Rough-Form Lipopolysaccharide Increases Apoptosis in Human CD4⁺ and CD8⁺ T Lymphocytes.

Immunosuppression induced by lymphocyte apoptosis is considered an important factor in the pathogenesis of sepsis and has been demonstrated in both animal models of lipopolysaccharide (LPS)-induced endotoxemia and septic patients. As rough-form LPS (R-LPS) has recently been shown to elicit a stronger immunological response than regular smooth-form LPS (S-LPS), we aimed to assess the apoptosis-inducing capabilities of R-LPS in different subsets of lymphocytes (CD4(+) T cells, CD8(+) T cell, B cells and NK cells). Using multicolour flow cytometry on human peripheral blood mononuclear cells, we found that R-LPS increased apoptosis in CD4(+) and CD8(+) T cells assessed by annexin V and propidium iodide (AV(+) PI(-)), compared with both S-LPS-stimulated and unstimulated cells. 7-Amino-actinomycin D and staining for intracellular active caspase-3, which are considered later signs of apoptosis, did not reveal the same results. Both forms appeared to inhibit apoptosis in B cells, but no LPS-form-specific effect was seen on B or NK cells. Our results indicate that R-LPS induces a stronger AV(+) PI(-)-assessed apoptotic response in T cells than S-LPS. Our findings emphasize the importance of T cell apoptosis in endotoxemia and advocates for control of LPS form in both endotoxemia research and clinical trials with Gram-negative infections.

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells.

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.

Growth hormone antiserum suppresses the growth effect of diabetic serum. Studies on rabbit aortic medical cell cultures.

Growth medium containing serum from young diabetic subjects caused a significant stimulation both of cell proliferation and of the outgrowth in cultures of rabbit aortic medial cells above that noted with normal human sera. The addition to the sera of guinea-pig human growth hormones antibody caused a marked inhibition of these stimulatory effects. The growth effect of rabbit serum was not affected by the human growth hormone antiserum. Reinvestigation of the effect of human growth hormone disclosed that the same increase as observed in growth with the diabetic sera could be obtained with a growth hormone concentration of 0.2 ng. per milliliter medium. The present results strongly suggest that the increased stimulatory effect of normolipemic human diabetic serum on growth and cell proliferation of aortic medial cell cultures is due to increased serum growth hormone concentration.

Diabetic macroangiopathy and growth hormone.

The large vessel disease develops slowly and progressively among most diabetics. According to the concept of a specific diabetic macroangiopathy, the alterations in the large vessels are part of the general diabetic angiopathy and are different from the spotty atherosclerosis. This hypothesis proposes that the changes develop a consequence of the metabolic situation in diabetes. The concept is based on epidemiologic, clinical, and patho-anatomical observations. A model of large-vessel disease in diabetes is briefly described. Diabetic serum causes proliferation of the aortic myomedial cells in culture. Growth hormone causes a similar proliferation. Type 1 procollagen and fibronectin elaboration is enhanced by diabetic serum. The same effect has been found with growth hormone. Insulin treatment in experimental diabetes prevents the proliferation of arterial myomedial cells in the coronary arteries. The presented data are compatible with the concept of a diabetic macroangiopathy distinct from atherosclerosis.

Growth hormone stimulating the growth of arterial medial cells in vitro. Absence of effect of insulin.

Earlier studies have shown a stimulatory effect of diabetic serum on the growth of rabbit aortic medial cell cultures. Growth media supplemented with normal serum with added insulin (50-2,000 muU./ml. serum) did not enhance the growth of the medial cell cultures. Control media containing serum from recent diabetics with low insulin concentration stimulated the growth (2p less than 0.01). Supplementation of normal serum with human growth hormone (final concentration 1-5 ng./ml. medium) resulted in a significant enhancement of growth (2p less than 0.005). The growth-promoting effect of growth hormone was not detectable with lower concentrations (0.5 ng. and 0.1 ng./ml. medium). The growth effect of the low concentration of growth hormone could not be augmented by increasing the concentration of glucose in the incubation medium. Growth hormone in an amount of 1 ng./ml. medium increased both the number of 3H-thymidine-labeled cells as identified by autoradiography and the number of mitotic bodies (2p less than 0.005 and 2 p less than 0.025). The present results demonstrate that the growth-stimulating factor(s) in diabetic human serum described earlier is not insulin but may well be growth hormone.

Diabetic macroangiopathy and atherosclerosis.

