In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets.

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.

Impaired frequencies and function of platelets and tissue remodeling in chronic Chagas disease.

Chronic inflammation, as a consequence of the persistent infection with Trypanosoma cruzi, leads to continuous activation of the immune system in patients with chronic Chagas disease. We have previously shown that increased sera levels of soluble P-selectin are associated with the severity of the cardiomyopathy distinctive of chronic Chagas disease. In this study, we explored the expression of biomarkers of platelet and endothelial activation, tissue remodeling, and mediators of the coagulation cascade in patients at different clinical stages of chronic Chagas heart disease. The frequencies of activated platelets, measured by the expression of CD41a and CD62P were decreased in patients with chronic Chagas disease compared with those in uninfected subjects, with an inverse association with disease severity. Platelet activation in response to adenosine diphosphate was also decreased in T. cruzi-infected subjects. A major proportion of T. cruzi infected subjects showed increased serum levels of fibrinogen. Patients with severe cardiac dysfunction showed increased levels of endothelin-1 and normal values of procollagen I. In conclusion, chronic infection with T. cruzi induced hemostatic alterations, even in those patients who do not yet present cardiac symptoms.

Inactivation of Babesia microti in red blood cells and platelet concentrates.

BACKGROUND: With an increasing number of recognized transfusion-transmitted (TT) babesiosis cases, Babesia microti is the most frequently TT parasite in the United States. We evaluated the inactivation of B. microti in red blood cells (RBCs) prepared in Optisol (AS-5) using amustaline and glutathione (GSH) and in platelet components (PCs) in 100% plasma using amotosalen and low-energy ultraviolet A (UVA) light. STUDY DESIGN AND METHODS: Individual RBCs and apheresis PCs were spiked with B. microti-infected hamster RBCs (iRBCs) to a final concentration of 106 iRBCs/mL and treated with the respective inactivation systems according to the manufacturer's instruction. Samples were collected before (control) and after (test) each treatment. Dilutions of the control samples to 10-6 were inoculated into hamsters, while the test samples were inoculated neat or at 10-1 dilution. At 3 and 5 weeks postinoculation, hamsters were evaluated for B. microti infection by microscopic observation of blood smears and 50% infectivity titers (ID50 ) were determined. Log reduction was calculated as control log ID50 minus test log ID50 . RESULTS: Parasitemia was detected in hamsters injected with as low as 100,000-fold diluted control samples, while no parasites were detectable in the blood smears of any hamsters receiving neat test samples. Mean log reduction was more than 5 log/mL by amustaline/GSH for RBCs and more than 4.5 log/mL by amotosalen/UVA for PCs. CONCLUSION: B. microti was inactivated to the limit of detection in RBCs and PCs after the respective inactivation treatment. Complete inactivation of B. microti was achieved in this animal infectivity model, and pathogen reduction treatment inhibited transmission of infection.

Platelets activate a pathogenic response to blood-stage Plasmodium infection but not a protective immune response.

Clinical studies indicate that thrombocytopenia correlates with the development of severe falciparum malaria, suggesting that platelets either contribute to control of parasite replication, possibly as innate parasite killer cells or function in eliciting pathogenesis. Removal of platelets by anti-CD41 mAb treatment, platelet inhibition by aspirin, and adoptive transfer of wild-type (WT) platelets to CD40-KO mice, which do not control parasite replication, resulted in similar parasitemia compared with control mice. Human platelets at a physiologic ratio of 1 platelet to 9 red blood cells (RBCs) did not inhibit the in vitro development or replication of blood-stage Plasmodium falciparum The percentage of Plasmodium-infected (iRBCs) with bound platelets during the ascending parasitemia in Plasmodium chabaudi- and Plasmodium berghei-infected mice and the 48-hour in vitro cycle of P falciparum was <10%. P chabaudi and P berghei iRBCs with apoptotic parasites (TdT+) exhibited minimal platelet binding (<5%), which was similar to nonapoptotic iRBCs. These findings collectively indicate platelets do not kill bloodstage Plasmodium at physiologically relevant effector-to-target ratios. P chabaudi primary and secondary parasitemia was similar in mice depleted of platelets by mAb-injection just before infection, indicating that activation of the protective immune response does not require platelets. In contrast to the lack of an effect on parasite replication, adoptive transfer of WT platelets to CD40-KO mice, which are resistant to experimental cerebral malaria, partially restored experimental cerebral malaria mortality and symptoms in CD40-KO recipients, indicating platelets elicit pathogenesis and platelet CD40 is a key molecule.

