Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. While its etiology is not well understood, genetic factors are clearly involved. Until recently, most genetic studies in MS have been association studies using the case-control design testing specific candidate genes and studying only sporadic cases. The only consistently replicated finding has been an association with the HLA-DR2 allele within the major histocompatibility complex (MHC) on chromosome 6. Using the genetic linkage design, however, evidence for and against linkage of the MHC to MS has been found, fostering suggestions that sporadic and familial MS have different etiologies. Most recently, two of four genomic screens demonstrated linkage to the MHC, although specific allelic associations were not tested. Here, a dataset of 98 multiplex families was studied to test for an association to the HLA-DR2 allele in familial MS and to determine if genetic linkage to the MHC was due solely to such an association. Three highly polymorphic markers (HLA-DR, D6S273 and TNFbeta) in the MHC demonstrated strong genetic linkage (parametric lod scores of 4.60, 2.20 and 1.24, respectively) and a specific association with the HLA-DR2 allele was confirmed (TDT; P < 0.001). Stratifying the results by HLA-DR2 status showed that the linkage results were limited to families segregating HLA-DR2 alleles. These results demonstrate that genetic linkage to the MHC can be explained by the HLA-DR2 allelic association. They also indicate that sporadic and familial MS share a common genetic susceptibility. In addition, preliminary calculations suggest that the MHC explains between 17 and 62% of the genetic etiology of MS. This heterogeneity is also supported by the minority of families showing no linkage or association with loci within the MHC.      

Induction of sister chromatid exchanges by cypermethrin and carbosulfan in bone marrow cells of mice in vivo

The public health effects of pesticides cannot be denied. However, the undesired effects of chemical pesticides have been recognized as a serious public health concern during the past decades. The present study describes the genotoxic effects of two pesticides, namely cypermethrin and carbosulfan, in a murine test system in vivo. The test parameter used was analysis of sister chromatid exchanges (SCE) in bone marrow cells. Both cypermethrin (5, 10 and 20 mg/kg) and carbosulfan (1.25, 2.5 and 5 mg/kg) induced significant increases in the frequency of SCEs (P < 0.001). However, no significant dose-response correlation could be found for either of the pesticides. Carbosulfan induced a cell cycle delay, as evidenced by an increase in average generation time accompanied by accumulation of cells in the first division cycle, but cypermethrin did not induce any such response. The present study indicates that carbosulfan has a higher potential to cause genetic alterations than cypermethrin in mice and may also pose a mutagenic risk to human beings.     

Dendritic cells generated in the presence of GM-CSF plus IL-15 prime potent CD8+ Tc1 responses in vivo

Dendritic cells (DC) comprise a system of professional antigen-presenting cells, which induce the stimulation of very rare antigen-specific naive T cells. DC progenitors can be stimulated to differentiate into immature DC by various growth factors, including GM-CSF and IL-4. Here we show that IL-15, in combination with GM-CSF, is a growth factor for murine DC. Murine bone marrow cells, depleted of T cells, B cells, I-A+ cells and Gr-1+ granulocytes, and cultured in the presence of GM-CSF plus IL-15 (IL-15 DC), yielded DC expressing high levels of CD11c and MHC class II molecules, as well as CD11b. These cells expressed significant levels of CD40, CD80 and CD86, and could stimulate allogeneic CD4+ T cells efficiently. Interestingly, IL-15 DC were far superior to DC generated with GM-CSF plus IL-4 in stimulating allogeneic CD8+ T cells in vitro. Consistent with this, IL-15 DC induced much more potent antigen-specific CD8+ T cell responses with high levels of Th1 cytokines in vivo, compared to DC generated with GM-CSF plus IL-4, or with GM-CSF plus TGF-beta, or with GM-CSF alone. Together, these data suggest that IL-15 promotes the development of DC, which induce potent Th1 and Tc1 responses in vivo. This suggests potential roles for these IL-15 DC cells in the immunotherapy of tumors and infectious diseases.     

Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion

Submicroscopic deletions of the Y chromosome and polymorphisms of the androgen receptor (AR) gene in the X chromosome have been observed in men with defective spermatogenesis. To further define the subregions/genes in the Y chromosome causing male infertility and its relationship to polymorphisms of the AR polyglutamine tract, we screened the genomic DNA of 202 subfertile males and 101 healthy fertile controls of predominantly Chinese ethnic origin. Y microdeletions were examined with 16 sequence-tagged site (STS) probes, including the RBM and DAZ genes, spanning the AZFb and AZFc subregions of Yq11, and related to the size of trinucleotide repeat encoding the AR polyglutamine tract. Y microdeletions were detected and confirmed in three out of 44 (6.8%) of azoospermic and three out of 86 (3.5%) severely oligozoospermic patients. No deletions were detected in any of the patients with sperm counts of >0.5 x 10(6)/ml, nor in any of the 101 fertile controls. All six affected patients had almost contiguous Y microdeletions spanning the entire AZFc region including the DAZ gene. The AZFb region, containing the RBM1 gene, was intact in five of the six subjects. Y deletions were not found in those with long AR polyglutamine tracts. Our study, the first in a Chinese population, suggest a cause and effect relationship between Y microdeletions in the AZFc region (possibly DAZ), and azoospermia or near-azoospermia. Y microdeletions and long AR polyglutamine tracts appear to be independent contributors to male infertility.      

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.      

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine- to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.      

Cimetidine — antacid combination as premedication for elective Caesarean section

The effect of premedication with an oral antacid (magnesium trisilicate), oral cimetidine or a combination of both was studied in 80 patients undergoing elective Caesarean section. Twenty patients served as untreated controls. Seventy per cent of the patients were in the high risk range for acid aspiration Pneumonitis (pH < 2.5 plus gastric coment volume > 25 ml). Antacid therapy was effective in raising pH but the gastric volume remained high in 50 per cent of the patients. Cimetidine was effective in decreasing gastric volume and raising pH but the pH was still <2.5 in two patients. None of the patients given the cimetidine-antacid combination were in the high risk range for acid aspiration Pneumonitis. The combination of an oral dose of Cimetidine 400 mg, three to four hours before the operation followed by 20 ml of magnesium trisilicate one hour preoperatively proved to be the most efficacious regimen for prophylaxsis against Mendelson’s syndrome in elective Caesarean section. Recent reports have suggested that non-particulate antacids (e.g., sodium citrate) may be preferable to particulate antacids such as magnesium trisilicate.     

"Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa-II. Characterization by means of selective antagonists (L-364,718 and L-365,260).

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39-enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L-365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway.