Determination of optimal freezing parameters of human prostate cancer in a nude mouse model

Infants who present with multiple unexplained fractures pose a difficult diagnostic dilemma of child abuse versus intrinsic bone disease. Temporary brittle bone disease is a recently described disease characterized by a transient bone weakness in the first year of life which presents with multiple, unexplained fractures that can be confused with child abuse. The purpose of this study was to determine if there are common, historical features in infants with unexplained fractures that might suggest a basis for the fractures, and to determine if bone density measurements might indicate that such infants have low bone density. Medical records were reviewed in 33 infants who were referred for consultation for multiple unexplained fractures in which the parents and other caregivers denied wrongdoing. In 9 of the infants, radiographic absorptiometry and/or computed tomography bone density studies were performed. In 26 of these infants the diagnosis of temporary brittle bone disease was made. A normal collagen test was found in 17 of the 26 infants studied; 9 infants did not have a collagen test because the diagnosis of osteogenesis imperfecta was considered highly unlikely. In 25 of them there was a history of decreased fetal movement and/or intrauterine confinement. Bone density, as judged by plain X-ray films, was normal in all 26 cases, but when formally measured by radiographic absorptiometry or computed tomography, the bone density measurements were low in 8 of the 9 infants studied. These findings implicate decreased fetal movement and intrauterine confinement as contributing factors to temporary brittle bone disease and suggest that normal, unconstrained fetal movement during pregnancy is important for normal fetal bone formation. These findings support the model that bone formation and strength are dependent on the mechanical load placed on the bone. The results also demonstrate the usefulness of bone density measurements in evaluating the infant with multiple unexplained fractures to help distinguish nonaccidental injury from intrinsic bone disease.     

Antisense oligonucleotide intralesional therapy for human PC-3 prostate tumors carried in athymic nude mice

To address the question whether calretinin (CR) may protect cells against Ca2+ overload or trophic factor deprivation, PC12 cells were transfected with plasmids containing a CR coding region under control of a cytomegalovirus promoter. Nerve growth factor (NGF) treatment induced differentiation, increased transfection efficiency (at least 10-fold) and activated the CR gene (as found by RNase protection method and immunohistochemistry). Exogenous CR expression was identified either in living cells by fluorescence of green fluorescent protein (when the CR coding region was fused to this protein) or in fixed cells by CR immunoreactivity. Undifferentiated and NGF-differentiated populations of transfected cells were incubated in the presence of a Ca(2+)-ionophore or in media deprived of serum or NGF. Expression of exogenous CR in undifferentiated or NGF-treated cells (due to transfection) or endogenous CR (due to gene activation by NGF) did not render PC12 cells more resistant to insults such as Ca(2+)-overload and trophic factor deprivation.     

Generation of a nude mouse tumor model for in vivo replication of human cytomegalovirus

PURPOSE: Our purpose was to report the patterns of injury observed in five patients who suffered brain damage consequent to neonatal hypoglycemia. METHODS: The imaging studies and clinical records of five patients with brain damage caused by neonatal hypoglycemia were reviewed retrospectively. Patterns of injury were compared with those described in the literature and those seen in neonatal hypoxic-ischemic injury. RESULTS: Diffuse cortical and subcortical white matter damage was seen, with the parietal and occipital lobes affected most severely. Globus pallidus injury was present in one patient who had the most severe cortical injury. CONCLUSION: We found a specific pattern of injury that correlates well with the sparse pathologic and imaging reports on neonatal hypoglycemia. We speculate that the patterns of damage are the result of regional hypoperfusion and excitatory toxicity with cell-type-specific injury.     

