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1.
Immunity ; 11(4): 399-409, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10549622

ABSTRACT

Naive Itk-deficient CD4+ T cells were unable to establish stable IL-4 production, even when primed in Th2-inducing conditions. In contrast, IFNgamma production was little affected. Failure to express IL-4 occurred even among cells that had gone through multiple cell divisions and was associated with a delay in the kinetics and magnitude of NFATc nuclear localization. IL-4 production was restored genetically by retroviral reconstitution of Itk or biochemically by augmenting the calcium flux with ionomycin. In vivo, Itk-deficient mice were unable to establish functional Th2 cells. Development of protective Th1 cells was unimpeded. These data define a nonredundant role for Itk in modulating signals from the TCR/CD28 pathways that are specific for the establishment of stable IL-4 but not IFNgamma expression.


Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins , Protein-Tyrosine Kinases/deficiency , Th2 Cells/cytology , Transcription Factors/metabolism , Animals , Biological Transport , CD28 Antigens/immunology , Calcium Signaling/drug effects , Cell Differentiation , Cell Division , Disease Progression , Female , Gene Expression Regulation/drug effects , Interferon-gamma/biosynthesis , Interleukin-2/physiology , Interleukin-4/biosynthesis , Interleukin-4/deficiency , Ionomycin/pharmacology , Ionophores/pharmacology , Leishmania major , Leishmaniasis, Cutaneous/immunology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , NFATC Transcription Factors , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/physiology , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/physiology , Specific Pathogen-Free Organisms
4.
Immunity ; 6(5): 551-7, 1997 May.
Article in English | MEDLINE | ID: mdl-9175833

ABSTRACT

Resistance or susceptibility of inbred mouse strains to the parasite Leishmania major correlates with CD4+ T cell responses of the Th1 or Th2 subsets, respectively. To evaluate the genetic basis for this difference, resistant B10.D2 mice were backcrossed onto susceptible BALB/c mice for five generations with selection for resistance. Candidate resistance loci were identified by high frequency of heterozygosity in resistant N5 backcross mice. Loci on chromosomes 6, 7, 10, 11, 15, and 16 were associated with resistance, demonstrating the multigenic nature of this phenotype. The presence of all six loci was not necessary to confer resistance and no single locus was required. Rather, a variety of combinations of these loci may be capable of interacting to confer resistance.


Subject(s)
Chromosome Mapping , Crosses, Genetic , Leishmania major/genetics , Leishmaniasis, Cutaneous/genetics , Animals , Disease Susceptibility , Female , Genotype , Immunity, Innate , Leishmaniasis, Cutaneous/immunology , Linkage Disequilibrium , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Pedigree , Phenotype , Probability
5.
J Immunol ; 157(10): 4421-7, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8906817

ABSTRACT

Splenic CD4+NK1.1+ T cells have been shown to secrete large and transient amounts of IL-4 mRNA 90 min after i.v. injection of anti-CD3 Ab, suggesting that this novel subset of T cells may induce Th2 responses in the spleen by quickly providing IL-4 at the onset of an immune response. beta2-microglobulin-deficient (beta2m(o/o)) mice have been shown to contain strongly reduced numbers of NK1.1+ T cells and to be severely impaired in their capacity for rapid induction of IL-4 mRNA in response to anti-CD3, demonstrating that these cells are MHC class I dependent. To address the role of CD4+NK1.1+ T cells in the induction of Th2 responses against Leishmania major, we have dissected the onset and the outcome of the immune response elicited against the parasite in BALB/c-beta2m(o/o) mice and in anti-NK1.1-treated congenic BALB/c mice expressing the NK1.1 marker (BALB/c-NK1.1+). Both BALB/c-beta2m(o/o) and NK1.1-depleted BALB/c-NK1.1+ mice developed a progressive, nonhealing disease that was indistinguishable from wild-type mice. Upon infection, early induction of IL-4 mRNA in the lymph node was not affected in BALB/c-beta2m(o/o) and in NK1.1-depleted BALB/c-NK1.1+ mice, but was abrogated by injection of a CD4-depleting Ab. These data suggest that, in the lymph node, MHC class I-dependent CD4+NK1.1+ T cells do not play a major role in the generation of Th2 responses against L. major. To investigate whether the inability of NK1.1+ T cells to induce IL-4 production in the lymph node was specific to L. major Ag, mice were challenged with low doses of anti-CD3 Ab s.c. in the footpad. In contrast to the spleen, normal levels of IL-4 mRNA were expressed in the lymph nodes of BALB/c-beta2m(o/o) mice. Thus, the MHC class I-dependent CD4+NK1.1+ cell population that gives a rapid IL-4 response in the spleen appears not to contribute significantly to early induced IL-4 responses in the popliteal lymph nodes.


