Developmental distribution of plasma membrane Ca2+-ATPase isoforms in chick cerebellum.

The plasma membrane Ca(2+)-ATPase (PMCA) is highly expressed in the nervous system, but little information is available about its implication in neuronal development. We have analyzed the expression and localization of different isoforms of PMCA in membrane vesicles and sections of chick cerebellum from embryonic day 10 to hatching. We found that the relative amount of each PMCA isoform and their spatiotemporal distribution in the cerebellum are directly linked to precise cellular types during the cerebellar maturation, even in a non-neural tissue as choroid plexus. Purkinje cells contain the highest diversity of PMCA isoforms of the cerebellar cortex since the moment of its morphogenesis. From embryonic day 15, the PMCA2 was highly expressed in the whole Purkinje cell, while PMCAs 1 and 3 had a more restricted distribution in the soma and dendritic branches, and these distributions were evolving according with cell maturation. Other cellular types seem to contain a specific combination of isoforms, but with a well-defined distribution pattern at late moments of development. Thus, PMCAs 1 and 3 were located in the soma of molecular layer interneurons, and only the PMCA2 was observed in granule cells at hatching. Furthermore, PMCA isoforms are also expressed in cellular compartments characterized by a high amount of synapses, suggesting a key role of these proteins in synaptogenesis and in the maturation of neuronal electrophysiological properties.     

Effect of clarithromycin on cytokines and chemokines in children with an acute exacerbation of recurrent wheezing: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: Clarithromycin is postulated to possess immunomodulatory properties in addition to its antimicrobial activity. OBJECTIVE: To evaluate the effect of clarithromycin on serum and nasopharyngeal cytokine and chemokine concentrations in children with an acute exacerbation of recurrent wheezing. METHODS: Children with a history of recurrent wheezing or asthma and who presented with an acute exacerbation of wheezing were enrolled in a double-blind, randomized trial of clarithromycin vs placebo. Concentrations of tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, RANTES, eotaxin, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, and monocyte chemoattractant protein 1 were measured in serum and/or nasopharyngeal aspirates before, during, and after therapy. Mycoplasma pneumoniae and Chlamydophila pneumoniae infection were evaluated for by polymerase chain reaction and serologic testing. RESULTS: Nasopharyngeal concentrations of TNF-alpha, IL-1beta, and IL-10 were significantly and persistently lower in children treated with clarithromycin compared with placebo. There tended to be a greater effect of clarithromycin on nasopharyngeal cytokine concentrations in patients with evidence of M. pneumoniae or C. pneumoniae infection. No significant differences were detected in serum cytokines for children treated with clarithromycin compared with placebo. CONCLUSION: Clarithromycin therapy reduces mucosal TNF-alpha, IL-1beta, and IL-10 concentrations in children with an acute exacerbation of recurrent wheezing.     

FINGERPRINTING OF HLA CLASS I GENES FOR IMPROVED SELECTION OF UNRELATED BONE MARROW DONORS

The degree of matching of HLA genes between the selected donor and recipient is an important aspect of the selection of unrelated donors for allogeneic bone marrow transplantation (UBMT). The most sensitive methods currently used are serological typing of HLA class I genes, mixed lymphocyte culture (MLC), IEF and molecular genotyping of HLA class II genes by direct sequencing of PCR products. Serological typing of class I antigenes (A, B and C) fails to detect minor differences demonstrated by direct sequencing of DNA polymorphic regions. Molecular genotyping of HLA class I genes by DNA analysis is costly and work-intensive. To improve compatibility between donor and recipient, we have set up a new rapid and non-radioisotopic application of the ‘fingerprinting PCR’ technique for the analysis of the polymorphic second exon of the HLA class I A, B and C genes. This technique is based on the formation of specific patterns (PCR fingerprints) of homoduplexes and heterodu-plexes between heterologous amplified DNA sequences. After an electrophoretic run on non-denaturing polyacrylamide gel, different HLA class I types give allele-specific banding patterns. HLA class I matching is performed, after the gel has been soaked in ethidium bromide or silver-stained, by visual comparison of patients’ fingerprints with those of donors. Identity can be confirmed by mixing donor and recipient DNAs in an amplification cross-match. To assess the technique, 10 normal samples, 22 related allogeneic bone marrow transplanted pairs and 10 unrelated HLA-A and HLA-B serologically matched patient-donor pairs were analysed for HLA class I polymorphic regions. In all the related pairs and in 1/10 unrelated pairs, matched donor-recipient patterns were identified. This new application of PCR fingerprinting may confirm the HLA class I serological selection of unrelated marrow donors.     

Effect of challenge method on sensitivity, reactivity, and maximal response to methacholine.

BACKGROUND: Recent data suggest that the tidal breathing method may produce methacholine provocation concentration that caused a decrease in forced expiratory volume in 1 second of 20% (PC20) values significantly lower than the dosimeter method; however, the effect of the challenge method on the shape of the concentration-response curve has not been investigated. OBJECTIVE: To determine the effect of the challenge method on sensitivity, reactivity, and maximal response to methacholine. METHODS: We measured airway responsiveness to methacholine using dosimeter and tidal breathing methods in 30 individuals with suspected asthma. Concentration-response curves were characterized by their PC20 (sensitivity), slope (reactivity), and, if possible, level of plateau. RESULTS: Dosimeter PC20 values were significantly higher than tidal breathing values (geometric mean, 8.9 and 5.2 mg/mL, respectively); the mean difference in PC20 values obtained using each method was 0.78 doubling concentrations (P = .01). The mean slopes were 22.7%/log mg/mL using the tidal breathing method and 24.9%/log mg/mL using the dosimeter method; the mean difference in the slopes obtained using each method was -2.17%/log mg/mL (P = .18). In 10 individuals who showed a plateau with the 2 methacholine challenge tests, the mean level of plateau was 19.8% using the tidal breathing method and 19.5% using the dosimeter method; the mean difference in the plateau values obtained with each method was 0.3% (P = .87). CONCLUSIONS: Although the tidal breathing method produces methacholine PC20 values significantly lower than the dosimeter method, both methods provide similar values for slope and level of plateau. These results suggest that the technical factors that affect methacholine sensitivity and the shape of the curve are different.     

