Postnatal development of parvalbumin and calbindin D-28k immunoreactivities in the canine hippocampus

The calcium-binding proteins, parvalbumin and calbindin D-28k, are markers of different classes of GABAergic interneurons and display different functions. The present study was attempted to determine immunoreactivities and colocalization of the parvalbumin and calbindin D-28k in the developing canine hippocampus by immunohistochemistry. The calcium-binding protein-containing neurons showed different developmental patterns. The first appearance of parvalbumin immunoreactive nonpyramidal cells was observed at P7. Parvalbumin immunoreactivity was elicited by the sequence from CA3 to CA1 to reach an adult-like distribution pattern, which was reached at P60, while calbindin D-28k immunoreactivity appeared from P0, including pyramidal and nonpyramidal cells. The characteristic distribution of calbindin D-28k immunoreactive pyramidal cells was clarified by P28, and an adult-like distribution pattern was reached by the end of the second postnatal month. Double-labeled nonpyramidal cells were frequently seen in the subareas, CA3 of P14/CA1-CA2 of P28, where parvalbumin immunoreactive nonpyramidal cells were emerging. These data suggest that the colocalization of the two calcium-binding proteins during development is related closely to the area-specific maturation of parvalbumin expression, although either prenatal expression of calbindin D-28k or parvalbumin was not determined.     

Psychosocial correlates of alcohol intake among women aged 45 to 64 years: The Framingham Study

This study of 749 women, aged 45 to 64 years, investigates the psychological, behavioral, and social correlates of alcohol intake. These data from the Framingham Study are uniquely based on a community sample of women, which results in a normative study of drinking behavior in women. Two measures of alcohol intake were utilized in these analyses: (1) the frequency of alcohol intake over 1 week and (2) drinking vs abstaining from alcohol. Among this sample of women, increased socioeconomic status, worrying about aging, and being easily upset were positively associated with frequency of alcohol intake. The rigid attitude scale was the strongest discriminating variable for drinkers vs nondrinkers. Older women were more likely to be nondrinkers compared to younger women, however, among older women, being a homemaker was significantly associated with increased alcohol intake. Contrarily, younger women who were homemakers were more likely to be abstainers than women employed outside the home. As would be expected, cigarette smoking was associated with drinking alcohol.This research is dedicated in fond remembrance to our friend and colleague Joseph Stokes III, M.D.     

Identification of two phylogenetically related organisms from feces by PCR for detection of Salmonella spp

Two previously reported PCR methods were evaluated to determine whether they are as sensitive and specific as conventional culture methods in detecting Salmonella spp. from feces. Bovine and equine feces were enriched overnight in brain heart infusion broth and assayed using PCR methods and primer sets described by other investigators. A total of 774 fecal specimens were tested using a primer set (invE-A primer set) that amplifies a region spanning the invasin E and A genes of Salmonella enterica serovar Typhimurium. A subset of these fecal specimens (306 of the 774 total) were tested using primers (hisJ primer set) that amplify a portion of the histidine transport J gene. The PCR required 24 h to obtain results, whereas it took 5 to 7 days to identify Salmonella spp. by culture. PCR detection of Salmonella spp. using the hisJ primers and the invE-A primers had a sensitivity of 93.3 and 80%, respectively, and a specificity of 85.6 and 98.6%, respectively, compared with bacterial culture. Amplification of 42 culture-negative fecal specimens (of 306 total specimens) generated a DNA fragment that corresponded to the molecular weight of the amplified hisJ gene. The hisJ-generated amplicons from six culture-negative and six culture-positive specimens were sequenced and analyzed using DNA sequence alignment and phylogenetic analysis software. A neighbor-joining dendrogram of the DNA sequences of both sets of hisJ amplicons revealed two distinct groups-one group of amplicons from culture-positive specimens identical to the hisJ gene of S. enterica serovar Typhimurium and a second group of amplicons from culture-negative specimens that were more closely related to hisJ of S. enterica serovar Typhimurium than to other hisJ sequences present in nucleotide databases.     

