Toward gene therapy of premature ovarian failure: intraovarian injection of adenovirus expressing human FSH receptor restores folliculogenesis in FSHR(-/-) FORKO mice.

A homozygous missense mutation, C566T, in the follicle stimulation hormone receptor (FSHR) gene has been linked to premature ovarian failure. The disease leads to infertility in a normal karyotype female with an elevated follicle stimulating hormone (FSH) and decreased serum estrogen level. Female mice carrying mutated FSHR gene, called follitropin receptor knockout (FORKO), display similar phenotype and are sterile because of a folliculogenesis block at a primary stage. We investigated the effects of bilateral intra-ovarian injection of an adenovirus expressing a normal copy of human FSHR on the reproductive system of 6-10 weeks female FORKO mice. Ad-LacZ was injected directly into each ovary of the control group. Animals were sacrificed at 2, 4, 8 and 12 weeks post-injection and tissues collected for evaluation. Treated mice showed estrogenic changes in daily vaginal smear whereas control animals remained fixated in the diestrus stage. Histological evaluation showed on average 26 +/- 4 follicles/ovary in treated group with 8 +/- 2 follicles at the antral stage compared with only 5 +/- 2 with zero follicles at antral stage in Ad-LacZ control mice. There was no significant change in serum level of progesterone, however, estrogen level increased 2-3-fold (P < 0.02) and FSH decreased by up to 50% (P < 0.04) in treated animals. FSHR mRNA was detected in the ovaries of the treated group. In conclusion, intra-ovarian injection of an adenovirus expressing human FSHR gene is able to restore FSH responsiveness and reinitiate ovarian folliculogenesis as well as resume estrogen production in female FORKO mice. Ad-LacZ injections indicate the absence of systemic viral dissemination or germ line transmission of adenovirus DNA to offspring.

Spontaneous amyloidosis in twelve chimpanzees, Pan troglodytes.

Spontaneous amyloidosis was diagnosed in 11 male and 1 female chimpanzees and confirmed histologically and immunohistochemically. The chimpanzees were > or = 15 years of age when first diagnosed and averaged 22.4 years of age. The average survival time after diagnosis of systemic amyloidosis was 1.86 years with a standard deviation of 4.06 years (n = 7). The chimpanzees with amyloidosis were asymptomatic except for hepatomegaly, which became more detectable with age. Significant increases in clinical chemistry values, as compared with referenced normals and established normals, of blood urea nitrogen (BUN), asparate aminotransferase (AST), gamma-glutamyltransferase (GGT), globulin, total protein, creatinine phosphokinase (CPK), sedimentation rate, and triglycerides were found in animals 7 years of age or older with amyloidosis. These serum chemistry values, while increased in chimpanzees with amyloidosis, were generally within normal limits. Immunohistochemistry for both amyloid A protein and amyloid P component-labeled extracellular amyloid in all chimpanzees with amyloidosis was determined. Amyloid was deposited primarily in the liver. Amyloidosis in the chimpanzee is a chronic, intractable, progressive, fatal disease, and appears to be similar to secondary amy loidosis in other species.

Mutational and functional analysis of the C-terminal region of the C3H mouse mammary tumor virus superantigen.

The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vbeta) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vbeta chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+ Vbeta14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of Sag function may be compensated, at least partially, by loss of transcriptional suppression in certain tissues. Together these results confirm the importance of Sag for efficient milk-borne transmission and indicate that the entire C-terminal region is needed for complete Sag function.

Spontaneous anaplastic large cell lymphoma in a chimpanzee: a clinicopathological and immunohistochemical study.

An anaplastic large cell lymphoma with disseminated abdominal metastases was diagnosed in a 35-year-old male chimpanzee. Clinically, the animal displayed lethargy, weight loss, ascites, and abdominal distention. Imaging studies showed several large abdominal masses. At necropsy, variably sized masses of neoplastic mesenteric lymph nodes that encompassed several intestinal loops were present throughout the abdomen. The largest mass measured 9 x 5 cm and had cauliflower-like protrusions into the jejunal lumen. The entire abdominal cavity was covered by a sheet of neoplastic tissue. Histopathologically, the tumor consisted of solid sheets of proliferating lymphoid cells forming a cohesive growth that filled the lymph node sinuses. The tumor had invaded the intestinal wall. Anaplastic large cell lymphoma was diagnosed from immunohistochemistry findings on the basis of positive reaction to the CD3 and CD30 markers and negative reaction to the CD20 marker. Serologic analysis revealed positive titers for Epstein-Barr, cytomegalo-, and varicella-zoster viruses. Both serologic and virologic studies showed no evidence of detectable retroviral infection. This type of tumor has not been reported before in the chimpanzee.

