p53 protein in odontogenic cysts: increased expression in some odontogenic keratocysts.

AIMS: To assess p53 protein expression in a range of odontogenic cysts arising in the mouth, including those of developmental and inflammatory origin. METHODS: p53 protein was identified using the polyclonal antibody CM-1, together with a standard immunoperoxidase technique. A total of 36 cystic lesions were examined, all of which were histologically benign. RESULTS: Expression of p53 protein was identified within the lining of five of 12 odontogenic keratocysts but was not detected in the other cystic lesions in the series. CONCLUSIONS: This is believed to be the first report that identifies increased expression of p53 protein in benign cystic epithelium. The increased expression of p53 protein in the nucleus is usually associated with malignant disease. These findings are relevant to the management of odontogenic keratocysts which have a tendency to recur, and also to Gorlin Goltz syndrome in which keratocysts and multiple basal cell carcinomas are features.     

The inflammatory radicular cysts have higher concentration of tnf-alpha in comparison to odontogenic keratocysts (odontogenic tumour)

TNF-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. We investigated levels of TNF-alpha in 43 radicular cysts and 15 odontogenic keratocysts, obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. TNF-alpha is elevated in both cysts' fluid, but higher values were found in radicular cysts in comparison to keratocysts. The significantly higher concentration of TNF-alpha was associated with smaller radicular cysts, higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, and the degree of vascularisation and cysts wall thickness (Mann-Whitney U-test, p < 0.05). No correlation was found based on these parameters in odontogenic keratocyst, but all cysts have detectable concentrations of TNF-alpha. We here for the first time present that a difference in the concentration of TNF-alpha exists between these two cystic types.     

Molecular markers demonstrate that the first described multidrug-resistant Mycobacterium bovis outbreak was due to Mycobacterium tuberculosis

We genetically characterized multidrug-resistant Mycobacterium tuberculosis complex strains which caused a nosocomial outbreak of tuberculosis affecting six human immunodeficiency virus (HIV)-positive patients and one HIV-negative staff member (E. Bouvet, E. Casalino, G. Mendoza-Sassi, S. Lariven, E. Vallée, M. Pernet, S. Gottot, and F. Vachon, AIDS 7:1453-1460, 1993). The strains showed all the phenotypic characteristics of Mycobacterium bovis. They presented a high copy number of IS6110, the spacers 40 to 43 in the direct repeat locus, and the mtp40 fragment. They lacked the G-A mutation at position 285 in the oxyR gene and the C-G mutation at position 169 in the pncA gene. These genetic characteristics revealed that these were dysgonic, slow-growing M. tuberculosis strains mimicking the M. bovis phenotype, probably as a consequence of cellular alterations associated with the multidrug resistance. Spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis confirmed that the outbreak was due to a single strain. However, the IS6110 RFLP pattern of the strain isolated from the last patient, diagnosed three years after the index case, differed slightly from the patterns of the other six strains. A model of a possible genetic event is presented to explain this divergence. This study stresses the value of using several independent molecular markers to identify multidrug-resistant tubercle bacilli.     

Expression of cytokeratin in the epithelium of dentigerous cysts and odontogenic keratocysts: an aid to diagnosis.

Sections of tissue embedded in paraffin wax from 18 selected odontogenic cysts were studied both histologically and immunohistochemically with antibodies to cytokeratins using the indirect peroxidase technique. The cysts were divided on a clinical and histological basis into two equal groups comprising dentigerous cysts and odontogenic keratocysts. It was possible to differentiate the two cyst types in every case by the pattern of staining using the monoclonal antibody LP34 for cytokeratins of intermediate molecular weight. The monoclonal antibody CAM 5.2 for cytokeratins of low molecular weight was not discriminatory. Such a clear distinction may prove useful diagnostically in distinguishing between two cysts of similar appearance but very different behaviour.     