Replication of Toxoplasma gondii in chicken erythrocytes and thrombocytes compared to macrophages.

Toxoplasma (T.) gondii is able to infect various cell types in different hosts. The replication of this parasite within different peripheral mononuclear blood cell populations in chicken has not yet been fully understood. Aim of the present study was to investigate the impact of chicken erythrocytes and thrombocytes as potential host cells for T. gondii. Cultures of primary avian erythrocytes and thrombocytes were inoculated with tachyzoites of T. gondii type II strain ME49. Parasite replication was detected by a quantitative real-time PCR at different times postinoculation until 24 or 48 h, respectively, displaying long-term investigations for the chosen cultures. The parasite replication curve showed a continuous decrease of parasite stages in erythrocytes and thrombocytes. Observations by light microscopy showed massive destruction for both cell populations. Few macrophages in between the infected thrombocytes were viable during the investigation period and showed internalised tachyzoites by confocal laser scanning microscopy. These findings show that T. gondii is not capable of replication in chicken erythrocytes and thrombocytes; therefore, both cannot be considered as potential host cells. In further consequence, monocyte-derived macrophages seem to be the key to the dissemination mechanisms for T. gondii in chicken.

Proteome changes in platelets after pathogen inactivation--an interlaboratory consensus.

Pathogen inactivation (PI) of platelet concentrates (PCs) reduces the proliferation/replication of a large range of bacteria, viruses, and parasites as well as residual leucocytes. Pathogen-inactivated PCs were evaluated in various clinical trials showing their efficacy and safety. Today, there is some debate over the hemostatic activity of treated PCs as the overall survival of PI platelets seems to be somewhat reduced, and in vitro measurements have identified some alterations in platelet function. Although the specific lesions resulting from PI of PCs are still not fully understood, proteomic studies have revealed potential damages at the protein level. This review merges the key findings of the proteomic analyses of PCs treated by the Mirasol Pathogen Reduction Technology, the Intercept Blood System, and the Theraflex UV-C system, respectively, and discusses the potential impact on the biological functions of platelets. The complementarities of the applied proteomic approaches allow the coverage of a wide range of proteins and provide a comprehensive overview of PI-mediated protein damage. It emerges that there is a relatively weak impact of PI on the overall proteome of platelets. However, some data show that the different PI treatments lead to an acceleration of platelet storage lesions, which is in agreement with the current model of platelet storage lesion in pathogen-inactivated PCs. Overall, the impact of the PI treatment on the proteome appears to be different among the PI systems. Mirasol impacts adhesion and platelet shape change, whereas Intercept seems to impact proteins of intracellular platelet activation pathways. Theraflex influences platelet shape change and aggregation, but the data reported to date are limited. This information provides the basis to understand the impact of different PI on the molecular mechanisms of platelet function. Moreover, these data may serve as basis for future developments of PI technologies for PCs. Further studies should address the impact of both the PI and the storage duration on platelets in PCs because PI may enable the extension of the shelf life of PCs by reducing the bacterial contamination risk.

Papel do fator de ativação de plaquetas na infecção de macrófagos por leishmania infantum e identificação de uma Paf-Acetilhidrolase no parasita / Role of platelet-activating factor in macrophage infection by Leishmania infantum and identification of the parasite PAF acetylhydrolase

