Lung regions differently modulate bronchial branching development and extracellular matrix plays a role in regulating the development of chick embryo whole lung.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.

Morphometric analysis of primary graft non-function in liver transplantation.

AIMS: Primary graft non-function (PNF) is a life-threatening condition that is thought to be the consequence of microcirculation injury. The aim of the present study was to assess, with a computerized morphometric model, the morphological changes at reperfusion in liver biopsy specimens from patients who developed PNF after liver transplantation. METHODS AND RESULTS: Biopsy specimens were obtained at maximum ischaemia and at the end of reperfusion. Morphology included many stereological parameters, such as volumes of all parenchymal components, surface density, size distribution and mean diameter of hepatocytes. Other variables examined were intensive care unit stay, degree of steatosis, serum liver function tests and ischaemic time. In the postoperative period, the PNF group showed elevated serum levels of alanine transferase, decreased daily rate of bile production and prothrombin activity. Blood lactates were significantly higher in the PNF group than in a control group. When comparing groups, the volumetric parameters related to hepatocytes and sinusoids and the surface densities of the hepatic cells showed an inverse relationship. At the end of reperfusion, in PNF group the volume fraction of hepatocyte cytoplasm was decreased; in contrast, the volume fraction of sinusoidal lumen was markedly increased. The cell profiles showed the same inverse trend: the surface density of the parenchymal border of hepatocytes was decreased in PNF when compared with the control group, while the surface density of the vascular border was increased. In the PNF group, the surface density of the sinusoidal bed was directly correlated with alanine transferase, daily rate of bile production, prothrombin activity and cold ischaemic time. CONCLUSIONS: The alterations in hepatic architecture, as demonstrated by morphometric analysis in liver transplant recipients that developed PNF, provide additional information that may represent useful viability markers of the graft to complement conventional histological analysis.

Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

Extracellular glycosaminoglycan changes in healthy and overgrown gingiva fibroblasts after cyclosporin A and cytokine treatments.

BACKGROUND: It has been demonstrated that cyclosporin A (CyA) blocks the immune system, acts on cytoskeleton and stimulates the production of extracellular matrix (ECM) and transforming growth factor-beta1 (TGF-beta1). This cytokine, such as transforming growth factor-alpha (TGF-alpha), induces deposition of glycosaminoglycans (GAG), proteoglycans and collagen fibres in the ECM. METHODS: In this work, we examined the effect induced by CyA, TGF-beta1 and TGF-alpha on cultures of healthy and overgrown human gingival fibroblasts in order to evaluate the glycosaminoglycan, cytoskeletal changes and the behaviour of fibroblasts after concanavalin A (Con A) treatment. Moreover, we examined gingival biopsies by Alcian blue histochemical staining and electron transmission microscopy. RESULTS: Total and extracellular sulphated GAG in overgrown gingiva specimens and in derived fibroblast cultures treated with CyA and cytokines were significantly higher than controls. The action of cytokines was increased (P < or = 0.01) compared with CyA with a greater effect of TGF-alpha in comparison with TGF-beta1; the electron microscopy showed ECM accumulation. The agglutinations showed the heterogeneity of fibroblast populations. CONCLUSIONS: Stimulation with Con A showed that the fibroblast population had cell surface heterogeneity, and could respond in a different way to both CyA and cytokine stimulus. Moreover, increased synthesis of GAG in overgrown gingiva compared with synthesis in normal fibroblasts before CyA treatment suggests a possible genetic origin of damage. As not all CyA-treated patients develop gingival overgrowth, a genetic predisposition may explain the different responses of gingival fibroblast populations.

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Polyamine levels and ornithine decarboxylase activity in blood and erythrocytes in human diseases.

Serum and erythrocyte levels of the polyamines spermine, spermidine and putrescine, as well as ornithine decarboxylase in erythrocytes, were studied in patients with different neoplasms (breast, lung and colon cancer) and in those with a nonmalignant proliferative disease (familial polyposis). The blood levels of polyamines and the spermine/putrescine ratio were significantly higher in all tumors and in nonmalignant colon polyposis. In erythrocyte ornithine decarboxylase activity, spermine and spermidine levels, as well as spermidine/putrescine and spermine/putrescine ratios showed a significant decrease after surgery and chemotherapy. Our data suggest that high levels of blood polyamines and erythrocyte ornithine decarboxylase activity are related to cell proliferation and cancer treatment, but that levels of polyamines in serum and erythrocytes are still significantly high after cancer treatment and are similar to those in polyposis disease. Polyamines are related to nuclear activity during differentiation; therefore, the altered turnover of polyamines could be a sign of abnormal nuclear function. Since polyamines stimulate protooncogene expression, their high levels could be considered an important cofactor in malignant cell transformation.

Liver transplantation in man: morphometric analysis of the parenchymal alterations following cold ischaemia and warm ischaemia/reperfusion.

Ischaemia and reperfusion phases represent critical events during liver transplantation. The purpose of this study was to describe morphological alterations of both vascular and parenchymal compartments after ischaemia and reperfusion and to evaluate the possible relationship between morphometric parameters and biochemical/clinical data. Three needle biopsies were drawn from 20 patients who underwent orthotopic liver transplantation. The first biopsy was taken before flushing with preservation solution, and the second and the third to evaluate respectively the effects of cold ischaemia and of warm ischaemia/reperfusion. Biopsies were examined by an image analyser and morphometric parameters related to the liver parenchyma were evaluated. At the second biopsy we observed a decrease of the endothelium volume fraction while the same parameter referred to the sinusoidal lumen achieved a peak value. The hepatocytes showed a lower surface parenchymal/vascular sides ratio. This parameter was reversed at the end of the reperfusion phase; furthermore the third biopsy revealed endothelial swelling and a decreased volume fraction of the sinusoidal lumen. The results quantify the damage to the sinusoidal bed which, as already known, is one of the main targets of cold ischaemia; warm ischaemia and reperfusion accentuate endothelial damage. The end of transplantation is characterised by damage chiefly to parenchymal cells. Hepatocytes show a rearrangement of their surface sides, probably related to the alterations of the sinusoidal bed. In addition, the fluctuations of morphometric parameters during ischaemia/reperfusion correlate positively with biochemical data and clinical course of the patients.

Effect of testosterone on purine metabolism and morphometric parameters in the rat liver.

The effect of testosterone on the morphology and biochemistry of adult castrated rat liver is described. Castration decreases mean weight and volume of hepatocytes, volume and surface area of sinusoidal lumen, and apparently increases cell number per g of tissue. These variations indicate cell distress. Testosterone administration restored sinusoidal volume and surface area, indicating a true hyperplastic response and improved trophic conditions. Acid soluble nucleotides, RNA and DNA content were lower after castration, being partially restored after testosterone treatment. This restoration, however, was only statistically significant for total guanylate. We concluded that testosterone deficiency and administration exerts a specific effect on the liver in terms of morphological and biochemical changes. Purine nucleotide metabolism is probably the first target of hormonal action, since its changes are the most significant and useful to explain all the other observations.

Morphometric evaluation of rat liver after castration: effects of testosterone.

In men and animals subject to orchiectomy, varying degrees of hypotrophy have often been observed, even involving non-reproductive organs. Liver hypotrophy appears particularly interesting in view of the metabolic alterations that could possibly ensue. We have applied the morphometric approach to the study of this kind of hypotrophy in castrated rats and in animals receiving a substitutive, testosterone-based treatment after castration. The morphometric model adopted was built on the basis of an interactive approach, using an image analyser (IBAS) with specially-designed software, and consists of number and size, as well as surface and volume fractions, of hepatocytes, their nuclei, cytoplasm, and the relevant relation to the sinusoidal bed. The effects of castration were revealed by a reduction of the mean volume of hepatocytes, associated with a reduction of the sinusoidal bed. Such reduction is correspondingly associated with an increase of the extraparenchymal components (endothelia, Glisson capsule). In the testosterone-treated group, while no appreciable symptoms of retrieval were yet observable in the volume of hepatocytes, the sinusoidal bed was instead superimposable onto the control group data as far as both volume and blood/hepatocyte interface were concerned. In the castrated animals the number of hepatocytes per mm3 was found to be increased. The group of testosterone-administered rats also showed a high level for this parameter which, associated with the spread of the capillary bed, could be compatible with a picture of precocious hyperplastic response to the therapy. The correlation between the available morphometric data and the biochemical results which are being currently sought will further clarify the meaning of this phenomenon.

