Immunohistochemical and molecular analysis of caveolin-1 expression in canine mammary tumors.

Caveolin-1 (Cav-1) is a structural protein present in invaginations of the cell membrane. In human breast cancer, the cav-1 gene is believed to be a tumor suppressor gene associated with inhibition of tumor metastasis. However, little is known about its expression, regulation and function in canine mammary tumors. Expression levels of cav-1 were investigated using real-time PCR and immunohistochemical detection with an anti-human Cav-1 antibody. Gene expression stability of different samples was analyzed using the geNorm software. Mammary tumors from 51 female dogs were compared to normal mammary tissue from 10 female dogs. Malignant mammary cells showed a loss of Cav-1 expression by quantitative RT-PCR and weak Cav-1 staining by immunohistochemistry compared to normal mammary gland tissue. There was a significant relationship between outcome and immunostaining as well as with tumor size, indicating that caveolin subexpression has a positive predictive value and is related to higher survival and smaller tumor size. Our findings indicate that Cav-1 is a potential prognostic marker for canine mammary tumors.

Interleukin-8 expression associated with canine mammary tumors.

The use of prognostic markers for mammary cancer is important for routine diagnosis and research. Interleukin-8 (IL-8) is a chemotactic cytokine, produced by several cell types in response to inflammation. The expression, regulation and function of IL-8 in dogs are little known. Recent studies have associated angiogenesis and inflammatory processes with tumor malignancy. We investigated a possible correlation between IL-8 expression and mammary tumor prognosis in female dogs. IL-8 expression was measured in 50 dogs with mammary neoplasia by immunohistochemistry and real-time PCR. Immunohistochemical staining was done with anti-IL-8 antibodies and PCR amplifications were performed in a 7500 Fast Real-Time PCR system. Gene expression stability was analyzed by the geNorm software. Quantitative real-time PCR showed that IL-8 expression decreased in malignant mammary cells compared to normal mammary tissue, while weak immunostaining was associated with a diagnosis of carcinoma. Complementing earlier studies on IL-8 expression in several types of cancer, including mammary cancer, we conclude that IL-8 has potential for use as a prognostic marker for canine mammary neoplasia.

Expressão imunoistoquímica e molecular da laminina-332 cadeia gama-2 em tumores mamários de cadelas / Immunohistochemical and molecular expression of laminin-332 gamma-2 chain in canine mammary tumors

Forty-eight cases of canine mammary cancer were investigated to evaluate the immunohistochemical distribution of the γ2 chain of laminin-332. Tumor cells were compared to a pool of normal mammary tissues using quantitative RT-PCR. The western blot was performed in eight tumor samples as complementary test to evaluate protein integrity. Immunohistochemistry experiments showed negative, focal, and weak expression of laminin-332 γ2 in tumors with the worst prognosis. Quantitative PCR revealed downregulation of the gene in 27 (56.2 percent) of the animals. Out of the 16 dogs with γ2 chain overexpression, seven were still alive. The western blot results showed bands generation of 36, 50, and 98kDa, suggesting degradation of laminin-332 γ2 in malignant tumors. The results suggest that, in the future, low expression and/or degradation of laminin-332 γ2 chain in canine mammary tumors may be used as an indicator of malignant potential. However, further studies are necessary to corroborate these results.

Para avaliar a expressão imunoistoquímica da cadeia γ 2 da laminina-332, foram investigados 48 casos de câncer mamário canino. Comparou-se, por RT-PCR, a expressão gênica nas células tumorais com um pool de tecido mamário. Como teste complementar, em oito amostras tumorais, realizou-se western blot para avaliar a integridade da proteína laminina-332 γ 2. A imunoistoquímica demonstrou expressão negativa, focal e fraca da laminina-332 γ 2 nos tumores com pior prognóstico. A RT-PCR quantitativa revelou a baixa expressão do gene em 27 (56,2 por cento) dos animais. Das 16 cadelas com superexpressão da cadeia γ 2, sete ainda estavam vivas no final do estudo. O resultado do western blot mostrou bandas de 36, 50 e 98kDa, sugerindo uma degradação da laminina-332 γ 2 em tumores malignos. Os resultados sugerem que, no futuro, a baixa expressão e/ou degradação da laminina-332 γ 2 nos tumores mamários caninos podem ser utilizadas como indicadores de potencial maligno. No entanto, mais estudos são necessários para confirmar estes resultados.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: homologous erythrocytes as a delivery system for preferential immunization with putative protective antigens.

Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 microg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.

Preparation and characterization of biotinylated red blood cells.

