Polymorphic microsatellite loci for the common marmoset (Callithrix jacchus) designed using a cost- and time-efficient method.

We describe a cost- and time-efficient method for designing new microsatellite markers in any species with substantial genomic DNA sequence data available. Using this technique, we report 14 new polymorphic dinucleotide microsatellite loci isolated from the common marmoset. The relative yield of new polymorphisms was higher with less labor than described in previous marmoset studies. Of 20 loci initially evaluated, 14 were polymorphic and amplified reliably (70% success rate). The number of alleles ranged from 3 to 9 with heterozygosity varying from 0.48 to 0.83.

Germ-line chimerism and paternal care in marmosets (Callithrix kuhlii).

The formation of viable genetic chimeras in mammals through the transfer of cells between siblings in utero is rare. Using microsatellite DNA markers, we show here that chimerism in marmoset (Callithrix kuhlii) twins is not limited to blood-derived hematopoietic tissues as was previously described. All somatic tissue types sampled were found to be chimeric. Notably, chimerism was demonstrated to be present in germ-line tissues, an event never before documented as naturally occurring in a primate. In fact, we found that chimeric marmosets often transmit sibling alleles acquired in utero to their own offspring. Thus, an individual that contributes gametes to an offspring is not necessarily the genetic parent of that offspring. The presence of somatic and germ-line chimerism may have influenced the evolution of the extensive paternal and alloparental care system of this taxon. Although the exact mechanisms of sociobiological change associated with chimerism have not been fully explored, we show here that chimerism alters relatedness between twins and may alter the perceived relatedness between family members, thus influencing the allocation of parental care. Consistent with this prediction, we found a significant correlation between paternal care effort and the presence of epithelial chimerism, with males carrying chimeric infants more often than nonchimeric infants. Therefore, we propose that the presence of placental chorionic fusion and the exchange of cell lines between embryos may represent a unique adaptation affecting the evolution of cooperative care in this group of primates.

A near fatal case of the dapsone hypersensitivity syndrome in a patient with urticarial vasculitis.

Dapsone (4,4'-diaminodiphenyl sulphone) is used for a variety of dermatological conditions including immunobullous diseases and urticarial vasculitis. Side-effects are common and include lethargy, headaches, methaemoglobinaemia and haemolysis. Severe adverse effects are rare but the dapsone hypersensitivity syndrome is well recognized. Symptoms include fever, haemolytic anaemia, lymphocytosis and hepatitis. We report a near fatal case of the dapsone hypersensitivity syndrome in a patient with urticarial vasculitis. This diagnosis should be remembered in any patient who becomes unwell whilst taking dapsone.

A single-chain Fv reactive with the Goodpasture antigen.

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

Microangiopathic haemolytic anaemia and systemic vasculitis.

Two cases of systemic vasculitis complicated by microangiopathic haemolytic anaemia (MAHA) are described: this association has not previously been reported. Both patients had atypical presentations of their primary disease, one with parotitis and one with a Guillain-Barré syndrome. Other causes of MAHA were excluded and a possible link with macromolecular von Willebrands factor is speculated upon.

Analysis of T cell responses to the autoantigen in Goodpasture's disease.

Goodpasture's disease is a rare form of glomerulonephritis characterized by the production of autoantibodies to the glomerular basement membrane (GBM). In order to understand the development of autoimmunity to the GBM, it is important to examine mechanisms underlying T cell responses to the autoantigen. A MoAb P1, with the same specificity as patients' autoantibodies, was used to affinity-purify the antigen from collagenase-digested human GBM. This material was enriched in the NC1 domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1), known to be the principal target of anti-GBM antibodies, but also contained lower quantities of alpha 4(IV)NC1. In proliferation assays, T cells from 11/14 patients with Goodpasture's disease showed significant responses (SI > or = 2.0) to affinity-purified human GBM. Peak responses were demonstrated at 7 or 10 days at antigen concentrations of 10-30 micrograms/ml. As in other autoimmune disorders, the presence of autoantigen-reactive T cells was also demonstrated in 5/10 healthy volunteers. Tissue typing revealed that all patients possessed HLA-DR2 and/or -DR4 alleles, while normal individuals whose T cells responded possessed DR2 and/or DR7 alleles. The specificity of the T cell response in Goodpasture's disease was further investigated using monomeric components of human GBM purified by gel filtration and reverse phase high performance liquid chromatography (HPLC). Two antigenic monomer pools were obtained, which were shown by amino-terminal sequence analysis to contain alpha 3(IV)NC1 and alpha 4(IV)NC1, respectively. In all patients tested, significant T cell proliferation was observed in response to one or both of these alpha (IV)NC1 domains. These results demonstrate that patients with Goodpasture's disease possess T cells reactive with autoantigens known to be recognized by anti-GBM antibodies.

Development and application of an ELISA for Goodpasture's disease based on sheep alpha 3(IV)NC1 domains.

