Redifferentiation of dedifferentiated human chondrocytes in high-density cultures.

High-density cultures are widely used as an in vitro model for studies of embryonic cartilage formation. In the present study we investigated the suitability of high-density cultures for the redifferentiation of dedifferentiated chondrocytes. When primary human chondrocytes were cultured in alginate beads, some cells emigrated into Petri dishes. These cells were cultured for one to eight passages (each passage lasting about 3 days) in monolayer culture. At each passage, monolayer cells were removed and allowed to grow in high-density cultures at the medium-air interface and subsequently investigated with morphological, immunolocalization and biochemical methods for the production of cartilage-specific markers, i.e. collagen type II and cartilage-specific proteoglycans. When such dedifferentiated chondrocytes from monolayer passages P1-P4 were introduced in high-density culture, they regained a chondrocyte phenotype and formed cartilage nodules surrounded by fibroblast-like cells. Cells were interconnected by typical gap junctions and after a few days in culturing produced cartilage-specific extracellular matrix, notably collagen type II and cartilage-specific proteoglycans. In contrast, cells taken from monolayer passages P5-P8 did not produce these chondrocyte-specific extracellular materials when grown in high-density culture. We conclude that the growth of dedifferentiated chondrocytes in high-density culture promotes their redifferentiation and reveals their chondrogenic potential. Such high-density cultures might serve as a model system to initiate and promote the redifferentiation of chondrocytes and to provide sufficient quantities of differentiated chondrocytes for autologous chondrocyte transplantation.

[Response of fetal macrophages in the placenta of pregnant HIV-positive patients with and without antiretroviral treatment]. / Respuesta de los macrófagos fetales en placentas de pacientes embarazadas VIH-1 positivas con tratamiento y sin tratamiento antiretroviral.

We studied the cellular answer of placentary macrophages in pregnant women seropositive to the virus of human immunodefficiency (VIH-1) treated with zidovudina (AZT) and didanosine (ddl). Twenty eight pregnant women were studies; there were four groups of seven patients each: The control group; the group with seropositive women without treatment; the group given AZT, and the group that recieved AZT and ddl. Placentary specimens were obtained immediately after delivery. One hundred and fifty chorionic vellosities of cells. The control group showed an average of 26 Hofbauer cells; the seropositive women without antiretroviral treatment, was 115; the patients who received only AZT, the average was 65; and the ones who received a combine therapy AZT and ddl, cellular average was 44. There were no differences in the weight of the products in all the groups, nor congenital malformations in the newborns. The use of medication antiretroviral suppress viral replication, and so, there is a significant answer in the amount and size of Hofbauer cells. The administration of two medicaments is more effective in the cellular immune answer.

Transmission electron microscopy findings in the respiratory epithelium of guinea pigs exposed to the polluted air of southwest Mexico City.

We evaluated the nature and the extent of the damage to the respiratory epithelium of guinea pigs after a 4-month exposure to the mixture of air pollutants in southwest Mexico City. Guinea pigs were placed outdoors on the roof of our facility, 8 hours daily, from February to May 1995. At the same time, control guinea pigs were kept indoors breathing filtered air. Air pollutants, temperature, and humidity data were obtained from the nearest station of the Environmental Monitoring Net. The airways and lung parenchyma were analyzed after 120 days using transmission electron microscopy (TEM). During the 4-month exposure period, ozone (O3) exceeded the norm during 511 hours, and suspended particles less than 10 microm in diameter (PM10) during 52 hours. Both pollutants reached peak levels of more than twice the norm. TEM revealed no important abnormalities in the control group. In the exposed group, there was loss of cilia, detachment of epithelial cells, and eosinophil and macrophage migration toward alveolar spaces through type I pneumocytes with destruction of their basal membranes. In six guinea pigs in the exposed group, we noted bacteria along the airways, with associated inflammatory response. We explain the colonization of the respiratory epithelium by bacteria as the result of the impairment on the defense mechanism caused by the exposure to environmental O3 and PM10.

