Zilton A. Andrade

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

Schistosomiasis and liver fibrosis

Schistosoma mansoni infection invariably results in liver fibrosis of the host. This fibrosis may be represented by small focal areas of chronic inflammation and excess extracellular matrix deposited in periovular granulomas, distributed in variable numbers at the periphery of the portal vein system. This is the outcome of 90% of the infected population in endemic areas. Conversely, a minority of infected individuals develop extensive disease with numerous granulomas along the entire extension of the portal spaces. This latter situation is mainly dependent on special hemodynamic changes created by a heavy worm load, with the subsequent production of numerous eggs and represents a severe form of a peculiar chronic hepatopathy. Thus, host–parasite interactions in schistosomiasis help us to understand a number of important features of liver fibrosis: its initiation and regulation, the significance of accompanying vascular changes, the dynamics of fibrosis formation and regression with antiparasitic treatment; host genetic and immunological contributions, and the pathophysiology of portal hypertension.

Pathological lesions associated with Schistosoma mansoni infection in man

Abstract The pathological lesions associated with schistosome infection in Bahia, Brazil, were reviewed in an attempt to clarify the nature and prevalence of the lesions caused by S. mansoni , and to define better our material for comparison with that from other centres. The prevalence of various lesions possibly caused by schistosome infection was determined in cadavers with and without S. mansoni infection. The records of 1435 consecutive autopsies were reviewed, 502 of which showed the infection. In 105 of these, Symmerss clay-pipe-stem fibrosis of the liver was present. All cases of portal hypertension attributed to schistosomiasis had Symmerss fibrosis, and nearly all cases of Symmerss fibrosis had symptoms and signs of marked portal hypertension. Schistosomal pulmonary arteritis and cor pulmonale were frequent in cases of Symmerss fibrosis, but in those without Symmerss fibrosis no unequivocal cases of cor pulmonale caused by schistosomiasis were identified. Another positive finding was segmental fibrous thickening of the colonic serosa and of the mesocolon, lesions most frequently seen in cases with Symmerss fibrosis. Fibrosis of the intestinal submucosa was rare. The absence of severe or diffuse fibrosis of the intestinal submucosa and the decreasing incidence of infection with increasing age in our cases, diagnosed by tissue examination, indicate that increased egg retention with decreased excretion of eggs in the faeces is, by itself, an unlikely explanation for decreased egg excretion noted in older persons. Death of worms or a decrease in egg production are hypotheses more consistent with the present observations. A number of other lesions showed no positive correlation with schistosome infection. These included cirrhosis, hepatoma, ulcerative intestinal lesions and clinically significant colonic polyposis. Thus Symmerss fibrosis was the most significant lesion caused by schistosome infection were found almost exclusively in the cases also having Symmerss fibrosis. Compared with subjects with cirrhosis, more of those with Symmerss fibrosis died from rupture of oesophago-gastric varices and showed lower indices of hepatic coma, jaundice, oedema and ascites. Liver and spleen sizes were larger in subjects with Symmerss fibrosis than in those with cirrhosis. Portal vein thrombosis was more frequent in cases with. Symmerss fibrosis, and was very frequent in patients who had been subjected to surgery.

Immunopathology of Chagas disease

The main clinical forms of Chagas disease (acute, indeterminate and chronic cardiac) present strong evidences for the participation of the immune system on pathogenesis. Although parasite multiplication is evident during acute infection, the intense acute myocarditis of this phase exhibits clear ultrastructural signs of cell-mediated immune damage, inflicted to parasitized and non-parasitized myocardiocytes and to the endothelium of myocardial capillaries (microangiopathy). Inflammation subsides almost completely when immunity decreases parasite load and suppressor factors modulate host reaction, but inflammation does not disappear when the disease enters the indeterminate phase. Inflammation becomes mild and focal and undergoes cyclic changes leading to complete resolution. However, the process is maintained because the disappearance of old focal lesions is balanced by the upsurge of new ones. This equilibrium allows for prolonged host survival in the absence of symptoms or signs of disease. The chronic cardiac form is represented by a delayed-type, cell-mediated diffuse myocarditis, that probably ensues when the suppressive mechanisms, operative during the indeterminate phase, become defaulted. The mechanism responsible for the transition from the indeterminate to the cardiac form, is poorly understood.

Enhanced helminthotoxic capacity of eosinophils from patients with eosinophilia.

To determine whether eosinophils from patients with eosinophilia have an enhanced capacity to kill parasites, we compared purified eosinophils (mean purity, 89 per cent) from 30 patients with various degrees of eosinophilia and with or without infection with Schistosoma mansoni for the capacity to kill schistosomula, the larval stage of S. mansoni, in vitro. There was a significant correlation between peripheral eosinophil count and antibody-dependent, eosinophil-mediated death of parasites after 40 hours of culture (P < 0.0001). Antibody-dependent adherence of eosinophils, measured after two hours of incubation, also correlated with the capacity of the eosinophils to kill the parasites. The correlation between the killing capacity of eosinophils and their peripheral-blood count was observed in patients both with and without S. mansoni infection. We suggest that eosinophilia involves not only a quantitative change in eosinophil numbers but also a qualitative change in functional capacity that renders circulating eosinophils more effective in resisting parasitic infections.

