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1.
Clin Exp Immunol ; 145(3): 420-7, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16907909

ABSTRACT

Monocyte-derived dendritic cells (MdDCs) from many patients with common variable immunodeficiency (CVID) have been shown recently to have reduced expression of surface molecules associated with maturity. Using flow cytometry and confocal microscopy, we now show that this is due to a partial failure to fix Class II DR molecules on the surface during procedures that induce full maturation in vitro in cells from normal subjects. Major histocompatibility complex (MHC) class I, CD86 and CD83 expression were expressed normally, but CD40 was reduced. These abnormalities are unlikely to be due to prior in vivo exposure of monocytes to lipopolysaccharide (LPS), as addition of LPS to monocytes from normal subjects in vitro caused a different pattern of changes. CVID MdDCs retained Class II DR in the cytoplasm during maturation, showed increased internalization of cross-linked Class II DR surface molecules and were unable to polarize DR within a lipid raft at contact sites with autologous lymphocytes. These cells retained some features of monocytes, such as the ability to phagocytose large numbers of fixed yeast and fluorescent carboxylated microspheres and expression of surface CD14. These abnormalities, if reflected in vivo, could compromise antigen presentation and may be a fundamental defect in the mechanism of the antibody deficiency in a substantial subset of CVID patients.


Subject(s)
Common Variable Immunodeficiency/immunology , Dendritic Cells/immunology , Adult , Aged , Biomarkers/analysis , CD40 Ligand/immunology , Case-Control Studies , Cell Differentiation , Cells, Cultured , Female , Flow Cytometry/methods , HLA-DR Antigens/analysis , Humans , Lipopolysaccharides/pharmacology , Male , Microscopy, Confocal , Middle Aged , Phagocytosis , T-Lymphocytes/immunology
2.
Clin Exp Allergy ; 35(11): 1473-80, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16297145

ABSTRACT

BACKGROUND: The contribution of different T cell subsets to the overall measured cytokine response to food allergens is largely unexplored. METHOD: The patterns of cytokine production of peripheral blood-derived T cells after allergen stimulation were studied in 22 children with multiple food allergies and in 20 non-allergic children as controls, using flow cytometry. RESULTS: Proportions of T cells of food-sensitized children spontaneously secreting IFN-gamma and IL-10 (without antigen stimulation) were lower than non-atopic children and adult controls (P

Subject(s)
Allergens/immunology , Cytokines/immunology , Food Hypersensitivity/immunology , T-Lymphocytes/immunology , Cells, Cultured , Child , Child, Preschool , Dendritic Cells/immunology , Female , Humans , Immunoglobulin E/blood , Interferon-gamma/immunology , Interleukin-10/immunology , Interleukin-2/immunology , Interleukin-4/immunology , Lactoglobulins/immunology , Leukocyte Common Antigens/immunology , Male , Ovalbumin/immunology , Peanut Hypersensitivity/immunology
3.
Clin Exp Immunol ; 138(3): 484-90, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15544626

ABSTRACT

The phenotype and function of monocyte derived dendritic cells (MdDC) were investigated in 25 patients with common variable immunodeficiency (CVID) to test for abnormalities that might help explain the failure of antibody production. Using MHC class II DR and CD86 as markers of maturation, DCs from the majority of CVID patients were normal. However 5 patients, the majority of whom had affected family members who had previously been shown to have a susceptibility genetic locus in the MHC region, expressed abnormally low levels of DR on repeated testing, in some cases associated with a reduced capacity to support antigen stimulated T cell proliferation; nevertheless costimulatory molecules for production of IL-13, IL-10 and IFN-gamma from T cells were intact. In contrast to DCs from healthy donors, DCs from many CVID patients had high spontaneous production of IL-8 and lipopolysaccharide stimulation often caused a reduction in DR expression. Expression of other cytokines (IL-1a, IL-6 and IL-12), either before or after LPS stimulation, was normal. The data suggests there is a fundamental defect in the maturation of MdDCs in a subset of CVID patients that may compromise antigen presentation and subsequent antibody production.