[Radiologic, densitometric, morphologic and electron microscopic study of osteoporosis and osteopenia in the third lumbar vertebra in men and women in the city of Mexico]. / Estudio de la osteoporosis y osteopenia por medio de radiología, densitometría, morfometría y microscopia de barrido en la tercera vértebra lumbar en una muestra de hombres y mujeres en la ciudad de México.

OBJECTIVE: To determine bone density in the third lumbar vertebra in a group of men and women from the Mexico City correlating the morphometric characteristics of vertebral body by specific techniques and to detect groups with alterations like osteoporosis or osteopenia. METHODOLOGY: The study was made in third lumbar vertebra from 23 patients dead of the Medical Service Forense-México, 8 women between 31 to 72 years old and 15 men between 25 yo 62 years old. The studies were: Radiological, bone densitometry and by image in which was determined density of vertical and horizontal trabeculaes. With x-ray technique in scanning electron microscopy was determined in form semiquantitative the presence of Ca, P, Mg and Na, and Ca distribution by technique of x-ray energy dispersed. RESULTS: In the radiological trail all vertebras of the female group were abnormal, the major finding was degenerative feature; 5 Showed trabeculation increased 3 fractured; in the males group 7 patients were normal, degenerative fracture in 8; of these six had densitometry with DEXA; in the females group only one was normal, 3 osteopenia, 5 osteoporosis; in the male group: 5 osteopenia and 5 osteoporosis. Scanning electron microscopy examination exhibit a homogeneous and dense expression pattern, wherever SEM demonstrated the absence or decrease of deposit calcium in osteoporosis. A morphometric examination of the trabecular thickness demonstrates a difference between the two sexes. The normal median was 222.1 microns, the osteoporosis range was 126.3 to 156.2 microns in the female osteoporosis, in male group was found two normal values with a trabecular density mean of 249.7 microns, in the last two cases one with osteopenia and the other with osteoporosis, the mean value was 186.4 microns. CONCLUSIONS: In the present study, was demonstrated that the degenerative alterations in osteoporosis and osteopenia in young people are more frequent to respect other reports. The following observations require special emphasis either because they provide clues to the mechanism of altered expression of calcium: Genetic, ethnic, consume, sedentary and others.

[Bacterial vaginosis. Relation between the vaginal flora and the vaginal epithelial cells under different treatments. Ultrastructural study]. / Vaginosis bacteriana. Relación de la flora vaginal con las células epiteliales de vagina, con diferente tratamiento. Estudio ultraestructural.

At this study we analyzed morphologic relation with vaginal ephitelium, in women with bacterial vaginosis, in pre-treatment; first post-treatment and second post-treatment using metronidazol (oral), and clindamycine (intra-vaginally). From 20 patients with bacterial vaginosis, 10 received oral metronidazol, 500 mg/b.i.d. during seven days; the other patients received, clindamycine 2% intravaginal, once a day, during seven days. Couples received metronidazol. In pre-treatment, cellular prolongations produced greater adhesion of bacteria, and rests of cellular unions with adhered bacteria. There was penetration of some bacteria to epithelial cells, confirming this with serial cuts and discarding superposition in the cells. This penetration was found in five cases y persisted during the first and second post-treatment. In the first post-treatment, cellular groups without bacteria, were found. The presence of lactobacillus was low, increasing afterwards. The presence of yeasts was in the first post-treatment, and in some cases persisted. It is important to differentiate intracellular bacteria and to know the related characteristics with bacterial penetration, for an adequate prescription and a better use of medication, avoiding possible damage. The presence of bacteria may be one of the causes for bacterial vaginosis reincidence.

[Ultrastructure of the tracheal epithelium in preterm neonates treated with mechanical ventilation]. / Ultraestructura del epitelio traqueal en recién nacidos pretérmino sometidos a ventilación mecánica.

