False positive results of mitochondrial DNA depletion/deletion due to single nucleotide substitutions causing appearance of additional PvuII restriction sites.

Human mitochondrial diseases are usually caused by dysfunction of mitochondrial DNA (mtDNA), particularly by point mutations, deletions, or depletions. In commonly used procedures for molecular diagnostics of mitochondrial dysfunction, one of the first steps is linearization of circular mitochondrial genomes with either BamHI or PvuII restriction endonulease, which cuts human mtDNA at a unique site. Here, we describe a case of false positive results, which suggested mtDNA depletion or a large deletion in a patient's tissue sample. More detailed analysis (mtDNA sequencing) revealed that these false positive results were caused by the presence of the 12753A>G substitution in the gene coding for NADH dehydrogenase subunit 5 (ND5). This substitution results in no change in amino acid sequence of the gene product but creates an additional PvuII site. Investigating a population of 200 patients not affected by mitochondrial diseases, we found an additional case of 12753A>G, and also another substitution, 12804T>C, which also results in no change in amino acid sequence of ND5 but creates an additional PvuII site. A few cases of 12753A>G and 12804T>C substitutions were found previously in Asian, American, African, and European populations (though they were not reported to date in the MITOMAP), but those samples were used in population studies and not tested for mtDNA deletion or depletion. Therefore, we present a cautionary report indicating that these mtDNA polymorphisms exist in various human populations (and thus, they are panethnic) and may cause false positive results of standard molecular analyses, including molecular diagnostics, of human mtDNA.     

Expression of the CD44 receptor in the blood vessel system: an experimental study in rat

The CD44 receptor is a transmembrane glycoprotein expressed on a variety of cells like endothelial, epithelial and smooth muscle cells. This molecule has many important functions, e.g. in cell-cell and cell-matrix interactions and signal transduction. The main ligand for CD44 is hyaluronan (HYA). HYA is a glycosaminoglycan with structural and cell biological properties. The localization of HYA in the vessel wall of arteries and veins in the healthy adult and newborn rat has been described earlier. In this study the occurrence of the CD44 receptor was investigated in the same vessels and compared to the localization of HYA. Both CD44 and its ligand showed an increased expression in the vessel wall of newborn rats compared to that of adult rats. Although HYA is abundant in the adventitia of adult rats, virtually no expression of CD44 was observed. Our results indicate that the CD44 receptor expression is increased during the stage of maturation of the vessel tree whereas the CD44 receptor is less needed by HYA in the healthy vessel wall.     

Magnetic resonance imaging of brain abnormalities in patients with the Nijmegen breakage syndrome

The results of brain MRI are presented in 22 patients with documented Nijmegen breakage syndrome (NBS), aged from 1 and 9/12 to 20 years. T1-, PD or FLAIR and T2-weighted SE/TSE images in three planes were obtained. Twenty-one patients showed microcephaly. Decreased size of frontal lobes and narrow frontal horns of the lateral ventricles was observed in all cases. In 6 patients agenesis of the posterior part of the corpus callosum was found as well as colpocephaly and temporal horn dilatation. In 2 patients callosal hypoplasia was accompanied by other anomalies: abnormal cerebrospinal fluid spaces. Sinusitis was present in all patients as a result of primary immunodeficiency. As in ataxia teleangiectasia and other breakage syndromes, NBS patients show inherited malignancy susceptibility and hypersensitivity to X and gamma radiation. Because of that computed tomography is contraindicated in these patients and MRI should be the method of choice in diagnostic imaging.     

Significance of PBMC response of rheumatic fever patients and control individuals to immunodominant streptococcal M5 protein epitopes

β-hemolytic streptococcal infection in developing countries still causes thousands of cases of Rheumatic Fever (RF). Molecular mimicry between streptococcal M protein (strep M) and heart components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility, which is linked to different HLA-DR alleles in different populations. In our hands, RF was significantly associated to HLA-DR7/53. Previous work in our lab has shown that heart-infiltrating T cells that simultaneously recognize strep M and heart proteins. Further, such T cells predominantly recognized the 81-103 strep M5 epitope. In this work, we analysed the proliferative response of peripheral blood mononuclear cells of 99 RF patients and 40 normal controls. Eighty-nine of the RF patients were HLA-typed. As among heart-infiltrating T cells, the 81-103 strep M5 protein epitope is the most frequently recognized epitope among RF PBMC (35.4%), against a 7.5% frequency of proliferation among normal controls (p=0.0018, chi square). However, the 81-103 epitope was as frequently recognized by HLA-DR7,53 positive as by negative individuals (45.2% vs 54.8%, respectively). Taken together, the results suggest that the 81-103 strep M5 epitope may be the immunodominant epitope, “promiscuously” recognized by T cells in a genetically diverse population. The demonstration that molecular mimicry is targeted to a discrete immunodominant “promiscuous” epitope in strep M5 may allow the development of a safe anti-streptococcal synthetic vaccine devoid of such epitopes.     

gp49B1 suppresses stem cell factor-induced mast cell activation-secretion and attendant inflammation in vivo

We report that gp49B1, a mast cell membrane receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIM), constitutively inhibits mast cell activation-secretion induced by stem cell factor (SCF), a tissue-derived cytokine that also regulates mast cell development. The intradermal injection of SCF into the ears of gp49B1 null (gp49B(-/-)) mice elicited approximately 4- and 2.5-fold more degranulating mast cells and tissue swelling caused by edema, respectively, than in gp49B(+/+) mice. SCF did not induce tissue swelling in mast cell-deficient mice, and the responsiveness of gp49B(-/-) mice to mast cell-associated amine and lipid mediators was unaltered. When gp49B(+/+) and gp49B(-/-) mice were pretreated with antagonists of the amines, SCF-induced tissue swelling was reduced by >90% and 60%, respectively, and it was reduced by >90% in both genotypes when a cysteinyl leukotriene receptor antagonist was also provided. Hence, the dominant contribution of secretory granule amines to SCF-induced tissue swelling is the result of gp49B1-mediated inhibition of the production of cysteinyl leukotrienes by mast cells. Our findings also provide the first example of an ITIM-bearing receptor that constitutively suppresses inflammation generated in vivo independently of the adaptive immune response by a receptor that signals through intrinsic tyrosine kinase activity rather than immunoreceptor tyrosine-based activation motifs.     

Development of an ELISA test for determination of the urinary trypsin inhibitor: analytical performance and applications

Increased urinary excretion of urinary trypsin inhibitor (UTI) has been reported in various inflammatory conditions and in Alzheimer's subjects, but its diagnostic potential remains to be elucidated. A reliable and specific enzyme-linked immunosorbent assay (ELISA) test for the determination of the UTI in human urine was developed. This assay was performed using 96-well microtiter plates. The plate surface is coated with an anti-UTI polyclonal antibody, the urine sample was added in a dilution range, and the detection was achieved using the enzyme-conjugated antibody. The assay was quantified by the build-up of colored product upon the addition of the substrate. Recoveries were 93%, and the intra- and inter-assay CVs were 4.25% and 21%, respectively. The ELISA showed parallelism of standard and urine samples and no significant interference by the biological matrix. The usefulness of the assay has been demonstrated by applying it to urine samples from Alzheimer's disease patients, and comparing with negative controls. UTI urinary levels are significantly increased in Alzheimer's subjects.     

Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.