Guinea pig maximisation test for skin sensitisation: the use of fewer test animals

Current European Community (Annex V) guidelines recommend the use of 20 test animals in the guinea pig maximisation test for skin sensitisation. The suitability, for classification and labelling purposes, of reducing the number of test animals has been examined by analysing the results of 40 studies submitted to the Health and Safety Executive, and by the use of a mathematical model. Our results suggest that in most cases an experiment with ten test animals can be used to determine satisfactorily whether a substance should be labelled with the risk phrase may cause sensitisation by skin contact. However, serious consideration should be given to the need for additional investigation if two or three of the ten test animals show a sensitisation response. The highest nonirritant concentration of a substance should be used at challenge. Clearer guidance in Annex V on evaluating challenge responses would be beneficial.     

Immunological senescence. I. The role of suppressor cells.

The in vitro anti-SRBC response of several murine strains declined markedly with age in parallel with an increase in the activity of suppressor cells in the spleen and bone marrow which prevented early events during the induction of the immune response. These suppressor cells released soluble mediators and lacked the characteristics of mature T cells or macrophages. In addition the suppressor cell in the bone marrow could be removed on anti-Ig columns and fractions of old splenic suppressor cells sedimenting at 0.32 cm/h were greatly enriched in surface Ig bearing cells. Old immunodepressed mice did not lack potentially immunocompetent cells since the antibody response of old spleen cells could be restored by specifically activated T cells or lipopolysaccharide which act on B cells. These results suggest that a rise in the activity of non-T suppressor cells in the spleen and bone marrow may account, in part, for the depression in humoral immunity observed in aging mice.     

Chenodeoxycholic acid treatment of gallstones. A follow-up report and analysis of factors influencing response to therapy.

We treated 70 patients with gallstones with chenodeoxycholic acid over 3 1/2 years and analyzed the factors influencing the outcome of therapy. This treatment was unsuccessful in 11 patients with radiopaque gallstones and in seven with nonfunctioning gallbladders, but 64 per cent with radiolucent gallstones treated for six months or more showed partial or complete gallstone dissolution, and of those whose bile became unsaturated with cholesterol, 100 per cent had evidence of dissolution. In patients with partial or complete gallstone dissolution, the mean post-treatment biliary cholesterol saturation index--0.78 +/- 0.04 (S.E.M.)--was significantly less (P less than 0.001), and the dose of chenodeoxycholic acid (14.4 +/- 1.0 mg per kilogram of body weight per day) significantly more (P less than 0.025) than in those whose gallstones did not change (1.15 +/- 0.04 and 10.6 +/- 1.2 respectively). In patients with radiolucent gallstones, the dose of chenodeoxycholic acid should be based on body weight; 14 to 15 mg per kilogram of body weight per day effectively lowers the saturation index and dissolves gallstones.     

Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable,epigenetic gene silencing: a mammalian cell culture model of gene variegation

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.     

Localization of a gene for otosclerosis to chromosome 15q25-q26

Among white adults otosclerosis is the single most common cause of hearing impairment. Although the genetics of this disease are controversial, the majority of studies indicate autosomal dominant inheritance with reduced penetrance. We studied a large multi- generational family in which otosclerosis has been inherited in an autosomal dominant pattern. Five of16 affected persons have surgically confirmed otosclerosis; the remaining nine have a conductive hearing loss but have not undergone corrective surgery. To locate the disease- causing gene we completed genetic linkage analysis using short tandem repeat polymorphisms (STRPs) distributed over the entire genome. Multipoint linkage analysis showed that only one genomic region, on chromosome 15q, generated a lod score >2.0. Additional STRPs were typed in this area, resulting in a lod score of 3.4. STRPs FES (centromeric) and D15S657 (telomeric) flank the 14. 5 cM region that contains an otosclerosis gene.      

Inflammatory Bowel Disorders

Infiltration of leucocytes into the mucosa is a hallmark feature of a number of inflammatory bowel disorders, most notably Crohn's disease and ulcerative colitis. The interactions between circulating leucocytes and the vascular endothelium that permit leucocyte migration to a site of injury or infection are mediated via a variety of adhesion molecules. There is now ample evidence for alterations in adhesion molecule expression and function in inflammatory bowel disorders. This raises the possibility that adhesion molecules could be targets for novel therapies. Indeed, many existing anti-inflammatory drugs are capable of modulating adhesion molecule expression or function. Moreover, intensive research is under way to develop more selective and effective modulators of adhesion molecules, in the hope that they will be useful for treating various inflammatory disorders.     