Tracheal epithelial damage, was evaluated in prematures that where intubated and with mechanical ventilation; biopsies were taken in seven preterm neonates; weight was 1100 to 2350; five were by cesarean section, y one via vaginal. Bronchial culture was negative in five, in one Lysteria monocitogena, and in another one Pseudomonas spiralis; biopsy was taken at different times o intubation. Five died because of pneumonia associated to other diseases. In optical and ultrastructural study it was found that with one day of intubation there was cilia loss; with three days of ventilation there was ciliary cells loss, and not ciliary cellular death and there were no cellular unions. In one case with three days of ventilation and 17 days of post-extubation with infection by Pseudomonas, ulcerated zones and edema were found. At 14 days there necrosis zones, increase in collagene. At 17 days there were ulcerated zones to the muscular layer with fibrosis and cellular rests at tracheal path. So, mechanical ventilation and the presence of a catheter, damage bronchial epithelium since the first 24 hours.

[Comparative study of the placenta from HIV+ mothers. Ultrastructural analysis]. / Estudio comparativo de la placenta de madres seropositivas a VIH. Análisis ultraestructural.

Perinatal vertical transmission has increased in all the world; it is considered that at the present time there are about one million of children with HIV. Variation goes from 12 to 40%, at different countries. During the last years antiretroviral drugs as AZT, ddI and others have been used to diminish the virus passage via transplacentary. Eighteen placentaes from HIV seropositive women, three corresponded to first trimester, and 15 to the third trimester of gestation; in four cases they were treated with AZT in weeks fourteen (two patients), 26 and 35 of gestation; and one patient received AZT and ddI at week 28. Control group was with ten normal placentaes. Ultraestructural analysis and immuno-peroxidase and immuno-oro with antibody anti gp 41, were done. Ultraestructurally there were different localizations of HIV virus, at sincitiotrophoblast, decidual cells and umbilical vessels (six cases). In 13 cases there was hyperplasia and hypertrophy of macrophages containing a great amount of lysosomes. In one case, where a girl was seropositive many viriones HIV, were identified in macrophages. With immuno-oro viral proteins were seen in cytoplasm an plasmatic membrane, in endothelium of fetal capillars and trophoblast. With immunoperoxidase, four cases were positive. Placentaes with antiretroviral treatment since week 14, trophoblast was more dense by philaments increment. Placentaes with treatment during the third trimester, showed normal morphology with slight increase of philaments. In the cases treated with AZT and ddI, there were not macrophages hyperplasia and hypertrophy, nor viral particles. It is concluded that in seropositive mothers without treatment, the virus may be present in any part of chorionic villi, and in patients with treatment, virus is not identified, but a viral proteins synthesis.

[Ultrastructural and immunocytochemical analysis of the synaptonemal complex at the initiation of synapsis]. / Análisis ultraestructural e immunocitoquímico del complejo sinaptonémico al inicio de la sinapsis.

The synaptonemal complexes (SCs) are nuclear structures specific for meiosis. They have a central role in homolog chromosomes coupling; they are essential in crossing over events and chromosomic segregation during the first meiotic division. When its joining ends in pakiteno stage, each synaptonemal extends along the bivalent joining the ends to nuclear wrapping. The SCs are characterized by the presence of two lateral elements and a central region. The lateral elements are parallel and equidistant. The chromatine of homolog chromosomes fixes in a series of loops to these elements. The central region is between the lateral elements. It is formed by the latero-medial fibers and the medial element. The first ones are perpendicularly oriented to the longitudinal axis of CS and connect lateral elements with the medial element. The recombination modules have an active role in recombination processes and quiasma formation, they are associated, at intervals, with the central region among the homolog chromosomes. The localization and function of nucleic acids in formation and coupling of synaptonemal complex is little known, so methodologic alternatives are looked for to resolve this type of problems. In this work, ADN distribution in chicken ovocytes in cigotene, using techniques for electronic microscopy of immuno-oro, were studied. Besides, cytochemical techniques, were used as preferential contrast for ADN or preferential for ribonucleoproteins (RNPs). The combination of preferential tincture for RNPs and immunolocalization of ADN show that chromatin accumulates jointly with ribonucleoproteins in nor coupled lateral elements and the presence of numerous RNPs fibers distributed around lateral elements. Recombination nodules were found among lateral elements during the coupling, these nodules are PTA positives, which means ADN presence, and so, ADN presence among lateral elements. THe presence of a bridge of marked fibers with coloidal gold (ADN) uniting not coupled lateral elements, suggests ADN as a sort of macromollecule forming synapsis sites.