A leishmaniose visceral é causada pelo parasita Leishmania infantum. A infecção ocorre quando flebótomos infectados se alimentam na derme do hospedeiro vertebrado, inoculando o parasita. A infecção produz uma resposta com diversas moléculas inflamatórias, como os mediadores lipídicos. O fator de ativação de plaquetas (PAF) é um potente mediador lipídico derivado de um lisofosfolipídio. PAF participa da fisiologia normal da célula e possui um perfil pró-inflamatório. A participação de mediadores lipídicos, como eicosanóides e PAF, já foi identificada na imunopatogênese das leishmanioses. PAF gerado pelo hospedeiro tem efeito leishmanicida e de controle da infecção por L. amazonensis. PAF-acetilhidrolases (PAF-AH) são fosfolipases A2 que hidrolisam PAF e foi demonstrado que PAF-AH podem ser um fator de virulência devido a essa habilidade. O objetivo desse estudo foi avaliar o papel do PAF e de uma PAF-AH na infecção de macrófagos por L. infantum. Foi observado que PAF 1μM, quando adicionado durante e após a infecção, foi capaz de diminuir 50% da infecção após 72 horas, bem como a viabilidade dos parasitas dentro dos macrófagos num mecanismos independente do seu receptor PAFR e da produção de óxido nítrico. PAF 10μM interrompeu o crescimento de promastigotas de L. infantum em cultura axênica. Uma PAFAH, com elevada identidade e semelhança com PLA2/PAF-AH de outros tripanossomatídeos, foi identificada no genoma de L. infantum. A clonagem e expressão recombinante produziu uma proteína de cerca de 69kDa, com atividade PAF-AH. Frações celulares do parasita, enriquecidas com estruturas de membrana também apresentaram atividade PAF-AH. Os resultados indicam que PAF é capaz de diminuir a infecção de macrófagos por L. infantum e que o parasita possui uma PAF-AH funcional possivelmente envolvida com sua virulência.

Visceral leishmaniasis is caused by Leishmania infantum parasites. Infection occurs when infected sandflies feed on vertebrate host skin delivering the parasite which survive, multiply and spread on the parasitophorous vacuoles of macrophages. The inflammatory response during the infection leads to the production of diverse bioactive molecules, as lipid mediators. The platelet activating factor (PAF) is a lipid mediator derived from a lysophospholipid. PAF has a role in normal cellular physiology, acting as proinflamatory molecule. The participation of some lipid mediators, as eicosanoids and PAF has been identified in leishmaniasis. PAF produced by the host is able to kill the parasite and control the infection by L. amazonensis. PAF-acetylhydrolases (PAF-AH) are phospholipases A2 (PLA2) that hydrolyse PAF, and possibly involved in pathogen virulence. The aim of this study was to evaluate the role of PAF on macrophages infection by L. infantum and identify a PAF-AH expressed by the parasite. PAF 1μM, added during and after the infection, was able to reduce approximately 50% of infection, as well as, the viability of parasites inside macrophages. Apparently this reduction occurs by an classical PAF receptor and nitric oxide production independent mechanism. PAF 10μM inhibited L. infantum promastigotes growing in axenic culture. A PAF-AH with high identity to PLA2/PAF-AH of others trypanosomatids was identified in L. infantum genome. The cloning and recombinant expression produced a 69kDa protein with PAF-AH activity. Cellular fractions from parasites, with membrane structures also presented PAF-AH activity. The results suggest that PAF is able to decrease machophage infection by L. infantum witch has a functional PAFAH possibly related to its virulence.

Evaluation of the Mirasol pathogen [corrected] reduction technology system against Babesia microti in apheresis platelets and plasma.

BACKGROUND: Babesia microti is an intraerythrocytic parasite, transmitted naturally to humans by infected ixodid ticks, that causes babesiosis. In recent years, B. microti has been identified as a growing public health concern that has also emerged as a critical blood safety issue in the absence of appropriate interventions to reduce transmission by blood transfusion. Thus, we evaluated the ability of the Mirasol pathogen reduction technology (PRT; CaridianBCT), which uses riboflavin (RB) and ultraviolet (UV) light, to diminish the presence of B. microti in apheresis plasma and platelets (PLTs). STUDY DESIGN AND METHODS: Apheresis plasma and PLT units were spiked with B. microti-infected hamster blood and subsequently treated using the Mirasol PRT system. Control and experimental samples were collected at different stages during the treatment process and injected into hamsters to detect the presence of viable parasites. Four weeks postinoculation, hamster blood was tested for B. microti infection by blood smear and real-time polymerase chain reaction analysis. RESULTS: None of the blood smears from animals injected with samples from PRT-treated plasma or PLT units were positive by microscopy, while all the non-PRT-treated plasma and PLT units were demonstrably parasitemic. Parasite load reduction in hamsters ranged between 4 and 5 log in all PRT-treated units compared to untreated controls. CONCLUSION: The data indicate that the use of RB and UV light efficiently reduces the presence of viable B. microti in apheresis plasma and PLT products, thereby reducing the risk of transfusion-transmitted Babesia potentially associated with these products. Based on this observed "proof of principle," future studies will determine the efficacy of the Mirasol PRT in whole blood.

