Hormone receptor expression, clinical and histopathological analysis in feline injection site sarcomas.

Feline injection site sarcomas (FISS) are aggressive, with high recurrence and rarely metastasising. The objective of this study was to evaluate, by immunohistochemistry, the expression of oestrogen (ER) and progesterone (PR) receptors in FISS and correlate them with clinical and histopathological aspects. This was a retrospective study with 51 cases of FISS. Immunohistochemistry was performed to detect vimentin, ER, PR and Ki67 expression. Clinical, histopathological and immunohistochemical characteristics were predictor variables and the expression of ER and PR were the dependent ones. Twenty-eight (55%) of the 51 FISS cases were female and 23 (45%) male with 10.7 ± 4.2 years and median tumour size of 3 cm (2.0-5.4). The trunk was the most affected site, with 38 cases (84%). Histological grade III was observed in 57% of the cases, considering differentiation score, necrosis and mitotic index. ER expression, positive in 64% of cases, was associated with the mitotic index (P = .05) and degree of pleomorphism (P = .04). PR was not associated with the variables and 63% of cases were negative for this receptor. Thus, ER expression can affect tumour growth. The knowledge on the FISS hormonal expression is important to clarify the pathophysiological mechanisms. Further studies are needed to predict the value of ER expression in the prognosis of FISS.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination.

Potency determination of recombinant IFN-alpha based on phosphorylated STAT1 using flow cytometry.

The interferon (IFN) family of cytokines is recognized as a key component of the innate immune response and the first line of defense against viral infection. The usage of the IFN-alpha as a biopharmaceutical has been mainly applied in the treatment of chronic hepatitis C. In the literature it is possible to find a great variety of methods to determine the potency of these cytokines, and many efforts have been made in order to develop practical bioassays to study the biological activity of IFNs. In this technical note, we present a different approach to determine the potency of a recombinant IFN-alpha preparation based on the activation of the signal transducers and activators of transcription 1 (STAT1) using flow cytometry technique. Under the conditions of this study, this new approach proved to be useful and promising to assess the potency of these biopharmaceuticals and may also be used as an important tool in the quality control of such biological products.