI) levels remained stable and in the normal range. Although rFVIIa is an effective new therapy for the treatment of ICH in hemophilia patients with inhibitors, its optimal use with respect to safety and efficacy requires further clinical study.

To assess the possibility of substituting our routine method (dextran-coated charcoal, DCC) of determining estrogen (ER) and progesterone receptors (PR) for an enzyme immunoassay technique (EIA), we compared the two methods for determination of the two types of receptor in breast cancer specimens. In terms of sample positivity or negativity, the two techniques agreed in 76 of the 82 samples analyzed for ER (92.7%; p < 0.001), and in 65 out of 75 samples assayed for PR (86.6%; p < 0.001). Quantitative analysis of the data showed a significant correlation between DCC and EIA for both ER (r = 0.84; p < 0.0001) and PR (r = 0.77; p < 0.0001). The results suggest the usefulness of EIA in substituting DCC, although further clinical studies are required to fully evaluate this new method.

<h4>Background</h4>The incidence of estrogen receptor (ER)-positive breast cancer apparently is increasing. It remains unclear whether this increase is due to an improvement in receptor assay sensitivity, a change in patient characteristics, or a change in tumor biology.<h4>Methods</h4>The distribution of ER, tumor size, and patient age for 11,195 tumor specimens gathered from patients nationwide from 1973 to 1992 were analyzed. All assays were performed in a single laboratory. A single-label, dextran-coated charcoal (DCC) method was used from 1973 to 1984, and a dual-label, DCC method, which allows the determination of both ER and progesterone receptor levels in the same assay, was used from 1985 to 1992.<h4>Results</h4>The median level of ER has increased steadily from 14 fmol per milligram of protein in 1973 to 58 fmol per milligram of protein in 1992 (P < 0.0001). The percentage of ER-positive tumors also rose from 73-78% during the same period (P = 0.008). When the assay method was modified from single to dual label, no abrupt or stepwise increase occurred. Tumor size decreased over the same period (P < 0.0001). From 1973 to 1977, 48% of tumors were larger than 2 cm, and 15% were larger than 5 cm, compared to 60% and 9%, respectively, from 1988 to 1992. The percentage of women older than 50 years of age remained relatively constant over time. After adjusting for tumor size, age, number of positive lymph nodes, and change in assay method, a sustained rise in ER level remained. In a multivariate analysis that included age, age group, year of biopsy, tumor size, and number of positive nodes, the year of biopsy still was independently predictive of ER level (P < 0.0001).<h4>Conclusion</h4>The measured level of ER in primary breast cancers has increased during the last 2 decades. It is unlikely that technical improvements or changes in tumor size, age, or nodal status fully explain this increase. The rising level of ER may reflect a change in breast cancer biology and in hormonal events that influence breast cancer genesis and growth.

We have cloned overlapping cDNAs encoding alpha 1E Ca2+ channel subunits from mouse and human brain. We observed that these alpha 1E transcripts were widely distributed in the central nervous system. We also demonstrated the existence of two variants of the human alpha 1E subunit. Comparison of the sequence of these alpha 1E subunits to those from other species suggests that at least four alternatively spliced variants of alpha 1E exist. Expression of human alpha 1E in HEK293 cells and Xenopus oocytes produced high voltage-activated Ca2+ currents that inactivated rapidly (tau approximately 20 ms at 0 mV). The size of the currents obtained were enhanced approximately 40-fold by co-expression with human neuronal alpha 2 and beta Ca2+ channel subunits. alpha 1E currents were insensitive to the drugs and toxins previously used to define other classes of voltage-activated Ca2+ channels. Thus, alpha 1E-mediated Ca2+ channels appear to be a pharmacologically distinct class of voltage-activated Ca2+ channels.

A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.

We have examined a series of human colorectal adenomas, carcinomas and cell lines derived from human colorectal cancer for loss of heterozygosity (LOH) on chromosome 11q22-23 by polymerase chain reaction (PCR) amplification of a microsatellite polymorphism of the dopamine D2 receptor (DRD2) locus. LOH was demonstrated in 5/30 (16.7%) adenomas and 23/68 (33.8%) carcinomas. Only 2/20 (10%) cell lines showed homozygosity which could potentially be as a consequence of LOH. This moderate level of loss in the tumour samples was probably not an underestimation as a result of excessive stromal contamination because high rates (68-77%) have been detected in the same samples on chromosomes 17 and 18. In contrast to a previous report, LOH in carcinomas at 11q22-23 occurred at a lower frequency and was not associated with Dukes' stage, degree of differentiation, mucin production or the location of the cancer. However, a significant association was found between LOH on chromosome 11 and chromosome 14. Thus, inactivation of any putative tumour-suppressor gene at 11q22-23 by LOH is not a very common event in the development of colorectal tumours, but may be biologically significant if accompanied by chromosome 14 deletions.

We have found several genetic changes in the TGF-beta-type II receptor gene in human gastric cancer cell lines resistant to the growth inhibitory effect of TGF-beta. Southern blot analysis showed deletion of the type II receptor gene in two of eight cell lines and amplification in another two lines. The single cell line we studied that is sensitive to growth inhibition by TGF-beta showed no structural abnormalities of the type II receptor gene. Some of the gastric cancer cells resistant to the growth inhibitory effect of TGF-beta express either truncated or no detectable TGF-beta type II receptor mRNAs, whereas the one that retains responsiveness to the growth inhibitory effect of TGF-beta expresses a full-size type II receptor mRNA. Immunoprecipitation followed by Western blot analysis showed parallel changes in TGF-beta type II receptor expression. Our results suggest that one of the possible mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve genetic changes in the TGF-beta type II receptor gene itself or altered expression of its mRNA.

Germ-line mutations in the human adenomatous polyposis coli (APC) gene result in familial adenomatous polyposis, an autosomal dominant disorder characterized by the early onset of multiple adenomatous polyps in the large bowel with a high likelihood of developing colorectal carcinomas. To understand the role of APC in intestinal tumor formation, we have introduced a chain-termination mutation in the 15th exon of the mouse Apc gene and employed it to modify the endogenous gene by homologous recombination in embryonic stem cells. Mice which are heterozygous for the Apc gene modification progressively develop intestinal tumors in a manner that is similar to that observed in patients with familial adenomatous polyposis and in mice which carry a mutation called multiple intestinal neoplasia (Min). Our results indicate that the Apc gene modification is a critical event in the initiation of intestinal tumor formation and results in an autosomal dominant predisposition toward development of spontaneous colonic and intestinal tumors in mice.