Photochemical inactivation with amotosalen and long-wavelength ultraviolet light of Plasmodium and Babesia in platelet and plasma components.

BACKGROUND: Transfusion-transmitted cases of malaria and babesiosis have been well documented. Current efforts to screen out contaminated blood products result in component wastage due to the lack of specific detection methods while donor deferral does not always guarantee safe blood products. This study evaluated the efficacy of a photochemical treatment (PCT) method with amotosalen and long-wavelength ultraviolet light (UVA) to inactivate these agents in red blood cells (RBCs) contaminating platelet (PLT) and plasma components. STUDY DESIGN AND METHODS: Plasmodium falciparum- and Babesia microti-contaminated RBCs seeded into PLT and plasma components were treated with 150 micromol per L amotosalen and 3 J per cm2 UVA. The viability of both pathogens before and after treatment was measured with infectivity assays. Treatment with 150 micromol per L amotosalen and 1 J per cm2 UVA was used to assess the robustness of the PCT system. RESULTS: No viable B. microti was detected in PLTs or plasma after treatment with 150 mol per L amotosalen and 3 J per cm2 UVA, demonstrating a mean inactivation of greater than 5.3 log in PLTs and greater than 5.3 log in plasma. After the same treatment, viable P. falciparum was either absent or below the limit of quantification in three of four replicate experiments both in PLTs and in plasma demonstrating a mean inactivation of at least 6.0 log in PLTs and at least 6.9 log in plasma. Reducing UVA dose to 1 J per cm2 did not significantly affect the level of inactivation. CONCLUSION: P. falciparum and B. microti were highly sensitive to inactivation by PCT. Pathogen inactivation approaches could reduce the risk of transfusion-transmitted parasitic infections and avoid unnecessary donor exclusions.

Pathogen inactivation of Trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND: Emigration of people infected with Trypanosoma cruzi to non-endemic areas has resulted in transfusion transmission to immunocompromised recipients. We studied the feasibility of inactivating T. cruzi using a new technology which utilizes riboflavin as a photosensitizer in combination with UV light, Mirasol PRT. METHODS: One billion T. cruzi organisms and 30 mL of 500 microM riboflavin were added to each of six units of human plasma and six units of platelets. To determine the level of detection of organism, a sample of each unit was cultured in tenfold serial dilutions beginning with 100 billion/250 mL as the starting culture. After 30 min, each unit was illuminated with 5.9 J/cm(2) of UV light (6.24 J/mL). The units were then cultured again in tenfold serial dilutions post-treatment. RESULTS: A 6 log reduction of pathogens was demonstrated in 5 of 6 units of plasma, and a 7 log reduction of pathogens was demonstrated in one unit. A 6 log reduction of pathogens was demonstrated in 3 of 6 units of platelets, a 7 log reduction was demonstrated in 2 of 6 units of platelets, and an 8 log reduction of pathogens was demonstrated in 1 of 6 units. CONCLUSIONS: Mirasol PRT treatment demonstrated an ability to inactivate 5-7 logs of T. cruzi in plasma and platelets.

Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND AND OBJECTIVES: Leishmania is transmitted by the bite of the phlebotomine sandfly or by transfusion of infected blood products. Leishmaniasis currently poses a significant problem in several parts of the world, and is an emerging problem in others. The Mirasol PRT technology is based on the use of riboflavin and ultraviolet light to generate chemical reactions in the nucleic acids of pathogens, which prevents replication and leads to inactivation. The intent of this study was to examine the ability of the Mirasol PRT System to kill the Leishmania parasite in human plasma and platelet concentrates. MATERIALS AND METHODS: In visceral Leishmaniasis, amastigotes are present in the blood and in the reticuloendothelial system within monocytes. For each unit of plasma or platelets treated, isolated mononuclear cells obtained from 100 ml of normal donor whole blood were incubated with 1.0 x 10(8) Leishmania donovani infantum promastigotes to produce amastigote-laden macrophages. The infected macrophages were added to 250 ml of human plasma or to 250 ml of platelet concentrates. Infected units were cultured pretreatment in 10-fold serial dilutions to determine the limits of detection. Thirty millilitres of 500 microM riboflavin was added to each unit, which was then illuminated with 5.9 J/cm2 of ultraviolet light (6.24 J/ml). After treatment and after 2 months of frozen storage, plasma units were cultured in 10-fold serial dilutions. Platelets were cultured on the day of treatment and on day 5 of storage post-illumination. RESULTS: A 5 log reduction of Leishmania was demonstrated in five of six units of plasma, and a 7 log reduction of Leishmania was demonstrated in one plasma unit. A 5 log reduction of Leishmania was demonstrated in five of six units of platelets, and a 6 log reduction of Leishmania was demonstrated in one unit. CONCLUSIONS: There is no donor screen for Leishmania and other pathogens constantly emerging in our blood supply. The Mirasol PRT System for Platelets and Plasma is an effective means of killing Leishmania and other emerging pathogens in these blood products.

Leishmania inactivation in human pheresis platelets by a psoralen (amotosalen HCl) and long-wavelength ultraviolet irradiation.

BACKGROUND: Leishmania spp. are protozoans that cause skin and visceral diseases. Leishmania are obligate intracellular parasites of mononuclear phagocytes and have been documented to be transmitted by blood transfusion. STUDY DESIGN AND METHODS: This study examines whether Leishmania can be inactivated in human platelet (PLT) concentrates by a photochemical treatment process that is applicable to blood bank use. Human PLT concentrates were contaminated with Leishmania mexicana metacyclic promastigotes or mouse-derived Leishmania major amastigotes and were exposed to long-wavelength ultraviolet (UV) A light (320-400 nm) plus the psoralen amotosalen HCl. RESULTS: Neither treatment with amotosalen nor UVA alone had an effect on Leishmania viability; however, treatment with 150 micromol per L amotosalen plus 3 J per cm(2) UVA inactivated both metacyclic promastigotes and amastigotes to undetectable levels, more than a 10,000-fold reduction in viability. CONCLUSIONS: This study demonstrates the effectiveness of photochemical treatment to inactivate Leishmania in PLT concentrates intended for transfusion. Both metacylic promastigotes, which represent the infectious form from the sand fly vector, and amastigotes, which represent the form that grows in mononuclear phagocytes, were extremely susceptible to photochemical inactivation by this process. Thus, the photochemical treatment of PLT concentrates inactivates both forms of Leishmania that would be expected to circulate in blood products collected from infected donors.

Parasitismo de leucocitos y trombocitos de gallus gallus l. por plasmodium (novyella) juxtanucleare (apicomplexa: plasmodiidae) / Parasitism of leukocytes and thrombocytes of gallus gallus L. by plasmodium (novyella) juxtanucleare (apicomplexa: plasmodiidae)

Se realizó un investigación del parasitismo de plasmodium juxtanucleare en gallinas sin razas definida, provenientes de criaderos rústicos en el municipio de Seropédica, estado de Río de Janeiro, de Brasil. Se realizaron frotis sanguíneo periféricos, los cuales fuero coloreados con giemsa diluido en tampón sorensen pH6,8. En el examen hemoscópico se puede observar en aves con alto índice de (< 10 por ciento ) formas parasitarias de trofozoítas y esquizontes en el citoplasma de células de la línea leucocitica y trofozoítas en células de la línea trombocítica. Las observaciones en el presente estudio hacen inferir que la cepa de P. juxtanuclear que ocurre en Seropédica realiza esquizogonia fanerozóica. Este trabajo constituye el primer hallazgo de formas de P. juxtanucleares en leucocitos

