A lifetime aging study of human CD19 transgenic mice.

Mice transgenic for human CD19 have been an important animal model to help understand the role of this molecule in B lymphocyte function. Previously, no lifetime studies had been performed to understand the effects of this CD19 over expression on the survival or spontaneous pathology within the C57BL/6J background strain. We conducted a lifetime study with interim sacrifices to understand the transgenic effects on clinical signs, body weight, survival, and spontaneous pathology. Blood and urine samples were collected from select animals at various time points during the study for measurement of clinical pathology parameters and groups of animals were euthanized and examined at predetermined intervals. There was fair survival with some animals living to 108 weeks of age. Clinical pathology evaluations revealed a declining red cell mass with a regenerative anemia, increasing total white blood cell counts and decreasing glucose level. Total protein, albumin, and globulin levels increased to 52 weeks of age and then declined to or below baseline with advancing age. Increased urinary microalbumin levels correlated with the severity of a glomerulopathy at 76 and 84 weeks of age. Mean body weight increased through 70 weeks and then declined to weights similar to week 28 at 108 weeks. Macroscopic observations included pale kidneys, enlarged seminal vesicles, and enlarged spleens (at 108 weeks of age). The most common neoplasms in this study were bronchiolar alveolar adenomas in the lung, histiocytic sarcoma in several different tissues, and hepatocellular adenomas. The most common non-neoplastic lesions were renal glomerulopathy, and pulmonary lymphocytic infiltrates with increased numbers of alveolar macrophages.

Comparative 90-day dietary study of paraffin wax in Fischer-344 and Sprague-Dawley rats.

Highly refined mineral hydrocarbons (MHCs) such as low melting point paraffin wax (LMPW) and low viscosity white oils can cause inflammatory changes in the liver and mesenteric lymph nodes (MLNs) of the Fischer-344 (F-344) rat. In contrast, only minimal MLN changes are seen in the Sprague-Dawley (S-D) rat with no changes in the liver. In this study, the response of female F-344 and S-D rats was compared after 90days dietary treatment with 0%, 0.2% or 2% LMPW. Effects in the F-344 rats were significantly greater than in the S-D rats: increased liver and splenic weights and inflammatory changes (hepatic microgranulomas) in these tissues were observed only in the F-344 rats. Microgranulomas in the MLNs were observed in both strains but the effects were substantially greater in the F-344 rats. Cellular markers of inflammation were examined in a subset of rats from each group using immunohistochemical staining. An increase in staining for CD3 (T-cells), CD8a (suppresser/cytotoxic T-cells) and CD4 (helper T-cells) correlated with an increase in lymphoid cells in the livers of treated F-344 rats. The majority of macrophages in the hepatic microgranulomas of treated F-344 rats were negative for the ED2 marker, indicating a likely origin from non-resident macrophages. Electron microscopy showed Kupffer cell hypertrophy and hyperplasia in treated F-344 rats. However, lysozyme staining (indicating activation of epithelioid macrophages) decreased with increasing granuloma size. Non-ED2 expressing cells may have been recruited but not sufficiently activated to be lysozyme positive. Inflammatory changes in the cardiac mitral valve noted in previous studies of LMPW were also seen in the F-344 rats in this study but not in the S-D rats. Chemical analysis showed that MHC accumulated in livers from treated F-344 but not S-D rats and the concentration was more than 2-fold greater in MLNs from the F-344 than from the S-D rats. The F-344 appears to be more immunologically sensitive to a number of agents than other rat strains and the results of this study suggest that this may contribute, along with pharmacokinetic differences, to the inflammatory response of F-344 rats to dietary MHCs.

Mercury induces regional and cell-specific stress protein expression in rat kidney.

