Plasma of Acute Lymphoblastic Leukemia Patients React to the Culture of a Mycovirus Containing Aspergillus flavus.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and is also seen in adults. Currently, no plasma-based test for the detection of ALL is available. We have cultured the home of a patient with ALL and isolated a mycovirus containing Aspergillus flavus. This culture was subjected to electron microscopy, purification, and mass spectrometry. Using enzyme-linked immunosorbent assay technique, plasma of patients with ALL and long-term survivors of this disease were tested for antibodies, utilizing supernatant of the culture of this organism. The results were compared with 3 groups of controls, including healthy individuals, patients with sickle cell disease, and solid tumors. Using electron microscopy, the isolated A. flavus contained mycovirus particles. In chemical analysis, this organism did not produce any aflatoxin. Using an enzyme-linked immunosorbent assay technique, the supernatant of the culture of the mycovirus containing A. flavus could differentiate ALL patients from each group of controls (P<0.001). These studies provide a new technique for the detection of ALL and may add information for future research regarding leukemogenesis.

Exposure to a mycovirus containing Aspergillus Flavus reproduces acute lymphoblastic leukemia cell surface and genetic markers in cells from patients in remission and not controls.

The etiology of acute lymphoblastic leukemia (ALL) remains unknown. A recent "two-hit" model for the occurrence of precursor B cell acute lymphoblastic leukemia propose that this disease arises through a two-step process, including predisposing genetic mutation and exposure to infections. While several genetic mutations are proposed, no infection category has been suggested. We have isolated a certain Aspergillus Flavus from residence of an ALL patient. This organism contains mycovirus and does not produce aflatoxin. The supernatant of culture of this mycovirus containing Aspergillus Flavus (SAF) was tested on the PBMCs of ALL patients in remission and controls. Cell surface phenotypes and genetic markers were examined. The effects of its combination with Epstein-Barr virus (EBV) was also investigated. For the SAF, positive and negative controls were aflatoxin and culture of Mycocladus corymbifer, respectively. Controls for ALL were sickle cell patients undergoing exchange transfusion. Incubation of the PMBCs from ALL patients in remission, or controls, with SAF resulted in re-development of ALL cell surface phenotypes and genetic markers in ALL patients in remission and not controls. These differentiating effects were not seen with aflatoxin or culture of Mycocladus Corymbifer. Addition of EBV did not alter effects of SAF. Currently, there are no techniques to discriminately reproduce characteristic leukemic genetic markers and cell surface phenotypes in cells from ALL patients in remission and not controls. These studies may provide a test for recognition of ALL patients in remission and new prospects for the investigation of leukemogenesis.

An in utero allotransplantation model of metastatic breast cancer in the cat.

BACKGROUND: Breast cancer represents one of the most significant health risks in the human female population due both to its frequency and metastatic potential. Current therapies, while improving the overall survival rate of women with mammary neoplasias, have not eliminated this disease as an important cause of morbidity and mortality. MATERIALS AND METHODS: We have investigated transplantation of a feline mammary adenocarcinoma cell line into fetal cats as a model of human metastatic breast cancer. Fetal cats injected in utero with an allogeneic mammary adenocarcinoma cell line were born with palpable masses at the site of injection and developed widespread metastases over the following 6-10 weeks. Tumor foci were seen most commonly in the lung and in the local tissues adjacent to the primary injection site. This distribution of metastases mimics that seen in human breast cancer. Thus, in utero transplantation in cats is a reproducible experimental model of metastatic breast cancer in women.

Alterations in CDK1 expression and nuclear/nucleolar localization following induction in a spontaneous canine mammary cancer model.

Transcription of CDK1 is induced as cells re-enter the cell cycle from quiescence and these early cell cycle re-entry events have been modeled by okadaic acid treatment due to its activity on specific enhancer sequences in the human CDK1 promoter. To investigate heterogeneity of control of this mechanism in the context of neoplastic transformation, a cellular model derived from spontaneous canine mammary cancer (CMT) was developed that includes six cell lines derived from different animals. Notable heterogeneity in response to okadaic acid was observed in expression of CDK1 mRNA and protein. In response to okadaic acid treatment, two CMT cell lines exhibited a CDK1 mRNA induction while one cell line exhibited CDK1 mRNA suppression, and three remained unchanged. Despite this variability, three CMT cell lines arrested in S or G2/M phase and five exhibited marked increases in apoptosis. Moderation of some of these differences were observed at the level of CDK1 protein as three of six CMT cell lines exhibited only moderate enhancement in CDK1 protein levels while three remained essentially unchanged. Some additional differences in distribution of CDK1 protein, favoring enhanced nuclear over cytoplasmic CDK1 localization, were observed in treated cells in the form of concentrated nuclear CDK1 labeled foci. Confocal microscopy revealed the presence of brightly labeled punctate foci containing CDK1 protein within nuclei as well as nucleoli in okadaic acid treated non-mitotic cells suggesting a role for this kinase outside the normal G2/mitotic phase of the cell cycle and suggesting a possible new function within the nucleolus.

Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA.

BACKGROUND: Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice. METHODS: An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated. RESULTS: After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles. CONCLUSIONS: Overall, the previously reported 'resistance' of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA.

Efficacy of gene therapy for a prototypical lysosomal storage disease (GSD-II) is critically dependent on vector dose, transgene promoter, and the tissues targeted for vector transduction.

Lysosomal storage diseases are an intriguing target for gene therapy approaches, as transduction of a "depot" organ with a transgene encoding a lysosomal enzyme can be followed by secretion, systemic distribution, downstream uptake, and lysosomal targeting of the enzyme into non-transduced tissues. These benefits are of utmost importance when considering gene therapy approaches for glycogen storage disease type-II (GSD-II). GSD-II is a prototypical lysosomal storage disorder caused by lack of intralysosomal acid alpha-glucosidase (GAA) activity. Lack of GAA can result in a proximal limb myopathy and respiratory and cardiac failure, each due to abnormal glycogen accumulation in the skeletal muscles or cardiac tissues, respectively. After converting the liver into a "depot" organ, we found that intravenous injection of the [E1-,polymerase-]AdGAA vector allowed for hepatic secretion of GAA over an at least 20-fold dosage range. We noted that very low plasma GAA levels (derived from hepatic secretion of GAA) can allow for GAA uptake by muscle tissues (skeletal or cardiac), but significantly higher plasma GAA levels are required before glycogen "cross-correction" can occur in these same tissues. We also demonstrated that liver-specific enhancer/promoters prolonged GAA transgene expression from persistent [E1-,polymerase-] adenovirus based vector genomes for at least 180 days, and significantly diminished the amounts of neutralizing anti-GAA antibodies elicited in this animal model. Finally, we demonstrated that skeletal muscles can also serve as a "depot" organ for GAA secretion, allowing for secretion of GAA and its uptake by noninfected distal tissues, although glycogen reductions in non-injected muscles were not achieved by the latter approach.