Molecular typing of Mycobacterium tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis, a more accurate method for identifying epidemiological links between patients with tuberculosis

IS6110 fingerprinting of Mycobacterium tuberculosis is the standard identification method in studies on transmission of tuberculosis. However, intensive epidemiological investigation may fail to confirm transmission links between patients clustered by IS6110-restriction fragment length polymorphism (RFLP) typing. We applied typing based on variable numbers of tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) to isolates from 125 patients in 42 IS6110 clusters, for which thorough epidemiological data were available, to investigate the potential of this method in distinguishing epidemiologically linked from nonlinked patients. Of seven IS6110 clusters without epidemiological links, five were split by MIRU-VNTR typing, while nearly all IS6110 clusters with proven or likely links displayed conserved MIRU-VNTR types. These results provide molecular evidence that not all clusters determined on the basis of multibanded IS6110 RFLP patterns necessarily reflect transmission of tuberculosis. They support the use of MIRU-VNTR typing as a more reliable and faster method for transmission analysis.     

Characterization of a human-human hybridoma antibody, C-OU1, directed against a colon tumor-associated antigen.

The human hybridoma cell line, B9165, was obtained after fusion of lymphocytes from lymph nodes draining the tumor region in a patient with adenocarcinoma of the colon with the human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2). B9165 secretes the human monoclonal antibody, C-OU1 (IgM, kappa). Immunocytochemical and immunohistochemical analysis showed that the antibody bound to a differentiation antigen. Electron microscopy of colonic adenocarcinoma cells, intact tumor and colonic epithelium by the immunogold technique demonstrated that the C-OU1 antibody reacted with a molecule associated with areas of disruption of the intermediate filaments in the cytoplasm of the tumor cells. No reaction was seen with intermediate filaments in normal colonic epithelium. The molecular weight of the antigen was shown to be 43 Kda by SDS-PAGE and Western blotting of tumor extracts, and isoelectric focusing of sonicated extracts demonstrated reaction with molecular species of pI 5.4-6.2. These findings suggest that the C-OU1 antigen is a modified cytokeratin 18. The B9165 cell line has proved to be quite stable, and the antibody is of potential clinical value. Its usefulness for localizing tumors in patients is being investigated.     

Expression of IL-18 in patients with head and neck squamous cell carcinoma

Interleukin-18 (IL-18), a recently described cytokine secreted mainly by macrophages, stimulates interferon-gamma (IFN-gamma) production by natural killer cells and T cells. The purpose of this study was to determine tissue expression and serum levels of IL-18 in head and neck squamous cell carcinoma (HNSCC) and to evaluate ethanol and endotoxin-driven cytokine secretion. In 24 patients with primary HNSCC and 28 healthy controls, PBMC were isolated and incubated with 50 mM ethanol, LPS (doses 25 ng/ml, 250 ng/ml, 2500 ng/ml) and both agents for 24 h. Levels of IL-18 in serum, and cell supernatants were analysed by capture ELISA, IL-18 tissue level by immunoblotting. Serum levels of IL-8, IL-10 and IL-12, IFN-gamma, and endotoxin plasma levels were also determined. Statistical analysis involved Welch t-test and Page's test for trend. The majority of patients with HNSCC had high concentrations of serum IL-18. The level of IL-18 in the sera of these patients had a mean level of 271.7 pg/ml, while the mean IL-18 serum level in healthy controls was 174,0 pg/ml (p<0.001). Levels of IL-10 and IL-12, IFN-gamma were not increased in patients. Endotoxin was not detectable in either group. LPS stimulated dose-dependently IL-18 secretion from PBMC of patients and controls in vitro (p<0.05). Incubation with ethanol alone did not affect basal IL-18 secretion, but ethanol reduced LPS-stimulated IL-18 secretion compared to LPS stimulation alone. The mRNA expression of IL-18 in unstimulated PBMC and the response of PBMC to ethanol and LPS was similar in patients and controls. Our data on elevated serum levels of IL-18 in the majority of HNSCC cancer patients, irrespective of its biological activity, suggest that serum IL-18 might be a candidate for a new marker for HNSCC. The pathways for IL-18 production and its mechanisms of action in patients with HNSCC remain to be determined. Understanding of the immunological pathways might offer new therapeutic options in head and neck cancer in the future.     

In serous ovarian neoplasms the frequency of Ki-ras mutations correlates with their malignant potential

Ki-ras mutations by denaturing gradient gel electrophoresis (DGGE) and direct sequencing after microdissection. Point mutations at codon 12 were found in 7 of 20 tumours of low malignant potential (LMP) (35%) and in 2 of 6 well-differentiated carcinomas (33%). In contrast, no mutations were detected in the 11 poorly differentiated ovarian carcinoma samples or in the 7 serous cystadenomas. The frequency of Ki-ras mutations in serous ovarian tumours seems to correlate with the malignant potential of the neoplasms. The data favour the hypothesis of a de novo development of poorly differentiated ovarian carcinomas and do not support an evolution from LMP tumours or well-differentiated carcinomas. Received: 8 June 1998/Accepted: 8 October 1998     

Histamine excites GABAergic cells in the rat substantia nigra and ventral tegmental area in vitro

We have investigated the effect of histamine (HA) on spontaneous firing of dopaminergic (DA) and GABAergic neurons in the substantia nigra (SN) and the ventral tegmental area (VTA) of the rat in vitro. Single-unit extracellular recordings were obtained and drugs were bath applied. In both regions application of HA (10 and 100 μM) did not affect the firing frequency of DAergic cells, but increased the firing of GABAergic neurons. The histamine-induced excitation was blocked by the H₁ receptor antagonist mepyramine (1 μM), but was unaffected by application of the H₂ antagonist cimetidine (50 μM) or the H₃ antagonist thioperamide (10 μM). Our results suggest that histamine does not directly inhibit dopaminergic neurons in SN and VTA, but rather that this inhibition is mediated through histamine-induced excitation of GABAergic neurons.     

Is trisomy 22 in acute myeloid leukemia a primary abnormality or only a secondary change associated with inversion 16?

In an attempt to confirm the existence of acute myeloid leukemia (AML) with trisomy 22, we studied three patients in whom trisomy 22 imposed as the sole karyotype abnormality. After revision of the karyotypes, however, we were able to identify an inv(16) as the important primary abnormality in all of them. Based on this experience, we investigated whether at least some of the 17 AML cases with trisomy 22 reported so far might possibly have been misinterpreted. Interestingly, ten out of 16 evaluable cases were classified as M4, some of them with bone marrow eosinophilia. As in cases with inv(16), only few metaphases contained trisomy 22. Furthermore, in at least two out of the only four published karyotypes of cases with trisomy 22, an inv(16) is evident and in the other two cases it cannot be ruled out. We therefore believe that at least some of the trisomy 22 cases mentioned in the literature are in fact only secondary changes occurring in AML with an inv(16) and suggest that future reports of AML with trisomy 22 as a specific primary abnormality can only be accepted as such if inv(16) has been excluded with appropriate methods.     

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Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.