Cells respond to physiologic stress by enhancing the expression of specific stress proteins. Heat-shock proteins (hsps) and glucose-regulated proteins (grps) are members of a large superfamily of proteins collectively referred to as stress proteins. This particular stress-protein response has evolved as a cellular strategy to protect, repair, and chaperone other essential cellular proteins. The objective of this study was to evaluate the differential expression of four hsps in the renal cortex and medulla during experimental nephrotoxic injury using HgCl2. Male Sprague-Dawley rats received single injections of HgCl2 (0.25, 0.5, or 1 mg Hg/kg, i.v.). At 4, 8, 16, or 24 h after exposure, kidneys were removed and processed for histopathologic, immunoblot, and immunohistochemical analyses. Nephrosis was characterized as minimal or mild (cytoplasmic condensation, tubular epithelial degeneration, single cell necrosis) at the lower exposures, and progressed to moderate or severe (nuclear pyknosis, necrotic foci, sloughing of the epithelial casts into tubular lumens) at the highest exposures. Western blots of renal proteins were probed with monoclonal antibodies specific for 4 hsps. In whole kidney, Hg(II) induced a time- and dose-related accumulation of hsp72 and grp94. Accumulation of hsp72 was predominantly localized in the cortex and not medulla, while grp94 accumulated primarily in the medulla but not cortex. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure, and it was equally localized in cortex and medulla. Hsp90 was not detected in kidneys of control or Hg-treated rats. Since hsp72 has been shown involved in cellular repair and recovery, and since Hg(II) damage occurs primarily in cortex, we investigated the cell-specific expression of this hsp. Hsp72 accumulated primarily in undamaged distal convoluted tubule epithelia, with less accumulation in undamaged proximal convoluted-tubule epithelia. These results demonstrate that expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury. While the role of accumulation of hsps and other stress proteins in vivo prior to or concurrent with nephrotoxicity remains to be completely understood, these stress proteins may be part of a cellular defense response to nephrotoxicants. Conversely, renal tubular epithelial cells that do not or are unable to express stress proteins, such as hsp72, may be more susceptible to nephrotoxicity.

Mercuric chloride-induced apoptosis is dependent on protein synthesis.

Apoptosis is a mode of cell death with morphologic and biochemical features that distinguish it from necrosis. Recent studies demonstrating that mercury compounds initiate apoptosis in cultured cells did not elucidate if the biochemical mechanism of apoptosis involved a dependence on macromolecular synthesis post-insult, i.e. programmed cell death. The objectives of this in vitro study were (1) to determine if HgCl2 cytotoxicity includes an apoptotic component, and (2) to determine if apoptosis is dependent on protein synthesis, i.e. proceeds by an inducible mechanism. Suspensions of mouse lymphoma (L5178Y-R) cells were exposed to 0, 1, 5, or 10 microM HgCl2 and apoptosis was evaluated utilizing qualitative and quantitative methods. At 24 h after exposure, transmission electron microscopy revealed a concentration-related increase in morphologic changes typical of apoptosis: margination of condensed chromatin to the nuclear membrane, dilation of the rough endoplasmic reticulum, cytoplasmic condensation and vacuolation, nuclear dissolution, and plasma membrane blebbing. An increase in Hg-induced DNA fragmentation (DNA 'ladder') was observed using agarose gel electrophoresis. Time- and concentration-dependent increases in the percent of apoptotic cells were observed at 1, 6, 12, and 24 h after HgCl2 exposure using a flow cytometric method that discriminates between cells according to size and granularity. Pretreatment of cells with cycloheximide (CHX), an inhibitor of translation, prior to HgCl2 exposure resulted in a 25-50% reduction in apoptotic cells 24 h after exposure to 10 and 20 microM HgCl2, and concomitantly reduced the overall cytotoxicity compared to HgCl2 alone. These results, although limited to a single cell line, support the hypothesis that HgCl2 induces apoptosis that is dependent, at least in part, upon protein synthesis.

Correlation of toxicity and pharmacokinetic properties of a phosphorothioate oligonucleotide designed to inhibit ICAM-1.

