Modulation of experimental autoimmunity: treatment of adjuvant arthritis by immunization with a recombinant vaccinia virus.

Live recombinant vaccinia viruses, expressing antigens from pathogenic microorganisms, are studied for their use as vaccines designed for the protection against infectious diseases. Infections with these vaccinia virus recombinants, expressing proteins or epitopes from viruses, parasites, or bacteria, have resulted in the development of specific neutralizing antibodies or cytotoxic T lymphocytes. Here, we describe the generation of a recombinant vaccinia virus expressing the mycobacterial 65-kDa heat shock protein (HSP65). A vaccinia recombinant virus was constructed by placing the gene for the Mycobacterium bovis BCG HSP65 under control of a vaccinia virus promoter and inserting this mycobacterial gene in the thymidine kinase locus of the vaccinia virus genome. Mycobacterial HSP65 is a critical antigen in the autoimmune model of adjuvant arthritis induced in Lewis rats by the immunization with Mycobacterium tuberculosis. We report the induction of immunity directed to this mycobacterial HSP65 by testing for the presence of specific antibodies and T-cell proliferation. Furthermore, induction of such immunity resulted in a reduction of arthritis severity when given to rats before or, even more interestingly, during development of arthritis. Disease reduction was not found after administration of HSP65 in the absence of vaccinia virus as a vector when given during arthritis development. Therefore, recombinant vaccinia virus may offer new prospectives for specific intervention in autoimmunity.     

Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism,multilocus sequence typing,and short repeat sequencing: strain diversity,host range,and recombination

Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.     

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.     

K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung

BACKGROUND. The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas. METHODS. We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes. RESULTS. Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001). CONCLUSIONS. The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.     

Alcohol sales to pseudo-intoxicated bar patrons.

OBJECTIVES: Many establishments serve alcoholic beverages to obviously intoxicated patrons despite laws against such sales. To guide the development of interventions to reduce these illegal alcohol sales, this study used actors feigning intoxication to determine whether servers recognized obvious signs of intoxication and to assess the tactics servers used when dealing with intoxicated patrons. METHODS: Male actors ages 30 to 50 acted out signs of obvious intoxication as they attempted to purchase alcoholic beverages. If served during the first attempt, these pseudo-intoxicated buyers made second purchase attempts during the same visit. Observers accompanied the actors; after each visit, actors and observers recorded the servers' behavior and comments. RESULTS: Alcoholic beverages were served to actors portraying intoxicated patrons at 68% of first purchase attempts and 53% of second purchase attempts (62% of a total of 106 purchase attempts). The most common refusal technique was a direct refusal (68% of refusals), made with either no excuse or with reference to the actors' apparent intoxication level. Servers' second most commonly used refusal technique was offering alcohol-free beverages, such as coffee or water (18% of refusals). CONCLUSIONS: Further research is needed to determine why servers who recognize intoxication serve alcoholic beverages and what training, outlet policies, and external pressures are needed to reduce illegal alcohol sales to obviously intoxicated patrons.     

Role of blood-brain barrier P-glycoprotein in limiting brain accumulation and sedative side-effects of asimadoline, a peripherally acting analgaesic drug.

Studies with knockout mice lacking mdr1a P-glycoprotein (P-gp) have previously shown that blood-brain barrier P-gp is important in preventing the accumulation of several drugs in the brain. Asimadoline (EMD 61753) is a peripherally selective kappa-opioid receptor agonist which is under development as a therapeutic analgaesic. From the structural characteristics of this drug and its peripheral selectivity, we hypothesized that it is transported by P-gp. Using a pig-kidney polarized epithelial cell line transfected with mdr cDNAs, we demonstrate that asimadoline is transported by the mouse mdr1a P-gp and the human MDR1 P-gp. Furthermore, we show that in mdr1a/1b double knockout mice, the absence of P-gp leads to a 9 fold increased accumulation of asimadoline in the brain. In line with this accumulation difference, mdr1a/1b (-/-) mice are at least 8 fold more sensitive to the sedative effect of asimadoline than wild-type mice. Interestingly, the oral uptake of asimadoline was not substantially altered in mdr1a/1b (-/-) mice. Our results demonstrate that for some drugs, P-gp in the blood-brain barrier can have a therapeutically beneficial effect by limiting brain penetration, whereas at the same time intestinal P-gp is not a significant impediment to oral uptake of the drug.     

Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study

We examined assembly and expression of the factor X activating complex on human and rabbit alveolar macrophages. Kinetic parameters of the factor X activating reaction were determined by functional titrations of factors VII and X with macrophage tissue factor (TF) added. We found rapid activation of factor X to Xa on alveolar macrophage surfaces. Detection of rapid factor Xa formation on macrophages required addition of exogenous factors VII and X. At plasma concentrations of the purified factors, factor Xa was formed on freshly isolated macrophages at approximately 5.4 pmol/min/10(6) cells. After macrophage maturation in culture for 20 hours with LPS (endotoxin) added, the factor X activation rate was increased two- to sixfold. The km' (apparent km) of TF-factor VII enzymatic complexes assembled on alveolar macrophages for factor X were (258 +/- 55 and 475 +/- 264 nmol/L for human and rabbit cells, respectively). The km' did not change during macrophage maturation in culture, but V'max (apparent Vmax) was consistently increased. The K1/2 of human factor VII (concentrations giving half maximal rates of factor X activation) for the interaction with human and rabbit alveolar macrophage TF were 0.191 +/- 0.096 and 1.7 +/- 0.7 etamol/L, respectively. The K1/2 were not significantly changed after maturation, whereas rates of Xa formation at saturation with factor VII were increased. The fast rates of factor X activation observed at physiologic concentrations of plasma-derived factors VII and X indicate that TF on alveolar macrophages is likely to provide sites for binding of factor VII and activation of factor X in vivo during clotting reactions associated with alveolar edema and inflammation.