Laboratory diagnosis of variant Creutzfeldt-Jakob disease

The neuropathological and biochemical features of 33 cases of variant Creutzfeldt-Jakob disease (vCJD) diagnosed up to the end of 1998 are analysed in relation to the 646 cases of suspected CJD referred to the CJD Surveillance Unit laboratory from 1990 to 1998. Morphological studies of the central nervous system, lymphoid tissues and other organs were accompanied by immunocytochemistry; Western blot analysis of PrPRES was performed on frozen brain tissue. The findings were analysed in relation to clinical and genetic data. The pathology of vCJD showed morphological and immunocytochemical characteristics distinct from other cases of CJD. PrP accumulation was widespread in lymphoid tissues in vCJD, but was not identified in other non-neural tissues. PrPRES accumulation in vCJD brain tissue showed a uniform glycotype pattern distinct from sporadic CJD. All analysed cases of vCJD were methionine homozygotes at codon 129 of the PrP gene. No evidence currently exists to suggest that cases of CJD diagnosed in individuals who are MV or VV at codon 129 of the PrP gene represent 'human bovine spongiform encaphalopathy (BSE)'. Continued surveillance is required to further investigate this possibility, with the need to investigate autopsy tissues from suspected cases by histological and biochemical techniques.     

T-helper subset function in the gut of rats: differential stimulation of eosinophils, mucosal mast cells and antibody-forming cells by OX8- OX22- and OX8- OX22+ cells.

Thoracic duct lymphocytes (TDL) collected 3 days after infection of rats with Trichinella spiralis (TS) and adoptively transferred into normal, uninfected recipients, increased the numbers of both mucosal mast cells (MMC) and eosinophils (EOS) in the intestine. The CD4+ T-helper cell population was separated into two subsets (OX22+ and OX22-) using OX22 monoclonal antibody (mAb) and panning techniques. After adoptive transfer of these T-helper subsets i.v., rats were challenged with TS 24 hr later. The intestine of recipient rats was examined histologically at intervals from Day 3 to Day 21. On Day 9 after transfer, OX22+ T helpers induced a substantial mastocytosis [94 +/- 3, mean +/- SE/villus crypt unit (VCU)], whereas the OX22- T-helper subset increased resident EOS numbers (60 +/- 2/VCU) compared to the challenge control (18 +/- 1 MMC, 27 +/- 1 EOS/VCU). The time of peak eosinophilia was advanced by 3-6 days for recipients of OX22- cells and that of mast cells by 9-12 days for recipients of OX22+ cells. The recipients of OX22-, but not OX22+, cells also showed a large increase in the numbers of B cells in the spleen and mesenteric lymph node (MLN) secreting antibody against adult TS. Recipients of OX22- cells displayed an even increase in EOS throughout the villi, lamina propria (LP) and muscularis, whereas in OX22+ cell recipients mast cells were only present in the lower villus and the epithelium just above the crypt as well as the muscularis layer. Only the CD4+ OX22- cell subset conferred protection against TS in the intestine. We conclude that the OX22+ and OX22- T-helper cells exert distinctive effects in the intestine on MMC and EOS. Because protection was established in the presence of an OX22- T-helper-induced eosinophilia but without a concurrent mastocytosis, the results suggest that MMC are probably not involved in expulsion of TS to terminate the primary infection.     

A scale for the assessment of object relations: reliability, validity, and factorial invariance

Factor analysis of the Bell Object Relations Inventory items produced four subscales interpreted to be underlying dimensions of object relations. Replication factor analysis confirmed the factor structure. Subscales had high internal consistency and were free of age, sex, or social desirability response bias. Subscales had low intercorrelations with Brief Psychiatric Rating Scale (BPRS) sum scores, Global Assessment Scale scores, and most BPRS symptoms. Subscales appear to represent common features of personality and to sample a domain that is distinct from symptomatology, but related to variations in psychopathology. Percentage of high scoring subjects and subscale mean values are compared for seven criterion groups. High scores were least frequent among community active adults and most frequent among borderlines. Selected findings from the group comparisons are discussed to illustrate the potential of the instrument for empirical examination of theoretical assumptions about the object relations ego function and its components.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.