Inhibiciòn de la agregaciòn plaquetaria por extractos de plantas de uso medicinal en Paraguay / Annual reports 1995 / Inhibition of platelet aggregation by medicinal plants usein Paraguay

Con el propòsito de identificar extractos y luego aislar productos on actividad inhibidoria sobre la agregaciòn plaquetaria in vitro fueron probados 21 extractos alcohòlicos de plantas antitrombòlicos y en transtornos circulatorios e inmunològicos. La inhibiciòn de la agregaciòn se determinò mediante el mètodo turbo-densitomètrico (coagulòmetro AccuStasisTM 1000) midiendo el tiempo de agregaciòn del plasma rico en plaquetas inducido por adenosina 5 difosfato (ADP), colàgeno y epinefrina a las concentraciones iniciales de 2x10-4mo1/L, 2 mg/ml y 1x10-4mo1/L respectivamente y como control positivo de la inhibiciòn de la agregaciòn plaquetaria se usò aspirina a la concentraciòn de 0,1g/ml. La evaluaciòn cualitativa se realizò por apreciaciòn microscòpica (A 100x) de frotis teñidos con Giemsa del agregado plaquetario o nò por los extractos y los controles. Del screening resultò activo el extracto de una planta medicinal perteneciente a la familia Myrtaceae a la concentraciòn de 272ug/ml con los porcentajes de inhibiciòn de 96 por ciento a la concentraciòn de 0,18x10-4mo1/L de ADP y 94,4 por ciento a la concentraciòn de 0,045x10-4mo1/L de ADP. Tambièn resultò activo el extracto de una planta medicinal perteneciente a la familia Flacourtiaceae con un porcentaje de inhibiciòn de 92 por ciento al ser inducido por ADP a la concentraciòn de 0,045x10-4mo1/L. Este estudio indica el potencial de algunos extractos estudiados y justifica el aislamiento de compuestos puros de los extractos activos con mejores propiedades.

Inhibiciòn de la agregaciòn plaquetaria por extractos de plantas de uso medicinal en Paraguay / Annual reports 1995 / Inhibition of platelet aggregation by medicinal plants usein Paraguay

Con el propòsito de identificar extractos y luego aislar productos on actividad inhibidoria sobre la agregaciòn plaquetaria in vitro fueron probados 21 extractos alcohòlicos de plantas antitrombòlicos y en transtornos circulatorios e inmunològicos. La inhibiciòn de la agregaciòn se determinò mediante el mètodo turbo-densitomètrico (coagulòmetro AccuStasisTM 1000) midiendo el tiempo de agregaciòn del plasma rico en plaquetas inducido por adenosina 5 difosfato (ADP), colàgeno y epinefrina a las concentraciones iniciales de 2x10-4mo1/L, 2 mg/ml y 1x10-4mo1/L respectivamente y como control positivo de la inhibiciòn de la agregaciòn plaquetaria se usò aspirina a la concentraciòn de 0,1g/ml. La evaluaciòn cualitativa se realizò por apreciaciòn microscòpica (A 100x) de frotis teñidos con Giemsa del agregado plaquetario o nò por los extractos y los controles. Del screening resultò activo el extracto de una planta medicinal perteneciente a la familia Myrtaceae a la concentraciòn de 272ug/ml con los porcentajes de inhibiciòn de 96 por ciento a la concentraciòn de 0,18x10-4mo1/L de ADP y 94,4 por ciento a la concentraciòn de 0,045x10-4mo1/L de ADP. Tambièn resultò activo el extracto de una planta medicinal perteneciente a la familia Flacourtiaceae con un porcentaje de inhibiciòn de 92 por ciento al ser inducido por ADP a la concentraciòn de 0,045x10-4mo1/L. Este estudio indica el potencial de algunos extractos estudiados y justifica el aislamiento de compuestos puros de los extractos activos con mejores propiedades

Inhibition by platelets of in vitro blastogenic responses of canine PBMC by a PAF-dependent mechanism.