Molecular evidence supporting the neoplastic nature of odontogenic keratocyst: a laser capture microdissection study of 15 cases

AIMS: The bland histology of odontogenic keratocyst (OKC) belies its capacity for aggressive behaviour. Genetic alterations of OKC have not been well studied. We examined the frequency and pattern of allelic imbalance on five different chromosome regions from 15 patients with OKC. METHODS AND RESULTS: Laser-assisted microdissection was performed on formalin-fixed paraffin-embedded tissue. Polymerase chain reaction analysis of extracted DNA targeted five polymorphic DNA markers (D3S1285, D9S161, D11S1316, D13S290, and TP53) representing chromosome regions 3p14, 9p21, 11q23, 13q12.1 and 17p13, respectively. All 15 cases of OKC were informative at a minimum of three of five loci, with 11 informative on all five loci. Twelve of 15 cases (80%) demonstrated loss of heterozygosity (LOH). Seven cases (47%) showed LOH at more than two DNA loci. The frequency of LOH was 5/11 (45%) at D3S1285, 3/15 (20%) at D9S161, 4/14 (29%) at D11S1316, 8/14 (57%) at D13S290 and 3/15 (20%) at TP53. CONCLUSIONS: The majority of OKCs harbour chromosomal abnormalities. This finding supports the supposition that OKCs are neoplastic. Furthermore, OKCs harbour allelic loss at some of the same loci identified in squamous cell carcinoma. This may aid in explaining the rare occurrence of squamous cell carcinoma arising in OKC.     

CD8+ T lymphocytes are recruited to neoplastic cervix

To^liicIV distinguish normal cervical lymphocyte populations from phenotypes recruited to the cervix in response to cervical neoplasia, lymphocytes were isolated from normal and neoplastic cervix. A portion of the cervical transformation zone was obtained from 19 patients with pathologically confirmed cervical intraepithelial neoplasia and from 20 patients with normal cervices undergoing hysterectomy for benign indications. Mononuclear cells were harvested from cervical tissue using a serial, multienzymatic digestion procedure and enriched by density gradient centrifugation. Isolated cell populations were stained with surface marker-specific monoclonal antibodies and analyzed by fluorescent activated cell sorter to determine the percentage of B cells, total T cells, CD4⁺ T cells, CD8⁺ T cells, and natural killer (NK) cells. The distribution of circulating peripheral blood lymphocyte phenotypes was similar for both patients with neoplasia and normal controls. A marked disparity in the proportions of NK cells and T cells was demonstrated among lymphocyte phenotypes infiltrating the cervix. The percentage of CD4⁺ T cells and NK cells was significantly depressed (P=0.04,P=0.03, respectively) in dysplastic tissue as compared to normal cervical tissue. In contrast, the proportion of CD8⁺ T cells was significantly increased in the dysplastic tissue (P=0.0001). Analysis of immunocompetent cells in the circulation appears to have little correlation with immunocytes present in the dysplastic epithelium. The depression in the proportion of CD4⁺ T lymphocytes and NK cells at the cervical squamocolumnar junction reflects a local recruitment of CD8⁺ T cells to the site of neoplasia in the cervix.     

Expression of calretinin in odontogenic keratocysts and basal cell carcinomas: A study of sporadic and Gorlin-Goltz syndrome-related cases

