Nucleic acid hybridization studies of mycobactin-dependent mycobacteria.

Using molecular techniques, specifically, DNA-DNA hybridization in solution and measurement of the thermal stabilities of hybrids, we examined the genetic relationships among mycobactin-dependent mycobacteria and between such organisms and other (mycobactin-independent) mycobacteria. The mycobactin-dependent mycobacteria examined included five strains of Mycobacterium paratuberculosis, two wood pigeon isolates, and one bovine isolate that was biochemically identified as Mycobacterium avium. All mycobactin-dependent mycobacteria were found to belong to the same hybridization group and to be closely related to organisms in M. avium complex serovars 2, 8, and 9, but more distantly related to organisms in serovars 7 and 19. Relatively low levels of hybridization were observed with other mycobacterial species examined. Thus, these results provide genetic evidence to support previous biochemical and cultural evidence that indicated that mycobactin-dependent mycobacteria constitute a closely related group of organisms within the M. avium complex.     

The interleukin-25 gene located in the inflammatory bowel disease (IBD) 4 region: no association with inflammatory bowel disease.

Genetic predisposition has been suggested to play an important role in the pathogenesis of inflammatory bowel diseases (IBDs). Linkage studies have identified a Crohn's disease susceptibility locus on chromosome 14 (14q11-12; IBD4). Interleukin-25 (IL-25) is a newly identified proinflammatory cytokine that has been shown to promote Th2 responses by inducing cytokines such as IL-4, IL-5 and IL-13. The IL-25 gene is located within this susceptibility region at 14q11.2. As IBDs are characterized by an imbalance of the Th1/Th2 cytokine response, we hypothesized that genetic alterations within the IL-25 gene might contribute to IBD. First, direct sequencing of the coding regions of the IL-25 gene in 40 patients with Crohn's disease or ulcerative colitis revealed only a newly reported polymorphism (c424C/A) in exon 2. Next, the frequency of this polymorphism was further investigated in 151 patients with Crohn's disease, 111 patients with ulcerative colitis, and 119 healthy controls to determine its clinical relevance. The genotypes of the c424C/A polymorphism did not reveal any significant differences between patients with Crohn's disease or ulcerative colitis and controls. Genoytype-phenotype relations in patients with Crohn's disease showed a comparable distribution of the c424C/A polymorphism in all subgroups of the Vienna classification. In summary, our data indicate that genetic alterations in the coding regions of the IL-25 gene are unlikely to play a role in IBDs, but the c424C/A polymorphism in the IL-25 gene should be investigated for a potential association with other chronic inflammatory and inherited disorders such as autoimmune diseases.     

Detection of Fiji disease virus in infected sugarcane by nucleic acid hybridization

Clones of Fiji disease virus were obtained by cDNA synthesis from isolated viral genomic double-stranded RNA. Nick-translated probes made from a mixture of clones specifically detected Fiji disease virus in infected sugarcane galls, leaves and growing tips. This method for detection of Fiji disease virus is extremely sensitive, and can be used as a rapid screening procedure for the virus with very small tissue samples.     

Breed-independent toll-like receptor 5 polymorphisms show association with canine inflammatory bowel disease

Inflammatory bowel disease (IBD) is thought to be the most common cause of vomiting and diarrhoea in dogs. Although IBD can occur in any canine breed, certain breeds are more susceptible. We have previously shown that polymorphisms in the TLR4 and TLR5 (toll-like receptor) genes are significantly associated with IBD in German Shepherd dogs (GSDs). In order to allow for the development of novel diagnostics and therapeutics suitable for all dogs suffering from IBD, it would be useful to determine if the described polymorphisms are also significantly associated with IBD in other breeds. Therefore, the aim of this study was to investigate whether polymorphisms in the canine TLR4 and TLR5 genes are associated with IBD in other non-GSD canine breeds. The significance of the previously identified non-synonymous single nucleotide polymorphisms (SNPs) in the TLR4 (T23C, G1039A, A1571T and G1807A) and TLR5 genes (G22A, C100T and T1844C) were evaluated in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 85 unrelated dogs with IBD consisting of 38 different breeds was compared with a breed-matched control group consisting of 162 unrelated dogs. Indeed, as in the GSD IBD population, the two TLR5 SNPs (C100T and T1844C) were found to be significantly protective for IBD in other breeds (P = 0.023 and P = 0.0195 respectively). Our study suggests that the two TLR5 SNPs, C100T and T1844C could play a role in canine IBD as these were found to be protective factors for this disease in 38 different canine breeds. Thus, targeting TLR5 in the canine system may represent a suitable way to develop new treatment for IBD in dogs.     