It was demonstrated that platelets can inhibit in vitro blastogenic responses by a platelet activating factor (PAF)-dependent mechanism. A procedure for the isolation of virtually platelet-free canine peripheral blood mononuclear cells (PBMC), using Ficoll density gradient centrifugation followed by Percoll density centrifugation, was developed to investigate the mechanism by which platelets inhibit the in vitro blastogenic response of PBMC. It was shown that PBMC purified on Ficoll gradients alone are contaminated with platelets and proliferate weakly compared with platelet-free PBMC purified on an additional Percoll gradient. Addition of platelets to PBMC cultures in the presence of PAF receptor antagonist resulted in a proliferative response similar in intensity to that of platelet-free PBMC cultures, whereas the addition of platelets to PBMC cultures in the absence of PAF receptor antagonist resulted in marked inhibition of the mitogen-induced proliferative response. Therefore, PAF is likely to be involved in the inhibition of in vitro proliferative responses of platelet-contaminated canine PBMC.

Inactivation of Trypanosoma cruzi trypomastigote forms in blood components with a Psoralen and Ultraviolet A light.

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 micrograms/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi, the trypomastigote, was achieved at a UVA (320-400 nm radiation) fluence of 4.2 J/cm2. We note that while the infectivity of the parasite is eliminated at 4.2 J/cm2 the trypomastigote motility continues for at least 16 h-post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of 3H-AMT indicated that at the lethal UVA influence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi, the causative agent of Chagas disease.

Treatment of T. cruzi infected human platelet concentrates with aminomethyltrimethyl psoralen (AMT) and ultraviolet A (UV-A) light: preliminary results

The present measures adopted to prevent transfusion-associated Chagas' disease include screening of blood donors, and/or the inactivation of T. cruzi in collected blood using gentian violet (GV), as a trypanocidal agent. In this study, we investigated the efficacy of the combined use of AMT and UV-A in inactivating T. cruzi in infected human platelet concentrates. Human platelet concentrates were infected with T. cruzi (2 x 10(8)/ml) of the Y strain, transfered to PL 269 (Fenwal Laboratories) containers, and treated with GV (250 micrograms/ml), and ascorbic acid (1 mg/ml); GV, ascorbic acid and UV-A; GV and UV-A; AMT (40 microG/ml) and ascorbic acid; AMT, ascorbic acid and UV-A; AMT and UV-A; UV-A alone; and untreated (control). All UV-A treated platelet concentrates were exposed to UV-A doses of 24, 92, 184, 276, 368 and 644 kJ/m2, and the microscopical research of active T. cruzi was performed, using the microhematocrit technique, 1, 6 and 24 hours after each treatment. A high number of active forms of T. cruzi was observed in all condictions, except when GV was used as the trypanocidal agent, providing evidence of the failure of AMT and UV-A in inactivating T. cruzi in infected human platelet concentrates.

Treatment of T. cruzi infected human platelet concentrates with aminomethyltrimethyl psoralen (AMT) and ultraviolet A (UV-A) light: preliminary results.

The present measures adopted to prevent transfusion-associated Chagas' disease include screening of blood donors, and/or the inactivation of T. cruzi in collected blood using gentian violet (GV), as a trypanocidal agent. In this study, we investigated the efficacy of the combined use of AMT and UV-A in inactivating T. cruzi in infected human platelet concentrates. Human platelet concentrates were infected with T. cruzi (2 x 10(8)/ml) of the Y strain, transfered to PL 269 (Fenwal Laboratories) containers, and treated with GV (250 micrograms/ml), and ascorbic acid (1 mg/ml); GV, ascorbic acid and UV-A; GV and UV-A; AMT (40 microG/ml) and ascorbic acid; AMT, ascorbic acid and UV-A; AMT and UV-A; UV-A alone; and untreated (control). All UV-A treated platelet concentrates were exposed to UV-A doses of 24, 92, 184, 276, 368 and 644 kJ/m2, and the microscopical research of active T. cruzi was performed, using the microhematocrit technique, 1, 6 and 24 hours after each treatment. A high number of active forms of T. cruzi was observed in all condictions, except when GV was used as the trypanocidal agent, providing evidence of the failure of AMT and UV-A in inactivating T. cruzi in infected human platelet concentrates.