A high titre of antibodies to collagenase-solubilised human glomerular basement membrane (CS-GBM) is almost pathognomic of Goodpasture's (anti-GBM) disease. In order to develop an assay independent of scarce human material, a molecule of approximately 26 kD corresponding to the C-terminal NC1 domain of the alpha 3 chain of type IV collagen was purified from sheep GBM by gel filtration and reverse phase HPLC. This molecule was antigenic when assessed by inhibition studies, by immunoblotting, and as a ligand on ELISA plates. An ELISA using this sheep alpha 3(IV)NC1 preparation to detect circulating anti-GBM antibodies gave comparable results to the standard RIA using crude CS-GBM. Sera from patients with a variety of nephropathies other than Goodpasture's disease gave negative results. In a prospective study, 170 consecutive sera were analysed by both the ELISA and the RIA. Twenty seven specimens gave positive results in one or both of the assays. Eleven of these were confirmed as true positive results and all were correctly identified by the RIA. Two false negative results in the ELISA occurred in previously treated cases, and both sera were only weakly positive by RIA. The RIA gave 13 false positive results compared with five by ELISA. The ELISA using highly purified sheep antigen is a robust, reliable, and more specific alternative to immunoassays based on crude human antigen preparations.

Renal transplantation following immunoadsorption in highly sensitized recipients.

Five highly sensitized patients, with panel reactivity greater than 80% for 1.75-5 years, were treated by extracorporeal staphylococcal protein-A immunoadsorption, prednisolone, and cyclophosphamide. The five patients underwent treatment of 18-40 (mean 31) liters of plasma, respectively in 4-7 (mean 5.6) sessions. This reduced the titer of cytotoxic antibodies to sensitizing antigens to < 1/8 in all cases and abolished reactivity to crossreacting antigens. Two patients required retreatment following resynthesis of cytotoxic antibodies. All five patients have been transplanted, and four of these now have stable serum creatinines of 168 mumol/L at 34 months, 208 mumol/L at 29 months, 96 mumol/L at 5 months, and 125 mumol/L at 3 months posttransplantation. One patient had primary graft dysfunction due to acute tubular necrosis; the kidney was removed after eight weeks and showed cortical necrosis without evidence of acute rejection.

An investigation of cognitive style and alcohol/work-related problems among Naval personnel.

This study examines the relationship between cognitive style (i.e., information, normative, and diffuse orientation, and commitment) and alcohol/work-related problems. A random sample of Naval personnel (n = 2000) with less than two years of service toward their first enlistment was sent a questionnaire to assess cognitive style [1] and alcohol/work-related problems. A total of 899 respondents (45.0%) returned their questionnaires. Findings support prevention strategies that incorporate problem-solving, decision-making, and coping strategies, particularly among participants who employ diffuse problem-solving/coping strategies (lack of exploration and commitment, avoidance of problems). Correlations between cognitive style and alcohol/work-related problems are theoretically compatible. Diffuse orientation scores are positively related to both alcohol- and work-related problems, while norm and information orientation scores are negatively related to both measures. Collectively, cognitive style measures explain 11.4 percent of the variability in alcohol-related problems, and 14.0 percent of the variability in work-related problems.

Ganciclovir treatment for cytomegalovirus infection in immunocompromised patients with renal disease.

The safety and efficacy of a 10-day course of ganciclovir therapy was assessed in 17 consecutive patients with proven cytomegalovirus infection. The patients were receiving immunosuppressive therapy for a variety of non-malignant renal conditions, including renal transplantation (seven patients), small vessel vasculitis (six patients), systemic lupus erythematosus (three patients) and Goodpasture's disease (one patient). Fifteen patients were pyrexial at the time of their cytomegalovirus infection. Twelve patients had pneumonitis manifesting as a pulmonary parenchymal infiltrate or a reduction in gas transfer. Fourteen patients had a significant lymphopenia (lymphocyte count less than 1 x 10(9)/l), nine were leucopenic (white cell count less than 3.5 x 10(9)/l) and nine had abnormal liver biochemistry. One patient had an infection of the ileum and one an infection of the larynx. All these disease manifestations responded completely to a single course of ganciclovir therapy. There were no clinical relapses and no side effects were observed. Ganciclovir is a safe and effective therapy when administered early in the course of cytomegalovirus infection in immunosuppressed patients with renal impairment.

Proximal cutaneous necrosis associated with small vessel calcification in renal failure.

Cutaneous necrosis with microvascular calcification is a rare and serious complication of chronic renal failure and has been given the sobriquet of 'calciphylaxis'. We describe four dialysis-dependent patients with proximal cutaneous necrosis who presented with this distinctive clinical syndrome. All of the patients were women aged between 40 and 68, and all developed widespread livedo reticularis followed by painful subcutaneous nodules which progressed to eschar-like lesions of the skin. Microvascular calcification was seen on radiographs of the limbs, especially at the sites of the cutaneous lesions. Serum phosphorus concentrations were increased in all the patients (maximally to between 2 and 3.6 mmol/l), but serum calcium concentrations were mildly increased in only two patients and only one patient had hyperparathyroidism. Histological examinations of skin biopsies in two patients showed cutaneous infarcts. Three patients died despite a reduction in serum phosphorus concentration and one patient improved. The proximal form of 'calciphylaxis' constitutes a distinct syndrome which can be recognized clinically.