Feline Thymus Research Articles

Molecular cloning of the feline thymus and activation-regulated chemokine cDNA and its expression in lesional skin of cats with eosinophilic plaque.

Thymus and activation-regulated chemokine (TARC) is a member of CC chemokine and plays an essential role in recruitment of CC chemokine receptor 4 positive Th2 cells to allergic lesion. To investigate the association of TARC in allergic inflammation of cats, a TARC cDNA was cloned from feline thymus by RT-PCR with 3' rapid amplification of cDNA ends (RACE) method. The feline TARC clone contained a full length open reading frame encoding 99 amino acids which shared 80.8%, 72.5%, 65.6% and 67.8% homology with dog, human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lung, lymph node, kidney, small intestine, colon and skin of the normal cat tissues examined. Furthermore, it was found that TARC mRNA was strongly expressed in lesional skin of cats with eosinophilic plaque. The present results demonstrated that TARC might be involved in the pathogenesis of eosinophilic plaque in cats.

Biphasic thymus response by kittens inoculated with feline immunodeficiency virus during fetal development.

The objective of this study was to assess the response of the feline thymus to fetal infection with feline immunodeficiency virus (FIV), an animal model for human immunodeficiency virus infection. Thirteen feline embryos from four litters were directly inoculated with FIV during the sixth week postbreeding, a period corresponding to the late second trimester of pregnancy. Thymus tissue was collected and analyzed from randomly selected kittens at 2, 4, and 16 weeks postinoculation (PI) and compared to age-matched control kittens that did not receive fetal inoculations. Of three kittens evaluated at 2 weeks PI (week 8 of gestation), neither thymus:body weight ratio nor histologic structure differed from five age-matched control animals. However, analysis of thymocyte subpopulations by flow cytometry revealed a significant (P = 0.011) reduction in the percentage of cluster of differentiation (CD)4+/CD8+ cells from an average of 66% in control fetuses to 45% in infected fetuses. FIV RNA transcription, assessed by in situ hybridization using an FIVgag RNA probe, was widely distributed throughout the thymus in patterns suggestive of both stromal and parenchymal infection. By 4 weeks PI (week 1 postpartum), the thymus:body weight ratio was significantly reduced (P = 0.007) from 0.36% in five control kittens to 0.13% in four fetal inoculates. Severely atrophied thymus lobules supported minimal virus transcription and mean CD4+/CD8+ thymocyte percentages were lower (P = 0.021) in infected kittens (15%) compared to age-matched controls (66%). By 16 weeks PI (week 12 postpartum), thymus:body weight ratios of six inoculated kittens were not significantly different from six age-matched controls, suggesting that partial postnatal thymus regeneration had occurred. However, despite similar size, the regenerative thymus contained reduced percentages of CD4+/CD8+ thymocytes (infected: 40% versus control: 76%; P = 0.009) and increased percentages of CD4+/CD8- (11% versus 5%; P = 0.002) and CD4-/CD8+ (16% versus 9%; P = 0.035) lymphocytes. These changes were associated with widespread FIV transcription within thymic lymphocytes. Thus, the thymus of kittens infected with FIV during late fetal development is characterized by two distinct changes: neonatal atrophy and postnatal regeneration. Despite a recovery in thymus weight, thymus regeneration ineffectively restores the normal phenotypic distribution of thymocytes and supports FIV transcription.

Immunopathologic changes in the thymus during the acute stage of experimentally induced feline immunodeficiency virus infection in juvenile cats.

The feline thymus is a target organ and site of viral replication during the acute stage of feline immunodeficiency virus (FIV) infection. This was demonstrated by histologic, immunohistologic, flow cytometric, and virologic tests. Thymic lesions developed after 28 days postinoculation (p.i.) and included thymitis, premature cortical involution, and medullary B-cell hyperplasia with germinal center formation and epithelial distortion. Alterations in thymocyte subsets also developed. Fewer CD4+ CD8- cells were detected at 28 days p.i., while an increase in CD4- CD8+ cells resulted in an inversion of the thymic CD4/CD8 ratio of single-positive cells, similar to events in peripheral blood. Provirus was present in all thymocyte subpopulations including cortical CD1(hi), CD1(lo), and B cells. The CD1(hi) thymocyte proviral burden increased markedly after 56 days p.i., coincident with the presence of infiltrating inflammatory cells. Increased levels of provirus in the CD1(lo) thymocyte subpopulation were detected prior to 56 days p.i. This was likely due to inclusion of infected infiltrating inflammatory cells which could not be differentiated from mature, medullary thymocytes. Proviral levels in B cells also increased from 70 days p.i. Morphologic alterations, productive viral infection, and altered thymocyte subpopulations suggest that thymic function is compromised, thus contributing to the inability of FIV-infected cats to replenish the peripheral T-cell pool.

