B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months.

Xenotransplantation of genetically modified pig organs offers great potential to address the shortage of human organs for allotransplantation. Rejection in Gal knockout (GTKO) pigs due to elicited non-Gal antibody response required further genetic modifications of donor pigs and better control of the B-cell response to xenoantigens. We report significant prolongation of heterotopic alpha Galactosyl transferase "knock-out" and human CD46 transgenic (GTKO.hCD46Tg) pig cardiac xenografts survival in specific pathogen free baboons. Peritransplant B-cell depletion using 4 weekly doses of anti-CD20 antibody in the context of an established ATG, anti-CD154 and MMF-based immunosuppressive regimen prolonged GTKO.hCD46Tg graft survival for up to 236 days (n = 9, median survival 71 days and mean survival 94 days). B-cell depletion persisted for over 2 months, and elicited anti-non-Gal antibody production remained suppressed for the duration of graft follow-up. This result identifies a critical role for B cells in the mechanisms of elicited anti-non-Gal antibody and delayed xenograft rejection. Model-related morbidity due to variety of causes was seen in these experiments, suggesting that further therapeutic interventions, including candidate genetic modifications of donor pigs, may be necessary to reduce late morbidity in this model to a clinically manageable level.

Surgical and nonsurgical complications of a pig to baboon heterotopic heart transplantation model.

A modified immunosuppressive regimen, developed at the National Institutes of Health, has been employed in a large animal model of heterotopic cardiac xenotransplantation. Graft survival has been prolonged, but despite this, our recipients have succumbed to various surgical or nonsurgical complications. Herein, we have described different complications and management strategies. The most common complication was hypercoagulability (HC) after transplantation, causing thrombosis of both small and large vasculature, ultimately leading to graft loss. While managing this complication we discovered that there was a delicate balance between HC and consumptive coagulopathy (CC). CC encountered in some recipient baboons was not able to be reversed by stopping anticoagulation and administering multiple blood transfusions. Some complications had iatrogenic components. To monitor the animals, a solid state left ventricular telemetry probe was placed directly into the transplanted heart via the apex. Induction of hypocoagulable states by continuous heparin infusion led to uncontrollable intra-abdominal bleeding in 1 baboon from this apical site. This occurrence necessitated securing the probe more tightly with multiple purse strings and 4-quadrant pledgeted stay sutures. One instance of cardiac rupture originated from a lateral wall infarction site. Earlier studies have shown infections to be uniformly fatal in this transplant model. However, owing to the telemetry placement, infections were identified early by temperature spikes that were treated promptly with antibiotics. We had several cases of wound dehiscence due to recipients disrupting the suture line. These complications were promptly resolved by either re-approximating the wound or finding distractions for the baboon. A few of the most common problems we faced in our earlier experiments were related to the jacket, tether, and infusion pumps. It was difficult to keep the jackets on some baboons and the tether had to be modified several times before we assured long-term success. Infusion catheter replacement resulted in transplant heart venous obstruction and thrombosis from a right common femoral venous line. Homeostatic perturbations such as HC and CC and baboon-induced wound complications comprised most complications. Major bleeding and death due to telemetry implantation and infarct rupture occurred in 2 baboons. Despite the variety of complications, we achieved significant graft prolongation in this model.

Amelanotic melanoma in a New Zealand White Rabbit (Oryctolagus cuniculus).

A 3.5-year-old intact male double-transgenic New Zealand white rabbit (Oryctolagus cuniculus), apoA-I and LCAT (apolipoprotein and lecithin:cholesterol acyltransferase), was presented with a discrete, raised facial mass (0.5 x 1.0 x 1.0 cm). The mass was surgically excised, with reoccurrence to the same site 88 days later. A second surgical excision was performed, and the rabbit died 3 weeks later from respiratory distress. At necropsy, multiple varying-sized masses were observed in the ventral mandibular region and throughout the lungs, pleura, and diaphragm. On histopathology, the masses were composed of moderately anisocytotic and anisokaryotic polygonal to spindloid cells with moderate finely granular, lightly eosinophilic cytoplasm, having round to oval nuclei with one to several nucleoli and finely stippled chromatin. Mitotic figures were frequent. Lymphatic and venous invasion were noted with neoplastic cells metastasized to the submandibular lymph nodes, lungs, liver, and adventitial surface of the aorta. Fontana-Masson stain was negative for melanin, thereby necessitating immunohistochemistry and transmission electron microscopy. Positive staining with MART-1 (a melanocyte protein marker) combined with transmission electron microscopy revealing type II melanosomes confirmed the diagnosis of an amelanotic melanoma.

Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands.

OBJECTIVES: Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands. METHODS: A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval. RESULTS: AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal. CONCLUSION: Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

AAV5-mediated gene transfer to the parotid glands of non-human primates.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.

Transforming growth factors-beta are not good biomarkers of chemopreventive efficacy in a preclinical breast cancer model system.

Using a carcinogen-initiated rat model of mammary tumorigenesis, we tested the hypothesis that transforming growth factor (TGF)-betas are useful biomarkers of chemopreventive efficacy in the breast. The chemopreventive agents tested were tamoxifen and the retinoids 9-cis-retinoic acid (9cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR), because both antiestrogens and retinoids have previously been shown to upregulate TGF-betas in vitro. Despite demonstrable chemopreventive efficacy in this model, none of these agents, alone or in combination, had any significant impact on the expression of TGF-betas in the mammary ductal epithelium or periductal stroma as determined by immunohistochemistry. These data suggest that TGF-betas are not likely to be useful biomarkers of chemopreventive efficacy in a clinical setting.

Manometric changes during retrograde biliary infusion in mice.

The manometric, ultrastructural, radiographic, and physiological consequences of retrograde biliary infusion were determined in normostatic and cholestatic mice. Intraluminal biliary pressure changed as a function of infusion volume, rate, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Retrograde infusion with significant elevations in pressure led to paracellular leakage of lanthanum chloride, radiopaque dye, and [(14)C]sucrose with rapid systemic redistribution via sinusoidal and subsequent hepatic venous drainage. Chronic extrahepatic bile duct obstruction resulted in significantly smaller peak intrabiliary pressures and lower levels of paracellular leakage. These findings indicate that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in an acute pressure-dependent physical opening of tight junctions, permitting the movement of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intraluminal pressure may potentially permit the selective delivery of macromolecules >18-20 A in diameter to specific histological compartments.

Recombinase-activating gene (RAG) 2-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice.

The majority of Atm-deficient mice die of malignant thymic lymphoma by 4-5 mo of age. Cytogenetic abnormalities in these tumors are consistently identified within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA breaks that occur during V(D)J recombination. Since V(D)J recombination is a recombinase-activating gene (RAG)-dependent process, we generated Rag2(-/-)Atm(-/-) mice to assess the requirement for RAG-dependent recombination in thymic lymphomagenesis. In contrast to expectation, the data presented here indicate that development of malignant thymic lymphoma in Atm(-/-) mice is not prevented by loss of RAG-2 and thus is not dependent on V(D)J recombination. Malignant thymic lymphomas in Rag2(-/-)Atm(-/-) mice occurred at a lower frequency and with a longer latency as compared with Atm(-/-) mice. Importantly, cytogenetic analysis of these tumors indicated that multiple chromosomal abnormalities occurred in each tumor, but that none of these involved the Tcr alpha/delta locus. Nonmalignant peripheral T cells from TCR-transgenic Rag2(-/-)Atm(-/-) mice also revealed a substantial increase in translocation frequency, suggesting that these translocations are early events in the process of tumorigenesis. These data are consistent with the hypothesis that the major mechanism of tumorigenesis in Atm(-/-) mice is via chromosomal translocations and other abnormalities that are secondary to aberrant responses to double-stranded DNA breaks. Furthermore, these data suggest that V(D)J recombination is a critical, but not essential, event during which Atm-deficient thymocytes are susceptible to developing chromosome aberrations that predispose to malignant transformation.

An immunoablative regimen of fludarabine and cyclophosphamide prevents fully MHC-mismatched murine marrow graft rejection independent of GVHD.

