Limiting dilution analysis of CD4 T-cell cytokine production in mice administered native versus polymerized ovalbumin: directed induction of T-helper type-1-like activation.

Polarized expression of T-helper type-1 (Th1)- or Th2-like patterns of cytokine production frequently correlates with disease outcome. Previously, we have described the long-lived reciprocal regulation of ovalbumin (OVA)-specific IgE (> 95% inhibition) and IgG2a (300-800-fold increased) production following administration of high MW OVA polymers (OVA-POL), in both de novo and ongoing OVA (alum)-induced responses. Here, limiting dilution analysis (LDA) was used to compare precursor frequencies of CD4 T cells producing interferon-gamma (IFN-gamma), interleukin-4 (IL-4) or IL-10 following OVA versus OVA-POL exposure in vivo. Adjuvants were not used, so as to circumvent their impact on measurement of precursor frequencies. We found that the two forms of antigen elicited T-cell activation of comparable intensity, as indicated by equivalent precursor frequencies of clonogenic antigen-specific CD4 T cells. However, they elicited qualitatively different cytokine responses. OVA-POL treatment led to 10-fold higher (mean of six independent LDA experiments) frequencies of IFN-gamma-producing cells, and a mean fivefold lower frequency of IL-10-producing cells, than was observed following in vivo administration of unmodified OVA. Thus, the high MW polymerized form of antigen acted to steer commitment of naive (for this antigen) CD4 T-cell activation from a situation in which IL-10 producers outnumbered IFN-gamma-producing cells by a factor of 4:1 (found in mice administered OVA), to one where IFN-gamma producers dominated by a factor of 11:1 (in mice given OVA-POL), i.e. a qualitative shift in the nature of the OVA-specific response induced from Th2-like to Th1-like. In vivo co-administration of anti-IFN-gamma monoclonal antibody (mAb) abolished the capacity of OVA-POL to preferentially elicit Th1-like dominance. Interestingly, although the ratios of IFN-gamma:IL-4 and IFN-gamma:IL-10 OVA-specific precursor frequencies were strongly increased following OVA-POL exposure (mean 18- and 47-fold higher), the frequency of IL-4-producing CD4 T cells did not differ significantly. The data suggest that this modified antigen promotes in vivo commitment of naive T cells towards a Th1-like response, with consequent inhibition of IgE and enhancement of IgG2a responses, not through direct effects on IL-4 production, but via decreased frequencies of IL-10 and increased frequencies of IFN-gamma-producing OVA-specific CD4 cells. Collectively, the data (1) demonstrate the ability to manipulate commitment of antigen-driven CD4 T-cell populations in naive mice to specific patterns of cytokine gene expression, and (2) provide in vivo evidence of the regulatory role played by IFN-gamma in limiting induction and/or expansion of IL-4- and IL-10-producing CD4 cells to protein allergens.     

Benzbromarone disposition and uricosuric action; evidence for hydroxilation instead of debromination to benzarone

Summary Benzbromarone is one of the main uricosuric drugs currently used. We determined plasma concentrations of benzbromarone, bromobenzarone, and benzarone and 24 hour uric acid excretion in ten healthy individuals following fasting application of two different non-micronised benzbromarone brands. In addition we explored the influence of adjusting urinary pH to near neutral values and of concomitant food intake. Benzbromarone was more rapidly absorbed from the test preparation than from the reference preparation; the extent of systemic availability did not differ significantly. Urinary pH adjustment had no clearcut effect, whereas food intake retarded drug absorption (even though not significant because of the variability of the data). Binding of benzbromarone to plasma proteins exceeded 99%. Bromobenzarone and benzarone were not detectable and are unlikely to be major metabolites of benzbromarone. Instead we found two other compounds suggestive of metabolites, one of them being monohydroxilated benzbromarone. The plasma concentrations of the parent compound in one subject exceeded those of the rest of the group, possibly indicating genetic differences in drug metabolism. The uricosuric effect was not related to benzbromarone plasma concentrations.Abbreviations AUC area under the (plasma concentration time) curve - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - t1/2 plasma elimination half life Dedicated to Professor Dr. N. Zöllner on the occasion of his 65th anniversary     

Numerical and structural chromosomal abnormalities detected in human sperm with a combination of multicolor FISH assays

