Diversity of genomic breakpoints in TFG-ALK translocations in anaplastic large cell lymphomas: identification of a new TFG-ALK(XL) chimeric gene with transforming activity

Anaplastic large cell lymphomas are associated with chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene at 2p23 that result in the expression of novel chimeric ALK proteins with transforming properties. In most of these tumors, the t(2;5)(p23;q35) generates the NPM-ALK fusion gene. However, several studies have now demonstrated that genes other than NPM may be fused to the ALK gene. We have recently described two different ALK rearrangements involving the TRK-fused gene (TFG) in which the same portion of ALK was fused to different length fragments of the 5' TFG region. These two rearrangements encoded chimeric proteins of 85 kd (TFG-ALK(S)) and 97 kd (TFG-ALK(L)), respectively. In this study, we have identified a new ALK rearrangement in which the catalytic domain of ALK was fused to a larger fragment of the TFG gene (TFG-ALK(XL)), encoding for a fusion protein of 113 kd. Genomic analysis of these three TFG-ALK rearrangements revealed that the TFG breakpoints occur at introns 3, 4, and 5, respectively, whereas the ALK breakpoints always occur in the same intron. No homologous regions or known recombination sequences were found in these regions. Transfection experiments using NIH-3T3 fibroblasts showed a similar transforming efficiency of TFG-ALK variants compared with NPM-ALK. In addition, in common with NPM-ALK, the TFG-ALK proteins formed stable complexes with the signaling proteins Grb2, Shc, and PLC-gamma. In conclusion, these findings indicate that the TFG may use a variety of intronic breakpoints in ALK rearrangements generating fusion proteins of different molecular weights, but with similar transforming potential than NPM-ALK.     

Cytokine profiles of patients infected with Mycobacterium ulcerans and unaffected household contacts

Mycobacterium ulcerans, the cause of Buruli ulcer, is an environmental mycobacterium with a distinct geographic distribution. The reasons why only some individuals who are exposed to M. ulcerans develop ulcers are not known but are likely to reflect individual differences in the immune response to infections with this bacterium. In this study, we investigated cytokine profiles of peripheral blood mononuclear cells (PBMC) from 23 Buruli ulcer patients and 25 household contacts in a region of Australia where Buruli ulcer is endemic. The results showed that following stimulation with M. ulcerans or Mycobacterium bovis BCG, PBMC from Buruli ulcer patients mounted a Th2-type response, which was manifested by the production of mRNA for interleukin 4 (IL-4), IL-5, IL-6, and IL-10, whereas unaffected contacts responded mainly with the Th1 cytokines gamma interferon (IFN-gamma) and IL-12. For example, mRNA for IL-4 was detected in 18 of 23 patients but in only 3 of 25 control subjects (P < 0.0001). By contrast, PBMC from 21 of 25 unaffected individuals produced IFN-gamma compared with 3 of 23 patients (P < 0.0001). IFN-gamma release following stimulation with mycobacteria was markedly reduced in affected subjects. Frequencies of antibodies to M. ulcerans in serum samples from affected and unaffected subjects were similar, indicating that many of the control subjects had been exposed to this bacterium. Together, these findings suggest that a Th1-type immune response to M. ulcerans may prevent the development of Buruli ulcer in people exposed to M. ulcerans, but a Th-2 response does not.     

Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.     

Transport, enzymatic activity, and stability of mutant sulfamidase (SGSH) identified in patients with mucopolysaccharidosis type III A