The prevention of graft rejection in the setting of nonmyeloablative transplant approaches might be mediated by chemotherapy-induced host immunoablation and by the graft-promoting effects of graft-versus-host disease (GVHD). To evaluate whether host immunoablation alone might allow for alloengraftment, we developed an F1-into-parent murine marrow rejection model using host preparative regimens of lethal total body irradiation (TBI; 950 cGy), sublethal irradiation (600 cGy), or combinations of fludarabine (Flu) and cyclophosphamide (Cy). A preparative regimen selectivity index (SI) was calculated to determine whether host lymphocytes were preferentially depleted relative to myeloid cells (SI = number of host myeloid/number host T lymphoid cells remaining after preparative regimen administration). Saline-treated recipients were assigned an SI value of 1.0. Recipients of lethal TBI had reduced myeloid cells relative to T cells (SI = 0.6). In contrast, all Flu/Cy regimens preferentially depleted host T cells: recipients of Flu (100 mg/kg per day)/Cy (50 mg/kg per day) for 10 days (SI = 28.1); recipients of Flu (100 mg/kg per day)/Cy (100 mg/kg per day) for 10 days (SI = 64.1); and recipients of Flu (100 mg/kg per day)/Cy (50 mg/kg per day) for 19 or 27 days (SI = 74.6). The 10-day regimen of Flu/Cy (50 mg/kg per day) did not severely reduce host T cell numbers, nor did it prevent F1 marrow rejection (<1% chimerism, n = 14). In contrast, the 10-day regimen of Flu/Cy (100 mg/kg per day) reduced T-cell numbers below that of lethal TBI recipients and prevented F1 marrow rejection (11.4% chimerism, n = 15); donor chimerism was predominant in lymphoid cells and was stable through day 240 post-BMT. Additionally, the 19- or 27-day regimen of Flu/Cy, which most selectively depleted host T cells, also prevented F1 marrow rejection (6.3% chimerism, n = 15). These results therefore demonstrate that optimized Flu-containing, immunoablative preparative regimens can prevent fully MHC-disparate marrow rejection independent of GVHD.

Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice.

In lipoatrophic diabetes, a lack of fat is associated with insulin resistance and hyperglycemia. This is in striking contrast to the usual association of diabetes with obesity. To understand the underlying mechanisms, we transplanted adipose tissue into A-ZIP/F-1 mice, which have a severe form of lipoatrophic diabetes. Transplantation of wild-type fat reversed the hyperglycemia, dramatically lowered insulin levels, and improved muscle insulin sensitivity, demonstrating that the diabetes in A-ZIP/F-1 mice is caused by the lack of adipose tissue. All aspects of the A-ZIP/F-1 phenotype including hyperphagia, hepatic steatosis, and somatomegaly were either partially or completely reversed. However, the improvement in triglyceride and FFA levels was modest. Donor fat taken from parametrial and subcutaneous sites was equally effective in reversing the phenotype. The beneficial effects of transplantation were dose dependent and required near-physiological amounts of transplanted fat. Transplantation of genetically modified fat into A-ZIP/F-1 mice is a new and powerful technique for studying adipose physiology and the metabolic and endocrine communication between adipose tissue and the rest of the body.

ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

Transgenic mice lacking white fat: models for understanding human lipoatrophic diabetes.

The human disease lipoatrophic (or lipodystrophic) diabetes is a rare syndrome in which a deficiency of adipose tissue is associated with Type 2 diabetes. This disease is an interesting contrast to the usual situation in which diabetes is associated with obesity, an excess of fat. Aside from obesity, patients with lipodystrophic diabetes have the other features associated with Metabolic Syndrome X, including hypertension and dyslipidemia. The contrast between diabetes with a lack of fat and diabetes with an excess of fat provides an opportunity to study the mechanisms causing Type 2 diabetes and its complications. Recently, three laboratories have produced transgenic mice that are deficient in white adipose tissue. These mice have insulin resistance and other features of lipoatrophic diabetes, and are a faithful model for the human disease. Here we review the different murine models of fat ablation and compare the murine and human diseases, addressing the questions: Is the lack of fat causative of the diabetes, and if so by what mechanism? How could the other clinical features be explained mechanistically? And finally, what can be gleaned about insight into treatment options?

Life without white fat: a transgenic mouse.

