Genomic organization and differential expression of channel catfish MHC class I genes.

Two clones, designated Icpu-UA/3 and Icpu-UA/26, were isolated from a genomic library prepared from a single homozygous gynogenetic channel catfish. Sequence analysis showed that each clone encoded a gene product containing features conserved among MHC class I molecules. The genomic organization of both clones indicated that each domain, with the exception of the cytoplasmic, was encoded by a separate exon. Moreover, like mammals, catfish cytoplasmic regions were encoded by three exons rather than two as previously described for other teleost MHC class I genes. Analysis of nucleotide sequences upstream of catfish class I genes revealed the presence of several regulatory motifs similar to those seen in mammalian class I genes. These included a TATA box, Enhancer B, Site alpha, ISRE, and GAS elements. To determine the functional significance of these elements, EMSAs and tissue expression assays were performed. EMSAs demonstrated that an Enhancer B element within Icpu-UA/26, and an imperfect Enhancer B element and/or a GC-rich region within Icpu-UA/3 were responsible for formation of specific DNA/protein complexes. Expression studies detected Icpu-UA/26 transcripts in all tissues tested, whereas Icpu-UA/3 encoded messages were seen in a limited number of tissues. These results define the intron/exon organization of catfish MHC class I genes, suggest that Icpu-UA/3 encodes a nonclassical gene, and provide the first functional evidence that upstream sequences, similar to those seen in mammalian class I genes, play important roles in regulating teleost MHC gene expression.

Molecular and Immunologic Characterization of Gynogenetic Channel Catfish (Ictalurus punctatus).

: Second-generation gynogenetic channel catfish were characterized by molecular and immunologic assays to determine if they were isogenic at major histocompatibility complex loci. Southern blot analyses, using channel catfish MHC class II B and class I A gene probes, revealed identical banding patterns among second-generation gynogenetic fish. In contrast, banding patterns from outbred fish differed not only from gynogenetic animals, but also among themselves. Nucleotide sequence analysis of the MHC class II beta(1) domain, which encompasses the peptide binding region, from four randomly selected gynogenetic fish showed a single DNA sequence. In contrast, analysis of the same region from three outbred fish showed sequences that differed not only among themselves, but also from those of gynogenetic animals. In cytotoxic assays, peripheral blood leukocytes from outbred fish lysed both gynogenetic and allogeneic targets, whereas those from gynogenetic fish lysed only allogeneic targets. Taken together, these results suggest that this group of second-generation gynogenetic channel catfish is isogenic at MHC loci and may provide an excellent system with which to study cell-mediated immunity in teleosts.

MHC class I genes of the channel catfish: sequence analysis and expression.

Four cDNAs encoding the major histocompatibility complex (MHC) class I alpha chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I alpha chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I alpha and beta2-microglobulin (beta2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I alpha) was associated with beta2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of beta2m with the MHC class I alpha chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.

The association between retinal ischaemia and end-stage renal damage in thrombotic microangiopathy.

Haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are conditions in which thrombocytopenia and microangiopathic haemolytic anaemia are associated with organ damage due to thrombotic microangiopathy (TMA). The prognosis for adults with HUS is variable, with some patients presenting with an acute illness from which a full recovery is made, whilst others develop irreversible renal failure. Evidence of retinal ischaemia was noted on presentation in a normotensive patient with irreversible renal failure due to HUS. This study examined prospectively the optic fundi of all patients who subsequently presented to a single adult renal unit (catchment population approximately 700,000) with HUS. Eleven patients presented with HUS over 12 years (1985-1997). Six patients with irreversible renal failure had retinal abnormalities either at the time or within a few days of initial presentation. The other five patients whose renal function recovered did not develop retinal changes during the course of their illness. Retinal ischaemia associated with TMA is a poor prognostic sign in adults with HUS.

Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor.

Catfish reovirus (CRV), a double stranded RNA virus, inhibited channel catfish herpes-virus (CCV) replication by 2 different mechanisms: (1) directly as a consequence of its own replication, and (2) indirectly due to the induction of an anti-viral factor. In the former, prior infection with CRV significantly reduced subsequent CCV protein synthesis and virus yield. CRV mediated-interference was greatest when CRV infection preceded CCV infection by 16 h, and was least when cell cultures were simultaneously infected with both viruses. in the latter case, the infection of channel catfish ovary (CCO) cultures with UV-inactivated CRV resulted in the synthesis (or release) of an anti-viral factor. Cells producing the factor were protected from CCV infection, as were cells which had been treated with spent culture medium containing anti-viral activity. Interestingly an anti-viral activity was constitutively present in long-term cultures of catfish T-cells and macrophages. Whether this factor and the one induced by UV-inactivated CRV are identical is not known, but analogy to mammalian systems suggests that the former may be similar to type II interferon, whereas the latter may be the piscine equivalent of type I interferon. These results suggest that UV-inactivated CRV may prove useful in the induction and characterization of interferon-like anti-viral proteins in the channel catfish and that long-term cultures of catfish T-cells and monocytes may serve as a ready source of additional anti-viral factors.

MHC class II B genes in the channel catfish (Ictalurus punctatus).

Two different cDNA sequences for major histocompatibility complex (MHC) class II beta chains from the channel catfish (Ictalurus punctatus) have been identified. Homology between these sequences and those previously identified as MHC class II B genes in other teleosts suggests they represent alleles of the DAB locus. The inferred amino acid sequences show strong evidence for a functional polypeptide chain with a peptide binding region. Southern blot analysis reveals polymorphism in the MHC class II B gene(s) of the channel catfish and suggests the presence of two to four genes.

Referral of elderly patients with end-stage renal failure for renal replacement therapy.

An increasing number of patients with end-stage renal failure is being treated in the United Kingdom and in 1987 approximately 50 new patients per million started renal replacement therapy. Nevertheless comparatively fewer patients are treated in this country than in some other European countries and in North America. During the 3 years 1985 to 1988, 95 patients from West Glamorgan started chronic renal replacement therapy (87 patients/million population/year). Of these, 16 were aged 75 years or over on starting treatment. In addition two others attained the age of 75 years during the study period. The proportion of elderly new patients is increasing and in the year 1987 to 1988, 59 per cent were older than 65 years and 32 per cent were older than 75 years. Throughout the United Kingdom many elderly patients are being denied treatment, due partly to under-referral.

Imported epidemic non-A, non-B hepatitis in Qatar.

During one year, 198 patients were admitted to the Hamad General Hospital, Qatar, with acute viral hepatitis. Sera from 126 of these were tested for HBsAg, IgM anti-HBc, IgG anti-HAV, IgM anti-HAV, and delta antibody in those positive for HBsAg. Only 6% of the patients were Qatari nationals and the remainder were immigrants. Of the 126 patients tested, 7 had acute hepatitis A, 29 had acute hepatitis B (none were positive for delta antibody), and the remaining 91 were regarded as having had acute non-A, non-B hepatitis. Of this latter group, 75% were Indian immigrants of whom 59% presented within six weeks of arrival in Qatar and only 2 patients presented later than eight weeks. These patients were thought to have contracted the infection in transit camps in India before immigration to Qatar.