Cell death in the rat thymus: a minireview.

During the last decades, the literature has clearly established the fundamental role of the thymus in the development of an effective immune system. During thymocyte development and maturation, potentially autoreactive thymocytes are eliminated by a process known as apoptosis or programmed cell death responsible for the negative selection occurring within the thymus. This process is in sharp contrast to other types of cell death referred to as necrosis. Actually, three different types of cell death have been recently observed morphologically in the rat thymus, i.e. necrosis, apoptosis and clustered cell death. Moreover, among the numerous factors influencing thymocyte cell death, particular attention has been paid to hormones, chemicals, biological compounds and physical agents that may influence the type and/or the extent of cell death. Finally, a brief overview has been devoted to the contribution of mitochondria, nitric oxide, glutathione and intracellular levels of cations in addition to the activity of genes as cdk2, p53, Fas and members' of the Bcl2 family in modulating rat thymus cell death.

Morphological analysis of articular cartilage biopsies from a randomized, clinical study comparing the effects of 500-730 kDa sodium hyaluronate (Hyalgan) and methylprednisolone acetate on primary osteoarthritis of the knee.

OBJECTIVE: Histomorphometric study on cartilage samples taken from osteoarthritic human knees before and 6 months after intraarticular injections of a specific fraction (500-730 kDa) of hyaluronan. The results obtained with hyaluronan were compared with the results of methylprednisolone acetate treatment. METHODS: Twenty-four subjects with primary osteoarthritis (OA) of the knee were considered. Eleven patients were treated with Hyaluronan (Hyalgan), 20 mg/2 ml once a week for 5 weeks) and 13 with methylprednisolone (Depo-Medrol, 40 mg/1 ml once a week for 3 weeks). At the time of baseline and after 6 months from the start of treatment, biopsies of cartilage were taken and processed for electron microscopy. Articular surface morphology, territorial matrix, chondrocyte number and ultrastructure were characterized by a set of morphometric parameters. Samples from 19 informed patients showing no arthroscopic sign of OA were also used for comparison. RESULTS: Six months after hyaluronan treatment a significant reconstitution of the superficial layer were observed together with an improvement in chondrocyte density and territorial matrix appearance. Furthermore, chondrocytes appeared significantly improved in their metabolism, as indicated by the increased extension of the synthetic structures and mitochondria with respect to the organelles having catabolic or storage functions. Hyaluronan treatment produced results that were significantly superior to those delivered with Methylprednisolone in almost all the morphometric estimators. CONCLUSIONS: These results cannot be explained simply by temporary restoration of the synovial fluid viscoelasticity, and provide further evidence that the specific fraction of hyaluronan used in this study is a useful tool in OA treatment, with a potential structure-modifying activity.

Modulation of cell death in the rat thymus. Light and electron microscopic investigations.

In mammals, the thymus is the primary central organ of the lymphoid system; after birth, it progressively diminishes in size, undergoing gradual atrophy. Physiological maturation and/or involution of the thymus may be accelerated by endogenous or exogenous factors. Exposure to extremely low frequency EMF seems to interfere with thymic cell death. Data suggest that, in the rat model, a prolonged exposure to 50 Hz electric and magnetic fields, independently from field strength, seems to affect thymic cell death and possibly thymic physiology, since alterations in the balance of cell death and other parameters such as mitoses might interfere with the positive and negative selection of thymocytes and with the immunosurveillance properties of the thymus.

Identification of heterozygote carriers in families with a recessive form of pseudoxanthoma elasticum (PXE).