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus.

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.

Ventricular septal defect in an infant chimpanzee (Pan troglodytes).

During clinical examinations and imaging studies of a prematurely born chimpanzee, a heart murmur, tachypnea, dyspnea, and disturbances of blood flow were observed. At necropsy, cardiomegaly, ventricular hypertrophy, and septal defects confirmed the presence of congenital VSD.

Hydranencephaly in two rhesus monkeys (Macaca mulatta).

Spontaneously occurring hydranencephaly was diagnosed at necropsy and confirmed histologically in two stillborn fetuses that were delivered from young female rhesus macaques (Maccaca mulatta).

Comparative susceptibility of African green monkeys (Cercopithecus aethiops) to experimental infection with Leishmania leishmania donovani and Leishmania leishmania infantum.

The leishmaniases are global health problems that affect both humans and animals. The availability of nonhuman primate models is desirable for such important areas as testing candidate vaccines and newly developed chemo- and immunotherapeutic agents. Visceral leishmaniasis was experimentally induced in African green monkeys (Cercopithecus aethiops) by intravenously inoculating 10(7) amastigotes/kg of body weight of either Leishmania leishmania donovani of human origin (group 1) or L. l. infantum of canine origin (group 2). The infected monkeys were monitored for 12 weeks. The monkeys developed persistent infections, became emaciated, and lost between 9 and 22% of their body weights. Splenomegaly developed by 6 to 10 weeks postinfection. All infected monkeys developed normocytic, normochromic anemia (3.5 to 3.8 x 10(6)/microliters), leukopenia (3,000 to 3,700/microliters), and neutropenia of varying severity. Hyperproteinemia with hyperglobulinemia (5.22 to 6.12 g/dl) was present in all monkeys to various degrees. Antibody responses gradually increased to peak values at 2 weeks postinfection in the L. l. donovani group and by 6 weeks postinfection in the L. l. infantum group. Lymphocyte blastogenesis proliferation responses were mildly decreased in all infected monkeys at 10 to 12 weeks postinfection. Parasite numbers were consistently higher in the livers than in spleens, and parasites were present in smears or cultures of the liver, spleen, bone marrow, and lymph nodes. Contrasting data between the two groups included 20-fold-higher parasite numbers in the livers (3.23 to 9.48 x 10(9)) and 39-fold-higher parasite numbers in the spleens (6.7 x 10(8) to 2.69 x 10(9)) of group 1. Granulomatous inflammatory reactions of various severity and intensity were observed in the liver, spleen, lymph nodes, thymus, and bone marrow of all infected monkeys. Within the granulomatous inflammatory reactions, clusters of macrophages, often containing amastigotes, were present. The morphologic changes in the bone marrow suggested a myelophthisic disease and those in lymph nodes and spleen suggested a B-cell proliferation. The clinicopathologic changes, mild suppression of cell-mediated immunity, and high antibody response in all infected monkeys indicated that African green monkeys can be a useful laboratory model for studying the clinicopathologic and immunopathologic changes induced by both L. l. donovani and L. l. infantum.

Determination of virulence and pathogenesis of a canine strain of Leishmania leishmania infantum in hamsters and dogs.

Visceral leishmaniasis was experimentally induced in hamsters by the intracardiac inoculation of 10(7) amastigotes of Leishmania leishmania infantum of canine origin. At postinoculation (PI) days 7, 21, 42, and 63, hamsters were euthanatized. Body weights and total parasite numbers of the liver and spleen were determined. Gross and histologic evaluations of tissues were done. Dogs also were inoculated IV with 10(8) amastigotes/kg of body weight. Samples were obtained from dogs prior to infection and at biweekly PI intervals for CBC and serum chemical analysis, for lymphocyte blastogenic assay by use of blood leukocytes, and for ELISA to determine antileishmanial antibody titers. At PI week 12, dogs were necropsied; organ weights, tissue imprints of the liver and spleen, and histologic interpretations of tissues were obtained. Hamsters developed high parasite numbers within 7 days after inoculation, at which time the total parasite numbers in the liver (3.51 x 10(7) amastigotes) was observed to be approximately 11 times that in the spleen (2.93 x 10(6)). The liver had the highest parasite numbers throughout the infection period. Some infected hamsters became either cachectic and emaciated or ascitic. Two of the 10 infected hamsters died at PI days 54 and 58. Moderate to severe hepatosplenomegaly with granulomatous inflammatory reactions characterized by the presence of varied numbers of parasitized macrophages, giant cells, and hepatic Schaumann bodies were observed in infected hamsters. Infected dogs developed significantly altered hematologic values consisting of mild anemia and moderate leukopenia at PI weeks 8 to 12. Hyperproteinemia characterized by hyperglobulinemia (4.5 g/dl) was noticed at PI week 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Canine leishmaniasis caused by Leishmania leishmania infantum in two Labrador retrievers.