Subject(s)
Antigens/analysis , CD4 Antigens/analysis , Histocompatibility Antigens Class I/physiology , Interleukin-4/biosynthesis , Lymphocyte Activation , Proteins/analysis , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th2 Cells/immunology , Animals , Antigens, Ly , Antigens, Surface , Female , Lectins, C-Type , Leishmania major/pathogenicity , Leishmaniasis, Cutaneous/immunology , Lymph Nodes/cytology , Lymph Nodes/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily B , Time Factors
6.
Blood ; 85(4): 941-51, 1995 Feb 15.
Article in English | MEDLINE | ID: mdl-7849316

ABSTRACT

Severe neutropenia and bone marrow (BM) morphologic abnormalities occur during experimentally induced primary infection with feline immunodeficiency virus (FIV), a lentivirus biologically similar to human immunodeficiency virus (HIV). To further characterize the mechanisms involved in this acute infection model of lentivirus-induced BM suppression, peripheral blood counts, histologic BM studies, and BM culture assays were performed on 12 cats that underwent necropsy at regular intervals postinoculation (PI) with FIV Petaluma. Plasma viremia developed at week 3 PI and neutropenia was initially detected at week 6 PI. Low neutrophil counts, but normal hematocrits and platelet counts, persisted through week 12 PI. Infected BM mononuclear cells and megakaryocytes were identified by in situ hybridization assays for FIV nucleic acids in BM sections of cats that underwent necropsy at weeks 4 to 12 PI, correlating with detection of soluble FIV p24 antigen and identification of infected mononuclear and macrophage cells in BM buffy-coat cell cultures from these cats. At weeks 1.5 to 4 PI, the mean frequencies (number per 10(5) BM mononuclear cells) of erythroid progenitors (erythroid colony-forming units [CFU-E] and erythroid burst-forming units [BFU-E] and granulocyte/macrophage progenitors (CFU-granulocyte/macrophage [CFU-GM]) were increased to 508 +/- 74, 143 +/- 24, and 110 +/- 17, respectively (n = 5 cats) as compared with controls (172 +/- 24, 86 +/- 26, and 44 +/- 10; n = 3 cats; P < .02), and the percentages of progenitors in the DNA-synthetic phase of the cell cycle were equivalent to controls. In contrast, the progenitor frequencies at weeks 6 to 12 PI were significantly decreased (72 +/- 16, 43 +/- 6, and 19 +/- 4, respectively; n = 7 cats; P < .01), and these progenitors were more frequently in S-phase. Autologous serum significantly inhibited (P < .05) the growth of CFU-GM in 6 of 9 cats and failed to support the maximal growth of BFU-E in 4 of 9 cats studied at weeks 4 to 12 PI, whereas no such abnormalities were observed in colony assays containing autologous sera from control cats (n = 3) or cats studied at weeks 1.5 or 3 PI (n = 3). In comparison, sera from FIV-infected cats did not inhibit the growth of normal, allogeneic progenitors. However, FIV serum frequently failed to support maximal in vitro growth of normal CFU-GM as compared with uninfected allogeneic sera, further suggesting a lack of progenitor growth-promoting substances in infected cat sera.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Bone Marrow/virology , Feline Acquired Immunodeficiency Syndrome/pathology , Feline Acquired Immunodeficiency Syndrome/physiopathology , Hematopoiesis , Hematopoietic Stem Cells/pathology , Immunodeficiency Virus, Feline/physiology , Neutropenia/etiology , Animals , Bone Marrow/pathology , Bone Marrow/physiopathology , Cats , Cell Cycle , Cells, Cultured , DNA, Viral/analysis , Feline Acquired Immunodeficiency Syndrome/blood , Immunodeficiency Virus, Feline/isolation & purification , In Situ Hybridization , Kinetics , RNA, Viral/analysis , Time Factors , Viremia/blood , Viremia/pathology , Viremia/physiopathology
7.
Virology ; 205(2): 546-53, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7975256