Genetic variation in the IGSF6 gene and lack of association with inflammatory bowel disease.

The immunoglobulin superfamily 6 gene (IGSF6) on chromosome 16p11-p12 has been investigated as a positional and functional candidate for inflammatory bowel disease (IBD) susceptibility. Screening of the six exons of IGSF6 for single nucleotide polymorphisms (SNPs) detected four novel SNPs, and validated three of six SNPs listed in the international SNP database (dbSNP). The seven SNPs in IGSF6 formed five distinct linkage disequilibrium groups. There was no evidence for association of the common SNPs with disease in a large cohort of patients with IBD. The novel SNPs and the linkage disequilibrium map will be a useful resource for the analysis of IGSF6 in other immune disorders.     

Diagnosis of fetal rubella infection by nucleic acid hybridization

The efficacy of nucleic acid hybridization for the diagnosis of rubella infection in experimental and clinical materials was compared with immunoblot and virus isolation techniques. Our results showed that nucleic acid hybridization is specific and rapid but gives false-negative results when compared with conventional virus isolation in some experimental although not in clinical materials so far examined. For this reason, a failure to demonstrate rubella virus in fetal specimens by this method alone cannot yet be taken as a sole criterion for ruling out fetal rubella infection.     

The interleukin‐25 gene located in the inflammatory bowel disease (IBD) 4 region: no association with inflammatory bowel disease

Genetic predisposition has been suggested to play an important role in the pathogenesis of inflammatory bowel diseases (IBDs). Linkage studies have identified a Crohn's disease susceptibility locus on chromosome 14 (14q11–12; IBD4). Interleukin‐25 (IL‐25) is a newly identified proinflammatory cytokine that has been shown to promote Th2 responses by inducing cytokines such as IL‐4, IL‐5 and IL‐13. The IL‐25 gene is located within this susceptibility region at 14q11.2. As IBDs are characterized by an imbalance of the Th1/Th2 cytokine response, we hypothesized that genetic alterations within the IL‐25 gene might contribute to IBD. First, direct sequencing of the coding regions of the IL‐25 gene in 40 patients with Crohn's disease or ulcerative colitis revealed only a newly reported polymorphism (c424C/A) in exon 2. Next, the frequency of this polymorphism was further investigated in 151 patients with Crohn's disease, 111 patients with ulcerative colitis, and 119 healthy controls to determine its clinical relevance. The genotypes of the c424C/A polymorphism did not reveal any significant differences between patients with Crohn's disease or ulcerative colitis and controls. Genoytype–phenotype relations in patients with Crohn's disease showed a comparable distribution of the c424C/A polymorphism in all subgroups of the Vienna classification. In summary, our data indicate that genetic alterations in the coding regions of the IL‐25 gene are unlikely to play a role in IBDs, but the c424C/A polymorphism in the IL‐25 gene should be investigated for a potential association with other chronic inflammatory and inherited disorders such as autoimmune diseases.     

Arthritic manifestations of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is commonly associated with arthritic manifestations. They are divided into three clinical categories; peripheral arthritis, spondylitis, and sacroiliitis. To evaluate the incidence of arthritis associated with IBD in Korea, we retrospectively reviewed one hundred and twenty-nine patients with IBD, 77 with ulcerative colitis (UC) and 52 with Crohn''s disease (CD). Arthritis occurred in twenty-two patients (17.1%); 15 with UC(19.6%), 7 with CD (13.5%). Patients with arthritis had more active inflammations and all were seronegative except one patient. Peripheral arthritis was found in twenty patients (15.5%) and more common in UC (19.6%) than in CD (9.6%). Joint involvements tended to be monoarticular or pauciarticular, and most frequently developed in the knee and ankle. Spondylitis was diagnosed in one patient (1.6%) who showed HLA B27 positivity. Radiographic sacroiliitis was observed in eight patients (6.2%) who revealed HLA B27 negativity. Both peripheral arthritis and sacroiliitis were found in six patients (4.6%). In CD, arthritis occurred in 20% of the patients with colonic involvement but in none of the patients without colonic involvement. In conclusion, arthritis was frequent in patients with IBD. Peripheral arthritis was more common in patients with UC than CD. All the patients with CD and arthritis had colonic involvement.     