ISIS 2302 is a phosphorothioate oligodeoxynucleotide with a sequence complementary to the mRNA of human intercellular adhesion molecule 1 (ICAM-1). Hybridization of ISIS 2302 to the mRNA inhibits expression of the ICAM-1 protein in response to inflammatory stimuli. A murine active antisense oligonucleotide, ISIS 3082, has been used for in vivo pharmacology studies and has anti-inflammatory activity in models of organ transplant rejection, ulcerative colitis, and collagen-induced arthritis at doses ranging from 0.03 to 5 mg/kg. The safety assessment for ISIS 2302 includes general toxicity studies up to 6 mo in duration in mice and monkeys, genetic toxicity studies, and reproductive/fertility studies. ISIS 3082 was examined in parallel with ISIS 2302 in mouse toxicity and reproductive studies. The toxicities observed following systemic administration of ISIS 2302 and ISIS 3082 were similar and consistent with those observed for other compounds in this chemical class and, therefore, are independent of the suppression of ICAM-1 expression. Toxicokinetic evaluation demonstrated that toxicities occurred in organs containing the highest concentrations of ISIS 2302. Evidence of immune stimulation. including dose-dependent splenomegaly, lymphoid hyperplasia, and multiorgan mixed mononuclear cell infiltrates, was the most common finding in rodent studies. Monkeys were much less sensitive than mice to immune stimulation. Kidney contained the highest concentrations of ISIS 2302. Morphologic changes observed in kidney included atrophic and regenerative changes in proximal tubular epithelium; however, there was no evidence of functional abnormalities. Additional histologic changes noted in proximal tubular epithelium included basophilic granules, which were reflective of oligonucleotide distribution and uptake in these cells. Liver also contained high concentrations of oligonucleotide, which were associated with Kupffer cell hypertrophy in mice. Changes in serum transaminases, cholesterol, and triglycerides were reflective of hepatic alterations. In monkeys, high concentrations of oligonucleotide caused a transient increase in clotting times and activation of the alternative complement pathway. All toxicities associated with ISIS 2302 were reversible and occurred at doses well above those required for pharmacologic activity or currently used in clinical trials. In addition, there has been no evidence of genetic toxicity associated with ISIS 2302, and no changes in reproductive performance, fertility, or fetal development have been noted in animals treated with ISIS 2302 or ISIS 3082.

An oligopeptide of the feline leukemia virus envelope glycoprotein is associated with morphological changes and calcium dysregulation in neuronal growth cones.

Neuropathogenic processes that affect the pathfinding properties of neuronal growth cones could account for many of the dysfunctions unique to retroviral infection of developing nervous systems. Pediatric HIV-1 infection, for example, is associated with a distinctive neuropathogenesis that includes marked cortical atrophy, cognitive disorders, and pyramidal dysfunction. The ability of HIV's envelope glycoprotein, gp120, to produce increased intracellular free calcium ([Ca2+]i) leading to neuronal death has been documented. We hypothesize that gp120 and the envelope glycoproteins of other retroviruses may have similar calcium-increasing effects in advancing growth cones, a property which could disrupt the orderly development of the nervous system. To explore this possibility, we exposed chick ciliary ganglion neurons in culture to a known cytopathic region (CVR5) of the feline leukemia virus' envelope glycoprotein. CVR5 produced [Ca2+]i increases and dose-dependent morphological changes in growth cones isolated from their cell bodies by axotomy. These responses of growth cones to CVR5 suggest that the neurotoxic effects of retroviruses could be mediated at the level of the individual growth cone through exposure to envelope glycoproteins and could constitute one mechanism by which these viruses perturb the normal development of the nervous system.

FeLV envelope protein (gp70) variable region 5 causes alterations in calcium homeostasis and toxicity of neurons.

