Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo.

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2(-/-)IL-2Rgamma(c)(-/-) immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development.

We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a- cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR alpha beta+CD4+ SP cells, indicating that mouse MHC class II can positively select TCR alpha beta +CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

Id2 and Id3 inhibit development of CD34(+) stem cells into predendritic cell (pre-DC)2 but not into pre-DC1. Evidence for a lymphoid origin of pre-DC2.

We found previously that Id3, which inhibits transcriptional activities of many basic helix-loop-helix transcription factors, blocked T and B cell development but stimulated natural killer (NK) cell development. Here we report that ectopic expression of Id3 and another Id protein, Id2, strongly inhibited the development of primitive CD34(+)CD38(-) progenitor cells into CD123(high) dendritic cell (DC)2 precursors. In contrast, development of CD34(+)CD38(-) cells into CD4(+)CD14(+) DC1 precursors and mature DC1 was not affected by ectopic Id2 or Id3 expression. These observations support the notion of a common origin of DC2 precursors, T and B cells. As Id proteins did not block development of NK cells, a model presents itself in which these proteins drive common lymphoid precursors to develop into NK cells by inhibiting their options to develop into T cells, B cells, and pre-DC2.

Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model.

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

Prognostic factors in feline mammary carcinoma.

Feline mammary carcinoma and canine mammary cancer were evaluated as models for future experimental therapy. Those tumor characteristics known to be of special prognostic significance in human mammary cancers were tested for their prognostic significance in the cat and were compared with those in the dog. The statistical analysis presented is based on a prospective follow-up study of 202 cats treated surgically by mastectomy and by block dissection. Thirty-five factors (general, anamnestic, clinical and histologic data, and data on therapy) were analyzed for relationships with survival, with local recurrence, and with each other. Of the 17 significant relationships found between survival and the direct factors, only 7 remained significant after correction. The factors that related to survival independently of each other were age, diameter of the primary tumor, presence of tumor-positive lymph nodes as judged by microscopic examination, number of mitotic figures, necrosis of the primary tumor, and histologic verification of completeness of surgical treatment. The value of statistical analysis for use in prospective studies of human mammary cancer is discussed.

Characterization of CD34+ thymic stromal cells located in the subcapsular cortex of the human thymus.

In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with anti-CD34 and Thy-1 antibodies revealed that the double-labeled cells were located in the pericortical area. In addition, it was found that the CD34+Thy-1+ cells lacked CD45 and CD50, indicating that these cells are not of hematopoietic origin; this was confirmed by the finding that these cells could be cultured as adherent cells in a medium with cholera toxin and dexamethasone, but failed to grow in mixtures of hematopoietic growth factors. Further analysis indicated that most cultured CD34+Thy-1+ cells expressed cytokeratin (CK) 14 but lacked CK 13, suggesting that these cells are immature epithelial cells. Cultured CD34+Thy-1+ cells were able to induce differentiation of CD1-CD34+CD3-CD4-CD8- thymic precursors into CD4+CD8+ cells in a reaggregate culture in the absence of exogenous cytokines. The CD4+CD8+ cells that developed in these cultures did not express CD3, indicating that CD34+Thy-1+ thymic stromal cells are not capable of completing full T cell differentiation of thymic hematopoietic progenitor cells.

Induction of anti-viral immune response by immunization with monoclonal anti-idiotype antibodies directed to private idiotopes.

Syngeneic monoclonal anti-idiotypic antibodies were raised against idiotopes on neutralizing monoclonal antibodies with specificity for feline leukemia virus and canine parvovirus. The anti-idiotypic antibodies were shown to recognize paratope-related private idiotopes. Mice were injected with the monoclonal anti-idiotypic antibodies and the sera of these mice were screened for antiviral reactivities. Antibodies to both feline leukemia and canine parvovirus could be induced as determined by ELISA. These results suggest that anti-idiotypic antibodies which detect private idiotopes and thus do not represent internal images of viral antigens may be considered as candidates for the induction of antiviral immunity.

Feline immunodeficiency virus (FIV) infection in the cat as a model for HIV infection in man: FIV-induced impairment of immune function.