ABSTRACT

Molecularly cloned viruses are considered essential reagents for characterizing viral domains responsible for infectivity and disease pathogenesis in the host. The infectivity and hematological alterations associated with two molecularly cloned isolates of feline immunodeficiency virus (FIV-pPPR and FIV-pF34) and an uncloned isolate (FIV-PPR) were assessed by inoculation of cats. Inoculated cats were tested for viral antibody, viremia, and clinical pathological disease. Peripheral blood mononuclear cells isolated from inoculated cats were assayed for virus infection by virus isolation, amplification of proviral DNA (by polymerase chain reaction), and in situ hybridization for viral RNA. Over 50% of the cats inoculated with cloned virus FIV-pF34 failed to seroconvert even when coinfected with feline leukemia virus; these cats were consistently virus positive only by amplification of proviral DNA. All cats inoculated with cloned virus FIV-pPPR seroconverted and were found virus positive by at least two of three virus detection assays. Both cloned viruses were less capable of suppressing CD4:CD8 ratios when compared to the biological isolates from which they were cloned. Isolates which replicate efficiently in feline peripheral blood mononuclear cells (PBMC), i.e., FIV-pPPR or biological FIV-PPR, caused greater virus load and lower CD4:CD8 ratios when compared to cloned FIV-pF34, which replicates efficiently in feline adherent cell lines and macrophages but poorly in primary feline PBMC. Molecular clones FIV-pF34 and FIV-pPPR will be useful reagents for characterization of viral determinants of virus load and possibly, cell tropism in vivo.


Subject(s)
Feline Acquired Immunodeficiency Syndrome/virology , Immunodeficiency Virus, Feline/pathogenicity , Animals , Antibodies, Viral/biosynthesis , CD4-CD8 Ratio , Cats , Cell Line , Cloning, Molecular , Disease Models, Animal , Feline Acquired Immunodeficiency Syndrome/immunology , Immunodeficiency Virus, Feline/immunology , Immunodeficiency Virus, Feline/isolation & purification , Leukocytes, Mononuclear/virology
8.
AIDS ; 8(7): 873-8, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7946095

ABSTRACT

OBJECTIVE: To determine the potential mechanisms for disease potentiation where feline immunodeficiency virus (FIV) infection of persistently feline leukemia virus (FeLV)-infected cats results in more severe FIV disease and increased mortality than FIV infection of specific pathogen-free cats. DESIGN AND METHODS: To determine whether pseudotype formation resulting in expanded cell tropism may be an important mechanism, cellular targets and tissue distribution of FIV and FeLV were determined by in situ hybridization and/or immunohistochemistry. To determine whether FeLV can transactivate the FIV long terminal repeat (LTR) resulting in increased FIV expression, in vitro transient expression assays were performed. To examine whether persistent FeLV infection can cause the deletion of a suppressive T-lymphocyte population, peripheral blood mononuclear cell (PBMC) cultures from persistently FeLV-infected cats were infected with FIV and monitored for FIV antigen levels. RESULTS: Macrophages were the predominant target of FIV infection and were disseminated in a similar pattern in lymphoid and nonlymphoid tissues of both FIV-infected and FeLV/FIV-coinfected cats. FeLV-infected cells expressing FIV RNA were not present. Significant transactivation of the FIV LTR in FeLV-infected cells was not demonstrated. FIV antigen production was similar upon in vitro infection of PBMC from FeLV-infected and uninfected cats. CONCLUSIONS: Neither direct virus/virus interactions, such as FeLV/FIV pseudotype formation or transactivation of the FIV LTR in FeLV-infected cells, nor deletion of a regulatory cell subset from the blood of FeLV-infected cats, was found to be the mechanism of disease potentiation.


Subject(s)
Feline Acquired Immunodeficiency Syndrome/complications , Immunodeficiency Virus, Feline/physiology , Leukemia Virus, Feline/physiology , Leukemia, Feline/complications , Animals , Cats , Cells, Cultured , Feline Acquired Immunodeficiency Syndrome/microbiology , Gene Expression Regulation, Viral , Immunodeficiency Virus, Feline/genetics , In Situ Hybridization , Leukemia Virus, Feline/genetics , Leukemia, Feline/microbiology , Macrophages/microbiology , RNA, Viral/analysis , Repetitive Sequences, Nucleic Acid , T-Lymphocytes/microbiology , Transcriptional Activation , Viral Interference
9.
J Virol ; 68(5): 3080-91, 1994 May.
Article in English | MEDLINE | ID: mdl-8151773

