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Biomedical subjects

C I Smith

Publications and source records attributed to C I Smith.

337 records · Page 19Linked to original sources
PubMedDate not supplied

Intercellular spread of GFP-VP22.

BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 polypeptide has been reported to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus, a feature making it a useful tool in bystander cell-targeted gene therapy. METHODS: Here we show, by detection of Green Fluorescent Protein (GFP) fused to VP22, its subcellular distribution in living producer (transfected) and recipient (non-transfected) cells as well as in cells after fixation. Four cell lines from different species were used. RESULTS: Different fractions of translocated GFP-VP22 fusion protein could be detected in fixed recipient cells by two different methods of fixation. Functional GFP in live recipient cells could not be detected. CONCLUSIONS: Our study indicates that the VP22-chimeric protein transfer in its present form is suboptimal in terms of protein function. However, after fixation, the GFP signal in 100% of cells in a monolayer can be detected even at moderate transfection efficiency.

Animals↗
PubMedDate not supplied

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones.

BACKGROUND: It has been shown previously that mutations in the cytoplasmic protein kinase, Bruton's tyrosine kinase (BTK) lead to X-linked agammaglobulinemia, an inherited primary immunodeficiency, thus making it a potential candidate for gene therapy. METHODS: Producer cell lines using retroviral BTK constructs were generated and retroviral titers determined. Southern blot analysis was performed to check for pro-viral integrity in the respective clones. Furthermore, cotransfection of green fluorescent protein (GFP) with BTK expression plasmids was used in HeLa cells to establish and characterize the role of BTK in apoptosis. RESULTS: Following the attempt to generate retroviral producer clones by conventional methods, we observed that the BTK gene is deleted from neomycin-resistant high titer clones. We show that BTK mediated apoptosis in GP#E86 and HeLa cells. Furthermore, membrane targeting and kinase activity are required for this effect. In addition, BTK induced apoptosis could be inhibited by using a specific inhibitor for p38 mitogen-activated protein kinase (MAPK), SB203580. CONCLUSION: Failure to generate retroviral producer clones may be caused by the induction of apoptosis mediated by the therapeutic gene product.

Agammaglobulinaemia Tyrosine Kinase↗
PubMedDate not supplied

Septic osteomyelitis and polyarthritis with ureaplasma in hypogammaglobulinemia.

We describe a hypogammaglobulinemic woman with a one-year history of destructive septic osteomyelitis and polyarthritis with positive cultures for Ureaplasma urealyticum from joint exudate and blood. The clinical course was complicated by subcutaneous abscesses from which both U. urealyticum and Mycoplasma hominis were grown. Multiple routine cultures had been negative, except for sporadic findings of Staphylococcus epidermidis before specific cultures for mycoplasmas were performed. Therapy with beta-lactam antibiotics, clindamycin, rifampicin, fusidic acid and aminoglycosides had been given without obvious clinical effect. Intravenous doxycycline treatment instituted after microbiological diagnosis had a dramatic effect on the clinical course. The clinical suspicion of mycoplasma and ureaplasma as etiologic agents of orthopaedic infections in hypogammaglobulinemic patients is mandatory in order to perform appropriate cultures.

Adult↗
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Inversion of chromosome 14 marks human T-cell chronic lymphocytic leukaemia.

Rare cases of chronic lymphocytic leukaemia (CLL) in man stem from the malignant proliferation of T cells. The disease is usually more aggressive clinically than B-cell-derived CLL. Various haematological tumours are associated with specific chromosome aberrations (for example, refs 1, 2). Only limited numbers of T-cell CLL patients have so far been studied cytogenetically and, whereas chromosome 12 seems particularly to be involved in B-cell CLL, several markers have been found in T-cell tumours. Recently, by stimulating malignant clones with different mitogens, novel chromosome abnormalities have been detected in T-cell CLL. Using the same approach for additional cases of T-cell CLL, we now report that the most consistent chromosome change is an inversion of the long arm of chromosome 14, inv(14)(q11 q32), in four of five patients. Another remarkable chromosome aberration is trisomy for the long arm of chromosome 8, found in three of five patients.

Chromosome Aberrations↗
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IgA2 allotypes determined by restriction fragment length polymorphism in IgA deficiency. Re-expression of the silent A2m(2) allotype in the children of IgA-deficient patients.