Skin biopsies of 18 healthy relatives of patients with pseudoxanthoma elasticum (PXE), belonging to six different recessive families, have been examined by optical and electron microscopy in order to determine morphologic alterations potentially useful for the identification of carriers of this genetic disorder. These morphologic features have been compared with those observed in the same tissue areas of eight PXE patients belonging to the same families, with six normal subjects, and to the carrier status of these apparently unaffected relatives as determined by haplotype analysis using informative markers surrounding the locus of the PXE gene on chromosome 16p. The dermis of all the relatives of PXE patients, established by haplotype analysis to be heterozygote carriers of a mutation in the PXE gene, exhibited several alterations very similar, although less severe, to those typical in PXE patients. Alterations were present in the reticular dermis and consisted of irregular-sized collagen bundles and elastic fibers; elastic fibers fragmented, cribriform, and mineralized; numerous fibroblasts, larger than normal, and subendothelial elastin in small vessels. Strikingly, none of these dermal changes were noted in an unaffected relative in one family who was identified as a noncarrier by haplotype analysis. Although many of these alterations are not specific for PXE, the presence of these morphologic changes in unaffected relatives of PXE patients indicates alterations in skin that could be diagnostic for carriers of a subclinical phenotype of PXE.

The effect of caloric restriction on the aortic tissue of aging rats.

Connective tissue shows peculiar and complex age-related modifications, which can be, at least in part, responsible for altered functions and increased susceptibility to diseases. Food restriction has long been known to prolong life in rodents, having antiaging effects on a variety of physiologic and pathologic processes. Therefore, the aorta has been investigated in rats fed normal or hypocaloric diet, from weaning to senescence. Compared with controls, caloric-restricted animals showed less pronounced age-dependent alterations such as elastic fiber degradation, collagen accumulation and cellular modifications. Immunocytochemical analyses revealed that elastic fibers were positively labelled for biglycan, decorin, ApoB100 (LDL), ApoA1 (HDL) and elastase and that the intensity of the reactions was time- and diet-dependent. With age, the major changes affecting aortic elastic fibers were increased positivity for decorin, LDL and elastase. Compared with age-matched normal fed rats, caloric restricted animals revealed lower content of LDL, decorin and elastase and higher positivity for HDL. These data suggest that a caloric restricted diet might influence the aging process of the arterial wall in rats, delaying the appearance of age-related degenerative features, such as structural alterations of cells and matrix and modified interactions of elastin with cells and with other extracellular matrix molecules.

Age-dependent remodeling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age.

Structural and phenotypic modifications of rat thymocytes from birth up to one year of age, i.e. during maturation and at the beginning of the involutive process of the thymus are described. Since the biological significance and the mechanisms of thymic involution are still a matter of debate, this study aims at clarifying the complexity of the compensatory events occurring during this relatively neglected period of time. Thymuses from Sprague-Dawley rats were analyzed morphologically and morphometrically by light and electron microscopy. At the same time, thymocyte subsets, isolated from the same animals, were characterized by flow cytometry according to physical parameters and phenotypic markers. Results indicate that major changes occur during the first month from birth and from six months onward. In particular, already during the first weeks after birth, thymocytes undergo a slight reduction of mitoses associated with an increased number of apoptoses. Moreover, during the same period of time, flow cytometry revealed an expansion of small thymocytes and changes in thymocyte subsets such as increase of CD4+CD8+ and CD5+alpha(beta)TCR- and a decrease of CD4-CD8-, CD4-CD8+ cells. The thymus of adult rats was characterized by time-dependent decrease of both mitoses and apoptoses, progressive physical disconnection among cells, increase of necrotic areas and fibrosis. Around one year of age tissue changes were associated with a dramatic reduction of the population of large thymocytes and the rise of numerous small thymocytes that were unexpectedly negative for all tested markers. By contrast, medium-size thymocytes exhibited a marked decrease of CD4+CD8+ and CD5+alpha(beta)TCR- subsets. In conclusion, our data indicate that thymus undergoes, with time, a complex remodeling and suggest that thymic involution is not only a simple shrinkage of the organ but rather the result of a series of compensatory mechanisms among different cell populations in a setting of progressive involution.

Cell behavior and cell-matrix interactions of human palmar aponeurotic cells in vitro.

