Tattered (Td) is an X-linked, semi-dominant mouse mutation associated with prenatal male lethality. Heterozygous females are small and at 4-5 days of age develop patches of hyperkeratotic skin where no hair grows, resulting in a striping of the coat in adults. Craniofacial anomalies and twisted toes have also been observed in some affected females. A potential second allele of Td has also been described. The phenotype of Td is similar to that seen in heterozygous females with human X-linked dominant chondrodysplasia punctata (CDPX2, alternatively known as X-linked dominant Conradi-Hünermann-Happle syndrome) as well as another X-linked, semi-dominant mouse mutation, bare patches (Bpa). The Bpa gene has recently been identified and encodes a protein with homology to 3beta-hydroxysteroid dehydrogenases that functions in one of the later steps of cholesterol biosynthesis. CDPX2 patients display skin defects including linear or whorled atrophic and pigmentary lesions, striated hyperkeratosis, coarse lusterless hair and alopecia, cataracts and skeletal abnormalities including short stature, rhizomelic shortening of the limbs, epiphyseal stippling and craniofacial defects (MIM 302960). We have now identified the defect in Td mice as a single amino acid substitution in the delta8-delta7 sterol isomerase emopamil binding protein (Ebp; encoded by Ebp in mouse) and identified alterations in human EBP in seven unrelated CDPX2 patients.

Summary Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll‐like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8‐like gene with a 6‐kilobase insertion containing chicken repeat 1 (CR1) retroviral‐like insertion elements. The chicken TLR7 gene encodes a 1047‐amino‐acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine‐rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune‐related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin‐1β (IL‐1β) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up‐regulated both chicken IFN‐α and chicken IFN‐β mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up‐regulation of chicken IL‐1β, and chicken IL‐8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.

Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of Salmonella serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5 ؉ cells to flagellin induced elevated levels of chicken interleukin-1␤ (chIL-1␤) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar Salmonella enterica serovar Typhimurium fliM mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the fliM mutant induced less IL-1␤ mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated Salmonella into systemic sites of the chicken.The pattern recognition receptors (PRRs) play a central role in the rapid initiation of host immune responses and the generic identification of an invading pathogen (36, 43) by recognition of pathogen-associated molecular patterns. Toll-like receptors (TLRs) have emerged as a major component of the vertebrate PRR repertoire. Upon activation, TLRs induce the expression of a wide range of immunoregulatory and effector molecules (41, 51) and maturation of immune cell types (1,3,11,24,50).A range of TLR genes has been identified in nonmammalian vertebrates including chicken (10, 18, 32) and fish (6,26,37). To date, avian orthologues of TLR2 and TLR4 have been characterized and expressed sequence tags (ESTs) with sequence homologies to TLR1, -6, or -10; TLR3; TLR5; and TLR7 have been identified (34, 48; our unpublished results). Two chicken TLR2 (chTLR2) molecules (type 1 and type 2) were identified that lie in a tandem arrangement within a genomic region expressing conserved synteny to mammals (10, 18). The chTLR4 gene was also demonstrated to lie in a region of conserved synteny and has been associated with susceptibility to systemic infection with Salmonella enterica serovar Typhimurium in young chickens (32). Collectively, these data indicate that a range of distinct TLR genes, orthologous to the mammalian TLR repertoire, were present before the divergence of birds and mammals over 300 million years ago.The observation that nonflagellated Salmonella enterica serovars (Gallinarum or Pullorum) typically cause more acute systemic infection than flagellated serovars (Typhimurium or Enteritidis) provoked our interest in chTLR5. Our working hypothesis was that TLR5-flagellin interactions contribute to the broad biology of Salmonella serovars in the chicken. We identified a chicken orthologue for TLR5, determined expression ...

The 37-amino acid peptide called amylin is a major component of the islet amyloid deposited in the pancreases of persons with type 2 diabetes mellitus. We report the isolation of a partial cDNA clone and a phage A genomic clone of the coding region of the amylin gene. The DNA sequence encodes a protein sequence identical to that of amylin isolated from the amyloid found in the diabetic pancreas and shows that amylin is likely to be synthesized as a precursor peptide, now named proamylin. We have demonstrated that the amylin gene is present on chromosome 12 and that it is probably transcribed in the islets of Langerhans. The sequences of the genes for amylin and the calcitonin gene-related peptides (CGRPs) show strong similarity, especially over their 5' coding regions, where both peptides have a conserved intramolecular disulfide bridge, and also over their 3' coding regions, where the presence of a glycine codon strongly suggests that the carboxylterminal residue of amylin, like that of CGRP, is amidated. To examine the functional relevance of these posttranslational modifications, the biological activity of amylin synthesized with or without the disulfide bridge and/or amidation was measured. It was found that both features are necessary for full biological activity, thereby confirming the functional importance of those regions of the molecule whose sequences are conserved at both protein and genetic levels.

Christmas disease, or haemophilia B, is an inherited X-linked haemorrhagic disease which at present occurs in 798 known cases in the United Kingdom, corresponding to a frequency of about 1 in 30,000 males. Patients are deficient in the intrinsic clotting factor IX and are treated by replacement of this protein prepared from pooled plasma obtained from normal individuals. Occasionally treatment is complicated by the appearance of specific anti-factor IX antibodies. It seemed to us that this might be due to the absence of 'self' factor IX causing the immune system to regard the infused normal factor IX as foreign. The absence of all or part of the factor IX gene was an obvious possible reason for this, which we have now tested using our previously isolated gene probe. We have found four patients with gross gene defects.

Campylobacter jejuni is the leading cause of food-borne gastro-enteritis and infection can be followed by severe clinical complications, such as the autoimmune neuropathy Guillain-Barré syndrome. Poultry meat is considered to be a common source of infection, with most flocks infected from 2 to 3 weeks of age. We have examined the effect of host genetics on the colonisation levels of C. jejuni in chickens. Chicks from different inbred lines were challenged with 10(7) to 10(8) cfu of C. jejuni 14N or C. jejuni 81-176 on the day of hatch and levels of bacterial colonisation measured over a period of 2-3 weeks. We consistently observed a 10- to 100-fold difference between four inbred lines in the number of C. jejuni organisms present in the cloaca or in the caeca, with the greatest differences detected between line N, which carried relatively high bacterial levels, and line 6(1), which carried relatively low numbers of bacteria. Amongst the four lines studied, major histocompatibility complex did not appear to be a major factor in determining the resistance. The difference in numbers of cloacal bacteria was observed as soon as 24 h after challenge and was still present at the end of the experiment. Lines N and 6(1) were chosen to analyse the mode of inheritance of the genetic differences in response to this infection. Challenge of progeny from reciprocal (6(1) female x N male) and (6(1) female x N male) F1 crosses and from (N female x 6(1) male) F1 female x N male backcrosses with C. jejuni 14N revealed that the difference in bacterial numbers was inherited in a manner consistent with the resistance (low bacterial numbers) controlled by a single autosomal dominant locus. These data suggest that it might be possible to identify the genes responsible by genetic mapping and candidate gene analysis.

The identified mutation in Gja8 is both correlated and consistent with the cataract observed in the No2 mouse mutant, making it an ideal candidate for the cataract. This study provides the first evidence that a mutation in a lens connexin can result in congenital hereditary cataract, highlighting the importance of lens connexins in maintaining lens transparency.

No abstract