In humans and animals, retroviruses have been implicated in nervous system disease. Our objective was to characterize the neurotoxicity of a peptide sequence derived from an animal retrovirus, the feline leukemia virus (FeLV). Using a peptide sequence from the subtype FeLV-C envelope protein variable region 5 (VR5), cytotoxicity was demonstrated in studies that evaluated neuronal survival, neurite outgrowth, and alterations in intracellular calcium ion concentration. The FeLV subtype isolate FeLV-CSarma possesses an envelope protein VR5 amino acid sequence that varies by four amino acids from the VR5 amino acid sequence of subtype FeLV-AGlasgow. The polypeptide representing the VR5 of FeLV-CSarma (FeLV-CVR5) is significantly more neurotoxic than the polypeptide sequence representing the VR5 of FeLV-AGlasgow (FeLV-AVR5). FeLV-CVR5 (> or = 3 microM) exposure resulted in significant dose-dependent neurotoxicity. Antibodies to FeLV-CVR5 blocked this effect. Neurite outgrowth was significantly reduced at all tested concentrations (3-12 microM) of FeLV-CVR5, with a 92% reduction in neurite length at 12 microM. FeLV-AVR5 was significantly less neurotoxic with respect to neurite outgrowth than was FeLV-CVR5. The significant reduction in neurotoxicity for FeLV-AVR5 illustrates the importance of the 4-amino-acid difference between it and FeLV-CVR5. Alterations in intracellular calcium ion concentration were associated with this neurotoxicity.

Toxicological properties of several novel oligonucleotide analogs in mice.

The toxicological properties of ISIS 3082, a phosphorothioate oligonucleotide, and five structurally related analogs of ISIS 3082, were examined in Balb/c mice. Comparisons were made between the uniform phosphorothioate oligonucleotide (ISIS 3082), and a 2' propoxy modified phosphodiester (ISIS 9044), a 2' propoxy phosphorothioate (ISIS 9045), a chimeric oligonucleotide comprised of 2' propoxy diester wings and phosphorothioate deoxy center (ISIS 9046), a 5' C18 amine phosphorothioate (ISIS 9047), or a 5' cholesterol modified phosphorothioate (ISIS 8005) oligonucleotide. Oligonucleotides were administered at 50 mg/kg by i.v. bolus injection (tail vein) every other day for 14 days. In general, the spectrum of alterations observed for ISIS 3082 and all of the analogs were relatively similar. Balb/c mice treated with ISIS 3082 were observed to have increases in liver transaminases and a decrease in triglycerides consistent with results from previous studies performed in CD-1 mice. Spleen weights were also increased in ISIS 3082-treated mice, but no histopathological alterations were noted. ISIS 9046 resulted in a toxicity profile that was very similar to that described for ISIS 3082 with the exception of a slightly lower cholesterol level. Alterations induced by ISIS 9045, ISIS 9047 and ISIS 8005 were qualitatively similar to ISIS 3082, but in general more pronounced, with greater reductions in cholesterol and platelet counts, or increases in blood urea nitrogen relative to ISIS 3082. Red blood cell (RBC) counts and hematocrit were also reduced in mice treated with ISIS 9046, ISIS 9047 and ISIS 8005 relative to the ISIS 3082 treatment group. Kupffer cell hypertrophy and basophilic inclusions in Kupffer cells were observed in mice treated with ISIS 9045, ISIS 9047 and ISIS 8005, but not in ISIS 3082-treated mice. A unique renal lesions was noted in mice treated with ISIS 9044 only that was characterized as mild atrophy of proximal convoluted tubules associated with interstitial fibrosis. With the exception of the renal lesions observed in ISIS 9044 treated mice, the toxicity profiles of various oligonucleotide analogs examined in this study were similar to that observed for ISIS 3082.

Cytopathic feline leukemia viruses cause apoptosis in hemolymphatic cells.