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren's affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren's aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha 2, alpha 1, and alpha 5 integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren's patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren's patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren's contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren's disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.

Earthworm coelomocytes in vitro: cellular features and "granuloma" formation during cytotoxic activity against the mammalian tumor cell target K562.

Earthworms possess specific, adaptive, cellular immunodefense as well as non-specific responses found in other complex metazoans. Here we characterized coelomocytes from the earthworm Eisenia foetida by electron microscopy and cytofluorimetric analyses, and investigated structural changes that occur when effector coelomocytes and target K562 erythromyeloid human tumor cells interact during cytotoxic activity. In in vitro cultures 1) the two earthworm cell types (i.e. small and large coelomocytes) retained their morphological features; 2) their DNA content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line K562; 3) significant percentages of coelomocytes were found to be in S or G2/M phases of the cell cycle. When cultivated alone for up to 3 h, coelomocytes formed no aggregates. However, when mixed with K562, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. These results are the first to describe this type of earthworm non-specific "inflammatory" response in vitro against tumor cells.

Matrix proteins with high affinity for calcium ions are associated with mineralization within the elastic fibers of pseudoxanthoma elasticum dermis.

Ultrathin sections from the dermis of five normal subjects and from 10 patients suffering from pseudoxanthoma elasticum (PXE) were analyzed by immunoelectron microscopy with the aim of identifying and localizing proteins associated with the mineral precipitates within the altered elastic fibers. Serial sections were processed by indirect immunogold cytochemistry using primary antibodies against human fibronectin, vitronectin, bone sialoprotein, alkaline phosphatase, osteonectin, and osteopontin. In the latter two cases, antibodies against synthetic peptides were also used. The results indicate that normal elastic fibers contained osteopontin, and that this protein was associated with the apparently normal elastin as well as with the needle-shaped mineral precipitates in the elastic fibers of patients. On the contrary, significant amounts of vitronectin, alkaline phosphatase and, less, of bone sialoprotein were associated with the polymorphous mineral precipitates inside the elastic fibers. Large amounts of osteonectin and fibronectin, together with vitronectin, were localized on the microfilament aggregates, which were often associated with altered elastic fibers in PXE dermis and were never visualized in the dermis of control subjects. The results seem to indicate once more that PXE is a complex disorder of the fibroblast synthetic control. Elastic fiber mineralization might be considered a secondary event, which could depend on the abnormal synthesis and accumulation within the elastic fibers of proteins that are normally involved in mineralization processes.

Ultrastructural and immunocytochemical study on normal human palmar aponeuroses.

BACKGROUND: Human palmar aponeurosis can be affected by a fibrotic process whose aetiopathology is unknown. As the organization of that normal tissue has not been completely investigated, the aim of the present study was to define the ultrastructure of the aponeurosis in order to better understand its biology and behaviour in pathology. METHODS: Bioptic samples from normal subjects of different ages were analysed by optical and electron microscopy and by immunocytochemistry. RESULTS: The aponeurotic branches consisted of thick, almost parallel collagen bundles containing columns of prominent cells, characterized by long cytoplasmic projections. Cells did not change in number and distribution with age and appeared longer and slighter in the old than in the young subjects. They exhibited plasma membrane almost completely decorated by pinocytic vesicles, intracytoplasmic bundles of thin filaments with zonal thickenings close to the cell membrane, and well-developed subcellular structures. Cells expressed smooth muscle cell alpha-actin, as revealed by immunostaining. The external surface of the plasma membrane was underlined by a discontinuous basement membrane-like structure and by a thick coat of interwoven filaments, highly positive to hyaluronan-recognizing antibodies. Immunocytochemical analyses revealed that collagen fibrils were positive for collagen types I, III, and VI and that elastin fiber composition was rather complex. CONCLUSIONS: Independently of the age, normal palmar aponeurotic cells show peculiar morphological features and peculiar cell-matrix interactions, very likely mediated by hyaluronan. These findings indicate that normal aponeurotic cells cannot be regarded as typical tenocytes and suggest the need for a better definition of their phenotype in order to understand their behaviour in pathological processes.