The influence of low-level laser therapy on biomodulation of collagen and elastic fibers

The study of low-level laser therapy upon extracellular matrix elements is important to understand the wound healing process under this agent. However, little is known about the interference of laser light in relation to collagen and elastic fibers. Cutaneous wounds were performed on the back of 72 Wistar rats and a Ga-Al-As low-level laser was punctually applied with different energy densities. The animals were killed after 24, 48, 72 hours and 5, 7 and 14 days. Tissues were stained with hematoxilin-eosin, sirius red fast green and orcein and then analyzed. It was observed that the treated group exhibited larger reduction of edema and inflammatory infiltrate. The treated animals presented a larger expression of collagen and elastic fibers, although without statistical significance (p > 0.05). Treatment with a dosage of 4 J/cm(2) exhibited more expressive results than that with 8 J/cm(2). In this study, the authors concluded that low-level laser therapy contributed to a larger expression of collagen and elastic fibers during the early phases of the wound healing process.

Experimental models of Schistosoma mansoni infection

Experimental models of Schistosoma mansoni infections in mammals have contributed greatly to our understanding of the pathology and pathogenesis of infection. We consider here hepatic and extrahepatic disease in models of acute and chronic infection. Experimental schistosome infections have also contributed more broadly to our understanding of granulomatous inflammation and our understanding of Th1 versus Th2 related inflammation and particularly to Th2-mediated fibrosis of the liver.

Hepatitis B Surface Antigen Carrier State in Hepatosplenic Schistosomiasis

The prevalence of hepatitis B surface antigen (HBsAg) was studied in 103 cases of hepatosplenic schistosomiasis (HSS), 134 control cases with a variety of illnesses including hepatointestinal schistosomiasis, and 600 blood donors, in an area endemic for both schisfosomiasis and viral hepatitis. The patients with HSS proved to be persistent carriers for HBsAg in a significantly higher proportion than the other two groups of cases. The HSS cases who were carriers of HBsAg had more clinical signs of chronic liver disease and strikingly more chronic inflammation of the portal spaces on liver biopsy. It is suggested that abnormal immunological responses in patients with HSS makes them more susceptible to become carriers of HBsAg and that the addition of this injurious factor makes their basic disease worse, and may be responsible for the development of cirrhosis in some cases.

INFLUENCE OF LASER PHOTOBIOMODULATION UPON CONNECTIVE TISSUE REMODELING DURING WOUND HEALING

The modulation of collagen fibers during experimental skin wound healing was studied in 112 Wistar rats submitted to laser photobiomodulation treatment. A standardized 8mm-diameter wound was made on the dorsal skin of all animals. In half of them, 0.2ml of a silica suspension was injected along the border of the wound in order to enhance collagen deposition and facilitate observation. The others received saline as vehicle. The treatment was carried out by means of laser rays from an aluminum-gallium arsenide diode semiconductor with 9mW applied every other day (total dose=4J/cm2) on the borders of the wound. Tissue sections obtained from four experimental groups representing sham-irradiated animals, laser, silica and the association of both, were studied after 3, 7, 10, 15, 20, 30 and 60 days from the laser application. The wounded skin area was surgically removed and submitted to histological, immunohistochemical, ultrastructural, and immunofluorescent studies. Besides the degree and arrangement of collagen fibers and of their isotypes, the degree of edema, the presence of several cell types especially pericytes and myofibroblasts, were described and measured. The observation of Sirius-red stained slides under polarized microscopy revealed to be of great help during the morphological analysis of the collagen tissue dynamic changes. It was demonstrated that laser application was responsible for edema regression and a diminution in the number of inflammatory cells (p<0.05). An evident increase in the number of actin-positive cells was observed in the laser-treated wounds. Collagen deposition was less than expected in silica-treated wounds, and laser treatment contributed to its better differentiation and modulation in all irradiated groups. Thus, laser photobiomodulation was able to induce several modifications during the cutaneous healing process, especially in favoring newly-formed collagen fibers to be better organized and compactedly disposed.

Capillaria hepatica: a cause of septal fibrosis of the liver

Fine, long, fibrous septa were observed as a late change developing in the acinar zone III of the liver of rats experimentally infected with the helminth Capillaria hepatica. Hepatic septal fibrosis begun 30 days after inoculation of embryonated eggs into the stomach of rats and became clearly evident from the 40th day onwards. Experimental observation was undertaken for 170 days. Septal fibrosis increased progressively with time and was most marked when the parasitic nodules formed around larvae, disintegrating worms and eggs were involving. Septal fibrosis of the liver has not been previously recognized as a manifestation of hepatic capillariasis. The presence of sequestered parasite antigens, probably being slowly released within the liver, appears to be a major factor in the pathogenesis of hepatic septal fibrosis observed in rats with C. hepatica infection.