A cluster analysis of the neurons of the rat interpeduncular nucleus.

The morphometric characteristics of the neurons of the interpeduncular nucleus (IPN) in the rat were investigated by cluster analysis in order to identify neuronal groups which are morphometrically homogeneous, and to define their position and density in the IPN subnuclei. Two clusters of cells were detected. Cluster 1 neurons had a larger perikaryal size with a mean cross-sectional area of 170 microns2 and a high nuclear/cytoplasmic ratio. They were located mainly in the pars dorsalis (37%) and pars medialis (34%) rather than in the pars lateralis (29%). Cluster 1 neurons were also more frequent at the rostral (31%) and caudal (57%) poles than in the central part of the IPN. Cluster 2 cells showed a smaller mean perikaryal area (110 microns2), a small nucleus and abundant cytoplasm. They were equally distributed throughout the whole IPN. These findings suggest the existence of a magnocellular region at the rostral pole of the IPN which has not been described previously. The presence of IPN regions endowed with specific cytoarchitectural characteristics is discussed with respect to the complex neurochemical organisation of the nucleus.

Morphometric study of the human neonatal ovary.

A morphometric analysis, based on mathematical evaluations and stereological methods, has been used to study five left neonatal ovaries, removed from full-term neonates with a 46,XX karyotype free from malformations of the genital apparatus. Each ovary was completely cut obtaining serial sections and one 1-micron-thick section every 1,000 microns was examined. Ovarian length ranged from 9 to 17 mm (mean 13 mm), width from 3.5 to 7 mm (mean 5.7 mm), thickness from 2.5 to 5 mm (mean 4 mm), and volume from 82.23 to 198.3 mm3 (mean 125.88 mm3). In the ovarian cortex, primitive cortical tissue accounted for 10-20% of the total volume, follicles for 10-25% and interstitium for 35-45%; 10-30% of the organ consisted of inner medulla. The total follicle number ranged from 130,000 to 385,000 per ovary, with an average of 266,000 with 95% being represented by primordial follicles. In all ovaries examined follicular growth was still in process, with follicles at different stages of development.

Different neuronal types in transneuronally WGA-HRP-labeled premotor interneurons of the rat spinal cord.

Interneurons presynaptic to motoneurons were labeled by retrograde transneuronal transport of WGA-HRP. The tracer was injected either into the quadriceps muscles or into the posterior biceps muscles, thus labeling interneurons presynaptic to the quadriceps motoneurons (QINs) or interneurons presynaptic to posterior biceps motoneurons (PBINs). Statistical cluster analysis of area, perimeter, equivalent diameter and form factor of the labeled interneurons permitted the identification of 4 different types of premotor interneurons in the lumbar enlargement of the rat. Type I are small elongated interneurons which prevail in PBINs. Type II are medium-sized ellipsoidal cells prevailing in the QINs. Type 3 are small ellipsoidal neurons, slightly more frequent in PBINs. Type 4 is the smallest group and it is composed of large multipolar neurons. A different distribution of the 4 morphological neuronal types was found between the population of the QINs and the PBINs and in the laminae of ventral horn for each group.

Circadian rhythmicity in the rat exocrine pancreas: chronomorphological patterns.

Circadian rhythmicity of the structural morphometric model of rat endocrine pancreas has been studied in 24 Wistar female rats, four months old, kept in LD 12:12. The following parameters were evaluated: the volume fractions of nucleus and cytoplasm of exocrine cells, the size distribution and number in unit tissue volume of acinar cell nuclei, the mean nuclear diameter, the shape coefficient of glandular acini (that is the ratio acinar area/perimeter2 which indicates the shifting of structures from circularity). A statistically significant circadian rhythm was demonstrated for the shape coefficient of glandular acini. Results obtained in the present experiment are compared with data recorded in a previous study.

Characterization by morphometric model of liver regeneration in the rat.