ABSTRACT

The objective of this study was to identify cellular and organ targets of acute feline immunodeficiency virus (FIV) infection in vivo. Tissues of FIV-infected cats were studied at eight time points during the first 3 months after experimental infection. FIV nucleic acids were first detected by in situ hybridization 21 days after infection, approximately 1.5 weeks after lymph node enlargement was first observed and 3 weeks before the primary acute flu-like illness. The majority of FIV-infected cells were present in lymphoid organs, though low numbers of infected cells were noted in nonlymphoid organs as well. Germinal centers harbored many of the FIV-infected cells within lymphoid tissues. The thymic cortex was also a major site of early infection. Combined in situ hybridization and immunohistochemistry revealed that T lymphocytes were the primary target of early FIV infection in tissues of cats before the onset of clinical signs of acute illness. An unidentified population of mononuclear cells and a few macrophages were also infected. During the ensuing acute flu-like illness, the proportion of FIV-infected macrophages in tissues increased dramatically. This early shift in the predominant cellular localization of FIV from T lymphocytes to macrophages may be important for establishing viral persistence.


Subject(s)
Feline Acquired Immunodeficiency Syndrome/microbiology , Immunodeficiency Virus, Feline/growth & development , Animals , Cats , DNA, Viral/analysis , Feline Acquired Immunodeficiency Syndrome/blood , Feline Acquired Immunodeficiency Syndrome/pathology , Female , In Situ Hybridization , Intestines/microbiology , Intestines/pathology , Lymph Nodes/microbiology , Lymph Nodes/pathology , Macrophages/microbiology , Male , RNA, Viral/analysis , Spleen/microbiology , Spleen/pathology , T-Lymphocytes/microbiology , Thymus Gland/pathology , Time Factors , Tissue Distribution
10.
Vet Immunol Immunopathol ; 35(1-2): 37-49, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1337401

ABSTRACT

Natural or experimental feline immunodeficiency virus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness.


Subject(s)
Bone Marrow/microbiology , Feline Acquired Immunodeficiency Syndrome/microbiology , Immunodeficiency Virus, Feline/isolation & purification , Megakaryocytes/microbiology , Animals , Cats , DNA Probes , DNA, Viral/genetics , Female , Immunodeficiency Virus, Feline/genetics , In Situ Hybridization , Leukemia Virus, Feline/genetics , Leukemia Virus, Feline/isolation & purification , Leukemia, Feline/microbiology , Leukocytes, Mononuclear/microbiology , Male , RNA, Viral/analysis , Specific Pathogen-Free Organisms
11.
J Virol ; 66(7): 4040-9, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1318395

ABSTRACT

A study was undertaken to determine the rate of viral transmission among naive specific-pathogen-free (SPF) cats living in close contact with feline immunodeficiency virus (FIV)-infected cats. Twenty SPF cats were housed in the same rooms with experimentally FIV-infected seropositive and virus culture-positive cats for 2 to 4 years and were monitored for the presence of FIV nucleic acids and antibodies. Only 1 of the 20 cats became seropositive and virus culture positive and developed signs of disease. Genomic DNA from bone marrow and peripheral blood mononuclear cells (PBMC) of 10 of 19 healthy-appearing seronegative cats became positive for FIV DNA by the polymerase chain reaction. Twenty-eight SPF cats housed as groups in separate quarters and never exposed to FIV-infected cats were uniformly negative for FIV DNA. FIV RNA transcripts were detected in concanavalin A-stimulated PBMC cultures from 4 of 10 FIV DNA-positive, seronegative cats by in situ hybridization. PBMC from three of four naive SPF cats acquired FIV nucleic acids after the cats were transfused with blood and bone marrow from FIV genome-positive, seronegative donors. Three of five FIV-seronegative cats housed for years with naturally FIV-infected cats in a private household were also found to harbor FIV DNA, indicating that the same phenomenon occurred in the field. These findings demonstrate that cats living in close contact with FIV-infected seropositive cats can acquire FIV nucleic acids without developing detectable levels of serum antibodies or disease.


Subject(s)
Antibodies, Viral/blood , DNA, Viral/isolation & purification , Feline Acquired Immunodeficiency Syndrome/microbiology , Immunodeficiency Virus, Feline/isolation & purification , RNA, Viral/isolation & purification , Animals , Bone Marrow/microbiology , Cats , Feline Acquired Immunodeficiency Syndrome/blood , Feline Acquired Immunodeficiency Syndrome/immunology , Female , Genome, Viral , Immunodeficiency Virus, Feline/genetics , Immunodeficiency Virus, Feline/immunology , Immunologic Techniques , Leukocytes, Mononuclear/microbiology , Male , Nucleic Acid Hybridization , Polymerase Chain Reaction , Specific Pathogen-Free Organisms , T-Lymphocyte Subsets/microbiology , T-Lymphocytes, Regulatory/immunology
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