Quantification by image analysis of the Gallus gallus lung elastic fibres from embryonic to adult birds.

The organization of the lung's elastic fibres is amazingly uniform in all vertebrates, with the possible exception of birds, whose pulmonary architecture and air movement are unique. The overall goal of this work was to study and quantify elastic fibre distribution patterns and relative amounts in the parabronchi, during the incubation period until the 42nd day after hatching. Chick embryo lungs were examined on the 14th, 16th, 18th and 20th days of incubation and chick lungs on the 1st, 2nd, 7th, 14th, 35th and 42nd days after hatching. Four animals were used daily, and the observations were randomly performed on both lungs. A morphometric study was carried out focusing on the computerized image analysis of histological sections stained according to a modified Gomori technique. The values obtained for each day result from the observation and processing of 20 images. Complementary studies were performed using transmission electron microscopy, as on the 14th embryonic day the fibres were not visible on light microscopy. The results show that the area occupied by the elastic fibres increases gradually from the 16th day of incubation up till the 7th day after hatching and decreases slowly in the following days of the study. A prominent increase takes place before hatching, which points out to the adequate and essential structural roles played by the elastic fibres in the pulmonary maturation process.

Human chorionic gonadotropin in human placentas from normal and preeclamptic pregnancies.

OBJECTIVE: The aim of this study was to examine the distribution of human chorionic gonadotropin (hCG) in the human placenta of normotensive and preeclamptic pregnancies and to determine by computer image analysis, whether differences in hCG immunoreactivity occurred in preeclamptic as apposed to normotensive pregnancies. We discuss how far elevated maternal serum levels of hCG normally observed in preeclamptic patients reflect an increased secretory activity of the syncytiotrophoblast. METHODS: We used the immunoperoxidase technique to locate hCG. Quantification of immunostaining intensity was done by computer image analysis. RESULTS: In normotensive placentas from all the gestational ages human chorionic gonadotrophin immunoreactivity was specifically detected in the syncytiotrophoblast. There is an apparent decrease in the intensity of the hCG immunostaining in the syncytiotrophoblast from the 29th to 36th week of gestation in normotensive placentas. No hCG immunostaining was observed in the villous or extravillous cytotrophoblast of all placentas. In preeclamptic placentas the expression of hCG was homogeneous with a moderate to intense immunoreactivity in the syncytiotrophoblast. Microdensitometric analysis of the section from normotensive and preeclamptic placentas indicated that there is a statistically significant preeclampsia-induced increase in immunohistochemical reaction intensity for hCG (p < 0.05). CONCLUSION: This study seems to demonstrate that increased production of hCG by preeclamptic placentas is associated with strong hCG immunostaining of the syncytiotrophoblast.

Quantification of elastin from the mouse lung during postnatal development.

Elastic fibers play a vital role in secondary septal development during the process of lung alveolization. The authors present a quantitative study by image analysis and by high-performance liquid chromatography (HPLC) of the mouse lungs'elastic fibers throughout postnatal development. Five mice from birth up to the 21st day of postnatal development and on the 30th day of life were used daily. Their left lungs were collected and processed for observation under light microscopy. The staining was carried by a modification of the Gomori technique. The right lung of each animal was processed for elastin quantification by HPLC. Analysis by linear regression of the two methods' results showed there was a significant positive linear correlation. With this methodology of staining and image analysis we evaluated the normal establishment of the elastic network of the respiratory portion of the lung from birth to adult life. We now have a good model for studying the variations induced by exogenous agents in the synthesis and degradation of the pulmonary elastic fibers.

Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells.

Attachment of Giardia lamblia trophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for parasite-induced enterocyte dysfunction and clinical disease. In this work, coincubation of Giardia with Int-407 cells, was used as an in vitro model to study the role of cytoskeleton and surface lectins involved in the attachment of the parasite. This interaction was also studied by scanning and transmission electron microscopy. Adherence was dependent on temperature and was maximal at 37 degrees C. It was reduced by 2.5 mM colchicine (57%), mebendazole (10 microg/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 microg/ml) (21%). No significant modification was observed when Giardia was pretreated with cytochalasins B and D and with EDTA. Giardia attachment was also diminished by preincubating Int-407 cells with cytochalasin B and D (5 microg/ml) (16%) and by glutaraldehyde fixation of intestinal cells and of G. lamblia trophozoites (72 and 100%, respectively). Ultrastructural studies showed that Giardia attaches to the Int-407 monolayer predominantly by its ventral surface. Int-407 cells contact trophozoites with elongated microvilli, and both trophozoite imprints and interactions of Giardia flagella with intestinal cells were also observed. Transmission electron microscopy showed that Giardia lateral crest and ventrolateral flange were important structures in the adherence process. Our results suggest a combination of mechanical and hydrodynamic forces in trophozoite attachment; surface lectins also seem to mediate binding and may be involved in specific recognition of host cells.