Certain isolates of the oncoretrovirus feline leukemia virus (FeLV) are strongly cytopathic for hemolymphatic cells. A major cytopathicity determinant is encoded by the SU envelope glucoprotein gp70. Isolates with subgroup C SU gp70 genes specifically induce apoptosis in hemolymphatic cells but not fibroblasts. In vitro exposure of feline T-cells to FeLV-C leads first to productive viral replication, next to virus-induced cell agglutination, and lastly to apogenesis. This in vitro phenomenon may explain the severe progressive thymic atrophy and erythroid aplasia which follow viremic FeLV-C infection in vivo. Inappropriate apoptosis induction has also been hypothesized to explain the severe thymico-lymphoid atrophy and progressive immune deterioration associated with isolates of FeLV containing variant envelope genes. The influence of envelope hypervariability (variable regions 1 [Vr1] and 5 [Vr5] on virus tropism, viremia induction, neutralizing antibody development and cytopathicity is discussed. Certain potentially cytopathic elements in FeLV SU gp70 Vr5 may derive from replication-defective, poorly expressed, endogenous FeLVs. Other more highly conserved regions in FeLV TM envelope p15E may also influence apoptosis induction.

The use of immunohistochemistry for evaluating the liver.

Immunohistochemistry has been utilized in recent years primarily for diagnosis of infectious diseases of the liver, especially in humans. The utility of immunohistochemistry has extended to experimental and toxicologic pathology in a variety of areas: identification of cell phenotype, cell receptors, cytokine and chemikine production, and functional cell changes such as enzyme induction. In addition, markers for experimental carcinogenesis studies are detectable by immunohistochemical changes as well as novel antigen induction such as placental glutathione-S-transferase, oncofetal proteins, oncogene products, and typing of neoplasms. Immunohistochemistry is also used to detect the origin and function of various cell types in developmental and toxicity studies. Careful use of immunohistochemical procedures in conjunction with routine pathology and molecular techniques enhance the ability of the toxicologic pathologist to diagnose unique conditions and to understand mechanisms of lesion development.

Acute feline leukemia virus infection causes altered energy balance and growth inhibition in weanling cats.

Naturally occurring retroviral infections cause progressive weight loss, immune suppression, invasion by opportunistic organisms, and eventual death. Feline leukemia virus (FeLV) inhibited growth and decreased energy intake in seven experimentally infected weanling cats compared with age- and sex-matched controls. Remarkably, changes in energy intake, energy expenditure, and weight gain occurred in the acute phase of infection prior to the systemic/productive bone marrow phase of FeLV infection. In other words, growth inhibition developed before FeLV infection was clinically detectable with use of standard enzyme-linked immunosorbent assay or fixed-cell immunofluorescence assays of circulating neutrophils and platelets. Acutely infected, previremic cats consumed 25% less energy [Day 4 postinoculation to Day 16 postinoculation (p < 0.05)] and expended 20% less energy [Day 8 postinoculation to Day 18 postinoculation (p < 0.05)] compared with control cats. Growth stunting of inoculated cats began by Day 11 postinoculation (p < 0.05) and was not corrected during the remaining 4 months of the study. Thus, experimental FeLV infection causes perturbations of metabolism and energy balance resulting in permanent growth impairment. Secondly, detrimental metabolic effects begin in the acute phase of retroviral infection, prior to the clinically detectable phase of FeLV infection.

Increased insensible water loss in feline retrovirus-infected cats.

Feline retroviruses such as feline leukemia virus (FeLV) adversely affect the regulation of many vital host systems such as the immune response, erythropoiesis, and nutrient metabolism. In this paper, we describe the disruption of an additional homeostatic mechanism-evaporative water loss-by FeLV. Viremic cats had greater evaporative water losses (24.0 +/- 1.4 gm water/kg per day) at low relative humidity levels (19% to 25% relative humidity) when compared to age- and sex-matched control cats (19.7 +/- 1.4 gm of water/kg per day [P < 0.05]). At relative humidity levels greater than 50%, viremic and control cats had similar evaporative water losses. Viremia also resulted in an elevation in the average body temperature (39.1 +/- 0.5 degrees C) compared to control cats (38.4 +/- 0.3 degrees C) (P < 0.001). However, the energy expenditure of viremic cats (17.14 +/- 1.60 kJ/kg/h) was not significantly different from the energy expenditure of control cats (17.02 +/- 2.22 kJ/kg/h). The elevated body temperature of viremic cats likely causes a greater increase in evaporative water loss at low relative humidity levels and suggests further study of water balance in retroviral infection is warranted.