[Gardnerella vaginalis infection in heterosexual couples. Ultrastructural study of desquamation cells of the stratified epithelium]. / Infección por Gardnerella vaginalis en parejas heterosexuales. Estudio ultraestructural en células de descamación del epitelio estratificado.

Bacterial vaginosis is one of the most frequent infections during the woman's reproductive age. Lactobacilli normal flora is substituted by relatively elevated Gardnerella vaginalis (GV), anaerobic bacteroids, Mobiluncus and Mycoplasma. The purpose of this study is to perform a morphological analysis of possible mechanisms for adhesion and penetration of GV in the heterosexual couple, in squamous epithelium at vaginal wall, as well as in seminal fluid. Ten couples with positive GV culture with three to four days of abstinence, were studied. The women presented with at least three of the four Amsel's criteria. Samples were obtained from vaginal lateral walls and from seminal fluid; these were divided in two parts: 1. To realize cultures for GV. Ureaplasma urealyticum and Mycoplasma hominis; and 2. For an ultrastructural analysis. Samples were processed with common techniques for electronic microscopy. In vaginal cells, bacteria similar to GV free form fixed to plasmatic membrane and inside the cellular cytoplasm. In the seminal fluid there were found numerous urethral cells of desquamation, which presented, as well as in woman, free form bacteria, fixed to plasmatic membrane and inside the cytoplasm. In four cases, bacteria similar to Mycoplasma, were found; and one case with particles suggesting cytomegalovirus. It is concluded in this study that: 1. The male presents with urethral cells similar to vaginal "guide cells". 2. GV colonizes urethral epithelium in the male. 3. Male is capable of infecting and/or re-infecting the woman.

[Typification of a subgroup of cervical papilloma by in situ hybridization. Preliminary study]. / Tipificación del subgrupo de papiloma cervical por hibridización in situ. Estudio preliminar.

The purpose of this study was to analyze 19 patients infected with Human Papilloma Virus (HPV), in order to determine the most frequent subtype. Subjects were patients of the National Institute of Perinatology (INPer), between 1991-1992. All the patients had positive cytology, cervical biopsy was done through colposcopy control. Sample characteristics were: Mean age 29 years, married; most of them multiparous (9/19) and 7 were monogamous. Six out of 12 cytologies were positive for HPV, 12 out of 19 had an inespecific inflammatory reaction and one patient had NICIII. The histopathologic study showed in 15 cases a positive reaction to HPV, 2 out of 19 had both dysplasia and condyloma, two cases had moderate and severe dysplasia. All the cases were studied with in situ hybridization with biotinilaty probes, two cases were positive to Subtype 18 and one to Subtype 16. Positive cases were selected for electron microscopy in order to confirm viral particles. This study suggests that subtype 18 and 16 positivity must be considered as high risk of cancer.

[Ultrastructural study of placentas from HIV seropositive women]. / Estudio ultraestructural de placentas en mujeres seropositivas para el VIH.

Eleven placentas from seropositive women for HIV, were analyzed. In three cases the material came from first trimester abortions and the other eight from term pregnancies. In five cases retroviruses were identified, similar to HIV in the placental tissue. It was demonstrated for the first time the internalization of a retrovirus and its presence in the syncytiotrophoblast. It is communicated for the first time the presence of one cell in the placental stroma different to Hofbauer's by its granules type.

[Estrogen therapy in postoperative gynecologic cancer patients]. / Estrogenoterapia en pacientes postoperadas de cáncer ginecológico.

Bilateral oophorectomy is an essential part in the treatment of gynecologic cancer, and is often performed at the time of hysterectomy to prevent ovarian or/and breast cancer premenopausic women. In our study, we analyzed results from previous reports to determine the influence of estrogen on coronary heart disease, osteoporotic fracture and life expectancy in young patients with medical records of ovarian or breast cancer.