We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. The liver is engorged with lipid, and the internal organs are enlarged. The mice are diabetic, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50- to 400-fold), free fatty acids (2-fold), and triglycerides (3- to 5-fold). The A-ZIP/F-1 phenotype suggests a mouse model for the human disease lipoatrophic diabetes (Seip-Berardinelli syndrome), indicating that the lack of fat can cause diabetes. The myriad of consequences of having no fat throughout development can be addressed with this model.

Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene.

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein alpha-subunit (Gsalpha) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous Gs deficiency is embryonically lethal. Heterozygotes with maternal (m-/+) and paternal (+/p-) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m-/+, but not +/p-, mice. Gsalpha expression in the renal cortex (the site of PTH action) is markedly reduced in m-/+ but not in +/p- mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m-/+ and +/p- mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.

Effects of energy-matched pulsed and continuous ultrasound on tumor growth in mice.

BACKGROUND AND PURPOSE: A diagnosis of cancer is a contraindication for the use of therapeutic ultrasound (US). Continuous US applied to murine tumors has resulted in larger and heavier tumors compared with controls. We compared tumor growth using low-power continuous US and energy-matched pulsed US. SUBJECTS: Female C57BL/6 mice (N = 174) were used. METHODS: Animals received subcutaneous injections of methylcholanthrene tumor cells. The mice were randomly divided into three groups: 60 mice that received low-power continuous US for 5 minutes at 0.75 W/cm2 (LC US group), 63 mice that received pulsed US for 12.5 minutes at 1.5 W/cm2 (pulsed US group), and 51 mice that served as a control group. The LC and pulsed US groups received equal US energy. Both experimental groups received 10 treatments of 3-MHz US, which was applied directly over the tumor. The control group received identical handling but no US. After treatment, the tumors were excised, weighed, and measured. A one-way analysis of variance, followed by Newman-Keuls post hoc testing, was used to analyze the data. RESULTS: Mean tumor weights (in grams) and volumes (in cubic millimeters) were 0.563 g and 564 mm3 for the LC US group, 0.560 g and 525 mm3 for the pulsed US group, and 0.516 g and 406 mm3 for the control group. CONCLUSION AND DISCUSSION: Reducing total US energy will result in less growth of murine tumors. When infusing equal energy, continuous and pulsed US will produce similar effects on tumor growth.

Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice.

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein epsilon (C/EBP epsilon) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBP epsilon develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3-5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBP epsilon is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graft-versus-host disease.

Allogeneic CD8+ T cells mediate both a graft-vs-leukemia (GVL) effect and graft-vs-host disease (GVHD). To evaluate whether CD8 cells of defined cytokine phenotype differentially mediate these processes, alloreactive donor CD8+ T cells preferentially secreting type I or type II cytokines were generated by alloantigenic priming in vitro in the presence of IL-12 or IL-4, respectively. Both cytokine-secreting subsets lysed allogeneic tumor targets in vitro ("Tc1" and "Tc2" subsets). A transplantation model was established (B6 into B6C3F1, 1050 cGy host irradiation) using the 32Dp210 myeloid line (bcr/abl transfected, H-2k; 1 x 10(4) tumor cells/recipient). Compared with leukemia controls (death at 12.9 days post-bone marrow transplantation), both Tc1 and Tc2 recipients were conferred a survival advantage. At cell doses of 2 to 2.5 x 10(7), the Tc1-mediated GVL effect (mean survival of 34.2 days) was more potent than the Tc2-mediated GVL effect (mean survival of 20.5 days; Tc1 > Tc2, p = 0.009). On day 15, histologic examination showed that Tc1 recipients had undetectable tumor burdens, whereas Tc2 recipients had extensive leukemic infiltrates. However, Tc2 recipients had essentially no histologic evidence of GVHD, whereas Tc1 recipients had mild to moderate GVHD (average GVHD scores of 1/40 and 9.3/40, respectively). In contrast, recipients of uncultured CD8+ donor T cells developed severe GVHD (average GVHD score of 26.7/40). Because in vitro-generated, alloreactive Tc1 and Tc2 populations mediated GVL with reduced GVHD, we conclude that both subsets may improve the therapeutic outcome of allogeneic T cell transfers in patients with leukemia.