Elastin production and degradation in cutis laxa acquisita.

A case of cutis laxa acquisita was studied with the aim of defining the molecular defects involved and comparing them with those of an inherited form of cutis laxa. In the acquisita form of cutis laxa ultrastructural and biochemical observations confirmed a dramatic reduction of dermal elastin, whereas collagen content was normal. Elastin mRNA expression as well as tropoelastin production by dermal fibroblasts, in vitro, were normal compared with control cells, as revealed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Lysyl oxidase activity, measured on cultured fibroblasts, was reduced to 60% compared with age-matched control subjects. Unlike control skin fibroblasts or fibroblasts from inherited cutis laxa, the affected skin cells from cutis laxa acquisita predominantly expressed an elastolytic activity identified as cathepsin G. Patient serum also has reduced elastase inhibitory capacity and reduced levels of alpha 1-antiproteinase inhibitor (alpha 1-antitrypsin). Although cutis laxa acquisita is a heterogeneous group of disorders, findings in this patient were consistent with excessive loss of cutaneous elastin due to the combined effects of several factors, such as low lysyl oxidase activity together with high levels of cathepsin G and reduction of circulating proteinase inhibitor(s).

Effects of dehydroepiandrosterone and carnitine treatment on rat liver.

It is well established that DHEA treatment is associated in the rat to an increase in fatty acids metabolism. This condition would require levels of L-carnitine much higher than those physiologically present in the liver. The possibility thus exist that during DHEA treatment the concentration of L-carnitine may become a limiting factor for fatty acids oxidation and therefore responsible of some of the effects observed after administration of the hormone. The present experiments were designed to test this hypothesis. The results show that the increase in the levels of peroxisomal enzymes induced in hepatocytes by DHEA, is greatly reduced by parallel administration of L-carnitine. Furthermore, L-carnitine administration counteracts the effect of DHEA on mitochondrial structure. On the contrary, carnitine has no significant effect on the reduction in weight gain observed upon short- or long-term treatment with DHEA.

Epidemiological and structural findings supporting the fibromatous origin of dorsal knuckle pads.

Epidemiological, optical and electron microscopical findings suggest that dorsal knuckle pads and Dupuytren's disease are fibrosing disorders with common features. In all cases examined, knuckle pads were always associated with Dupuytren's contracture and, in a significant number of cases, with bilateral Dupuytren's contracture. In a statistically significant number of patients with knuckle pads, Ledderhose's and Peyronie's diseases were also present (P less than 0.001). Optical and electron microscopical studies showed that cell types and extracellular matrix were identical in knuckle pads and Dupuytren's nodules in different patients.

Elastofibroma: an in vivo model of abnormal neoelastogenesis.

Thirteen cases of elastofibroma have been studied by conventional light and electron microscopy, as well as by histochemistry and immunohistochemistry. By light microscopy elastinophilic material appeared as huge fibers crossing collagen bundles. Immunohistochemistry demonstrated a strong positivity for elastin in numerous and circumscribed areas of the extracellular matrix. By electron microscopy, collagen consisted of 40-50-nm wide fibrils, and elastin was made of large aggregates of moderately electron-dense material surrounding a very thin, apparently normal, elastin core. At high magnification these aggregates consisted of short tubules, often in regular arrays, surrounded by microfibrils and microfilaments. These data, associated with selective digestions on thin sections with elastase, purified collagenase, hyaluronidase, and chondroitinase ABC, revealed that elastic fibers in elastofibroma seem to be made of true elastin surrounded by an enormous amount of hydrophilic material, in which some elastin, chondroitin sulfates, and collagenase type-VII sensitive material are aggregated forming a rather ordered array of short tubules.

