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.

In vitro human osteoblast and extracellular matrix changes after transforming growth factor beta 1 treatment.

AIMS: Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia. METHODS: In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined. RESULTS: Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (p < or = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities. CONCLUSION: TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.

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.

Diphenylhydantoin affects glycosaminoglycans and collagen production by human fibroblasts from cleft palate patients.

During embryonic development, the proper production of extracellular matrix molecules mediates morphogenetic processes involved in palatogenesis. In the present study, we investigated whether any differences exist in glycosaminoglycan (GAG) and collagen synthesis between palate fibroblasts from infants, with or without cleft palate, in two age ranges. Subsequently, the effects of diphenylhydantoin (PHT), a teratogen known to induce cleft palate in human and mammalian newborns, on extracellular matrix (ECM) production were studied. We found that cleft palate fibroblasts (CPFs) synthesize greater amounts of GAG and collagen than normal fibroblasts (NFs). CPFs produced less cellular hyaluronic acid (HA) and more sulphated GAG. HA was the principal GAG species in the medium, and its percentage was lower in one- to three-year-old CPFs. Cleft palate fibroblasts produced more extracellular chondroitin 4- and 6-sulphate (CS) and dermatan sulphate (DS). Associated with a higher production of sulphated GAG, we observed a higher synthesis of type III and type I collagen with a normal ratio of alpha2(I) to alpha1(I) chains. PHT treatment of NFs reduced collagen and GAG synthesis, with a marked effect on sulphated GAG. The drug changed collagen synthesis, whereas it did not affect GAG production in CPFs whose phenotype may already be impaired. These findings indicate that, in CPFs, modifications in the pattern of ECM components, which are most likely responsible for the anomalous development, persist in infants. In addition, NFs and CPFs with a different phenotype respond differently to PHT treatment.

Chick embryo back skin organ and fibroblast cultures. Extracellular matrix changes induced by dialysate fluid and uraemic toxins in relation to proliferation and differentiation processes.

AIM: During uraemia, an increase of middle molecules and acetylpolyamines occurs. In vitro the middle molecules produce cell toxicity, while the acetylpolyamines stimulate cell proliferation and differentiation. These phenomena are related to protein and extracellular glycosaminoglycan production. The aim of the present study was to evaluate the activity of dialysate, dialysate fluid and the chromatographic peaks of dialysate fractionated by G-15 Sephadex column on chick embryo skin development. METHODS: We evaluated the effects of protein and glycosaminoglycan synthesis using monolayer and organotypic cultures. RESULTS: Our data show that dialysate, chromatographic peak II, and 2 x 10(-8)M N1-acetylspermine cause inhibition of chick embryo skin culture development. On the contrary, 10(-8)M N-acetylornithine and dialysate fluid increase protein and extracellular glycosaminoglycan synthesis, whereas chromatographic peak I does not reveal differences when compared to controls. CONCLUSIONS: These extracelluar changes are related to cell proliferation and feather formation in chick embryo organotypic culture. Moreover, the pH changes of culture medium do not influence the biological action of acetylpolyamines and dialysate fluid on protein and extracellular glycosaminoglycan synthesis. Cell death in the presence of N1-acetylspermine, dialysate and peak II appears unrelated to the apoptotic process. The data show that acetylpolyamines, dialysis fluid, dialysate and chromatographic peaks act on fibroblasts, and are able to modify glycosaminoglycan synthesis. The organotypic cultures of chick embryo back skin could represent a model for studying the modifications of the extracellular matrix induced by these substances.

Urinary glycosaminoglycans in recurrent urinary tract infections in kidney transplant patients.

Glycosaminoglycans have generalized antibacterial anti-adherent activity, and cooperate with secretory immunoglobulin-A in anti-infection defense mechanisms of the urinary tract. Cyclosporin A modulates T-lymphocytes and fibroblast functions. In this report we analyze urinary glycosaminoglycans and secretory immunoglobulin-A in renal transplant patients with recurrent urinary tract infections treated with cyclosporin. The results show a significant decrease of total glycosaminoglycans and secretory immunoglobulin-A in recurrent urinary tract infections which is unrelated to cyclosporin treatment. The data support the hypothesis that recurrent urinary tract infections may be the consequence of a genetic pathology rather than cyclosporin-induced alterations.

Megaesophagus in an asthmatic patient and beta2 stimulant treatment by inhalation.

Megaesophagus is a severe esophageal malformation. We report a case of megaesophagus in an asthmatic patient affected by congenital non-haemolytic anaemia and undergoing beta2 stimulant treatment by inhalation. Our case could be due to chronic beta2 receptor stimulation with imbalance of alpha and beta receptor, without any implication of favism.

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.

Relation between the osmolality trend and ornithynedecarboxylase activity in red blood cells of uremic patients during hemodialytic treatment.