Subject(s)
Common Variable Immunodeficiency/immunology , Dendritic Cells/immunology , Monocytes/immunology , Adult , Aged , Antigen Presentation/immunology , Antigens, CD/immunology , B7-2 Antigen , Cells, Cultured , Female , HLA-DR Antigens/immunology , Humans , Interferon-gamma/immunology , Interleukin-10/immunology , Interleukin-12/immunology , Interleukin-13/immunology , Male , Membrane Glycoproteins/immunology , Middle Aged , T-Lymphocytes/immunology
4.
Expert Opin Investig Drugs ; 9(3): 471-80, 2000 Mar.
Article in English | MEDLINE | ID: mdl-11060688

ABSTRACT

The premise that DNA coding for antigens produces proteins to stimulate the immune system when inoculated directly into muscle tissues, has the immense attractions of simplicity, versatility and economy. When other vaccination approaches are experiencing practical problems, meeting such challenges as AIDS and malaria, considerable attention has focused on DNA vaccines with entire conferences and a flood of commercial companies devoted to exploring the possibilities. A number of clinical trials for both infectious diseases and cancer have already commenced, even though a number of major issues have to be resolved.


Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Malaria/immunology , Neoplasms/immunology , Vaccines, DNA , Acquired Immunodeficiency Syndrome/prevention & control , Acquired Immunodeficiency Syndrome/therapy , Cancer Vaccines/therapeutic use , Clinical Trials as Topic , Humans , Malaria/prevention & control , Malaria/therapy , Neoplasms/prevention & control , Neoplasms/therapy , Vaccines, DNA/therapeutic use
5.
Biochim Biophys Acta ; 1500(3): 265-79, 2000 Mar 17.
Article in English | MEDLINE | ID: mdl-10699368

ABSTRACT

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.


Subject(s)
Cancer Vaccines , Cell Fusion , Hybrid Cells , Cell Line , Dendritic Cells , Electroporation , Humans , Polyethylene Glycols , Tumor Cells, Cultured
6.
Gene Ther ; 5(5): 621-9, 1998 May.
Article in English | MEDLINE | ID: mdl-9797866

ABSTRACT

Retroviral infection is restricted by the expression of a viral receptor on the surface of the target cell. Retrovirus-mediated gene transfer is therefore not possible in cells that fail to express sufficient levels of the appropriate receptor, representing one major obstacle to the use of recombinant retroviruses in experimental and clinical applications. In this study, we utilized an adenoviral vector to express transiently the receptor for the ecotropic murine leukemia virus in a panel of human cell lines. Following adenoviral infection, the susceptibility to ecotropic retroviral particles of A549, HeLa, RC39 and Meso 33 cells, derived from human lung epithelium, cervical epithelium, kidney and mesothelium, respectively, was measured on a single-cell basis by the detection of a cell surface marker encoded by the recombinant retrovirus. The marker, termed NTP, was found in 10-30%, 25-50% and 50-90% of cells infected at 5, 50 and 250 adenovirus multiplicity of infection, respectively. Southern blot analysis demonstrated the integration of intact retroviral DNA. The integrated vector copy number increased with the adenoviral multiplicity of infection, suggesting that retrovirus infection is proportional to receptor expression by the target cell, albeit not in a linear fashion. Susceptibility to ecotropic retroviral infection was maintained undiminished for at least 3 days, indicating the persistent expression of ecotropic receptor by the adenovirus-transduced cells in that time period and the lack of a major cellular defense triggered by adenovirus infection against the subsequent retroviral infection. Thus, the infection of human cells of various tissues with a recombinant adenovirus expressing the ecotropic murine leukemia virus receptor generates a window of susceptibility where a high retroviral infection rate can be achieved. Increased efficiency of retroviral infection obtained in this fashion is amenable to specific regulation via the controlled expression of the adenovirus-encoded retroviral receptor.


Subject(s)
Adenoviridae/genetics , Moloney murine leukemia virus/genetics , Receptors, Virus/genetics , Transfection/methods , Animals , Blotting, Southern , Cells, Cultured , DNA, Viral , Female , Genetic Therapy/methods , Genetic Vectors , Humans , Mice , Moloney murine leukemia virus/metabolism , Transduction, Genetic , Tumor Cells, Cultured
8.
J Med Virol ; 45(3): 331-8, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7775957