Altered expression of G1 regulatory proteins in human soft tissue sarcomas

CONTEXT: Soft tissue sarcomas constitute a heterogeneous group of tumors for which tumorigenesis is not fully understood. Altered cell-cycle regulation may underlie the development and/or progression of human malignancies. However, data concerning the occurrence of cell-cycle aberrations in soft tissue sarcomas are very limited. OBJECTIVES: To detect the abnormal features of cell-cycle regulatory proteins in soft tissue sarcomas and to determine the potential role of these proteins in clinical behavior. DESIGN: The p53 and Rb-cyclin D pathways were investigated by immunohistochemical studies of p53, mdm2, pRb, p16, cyclin D1, and cdk4 proteins, respectively. RESULTS: Of the 67 sarcomas analyzed, nuclear accumulation of p53 was detected in 25 samples (37%), and overexpression of mdm2 was found in 16 samples (24%). Both p53 and mdm2 expression correlated with tumor grade. Abnormalities involving the Rb-cyclin D pathway were identified in all of the tumors by the altered expression of either pRb (72%) or p16 (94%). Fourteen (21%) and 64 (96%) cases demonstrated cyclin D1 or cdk4 expression, respectively. Overexpression of cyclin D1 showed an association with pRb and p53. There was no correlation between pRb, p16, cyclin D1, or cdk4 and tumor grade or relapse. CONCLUSION: Disturbance in the cell-cycle regulatory system involving the p53 pathway and the Rb-cyclin D pathway is relatively frequent in soft tissue sarcomas and may be a contributing factor in the tumorigenesis of these tumors. The alterations in the Rb-cyclin D pathway probably constitute an early event, whereas the abnormalities in the p53 pathway seem to be involved in tumor progression. It is noteworthy that cyclin D1 may play a key role in linking both pathways.     

Effect of bleaching agents on the fluoride release and microhardness of dental materials

The use of bleaching agents has become a popular procedure for whitening teeth. Recently introduced polyacid-modified composite resins (compomers) have several favorable features, such as improved physical properties and fluoride release. Because these two materials have many possibilities to interact in the oral cavity during dental treatment, it is necessary to understand such interaction. To evaluate the effect of a bleaching agent on dental restoratives, three compomers were photopolymerized and then bleached for 1, 2, 3 and 5 days with the use of 30% hydrogen peroxide. Fluoride release, surface microhardness, and surface modifications were evaluated. It was found that the cumulative fluoride release was found to be linearly correlated to the tested periods of bleaching in all compomers. Among the tested compomers, F2000 showed the highest cumulative fluoride release. Bleached compomers became soft because of surface degradation, so the surface microhardness decreased. F2000 showed an apparent crack formation that was not observed in other compomers. The nearly linear correlation between the filler content and microhardness was found in the control samples. However, the same correlation was not observed after the compomers were stored in a bleaching agent or distilled water.     

Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters.

In this investigation, the role of antibodies against Clostridium difficile toxins A and B in protecting hamsters against C. difficile-associated ileocecitis was examined. We also studied the transfer of protection against C. difficile-associated intestinal disease from immunized female hamsters to their infants. Adult female hamsters immunized parenterally with toxoid A or a mixture containing both toxoids A and B were protected against clindamycin-induced C. difficile-associated fatal ileocecitis. On the other hand, hamsters immunized with toxoid B or a broth filtrate from a nontoxigenic strain of C. difficile were not protected against C. difficile-induced ileocecitis. Antibody against the immunizing toxoid could be demonstrated in both the serum and the cecal contents of hamsters. Some infant hamsters from mothers immunized with toxoid A or AB were protected against C. difficile-associated ileocecitis, while infant hamsters from mothers immunized with toxoid B or a nontoxigenic broth filtrate were not protected against disease. Neutralizing antibodies to toxins A and B could be demonstrated in both maternal milk and serum, as well as in infant serum and intestinal contents. Foster-mothering experiments demonstrated that maternal protection of infants against C. difficile-associated ileocecitis was transferred to infant hamsters through breast milk. These results suggest that toxin A may play a more important role in the pathogenesis of C. difficile-associated ileocecitis in hamsters than toxin B. Furthermore, variations in the severity of C. difficile-associated illness in infants and adults may reflect the lack or presence of passively or actively acquired immunity against C. difficile toxins.     