Defective endogenous proviruses are expressed in feline lymphoid cells: evidence for a role in natural resistance to subgroup B feline leukemia viruses.

Endogenous feline leukemia virus (FeLV)-related sequences (enFeLV) are a family of proviral elements found in domestic cats and their close relatives. These elements can recombine with exogenous, infectious FeLVs of subgroup A (FeLV-A), giving rise to host range variants of FeLV-B. We found that a subset of defective enFeLV proviruses is highly expressed in lymphoma cell lines and in a variety of primary tissues, including lymphoid tissues from healthy specific-pathogen-free cats. At least two RNA species were detected, a 4.5-kb RNA containing gag, env, and long terminal repeat sequences and a 2-kb RNA containing env and long terminal repeat sequences. Cloning of enFeLV cDNA from two FeLV-free lymphoma cell lines (3201 and MCC) revealed a long open reading frame (ORF) encoding a truncated env gene product corresponding to the N-terminal portion of gp70env. Interestingly, all of three natural FeLV-B isolates include 3' env sequences which are missing from the highly transcribed subset and hence must be derived from other enFeLV elements. The enFeLV env ORF cDNA clones were closely similar to a previously characterized enFeLV provirus, CFE-16, but were polymorphic at a site corresponding to an exogenous FeLV neutralization epitope. Site-specific antiserum raised to a C-terminal 30-amino-acid peptide of the enFeLV env ORF detected an intracellular product of 35 kDa which was also shed from cells in stable form. Expression of the 35-kDa protein correlated with enFeLV RNA levels and was negatively correlated with susceptibility to infection with FeLV-B. Cell culture supernatant containing the 35-kDa protein specifically blocked infection of permissive fibroblast cells with FeLV-B isolates. We suggest that the truncated env protein mediates resistance by receptor blockade and that this form of enFeLV expression mediates the natural resistance of cats to infection with FeLV-B in the absence of FeLV-A.

Chronic feline leukemia virus infection alters arachidonic acid proportions in vivo and in vitro.

The polyunsaturated omega-6 fatty acid, arachidonic acid ([AA] 20:4n-6), is both the key of the immunoregulatory substances, prostaglandins, and leukotrienes, and an essential component of immune cell membrane phospholipids, providing stability and flexibility to ensure cellular function. To explore possible effects of the physiological burden of viral replication in chronic viral infections on AA availability, plasma total esterified fatty acid (FA) proportions were measured in the feline leukemia (FeLV) model. Plasma FA profiles of 12 specific-pathogen-free cats with chronic infections with Rickard strain feline leukemia virus (FeLV-R) were compared with 12 age- and sex-matched uninfected specific-pathogen-free cats at 4 months after infection. A significant decrease from normal of average AA proportion was found in FeLV-R-infected cat plasma, while other major FA (palmitic, stearic, and oleic and omega-3 FA normally remained present until near death. Since plasma FA content rapidly affects circulating immune cell membrane composition and since FeLV infection also targets immune cells, we compared FA profiles of feline T4-thymic lymphoma 3201 cell membranes that were infected with virulent FeLV-R or less virulent FeLV-A, at 20 days after viral inoculation with sham-inoculated uninfected 3201 cells. Significantly altered FA proportions and ratios of saturated to unsaturated FA found in infected cell membranes were similar to plasma FA changes and paralleled the virulence of the FeLV inoculum. Altered postinfection FA proportions may impart serious functional defects to the immune cells of chronic FeLV-infected cats, contributing to the inability of their immune systems to eliminate FeLV by depleted plasma AA stores and modified cell membrane composition. Decreased AA availability may be an important factor in the cachexia and fatal outcome of FeLV infection.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes.

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.

Lymphoma involving large granular lymphocytes in cats: 11 cases (1982-1991).

