Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae).

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid-filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron-lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs.

Detección del virus de la tristeza de los cítricos por serología, microscopía e hibridación in situ / In situ serological, microscopy and hybridization detection of CTV

El virus de la tristeza de los cítricos (CTV) es perjudicial para la citricultura y causa la enfermedad llamada tristeza de los cítricos. Infecta las especies del género Citrus ocasionando la muerte de millones de árboles. Los síntomas son decaimiento rápido (QD) y acanalamiento de tallo (SP). En el trabajo se diagnosticó molecular y serológicamente al CTV en aislados provenientes de Citrus aurantifolia o Lima Tahití (LT) y Citrus madurensis (Lour) o Calamondino (Ca), y se realizaron estudios preliminares de detección viral por medio de microscopía óptica e hibridación in situ. Se utilizó IC-RT-PCR e inmunoimpresión de tejido (IMI) expuesto a los anticuerpos monoclonales 3DF1+3CA5, y con el anticuerpo discriminante MCA 13 con técnica de Enzyme Linked Inmunossorbent Assay Doble Sándwich (Elisa-DAS). La detección por microscopía se realizó sobre secciones de pecíolo de LT y C que se tiñeron con Azure A, y con acetato de uranilo y citrato de plomo. Para la hibridación in situ se empleó una sonda marcada con digoxigenina dirigida hacia el gen de la proteína mayor de la cápside. Los resultados de IC-RT-PCR, IMI y Elisa fueron positivos para LT y C, indicando la presencia de variantes virales de tipo severo. Con microscopía de luz se detectaron inclusiones citoplasmáticas en las células acompañantes y del floema, confirmado con IMI y por hibridación in situ. Se visualizaron inclusiones de partículas virales en el tejido vegetal con microscopía electrónica con cambios en la ultraestructura celular como presencia de grandes vacuolas propias de la infección viral. Este trabajo integra distintas técnicas diagnósticas sobre dos especies cítricas exóticas.

Citrus tristeza virus (CTV) is deleterious for citriculture and causes citrus tristeza disease. CTV infects all citrus species thereby causing the death of millions of trees. Its main symptoms are quick decline (QD) and stem pitting (SP). Serological, molecular and microscopy techniques were used in this work for diagnosing CTV in Citrus aurantifolia or Tahiti Lime (Citrus latifolia Tanaka) (TL) and Citrus madurensis (Lour) or Calamondin (Ca) isolates. Petioles were tissue printed (IMI) and exposed to 3DF1+3CA5 monoclonal antibodies; they were then ELISA buffer extracted and exposed to a discriminant MCA 13 monoclonal antibody in a double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA). Immunocapture reverse transcriptase-polymerase chain reaction (IC-RT-PCR) amplification, using specific major coat protein gene (CPG) primers, was used on the ELISA buffer extracts as template. Optical and electron microscopy were used for detection on transversal sections of petiole and stained with azure A, uranyl acetate or lead citrate. Digoxygenin-labelled major CPG CTV probes were used for in situ hybridisation of petioles printing. All IC-RT-PCR, IMI and ELISA results were positive for both LT and C, indicating the presence of severe viral variants. Light microscopy cytoplasm inclusions were detected in the phloem and accompanying cells, confirmed by IMI and in situ hybridisation. Electron microscopy analysis revealed cellular abnormalities with changes in ultrastructure and the presence of big vacuoles which are characteristic of cytoplasmic viral infection. This is the first work integrating all available diagnostic techniques on these two exotic citric species.

Invasive cells in the placentome of Andean populations of Mabuya: an endotheliochorial contribution to the placenta?

New world lizards of the genus Mabuya have the most specialized level of placentotrophy among reptiles known to date, and related to that, they have the most complex allantoplacenta characterized by a series of morphological specializations that converge with those known for eutherian mammals. One of these specializations is the placentome that is found in the embryonic pole of the incubation chamber. In the mature allantoplacenta, this structure is morphologically the most complex, which could support an important amount of nutrient exchange between mother and fetus. According to the relationship between the chorioallantois and the syncytial uterine epithelia, the placenta of Mabuya populations shows some interesting similarities to the synepitheliochorial type. Recently, cells of chorionic origin have been found invading the syncytial uterine epithelium, and in very close proximity with uterine blood vessels. In this study, we describe the relationship between these invasive chorionic cells, the uterine syncytium, and the subjacent blood vessels of several populations of this genus, by means of high resolution optical microscopy and transmission electron microscopy. Cell groups originating from the chorion, of variable size and shape, penetrate the uterine syncytial epithelium extending complex cytoplasmic projections that come in contact with uterine capillaries and form an extensive and complex double-membrane system that surrounds the capillary. The close relationship between the chorion and the maternal circulation suggests that the Mabuya placentome shows some characteristics of an endotheliochorial placenta. This finding constitutes so far the only documented example of an endotheliochorial placentation in Reptilia.

HIV type 1 drug resistance among naive patients from Venezuela.

