Immunolocalization of the prostaglandin E2 receptor subtypes in human bone tissue: differences in foetal, adult normal, osteoporotic and pagetic bone.

PGE(2) is an important mediator of bone metabolism, but the precise localization of its receptors in human bone remains unknown. The present study used specific antibodies against EP(1), EP(2), EP(3) and EP(4) receptors for immunolocalization in normal, osteoporotic and pagetic human adult bone and in human foetal bone. No labelling was obtained for the EP(1) and EP(2) receptors. The EP(3) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes, but only in osteoclasts and some osteoblasts from adult bone. The EP(4) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes and in adult osteoclasts and osteoblasts, but not in adult osteocytes. Our results show differences in PGE(2) receptor expression in foetal and adult human bone but no difference in adult normal compared to pathologic bone. Finally, these results show that the distribution of EP receptors in human osteoblasts in bone corresponds in part to what we recently described in human osteoblasts in culture.

Telomere dysfunction increases mutation rate and genomic instability.

The increased tumor incidence in telomerase null mice suggests that telomere dysfunction induces genetic instability. To test this directly, we examined mutation rate in the absence of telomerase in S. cerevisiae. The mutation rate in the CAN1 gene increased 10- to 100-fold in est1Delta strains as telomeres became dysfunctional. This increased mutation rate resulted from an increased frequency of terminal deletions. Chromosome fusions were recovered from est1Delta strains, suggesting that the terminal deletions may occur by a breakage-fusion-bridge type mechanism. At one locus, chromosomes with terminal deletions gained a new telomere through a Rad52p-dependent, Rad51p-independent process consistent with break-induced replication. At a second locus, more complicated rearrangements involving multiple chromosomes were seen. These data suggest that telomerase can inhibit chromosomal instability.

Frequency of human leukocyte antigen-A 24 alleles in patients with melanoma determined by human leukocyte antigen-A sequence-based typing.

The analysis of immune responses of patients with melanoma has led to the identification of melanoma-associated antigens targeted by T cells. Cytotoxic T lymphocytes recognize peptides from melanoma-associated antigens presented on the cancer cell surface in the context of HLA class I molecules. Immunodominant melanoma-associated antigen epitopes are being evaluated for their ability to immunize patients with advanced melanoma. However, these vaccination efforts are limited by the extensive polymorphism of the HLA class I heavy chain, which occurs in functional domains of the molecule. Patients with melanoma with the HLA-A-24 phenotype were recruited for vaccination with the peptide AFLPWHRLF from the melanoma-associated antigen tyrosinase. This peptide is recognized in association with HLA-A*2402. The HLA-A24 family includes at least 15 alleles whose frequency and ability to present the same peptide are unknown. The distribution of HLA-A24 alleles was studied in a melanoma population for the practical purpose of identifying patients suitable for vaccination with HLA-A*2402 epitopes. An HLA-A locus-specific polymerase chain reaction method followed by sequencing was developed to determine the HLA-A alleles in genomic DNA. HLA-A 24 was also typed in healthy persons of various ethnic backgrounds to further explore the HLA-A24 family. In white persons, the HLA-A*2402 allele was most common (in 85% of white persons and in 97% of the patients with melanoma). Fewer persons carried the HLA-A*2403 allele (13% in all samples, 3% in melanoma patients). Finally, two new alleles, HLA-A*2422 and HLA-A*24 null, were identified. These results suggest that vaccination with HLA-A*2402-associated epitopes has the potential for broad use in this patient population.

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model.

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of down-regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

Sulpiride-induced increases in serum prolactin levels in female rats exposed prenatally to alcohol.

We examined the impact of prenatal alcohol exposure on serum prolactin levels and on the ability of the D2 dopamine antagonist sulpiride to stimulate prolactin release in Long-Evans rats. Pregnant rats were intubated with alcohol (0, 3, or 5 g/kg/day) from gestational day 8 (GD8) to GD20. Adult female offspring were screened for estrous cycle stage. At diestrus, the rats were challenged with a single dose of sulpiride (0, 10, or 40 micrograms/kg) and trunk blood was collected 20 min later. After prenatal exposure to either dose of alcohol, mean basal serum levels of prolactin were about 65% less than the 0 g/kg group, and the 35-40% mean differences from an untreated control group were not significant. Sulpiride produced dramatic dose-dependent increases in serum prolactin levels in all prenatal treatment groups. Across all doses of sulpiride, the group given the higher dose of prenatal alcohol (5 g/kg/day) had significantly lower serum prolactin levels than all other groups. There was no significant interaction between prenatal treatment and sulpiride dose. Neither prenatal alcohol exposure nor sulpiride injections had significant effects on serum corticosterone levels in this study. Although the current results are unclear regarding a baseline decrease in prolactin levels after prenatal alcohol exposure, the overall results suggest that prenatal alcohol exposure decreases prolactin levels but there is no evidence that it does so by altering dopaminergic tone in hypothalamus of female rats.

Construction of K88- and K99-expressing clones of Salmonella typhimurium G30: immunogenicity following oral administration to pigs.

Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 10(11) vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Molecular cloning using immune sera of a 22-kDal minor outer membrane protein of Vibrio cholerae.

Using antisera prepared against live Vibrio cholerae we have selected several recombinant DNA clones, plasmids pPM440, pPM450 and pPM460, encoding the gene for a 22-kDal V. cholerae peptidoglycan-associated-outer-membrane protein. This is a minor protein in V. cholerae but is expressed in large amounts when the cloned gene is present in Escherichia coli K-12, where it is exposed on the cell surface as judged by ELISA. We have localized the gene within the cloned DNA by transposon mutagenesis and deletion analysis followed by analysis of whole cells and minicells to identify the plasmid-encoded proteins. The DNA region encoding the protein seems to be conserved between El Tor and Classical strains as judged by Southern DNA hybridization.

The mannophosphoinositides of Corynebacterium aquaticum.

Besides the monomannophosphoinositide previously reported in Corynebacterium aquaticum small amounts of other, apparently more glycosylated, mannophosphoinositides have been identified in stationary phase cells. Moreover, by labelling cells with [32P]Pi, phosphatidylinositol was found, comprising about 1.5% of the stationary-phase phospholipids. 2. Pulse-chase experiments performed on cells in the late exponential phase of growth further suggested the sequence phosphatidylinositol leads to monomannophosphoinositide as the first step in the biosynthesis of the mannophosphoinositides. 3. Di-and tri-mannophosphoinositides are apparently the main mannophosphoinositides present during exponential growth. Monomannophosphoinositide predominates only in late stationary phase; in the earlier stationary phase, phosphatidylinositol comprises 50% of the phosphoinositide lipid, and tetramannophosphoinositide constitutes much of the remainder. 4. The metabolism and functions of the mannophosphoinositides are discussed, particularly in relation to changes in their composition throughout the growth cycle.