In uremic patients during chronic hemodialysis an increase in the volume of red blood cells is observed. Contemporaneously there is an increase in intraerythrocytic ornithynedecarboxylase activity beyond the normal content (P < 0.01), a high level of seric and plasmatic polyamines (P < 0.01) and a decrease in seric osmolality (P < 0.01) with pH improvement. The trends of osmolality, ornithynedecarboxylase, mean cell volume and pH are significantly related. Our data support the hypothesis that, during hemodialysis, red blood cell volume changes and increased ornithynedecarboxylase activity are dependent on the general improvement of plasma tonicity. Moreover, the absence of inhibition of ornithynedecarboxylase activity by high levels of putrescine is noted.

Kaposi's sarcoma in renal transplant recipients: pathogenetic relation between the reduced density of Langerhans cells and cyclosporin-A therapy.

Patients treated with immunosuppressive drugs can develop cancers. The authors present two cases of Kaposi's sarcoma in kidney transplant patients who had been treated with azathioprine, steroids and cyclosporin-A; during this treatment the Langerhans cells decreased and Kaposi's sarcoma appeared. Discontinuation or reduction of the dosage of cyclosporin-A led to complete regression of the illness, and the Langerhans cells reappeared. We suggest that cyclosporin-A damages the immunological function of the epidermal Langerhans cells, and that this is the primary factor in the development of Kaposi's sarcoma.

Ultrastructural features of cyclosporine A-induced gingival hyperplasia.

The present work collected data on the ultrastructural features of the attached gingiva in kidney transplant patients who showed gingival hyperplasia following cyclosporin A (Cy A) treatment. Ultrastructural examination was carried out on biopsies of attached gingiva obtained from 8 male patients (30 to 60 years old) undergoing treatment at the Dental Clinic of the University of Ferrara. The data showed that, although many fibroblasts are present in Cy A-induced hyperplasia, there is a particular abundance of amorphous substance compared to fibrous, as well as marked plasma cell infiltration. On the basis of the data collected, we hypothesize that the morphological features of the dimensional increase in gingival tissue associated with Cy A treatment in kidney transplant patients may be considered local manifestations of a systemic phenomenon.

Ultrastructural and histochemical features of the ground substance in cyclosporin A-induced gingival overgrowth.

The overgrowth-affected gingiva of patients treated with cyclosporin A after kidney transplant was examined with ultrastructural and histochemical methods to evaluate the involvement of connective tissue. Gingival overgrowth has the same clinical signs as local edema. The ultrastructural study showed that the dimensional increase was largely due to increased production of amorphous ground substance by fibroblasts, possibly resulting from an increased release of histamine by mast cells. The histochemical data revealed that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin A induced comparably high levels of glycosaminoglycans in in vitro cultures of fibroblasts obtained from normal gingiva. The combination of ultrastructural and histochemical data, therefore, strongly suggests that the response of the connective tissue in gingival overgrowth cannot be ignored and may be the main cause of the observed pathological condition.

Glycosidases during chick embryo lung development and their colocalization with proteoglycans and growth factors.

During development, the epithelial component of the lung goes through a complex orderly process of branching, following strict patterns of space and time. Proteoglycans, glycosaminoglycans and growth factors are fundamental components of the extracellular matrix and perform a key role in differentiative processes. The embryonic chick lung shows a specific glycosaminoglycan composition at different levels of branching and at different embryonic stages. Proteoglycan and glycosaminoglycan accumulation is the result of secretion, absorption and degradation processes. In this pathway, enzymes, such as glycosidases, growth factors and cytokines are involved. We examined the behaviour of glycosidases, such as beta-hexosaminidases (beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase), beta-glucuronidase and beta-galactosidase, during the development of the lung bud. Our data show that the activity of the enzymes is closely linked to the processes of epithelial proliferation, bronchial tubule lengthening and infiltration of the surrounding mesenchyme. The glycosaminoglycans colocalize with transforming growth factor beta2 and interleukin-1 in the basement membrane and in the mesenchymal areas where the epithelium grows, and are complementary to the presence of the glycosidases. In conclusion, the activity of these glycosidases is spatially and temporally programmed and favors the release of the factors and the events which they influence.

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.

Experimental model for studying the effects of 2-ethylhexyl-phthalate and dialysate on connective tissue.

In order to have a model for studying the possible implications of 2-ethylhexyl-phthalate and dialysate on connective tissue, we evaluated their direct effects on the air pouch lining tissue and on fibroblast cultures. Air pouches were formed on the backs of 60 ten-week-old Wistar rats by subcutaneous injections of 10 ml sterile air. On the tenth day 2 ml sterile air, or 2 ml 5 microg/L or 2 ml 10 microg/L 2-ethylhexyl-phthalate in olive oil, or 2 ml olive oil alone, or 2 ml 5 mg/ml or 12 mg/ml lyophilized dialysate were injected into the air pouches. After sampling at seven or twenty-one days, the rats were killed. The biochemical data showed an increase in sulphated glycosaminoglycans with 2-ethylhexyl-phthalate and dialysate. Electron microscopy findings revealed cellular alterations such as vacuolation and cell remnants with 2-ethylhexyl-phthalate, while the cells of the air pouches treated with dialysate showed regular organelles with increased and dilated cisternae of rough endoplasmic reticulum. Moreover, an increase in collagen fibres surrounding the damaged zones was noticed in 2-ethylhexyl-phthalate and dialysate treated rats. The glycosaminoglycan modifications and collagen fibre increase seem to suggest that the morphological changes, with the features of fibrosis, could be the result of 2-ethylhexyl-phthalate and dialysate action on connective tissue. Moreover, the air pouch technique can be considered a good model for studying the direct effects of 2-ethylhexyl-phthalate and other substances, such as uremic toxins, on connective tissue.