Gorlin-Goltz syndrome (GGS), is an autosomal dominant inherited disorder related to germline mutation of PTCH1 gene, characterised by the presence of multiple developmental anomalies and tumours, mainly basal cell carcinomas (BCC) and odontogenic keratocysts (OKC).We analysed and compared the expression of calretinin in 16 sporadic OKCs, from 15 patients, and 12 syndromic OKCs from 11 patients; in 19 BCC's and 2 cutaneous keratocysts (CKC) belonging to 4 GGS patients, 15 sporadic BCCs and 3 steatocystomas (SC).Calretinin was negative in 10 of 12 syndromic OKCs, focally positive (<5% of cells) in 2; six sporadic OKCs were negative, 6 focally and 4 diffusely positive (p = .02, cases focally and diffusely positive vs. cases negative). All BCCs of 3 GGS patients were negative, the fourth patient presented two BCCs negative and 5 focally or diffusely positive; 7 sporadic BCCs were negative and 8 focally positive (p = NS). Two CKCs resulted negative in one GGS patient; 2 sporadic SCs were positive, and a third was negative.PTCH1 mutations produce an altered PTCH protein and an aberrant activation of Sonic hedgehog (SHH) pathway, leading to tumoral proliferation. It has been demonstrated that treatment of human foetal radial glia cells with SHH reduces, whereas the blockage of SHH increases calretinin expression. We found a lower expression of calretinin in syndromic OKCs compared to sporadic cases. Although calretinin's value in differential diagnosis between sporadic and syndromic tumours appears not crucial, our results shed light on the possible link between SHH dysfunction and calretinin expression in GGS-related tumours.     

Morphometric studies demonstrate that aromatase-immunoreactive cells are the main target of androgens and estrogens in the quail medial preoptic nucleus

The volume and cytoarchitectonic organization of the sexually dimorphic medial preoptic nucleus (POM) of the quail are sensitive to plasma levels of testosterone (T). We previously showed that, in castrated quail, T or its estrogenic metabolite, estradiol (E₂), increases the size of the large neurons located in the lateral part of POM. Embryonic treatments with estrogens are also known to affect permanently the size of these large neurons. Since the lateral POM also contains a dense population of aromatase-immunoreactive (ARO-ir) cells, and these are known to be a target for steroids, we hypothesized that the effects of steroids identified in previous experiments were primarily directed to these ARO-ir cells. This idea was tested in two experiments in which the size of these cells was measured in male quail under various endocrine conditions. In experiment 1, a detailed analysis of ARO-ir and of non-immunoreactive cells in the POM of adult, sexually mature males revealed that the immunoreactive perikarya are larger than the non-immunoreactive cells and that they constitute the vast majority of the large cells (area > 50 m²) in the POM. In experiment 2, it was shown that T and E₂ actually increase the size of ARO-ir cells in the POM while the androgenic metabolite of T, dihydrotestosterone has no effect at this level. Taken together, these data suggest that the sex differences and the steroid-induced changes in cell size previously described in the study of POM sections stained for Nissl material largely concern aromatase-containing cells. Since aromatization of T plays a limiting role in the activation of male copulatory behavior, these changes may represent the morphological signature of the mechanisms causally involved in the control of this behavior.     

Molecular characterization of disease-associated streptococci of the mitis group that are optochin susceptible

Eight optochin-susceptible (Opt(s)) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opt(s) isolates corresponded to streptococci of the mitis group. Besides, the Opt(s) streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opt(s) streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the F(o)F(1) H(+)-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.     

Molecular Cloning of Two Novel Transmembrane Ligands for Eph-Related Kinases (LERKS) that are Related to LERK-2

Abstract

A search of the nucleic acid database of expressed sequence tags (ESTs) revealed several partial cDNA sequences that could encode proteins homologous to the ligands for Eph-related kinases (LERKs). Oligonucleotides designed from the ESTs were used to probe a human brain cDNA library and obtain overlapping clones that encoded two different novel LERKS (NLERK-1 and NLERK-2). NLERK-1 and NLERK-2 are most closely related to human LERK-2/Elk-ligand and they form a subclass of LERKs that contain a transmembrane domain and a conserved cytoplasmic domain. Full-length NLERK-1 was expressed as a glycosylated membrane protein in COS cells and was not secreted into the medium. Full-length NLERK-2 was similarly expressed in COS cells but both membranebound and a truncated, proteolytically-released form were detected. Engineered forms of both NLERK-1 and NLERK-2 lacking transmembrane and cytoplasmic domains were also expressed in COS cells and each was detected in the extracellular medium.     