In the present study, we have compared and analyzed published data related to the pathogenesis of the large vessel disease in diabetes. The prevailing opinion appears to be that diabetes accelerates the mechanism that leads to the development of classical atherosclerosis. However, as an alternative, we have amassed data that point to the presence of a diabetic macroangiopathy. This phenomenon comprises a constellation of nonatherosclerotic large vessel abnormalities. Today, we know that accumulation of periodic acid-Schiff (PAS)-positive material, as laminin, fibronectin, and type IV collagen, occurs together with hyaluronic acid and various types of connective tissue and calcium deposition. All these changes occur independent of the presence of atherosclerosis in the large vessels of diabetic patients. It seems to us that these observations emphasize that the concept of a specific diabetic macroangiopathy is a more fruitful working hypothesis than the usual theory of a link between atherosclerosis and diabetes. It provides a causal relationship (although the mechanism is unknown) between such changes and the abnormal metabolism in diabetes and a background for research strategy and tactics, aiming finally at the possibility of prevention and/or treatment of this common and dangerous disease.

Growth of rabbit aortic smooth-muscle cells cultured in media containing diabetic and hyperlipemic serum.

Smooth-muscle cell cultures were grown from thoracic aortas of normal and diabetic rabbits. The effect of diabetic rabbit serum on the growth of these cultures was studied both in the first, rapid-growth phase and the following, more "stationary" phase of growth. Control experiments were carried out on normal sera to which glucose had been added. The concentrations of cholesterol, phospholipid, and triglyceride were same in both normal and diabetic sera. Media containing diabetic serum stimulated the growth of cultures significantly in both phases (2p less than 0.01). This occurred in experiments utilizing cells from normal as well as from diabetic rabbits. Control media containing normal serum with added glucose had no such effect. The growth-promoting effects of diabetic serum and of hyperlipemic serum from nondiabetic rabbits were of the same order of magnitude. Autoradiographic studies showed that the number of 3H-thymidine-labeled cells increased significantly after culture in diabetic serum (2p less than 0.005). Cells cultured from the very beginning in diabetic serum or normal serum with added glucose were significantly larger than cells grown in control serum (2p less than 0.05 and 2p less than 0.01, respectively). Cells grown in hyperlipemic serum were significantly smaller than those grown in normal serum (2p less than 0.01). These results indicate that diabetic serum contains a factor or factors that stimulate the arterial medial cell to excessive growth. This factor is not glucose, insulin, or lipid. The results may be of relevance for the understanding of human diabetic macroangiopathy.

Under control of the Ren-1c promoter, locally produced transforming growth factor-beta1 induces accumulation of glomerular extracellular matrix in transgenic mice.

Our purpose was to elucidate the hypothesis that paracrine-produced transforming growth factor (TGF)-beta1 regulates the accumulation of extracellular matrix (ECM) in renal glomeruli, a hallmark of diabetic nephropathy. To produce TGF-beta1 from the juxtaglomerular apparatus in mouse kidneys, we cloned a mouse Ren-1c promoter fragment (-4.100 to +6 base pairs) upstream of porcine TGF-beta1 (pTGF-beta1) cDNA, mutated to ensure secretion of biologically active TGF-beta beta1. The resulting transgenic mice had significantly more TGF-beta1 in their kidneys than was in those of nontransgenic controls, as confirmed by immunohistochemistry, and the production of TGF-beta1 was enhanced in vivo by captopril-induced stimulation of the Ren-1c promoter. Overproduction of pTGF-beta1 close to the glomerulus resulted in a local accumulation of ECM, composed partly of collagen type IV and laminin, and thickening of the basement membrane, characteristic features of diabetic nephropathy. Interstitial accumulation of ECM and signs of tubular atrophy were present only in older mice (>5 months of age). Results from in situ hybridization and immunohistochemistry suggest that pTGF-beta1 stimulated the production of endogenous TGF-beta1 along collecting ducts and connecting tubules. The increased amount of biologically active TGF-beta1, transgenic as well as endogenous, was corroborated by heightened proteoglycan synthesis from incubated kidney slices. This transgenic model demonstrates that sustained local expression of TGF-beta1 leads to glomerulopathy. We conclude that autocrine- or paracrine-produced TGF-beta1 may play a role in the development of glomerular diseases, such as diabetic nephropathy.

Gene transcription of receptors for growth hormone-releasing peptide and somatostatin in human pituitary adenomas.