Murine cytomegalovirus: detection of latent infection by nucleic acid hybridization technique.

The technique of nucleic acid hybridization was used to detect the presence of murine cytomegalovirus (MCMV)-specific deoxyribonucleic acid (DNA) in cell cultures and salivary gland tissues. The presence of approximately 4.5 and 0.2 genome equivalents per cell of MCMV-specific DNA was identified in cultures of salivary (ISG2) and prostate gland (IP) cells, respectively. These cells, derived from animals with experimentally induced latent infections, were negative for virus-specific antigens by immunofluorescence and on electron microscopy revealed no visible evidence of the presence of herpesviruses. A cell line derived from the salivary gland of an uninoculated animal (NSG2) was also found to possess MCMV-specific DNA (0.2 genome equivalents per cell). For this reason, salivary gland tissues from uninoculated animals supplied as "specific pathogen-free" mice by three commercial sources were tested upon arrival for the presence of MCMC-specific DNA. MCMV-specific DNA was detectable in pooled salivary gland extracts from uninoculated animals derived from two commercial sources. All of these animals were seronegative and virus negative by conventional infectivity assays.     

用核酸分子杂交技术产前诊断人巨细胞病毒感染

用ELISA法筛选早孕妇女和中孕引产妇女积压人巨细胞病毒IgM(HCMV-IgM)，早孕妇女阳性者在旱12．20周(中孕引产妇女阳性者在引产时)留羊水，用生物素标记的HCMV-DNA探针斑点杂吏检测其羊水中的HCMV-DNA。对胎儿巨细胞病毒惑染作出产前诊断。结果：早孕妇女384例．血HCMV-IgM阳性26例，此26例羊水HCMV-DNA阳性9例，其中7例出生时留脐血进一步检测HCMV-IgM和HCMV-DNA。证实有HCMV感染。中孕引产妇女125例。血HCMV-IgM阳性10例。此10例羊水HCMV-DNA阳性4例。     

Ultrastructural localization of viral nucleic acid by in situ hybridization

In situ hybridization has become a standard technique in the localization of viral nucleic acids in tissue sections and cytologic preparations at the light microscopic level. We have extended this technique to the electron microscopic level using human cytomegalovirus (CMV) infection in cultured human foreskin fibroblasts, and have shown for the first time that colloidal gold can be used to study intranuclear localization of viral replication. CMV-infected fibroblasts exhibiting early (4-day) and late (18-day) cytopathic effect were fixed in formalin, gently permeabilized with detergent and protease, and hybridized with a biotinylated CMV DNA probe. Hybridized sequences were localized by a pre-embedding technique using streptavidin-conjugated 15 to 20 nm colloidal gold particles. Ultrastructural nuclear and cytoplasmic architecture were well preserved through permeabilization and hybridization steps. Viral DNA was clearly detected in fibroblast nuclei containing nascent and well-formed electron-dense viral inclusions. Gold particles were localized to the periphery of electron-dense nuclear inclusions, occasionally in association with 70 nm nuclear dense bodies, but not with complete viral nucleocapsids. DNA hybridization was abolished by pretreatment of infected cells with DNase. Cross-hybridization of CMV DNA sequences with human DNA or with herpes simplex virus genome was not observed. The ultrastructural findings suggest that CMV DNA replication may occur at the margins of electron-dense regions in maturing viral inclusions, and that viral DNA associated with core dense bodies is available for hybridization with complementary nucleic acid sequences. This technique can be useful in studies of viral pathogenesis.     

Heparanase upregulation by colonic epithelium in inflammatory bowel disease.