Suppression of human prostate carcinoma metastases in severe combined immunodeficient mice by interleukin 2 immunocytokine therapy

Immunocytokines are antibody-cytokine fusion proteins that combine the unique targeting ability of antibodies with the multifunctional activities of cytokines to activate effector cells in the tumor microenvironment. Here, we demonstrate the therapeutic efficacy of a tumor-specific immunocytokine, huKS1/4-IL2, which effectively inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma in severe combined immunodeficient mice. This antitumor effect was specific and highly effective, irrespective of reconstitution of these mice with human lymphokine-activated killer cells. Survival times of mice treated with huKS1/4-IL2 were increased 4-fold as compared with animals treated with a mixture of the corresponding antibody and recombinant human interleukin-2 (rhIL2). A persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in B, T, and natural killer cell-deficient severe combined immuodeficient-BEIGE mice, depleted of granulocytes, implies a major role for macrophages in this treatment effect. Our data demonstrate that immunocytokine-directed interleukin-2 therapy to tumor sites is an immunotherapeutic approach with potent effects against disseminated metastases of human prostate carcinoma and suggest that this treatment could be effective in an adjuvant setting for patients with minimal residual disease.     

A clinical nude mouse metastatic model for highly malignant human pancreatic cancer

Pancreatic cancer is a highly aggressive and treatment-refractory cancer. A clinically-relevant animal model is necessary to develop therapy for metastatic pancreatic cancer. In this study we evaluated the efficacy of mitomycin C (MMC) and 5-FU against the human pancreatic adenocarcinoma cell line PAN-12 in an orthotopic human metastatic pancreatic cancer nude mice model. The model is constructed by surgical orthotopic implantation (SOI) of histologically intact tumor tissue in the tail portion of the pancreas near the spleen. PAN-12 grew very aggressively in the control group of nude mice with extensive local invasion and distant metastasis to various organs with a propensity for the lung but to other organs as well, including the liver, kidney and regional and distant lymph nodes. In a striking effect none of the mice in the MMC-treated group developed tumor. Although mice in the 5-FU treated group survived statistically significantly longer than those in the untreated control, the overall incidence of metastasis in these mice was equivalent to those in the control. However no liver or kidney metastases were found in the 5-FU treated animals perhaps accounting in part for their longer survival. This "clinical" nude mouse model of highly metastatic pancreatic cancer can now be used to discover new effective agents for this disease.     

Immunobiology of heterotransplanted human tumors in nude mice

The immunobiology of heterotransplanted human tumors was investigated following transplantation into nude mice of human bronchogenic, colon, rectal, ovarian, gastric, endometrial, vaginal, bladder, renal, esophageal, embryonic cell, pancreatic, and breast carcinoma, as well as fibrosarcoma, rhabdomyosarcoma, malignant melanoma, astrocytoma, Wilm's tumor, endometrial hyperplasia, and hydatidiform mole. Several of these tumors were passaged up to 15 generations. During these passages no changes in latency period for tumor development or in histology were noted. There were significant differences between several tumors in the minimum number of cells required for successful transplantation; such differences were independent of the basic biologic aggressiveness of the individual tumors. Nude mice that received transplants of fibrosarcoma and endometrial carcinoma had increased serum IgM and numbers of spleen cells and complement receptor lymphocytes. No such changes were noted for mice that received transplants of malignant melanoma, In contrast, there were no apparent differences in the responses of nude mice, who were given transplants of human tumors, to be T-cell mitogens concanavalin A or phytohemagglutinin or in the number of theta-bearing spleen cells. The success rate for transplantation was significantly improved when explants, rather than single-cell suspensions, were performed. Tumors transplanted to nude mice derived from strictly homozygous matings behaved like tumors transplanted to mice born of heterozygous mothers. Finally, despite the dramatic size of subcutaneous tumor nodules, there were no examples of invasion or distant metastases.     