DEHP-induced alterations in the lining tissue of the rat air pouch.

The direct effects of D(2-ethylhexyl)phthalate (DEHP), on subcutaneous tissue were studied in the rat, using the air pouch technique. Two ml of DEHP either undiluted or diluted in olive oil (10 microm/ml), were introduced into a previously created air pouch in the rats. The air pouch was removed and specimens of the lining tissue examined by optical and electron microscopy. The persistence of DEHP for 7 days is sufficient to cause alterations in the tissue. The major histological changes were the destruction of the tissue organization with cytoplasmic and nuclear alterations resulting in apoptotic bodies. The histochemical data showed an increase of sulphated glycosaminoglycans, showing proof of alterations in fibroblast functions. The authors suggest that the hyperplastic-type alteration induced by DEHP may be connected with the sclerosis of the peritoneal membrane described in uremic patients in CAPD.

Tissue expander: histological and histochemical study 6 months after transplant--our experience.

Implanting an expander in the subcutaneous layer causes gradual expansion and provides additional tissue for reconstruction of tissular defects. The force applied remodels the connective tissue and modifies dermis contractibility in additional tissue. Other authors confirm that parameters such as mitosis and hyaluronan influence the system in the tissue regeneration processes. We studied histochemical and morphological variations of tissue expanders before and 6 months after transplant. Our histochemical data do not show any changes in dermis glycosaminoglycans of the expanded and transplant-expanded skin when compared to controls. Morphological data demonstrate reorganization of connective fibers and disappearance of the papillar layer. The latter is not yet formed in the expanded skin 6 months after transplant. This suggests that a long time is required for biological reconstruction of epidermal-dermal interactions after transplant.

Estrogen-induced ultrastructural changes in cultured chick embryo hepatocytes.

Administration of estradiol-17-beta induces the synthesis of vitellogenin in primary cultures of chick embryo liver. The ultrastructural changes accompanying steroid induced vitellogenin synthesis were investigated in hepatocytes cultures incubated for 0, 12 and 24 h following hormonal treatment. Both electron microscopy and immuno-chemical techniques were used. The immuno-fluorescence data indicate that the cultures contain a cell population of 90--95% hepatocytes, both in control and estradiol-treated groups. Ultrastructurally, cultured hepatocytes are similar to in vivo ones, except for a reduced accumulation of storage materials, glycogen and lipids. In the estradiol-treated cultures hepatocytes show an accumulation of free ribosomes, enlargement of rough endoplasmic reticulum, and development of the Golgi apparatus.

Direct inhibitory effect of uremic toxins and polyamines on proliferation of VERO culture cells.

The dialysate fluid of uremic patients exhibits, in vitro, an inhibitory effect on cell growth, owing to urea, guanidino compounds, and substances named middle molecules. The polyamines are compounds which exhibit high levels in biological fluids during either normal development or disease such as psoriasis, uremia, and tumors. Dialysate and middle molecules show toxicity and degeneration of the organotype cultures, whereas the free polyamines and nonrecirculated dialysate do not have any toxic effect. The aim of this study is to analyze the effects of polyamines, nonrecirculated dialysate, and middle molecules of uremic patients in periodic hemodialysis on cultured VERO (fibroblast-like cells) growth. These cells show an inhibition of growth in middle molecules or 2 x 10(-4) M putrescine and a stimulation with nonrecirculated dialysate and 2 x 10(-8) M putrescine. The effect is different because the cultures with middle molecules begin growth again after 24 hr, whereas in the presence of 2 x 10(-4) M putrescine no further growth is observed. Cells maintained in middle molecules + 2 x 10(-8) M putrescine show an irreversible degeneration, attesting a toxic effect due to the low molarities of putrescine. The electron microscopy shows alteration of cytoplasmic, mitochondrial, and nuclear membranes, but no chromatin fragmentation with either middle molecules or 2 x 10(-4) M putrescine: this suggests that the cells do not die of apoptosis. In conclusion, during uremia the polyamines could cause toxic effects, even at low concentrations, on cells stressed by other toxic stimuli.

[Scanning electron microscope microanalysis of in-vitro cultured chick-embryo fibroblasts]. / Microanalisi al microscopio elettronico a scansione di fibroblasti di embrione di pollo coltivati in vitro.

This paper shows that cultured chick embryo fibroblasts derived from lung, heart and skin contain different amounts of sulphurated glycosaminoglycans (GAG). The sulphurated GAGs were detected by X-ray microanalysis at scanning electron microscopy. The results support the current hypothesis on the heterogeneity of the embryonic fibroblast populations.