Spontaneous obesity-linked type 2 diabetes in the absence of islet amyloid in a cynomolgus monkey infected with bovine spongiform encephalopathy.

A 9-year-old cynomolgus monkey (Macaca fascicularis) infected orally with bovine spongiform encephalopathy (BSE) was presented for necropsy following euthanasia 4 years post infection (p.i.). This macaque R984 was exposed to a BSE dose that causes a simian form of variant Creutzfeldt-Jakob disease (vCJD) within 5 years p.i. in other macaques. All orally BSE-infected macaques developed a significant weight gain within the first 2 years p.i. compared with non-BSE-infected age- and sex-matched control animals, suggesting increased risk of type 2 diabetes (T2D). In contrast, macaque R984 developed rapid weight loss, hyperglycemia, and glucosuria and had to be euthanatized 4 years p.i. before clinical signs of vCJD. Pancreas histopathological evaluation revealed severe islet degeneration but, remarkably, no islet amyloid deposits were present. Immunostaining of pancreas sections for insulin and glucagon confirmed the loss of endocrine cells. In addition, prions were present in the adenohypophysis but not in other areas of the brain, indicating centripetal prion spread from the gut during the preclinical phase of BSE infection. Plasma glucose and insulin concentrations of macaque R984 became abnormal with age and resembled T2D. This unusual case of spontaneous T2D in the absence of islet amyloid deposits could have been due to early prion spread from the periphery to the endocrine system or could have occurred spontaneously.

Partial protection by vaccination with recombinant feline immunodeficiency virus surface glycoproteins.

In an effort to induce a strong immune response that might protect against feline immunodeficiency virus (FIV) challenge infection, three groups of five specified pathogen-free (spf) cats each were immunized subcutaneously with different FIV antigen preparations. Immunizations were done at weeks 0, 2, and 4 with 100 microg of recombinant SU from an FIV Zurich 2 (FIV Z2) strain expressed by E. coli (group 1) or the baculovirus expression system (groups 2 and 3) adsorbed on aluminum hydroxyde and administered with QS-21 (groups 1 and 2) or Freund's adjuvant together with the recombinant nucleocapsid protein (protein NC) of rabies virus (group 3). Protein NC was described to act as an exogenous superantigen. Group 3 cats demonstrated the highest detectable antibody response to the vaccine antigen as determined by ELISA and Western blot analysis. All immunized cats together with seven control animals were challenged with 20 CID50 of cat lymphocyte-grown FIV Z2 3 weeks following the last immunization. Whereas virus was readily recovered from peripheral blood lymphocytes of seven of seven nonvaccinated control cats following this challenge dose, virus was not recovered from two cats of groups 1 and 2. All cats in groups 2 and 3 showed a provirus load significantly decreased to 3% of that of controls up to week 8 after challenge infection. Eleven of 15 vaccinated cats and 5 of 7 control cats developed virus-neutralizing antibodies by week 8 after challenge infection. The two cats negative on virus isolation remained seronegative, developed no detectable virus-neutralizing activities, but were repeatedly positive in provirus PCR. Moreover, starting at week 1 after challenge, both cats showed the lowest provirus load in their respective groups. These results indicate that immunization with recombinant FIV SU in conjunction with appropriate adjuvants may lead to partial protection against FIV challenge infection.

Female cats have lower rates of apoptosis in peripheral blood lymphocytes than male cats: correlation with estradiol-17beta, but not with progesterone blood levels.

During earlier study, we quantified by flow cytometry the rate of apoptotic feline lymphocytes after overnight culture. We found evidence that the sex of the animals influences the rate of apoptosis, intact females showed lower rates of apoptosis in lymphocytes cultured overnight than castrated male cats. This observation was also confirmed for cats that were previously experimentally infected with the feline immunodeficiency virus (FIV). In an attempt to find an explanation for these sexually determined differences, plasma estradiol-17beta and progesterone levels were measured by radio-immuno assay in the blood of these cats. The hormone levels were analyzed with respect to the rate of lymphocyte apoptosis. As expected, castrated males had lower blood levels of estradiol and progesterone than females. However, no overall correlation was found between hormone blood levels and rate of apoptosis under non-stimulating conditions. Interestingly, the rate of apoptosis found in lymphocytes collected from females and stimulated overnight in phytohaemaglutinin-containing medium, showed a strong negative correlation with the estradiol levels in the blood of these cats. To our knowledge, this is the first confirmation that estradiol in physiological concentrations may protect peripheral lymphocytes from apoptosis after stimulation. No correlation was found in male cats. In conclusion, these observations broaden the list of sexually determined differences of the immune system, sex and sex hormones predispose males and females for certain immune responses and dysfunctions. The present observations have to be taken into account when designing or interpreting experiments on apoptosis and, for example, evaluating the influence of a preexisting FIV infection on the rate of apoptosis. It would be highly advisable to include only spayed cats in studies on the immune system so as to minimize the influence of sex hormones.