Elimination of human leukemia by monoclonal antibodies in an athymic nude mouse leukemia model

A human acute myeloid leukemia model has been developed by i.v. transplantation of HL-60 myeloid leukemia cells into Swiss nude mice pretreated with cyclophosphamide. HL-60 cells disseminated into hematopoietic tissues as determined by flow cytometric analysis, fluorescence microscopy, fluorescence in situ hybridization analysis, and colony formation assay. Passive immunotherapy using murine anti-CD13 (F23) or anti-CD33 (M195) mAbs was able to eliminate completely the HL-60 cells in the mice, as determined by fluorescence in situ hybridization analysis, colony formation assay, and culture of mouse blood and tissue cells in vitro. Although F23 is able to inhibit completely CD13/aminopeptidase N enzymatic activity, actinonin, another potent inhibitor of CD13/aminopeptidase N, was not active as an antileukemic agent. HL-60 cell surface antigens, including CD13 (aminopeptidase N) and CD33 (p67), down-regulated over time, and murine anti-HL-60 antibody was generated while the cells grew in the mice. This response was suppressed by cyclophosphamide. These data suggest that leukemia cell elimination was antibody mediated.     

Prospects for gene therapy in human prostate cancer

Prostate cancer is the most common neoplasm in men and a significant cause of mortality in affected patients. Despite significant advances, current methods of treatment are effective only in the absence of metastatic disease. Gene therapy offers a renewed hope of using the differential characteristics of normal and malignant tissue in constructing treatment strategies. Several clinical trials in prostate cancer gene therapy are currently under way, using immunomodulatory genes, anti-oncogenes, tumor suppressor genes and suicide genes. A continued understanding of the etiological mechanisms involved in the establishment and progression of prostate cancer, along with advances in gene therapy technology, should make gene therapy for prostate cancer therapeutically valuable in the future.     

Inhibition of breast cancer in nude mouse model by anti-angiogenesis

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pell. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

NG-nitro-L-arginine methyl ester inhibits bone metastasis after modified intracardiac injection of human breast cancer cells in a nude mouse model

A retrospective evaluation of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated oestriol (uE3) levels in maternal blood in the second trimester was conducted for cases of aneuploid pregnancies identified from a series of women who underwent amniocentesis. Blood samples were collected from 1078 women just before genetic amniocentesis was performed, mainly for individuals of advanced maternal age (greater than 35 years). Twenty-five maternal serum samples from pregnant women with an aneuploid fetus, including 14 with Down's syndrome, were available for analysis of all three parameters. An algorithm to detect Down's syndrome was used for this analysis with a risk of > or = 1:299 classified as screen-positive, this being found for 20.4 per cent of the cases (220/1078). The actual Down's syndrome detection rate was 85.7 per cent (12/14), whereas the detection rate for all aneuploidies was 72.0 per cent (18/25). Those that were not detected were two cases of trisomy 21, one trisomy 18, two trisomy 13, three sex chromosome abnormalities, and one case of an additional marker chromosome. The data indicate that this tri-analyte test should be provided after thorough genetic counselling and informed decision-making regarding maternal serum screening for women who wish for a prenatal diagnosis.     

Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA

Telomerase is the RNA-protein complex which elongates telomeric DNA (TTAGGG)n and appears to play an important role in cellular immortalization. The almost exclusive expression of telomerase in tumor cells, and not in most normal cells, offers an exciting opportunity for therapy by inhibiting its function. Here, we have investigated the effect of inhibition of telomerase on the growth and survival of human malignant glioma cells in vitro and in vivo by using a 19-mer antisense oligonucleotide against human telomerase RNA linked to a 2',5'-oligoadenylate (2-5A). 2-5A antisense functions by activating the endoribonuclease, RNase L, resulting in the degradation of single stranded, targeted RNA. We have shown that the 2-5A antisense treatment effectively suppressed tumor cell growth and survival in vitro. Furthermore, treatment of tumors grown in nude mice with the antisense oligonucleotide inhibited survival of the tumor cells. TUNEL assays suggest that this effect is mediated through the induction of apoptosis. Targeting telomerase RNA with 2-5A antisense, therefore, may represent an effective and novel approach for treatment of a broad range of cancers.     