Medical records of 11 cats with lymphoma involving large granular lymphocytes were reviewed. All 9 cats tested were FeLV-negative. Ten cats had a history of anorexia, lethargy, vomiting, or diarrhea, and had lymphoma involving abdominal viscera. The most common site of tumor in these cats was the jejunum. One cat had cutaneous masses caused by dermal and epidermal infiltration with neoplastic large granular lymphocytes. The most common hematologic abnormality was leukocytosis, characterized by neutrophilia with a left shift (7 cats); 2 cats had a left shift without neutrophilia. None of the cats had lymphocytosis, but immature large granular lymphocytes were found in the blood of 4 cats. The most common serum biochemical abnormalities were hypoalbuminemia (10 cats), hypocalcemia (10 cats), hypoproteinemia (9 cats), high aspartate transaminase activity (9 cats), and hyperbilirubinemia (8 cats). Large granular lymphocytes were characterized by abundant cytoplasm containing distinct azurophilic granules that varied in size and number. The most common cytochemical staining pattern included detection of alpha-naphthyl butyrate esterase, acid phosphatase, and beta-glucuronidase activities. On examination of histologic sections, granules stained weakly eosinophilic with Giemsa and moderately with periodic acid-Schiff reaction. Ultrastructurally, the granules appeared membrane bound and contained an electron-dense matrix in 4 cats.

Lymphocytotoxic strains of feline leukemia virus induce apoptosis in feline T4-thymic lymphoma cells.

Feline leukemia retrovirus (FeLV) strains with subgroup C env genes kill feline T4 lymphoma 3201 cells by 7 to 12 days after in vitro inoculation, whereas FeLV strains with subgroup A env genes do not. Neither FeLV-A nor FeLV-C kill feline fibroblasts. FeLV-C, but not FeLV-A, is replicated to higher titer by 3201 cells and productive infection precedes death by 3 to 7 days. Transcriptional activity of the FeLV-C long terminal repeat, as assessed by chloramphenicol acetyltransferase activity, is high in feline lymphoid cells but low in feline fibroblasts. Activity of the FeLV-A long terminal repeat is moderate in both cell types. FeLV-C-infected cells form aggregates 1 to 4 days before dying; ultrastructurally, virus particles can be seen approximating the clustered cells. Dying cells demonstrate nuclear condensation, surface blebbing, and fragmentation. DNA fragmentation and laddering compatible with apoptosis occur 1 to 2 days before massive cell death. In FeLV-C-infected 3201 cells, a shift from phospholipid to neutral lipid incorporation of [14C]oleic acid, increases in palmitic acid proportions and decreases in linoleic acid proportions occur 1 to 2 days before peak killing. Exposure of 3201 cells to ultraviolet-inactivated FeLV-KT (200-800 micrograms/10(6) cells) causes cytostasis within 2 days and death within 4 days. Blebbing and nuclear condensation occur but clusters do not form. The induction of programmed cell death in feline thymic lymphoma cells by subgroup C feline retroviruses may be relevant to the pathogenesis of FeLV-induced thymic atrophy, paracortical lymphoid depletion and acquired immunodeficiency in vivo.

Incidence of localized feline leukemia virus infection in cats.

Anecdotal descriptions of atypical FeLV infections, wherein standard clinical ELISA or immunofluorescence testing fails to detect active infections, suggest that an unknown proportion of FeLV-infected cats may go undetected. In this study, 127 viremic and nonviremic cats experimentally inoculated with FeLV were evaluated at necropsy for atypical expression of FeLV antigen. Results from viremic cats were in accordance with results of earlier studies on the pathogenesis of FeLV infection in cats, wherein antigen was found in lymphoid and epithelial tissues. Differences in time course or tissue distribution of viral antigen in some cats appeared to be attributable to the challenge virus preparations, consisting of cell-free tumor homogenate or infectious plasma. It was discovered that 5 of 19 of the FeLV challenge-exposed cats that were nonviremic had FeLV-specific antigens in select tissues (bone marrow, spleen, lymph node, and small intestine) 6 to 75 weeks after inoculation. These results indicated an additional category of possible outcomes for cats exposed to FeLV. Localized FeLV infection, as described here, may explain the discordance between clinical disease and laboratory testing for FeLV.