In this study, we characterize proviral DNA of 20 HIV-1 asymptomatic antiretroviral-naive patients from Venezuela in env, gag, and pol genes regions. Results from both env/gag HMA subtyping and phylogenetic analysis of pol partial sequences led to the description of clade B in all cases. Nevertheless, the high prevalence of polymorphisms was particularly evident among the protease sequences. A 10% prevalence of major resistance mutations to RTIs was found. Our data also suggested that the protease polymorphisms I62T and V77T could be considered as molecular markers of the subtype B local epidemic. In addition, we show how proviral DNA can be used as a reliable tool to follow trends of resistance mutation transmission.

Allantoplacental ultrastructure of an Andean population of Mabuya (Squamata, Scincidae).

Mabuya species are highly matrotrophic viviparous lizards with Type IV epitheliochorial allantoplacenta. The allantoplacenta of an Andean population of this genus, currently assigned to Mabuya sp., possesses specializations related to histotrophic nutrition at the embryonic hemisphere (placentome, paraplacentome, and chorionic areolas), while at the abembryonic hemisphere it has a mixed function: histotrophic transfer (absorptive plaques) and hemotrophic nutrition (gas exchange in respiratory segments). These placental specializations were studied using high-resolution light microscopy and transmission electron microscopy, and were compared with those found in other squamate reptiles and eutherian mammals. Cytological features of the placentome suggest that this is an important region for nutritional provision; the paraplacentome also shows characteristics for nutrient transfer, especially lipids. Chorionic areolas allow the absorption of glandular products, as well as uterine and chorionic cellular debris produced by lysis of some cells of both epithelia during areola formation. In the absorptive plaques both uterine and chorionic epithelia are firmly attached and their cellular apices exhibit electron-dense granules that could be related to autocrine and paracrine functions. The short interhemal distance found in the respiratory segments confirms their role in gas exchange. A common feature of all regional specializations in the Mabuya sp. allantoplacenta is the presence of lipids in the interacting chorionic and uterine epithelia, suggesting that lipids are transferred throughout the entire embryonic chamber; placental transfer of lipids may be the principal fetal energy and lipid source in this species. In spite of this feature, each one of the specialized areas of the allantoplacenta has different features suggesting particular functions in the transfer of nutrients (as ions, lipids, proteins, amino acids, sugar, water, and gases), and in the possible synthesis of hormones and proteins. The placental complexity observed in this species of Mabuya is greater than in any other reptile, and resembles that of eutherian mammals: Each one of these specializations of the placental membranes in Mabuya sp. is similar to those found among different eutherian mammals, indicating a very impressive evolutionary convergence at the histological and cytological levels between both clades. However, no eutherian mammal species simultaneously displays all of these specializations in the embryonic chamber as does Mabuya sp.

Morphology of the male reproductive duct system of Caiman crocodilus (Crocodylia, Alligatoridae).

The male reproductive duct system of Caiman crocodilus in different reproductive stages was studied using light and electron microscopy, to determine whether shared morphological features exist between Crocodylia and Aves, in concurrence with the Archosauria hypothesis. The sexual duct system of Caiman crocodilus is constituted of the rete testis, ductuli efferentes, ductuli epididymides, ductus epididymidis, and ductus deferens. The morphology and histochemical properties of these ducts suggest their involvement in seminal fluid production and/or its modification. Three types of non-ciliated cells were identified along the duct system. 1. The noncliated cells of the ductuli efferentes contain electron dense worm-like structures and coated vesicles, both related to absorptive processes, as has been suggested in Aves. 2. The non-ciliated cells of the ductuli epididymides have apical electron dense granules suggesting a secretory role, and 3. The non-ciliated cells of the ductus epididymidis and ductus deferens did not exhibit conspicuous storage of secretory material, but have a prominent rough endoplasmic reticulum content indicating active protein production. The occurrence of abundant secretory granules in the epithelial cells of the distal ductus deferens during non-reproductive stages suggests its participation in the removal of luminal debris when the reproductive season ends. Additional ducts were observed running along the ductus deferens; they shared morphological characteristics with the ductuli epididymides. The maximum diameter and therefore the greatest sperm accumulation of the excurrent ducts were observed during the initial testicular regression. The comparative analysis suggests that the male reproductive system of the Crocodylia exhibits structural characteristics nearer to those of Aves than to Lepidosauria, Testudines, and Mammalia, additional data that support an Aves and Crocodylia sister group relationship.

Morphology of the ovary of Caiman crocodilus (Crocodylia: Alligatoridae).

We describe the ovarian histology and characterize the folliculogenesis of adult females of Caiman crocodilus in order to compare them with other species of Reptilia. The gonad has a thin irregular cortex and a well-developed reticular stroma, composed of vascularized chordae, drained by large lymphatic vessels and separated by extensive lacunae. Simple cuboidal to squamous epithelium, dense connective tissue with numerous elastin fibers, and smooth muscle constitute the ovarian wall. This morphology is similar to that of other Crocodylia, Aves and Testudines. Germinal nests are distributed in the ovarian cortex, some of them with oogonia. Oocytes leave the germinal nests with a single layered cuboidal granulosa, remaining simple during vitellogenesis, as in other Archosauria and Testudines and different from Squamata. As the oocyte grows, the theca is formed by numerous fibroblasts, collagen fibers, and smooth muscle fibers, becoming very thick, highly vascularized and developing muscle bands at advanced vitellogenesis. Folliculogenesis shares morphological features with other species of Reptilia. The corpus luteum is similar to that described for Archosauria; during luteolysis, the theca shrinks and the amount of luteinic cells decreases. The relationship of the corpus luteum to vitellogenesis and egg maintenance is unclear in Crocodylia. Follicular regression in Caiman crocodilus corresponds to an invasive atresia type. Histological features seen in the ovary of Caiman crocodilus are similar to those described in Alligator mississippiensis indicating that these features could be shared among Crocodylia. Also several of these morphological characteristics are observed in Aves suggesting a similar reproductive functional morphology in Archosauria.