Liposome-mediated therapy of intracranial brain tumors in a rat model

PURPOSE: Malignant brain tumors represent a serious therapeutic challenge, and survival often is low. We investigated the delivery of doxorubicin (DXR) to rat brain tumors in situ via liposomes, to test the hypothesis that intact liposomes undergo deposition in intracranial tumor through a compromised blood-tumor vasculature. Both therapeutic effect and intra-tumor drug carrier distribution were evaluated to identify variables in carrier-mediated delivery having impact on therapy. METHODS: The rat 9L gliosarcoma tumor was implanted orthotopically in Fischer 344 rats in the caudate-putamen region. The tumor-bearing rats were treated with DXR, either free or encapsulated in long-circulating, sterically-stabilized liposomes. Anti-tumor efficacy was assessed by survival time. In parallel, liposomes labeled with a fluorescent phospholipid analog were injected into tumor-bearing rats. At predetermined intervals, the brains were perfused with fixative, sectioned, and imaged with laser scanning confocal microscope (LSCM) to investigate the integrity of the tumor vascular bed and the intratumor deposition of liposomes. RESULTS: Free DXR given in 3 weekly iv injections was ineffective in increasing the life span of tumor-bearing rats at cumulative doses < or = 17 mg/kg, and at the highest dose (17 mg/kg) decreased survival slightly, compared to saline-treated controls. In contrast, DXR encapsulated in long-circulating liposomes mediated significant increases in life span at 17 mg/kg. Rats showed a 29% percent increase in median survival, respectively, compared to saline-control animals. The delay of treatment after tumor implantation was a major determinant of therapeutic effect. Fluorescent liposomes were deposited preferentially in tumor rather than normal brain, and were distributed non-uniformly, in close proximity to tumor blood vessels. CONCLUSIONS: Liposomes can be used to enhance delivery of drugs to brain tumors and increase therapeutic effect. The therapeutic effect may arise from release of drug from liposomes extravasated in discrete regions of the tumor vasculature and the extravascular space.     

Development of a nude mouse model of ras-mediated neoplasia using WR21 cells from a transgenic mouse salivary tumor

A novel cell line (WR21) was derived from a salivary tumor in a male wap-ras transgenic mouse. Salivary tumors in wap-ras transgenic mice are extremely aggressive and express high levels of oncogenic ras protein from the activated, human Ha-ras transgene. WR21 cells also expressed high levels of oncogenic ras protein in vitro and in vivo. They gave rise to aggressive, highly anaplastic solid tumors when injected subcutaneously into athymic nude mice and approximately 90% of the mice had lung metastases by the fifth week of tumor growth. WR21 tumors were inhibited by cyclophosphamide, 5-fluorouracil, adriamycin, mitomycin C and actinomycin D, but not methotrexate. Our results suggest that the WR21/nude mice model will be useful for testing the efficacy of drug therapies against ras-mediated neoplasias.     

Construction and radioimmunodetection of nude mouse xenograft of human choriocarcinoma

Fresh tissue of human choriocarcinoma was injected subcutaneously into the BALB/C nude mouse. The xenografts had grown up with 16-68 passages. The hCG level of the ascites or intratumoral blood was 400-20,000 mIU/ml. Radioimmunodetection was performed using 131I labelled murine anti-hCG McAb. The labelled antibody could be specifically accumulated in the tumor sites and the tumor imaging was acquired. The ideal imaging time was 72-96 hours after the injection of antibody.     

Human vestibular schwannoma growth in the nude mouse: evaluation of a modified subcutaneous implantation model