Immunocytochemical localization of endothelin-1 in human placenta from normal and preeclamptic pregnancies.

OBJECTIVE: The aim of this study was to examine the distribution of endothelin-1 (ET-1) in the human placenta at different gestational ages and to determine whether differences in ET-1 immunoreactivity occurred in preeclamptic compared with uncomplicated pregnancies. METHODS: Localization of ET-1 was investigated by the immunoperoxidase technique in first-trimester, second-trimester, and term human placentas from normal pregnancies and in placentas from preeclamptic pregnancies. RESULTS: In normal placentas from all gestational ages studied, endothelin-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the fetal vessels and in the syncytiotrophoblast. ET-1 IR was also expressed by the villous cytotrophoblast of first- and second-trimester normal placentas. The extravillous cytotrophoblast of the basal and chorionic plates also exhibited ET-1 IR, but with varying degrees of intensity. In preeclamptic placentas, the expression of ET-1 IR was uneven with a negative staining in all placentas from pregnancies between the 29th and 32nd weeks of gestation. The expression of ET-1 IR was most intense in some syncytiotrophoblast tissue in the terminal villi after the 33rd week of gestation. In placentas from preeclamptic pregnancies between the 35th and the 36th weeks of gestation, strong ET-1 IR expression was evident in the endothelium of fetal vessels and in the syncytiotrophoblast. Regardless of gestational age, ET-1 IR was also observed in the extravillous cytotrophoblast of the basal and chorionic plates of preeclamptic placentas. CONCLUSION: This study demonstrates that ET-1 IR is widely distributed in the human placenta and provides further evidence to support the concept that ET-1 plays an important role as a modulator of vascular tone in the uteroplacental and fetoplacental units and may participate in the pathogenesis of preeclampsia.

Menkes' kinky hair syndrome: ultrastructural cutaneous alterations of the elastic fibers.

Menkes' kinky hair syndrome is associated with the defective functioning of several copper-dependent enzymes due to impaired copper absorption, transport, or metabolism. Lysyl oxidase is a copper-requiring enzyme that catalyzes the oxidative deamination of lysyl residues linking two adjacent chains of tropoelastin polypeptides into an insoluble network. Elastin of the connective tissue is the responsible protein for the elastic properties of the skin. We report transmission electron microscopy findings concerning elastic fiber alterations of the skin in three patients with Menkes' syndrome. The reticular dermis showed marked changes in the elastic fibers with a paucity of the central amorphous component while retaining normal microfibrillary material. These ultrastructural observations, to the best of our knowledge, are reported for the first time in skin from these patients and may be readily interpreted in terms of a specific biochemical defect in elastogenesis.

Three-dimensional organization of the elastic fibres in the rat lung.

BACKGROUND: The elastic framework of the distal lung has been studied by light microscopy (LM) and transmission electron microscopy (TEM). The preservation of the elastic fibres, for the three-dimensional observation in their relative positions, is difficult because they lack support when the normal methods of tissue processing are used. The goal of the present study was to understand the three-dimensional ultrastructure and organization of the elastic fibres of the lung preserved in their relative positions. METHODS: A combination of intravascular resin injection and formic acid digestion was used. The resin cast of the microvasculature acted as a scaffold to preserve the in vivo arrangement of the elastic fibres that are, otherwise, easily collapsible. Scanning electron microscopy (SEM) samples were further processed for TEM in order to confirm that the fibres were indeed components of the elastic system. RESULTS: SEM demonstrated a fine framework of elastic fibres, representing remnants of the alveolar walls, with the casted capillaries interwoven with the network of elastin. Each individual elastic fibre is composed of a small bundle of discrete fibrils. Some of these fibrils emerge from the fibre and join other fibres, producing an anastomosing appearance. Several elastic fibres link the walls of the intrapulmonary conducting airways, the vessels walls and the alveolar network, thus establishing an interrelated and interlaced framework. CONCLUSIONS: The method we have applied to visualize the elastic fibres of the lung is a unique approach to define the spatial organization of the pulmonary elastic fibres. We have demonstrated here the close relationship between the elastic fibres and the capillaries of the septal alveoli. The arrangement of the interwoven network of elastin and its relationship with the capillaries offers the structural setting for the distending capacity of the alveolar wall.