Comparative analysis of equine lymphocyte subsets in whole blood and gradient-purified samples.

In the present study, two methods of lymphocyte preparation, whole blood lysis and Ficoll-Paque separation, prior to FACS analysis were compared. The comparison was done with single and dual-colour staining techniques. Monoclonal antibodies (mAb) against eCD4, eCD5, eCD8 and eMHC class II were used. There was no significant difference in the results obtained by these two methods.

FIV vaccine studies. II. Clinical findings, hematological changes and kinetics of blood lymphocyte subsets.

Five groups of cats were vaccinated with different recombinant feline immunodeficiency virus (FIV) SU vaccines expressed either in Escherichia coli or in the Baculovirus system. In Part I of this series, we described the humoral immune response and outcome of intraperitoneal FIV challenge exposure. Additionally, all cats were monitored for clinical and hematological changes and the course of blood lymphocyte subsets. These results are described in this present paper. A great increase of antibodies was found after vaccination with different recombinant FIV antigens, which did not protect the cats from intraperitoneal FIV challenge infection. This observation was paralleled by an increase of eosinophils during vaccination which was even more pronounced after challenge infection. After FIV challenge, infection lymphadenopathy, gingivitis, pharyngitis, changes in total leukocytes and neutrophils and a decrease in the CD4+:CD8+ ratio were found in cats of all groups and were considered as a sign of the FIV infection taking place, independent of vaccination. The following observations suggest that in these cats a TH2-like immune response was elicited: the high counts of eosinophils, the nature of antigen and adjuvant (aluminium hydroxide) and the high amounts of antigens used for immunization. Clearly, this type of immune response did not protect the animals from intraperitoneal FIV challenge infection.

Recombinant FeLV vaccine: long-term protection and effect on course and outcome of FIV infection.

The efficacy and the long-term protection of a recombinant feline leukemia virus (FeLV) vaccine were determined in 30 specified pathogen free cats for over 3 years. At the same time, in order to specify the effects of feline immunodeficiency virus (FIV) on the immune system, one half of the cats (n = 15) were previously infected with the Swiss isolate FIV Zurich 2. The second half of the animals (n = 15) served as non-infected controls. Eighteen (nine FIV-negative, nine FIV-positive) vaccinated and 12 (six FIV-negative, six FIV-positive) non-vaccinated cats were intraperitoneally challenged with FeLV A. Seventeen of 18 vaccinated cats were protected against persistent viremia, while ten of 12 non-vaccinated controls became infected. An increase of antibodies against FeLV SU was found in all protected cats after the challenge exposure. No difference in vaccine efficacy was found between FIV-negative and FIV-positive animals. The whole group of cats was observed for over 3 years. There were no further vaccinations during this period. CD4+ and CD8+ cell subsets, clinical outcome and time of survival of the cats were recorded. FIV-negative and FIV-positive animals were kept in two different rooms. However, FeLV-negative and FeLV viremic cats were housed together in both rooms in order to imitate a natural FeLV exposure situation. Anti-recombinant FeLV SU antibodies were measured by enzyme-linked immunosorbent assay. Although a continuous decline of antibodies was found in FeLV vaccinated cats, they remained protected against constant FeLV challenge for over 3 years. FIV infection had a stronger effect on the depression of the CD4+:CD8+ ratio than FeLV infection. Within the group of FIV-positive cats, the FeLV-vaccinated animals had significantly better survival rates as well as better clinical and laboratory parameters. FIV- and FeLV-coinfected cats showed the lowest CD4+:CD8+ ratio, mainly caused by decreased CD4+ lymphocyte counts. CD8+ lymphocytes with strong fluorescence (CD8(high)) disappeared and cells with weak fluorescence (CD8(low)) appeared instead. Prevention of coinfection by immunizing FIV-positive cats against FeLV infection improved the clinical outcome and prolonged the cat's life expectancy.

Vaccination with feline immunodeficiency virus induces CD4 epitope masking by soluble factors.

