Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Glycol methacrylate embedding for the histochemical study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum.

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy's solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs than controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than did controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail.