Biocompatibility analysis and chemical characterization of Mn-doped hydroxyapatite.

The present work studies the effect of Mn doping on the crystalline structure of the Hap synthesized by the hydrothermal method at 200 °C for 24 h, from Ca(OH)2 and (NH4)2HPO4, incorporating MnCl2 to 0.1, 0.5, 1.0, 1.5 and 2.0 %wt of Mn concentrations. Samples were characterized by the X-Ray Diffraction technique, which revealed the diffraction peaks that corresponded to the hexagonal and monoclinic phase of the Hap; it was observed that the average size of crystallite decreased from 23.67 to 22.69 nm as the concentration of Mn increased. TEM shows that in all samples, there are two distributions of particle sizes; one corresponds to nanorods with several tens of nanometers in length, and the other in which the diameter and length are very close. FTIR analysis revealed absorption bands corresponding to the PO4-3 and OH- groups characteristic of the Hap. It was possible to establish a substitution mechanism between the Mn and the ions of Ca+2 of the Hap. From the Alamar blue test, a cell viability of 86.88% ± 5 corresponding to the sample of Hap at 1.5 %wt Mn was obtained, considered non-cytotoxic according to ISO 10993-5. It also evaluated and demonstrated the good osteoinductive properties of the materials, which were verified by histology and immunofluorescence expression of osteogenic markers. Adhesion, viability, biocompatibility and osteoinductive properties, make these materials candidates for future applications in bone tissue engineering with likely uses in regenerative medicine.

GP35 ANOAL, an abundant acidic glycoprotein of female Anopheles albimanus saliva.

Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.

Flow cytometric analysis of cell-surface and intracellular antigens in the diagnosis of acute leukemia.

To evaluate the usefulness of flow cytometric detection of intracellular antigens (Ags) in establishing proper lineage affiliation and its contribution to the diagnosis of acute leukemia, we studied 100 consecutive patients in whom acute leukemia was diagnosed between January 1997 and July 1998. Immunological classification was assessed using a three-line panel of monoclonal antibodies for phenotypic characterization of leukemic blast cells as proposed at the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. We found 74 cases of B-cell lineage acute lymphoblastic leukemia (ALL), seven cases of T-cell ALL, and 19 cases of acute myeloid leukemia (AML). In this study cytoplasmic (cy) CD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML. Applied in combination with immunophenotyping this knowledge led to improvement in diagnostic precision and refinement of immunological classification, ensuring the selection of the most appropriate therapy for the patients studied. In conclusion, intracellular Ags detection was of utmost importance in establishing correct lineage affiliation in cases lacking expression of B, T, or myeloid surface Ags or disclosing equivocal or ambiguous immunophenotypic features and in identifying biphenotypic acute leukemia. In combination with FAB morphology and immunophenotyping, we were able to reliably classify all patients with acute leukemia in this study.

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites.

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.

Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico.

The geographic distribution of Plasmodium vivax circumsporozoite protein phenotypes from patient blood used to infect colonized Anopheles albimanus and An. pseudopunctipennis was investigated in southern Mexico. Parasite phenotype types were determined in blood samples by a polymerase chain reaction and oligoprobe hybridization or by immunofluorescent assay of sporozoites. The proportion of infected mosquitoes and the number of oocysts per mosquito confirmed previous in vitro observations indicating that An. albimanus is more susceptible to VK210 and that An. pseudopunctipennis is more susceptible to VK247. All patients living on the coast were infected with VK210 and most patients living above 170 meters above sea level had VK247. Both phenotypes infected patients from intermediate altitudes. These results concur with the distribution of the anophelines, indicating that An. albimanus is the main vector of the phenotype VK210, but that An. pseudopunctipennis transmits both phenotypes. These conditions have direct implications on parasite transmission rates and malaria epidemiology in Mexico.

Susceptibility of three laboratory strains of Anopheles albimanus (Diptera: Culicidae) to coindigenous Plasmodium vivax circumsporozoite protein phenotypes in southern Mexico.

The susceptibility to two coindigenous Plasmodium vivax Grassi & Feletti phenotypes VK210 and VK247 of three colonized Anopheles albimanus Wiedemann strains (white-striped, green and brown) from southern Mexico was investigated. Mosquitoes of the three strains were simultaneously fed with P. vivax-infected patient blood and examined 1 wk later for the presence of oocysts. The circumsporozoite protein phenotype type (VK210 and VK247) was determined by immunoflorescence of salivary gland sporozoites using monoclonal antibodies. The proportions of specimens infected and the number of oocyst per mosquito indicated that all mosquito strains were more susceptible to the phenotype VK210 than to VK247, but the white-striped strain was more susceptible to both parasite phenotypes than the other two strains.

Risk factors for Plasmodium vivax infection in the Lacandon forest, southern Mexico.

A study was conducted to characterize the risk of Plasmodium vivax infection in the Lacandon forest, southern Mexico. Blood samples and questionnaire data were collected in 1992. Malaria cases (n = 137) were identified by the presence of symptoms and a positive thick blood smear. The control group included individuals with negative antibody titres and no history of malaria (n = 4994). From 7628 individuals studied, 1006 had anti-P. vivax antibodies. Seroprevalence increased with age. Risk factors associated with infection included: place of birth outside the village of residence (odds ratio, OR 11.67; 95% CI 5.21-26.11); no use of medical services (OR 4.69, 95% CI 3.01-7.29), never using bed-nets (OR 3.98, 95 % CI 1.23-12.86) and poor knowledge of malaria transmission, prevention and treatment (OR 2.30, 95 % CI 1.30-4.07). Health education represents the best recommendation for controlling the disease in the area.

Differential susceptibilities of Anopheles albimanus and Anopheles pseudopunctipennis to infections with coindigenous Plasmodium vivax variants VK210 and VK247 in southern Mexico.

The susceptibilities to coindigenous Plasmodium vivax of colonized Anopheles albimanus and Anopheles pseudopunctipennis from southern Mexico were investigated by simultaneous feeding with infected blood obtained from patients. The genes encoding circumsporozoite protein variant types (VK210 and VK247) in blood samples were determined by PCR and oligonucleotide probe hybridization. A. albimanus was more susceptible to VK210, and A. pseudopunctipennis was more susceptible to VK247.

Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.

Effect of a cecropin-like synthetic peptide (Shiva-3) on the sporogonic development of Plasmodium berghei.

The effect of a synthetic cecropin-like peptide, Shiva-3, on in vitro ookinete development and on the early sporogonic stages of Plasmodium berghei in the midgut of Anopheles albimanus was investigated. Peptide concentrations of 75 and 100 microM were effective (P < 0.05) in reducing ookinete production and the number of infected mosquitoes in almost all experiments. These peptide concentrations in the midgut were not toxic for the survival of the mosquitoes. Complete inhibition was obtained if 100 microM Shiva-3 was applied in the first 8 hr of parasite development. The deleterious effect of the peptide on the parasite was effective after exposure for as short as 50 sec and the permanence of free peptide in the mosquito midgut was estimated to be of a minimum of 5 min. These observations indicate the possibility of using Shiva-like peptide genes to engineering malaria-resistant vectors as an alternative in malaria control strategies.

Anopheles albimanus (Diptera: Culicidae) host selection patterns in three ecological areas of the coastal plains of Chiapas, southern Mexico.

The host-feeding patterns of Anopheles albimanus Wiedemann were described and the effect of host availability on these patterns was assessed in three different ecological areas of coastal Chiapas, Mexico. Resting mosquitoes were collected indoors and outdoors during rainy seasons. A 20% sample of blood-fed mosquitoes was tested to determine the source of the blood meal using an ELISA. The unweighted human blood index (HBI) of An. albimanus in the three areas ranged from 0.11 to 0.21. This mosquito species fed more frequently on bovines than on any other host, but the forage ratio indicated that there was also a high preference for equines. Some females tended to rest or complete their gonotrophic cycle indoors after feeding on animals, but females also fed on man and rested outdoors. Host availability and ecological conditions appeared to be responsible for differences observed in the HBI among areas.

Some studies on acquired immunity to plasmodium chabaudi and plasmodium yoelii in mice

En este trabajo se estudió la duración de la inmunidad adquirida por ratones que curaron espontáneamente después de infectarlos con P. chabaudi y con la cepa no letal (17 X NL) de P. yoelii. Para ésto, se inocularon ratones con P. chabaudi y grupos de ellos fueron retados a diferentes tiempos después de la inoculación inical; unos con P. chabaudi y otros con la cepa letal (17 X L) de P. yoelii. En otros experimentos se inocularon ratones con la cepa 17 X NL de P. yoelii los cuales fueron retados con la cepa 17 X L de P. yoelii. En todas las combinaciones que fueron probadas se encontró un buen nivel de inmunidad aun cuando los ratones se retaron alrededor de 170 días despsués de la primera inoculación. Sin embargo, también se observó en todas las combinaciones que después de 50 o 60 días de la primera inoculación, algunos ratones tienden a perder inmunidad sólida. Se intentó, sin éxito, correlacionar esta pérdida de inmunidad con los niveles de anticuerpos anti-plasmodio. En el sistema de P. chabaudi, no se detectaron parásitos persistentes 180 días después de la inoculación en ninguno de los siguentes órganos: sangre, médula ósea, hígado, bazo o riñón