To assess the value of feline immunodeficiency virus (FIV) infection as a model for human immunodeficiency virus (HIV) infection in man, we studied the impairment of certain immunological functions following natural or experimental FIV infection. Proliferative responses of peripheral blood mononuclear cells (PBMC) from symptomatic and asymptomatic cats after naturally or experimentally acquired FIV infection, induced by activation with the mitogens concanavalin A, pokeweed mitogen, or lipopolysaccharide or by stimulation with human interleukin-2 (IL-2), were significantly lower than the proliferative responses found with PBMC from noninfected control cats. Also IL-2 production levels of mitogen-activated PBMC from naturally infected symptomatic cats were significantly reduced. These data confirm that the pathogenesis of FIV infection in the cat, like HIV infection in man, is characterized by a serious malfunction of the immune system.

Tyrosinase gene expression in clear cell sarcoma indicates a melanocytic origin: insight from the first reported canine case.

The aim of this study was to characterize a metastasizing soft tissue tumor in a dog, which clinically, grossly and histologically showed a close resemblance to human clear cell sarcoma, a soft tissue variant of malignant melanoma. Ultrastructurally, melanosomes were found, indicating a melanocytic origin of the tumor. Using reverse-transcription polymerase chain reaction, expression of the gene encoding tyrosinase was determined in tumor cells. With this first case of canine clear cell sarcoma, as well as the earlier report from our laboratory on amelanotic melanomas in the cat, we demonstrate that expression of the tyrosinase gene may occur in a broader range of less differentiated melanocytic tumors in different species, including man.

Feline mammary carcinomas as a model for human breast cancer. I. Sensitivity of mammary tumor cells in culture to cytostatic drugs. A preliminary investigation of a predictive test.

Feline mammary carcinomas were found to maintain well in short-term cultures. Principally the same types of nuclear DNA frequency distribution histograms were recognized in feline mammary carcinomas as in human mammary carcinomas. However, the more abnormal histograms are less frequent in feline than in human mammary carcinomas. Feline mammary carcinomas appeared, at least in vitro, most sensitive to Doxorubicin and 5-fluorouracil. Preliminary, thymidine incorporation studies indicate both cytotoxic and cytostatic effects of Doxorubicin and 5-FU. Methotrexate was found to stimulate thymidine incorporation.

gag- and env-specific serum antibodies in cats after natural and experimental infection with feline immunodeficiency virus.

In order to monitor the antibody response to feline immunodeficiency virus (FIV) in cats, following experimental and natural infection, enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant env and gag proteins and p24-specific monoclonal antibodies. It was shown that in experimentally infected cats an env protein-specific antibody response was directly followed by a gag protein-specific response. Furthermore, an ELISA for the detection of env protein-specific serum antibodies proved more sensitive in identifying experimentally and naturally infected cats than ELISAs demonstrating gag protein-specific antibodies. It was concluded that, like in HIV infection of humans, the detection of env protein-specific serum antibodies in addition to gag protein-specific antibodies is not only an important tool in the diagnosis of the infection but also in studies concerning the pathogenesis of the disease.

Control of feline leukaemia virus.

Feline leukaemia virus (FeLV) usually occurs in its natural species, the domestic cat. FeLV is also important to human individuals as a comparative model, as it may cause a variety of diseases, some malignant and some benign, such as immunosuppression, which bears a resemblance to AIDS (acquired immune deficiency syndrome) in man. FeLV is transmitted among cats by contagion. The main sources of infection are persistently infected carrier cats which continuously excrete virus. Dissemination of FeLV among cats may be prevented by identifying infected carrier cats and removing them from contact with non-infected cats. Removal programmes using indirect immunofluorescence antibody tests were applied successfully in The Netherlands. The proportion of FeLV-positive cats decreased from 9% in 1974 to approximately 3% in 1985 during such a programme. The results of a removal programme carried out in a catbreeders' society were even better: the incidence of cats positive for FeLV decreased from 11% in 1974 to less than 2% within 4 years. None of the cats tested in this society has been found to be positive for FeLV since 1984. Besides removal programmes, other methods of control, such as pre-exposure treatment, were developed to prevent the spread of FeLV. We attempted to protect kittens against oronasal infection with FeLV by treatment with virus-neutralizing (VN) monoclonal antibodies (MoAbs) directed against an epitope on the viral glycoprotein gp70. However, no protection was achieved. It is unlikely that the amount of VN antibodies, the mode and route of their application or the infectious dose of FeLV used can account for this failure. Other possible explanations for the lack of protective effect are that (i) the restricted epitope specificity of the MoAb preparation used may have led to selection of neutralization-resistant virus mutants, or (ii) other mechanisms than virus neutralization (complement-mediated lysis, antibody-dependent cell cytotoxicity), that may be involved in protection, function less efficiently with MoAb. However, in the light of our finding that an early anti-idiotypic response is observed in all cats following administration of the MoAb preparation, the rapid clearance of anti-FeLV MoAb from the circulation is a more likely explanation. Efforts were further made to develop a vaccine for controlling FeLV infection. The immunostimulating complex vaccine (FeLV-ISCOM vaccine), a subunit vaccine in which FeLV gp70 is presented in a particular manner, looks promising. The protective effect of FeLV-ISCOM vaccine was studied by vaccinating six 8-week-old SPF cats with ISCOM, followed by oronasal challenge with FeLV.(ABSTRACT TRUNCATED AT 400 WORDS)