Soluble factors are important effector mechanisms to control for lentiviral replication. Vaccination of cats with recombinant outer surface proteins (SU) of the FIV envelope protein in combination with complete Freund adjuvant (CFA) and rabies nucleocapsid (NC) protein led to significantly reduced viral loads [Leutenegger, C.M., Hofmann-Lehmann, R., Holznagel, E., Cuisinier, A.M., Wolfensberger, C., Duquesne, V., Cronier, J., Allenspach, K., Aubert, A., Ossent, P. , Lutz, H., 1998. AIDS Res. Hum. Retroviruses, 14(3) 275-283]. Lymphocytes from vaccinated and non-vaccinated cats were stained with two monoclonal antibodies, Fel7 and CAT30A, directed against the feline CD4 antigen. Peripheral blood lymphocytes from cats vaccinated with the SU glycoprotein, CFA and rabies NC protein showed a significantly reduced number of cells after staining with CAT30A, while the number in Fel7 positive lymphocytes remained unchanged. This decreased CAT30A fluorescent staining could be reproduced in vitro by pre-incubating FIV-negative lymphocytes with immune sera from cats in which reduced CAT30A staining was detected. Neither experimental infection nor vaccination with the unglycosylated SU protein alone resulted in this epitope masking. Furthermore, this masking phenomenon was negatively correlated with a decreased susceptibility to activation-induced cell death (AICD). These findings will be discussed based on the current knowledge of CD8(+) T-cell antiviral factors and their involvement in lentiviral infection and/or replication.

FIV vaccine studies. I. Immune response to recombinant FIV env gene products and outcome after challenge infection.

We have vaccinated five groups of cats (n = 25) four times with five preparations of recombinant feline immunodeficiency virus (FIV) env gene products; one group (n = 7) served as control. The vaccine formulations were as follows: (1) envelope glycoprotein of FIV Zurich 2 (FIV Z2) expressed in a Baculovirus system and isolated by gel electroelution (denatured form); (2) insect cells expressing FIV Z2 glycoprotein; (3) envelope glycoprotein of a Boston strain (FIV Bangston) expressed in insect cells and isolated by gel electroelution (denatured form); (4) glycosylated Bangston envelope protein made in insect cells and isolated in a native form; (5) non-glycosylated Bangston envelope protein made in Escherichia coli. All cats were challenged with 20 50% cat infective doses (CID50) of FIV Z2 previously titrated in cats. All vaccinated cats developed high enzyme-linked immunosorbent assay (ELISA) antibodies to the homologous antigen; crossreactivity to heterologous antigens was seen at a lower level. Virus neutralizing antibodies (tested with Petaluma virus) reached titers up to 32. After challenge, all cats seroconverted (as judged by anti gag antibodies in Western blot) and became infected (as judged by virus isolation and/or polymerase chain reaction) between 4 and 11 weeks with the exception of one cat. It is concluded that it is relatively easy to induce high ELISA antibody titers using recombinant env gene products, ELISA antibody titers do not correlate with virus neutralization or with protection.

Flow cytometric detection of activation-induced cell death (apoptosis) in peripheral blood lymphocyte subpopulations from healthy cats.

Human peripheral blood lymphocytes (PBLs) from healthy individuals are resistant to in vitro-induced apoptosis, but activated human lymphocytes can readily undergo apoptosis. The activation of human lymphocytes is accompanied by the upregulation of a cell surface antigen, the major histocompatibility complex (MHC) class II-antigen. Only a minority of PBLs are usually MHC class II-antigen-positive in healthy humans. In contrast, in healthy cats the majority of feline PBLs are MHC class II-antigen-positive. We have, therefore, investigated the sensitivity of feline peripheral blood lymphocytes obtained from specified pathogen free (SPF) cats to the induction of apoptosis. Feline PBLs from SPF cats (n = 16) and human PBLs from healthy donors (n = 2) were isolated. After short-term culture, cells were examined for the presence of fragmented DNA as a result of apoptosis by a DNA agarose gel electrophoresis method and for the presence of DNA double strand breaks by in situ 3' end labeling. In addition, relative DNA content per cell was flow cytometrically determined using propidium iodide (PI) or 7-actinomycin-D (7-AAD) and apoptotic cells were identified on the basis of a reduced DNA content. Cell surface antigens and cellular DNA were analyzed simultaneously by dual-color flow cytometric analyses in order to study lymphocyte subsets. Single- and dual-color analysis revealed that, in contrast to human lymphocytes, feline lymphocytes rapidly underwent apoptosis when cultured overnight in medium. Furthermore, the majority of apoptotic cells was found within the MHC II-positive cell subject.

Feline immunodeficiency virus (FIV) infection in cats at necropsy: a serological study.

Sera collected post mortem during a 6-month period from cats were tested for feline immunodeficiency virus (FIV)-specific antibodies by (1) an enzyme-linked immunosorbent assay (ELISA), (2) an indirect peroxidase-based immunocytological test (IP), (3) a Western immunoblotting (WB) method with FIV-infected cell lysates, and (4) a WB method with purified viral antigen. All four methods were capable of detecting FIV-specific antibodies in haemolysed sera. However, the ELISA showed the lowest "positive predictive value" (PVpos = 22%) followed by the IP (PVpos 50-60%). Serum was FIV antibody-positive in 6% (15/255) of all cats examined. The mean age of seropositive cats was 9 years (4 years among seronegative cases) and the male-to-female ratio in such cats was 1.8 to 1 (overall ratio 0.8 to 1). Forty per cent of the seropositive cats were in the final phase of acquired immune deficiency syndrome. Feline leukaemia virus (FeLV) predominated among viral co-infections. It was concluded that (1) a combination of the IP and WB reliably detected FIV-specific antibodies in sera collected post mortem, and (2) at post-mortem examination, cats from high-risk groups (male, > 5 years old, hypercellular bone marrow) were frequently infected with FIV.