Heparanase is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size ( approximately 5-7 kDa). Heparanase activity has long been detected in a number of cell types and tissues. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix barrier. Similarly, heparanase activity was implicated in neovascularization, inflammation and autoimmunity, involving migration of vascular endothelial cells and activated cells of the immune system. The involvement of heparanase in inflammatory processes of the gastrointestinal tract has not been examined. Here, we utilized immunohistochemical analysis to investigate heparanase expression in acute and chronic inflammatory conditions. Heparanase expression was not detected in specimens derived from normal colon tissue. In contrast, strong heparanase staining was observed in Crohn's disease and ulcerative colitis, but not in infectious colitis. Interestingly, heparanase staining was primarily observed in epithelial rather than immune cells. Importantly, un-fractionated as well as low molecular weight heparin (enoxaparin), which exhibit a strong inhibitory activity towards heparanase, have proven efficacious in ulcerative colitis and Crohn's disease patients, suggesting that heparanase is actively involved in these pathologies and thus may be considered as a target for the development of anti-inflammatory therapies.     

Relationship among selected Leptospira interrogans serogroups as determined by nucleic acid hybridization.

Leptospiral DNAs from a variety of Leptospira interrogans serogroups of veterinary significance, as well as a nonpathogenic leptospira, were compared by Southern blot hybridization of EcoRI-digested genomic DNA. The serogroups examined could be assigned to one of three groups on the basis of the degree of cross-hybridization between genomic DNAs. Only a few restriction fragments hybridized between the three groups, and most of these were shown to contain ribosomal DNA. The restriction fragment length polymorphism observed among the intergroup hybridizations allowed differentiation among serogroups and, in some cases, serovars. Under the hybridization conditions used, no hybridization was observed between leptospiral DNA and Leptonema, Escherichia coli, or porcine DNA.     

Mucin depletion in inflammatory bowel disease.

The mucin and gland content of 26 rectal biopsy specimens--five normal specimens, 10 from patients with ulcerative colitis, and 11 from patients with Crohn's disease--were measured using a Quantimet image analyser. There was significantly less mucin in the groups with ulcerative colitis compared with either those with Crohn's disease or the normal controls. The difference in the gland content between the groups with ulcerative colitis and Crohn's disease and between the group with Crohn's disease and the normal controls did not reach significance. The results suggest that it is worth while assessing the mucin content of rectal biopsy specimens from patients with inflammatory bowel disease. In routine practice this assessment can be made by eye using a suitably stained section.     

Lack of association of ACP1 gene with inflammatory bowel disease: a case-control study

The red cell acid phosphatease (ACP1) gene, which encodes a low molecular weight phosphotyrosine phosphatase (LMW-PTP), has been suggested as a common genetic factor of autoimmunity. In the present study, we aimed to investigate the possible influence of ACP1 polymorphisms in the susceptibility of inflammatory bowel disease (IBD). A total of 1271 IBD Spanish patients [720 Crohn's disease (CD) and 551 ulcerative colitis (UC)] and 1877 healthy subjects were included. Four single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247 and rs3828329, were genotyped using TaqMan SNP genotyping assays. Common ACP1 alleles (i.e. ACP1*A, ACP1*B and ACP1*C) were determined by two of these SNPs. After the analysis, no evidence of association of the ACP1 genetic variants was found with CD or UC. Therefore, our results suggest that the ACP1 gene may not play a relevant role in the development of IBD.     

Plasma viscosity in inflammatory bowel disease.

AIMS: To assess the relation of plasma viscosity to disease activity in patients with inflammatory bowel disease. METHODS: Crohn's disease (n = 60) and ulcerative colitis (n = 71) were diagnosed on the basis of typical histological or radiological features. Active Crohn's disease was defined as a Crohn's disease activity index of 150 or over. Active ulcerative colitis was defined as a liquid stool passed three times a day or more with blood. Blood samples were assessed for haemoglobin concentration, total white cell count, platelets, plasma viscosity, erythrocyte sedimentation rate, serum albumin, and C-reactive protein. RESULTS: Plasma viscosity was higher in those with active Crohn's disease compared with those with inactive Crohn's disease or active ulcerative colitis. Plasma viscosity correlated significantly with erythrocyte sedimentation rate, C-reactive protein, and platelet count in patients with Crohn's disease. In ulcerative colitis plasma viscosity correlated only with serum C-reactive protein. Plasma viscosity showed a low sensitivity for detecting active Crohn's disease, with 48% of those with active disease having a plasma viscosity within the laboratory reference range. CONCLUSIONS: Plasma viscosity is related to disease activity in Crohn's disease, but is insufficiently sensitive for it to replace erythrocyte sedimentation rate as a measure of the acute phase response in Crohn's disease.