HYPOTHESIS: Based on the hypothesis that vestibular schwannomas can be successfully implanted and grown in the nude mouse model, an in vivo experiment was designed for subcutaneous implantation of solid vestibular schwannoma tissue. BACKGROUND: Vestibular schwannomas are benign tumors arising from Schwann cells of cranial nerve VIII. Little in vivo research has been carried out with these tumors, due in part to the difficulty to grow cells in culture or maintain tumor in an animal model. Recently, vestibular schwannomas have been implanted in nude mice with moderate success. The current study evaluates a modification of prior techniques in an effort to establish a dependable research model. METHODS: Thirty-six nude mice were implanted with variable-sized vestibular schwannoma tissue from three human subjects. Volumes implanted ranged from 14-170 mm3. Mice were observed for 28 days and individual volumes recalculated. Eleven of the mice were observed for a total of 56 days with volumes re-evaluated, and tumors subsequently were removed for assessment of viability and vascularity. RESULTS: At 28 days, 36 tumors (100%) showed take with 34 tumors (94%) showing macroscopic growth. The 11 tumors observed for 56 days showed a trend of stable or decreased size at 56 days compared with that of the 28-day measurement. Overall growth from time of implantation to measurements at 56 days was noted in 8 (73%) of 11 tumors when measured at the skin and in 10 (91%) of 11 tumors when direct tumor volume was measured. One hundred percent of tumors evaluated microscopically at 56 days was viable. All tumors at the time of removal had significant vascularity with a mean of 70.68% (SD = 23.42) of surface covered with vessels. There were no significant differences in take and growth for the larger tumor specimens compared with those of smaller sizes. CONCLUSION: Human vestibular schwannomas successfully can be implanted and maintained in the subcutaneous pocket of the nude mouse. This in vivo tumor model provides a reliable, accessible base for further research with vestibular schwannomas.     

SCID-Hu mouse as a model for human lung HIV-1 infection

HIV induces a multi-organ infection with a dual tropism for both lymphocytes and monocytes/macrophages. The lung is a target both for HIV infection and HIV-related opportunistic infections. The SCID mouse has provided the opportunity to develop a small animal model for HIV infection. However, HIV-1 infection of the human fetal thymus and liver (SCID Liv/Thy) implanted in these mice occurred only after direct intraimplant injection of HIV-1 and the resultant HIV-1 infection was restricted to the human thymus. Here we report that human foetal lung can develop in SCID Liv/Thy mice resulting in the development of normal human alveolar and bronchiolar lung compartments which can be productively infected with cell-free HIV-1 virus, leading to a systemic and bifocal infection. This SCID-Hu model should be useful for studying AIDS physiopathology, human viruses with lung tropism and for helping to define gene therapy protocols in lung human cells in vivo.     

Systemic and gastrointestinal candidiasis of infant mice as model for antifungal therapy

Systemic and gastrointestinal infection was established in infant (15-19 days old) mice after oral-intragastric challenge with Candida albicans. All survivors retained high levels of organisms in the liver, kidney, spleen, stomach and intestine up to the 24th post infection day. These animals with persistent infections were used to study the efficacy of short term antifungal therapy. Drug treatment was initiated on 13th day for a two week period, treatment with fluconazole was compared with amphotericin B, and 5 fluorocytosine. The results suggest that fluconazole is a useful drug in the treatment of gastrointestinal candidiasis.     

Systemic therapy for breast cancer

Currently available information suggests that the optimal duration of adjuvant therapy for breast cancer patients with no involved axillary nodes is 5 years, though controversy about this recommendation persists. In postmenopausal patients, disease-free survival appears to be improved by the addition of combination chemotherapy to tamoxifen. Recently reported studies indicate that there is no benefit to dose escalation of cyclophosphamide in adjuvant therapy and that the risk of secondary leukemia may be increased. The combination of paclitaxel and doxorubicin has been reported in single-institution studies to produce high response rates but may also be cardiotoxic. Recent reports indicate that a pharmacokinetic interaction between these two drugs may cause these clinical findings. New agents that may be of utility in the management of advanced or primary breast cancer include novel hormonal agents, notably the aromatase inhibitors, and the bisphosphonates.     

Dietary modulation of carcinoma development in a mouse model for human familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is caused by a dominant mutation in the adenomatous polyposis coli (APC) gene. Individuals with FAP progressively develop adenomas and carcinomas of the colon and rectum. We developed a mouse model for this disorder by genetically modifying the Apc gene. The resulting mice Apc1638 progressively develop neoplasms in the colon and remainder of the gastrointestinal tract. In this study when Apc1638 mice were fed a Western-style diet, they developed an increased incidence of the end point of carcinomas and number of invasive tumors. The findings therefore demonstrated dietary modulation of carcinoma incidence in mice with a targeted mutation providing a model for the study of gene-environment interactions in cancer.