Study of the elastic fibres' framework of the rat lung.

A combination of intravascular resin injection and formic acid incubation was used to study the three-dimensional organization of the elastic fibres of the adult rat lung by scanning electron microscopy (SEM). After SEM observations, the same samples were further processed for transmission electron microscopy (TEM) in order to confirm the presence of the elastic fibres and to complement some aspects of its surface morphology observed under the SEM. Complementary studies by light microscopy (LM) and TEM using specific histochemical methods for the elastic fibres were also performed. The SEM study clearly demonstrated that the cast of the microvasculature acted as a scaffold to preserve the in vivo arrangement of the easily collapsible elastic tissue. The methodology used allowed the observation of a fine framework of elastic fibres representing remnants of the alveolar walls in close association with the capillaries interwoven with the network of elastin. Each thick elastic fibre was composed of a bundle of thin fibres. Some of these thin fibres separated from the main fibre, join other fibres, giving the appearance of an anastomosing net. The interwoven network of elastin and its proximity with the capillaries suggests that the distensibility of the alveolar wall should contribute to the subtle rhythmical change of the alveolar microcirculation at each respiratory movement. On the sub-pleural region of the lung, the elastic fibres were observed forming a continuous and fine mesh network. The elastic fibres linking the walls of the intrapulmonary conducting airways, the vessels wall and the alveolar and sub-pleural elastic network establish an interrelated and interlaced continuous framework, certainly with great physiological implications to the overall process of the mechanics of the lung respiratory function. The methodology applied was a useful tool in order to study the spatial organization of the pulmonary elastic fibres, its branching and close relation with the other lung structures.

Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temperature on fusion and viral inactivation.

We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecyl-rhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus-cell fusion. When the temperature was lowered from 37 degrees C to 25 degrees C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5-fold and 7-fold, respectively, as the temperature was lowered from 37 degrees C to 25 degrees C. The fusion process seemed essentially pH-independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37 degrees C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8-fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4 degrees C, 30 min) did not alter viral fusion activity. These acid-induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37 degrees C, 30 min). Changes in internal pH as well as endocytic activity of PC-12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.

Calcium stores in electropermeabilized HL-60 cells before and after differentiation.

Non-induced HL-60 cells (N-IND) and HL-60 cells induced to differentiate with 2 microM retinoic acid (IND) were electropermeabilized with electrical discharges, and the intracellular Ca2+ stores were measured in each type of cell. Both N-IND and IND cells accumulate Ca2+ in the presence of ATP after electropermeabilization. The Ca2+ is stored in at least two different compartments; accumulation in one of the compartments is inhibited by oligomycin and CCCP, and it is not releasable by Ins(1,4,5)P3. The maximal accumulation of Ca2+ by the Ins(1,4,5)P3 sensitive pool is about 0.3 nmol/10(6) cells and 0.9 nmol/10(6) cells for the N-IND and for the IND cells, respectively, and the half-maximal value occurs at a free Ca2+ concentration of 0.23 microM and 0.63 microM, respectively. The oligomycin + CCCP sensitive pool hardly accumulates any Ca2+ at this level of free Ca2+, but at higher free [Ca2+] (greater than microM) its maximal capacity is 80-100-fold higher than the Ins(1,4,5)P3-sensitive pool (about 17-18 nmol/10(6) cells). It is concluded that at physiological free Ca2+ concentrations, the non-mitochondrial Ca2+ pool is regulating the intracellular free Ca2+ in N-IND and IND HL-60 cells, and that this Ca2+ pool can be mobilized by Ins(1,4,5)P3. Furthermore, the capacity of this pool increases about 3-fold when the cells are induced to differentiate with retinoic acid.