Mutations and allelic deletions of the MEN1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms

Endocrine pancreatic tumors (EPT) and neuroendocrine tumors (NET) occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). We analyzed the frequency of allelic deletions and mutations of the recently identified MEN1 gene in 53 sporadic tumors including 30 EPT and 23 NET (carcinoids) of different locations and types. Allelic deletion of the MEN1 locus was identified in 18/49 (36.7%) tumors (13/30, 43.3% in EPT and 5/19, 26.3% in NET) and mutations of the MEN1 gene were present in 8/52 (15.3%) tumors (4/30 (13.3%) EPT and 4/22 (18.1%) NET). The somatic mutations were clustered in the 5' region of the coding sequence and most frequently encompassed missense mutations. All tumors with mutations exhibited a loss of the other allele and a wild-type sequence of the MEN1 gene in nontumorous DNA. In one additional patient with a NET of the lung and no clinical signs or history of MEN1, a 5178-9G-->A splice donor site mutation in intron 4 was identified in both the tumor and blood DNA, indicating the presence of a thus far unknown MEN1 syndrome. In most tumor groups the frequency of allelic deletions at 11q13 was 2 to 3 times higher than the frequency of identified MEN1 gene mutations. Some tumor types, including rare forms of EPT and NET of the duodenum and small intestine, exhibited mutations more frequently than other types. Furthermore, somatic mutations were not restricted to foregut tumors but were also detectable in a midgut tumor (15.2% versus 16.6%). Our data indicate that somatic MEN1 gene mutations contribute to a subset of sporadic EPT and NET, including midgut tumors. Because the frequency of mutations varies significantly among the investigated tumor subgroups and allelic deletions are 2 to 3 times more frequently observed, factors other than MEN1 gene inactivation, including other tumor-suppressor genes on 11q13, may also be involved in the tumorigenesis of these neoplasms.     

Colonoscope flexural rigidity measurement

A testing device is developed that determines the stiffness, or flexural rigidity, of an endoscope at specific locations down its length by subjecting it to a compressive axial force, a situation similar to the actual forces applied to the endoscope during a clinical procedure. The endoscope is made to deform in a similar fashion to a slender buckled column and the force causing this deformation is related to the flexural rigidity using column buckling theory. A direct relationship between the critical load needed to cause buckling and the square of column length L is demonstrated experimentally and is expected theoretically, giving confidence in the application of column buckling theory to endoscope testing. Additional confidence in the validity of the columnbuckling test results is obtained by their similarity to data obtained by subjecting the endoscope to a transverse load, determining deflection, and modelling the endoscope as a bent elastic beam. Several makes and models of endoscopes were tested, with flexural rigidity values typically ranging between 160 to 240 Ncm². The effect of a metal stiffener inserted in an endoscope's accessory channel is quantified, as is the change in flexural rigidity down the insertion shaft of a graded-stiffness endoscope. Significant differences in flexural rigidity were obtained between identical endoscopes, each sharing similar usage histories, indicating the need for flexural rigidity measurements for each individual endoscope of a particular model line, though a more extensive study is required to reliably determine scope-to-scope stiffness variations for a particular model line.     

The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (–) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV– patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCR+, CD4–CD8–, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ intermediate T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV– livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV– patients produced low levels of cytokines IL-1, IL-2, IL-6, TNF, and IFN- but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.     

Three highly polymorphic microsatellites at the human myelin oligodendrocyte glycoprotein locus, 100 kb telomeric to HLA-F : Characterization and relation to HLA haplotypes

The MOG locus, located on chromosomal bands 6p21.3-p22 and mapped about 100 kb telomeric to HLA-F, was isolated from cosmid ICRFcl09A2434 and shown to contain three microsatellites. These CA-repeat polymorphic markers were characterized in a sample of 173 healthy unrelated individuals and 84 DNAs from the HLA Workshop reference panel, by a method combining fluorescence labeling of PCR products and use of an automated DNA sequencer. For the three markers, frequencies of heterozygotes are well predicted from allele frequencies by the Hardy—Weinberg rule, which suggests that problems of allele nonamplification are unlikely. Typing of cell lines homozygous in the HLA region allowed unambiguous definition of 81 HLA-MOG haplotypes and showed that several HLA ancestral haplotypes extended to the MOG region. The high degree of polymorphism (59%, 51%, and 81% at the three loci, respectively, and 87% at the haplotype level) makes these new markers informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis, and for studies aimed at precisely delineating the site of crossover in chromosomes in which recombination occurred in the distal part of the HLA class I region.     

