The Prognostic Value of Interleukin-8 Expression in Feline Viral Immunodeficiency by IHC
D.A.P.C. Zuccari; R. Castro; C.L. da Silva; L.R. Pivaro; C.S. Frade; C.K. Accorsi; C. Meneghetti
Department of Molecular Biology, Faculty of Medicine of São José do Rio Preto, Av. Brigadeiro Faria Lima, Vila São Pedro, São José do Rio Preto, Brazil
The Feline Immunodeficiency virus, also called FIV, is an exogenous virus of the Retroviridae family, Lentivirinae subfamily. The infection of domestic cats by the FIV is one of the most promising models for the study of the virus of the human immunodeficiency that causes Acquired Immunodeficiency syndrome (AIDS) and vice versa. The FIV causes in cats a similar disease as AIDS, particularly with regard to the increased susceptibility to opportunistic infections (Caldas et al., 2000). The virus infection presents a prolonged course and it is characterized by an acute phase in which the viral replication in the host organism is followed by a period of less replication, in which the animal is almost asymptomatic. Years later, at the final stage of infection, the immunodeficiency syndrome of the cats is developed. The clinical signs are varied due to the immunosuppressive nature of the infection. In 25 to 50% of cases oral lesions as gingivitis, stomatitis and periodontitis occurs (Smith, 2003). The chronic symptoms are difficult to treat and the best results are obtained when the target is the virus and its action on the immune system of the animal. Some infected animals may develop the type polyclonal hypergammaglobulinemia, especially in chronic or ended stage of infection. This fact has been attributed to a disturbance in the production of cytokines, caused by viral infection of CD4 + T lymphocytes (Rodrigues et al. 2007). The IL-8, as the family member of the CXC chemokines, is a potent chemoattractant and activator of neutrophils and lymphocytes (Monton et al.1997, Snoussi et al. 2006). This study was conducted to verify the prognostic value of the cytokine IL-8 detection in chronic oral lesions in a cat FIV + .A male cat of 11 years was brought to medical care presenting sialorrhea, dyspnea, anorexia, gingivitis, halitosis and difficulty in swallowing. The animal was treated for periodontal disease for three consecutive times with a return of symptoms. With the worsening of the injuries it was decided to collect material for histopathological examination and PCR to confirm the diagnosis of FIV. After that, the fragment was submitted to IHC for the anti-interleukin-8 expression.
Materials and Methods
The material was submitted to routine procedures for histopathological examination antibody anti-Interleukin-8 (BD Biosciences®, 1:50). Sections 3μm thick were cut from a and then to the immunohistochemical procedure, using the representative block and collected onto silanized slides for adhesion of tissue sections. After this, the slides were deparaffined, rehydrated in graded alcohols, and incubated with 3% hydrogen peroxidase for 30 minutes to block endogenous peroxidase activity. Induced antigen retrieval in Pan Steam at 95 °C with citric acid (pH 6.0, 35 minutes) was used. After cooling down, the slides were covered with Bovine Serum Albumin (BSA, Sigma-aldrich) for 30 minutes before incubation with the primary antibody for 2 hours at room temperature, and the avidin-biotin-peroxidase complex (ABC, Erviegas) for 1 hour. The chromogen, 3,3' diaminobenzidine tetrahydrochloride 0.5% (Signet®) diluted in Phosphate-buffered Saline (PBS), was applied to the slides for 2-5 minutes at 20-22°C. Slides were counterstained with Harris's Hematoxylin. Negative controls were obtained by omitting the primary antibody, whereas an inflammatory tissue served as an internal positive control. Expression of the marker was verified in accordance with graduation of expression proposed by Allred et al. (1998).
Results and Discussion
Histopathological examination was negative for malignancy but a severe inflammation was confirmed with the positive PCR for FIV. The immunohistochemical procedure showed that the fragment of the animal's mouth lesion has shown strong IL-8 expression. With these results we can tell that the cells in question have a high amount of IL-8 protein in their cytoplasm. The literature shows an important relationship between IL-8 and FIV. According to Heit et al. (2006) the cats infected with FIV do not attract neutrophils to the desired location because the virus causes changes in the activity of IL-8. Already in humans, IL-8 is predominantly expressed in the female genital tract in certain infectious and inflammatory conditions. Some studies also show that IL-8 facilitates the transmission of HIV type 1 in cervical tissue explant. The IL-8 increases the replication of HIV-1 in T cells and a greater extent in monocytes / macrophages in vitro (Narimatsu et al. 2005, Lane et al. 2001). So, the studies show that increased levels of IL-8 are present in the lymphoid tissue of patients positive for HIV-1. Moreover, studies have shown that compounds that inhibit the actions of IL-8 also inhibit the replication of HIV-1 in macrophages and lymphocytes T. Considering the similarity between human and feline immunodeficiency and the established value of the presence of IL-8 in lesions cells caused by viruses we can infer that this cytokine could be used as an important prognostic marker and possible designate therapeutic drugs blocking its action for the treatment of these lesions. In addition, we can demonstrate that compounds which inhibit the action of IL-8 can also inhibit FIV replication in both T lymphocytes and macrophages, indicating potential therapeutic use for this compound in FIV infection.
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