ABSTRACT

Assessment of the current incidence of different adenovirus types in local gastroenteritis involved the examination of over 1,000 stool specimens annually from 1988-1992. Adenoviruses were detected by electron microscopy and/or cell culture in 32% of the specimens in which a viral pathogen was detected. The identification of every adenovirus isolate to type by neutralization with specific antisera against the first 6 types and by restriction analysis of nonneutralized isolates was started in 1990. Samples from 1988 and 1989 were examined retrospectively. Adenovirus strains were compared to those isolated in a study between 1980-1983. Enumeration of individual adenovirus types revealed a number of trends, demonstrating that rapid changes in the local incidence of several strains were occurring in Manitoba. The incidence of adenovirus type 40 (Ad40) as a cause of gastroenteritis was found to have fallen dramatically in recent years. The predominant cause of gastroenteritis in Manitoba is a variant strain of Ad41, increasing in predominance each year and now responsible for over a third of the symptomatic cases examined since 1990. The majority of restriction site differences of the Ad41 variant strain from the prototype strain Tak were mapped to the hexon and fiber genes, both of which code for the neutralizable external viral epitopes. The probability of the observed pattern of mutations occurring by chance was calculated as P < 0.0005, indicating a strong pressure for selection of these immunologically significant alterations to the viral proteins responsible for cell attachment.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Adenovirus Infections, Human/virology , Adenoviruses, Human/isolation & purification , Gastroenteritis/virology , Adenovirus Infections, Human/epidemiology , Adenoviruses, Human/classification , Adenoviruses, Human/genetics , Child , DNA, Viral/genetics , DNA, Viral/isolation & purification , Gastroenteritis/epidemiology , Genetic Variation , Humans , Longitudinal Studies , Manitoba/epidemiology , Mutation , Restriction Mapping
9.
Diagn Microbiol Infect Dis ; 18(3): 161-6, 1994 Mar.
Article in English | MEDLINE | ID: mdl-7924208

ABSTRACT

Fecal samples submitted for virus examination over July 1990 to June 1991 from children < 3 years of age were examined by electron microscopy (EM), virus culture (VC), and enzyme immunoassay [EIA, group-reactive and adenovirus (Ad) 40/41 specific; Cambridge BioScience] to compare the detection rate of adenovirus from pediatric fecal specimens. Ad isolates of serotypes 1-7 grown in HEp-2 or primary rhesus monkey kidney cells were identified by neutralization. Graham 293 cell cultures were used only when specimens were found to be positive for Ad by EM, type-specific Ad40/41 EIA, and for isolates not identified by neutralization. Ads grown in 293 cells were identified by DNA restriction endonuclease analysis. Of the 1187 specimens examined, 105 (9%) were found to be positive for Ad. VC detected 93, while 12 additional positives were detected by EM or EIA. The relative sensitivity of VC, EIA, and EM for the 105 specimens was 89% (93), 45% (47), and 35% (37), respectively. Among the 105 positive specimens, enteric Ad, nonenteric Ad, and untypeable Ad were 28% (29), 65% (68), and 7% (8), respectively. Of 37 EM positives, 62% (23) were enteric Ad; 27% (10) were nonenteric including serotypes 2, 3, 4, 5, 12, and 31, with 4, 1, 1, 2, 1, and 1 isolates of each type positive, respectively; and 11% (4) were detectable only by EM. Five isolates were identified as variant of Ad 2(3), Ad 3(1) and Ad 31(1). Over a 1-year period, a single Ad41 variant strain was the most frequently detected enteric Ad in Winnipeg, Manitoba, Canada.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Adenovirus Infections, Human/virology , Adenoviruses, Human/isolation & purification , Feces/microbiology , Adenoviruses, Human/classification , Cell Line , Child, Preschool , Humans , Immunoenzyme Techniques , Infant , Infant, Newborn , Microscopy, Electron , Restriction Mapping , Sensitivity and Specificity , Serotyping , Ultracentrifugation , Virus Cultivation
10.
J Med Virol ; 41(4): 328-37, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8106869

ABSTRACT

The abilities of hybridization probes to detect all human adenovirus types and to identify enteric adenovirus types were evaluated. The efficiency of hybridization was compared to other tests currently in routine laboratory use on clinical specimens from young children with gastroenteritis. Probes were derived from various regions of the adenovirus types 2 and 41 genomes, and were evaluated by hybridization with a series of DNA quantities from 1 microgram to 10 pg of one adenovirus type from each human subgenus, lambda phage, and HEp 2 cells. The sensitivity of hybridization with the HPII probe (92.7%), containing the conserved hexon gene, compared well with EM (54.6%), culture and neutralization (45.5%), and enzyme immunoassay (61.8%). The sensitivity of detection of enteric adenovirus isolates by the cloned Bg/II D fragment probe (92.9%) and by a synthetic probe (85.7%), manufactured from type-specific sequences of the Ad41 hexon gene were comparable to Ad40/Ad41 specific enzyme immunoassay (84.6%). Hybridization was found to be a sensitive method of adenovirus detection in comparison to traditional methods of laboratory diagnosis. Synthetic oligonucleotides enable specific detection of individual enteric adenovirus types. Hybridization had additional advantages over other tests in identifying cases of infection with more than one adenovirus type and in allowing an estimate of the concentration of adenovirus in the specimen.


