Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype.

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo.

Correlation of contrast-enhanced MR images with the histopathology of minimally invasive thermal and cryoablation cancer treatments in normal dog prostates.

Magnetic Resonance Imaging (MRI) is a promising tool for visualizing the delivery of minimally invasive cancer treatments such as high intensity ultrasound (HUS) and cryoablation. We use an acute dog prostate model to correlate lesion histopathology with contrast-enhanced (CE) T1 weighted MR images, to aid the radiologists in real time interpretation of in vivo lesion boundaries and pre-existing lesions. Following thermal or cryo treatments, prostate glands are removed, sliced, stained with the vital dye triphenyl tetrazolium chloride, photographed, fixed and processed in oversized blocks for routine microscopy. Slides are scanned by Trestle Corporation at .32 microns/pixel resolution, the various lesions traced using annotation software, and digital images compared to CE MR images. Histologically, HUS results in discrete lesions characterized by a "heat-fixed" zone, in which glands subjected to the highest temperatures are minimally altered, surrounded by a rim or "transition zone" composed of severely fragmented, necrotic glands, interstitial edema and vascular congestion. The "heat-fixed" zone is non-enhancing on CE MRI while the "transition zone" appears as a bright, enhancing rim. Likewise, the CE MR images for cryo lesions appear similar to thermally induced lesions, yet the histopathology is significantly different. Glands subjected to prolonged freezing appear totally disrupted, coagulated and hemorrhagic, while less intensely frozen glands along the lesion edge are partially fragmented and contain apoptotic cells. In conclusion, thermal and cryo-induced lesions, as well as certain pre-existing lesions (cystic hyperplasia - non-enhancing, chronic prostatitis - enhancing) have particular MRI profiles, useful for treatment and diagnostic purposes.

Dynamic nature of host-pathogen interactions in Mycobacterium marinum granulomas.

Mycobacterium marinum causes long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens). These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection. We examined organs of frogs with chronic M. marinum infection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection. Bacteria were always found within macrophage phagosomes. These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells by M. marinum. The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria. Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response. Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.

Suspected hypovitaminosis A in a colony of captive green anoles (Anolis carolinensis).

In a colony of 18 green anoles (Anolis carolinensis), 3 animals experienced focally thickened lips, ulcerative cheilitis, lethargy, depression, and weight loss over a 5-month period. In addition to crickets fed fresh fruit and leafy green vegetables, the diet of the green anoles consisted of a supply of mealworms that had been dusted with a commercial liquid vitamin supplement. The history, clinical findings, and histopathologic lesions were suggestive of hypovitaminosis A, which is known to cause squamous metaplasia of the mucus secreting glands and epithelial surfaces in many species.

Induction of angiogenesis by implantation of encapsulated primary myoblasts expressing vascular endothelial growth factor.

BACKGROUND: We previously demonstrated that intramuscular implantation of primary myoblasts engineered to express vascular endothelial growth factor (VEGF) constitutively resulted in hemangioma formation and the appearance of VEGF in the circulation. To investigate the potential for using allogeneic myoblasts and the effects of delivery of VEGF-expressing myoblasts to non-muscle sites, we have enclosed them in microcapsules that protect allogeneic cells from rejection, yet allow the secretion of proteins produced by the cells. METHODS: Encapsulated mouse primary myoblasts that constitutively expressed murine VEGF164, or encapsulated negative control cells, were implanted either subcutaneously or intraperitoneally into mice. RESULTS: Upon subcutaneous implantation, capsules containing VEGF-expressing myoblasts gave rise to large tissue masses at the implantation site that continued to grow and were composed primarily of endothelial and smooth muscle cells directly surrounding the capsules, and macrophages and capillaries further away from the capsules. Similarly, when injected intraperitoneally, VEGF-producing capsules caused significant localized inflammation and angiogenesis within the peritoneum, and ultimately led to fatal intraperitoneal hemorrhage. Notably, however, VEGF was not detected in the plasma of any mice. CONCLUSIONS: We conclude that encapsulated primary myoblasts persist and continue to secrete VEGF subcutaneously and intraperitoneally, but that the heparin-binding isoform VEGF164 exerts localized effects at the site of production. VEGF secreted from the capsules attracts endothelial and smooth muscle cells in a macrophage-independent manner. These results, along with our previous results, show that the mode and site of delivery of the same factor by the same engineered myoblasts can lead to markedly different outcomes. Moreover, the results confirm that constitutive delivery of high levels of VEGF is not desirable. In contrast, regulatable expression may lead to efficacious, safe, and localized VEGF delivery by encapsulated allogeneic primary myoblasts that can serve as universal donors.

Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice.

Loss-of-function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as Unverricht-Lundborg disease (EPM1). The primary cellular events and mechanisms underlying the disease are unknown. We found that mice lacking cystatin B develop myoclonic seizures and ataxia, similar to symptoms seen in the human disease. The principal cytopathology appears to be a loss of cerebellar granule cells, which frequently display condensed nuclei, fragmented DNA and other cellular changes characteristic of apoptosis. This mouse model of EPM1 provides evidence that cystatin B, a non-caspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis.

The role of the innate immune system in the reconstituted SCID mouse model of herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) has an immunopathological basis, thought primarily to involve a CD4+ T cell-mediated immune response to viral antigen. Other cell types, however, particularly those involved in nonspecific immunity, such as natural killer (NK) cells or neutrophils, may also contribute to tissue destruction in the cornea. The reconstituted SCID mouse model of HSK provides a powerful system in which to study the interactions of the innate and adaptive immune responses to herpes simplex virus type 1 corneal infection. In the present study, reconstituted SCID mice depleted of NK cells had a reduced incidence and severity of clinical and histopathological HSK. The levels of T cell cytokine protein and message in restimulated splenocytes and cytokine message in corneas did not differ between experimental groups. However, significantly fewer neutrophils were seen within the inflamed corneas of NK-depleted SCID mice. Therefore, endogenous NK cells may indirectly influence the severity of HSK in reconstituted SCID mice by affecting neutrophil migration into the cornea.

Lymphotoxin-alpha-deficient mice. Effects on secondary lymphoid organ development and humoral immune responsiveness.

Targeted mutagenesis in embryonic stem cells was used to generate mice deficient in lymphotoxin-alpha (LT-alpha). Mice lacking LT-alpha -/- (LT-alpha -/- mice) exhibit a phenotype dominated by defects in secondary lymphoid organ development. LT-alpha -/- mice lack lymph nodes and Peyer's patches, and possess spleens in which the usual architecture is disrupted. However, in a few of the mutants, abnormal lymph node-like structures were observed, mainly within the mesenteric fat. Abnormal clusters of lymphocytes were also found to accumulate in the periportal and perivascular regions of the liver and lung of LT-alpha -/- mice. Yet, lymphocytes from LT-alpha -/- mice appeared phenotypically normal, expressing the expected ratios of B and T cell surface markers as well as the lymphocyte homing marker, L-selectin. In addition, bone marrow cells from LT-alpha -/- mice were able to successfully reconstitute the lymphoid organs of severe combined immunodeficient mice. However, LT-alpha -/- mutant mice examined for humoral immune responsiveness were found to be impaired in their ability to respond to different Ag. These data illustrate the utility of this mouse model as a system for understanding lymphoid organ development and its effects on immune responsiveness.

Radiographic and scintigraphic evidence of focal pulmonary neoplasia in three cats with hyperthyroidism: diagnostic and therapeutic considerations.

Three cats were diagnosed as hyperthyroid based on clinical signs, historical findings, laboratory abnormalities, and basal serum thyroxine (T4) concentrations, and/or nuclear thyroid scans. Additionally, a presumptive diagnosis of thyroid carcinoma with pulmonary metastasis was made in each cat based on radiographic or scintigraphic evaluation. All three cats had solitary pulmonary nodules 1.5 to 2 cm in diameter on survey thoracic radiographs; one cat also had chylous pleural effusion and pulmonary lobar consolidation. Focal pulmonary accumulation of sodium pertechnetate (99mTcO4-) and/or radioiodine (131I) corresponding to radiographic lesions were seen in all cats. Two cats were treated with single ablative doses (1111 to 1480 MBq) of 131I; the remaining cat was euthanatized. One of the treated cats died 8 days later; the other cat was euthanatized 22 weeks following treatment. Histopathologic examination of tissue obtained at necropsy confirmed metastatic thyroid carcinoma in one cat and bronchogenic adenocarcinoma in two cats. Our findings indicate that increased radionuclide uptake in focal pulmonary lesions and cytologic evaluation of tissue obtained by fine-needle aspiration are not specific for thyroid tissue.