Feline mammary tumours and dysplasias conclusions based on personal studies and some suggestions for future research.

Summary Feline mammary tumours and dysplasias were studied by various methods: morphological, electron-microscopical, immunological and virological. The most important conclusion was that cats with mammary tumours (benign and malignant tumours and lesions, the significance of which is not known so far) may be suitable animal models for the study of certain features of human mammary tumours; for instance, the study of the relationship between particular histological and biological characteristics of mammary carcinoma and the prognosis; the study of the question whether a virus (or viruses) does (do) or does (do) not play a role in the pathogenesis or growth of mammary tumours; the study of the possible presence of specific cellular and/or humoral immunity to mammary tumours and the study of the possible effects of progestogens on the mammary gland. As the study can not be regarded as a self-contained entity, it should be continued; this applies particularly to those parts of the study, that offer the best prospects for comparative investigations.

A comparison of three methods of feline leukaemia virus diagnosis.

Samples of blood from pet cats were examined for evidence of feline leukaemia virus (FeLV) by three techniques: virus isolation, immunofluorescence and an enzyme-linked immunosorbent assay (ELISA) Leukassay F. There was good agreement between the results from virus isolation and immunofluorescence. However, about 30 per cent of cats which were positive for FeLV antigen by ELISA were negative by either of the other tests. The status of most of these cats was unchanged four or 12 weeks later.

[The incidence of lymphosarcoma (leukaemia) and feline leukaemia virus (FeLV) in cats in the Netherlands (author's transl)]. / Het voorkomen van lymfosarcoom/leukemie en katten-leukemievirus (FeLV) bij de kat in Nederland

Seven different groups of cats were examined to study the incidence and distribution of feline leukaemia virus (FeLV) in the Netherlands. The indirect fluorescent antibody (IFA) technique was used to detect FeLV antigen. Of the cats with lymphosarcoma (leukaemia), 73.2 per cent and of those with infectious peritonitis, 32.4 per cent were found to be positive for FeLV antigen. Of the sixty-six cats with other tumours, only one, a cat with carcinoma of the mammary gland; was positive for FeLV antigen. Of 557 cats with various lesions, forty-two (7.5 percent) were positive for FeLV antigen. The IFA-test was found to be a useful adjunct in establishing the correct diagnosis. Of all stud cats which had been in contact with FeLV-positive cats, 24.7 percent were positive for FeLV antigen, wheras all those which had not been in contact with these cats, were negative. There was a marked difference between the proportions of FeLV-positive cats in the groups of clinically normal cats which had (20.6 per cent) and which had not (0.4 per cent) been in contact with FeLV-positive cats. Follow-up studies showed that 67.8 percent of the clinically normal, FeLV-positive cats had died from or been sacrificed because of FeLV-associated diseases within twenty months.

Feline mammary tumours and dysplasias. Conclusions based on personal studies and some suggestions for future research.

Feline mammary tumours and dysplasias were studied by various methods: morphological, electron-microscopical, immunotlogical and virological. The most important conclusion was that cats with mammary tumours (benign and malignant tumours and lesions, the significance of which is not known so far) may be suitable animal models for the study of certain features of human mammary tumours; for instance, the study of the relationship between particular histological and biological characteristics of mammary carcinoma and the prognosis; the study of the question whether a virus (or viruses) does (do) or does (do) not play a role in the pathogenesis or growth of mammary tumours; the study of the possible presence of specific cellular and/or humoral immunity to mammary tumours and the study of the possible effects of progestogens on the mammary gland. As the study can not be regarded as a self-contained entity, it should be continued; this applies particularly to those parts of the study, that offer the best prospects for comparative investigations.

[Control of lymphosarcoma or leukaemia in cats and of feline leukaemia virus (author's transl)]. / De bestrijding van lymfosarcoom/leukemie en kattenleukemievirus.