A new stereological approach to the study of liver regeneration in the rat is described. The method employs a morphometric model consisting of number and size, as well as surface and volume fractions of hepatocytes, their nuclei, cytoplasm, and their relation to the sinusoidal bed. With this technique, it was found that 8 hr after partial hepatectomy, the number of nuclei increased as did the nucleus/cytoplasm ratio, suggesting an early hyperplastic phase. These data, which have not been reported in other studies, underline the importance of evaluating the complex phenomenon of liver regeneration using a stereological approach, which is more representative of all morphometric changes occurring inside and outside the hepatocyte.

A chronomorphological structural model of rat thyroid gland.

Circadian rhythmicity of the structural morphometric model of thyroid has been studied in 36 Wistar rats kept in LD 12:12. The parameters evaluated are: a. the volume fraction occupied by: 1. follicle epithelium, 2. colloid, 3. interstitium at 6 time points in 24h; b. the follicle size distribution; c. the number of follicles per unit tissue volume. The circadian rhythms of mean follicular diameter and of follicular cavity mean diameter have been demonstrated (p less than 0.03 and p less than 0.01 respectively) and show overlapping acrophases of -120 degrees (-64 degrees/-176 degrees) and -108 degrees (-99 degrees/-116 degrees). The synchronization between rhythms, shown for mean follicular diameter and for follicular cavity mean diameter, suggests a rhythmical pulsation of the whole follicle, while the thickness of the follicular epithelium does not undergo a statistically significant periodic variation.

Improvement in the haemostatic defect of uraemia after treatment with recombinant human erythropoietin.

Patients with uraemia have a defect of primary haemostasis expressed as long skin bleeding times and reduced platelet adhesion to the arterial subendothelium. Transfusion of red cells shortens the bleeding time and stops bleeding symptoms in uraemia. This study investigated whether the efficacy of recombinant human erythropoietin in correcting anaemia and the improvement in haemostasis are correlated. Recombinant human erythropoietin was given to seven consecutive patients with chronic uraemia, a history of bleeding, severe anaemia (haematocrit below 23%), and long bleeding times (above 19 min). The progressive rise in haematocrit induced by increasing doses of recombinant human erythropoietin was paralleled by a pronounced shortening of the bleeding time. Platelet adhesion to the subendothelium of human umbilical arteries, very low before the study, increased greatly in all patients and became normal in six. None of the patients bled during the study period.

Considerations on the dynamics of adipose cells in relation to ponderal variations: morphoquantitative aspects.

The sizes and numbers of subcutaneous adipose-tissue fat cells were determined in obese patients, some of whom had undergone severe weight reduction induced by a jejuno-ileal bypass operation, and others a moderate thinning by a reducing diet, for comparison with those in normals. The size-class distribution of the fat cells suggests a morphoquantitative, dynamic interpretation of the two known forms of obesity, one being of moderate degree and called hypertrophic, the other more severe and called hyperplastic. The hypertrophic obese condition is characterized by a bimodal size-distribution curve of the adipocytes, similar to the curve in normal individuals. In both the normal and the hypertrophic obese, ponderal variations displace this curve forwards or backwards without altering its shape. By contrast, the hyperplastic obese shows a flat size-distribution curve without discernible modes. The smallest size-class of fat cells are the most sensitive to weight-reduction process and hypotrophize so much that they can no longer be recognized as adipocytes in histological sections; thus the size-distribution curve for hyperplastic obese individuals losing weight changes in shape from uniform to bimodal. The relationship which expresses the mean fat cell diameter as a function simultaneously of the initial weight and of ponderal variation was also studied, and tested by multiple regression analysis.

Bile lipid output and composition in patients fitted with external biliary drainage: activity of cicloxilic acid.

In 7 patients with external biliary drainage for total occlusion of the extrahepatic bile ducts the bile output was measured in 3-day periods, with and without cicloxilic acid treatment (240 mg daily, per os). The output increased markedly during treatment in all cases. In the 3 patients in whom the bile lipid composition was analyzed also, the bile salt and phospholipid concentrations increased while the cholesterol concentration was unchanged--with an consequent lowering of the biliary cholesterol saturation index--during treatment with cicloxilic acid.