Molecular cloning of the feline CD8 beta-chain.

Human mouse and rat CD8 have been described as being disulphide-linked heterodimers of alpha and beta chains. More recently the chicken alpha and beta chains were described. In the bovine and feline immune system only the z-chain was reported. In this study we have cloned and determined the nucleotide sequence of a cDNA encoding the beta-chain of the feline T-cell surface antigen CD8. Using a nested polymerase chain reaction- (PCR) and two primer pairs designed from the human CD8 beta cDNA nucleotide sequence, we amplified a 430 base pair fragment from a feline thymus cDNA library which was used as a probe for screening the feline library at high stringency. After three rounds of screening, five clones were isolated. A clone, named FTb-6, containing a 3.8 kilobase pair insert was mapped, sequenced and compared with the published sequences of the genes encoding the human, mouse, rat and avian CD8 beta. We have determined the primary structure of the feline CD8 beta. The feline CD8 beta has an open reading frame, 630 nucleotides in length encoding a protein with 210 amino acid residues and its composition showed that the feline molecule is a member of the immunoglobulin gene super family.

Reduced provirus burden and enhanced humoral immune function in AZT-treated SCID-feline mice inoculated with feline immunodeficiency virus

The lack of a safe, economical murine lentivirus model for human immunodeficiency virus type 1 (HIV-1) infection of humans has hampered the preclinical evaluation of potential antiviral compounds, vaccines, and biological response modifiers. A small animal model that does not employ HIV-1 is needed to minimize risk of accidental human exposure, enhance efficient use of scarce experimental compounds, and reduce laboratory space necessary to conduct statistically significant in vivo trials. Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus of domestic cats, has been used extensively as an animal model for the pathogenesis and therapy of human HIV-1 infection. Cats, however, are not amenable to large-scale efficacy trials because of their relatively large size, high cost, and limited degree of physiologic characterization, particularly with regard to drug metabolism. To adapt the feline immune system to a small laboratory animal host, severe combined immunodeficient mice (SCID mice) were engrafted with feline lymphoid tissues (forming the SCID-fe mouse) and inoculated with FIV. Two quantitative parameters, the incidence of provirus detection in feline tissue grafts and the level of feline IgG in plasma, were used to demonstrate the antiviral efficacy of 3′-azido-3′-deoxythymidine (AZT, azidothymidine, Retrovir ®, zidovudine) in the SCID-fe system. Of 17 SCID-fe mice inoculated with 7 × 10 6 peripheral blood mononuclear cells (PBMC) from an FIV-infected cat, eight had detectable FIV provirus in both the feline thymus and feline lymph node implants, as measured by polymerase chain reaction (PCR)/Southern blot analysis. Treatment of these mice with AZT at a dose of 125 mg kg −1 day −1 in drinking water beginning 1 day prior to FIV inoculation and continuing throughout the study interval prevented the dual detection of provirus in feline lymph node and thymus grafts of all mice tested. In a separate experiment, the level of spontaneous feline IgG production was quantified by ELISA 2 weeks after FIV inoculation with and without AZT treatment. Mean plasma feline IgG level of five SCID-fe mice inoculated with 10 3 TCID 50 cell-free FIV was 2.23 mg ml −1. Mean feline IgG level of five mice inoculated with the same quantity of FIV and treated with AZT beginning 1 day prior to virus inoculation and continuing for 2 weeks thereafter was 14.98 mg ml −1. AZT significantly ( P < 0.05) enhanced feline humoral immune function at a virus inoculum titer of 10 3 TCID 50. These data indicate that a 4 week preclinical study using the FIV-infected SCID-fe model can generate quantitative indicators of provirus burden and immunologic function following treatment with an efficacious human antiretroviral compound.

Squamous cell carcinoma of the feline thymus with widespread apoptosis

The present report documents a case of squamous cell carcinoma originating from the thymus of a 12-year-old short-haired male cat. The tumour had metastasised to the sternal lymphoid tissue and to the left lung. To the authors' knowledge, thymic carcinoma has not been reported in domestic animals. Furthermore, extensive apoptosis was observed in the tumour mass.

