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SUMMARY: This study aimed to evaluate the microstructure of the buffalo's hoof capsule using hystomorphometry and computerized light microscopy. The length, thickness, gap and number of epidermal papillae and the morphology of the corneal tubules of the hoof were described in this paper. This study used 56 hoof capsules from hooves of 14 adults - age ranging from 24 to 60 months old - female buffaloes of the Jafarabadi breed; weighing around 650 kg. Fifty-six samples, 28 from thoracic limb and 28 from pelvic limbs, were analyzed, resulting in a total of 112 digits. Clinical specimens were collected at the coronary corium, laminar corium of the abaxial wall and pre-bulbar soles. The study concluded that the microstructure of the hoof capsule of buffalo hooves are made of epidermal papillae measuring 1,721.59 mm of length, 62.94 mm of thickness and 49.02 mm of gap between the papillae. This study determines that the coronary corium has more epidermal papillae than the laminar corium of the abaxial wall and pre-bulbar soles. The corneal tubules of the hoof capsules exhibit helical fashion and points to the possibility of applying this feature to the hooves of the biungulate species as well. In this research, we have been able to determine morphological parameters not yet described in scientific literature. These findings can be used in future comparative studies of healthy cattle and buffaloes and speculate evidence about the hooves' vulnerability facing different hoof diseases.

RESUMEN: El objetivo del estudio fue evaluar la microestructura de la cápsula del casco del búfalo mediante histomorfometría y microscopía óptica computarizada. Se describe la longitud, el espesor, el espacio y el número de papilas epidérmicas y la morfología de los túbulos corneales del casco. Utilizamos 56 cápsulas de cascos de 14 hembras adultas entre 24 a 60 meses de edad de la raza Jafarabadi, cuyo peso aproximado era de 650 kg. Analizamos 56 cascos, 28 del miembro torácico y 28 del miembro pélvico, con un total de 112 dedos. Se recogieron especímenes clínicos en el corion coronario, corion laminar de la pared abaxial y suelas pre-bulbares. Concluimos que la microestructura de la cápsula de los cascos de los búfalos está formada por papilas epidérmicas de 1.721,59 mm de longitud, 62,94 mm de espesor y 49,02 mm de espacio entre papilas. Se determinó que el corion coronario tiene más papilas epidérmicas que el corion laminar de la pared axial y las suelas pre-bulbares. Los túbulos córneos de las cápsulas de casco presentan una forma helicoidal y existe la posibilidad de que esta característica también se aplique a las pezuñas de otras especies de animales biungulados. También se determinaron algunos parámetros morfológicos no descritos en la literatura científica. Estos hallazgos pueden ser útiles en estudios comparativos de ganado bovino y bubalino saludable y en consideración de la 'vulnerabilidad' frente a diferentes enfermedades del casco.

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We have previously reported that epimastigote forms of Trypanosoma cruzi treated with the lysophospholipid analogues (LPAs) edelfosine (ET-18), ilmofosine, and miltefosine suffered alterations in plasma membrane, mitochondrion, and lipid synthesis. In this work, ET-18 induced membrane damage in trypomastigotes and amastigotes. Incubation of epimastigotes and trypomastigotes with the three LPAs led to membrane permeabilization, which was abolished by serum addition. Treatment for 24 h in culture medium interfered the with mitochondrial status of epimastigotes, with no effect in trypomastigotes, in agreement with ultrastructural data. LPAs induced alterations in the plasma membrane of the three forms of T. cruzi and in the mitochondria of epimastigotes, suggesting that these organelles are potential targets of these analogues.

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The potential activity of three new derivatives of 3-(4'-Y-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (2-PAMs) was assayed against Trypanosoma cruzi and Leishmania amazonensis. They showed higher activity against trypomastigotes and epimastigotes of T. cruzi than the standard drugs, crystal violet and nifurtimox. Besides these derivatives, a series of eleven 2-PAMs derivatives and the corresponding intermediates, biphenyl methanones (BPMs) were assayed against promastigotes of L. amazonensis, showing that the 2-PAMs were remarkably more active than the BPMs. The PAMs 2c, 2e and 2j were about 2-fold more active that pentamidine isothionate and between 27.2- and 46.4-fold less toxic to V79 mammalian cells. The present results encourage further studies, especially against intracellular parasites and in experimental animals.

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We investigated the mechanism of action of metabolically stable lysophospholipid analogues (LPAs), with potent anti-tumour and anti-protozoal activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Against the axenically grown epimastigote form of the parasite, the IC(50)s after 120 h for ET-18-OCH(3), miltefosine and ilmofosine were 3, 1 and 3 microM, respectively; at higher concentrations immediate lytic effects were observed. Eradication of the intracellular amastigote, grown inside Vero cells, was achieved at 0.1, 0.1 and 1 microM for ET-18-OCH(3), miltefosine and ilmofosine, respectively. Analysis of the lipid composition of epimastigotes exposed to LPAs at their IC(50) for 120 h showed that the ratio of phosphatidyl-choline (PC) to phosphatidylethanolamine (PE) changed from 1.5 in control cells to c. 0.67 in those treated with the analogues. A significant increase in the content of phosphatidylserine was also observed in treated cells. Intact epimastigotes efficiently incorporated radioactivity from L-[methyl-(14)C]methionine into PC, but not from [methyl-(14)C]choline. ET-18-OCH(3) inhibited the incorporation of L-[methyl-(14)C]methionine into PC with an IC(50) of 2 microM, suggesting that inhibition of the de novo synthesis through the Greenberg's pathway was a primary effect underlying the selective anti-parasitic activity of this compound. Antiproliferative synergism was observed as a consequence of combined treatment of epimastigotes with ET-18-OCH(3) and ketoconazole, a sterol biosynthesis inhibitor, probably due to the fact that a secondary effect of the latter is also a blockade of PC synthesis at the level of PE-PC-N-methyl-transferase.

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Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH(3), hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED(50)/24 h values were 13.4+/-2.8 microM and 11. 7+/-0.6 microM for amastigotes and epimastigotes, respectively. ET-18-OCH(3) (0.3 and 0.6 microM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40-57%. This drug (3.75-15 microM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED(50) values of 14.3+/-4.2, 8.9+/-1.9 and 6. 8+/-0.4 microM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs.

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The trypanocidal activity of several 3-(4'-bromo-[1,1-biphenyl]-4-yl) -3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine derivatives of the three evolutionary stages of Trypanosoma cruzi, namely the bloodstream trypomastigote form and both the proliferative epimastigote and amastigote forms, were studied. For both proliferative forms of T. cruzi, total lysis occurred at 10-60 microM for trypomastigotes at 40-200 muM. The following order of susceptibility was established: amastigotes > epimastigotes > trypomastigotes. The most were the bromo (X = g) and unsubstituted (X = b) compounds, which had 13- and 8-fold higher activity against trypomastigotes, respectively, than nifurtimox. Cytotoxicity in the Chinese hamster V-79 cell line, measured as inhibition of cell proliferation showed that all the compounds had the same range of IC50 (7.0-12.4 muM). The halogen (X = a,g,h) and the unsubstituted derivatives (X = b) were the least toxic in the series together with the compound (X = f).

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