[Index of cells obtainable from semen: indicator of fertility?]. / Indice de células recuperables del semen: indicador de fertilidad?

It has been recognized that semen analysis is not a sensible nor a specific tool for detecting infertility in the male. Its predictive value is limited by the subjectivity of manual analysis, a high variability of semen parameters in fertile men and lack of correlation between sperm characteristics and fertility indexes. In this paper we present evidence supporting the concept that calculating an index of potentially fertile cells from data obtained in a regular semen analysis might be useful for predicting fertility in the male.

Bacterial vaginosis associated with G vaginallis / Mobiluncus sp: ultrastructural parameters.

PIP: Physicians at the National Institute of Perinatology in Mexico City, Mexico used a Carl-Zeiss EM 10C electron microscope to examine genital secretion samples from 10 pregnant women (15-38 weeks' gestation) who had been diagnosed with Mobiluncus species and Gardnerella vaginalis infections to illustrate the form and structure of bacteria responsible for bacterial vaginosis. They were concerned that these bacteria induce preterm labor and premature rupture of membranes (PL/PROM). These bacteria have been present in the genital tract of 30% of pregnant women with a thick whitish discharge who have attended the Institute's prenatal outpatient clinic. Physicians noted on the microscope slides that bacteria surrounded vaginal squamous epithelial cells (clue cells). Numerous gardnerella-like bacteria surrounded elongated squamous epithelial cells with many plasma projections. An extensive area of lysis existed around the bacteria in the cytoplasm of many squamous epithelial cells with intact membrane and nonexistent microfilaments. This finding indicated that the bacteria invade and destroy the cells. Plasma membrane projections almost completely surrounded the gardnerella-like bacteria in certain areas. Since this study strengthened the theory that G. vaginalis enters the vaginal squamous epithelial cells, researchers should conduct more studies to determine its role in PL/PROM.^ieng

[Ultrastructural identification of human immunodeficiency virus in placenta, embryonic tissue and semen]. / Identificación ultraestructural del virus de la inmunodeficiencia humana en placenta, tejidos embrionarios y semen.

The objective of this paper was to evaluate, ultrastructurally the placental site, placenta, umbilical cord, embryonal tissues, and semen from 10 patients (5 females and 5 males), seropositive for the Human Immunodeficiency Virus. The tissue samples were processed by the routine techniques for electronic microscopy, and were examined with a Zeiss EM-10C Electronic, transmission microscope. Among the decidual cells of the placental site, macrophages were found, which contained vacuoles with particles similar to a virus, inside. Similar to virus, particles were found among microvilli of syncytiotrophoblast; it presented with a thick basal membrane and with calcifications. Hyperplasia of Hofbauer cells was seen, although there were not viral particles inside. In the membrane of fetal erythrocytes there particles, electron dense, possibly from viral origin. Viral particles were identified in the nuclei of endothelial cells of the brain and lung. In the semen budding areas were seen in immature spermatozoon; and similar particles were seen y spermatozoa, probable desquamation cells, and in seminal protein, in free form. It is concluded that the HIV may be transmitted via transplacental in very early stages of gestation. The hyperplasia of Hofbauer cells can be a response of local defense. And finally, the presence of viral particles in the different components of semen favor its transmission by sexual contact.

[The morphological aspects of the placenta, embryonic tissues and semen in human immunodeficiency virus infections]. / Aspectos morfológicos de placenta, tejidos embrionarios y semen en infecciones por virus de inmunodeficiencia humana.

Tissue samples from a therapeutic curettage performed in a woman with acquired immunodeficiency syndrome and a semen sample of the husband were studied with the electron microscope. The samples were processed according to routine technique for electron microscopy. Calcifications, basement membrane thickening and hyperplasia of Hofbauer cells were seen in the placenta villi. Electron-dense particles of unknown nature, probably of viral origin, were found on the fetal red blood cell membranes, virus-like particles were identified in the endothelial cell nucleus of the brain and lung. Retrovirus-like particles were found in the protein of the seminal plasma. These results suggest that the retrovirus pass through the placenta during early pregnancy.