Biotinylation of intact mammalian red blood cells (RBC) was performed either by attachment to the amino groups by means of biotin N-hydrosuccinimide ester (NHS-biotin) or by oxidation of the induced aldehyde groups of the RBC membrane by biotin hydrazide. Comparison of these different procedures showed that biotinylation by NHS-biotin provided the highest cell recovery (> 90%), the binding of approximately 1000 biotin molecules/cell (on mouse RBC) and the 24 h survival in circulation was unaffected. In contrast, biotin hydrazide produced cell recovery in the 5-30% range (depending on the extent of oxidation), an undetectable number of molecules of biotin/cell and negligible 24 h survival. Among the NHS derivatives of biotin, further studies were performed on those containing a spacer arm of 2.2 nm (22 A) [sulphosuccinimidyl-6-(biotinamido)-hexanoate]. In vitro this derivative was similar to, or better than, the NHS-biotin in terms of cell recovery and the number of molecules/cell. In vivo this derivative showed a 24 h circulation survival similar to that of NHS-biotin. Unfortunately, biotin bound with such a spacer arm is lost after a few days of RBC circulation, probably due to plasma biotinidase. Possible applications of biotinylated RBCs include the in vivo measurement of RBC volume, the RBC survival and the delivery of enzyme and antigens.

Methanol detoxification by enzyme-loaded erythrocytes.

Alcohol oxidase (AlOx) from Pichea pastoris (a methylotrophic yeast) was encapsulated into human and murine erythrocytes up to 2 units/ml of packed cells. This enzyme has a much higher affinity for methanol than for ethanol, thus making the loaded erythrocytes useful cellular bioreactors able to catabolize methanol. Enzyme-loaded erythrocytes showed an increased rate of the hexose-monophosphate-shunt activity and a significant methaemoglobin production. However, the in vivo survival of these cells does not seem to be significantly affected by methanol catabolism. In vivo, mice receiving AlOx-loaded erythrocytes were able to keep the blood methanol concentrations below values that were about 50% of those found in mice receiving unloaded cells and similar amounts of methanol. Thus AlOx-loaded erythrocytes may add an important contribution to the detoxification protocol against methanol poisoning.

Red blood cells as an antigen-delivery system.

The use of adjuvants is usually required to induce strong immunological responses to protein antigens. However, in many cases these adjuvants cannot be extensively applied in human and veterinary vaccinations because of associated inflammatory reactions or granuloma formation. We show here that protein antigens (bovine serum albumin, hog liver uricase, and yeast hexokinase), coupled to autologous red blood cells by way of a biotin-avidin-biotin bridge, elicit an immunological response in mice similar to or higher than that obtained by the use of Freund's adjuvant. Quantities as low as 0.5 micrograms/mouse are high enough to generate these immunological responses. Furthermore, splenocytes of mice immunized by red blood cell-coupled antigens can be used to generate hybridomas secreting monoclonal antibodies. Thus, the delivery of antigens by autologous red blood cells is an effective way to avoid the use of adjuvants for producing anti-peptide antibodies and possibly to generate peptide vaccines.

Human red blood cells as bioreactors for the inactivation of harmful xenobiotics.

Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione. This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione. By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state. These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells. These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles.

[Calcium-phosphorus changes in chronic anticonvulsant therapy: effects of the administration of 25-hydroxyvitamin D3 on secondary hyperparathyroidism]. / Alterazioni calcio-fosforiche nella terapia cronica con anticonvulsivi: effetti della somministrazione di 25-OHD3 sull'iperparatiroidismo secondario.

The present study represents a contribution to the knowledge of secondary hyperparathyroidism (SHP) in patients treated with anticonvulsant drugs (AC). In these subjects alterations of the calcium: phosphorus metabolism as rickets and osteomalacia are frequent; however literature data on SHP are scarce. Our research carried out on 29 adult patients under treatment with one or more AC for periods ranging from 9 months to 12 years confirmed that 25-OHD levels in the serum are low, especially in patients treated for longer times. The iPTH levels in the serum are increased with respect to normal controls, while blood calcium and phosphate levels are normal as are urine calcium and phosphate. The 25-OHD levels in serum present the same seasonal variations as the normal controls. The administration of 25-OHD3 (20 micrograms/day for 3 months) to 12 of these patients who had the lowest 25-OHD spring levels rendered the 25-OHD levels attain normal values. Cyclic AMP was normalized; serum and urine calcium and phosphorus and urinary hydroxyproline were not modified significantly. On the basis of the present data it is recommended that chronic AC treatment should be accompanied by long term administration of 25-OHD3 for prophylaxis and/or for treatment of SHP.