Subject(s)
Adenoviridae Infections/diagnosis , Adenoviruses, Human/classification , Oligonucleotide Probes , Adenoviridae Infections/epidemiology , Adenoviruses, Human/isolation & purification , Base Sequence , Child , DNA, Viral/analysis , Feces/microbiology , Gastroenteritis/microbiology , Humans , Immunoenzyme Techniques , Manitoba/epidemiology , Microscopy, Electron , Molecular Sequence Data , Neutralization Tests , Nucleic Acid Hybridization/methods , Oligonucleotide Probes/chemical synthesis , Sensitivity and Specificity , Virus Cultivation
11.
J Virol Methods ; 38(1): 25-37, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1322930

ABSTRACT

Enteric adenoviruses 40 and 41 (Ad40 and Ad41) are a prominent cause of gastroenteritis in young children. Diagnosis of these enteric types by conventional means is complicated by their fastidious growth characteristics. Enteric adenovirus growth was enhanced by cocultivation. Typing of enteric isolates currently entails analysis of the extracted viral DNA with restriction enzymes. Restriction endonuclease fragments of the Ad41 strain Tak genome were ordered by (i) double digestion, (ii) release of restriction fragments from plasmids containing 84% of the Ad41 genome in EcoRI fragments A, B and C, (iii) hybridization of Southern blotted Ad41 fragments with EcoRI fragment containing plasmids and various segments of the Ad2 genome, (iv) sequential reduction of the genome beginning with terminal restriction fragments with exonuclease III and S1 nuclease. The termini of adenovirus genomes are difficult to clone and the use of exonuclease III is a useful alternative to conventional restriction mapping. DNA restriction patterns, fragment sizes and restriction maps of the Ad4 1 strain Tak with enzymes BamHI, BglII, ClaI, EcoRI, HindIII, PstI, SalI, SmaI and XhoI are presented. Prototype strain restriction maps should enable better understanding of adenovirus type 41 and its epidemiology.


Subject(s)
Adenoviruses, Human/genetics , DNA, Viral/genetics , Adenoviruses, Human/isolation & purification , DNA Restriction Enzymes , Exodeoxyribonucleases , Gastroenteritis/microbiology , Humans , Restriction Mapping
12.
J Clin Microbiol ; 30(7): 1703-10, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1629324

ABSTRACT

The application of DNA hybridization directly to clinical specimens has the potential of improving the diagnosis of fastidious types of adenovirus. In this study, the genome of one adenovirus type from each human subgenus (A to F) was systematically evaluated by hybridization for homologous sequences to find the optimal common probe for detection of all human adenovirus types. The area of cross-hybridization, most closely defined with adenovirus type 2 (Ad2), mapped from map units 11.4 to 16.1 and 26.9 to 29.7 and, principally, to a central area of the genome between map units 47.5 and 65.2. The last area, enclosing the hexon gene, was highly conserved. Cloned probes generated from this area demonstrated the greatest homology to heterologous types by hybridization analysis. A HindIII-BglII clone containing the hexon gene of Ad2 within narrow confines reacted most evenly with all adenoviral types and detected the DNA of all other subgenera with a sensitivity 2 logs greater than that of a complete genomic Ad2 probe. The most homologous adenoviral gene sequences were observed in genes involved with DNA replication or intimately connected to the hexon in the early capsid formation. These results show that the hexon gene constitutes the best single region of the adenovirus genome for use as a genus-specific probe for the diagnosis of all human adenoviral subgenera by DNA hybridization.


Subject(s)
Adenoviruses, Human/genetics , DNA, Viral/genetics , Adenoviruses, Human/isolation & purification , Base Sequence/genetics , Cells, Cultured , DNA Probes , Humans , Nucleic Acid Hybridization
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