IgA2 allotyping was performed on DNA from 60 IgA-deficient Caucasian individuals. The frequency of A2m(2) was not statistically different from that of normal controls. Two informative families were selected for further studies. In both families, the A2m(2) allotype (derived from the IgA-deficient parent) was inherited by some of the children, as determined by Southern blotting experiments. In all cases the 'silent' IgA2m2 gene was re-expressed, as judged by conventional serological allotyping of serum proteins. These data strongly argue against structural gene deletions or mutations as a cause of IgA deficiency.

Alleles↗
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Pneumococcal antibodies of different immunoglobulin subclasses in normal and IgG subclass deficient individuals of various ages.

The distribution of pneumococcal antibody activity of the IgG and IgA subclasses was determined in healthy individuals of various ages using monoclonal antibodies in an enzyme linked immunosorbent assay (ELISA). High IgG 2 antibody activity was found in cord blood and in serum from adults. IgG 1 activity was most pronounced in serum from young children and virtually lacking in serum from adults. Anti-pneumococcal antibodies were found to be low in IgG 2 deficient donors and normal in IgG 3 deficient donors. A maturation from relatively low IgG 1 and IgG 2 antipneumococcal activity in young children to high IgG 2 levels in adults was found in healthy individuals. IgA subclass antipneumococcal activity was correlated to age. Evaluation of the distribution of pneumococcal antibody activity of various subclasses offers a new possibility for studying the immune response to infection and immunization.

Adolescent↗
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Effects of hormones on functional differentiation of mouse respiratory epithelium.

The effects of corticosteroids and thyroxine on morphogenesis and differentiation of fetal respiratory epithelium were tested in serum-free culture. Strips from the margins of 16-day fetal mouse lungs, containing future respiratory branches, were cultured in medium containing growth factors plus different concentrations of dexamethasone and/or thyroxine. After 5 days, some of the cultures were processed for electron microscopy and the rest were used to quantitate unsaturated and saturated phosphatidylcholine. In the absence of thyroxine and dexamethasone, the lung tissue developed the equivalent of one more day in utero during the 5 days of culture, perhaps because of residual hormone in the tissue. Thyroxine and dexamethasone in concentrations compatible with normal physiological conditions permitted normal development from the pseudoglandular stage to the saccular stage, differentiation of respiratory cell types, and normal surfactant accumulation. Thyroxine alone gave essentially the same lack of morphological maturation and accumulation of disaturated phosphatidylcholine as culture in the absence of all hormone, but columnar epithelial cells held increased numbers of lamellar bodies. Dexamethasone alone at 10(-6) M gave essentially normal development. Dexamethasone at 10(-9) M was sufficient to permit almost normal development when combined with 10(-7) M thyroxine. We conclude that dexamethasone is required for normal maturation of the fetal lung and that thyroxine acts synergistically to lower the dexamethasone requirement. The presence of apparently authentic lamellar bodies under some conditions of low disaturated phosphatidylcholine accumulation make this morphological criterion suspect as a test for differentiation of type II cells.

Animals↗
PubMedDate not supplied

Susceptibility to polyoma virus tumorigenesis in X-linked immunodeficient (XID) and B-cell deficient (microMT) mice is not increased.

Polyoma virus induced tumorigenesis is controlled by T-cells, while B-cells clear virus infection. In order to study if T-cells can override the tumorigenic effect of a long term disseminated viral infection, the tumorigenicity and persistence of polyoma virus in antibody deficient adult and newborn infected X-linked immunodeficient (XID) and microMT mice was followed. In newborn infected XID and CBA control mice (sensitive to tumorigenesis), the frequency of tumor development was similar, and viral DNA was persistent at least 10 months p.i. In polyoma-infected newborn and adult microMT, and control C57BL/6 mice (resistant to tumorigenesis) as well as in adult XID and CBA control mice, no polyoma tumors were observed. Nevertheless, viral DNA was detected in most tissues in all microMT mice throughout the 5-7 month observation period, whereas in the remaining groups of mice persistent viral infection was limited or not detected. We suggest that the tumorigenic potential of an extensive persistent polyoma virus infection can be overcome as long as a functional T-cell system is present.

Age Factors↗