Parameters of disease progression in long-term experimental feline retrovirus (feline immunodeficiency virus and feline leukemia virus) infections: hematology, clinical chemistry, and lymphocyte subsets.

After several years of latency, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) cause fatal disease in the cat. The aim of this study was to determine laboratory parameters characteristic of disease progression which would allow a better description of the asymptomatic phase and a better understanding of the pathogenesis of the two infections. Therefore, experimentally infected cats (FIV and/or FeLV positive) and control animals were observed over a period of 6.5 years under identical conditions. Blood samples were analyzed for the following: complete hematology, clinical chemistry, serum protein electrophoresis, and determination of CD4+ and CD8+ lymphocyte subsets. The following hematological and clinical chemistry parameters were markedly changed in the FIV-infected animals from month 9 onwards: glucose, serum protein, gamma globulins, sodium, urea, phosphorus, lipase, cholesterol, and triglyceride. In FeLV infection, the markedly changed parameters were mean corpuscular volume, mean corpuscular hemoglobin, aspartate aminotransferase, and urea. In contrast to reports of field studies, neither FIV-positive nor FeLV-positive animals developed persistent leukopenia, lymphopenia, or neutropenia. A significant decrease was found in the CD4+/CD8+ ratio in FIV-positive and FIV-FeLV-positive animals mainly due to loss of CD4+ lymphocytes. In FeLV-positive cats, both CD4+ and, to a lesser degree, CD8+ lymphocytes were decreased in long-term infection. The changes in FIV infection may reflect subclinical kidney dysfunction, changes in energy and lipid metabolism, and transient activation of the humoral immune response as described for human immunodeficiency virus (HIV) infections. The changes in FeLV infection may also reflect subclinical kidney dysfunction and, in addition, changes in erythrocyte and immune function of the animals. No severe clinical signs were observed in the FIV-positive cats, while FeLV had a severe influence on the life expectancy of persistently positive cats. In conclusion, several parameters of clinical chemistry and hematology were changed in FIV and FeLV infection. Monitoring of these parameters may prove useful for the evaluation of candidate FIV vaccines and antiretroviral drugs in cats. The many parallels between laboratory parameters in FIV and HIV infection further support the importance of FIV as a model for HIV.

High virus loads in naturally and experimentally SIVagm-infected African green monkeys.

A quantitative RT-PCR assay was developed for SIVagm and was used to measure the levels of viral RNA in the plasma of experimentally and naturally infected African green monkeys. The number of productively infected PBMCs and the number of cells carrying integrated provirus were also measured. Plasma virus loads in experimentally infected animals peaked at 2 weeks postinfection, ranging from 2.9 x 10(5) to 4.2 x 10(7) RNA copies/ml plasma. Set points of 2.1 x 10(3) to 2.8 x 10(6) RNA copies/ml plasma were maintained for one year. Similarly, the number of cells carrying integrated SIVagm provirus remained relatively stable in individual animals for one year with set points ranging from 73 to 810 proviral copies per 10(6) PBMC. However, the number of productively infected cells fluctuated considerably during this period. Virus loads in the 26 naturally infected AGMs ranged from 8.3 x 10(3) to 1.1 x 10(7) (mean 1.7 x 10(6)) RNA copies/ml plasma. These levels of viremia are similar to those seen in pathogenic systems (HIV-1, SIVmac), indicating that control of SIVagm replication is not the reason for the natural host's resistance to disease progression.

Placebo-controlled evaluation of a modified life virus vaccine against feline infectious peritonitis: safety and efficacy under field conditions.

A modified live virus vaccine against feline infectious peritonitis (FIP) was evaluated in a double blind, placebo-controlled field trial in two high-risk populations. The vaccine was found to be safe and efficacious in one population of cats that had low antibody titre against feline coronavirus (FCoV) at the time of vaccination. Although clinically healthy at the time of vaccination, retrospectively some vaccinees that later came down with FIP were found to be RT-PCR positive for FCoV in plasma and showed changes in blood parameters consistent with early stage of FIP. It is concluded that vaccination can protect cats with no or low FCoV antibody titres and that in some cats vaccine failure was probably due to pre-existing infection.

The role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression.

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4(+) T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4(+) cell count (P = 0. 002), bright CD8(+) cell count (P = 0.009), and CD4/CD8 ratio (P = 0. 01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.