A pair of multicolor FISH assays (X-Y-21 and A-M-16) was developed for human sperm to simultaneously measure sex ratios; aneuploidies involving chromosomes 1, 16, 21, X, and Y; meiotic diploidies; and structural aberrations involving chromosome 1p. Sex ratios in sperm were not significantly different from unity among healthy men. Baseline frequencies of disomic sperm for chromosomes 1, 8, and 21 were similar (6.7 per 10(4) sperm, 95% CI of 5.6-8.1), suggesting that among these three chromosomes, chromosome 21 was not especially prone to nondisjunction. Frequencies of disomy 16 sperm were significantly lower, however (3.5 per 10(4) sperm, 95% CI of 2.0-6.2; P < 0.02). The baseline frequencies of sperm disomy by FISH for chromosomes 16 and 21 were validated against aneuploidy data obtained by the hamster-egg technique for human sperm cytogenetics. The frequencies of X-X, Y-Y, X-Y ("Klinefelter") sperm and sex-null ("Turner") sperm were 5.5, 5.1, 5.5, and 7.8 per 10(4) sperm, respectively. For chromosomes 16 and 21, the frequencies of nullisomic and disomic sperm were similar, suggesting that gain and loss events occurred symmetrically. However, more gain than loss was reported for chromosomes 1, X, and Y. The frequency of MI and MII diploid sperm (with flagella) was approximately 12 per 10(4) (range 8.3-16.7 per 10(4) sperm). Based on flagella data, the frequency of somatic cells in the semen was estimated to be approximately 1.8 per 10(4) sperm. Loss or gain of a portion of chromosome-arm 1p occurred in 5.5 per 10(4) sperm, and the percentage of sperm carrying structural aberrations within the haploid genome as calculated from FISH (1.4%), was similar to that obtained with the hamster-egg technique. These complementary sperm FISH assays have promising applications in studies of chromosomally abnormal sperm after exposure to occupational, medical, and environmental toxicants.     

1例罕见桡侧腕伸肌变异与文献复习

作者于2004年在指导学生对一具90岁男性尸体标本进行解剖时,见其左前臂桡侧腕伸肌区存在有桡侧腕伸副肌(extensor carpi radialis accessorius,ECRA)合并桡侧腕长、短伸肌之间出现两条副肌腱并相互交叉连接的一种罕见的复合变异。     

Adhesion systems in normal breast and in invasive breast carcinoma.

To analyze the role of various elements of the adhesion system in the organization of the normal mammary gland and in breast carcinoma, we have studied simultaneously the expression of integrins, E- and P-cadherins, and cytoplasmic constituents of adherens junctions. In the normal gland, E-cadherin and alpha-catenin are present in luminal epithelial and myoepithelial cells, whereas integrins are more abundant in acinar epithelial and in myoepithelial cells. We demonstrate here that, in addition, myoepithelial cells express much more vinculin and alpha-actinin than luminal epithelial cells, whereas talin and focal adhesion kinase (pp125FAK) are restricted to the basal cell layer. In invasive carcinoma, E-cadherin is usually present although often in reduced amount; different integrin subunits are expressed either by a fraction or by all of the cells or are absent. However, the cytoplasmic components of adherens junctions, such as alpha-catenin, vinculin, alpha-actinin, talin, and pp125FAK, are expressed at low levels or cannot be detected in the carcinoma cells. Our data suggest that 1), in the normal mammary gland, the myoepithelial cells, being particularly rich in integrins and cytoplasmic components of the adherens junctions, play an important role in the maintenance of tissue integrity; 2), in invasive carcinoma, cell aggregates may be maintained due to varying levels of expression of E-cadherin and/or integrins; and 3), interaction of the transmembrane adhesion molecules with the cytoskeleton in carcinoma may be impaired as revealed by reduced levels of expression of alpha-catenin, vinculin, alpha-actinin, talin, and pp125FAK. Importantly, carcinoma cells, when exposed to stroma during invasion, do not acquire the adhesion apparatus characteristic of normal cells in contact with the extracellular matrix.     

维甲酸调变的巨噬细胞在体外对A549癌细胞的作用

The tumoricidal activity of mouse macrophage (M phi) activated with retinoic acid (RA) and the effect of RA in combination with CP on mouse M phi were studied with phase contrast microscopy, light microscopy, and 3HTdR labelling technique. The results of our studies suggest that: (1) RA can activate mouse peritoneal M phi both in vivo and in vitro, and the RA-activated M phi are cytostatic and cytolytic to A549 cells. The degree of activation is related to the concentration or dosage of RA given, and (2) RA can enhance the function of CP-activated M phi. A summation effect was obtained by giving RA in combination with CP. This effect was related to the dosage of RA. When optimal dosage was given in combination with CP, a synergistic action in activating M phi could be obtained. Morphological changes could be seen under phase contrast microscope and light microscope in killed tumor cells.     

Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.