Isolation of endothelial progenitor cells from cord blood and induction of differentiation by ex vivo expansion

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1 beta , fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.     

An anthrax lethal factor-neutralizing monoclonal antibody protects rats before and after challenge with anthrax toxin

Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [K(d)], 2.62 nM) and 3C16C3 (K(d), 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 microg/ml) was approximately one-third that of 3C16C3 (0.63 microg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.     

TEM-EDX study of mechanism of bonelike apatite formation on bioactive titanium metal in simulated body fluid

Bioactive titanium metal, which forms a bonelike apatite layer on its surface in the body and bonds to the bone through the apatite layer, can be prepared by NaOH and heat treatments to form an amorphous sodium titanate layer on the metal. In the present study, the mechanism of apatite formation on the bioactive titanium metal has been investigated in vitro. The metal surface was examined using transmission electron microscopy and energy dispersive X-ray spectrometry as a function of the soaking time in a simulated body fluid (SBF) and complemented with atomic emission spectroscopy analysis of the fluid. It was found that, immediately after immersion in the SBF, the metal exchanged Na(+) ions from the surface sodium titanate with H(3)O(+) ions in the fluid to form Ti-OH groups on its surface. The Ti-OH groups, immediately after they were formed, incorporated the calcium ions in the fluid to form an amorphous calcium titanate. After a long soaking time, the amorphous calcium titanate incorporated the phosphate ions in the fluid to form an amorphous calcium phosphate with a low Ca/P atomic ratio of 1.40. The amorphous calcium phosphate thereafter converted into bonelike crystalline apatite with a Ca/P ratio of 1.65, which is equal to the value of bone mineral. The initial formation of the amorphous calcium titanate is proposed to be a consequence of the electrostatic interaction of negatively charged units of titania, which are dissociated from the Ti-OH groups, with the positively charged calcium ions in the fluid. The amorphous calcium titanate is speculated to gain a positive charge and to interact with the negatively charged phosphate ions in the fluid to form the amorphous calcium phosphate, which eventually stabilizes into bonelike crystalline apatite.     

Defects in the differentiation and function of bone marrow-derived dendritic cells in non-obese diabetic mice

Due to their high immunostimulatory ability as well as the critical role they play in the maintenance of self-tolerance, dendritic cells have been implicated in the pathogenesis of autoimmune diseases. The non-obese diabetic (NOD) mouse is an animal model of autoimmune type 1 diabetes, in which pancreatic beta cells are selectively destroyed mainly by T cell-mediated immune responses. To elucidate initiation mechanisms of beta cell-specific autoimmunity, we attempted to generate bone marrow-derived dendritic cells from NOD mice. However, our results showed low proliferative response of NOD bone marrow cells and some defects in the differentiation into the myeloid dendritic cells. NOD dendritic cells showed lower expressions of MHC class II, B7-1, B7-2 and CD40, compared with C57BL/6 dendritic cells. In mixed lymphocyte reactions, stimulatory activities of NOD dendritic cells were also weak. Treatment with LPS, INF-gamma and anti-CD40 stimulated NOD dendritic cells to produce IL-12p70. The amount of IL-12, however, appeared to be lower than that of C57BL/6. Results of the present study indicated that there may be some defects in the development of NOD dendritic cells in the bone marrow, which might have an impact on the breakdown of self tolerance.