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein.

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 +/- 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 +/- 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.

Familial Ehlers-Danlos syndrome type II: abnormal fibrillogenesis of dermal collagen.

We examined a father and son affected by Ehlers-Danlos syndrome type II. Both patients had micrognathia together with ligament and skin hyperlaxity. The son exhibited complete cleft palate. Ultrastructural studies revealed abnormal collagen fibrils in the dermis of both patients. In the child the most striking alterations consisted of lateral fusion of an enormous number of collagen fibrils giving rise to huge polymorphic collagen masses. In the father's dermis the great majority of collagen fibrils appeared normal; however, lateral fusion of fibrils together with local abnormal collagen aggregation were occasionally seen. In both patients the dermal elastic network was well developed and elastic fibers appeared normal.

Alterations of elastin fibrogenesis by inhibition of the formation of desmosine crosslinks. Comparison between the effect of beta-aminopropionitrile (beta-APN) and penicillamine.

Experimental lathyrism was induced by feeding newborn chicks a diet containing 0.2 and 0.4% DL-Penicillamine, with or without CuSO4 (10 mg/Kg diet) and Vitamin B6 (100 mg/Kg diet), or 0.015 and 0.1% beta-aminopropionitrile fumarate (beta-APN). After 7, 15, 25 and 55 days of treatment the animals were killed, the aortas removed and processed for electron microscopy in the presence of markers for proteoglycans, and the elastic fibers were carefully examined. Penicillamine, which prevents the formation of desmosine crosslinks by binding to precursors, induced the production of numerous new elastin fibers which appeared normal from the ultrastructural point of view. It seems, therefore, that at least in chick aortas, desmosine crosslinks are not necessary for the aggregation of tropoelastin molecules into structurally normal fibers. On the contrary, beta-APN, a classical inhibitor of lysyl oxidase, induced the tropoelastin molecules to aggregate into abnormal protuberances on the old fibers. Moreover, the elastin deposited during beta-APN treatment was always permeated by cytochemically revealed proteoglycans, which were never observed after penicillamine treatment. It is speculated that, at least in the system under study, the epsilon-amino groups of tropoelastin molecules may offer the binding sites for matrix proteoglycans until they are removed by lysyl oxidase, and that matrix proteoglycans might play a role in elastin fibrogenesis by preventing spontaneous tropoelastin aggregation in areas far from growing elastin fibers.

Elastin-proteoglycans association revealed by cytochemical methods.

By using various cytochemical stains, proteoglycans are shown to be present inside elastic fibers in aortas of beta-aminopropionitrile-induced lathyritic chicks. Depending on the characteristics of the dyes, the shape, size and distribution of the proteoglycan-revealing precipitates are described. The monocationic dye toluidine blue O and the tetracationic dye Alcian blue in the presence of 0.3 M MgCl2 give the most detailed results. With these stains the proteoglycans inside lathyritic elastin appear to be lateral branches of matrix proteoglycans, lying on the external surface of the elastic fibers. A possible general biological significance of elastin-proteoglycan association is briefly discussed.

Contribution of cryotechniques to the study of elastin ultrastructure.

Cryomethods were used in order to investigate the ultrastructure of native elastin fibres from beef ligamentum nuchae. Filaments of diameter 5 nm, running almost in parallel in purified, negatively-stained elastin preparations, were also seen running along the elastin fibre both in freeze-fractured and etched elastin, that had been stretched up to 200%, and in cryo-sectioned elastin that had been stretched and chemically fixed before freezing. Interconnections between elastin filaments were revealed by the freeze-etching technique. Glycerol treatment, which probably leads to hydration of specimens, resulted, however, in disorganization of filaments and swelling of the elastin fibre. In conclusion, by the use of cryotechniques, it was convincingly demonstrated that elastin molecules are arranged in long interconnecting filaments of about 4-5 nm width.