Inmunopatología de la infección por el VIH / Immunopathology of the AIDS

Hemos resumido algunos de los mecanismos inmunológicos que ocurren durante la infección por VIH. Sin embargo, el establecimiento y la progresión de la enfermedad inducida por el VIH es el producto de una serie de eventos inmunopatogénicos complejos, donde debemos considerar diversos mecanismos genéticos, inmunológicos y virológicos que actúan en conjunto para determinar el curso de la enfermedad inducida por el VIH

Microsporidos en humanos / Microsporidiosis in humans

Se hace una revisión sobre la taxonomía, la biología y los métodos diagnósticos de los microsporidios encontrados en humanos. A partir de 1981-1982, con el advenimiento de la epidemia de SIDA, han sido muchos los informes sobre casos de microsporidiosis en humanos. Actualmente se han implicado especies correspondientes a los géneros Enterocytozoon, Encephalitozoon, Vittaforma, Pleistophora y Trachipleistophora, en diferentes patologías humanas no sólo en aquellos individuos que tienen algún compromiso del sistema inmune sino tambien en individuos sanos. Los métodos diagnósticos empledos se basan principalmente en coloraciones de cromotropo modificada y otras técnicas como microscopía electrónica de transmisión (TEM), análisis inmunogéneticos y de ácidos nucleicos. La forma de transmisión para los humanos es incierta, pero se han encontrado algunas especies parasitando animales lo que sugiere que puede tratarse de una zoonosis

Disminución de la respuesta proliferativa de los linfocitos frente a antígenos ambientales, como indicador precoz / Precocious indicator of the lympocytary injuries induce by the immunodeficiency virus human

A fin de evaluar las células CD4+/CD29+ y sus respuestas frente a diferentes antígenos en estadíos de la infección por el Virus de Imunodeficiencia Humana (VIH), estudiamos 26 pacientes seropositivos asintomáticos (SP) y 15 pacientes con Síndrome de Inmunodeficiencia Adquirida (SIDA) simultaneamente con 20 individuos controles sanos (CS) y 10 hombres homo y bisexuales seronegativos (SN) y 15 pacientes con Síndrome de Inmunodeficiencia Adquirida (SIDA) simultáneamente con 20 individuos controles sanos (CS) y 10 hombres homo y bisexuales seronegativos (SN). Los fenotipos CD3, CD4, CD29 y CD45RA fueron analizados por citometría de flujo de dos colores. Encontramos depleción significativa de los linfocitos T CD4+ y de las subpoblaciones de células memoria (CD4+/CD29+) y virgen (CD45/CD45RA+) en los pacientes SP y SIDA, en comparación con los grupos CS y SN. Las respuestas proliferativas con dosis óptimas de Candida Albicans, Estreptokinasa y Toxoide Tetánico fueron exploradas en células mononucleares de sangre periférica (CMSP) y en poblaciones enriquecidas de células CD4+ y CD4/CD29+. En pacientes SP, la respuesta ante C. Albicans de CMSP estaba significativamente disminuída con respecto a la de CS (15.308 vs 35.951 cpm). La reducción de la proliferación frente a estreptokinasa fue evidente solamente con preparaciones enriquecidas de linfocitos Cd4+/cd29+ (3.084 vs 10.367 cpm). Una vez analizados los resultados, evidenciamos entre los pacientes SP, 7 individuos respondedores a por lo menos un antígeno y 7 no respondedores. En este último grupo, la ausencia de proliferación era independiente del número absoluto de células memoria y virgen. La respuesta frente a Toxoide Tetánico, aunque disminuida en SP, no era significativamente diferente de la CS. Nuestros resultados sugieren que la respuesta defectuosa frente a antígenos ambientales comunes, no relacionada con el número absoluto de linfocitos CD4+/CD29+, es probablemente un indicador temprano de lesión linfocitaria inducida por el VIH

Tratamiento precoz de pacientes con infección por el virus de inmunodeficiencia humana / Early treatment of patients with infection by the human immunodeficiency virus

Dentro de las estrategias terapéuticas en pacientes con infección por el Virus de Inmunodeficiencia Humana, existen dos básicas: 1. El ataque directo al VIH a fin de prevenir y retardar la progresión de la enfermedad; 2. La prevención y tratamiento temprano de las infecciones oportunistas y neoplasias asociadas. En el siguiente trabajo se aborda el enfoque terapéutico con los antiretrovirales: Zidovudina, Didanosina y Zalcitabine