Canine leishmaniasis, a generally fatal parasitic disease, was diagnosed in 2 dogs with a medical history of foreign travel, lymphadenopathy, emaciation, anorexia, intermittent fever, and cutaneous lesions. Clinically, hyperproteinemia, proteinuria, azotemia, and glomerulopathy were evident. Isolation of Leishmania species was done using Schneider's Drosophila medium. Syrian hamsters were used for infectivity studies. Clear taxonomic identification was done biochemically by isoenzyme analysis and comparison of zymogram banding patterns with 6 World Health Organization reference strains. Based on the geographic origin of affected dogs, clinicopathologic presentation, visceralization with hepatosplenomegaly in hamsters, and isoenzyme analysis, a diagnosis of Leishmania leishmania infantum was made. This study, representing the first taxonomic identification of an isolate from canine leishmaniasis, demonstrates the zoonotic and epidemiologic implications of this disease.

Hydatid disease in a horse.

During routine necropsy of a 28-year-old horse with intestinal volvulus, numerous hydatid cysts were discovered in the liver. Viable protoscolices of Echinococcus granulosus were obtained from the cyst. As a 4-year-old, this horse had been imported from an area that is enzootic for equine hydatidosis.

Spontaneous external endometriosis in a De Brazza's monkey.

A spontaneous case of external endometriosis in a 22-year-old female De Brazza's monkey is described. During diagnostic laparotomy, peritoneal adhesions of the uterus to the urinary bladder, ovaries and abdominal wall were seen. A surgical biopsy of soft tissue attached to the ventral surface of the uterus was examined histologically and consisted of endometrial glands and stroma. These findings were compatible with a diagnosis of endometriosis.

Leishmania major: the suitability of East African nonhuman primates as animal models for cutaneous leishmaniasis.

The susceptibility of four species of East African nonhuman primates to experimental infection with Leishmania major was investigated. Four Syke's monkeys (Cercopithecus mitis), two vervet monkeys (Cercopithecus aethiops), two baboons (Papio cynocephalus), and two brown bushbabies (Galago garnettii) were each inoculated intradermally on the left eyelid, left ear, and nose with 0.1 ml of medium containing 1 x 10(7) promastigotes of a characterized L. major strain. All the nonhuman primates except the bushbabies developed erythema and conspicuous nodules on the eyelids and ears by 3 weeks PI. The nodules increased rapidly in size and ulceration was evident on the eyelids and ears by 49 days PI in the vervets, Syke's, and baboons. The aspirates were positive in culture or smears at 35, 49, 63, and 77 days PI. No parasites were observed in cultures or smears at 92, 105, 128, 147, and 161 days PI. The lesions in these animals began resolving by 84 days PI and were completely healed by 112 days PI. The exception was one baboon in which lesion healing did not start until around 147 days and was completely healed by 182 days PI. Cultures from the liver failed to demonstrate visceralization of the parasite in any of the animals throughout the 68 weeks of the experiment. Challenge with the same strain of L. major 6 months PI, corresponding to about 3 months after self cure, failed to produce infection in any of these experimental hosts. All the nonhuman primates except the bushbaby when challenged with the same strain of L. major at 12 months PI developed lesions and were positive for parasites at 14 and 28 days PI. Positive cultures were obtained from the eyelid and ear of one vervet up to 42 days PI. However, the lesion sizes in all these animals were smaller than in the initial infection and did not ulcerate. The nodules disappeared within 6 to 8 weeks as compared to 16 weeks in the initial infection. The histopathological appearance of the lesions varied from diffuse infiltration of plasma cells and lymphocytes which increased progressively to granulomata with epitheloid cells. This study shows that the vervets, Syke's, and the baboons are equally susceptible to L. major infection, while bushbabies are refractory. The vervets, Syke's, and baboons demonstrate a self-healing phenomenon within about 3 months which is comparable to that observed in humans infected with L. major. These three species of nonhuman primates are therefore considered as suitable models for drug or vaccine trials against human zoonotic cutaneous leishmaniasis.