Growth hormone (GH)-releasing peptides (GHRP) or secretagogs (GHS) constitute a family of synthetic compounds with potent and specific GH releasing activity. The receptor (GHS-R) has recently been cloned even though the endogenous ligand remains to be identified. GHRPs act both at the hypothalamic and the pituitary level through mechanisms involving amplification of GH-releasing hormone activity and functional somatostatin antagonism. In the present study we examined the co-expression of messenger RNA (mRNA) for GHS-R and all 5 somatostatin receptor subtypes (sstr 1-5) in 28 human pituitary tumors by RT-PCR. GHS-R transcription was detected in 11 out of 12 somatotroph adenomas and in 2 out of 2 prolactinomas, whereas GHS-R expression was detected in only 2 out of 14 clinically nonfunctioning adenomas (NFPA), and no expression was seen in the only ACTH secreting adenoma. Almost all tumors expressed sstr 2 mRNA (n = 24), whereas only 1 tumor expressed sstr 4 mRNA. The expression of sstr 3 mRNA was inversely associated with GHS-R expression (P < 0.001), which could be attributed to a high prevalence of sstr 3 expression in NFPA. This study suggests that GHS-R expression is predominantly observed in somatotroph adenomas and much less so in NFPA. Moreover, the presence of a distinct pattern of somatostatin receptor subtype co-expression is suggested, which may provide a molecular basis for the complex interaction between GHRPs and somatostatin.

Effect of growth hormone on steroidogenesis, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 production and DNA synthesis in cultured human luteinized granulosa cells.

Numerous clinical and experimental observations have suggested that GH is important in ovarian function. We have investigated the effect of GH alone and GH in combination with FSH on the secretion of oestradiol, progesterone, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) and on [3H]thymidine incorporation in cultured human luteinized granulosa cells. Granulosa cells from patients undergoing treatment for in vitro fertilization were isolated and cultured for 2 days in culture medium with 10% serum. After this preincubation, the medium was removed and the cells were incubated with GH (1, 10 and 100 micrograms/l) with or without FSH in serum-free medium and in the presence of [3H]methylthymidine (2 microCi/ml). GH alone resulted in a significant dose-dependent increase of oestradiol (P < 0.05) and in IGFBP-1 (P < 0.002) in the medium. The release of IGF-I was undetectable and there was no increase in [3H]thymidine incorporation with GH alone. Neither GH nor FSH alone stimulated granulosa cell proliferation or progesterone release, while the combination induced increases (P < 0.001) in both. The stimulatory effect of GH on steroidogenesis, IGFBP-1 production and granulosa cell proliferation supports a putative role for GH in the regulation of ovarian function.

The effect of human growth hormone on the carbohydrate units in arterial basement membrane-like material.

OBJECTIVE: The present study focuses on the pathogenesis of the large vessel disease in diabetes. The arterial wall from diabetic individuals displays characteristic alterations of the extracellular matrix. Other observations show that the metabolism is changed with increased levels of growth hormone in diabetes. DESIGN: The effects of growth hormone on the carbohydrate composition in the basement membrane around the arterial smooth muscle cells were investigated. METHODS: Basement membrane material was obtained from cultures of smooth muscle cells by sonication and differential centrifugation after labeling with either [(3)H]glucose or [(3)H]glucosamine. The proportions of galactose, glucose, mannose, xylose, fucose and glucosamine were evaluated after addition of 45.45pmol/l human growth hormone. Also, the proportion of glycopeptides generated from the basement membrane was analyzed after fractionation on a combination of a Concanavalin A and a Pea Sepharose column. RESULTS: The proportion of galactose and glucose was changed, and the incorporation of [(3)H]glucosamine was reduced. The proportion of glycopeptides containing high mannose moities was increased as well as that of triantinary glycopeptides with internal fucose residues. CONCLUSION: The current in vitro data indicates that growth hormone may change the carbohydrate composition of the arterial basement membrane.

Effect of insulin and growth hormone on the synthesis of radiolabelled proteoglycans from cultured human arterial smooth-muscle cells.