Presence of 5'-terminal cap structures in virus-specific RNA from feline leukemia virus-infected cells

The F-422 line of feline thymus tumor cells, chronically infected with the Rickard strain of feline leukemia virus (R-FeLV), was labeled with 32P, and the total cytoplasmic RNA was isolated. The RNA was centrifuged through sucrose gradients, and R-FeLV virus-specific RNA (vRNA) was located by hybridization of portions of the gradient fractions to R-FeLV complementary DNA. vRNA classes with average sedimentation coefficients of approximately 36S, 28S, 23S, and 15S were identified. Each class of RNA was recovered by hybridized with mercurated R-FeLV complementary DNA, and the hybrids were chromatographed on columns of sulfhydryl-Sepharose to separate them from unhybridized cellular RNA. Although insufficient amount of 36S and 28S vRNA were obtained for further analysis, the 23S and 15S VRNA classes were analyzed to determine the nature of their 5' termini. Each of these vRNA classes was found to contain stoichiometric amounts of cap structures per unit length of RNA, consistent with the presence of one cap per molecule. The structure of the 23S vRNA cap was found to be m7G5'ppp5'GmpAp, whereas that of the 15S vRNA cap was m7G5'ppp5'GmpGp.

Analysis of cytoplasmic RNA and polyribosmomes from feline leukemia virus-infected cells

Cytoplasmic virus-specific RNA and polyribosomes from a chronically infected feline thymus tumor cell line, F-422, were analyzed by using in vitro-synthesized feline leukemia virus (Rickard strain) (R-FeLV) complementary DNA (cDNA) probe. By hybridization kinetics analysis, cytoplasmic, polyribosomat, and nuclear RNAs were found to be 2.1, 2.6, and 0.7% virus specific, respectively. Size classes within subcellular fractions were determined by sucrose gradient centrifugation in the presence of dimethyl sulfoxide followed by hybridization. The cytoplasmic fraction contained a 28S size class, which corresponds to the size of virion subunit RNA, and 36S, 23S, and 15 to 18S RNA species. The virus-specific 36S, 23S, and 15 to 18S species but not the 28S RNA were present in both the total and polyadenylic acid-containing polyribosomal RNA. Anti-FeLV gamma globulin bound to rapidly sedimenting polyribosomes, with the peak binding at 400S. The specificity of the binding for nascent virus-specific protein was determined in control experiments that involved mixing polyribosomes with soluble virion proteins, absorption of specific gamma globulin with soluble virion proteins, and puromycin-induced nascent protein release. The R-FeLV cDNA probe hybridized to RNA in two polyribosomal regions (approximately 400 to 450S and 250S) within the polyribosomal gradients before but not after EDTA treatment. The 400 to 450S polyribosomes contained three major peaks of virus-specific RNA at 36S, 23S, and 15 to 18S, whereas the 250S polyribosomes contained predominantly 36S and 15 to 18S RNA. Further experiments suggest that an approximately 36S minor subunit is present in virion RNA.

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.

Analysis of intracellular feline leukemia virus proteins. I. Identification of a 60,000-dalton precursor of feline leukemia virus p30

The synthesis and release of feline leukemia virus p30 was studied using a permanently infected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions, a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40 but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular feline leukemia virus p30 was isolated from infected cells by immune precipitation with antiserum to p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the precipitated proteins. Cells labeled for 3 h with [35S]methionine contained equal amounts of p30 in both the CE and the PF. p30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cell pulse labeled for 2.5 min contained a labeled 60,000-dalton polypeptide (Pp60) in the PF and a polypeptide in the CE that comigrated with feline leukemia virus p30 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When cells were chased after a pulse label, there was a rapid loss of Pp60 in the PF and an accumulation of p30 in the CE within 30 min followed by distribution of p30 in both the PF and the CE. Estimation of intracellular and extracellular p30 levels during a 0.5- to 24-h chase period suggested that most of the newly synthesized p30 was incorporated into extracellular virus. Typtic peptide analysis of labeled Pp60 and p30 demonstrated the presence of 13 of 15 p30 peptides within the Pp60 molecule. The tryptic peptide analysis in concert with the pulse-chase labeling data provides strong evidence that Pp60 is a precursor of p30.