Characterization of cultured mast cells derived from Ws/Ws mast cell- deficient rats with a small deletion at tyrosine kinase domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.     

Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions.

The rate and nature of hypermutation of immunoglobulin genes are of prime importance in the affinity maturation of antibodies. Although a considerable body of information has been gathered for kappa light chains, there is much less data for lambda chains. We have derived a large data base of somatic mutants of mouse lambda 1 light chains from Peyer's patches germinal center B cells. The endogenous lambda 1 genes mutate at a rate comparable to that previously found for a kappa transgene (V kappa ox1). There are intrinsic hot spots of mutation common to both in-frame and out-of-frame rearrangements; these hot spots cluster in hypermutating domains. In contrast to the pattern seen for V kappa Ox1, the hot spot clusters are found not only in complementarity-determining region (CDR)1 but also in CDR2 and CDR3; mutations also cluster in the joining/constant region intron. The differences between the pattern of mutations in V kappa Ox1 and lambda 1 light chains are discussed.     

Effects of stem cell factor (kit-ligand) and interleukin-3 on the growth and serine proteinase expression of rat bone-marrow-derived or serosal mast cells

The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase- I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.     

Lack of somatic mutation in a kappa light chain transgene

Analysis of mice transgenic for immunoglobulin genes should allow definition of the cis-acting DNA sequences required to target somatic mutation to antibody V genes. We have looked for mutations in a chimeric kappa transgene encoding a V region specific for the hapten 2-phenyloxazolone (phOx) linked to a rat C kappa gene. Two independent lines of transgenic mice were hyperimmunized with phOx and splenic hybridomas established. In B cells that had been selected by antigen and which used mouse anti-phOx genes, the endogenous sequences were found to be mutated whereas the transgene remained unchanged. These results suggest either that (a) if the transgene is a "passenger" gene expressed at a low level, transgene mutation is a rare event, or that (b) sequences far from the kappa coding region are necessary to direct somatic mutation.     

Vagal neuropathy: evaluation with CT and MR imaging

The vagus nerve, as a result of its protracted course from the brain stem to the abdomen, can present a difficult imaging problem when it is compromised by a clinically occult lesion. The clinical and radiologic records of 48 patients with suspected vagus nerve dysfunction were reviewed to derive an efficient and effective approach to imaging this patient population. An imaging algorithm is proposed in which vagal neuropathies are divided both clinically and radiologically into proximal and distal categories. Proximal vagal lesions are part of a cranial neuropathy complex and have associated oropharyngeal signs and symptoms (e.g., abnormal gag reflex, uvular deviation). Distal vagal lesions occur as an isolated paralysis of the vagus nerve with no symptoms or signs referable to the oropharynx. Either computed tomography (CT) or magnetic resonance imaging can be used to diagnose proximal or distal lesions. However, CT will be insensitive in the detection of the more cephalic proximal lesions, especially those in the brain stem, basal cisterns, and skull base.     

Analysis of marginal donor parameters in liver transplantation for primary biliary cirrhosis

The shortage in cadaveric donor livers is pushing the transplant centers to expand the pool by using "marginal" donors. Primary biliary cirrhosis (PBC) remains an important indication for transplantation. We conducted a retrospective analysis of prospectively collected data in a well-defined group of patients with PBC where 301 consecutive donor-PBC recipient pairs transplanted were analyzed to identify donor and operative factors influencing recipient outcome. Mean follow-up was 56 months. The 1-, 3- and 5-year actuarial patient and graft survival was 93.97%, 90.64%, and 81.75%, and 85.49%, 82.57%, and 75.21%, respectively. Factors showing influence in decreased total patient survival were recipient old age (P = 0.003) and low recipient albumin (P = 0.01). However, the only variables showing an association with decreased patient survival within 90 days are old donor age (P = 0.002) and high donor body weight (P = 0.03) or high body mass index (BMI) (P = 0.055). Cold ischaemic time (CIT) of 18 hours showed statistical significance in patient survival (P = 0.025). Obesity did have a significant adverse impact on survival compared with normal or overweight donors (BMI < 30), decreasing survival by 50% at 5 years. In conclusion, this study of several factors considered "marginal" for transplantation in a recipient population with predictable liver disease (PBC), donor BMI and age were shown to be associated with decreased graft and patient survival.     

Cardiovascular morbidity and mortality after orthotopic liver transplantation

BACKGROUND: Hyperlipidemia and hypertension have been reported in liver allograft recipients and contribute to an increased risk of ischemic heart disease (IHD) after orthotopic liver transplantation (OLT). The aims of the study were (1) to determine the prevalence of risk factors for IHD in these patients and (2) to compare the observed incidence of cardiovascular events and related mortality in allograft recipients with a matched population. METHODS: One hundred ten consecutive adults (50 male) who attended for review after OLT (median follow-up 3.9 years; range 0.1-17.9) were assessed for cardiovascular risk factors using current blood pressure, diabetic status, and smoking history and measurements of total cholesterol, high-density lipoprotein cholesterol, and triglyceride concentrations. Cardiovascular events and cardiovascular mortality data were collected from the prospective database of all adult liver allograft recipients and compared to matched data from myocardial infarction registries and Office for National Statistics data, respectively. RESULTS: Raised serum cholesterol (>5.0 mmol/L) was found in 48 (44%) patients (18 male), and systolic hypertension (>140 mmHg) was found in 69 (63%) patients (27 male). The relative risk of ischemic cardiac events was 3.07 (95% [confidence interval] CI, 1.98-4.53) and the relative risk for cardiovascular deaths was 2.56 (95% CI, 1.52-4.05) in allograft recipients compared to an age-matched population without transplants. CONCLUSIONS: Liver allograft recipients have a greater risk of cardiovascular deaths and ischemic events than an age- and sex-matched population. The prevalence of raised cholesterol concentrations in patients after OLT is similar to those in previous reports. Moderate hypertension and hyperlipidemia may be more detrimental in patients after OLT compared to non-transplant patients without these risk factors.