The ribosomal DNA of the Zygomycete Mucor miehei

The ribosomal DNA from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70–80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi. Received: 21 December 1999 / 1 March 2000     

Detection and identification ofMycobacterium avium in the blood of AIDS patients by the polymerase chain reaction

One hundred fifty-three blood samples from patients positive for the human immunodeficiency virus (HIV) were analyzed by polymerase chain reaction (PCR) to detect the presence ofMycobacterium avium. Samples were collected from patients who also had blood cultures performed by a radiometric method. Blood samples were centrifuged on a Ficoll-Hypaque gradient to purify peripheral blood mononuclear cells. The purified cells were washed and incubated with a resin, boiled to release mycobacterial DNA, and then amplified. Polymerase chain reaction products were detected by a nonisotopic method. A 123 base-pair (bp) insertion sequence, namely IS6110, fromMycobacterium tuberculosis complex was also included in the reaction as an internal control ofTaq polymerase activity to exclude the presence of enzyme inhibitors. This IS6110 fragment can be distinguished from the 383 bp target product on ethidium bromide-stained agarose gel and may also be used in a colorimetric assay. Such results were compared with the results of culture and indicated that the assay is as sensitive as bacteriological methods, though faster.     

Mycoplasma pneumoniae induces host-dependent pulmonary inflammation and airway obstruction in mice

Respiratory tract infections result in wheezing in a subset of patients. Mycoplasma pneumoniae is a common etiologic agent of acute respiratory infection in children and adults that has been associated with wheezing in 20-40% of individuals. The current study was undertaken to elucidate the host-dependent pulmonary and immunologic response to M. pneumoniae respiratory infection by studying mice with different immunogenetic backgrounds (BALB/c mice versus C57BL/6 mice). After M. pneumoniae infection, only BALB/c mice developed significant airway obstruction (AO) compared with controls. M. pneumoniae-infected BALB/c mice manifested significantly elevated airway hyperresponsiveness (AHR) compared with C57BL/6 mice 4 and 7 d after inoculation as well as BALB/c control mice. Compared with C57BL/6 mice, BALB/c mice developed worse pulmonary inflammation, including greater peribronchial infiltrates. Infected BALB/c mice had significantly higher concentrations of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1beta, IL-6, IL-12, KC (functional IL-8), and macrophage inflammatory protein 1alpha in the bronchoalveolar lavage fluid compared with infected C57BL/6 mice. No differences in IL-2, IL-4, IL-5, IL-10, and granulocyte/macrophage colony-stimulating factor concentrations were found. The mice in this study exhibited host-dependent infection-related AO and AHR associated with chemokine and T-helper type (Th)1 pulmonary host response and not Th2 response after M. pneumoniae infection.     

Surface carbohydrates as recognition determinants in non-opsonic interactions and intracellular viability of group B Streptococcus strains in murine macrophages

Mononuclear cells have been found to play a key role in phagocytosis and eventual killing of group B streptococci (GBS). The rich array of sugars on bacterial surface plus the presence of membrane-associated lectin-receptors on the macrophage suggests that this is a likely means for GBS recognition by these host defense cells. Macrophages have been shown to bind GBS in the absence of serum components. However, participation of carbohydrate moieties in GBS intracellular survival had not been completely elucidated. The aim of this study was to assess the involvement of sugars on adherence and intracellular viability in murine macrophages of GBS serotypes Ia (85147 and 90222 strains), III (80340 and 90356 strains) and V (88641 and 90186 strains) isolated from assymptomatic carriers and patients, respectively. Most isolates showed higher adherence within 2-h incubation. Only 90222-Ia strain exhibited progressive adherence rate until 12-h incubation. All strains showed intracellular viability during first 0.5-h of incubation. Except for 90186-V strain that survived only for 2 h, strains of all serotypes tested were found to survive 24 h into macrophages. Treatments of bacteria by glycosidases inhibited macrophage interaction with GBS strains at varied levels. Neuraminidase inhibited 90-97% adherence and 100% intracellular survival of GBS strains (P<0.0001). Host cell treatments with Rhamnose, N-acetyl-D-glucosaminidase and Fucose (5 mg/ml) inhibited adherence and intracellular viability of GBS strains at varied levels. Removal of GlcNAc residues of invasive GBS isolates enhanced intracellular viability, suggesting that GlcNAc residues may act by intercepting the expression of hidden receptors probably related with invasiveness and survival within macrophages. Lastly, our results demonstrate involvement of sialic acid specific receptors on macrophages and lectinophagocytosis in non-opsonic interaction and survival of GBS invasive isolates.     

Wiedemann‐Steiner syndrome in two patients from Portugal

Wiedemann‐Steiner syndrome (WSS) is a rare genetic disorder characterized by growth retardation, facial dysmorphism, hypertrichosis cubiti and neurodevelopment delay. It is caused by pathogenic variants in the KMT2A gene. This report describes two unrelated Portuguese patients, age 11 and 17 years, with a phenotype concordant with WSS and clinical and molecular diagnosis of WSS by the identification of two novel frameshift variants in the KMT2A gene. This work also highlights the presence of certain clinical features in patients with growth retardation and development delay and should draw attention to the diagnosis of WSS, when hirsutism, particularly hypertrichosis cubiti is present.