An extended association screen in multiple sclerosis using 202 microsatellite markers targeting apoptosis-related genes does not reveal new predisposing factors

Apoptosis, the programmed death of cells, plays a distinct role in the etiopathogenesis of Multiple sclerosis (MS), a common disease of the central nervous system with complex genetic background. Yet, it is not clear whether the impact of apoptosis is due to altered apoptotic behaviour caused by variations of apoptosis-related genes. Instead, apoptosis in MS may also represent a secondary response to cellular stress during acute inflammation in the central nervous system. Here, we screened 202 apoptosis-related genes for association by genotyping 202 microsatellite markers in initially 160 MS patients and 160 controls, both divided in 4 sets of pooled DNA samples, respectively. When applying Bonferroni correction, no significant differences in allele frequencies were detected between MS patients and controls. Nevertheless, we chose 7 markers for retyping in individual DNA samples, thereby eliminating 6 markers from the list of candidates. The remaining candidate, the ERBB3 gene microsatellite, was genotyped in additional 245 MS patients and controls. No association of the ERBB3 marker with the disease was detected in these additional cohorts. In consequence, we did not find further evidence for apoptosis-related genes as predisposition factors in MS.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Assessment of phagocytic and antimicrobial activity of human granulocytes

A microassay for assessing two functions of polymorphonuclear leukocytes in the presence or absence of complement is presented. Requiring only minute amounts of blood (0.1 ml) and a minimum of laboratory equipment (microscope, incubator, and centrifuge), it allows the quantitation of phagocytosis and intracellular killing of microorganisms by leukocytes. To demonstrate the value of this assay, the phagocytic and microbicidal activity of leukocytes from healthy subjects and patients against Candida albicans was investigated. Apart from individual cases, no differences in the phagocytic activity between groups of healthy subjects and patients with candida vaginitis or different types of cancer could be found. However, the killing capacity of the leukocytes from women with recurrent candida vaginitis was reduced. The leukocytes of one patient showing a very low killing capacity lacked myeloperoxidase. Also, low values of killing were seen with leukocytes from three patients suffering from osteosarcoma, chronic lymphatic leukemia, or Hodgkins disease.     

Morphological analysis of degeneration and regeneration of syncytiotrophoblast in first trimester placental villi during organ culture

We have recently shown using dansyl-L-lysine exclusion studies that the release of human chorionic gonadotrophin (HCG) in conjunction with L- lactate dehydrogenase (LDH) from first trimester villi during organ culture is symptomatic of syncytiotrophoblast degeneration. The purpose of this study was to examine chorionic villi at the ultrastructural level in order to determine events occurring during organ culture. The tissue was sampled after 0, 24, 48 and 120 h in culture and processed for electron microscopy. In addition to confirming the previously recorded syncytial degeneration, the electron micrographs showed clearly the generation of a new syncytiotrophoblast layer. The new layer, derived from differentiating cytotrophoblast cells, was largely formed by 48 h and was maintained for at least 120 h in culture. This study demonstrates a model which provides an opportunity to study the differentiation of cytotrophoblast cells whilst they retain their anatomical relationships within the villous structure.      

Pharmacological control of leukotriene and prostaglandin production from mouse peritoneal macrophages

Leukotriene and prostaglandin production by mouse peritoneal macrophages was investigated. It could be shown that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate initiated the release of prostaglandin E₂ but had little effect on the release of leukotriene C₄-like immunoreactivity. The divalent cation ionophore A 23187 at concentrations between 10^–6 and 10^–8 mol/l initiated prostaglandin as well as leukotriene release. This prostaglandin and leukotriene release could be modulated by drugs. Non-steroidal anti-inflammatory drugs including benoxaprofen inhibited prostaglandin release but simultaneously enhanced leukotriene production. The analgesics paracetamol and 4-methylaminoantipyrine had similar effects at high concentrations. The experimental compound BW 755 c inhibited prostaglandin and leukotriene production while the antithrombotic compound nafazatrom inhibited the production of leukotriene C₄-like immunoreactivity but enhanced prostaglandin E₂ production. Nordihydroguaiaretic acid inhibited prostaglandin and leukotriene production. The results show that the metabolism of arachidonic acid in macrophages via the cyclooxygenase or the lipoxygenase pathway is dependent on the stimulus applied. Both pathways can be inhibited conjointly or selectively by drugs. Our results do not provide evidence that differences in anti-inflammatory activity claimed for some of the drugs tested can be explained by differential inhibition of either pathway. The experimental system described may be used for assessing the potency of drugs to inhibit the lipoxygenase and the cyclooxygenase pathway of arachidonic acid metabolism.     

A second locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region

Primary congenital glaucoma (gene symbol: GLC3) is an ocular disorder that occurs for 0.01-0.04% of blind people. In the majority of familial cases reported so far, this condition is inherited as an autosomal recessive trait. We have recently used a group of 17 GLC3 families with a minimum of two affected offspring and consanguinity in most of the parental generation and mapped the first GLC3 locus (GLC3A) to the 2p21 region. Six families did not show any linkage to the GLC3A locus and thus provided evidence for genetic heterogeneity of this disorder. A total of eight families unlinked to the 2p21 region were used to search for the chromosomal location of the second GLC3 locus. Herein, we describe mapping of a new locus (designated GLC3B) for primary congenital glaucoma to the short arm of chromosome 1 (1p36.2-36.1) that is situated centromeric to the neuroblastoma and Charcot-Marie-Tooth type 2A (CMT2A) loci. A total of 17 DNA markers were genotyped from this region of chromosome 1. Four families showed no recombination with the two markers D1S2834 and D1S402 with a maximum lod score of 4.510 and 4.157 respectively. Pairwise and multipoint linkage analysis and inspection of the haplotypes revealed that the remaining four families are not linked to this part of chromosome 1, thus providing further evidence that at least one more locus for the autosomal recessive form of GLC3 must exist in the genome. Based on the recombination events, the overall linkage map of this region is: tel-D1S1192-D1S1635-D1S1193 - (D1S1597/-D1S489/D1S228)- [GLC3B/D1S2834/D1S402] - (D1S1176/D1S507/D1S407) - D1S2728-(MFAP2/D1S170) - D1S1368 - D1S436- D1S1592-cen.      

Striated intramural gallbladder lucencies on US studies: predictors of acute cholecystitis

Ultrasound scans of 51 consecutive patients with gallbladder wall thickening were reviewed, and specific sonographic features were correlated with surgical and clinical follow-up. Two patterns of thickening were identified as specific indicators of the presence or absence of acute cholecystitis. "Striated" wall thickening, consisting of several alternating, irregular, discontinuous, lucent and echogenic bands, was seen in eight of 13 patients (62%) with acute cholecystitis. This pattern was not encountered in any of the patients who did not have acute cholecystitis. Conversely, "three-layer" thickening, consisting of a single circumferential lucent zone between two relatively uniform echogenic layers, was seen in only one of 13 patients (8%) with acute cholecystitis but in 11 of 38 patients (29%) with other diagnoses. Other abnormalities, including the presence of intramural echogenic foci and wall irregularities, were more frequently seen in patients with acute cholecystitis but were not as helpful. Use of these features may suggest or help exclude a diagnosis of acute cholecystitis in those patients in whom the cause of gallbladder wall thickening is otherwise not apparent.