A removal programme, specially designed for catteries or multiple cat households, to reduce the incidence of lymphosarcoma or leukaemia in cats and the spread of feline leukaemia virus (FeLV), is discussed. This removal programme includes annual testing of male stud cats, testing all contacts of FeLV-positive cats they had during the past two years, testing cats imported from abroad and isolation--or, if this is not possible, euthanasia--of cats found to be positive for FeLV-antigen. To detect the FeLV-antigen a simple indirect fluorescent antibody technique (FAT) was used to detect FeLV-antigen. During a period of four years, the following results were obtained when a removal programme was carried out by a large cat breeders' association in the Netherlands. The proportion of catteries, in which FeLV-positive cats were found to be present, decreased from 11.5 per cent in the former half of 1974 to 2.1 per cent in the latter half of 1977. The proportion of FeLV-positive cats and male studs decreased from 4.9 per cent to 1.2 per cent and from 5.9 per cent to 1.0 per cent respectively during this period. It is concluded that the recommended removal programme is a simple and successful procedure.

[Feline leukemia virus (FeLV) and FeLV-associated diseases in cats: a review]. / Kattenleukemievirus (FeLV) en FeLV-geassocieerde ziekten bij de kat: een overzicht.

Feline leukaemia virus (FeLV) usually occurs in its natural species, the domestic cat. FeLV is also important to human individuals as a comparative model, as FeLV may cause a variety of diseases which are partly malignant and partly benign, such as immunosuppression which bears a resemblance to AIDS (acquired immune deficiency syndrome) in man. Although FeLV is a common infective agent, the incidence of disease due to FeLV is much higher in cats kept in closed households in which several of them are present than it is in free-range cats. Consequently, diseases caused by FeLV are frequently diagnosed in pedigree cats which are often maintained in relatively large numbers. FeLV is transmitted among cats by contagion. The main sources of infection are persistantly infected FeLV carrier cats which continuously excrete virus from the mouth and in other secretions. The majority of cats infected with FeLV will produce neutralising antibodies. Cats which are unable to do so, will become permanently infected. The prognosis is bad in these cats: 90 per cent will die within five years. Various techniques are used to detect FeLV. The most common method, the indirect immunofluorescence (IFA) test, is performed on air-dried blood smears. The results of the IFA agree with that are almost completely identical to those of the virus isolation test. Another test is ELISA (enzyme-linked immunosorbent assay), which produces approximately 10 per cent more positive results which are probably due to circulating FeLV antigens. Dissemination of FeLV among cats may be prevented by identifying infected carrier cats and removing them from contact with non-infected cats. Removal programmes using indirect immunofluorescent antibody (IFA) tests were used successfully in the Netherlands. The proportion of FeLV-positive cats decreased from 9 per cent in 1974 to approximately 3 per cent in 1985 during such a removal programme. During the above period, the removal programme was carried out in the society of Dutch cat breeders 'Felikat', the programme being made compulsory on all members of the society. The incidence of cats positive for FeLV decreased from over 11 per cent in 1974 to less than 2 per cent within four years. None of the cats tested in this society was found to be positive for FeLV in 1984 and 1985. Besides removal programmes, other methods of control, such as vaccination, were developed to prevent the spread of FeLV. The FeLV-immunostimulating complex vaccine (FeLV-ISCOM vaccine) a subunit vaccine in which FeLV-gp70 is presented in a particular manner, seems to be promising.

[Prevalence of FTLV (feline T-lymphotropic lentivirus) infections in cats in The Netherlands and in West Germany]. / Voorkomen van FTLV-infecties onder katten in Nederland en West-Duitsland.

The prevalence in the Netherlands and the Free Republic of Germany (FRG) of the newly discovered retrovirus of cats, which causes an immunodeficiency syndrome in this species and is termed feline T-lymphotropic lentivirus (FTLV), was estimated by conducting a serological survey among cats with different histories of disease. In an enzyme-linked immunosorbent assay (Petcheck, Agritech Trademark, Portland, USA) 265 samples of cats with chronic disease symptoms, and 78 samples of clinically healthy cats in the Netherlands, and 138 samples of cats with chronic disease symptoms in the FRG, were tested for the presence of FTLV-specific antibodies. All these samples had been taken from cats which were negative for FeLV antigen in the immunofluorescence test. In the groups of chronically ill animals 18 (7%) and 6 (4.5%) seropositive animals were found respectively, whereas in the group of clinically healthy cats no seropositive animals could be demonstrated.