Mucopolysaccharidosis type IIIA (MPSIIIA) is an autosomal recessive lysosomal storage disease caused by mutations in the N-sulfoglucosamine sulfohydrolase gene (SGSH; encoding sulfamidase, also sulphamidase) leading to the lysosomal accumulation and urinary excretion of heparan sulfate. Considerable variation in the onset and severity of the clinical phenotype is observed. We report here on expression studies of four novel mutations: c.318C>A (p.Ser106Arg), c.488T>C (p.Leu163Pro), c.571G>A (p.Gly191Arg), and c.1207_1209delTAC (p.Tyr403del), and five previously known mutations: c.220C>T (p.Arg74Cys), c.697C>T (p.Arg233X), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) identified in MPSIIIA patients. Transient expression of mutant sulfamidases in BHK or CHO cells revealed that all the mutants were enzymatically inactive with the exception of c.318C>A (p.Ser106Arg), which showed 3.3% activity of the expressed wild-type enzyme. Western blot analysis demonstrated that the amounts of expressed mutant sulfamidases were significantly reduced compared with cells expressing wild type. No polypeptides were immunodetectable in extracts of cells transfected with the cDNA carrying the c.697C>T (p.Arg233X) nonsense mutation. In vitro translation and pulse-chase experiments showed that rapid degradation rather than a decrease in synthesis is responsible for the low, steady-state level of the mutant proteins in cells. The amounts of secreted mutant precursor forms, the cellular stability, the proteolytic processing, and data from double-label immunofluorescence microscopy suggest that the degradation of the majority of newly synthesized c.220C>T (p.Arg74Cys), c.571G>A (p.Gly191Arg), c.1297C>T (p.Arg433Trp), c.1026dupC (p.Leu343fsX158), and c.1135delG (p.Val379fsX33) mutant proteins probably occurs in the ER, whereas c.488T>C (p.Leu163Pro) mutant protein showed instability in the lysosomes.     

Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency

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Identification of a new cluster of T-cell receptor delta recombining elements

Within the human T-cell receptor delta (TCRD) gene we have identified a new cluster of seven delta recombining elements (deltaRec2.1-2.7), located 2.6-5.2 kilobases downstream of the Vdelta2 gene segment. The deltaRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Ddelta3 and Jdelta1 segments of the TCRD gene as well as with the psiJalpha of the TCRA gene. Rearrangements involving the deltaRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical deltaRec rearrangements. The total frequency of deltaRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the deltaRec2-Ddelta3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the deltaRec2-Jdelta1 rearrangements contained the Ddelta3 segment in the junctional region, we assume that they are derived from the deltaRec2-Ddelta3 rearrangements. In contrast, the majority of deltaRec2-psiJalpha rearrangements did not contain the Ddelta3 segment, indicating that they are single step rearrangements. The deltaRec2-Jdelta1 and deltaRec2-psiJalpha rearrangements seem to be T-lineage specific, but the deltaRec2-Ddelta3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that deltaRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Valpha-Jalpha rearrangements. We hypothesize, that in a similar manner to the classical deltaRec rearrangements, the deltaRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-alphabeta lineage.     

Electrical stimulation of rhesus monkey nucleus reticularis gigantocellularis

Saccade kinematics are altered by ongoing head movements. The hypothesis that a head movement command signal, proportional to head velocity, transiently reduces the gain of the saccadic burst generator (Freedman 2001, Biol Cybern 84:453-462) can account for this observation. Using electrical stimulation of the rhesus monkey nucleus reticularis gigantocellularis (NRG) to alter the head contribution to ongoing gaze shifts, two critical predictions of this gaze control hypothesis were tested. First, this hypothesis predicts that activation of the head command pathway will cause a transient reduction in the gain of the saccadic burst generator. This should alter saccade kinematics by initially reducing velocity without altering saccade amplitude. Second, because this hypothesis does not assume that gaze amplitude is controlled via feedback, the added head contribution (produced by NRG stimulation on the side ipsilateral to the direction of an ongoing gaze shift) should lead to hypermetric gaze shifts. At every stimulation site tested, saccade kinematics were systematically altered in a way that was consistent with transient reduction of the gain of the saccadic burst generator. In addition, gaze shifts produced during NRG stimulation were hypermetric compared with control movements. For example, when targets were briefly flashed 30 degrees from an initial fixation location, gaze shifts during NRG stimulation were on average 140% larger than control movements. These data are consistent with the predictions of the tested hypothesis, and may be problematic for gaze control models that rely on feedback control of gaze amplitude, as well as for models that do not posit an interaction between head commands and the saccade burst generator.     

Analysis of the behavior of eryC mutants of Brucella suis attenuated in macrophages

The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.     

Detection of DNA from infectious laryngotracheitis virus by colourimetric analyses of polymerase chain reactions

A combination of the polymerase chain reaction and a novel ELISA-type DNA colourimetric assay (developed from studies with a retrovirus from man) was used in a preliminary study to detect DNA from avian infectious laryngotracheitis virus. The method is sensitive, specific and easy to perform. Since it can be readily adapted for the detection of DNA from other sources it could be useful for the identification of a variety of pathogens from other species of veterinary importance.