Use of live cold-adapted influenza A H1N1 and H3N2 virus vaccines in seropositive adults.

To investigate the immunogenicity and protective efficacy of cold-adapted influenza vaccine in individuals with underlying immunity to influenza A virus, we administered cold-adapted H1N1 and H3N2 vaccines to adults with prevaccination serum hemagglutination inhibition antibody titers of 1:16 or more and challenged them 1 month afterwards with homologous wild-type influenza A virus. Both cold-adapted vaccines were immunogenic in seropositive adults. In addition, individuals receiving cold-adapted vaccines had lower rates of virus shedding and illness following challenge with wild-type influenza virus than did unvaccinated seropositive volunteers.     

Biotinylation: a simple method for labelling complement component C8 with preservation of functional activity

Biotinylation of human C8 with the water-soluble biotin derivative biotinylamidohexanoic acid, N-hydroxysulfosuccinimide ester is an excellent method for labelling this terminal complement component with preservation of its functional activity. The biotinylated product can be detected both in native form and also following its incorporation into the terminal complement complexes. Detection assays include Western blotting, crossed immunoblotting, ELISA, and immunocytochemistry. Biotinylation is an attractive alternative method for labelling C8 and may be used for detecting and quantifying C8 and C5b-9 complexes in their soluble and membrane-bound forms.     

Ultrastructural and immunohistochemical characterization of autonomic neuropathy in genetically diabetic Chinese hamsters

Selected portions of the prevertebral and paravertebral sympathetic and vagal parasympathetic nervous systems have been examined in the genetically diabetic Chinese hamster, an experimental animal model of diabetic gastrointestinal disease. The prevertebral sympathetic superior mesenteric/celiac ganglia, which provide much of the sympathetic innervation of the alimentary tract, developed large numbers of markedly dilated axons, many of which had the ultrastructural features of neuroaxonal dystrophy. Dystrophic axons, many involving presynaptic axonal elements, were increased in frequency in the prevertebral superior mesenteric/celiac ganglia, but not in the paravertebral superior cervical sympathetic ganglia, of chronically diabetic hamsters in comparison with age-matched controls. Dystrophic axons contained substance P- and gastrin-releasing peptide (gastrin-releasing peptide/bombesin)-like staining but were not labeled by antisera directed against vasoactive intestinal peptide, dynorphin-B, somatostatin, leu- and met-enkephalin and neuropeptide tyrosine. Substance P and gastrin-releasing peptide/bombesin containing subpopulations of presynaptic elements in prevertebral sympathetic ganglia are thought to participate in local reflex control of bowel motility and lesions preferentially involving these elements may contribute to bowel dysfunction. Immunohistologic techniques failed to demonstrate dystrophic axons in the superior cervical ganglia. Although morphometric studies failed to show significant axon loss in the abdominal vagus of chronically diabetic Chinese hamsters, evidence of markedly diminished numbers of axons comprising each Schwann cell unit and regenerative collections of Schwann cell processes devoid of axons are consistent with the participation of parasympathetic elements in the pathogenesis of alimentary dysfunction in this model system. These results suggest that selective subpopulations of neuropeptide containing axons are vulnerable to the diabetic condition and that these abnormalities may lead to physiologic dysfunction.     

Sedative effects of antihistamines

The central effects of a newly developed, long-acting H1 antihistamine, loratadine (10 and 40 mg), were compared with those of a standard H1 antihistamine, diphenhydramine (50 mg three times a day) with measures of performance and daytime sleepiness (multiple sleep latency test). Sixteen healthy adults (six women and 10 men), 19 to 35 years of age, received each of the drugs and placebo for 2 days, separated by 5 days at home. Each day, the drug or placebo was administered at 8 A.M. and 12 and 4 P.M. Diphenhydramine was administered in three equal doses (50 mg), and loratadine was administered in a single dose followed by two placebo doses. Mean latency to sleep on tests done at 9 and 11 A.M. and 1, 3, and 5 P.M. was reduced significantly with diphenhydramine compared to placebo, whereas neither loratadine dose reduced sleep latency. Performance measured at 9:30 P.M. and 1:30 P.M. with a battery of tests, including reaction time, vigilance, digit symbol substitution, and symbol copying tasks demonstrated a significant reduction in symbols copied and digits substituted after diphenhydramine compared to both loratadine doses. These results demonstrate that loratadine (10 and 40 mg doses) did not have clinically significant central nervous system activity, whereas diphenhydramine increases sleepiness and disrupts performance efficiency.