Mice that are congenic for the char2 locus are susceptible to malaria

A major advance has been made towards the positional cloning of char2 (a quantitative trait locus encoding resistance to Plasmodium chabaudi malaria). Mice congenic for the locus have been used to fine map the gene and to prove that char2 plays a significant role in the outcome of malarial infection, independently of other resistance loci.     

Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.     

Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host.

The importance of superoxide dismutase (SOD) in protecting cells of Nocardia asteroides from the oxidative killing mechanisms within the intact murine host was determined. Murine monoclonal antibodies specific for nocardial SOD and for another nocardial antigen were prepared. Both antibodies adhered to cell surface antigens, as shown by fluorescence-labeled-antibody staining. The anti-nocardial SOD antibody inhibited the effect of nocardial SOD on superoxide generated in vitro. Cells of N. asteroides GUH-2 in log phase of growth were incubated with monoclonal anti-nocardial SOD, another monoclonal antinocardial antibody (not reactive with SOD), or phosphate-buffered saline and then injected intravenously into mice. Total recovery of CFU and inhibition of growth were determined at 3, 24, and 48 h after infection. The brains, kidneys, spleens, lungs, and livers were weighed, homogenized, and plated in order to quantitate the number of organisms in each organ at each time period. There was an initial killing followed by enhanced clearance of N. asteroides from the lungs and livers of mice which had received anti-SOD antibody-treated nocardiae. There was also enhanced early killing in the spleen. At 48 h, there were fewer organisms recovered from the brains, kidneys, and livers of mice which had received anti-SOD antibody-treated nocardia. This was not true for mice which had received antinocardial antibody not specific for SOD. The data demonstrate that surface-associated SOD protects N. asteroides for oxidative killing in vivo during all stages of infection.     

Immunoblot analysis to demonstrate antigenic variability of clinical isolated. Pseudomonas pseudomallei.

Pseudomonas pseudomallei (Ps.ps.) is the causative organism of melioidosis, and is widely distributed in Southeast Asia and Northern Australia. Clinical manifestations range from subclinical infection to fulminant septicemia. To demonstrate the antigenic variability of Ps.ps., 62 clinical isolates from 31 blood, 13 sputum, 9 pus, 3 urine and 6 body fluid culture specimens were studied by SDS-PAGE and immunoblotting. In SDS-PAGE, there were approximately 20 antigenic components with molecular weights ranging from 14 to 66 kilodaltons (KD) which suggested that there was antigenic variability among these 62 clinical isolates of Ps.ps. Attempts to correlate immunoblot profiles with clinical illness or sources of specimens were not successful but 6 common antigens were identified with molecular weight of 17.5, 21, 33, 34, 40 and 45 KD, respectively. Among these antigens, the 45 KD component was recognised by all patients' sera. Thus, the 45 KD protein antigen may be useful for the future approach in immunodiagnosis of melioidosis.     

Vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type II collagen-induced arthritis in the same rat strain are different diseases.

The DA rat is highly susceptible to induction of arthritis after immunization with homologous type II collagen (CII) emulsified in Freund's incomplete adjuvant (FIA), resulting in collagen-induced arthritis (CIA). The DA rat also develops arthritis after injection of FIA alone (oil-induced arthritis (OIA)). This finding allows a direct comparison of two different models for rheumatoid arthritis; one induced with a defined auto-immunogen and one with a pure adjuvant. Both CIA and OIA develop approximately 2 weeks after induction but OIA is a self-limited acute disease whereas CIA induced with homologous CII follows a chronic disease course. Immunization with CII leads to a strong autoantibody response to CII while injection of FIA leads to no or very limited anti-CII antibody response. The Lewis rat develops neither CIA nor OIA while F1 (DA x Lewis) rats develop CIA but not OIA. Olive oil or CII emulsified in olive oil does not induce arthritis in DA rats. Pretreatment with CII in olive oil vaccinates against CIA but not OIA whereas pretreatment with FIA vaccinates against OIA but not CIA. These findings demonstrate that inclusion of CII in the adjuvant leads to a disease distinct from OIA which is characterized by a CII autoimmune response and chronicity of the disease course.