The present study focuses on the pathogenesis of increased frequency of large-vessel disease in diabetes. The diabetic arterial wall displays characteristic alterations of the extracellular matrix secreted by arterial smooth-muscle cells. The effects of insulin and growth hormone on the synthesis of sulphate-labelled proteoglycans and heparan sulphate proteoglycan were studied. Proteoglycans and heparan sulphate proteoglycan were obtained from the medium and the cell layer of cultured human arterial smooth-muscle cells grown in 5% human serum. Heparan sulphate proteoglycan was quantified using ethanol precipitation combined with specific enzyme degradation. Addition of insulin (0.01, 0.05 and 0.10 mU/ml) induced a significant accumulation of 35S-labelled proteoglycans in the cell layer (2p < 0.005 and 2p < 0.001). The relative amount of cell-associated heparan sulphate proteoglycan increased during insulin stimulation (2p < 0.05). Growth hormone stimulation (5.0 and 10.0 ng/ml) caused a significant decrease in the ratio between heparan sulphate proteoglycan and proteoglycan in the cell layer (2p < 0.02 and 2p < 0.01), whereas the distribution of proteoglycans between the cell layer and the medium was unaltered.

Production of hyaluronan and chondroitin sulphate proteoglycans from human arterial smooth muscle--the effect of glucose, insulin, IGF-I or growth hormone.

BACKGROUND: Although it is recognized that the extracellular matrix is important for cell proliferation, migration and metabolism of growth factors, the regulation of the synthesis of hyaluronan and chondroitin sulphate proteoglycan (CSPG) in the vessel wall is poorly understood. OBJECTIVE: To examine the role of glucose, insulin, IGF-I and human growth hormone (hGH) on the accumulation of hyaluronan and CSPG using cultures of human aortic smooth muscle cells. METHODS: The cultures were exposed for 36 h. The CSPG content in the incubation medium was measured by a combination of digestion with testicular hyaluronidase and precipitation of [35SO4(2-)]-labelled material with ethanol and trichloroacetic acid. Hyaluronan was estimated using a radiometric assay. RESULTS: Glucose and insulin reduced the amount of synthesized hyaluronan (2P<0.01). Stimulation of synthesis was seen with hGH (2P<0.01), whereas no effect was observed with IGF-I. The production of CSPG was increased with glucose and hGH (2P<0.01), but showed no change with insulin. CONCLUSIONS: The present data obtained with human arterial smooth muscle cells in vitro showed that glucose, insulin and hGH can influence the accumulation of hyaluronan and CSPG. These observations may be relevant for an understanding of diabetic macroangiopathy.

Serum from diabetic patients enhances synthesis of arterial basement membrane-like material in cultured smooth muscle cells.

The effect of serum from both type I and type II diabetic subjects on the metabolism of arterial basement membrane (BM)-like material was studied in cultures of rabbit aortic smooth muscle cells. The basement membrane-like material was isolated from the cell-layer by a combined sonication and centrifugation technique. Serum from type I diabetic persons added to the incubation medium increased statistically significantly the incorporation of L-[4,5]-3H-leucine into the basement membrane-like material as compared to serum from non-diabetic subjects (2P less than 0.05). The same effect was seen with serum from type II diabetic patients as compared to serum from nondiabetic subjects (2P less than 0.05). No effect of serum from type I diabetic persons was seen in degradation experiments. Incubation medium supplemented with normal serum and extra glucose neither changed the production of basement membrane-like material nor the disappearance rate of radioactive leucine from the basement membrane-like material in degradation experiments. The present study indicates that serum from diabetic subjects enhances the production of arterial basement membrane-like material from arterial smooth muscle cells in culture. The obtained data may be relevant for the understanding of the development of macroangiopathy among diabetic patients.

Octreotide and diabetes: theoretical and experimental aspects.

Diabetes is characterized by paradoxical hypersomatotropinemia and hyperglucagonemia. The latter appears to enhance the tendency in imperfect metabolic control to reduce nitrogen balance, and the former appears to accelerate the deterioration of carbohydrate and lipid metabolism, and also to induce peripheral insulin resistance and hyperinsulinemia. In addition to direct metabolic effects, increasing evidence points to an association between hypersomatotropinemia and a number of metabolically dependent, characteristic functional abnormalities linked to the development of late diabetic manifestations. These include increased capillary fragility, lipid and hemostatic aberrations, tissue hyperperfusion, including increased cardiac output and renal plasma flow, and kidney hypertrophy. In theory, octreotide's actions could reduce these aberrations, and, in fact, this has been confirmed in recent experimental trials.

Oligosaccharides of arterial basement membrane-like material studied on rabbit aortic myomedial cells in culture.

Oligosaccharides from myomedial basement membrane-like material were characterized in respect of size, attachment to protein and charge heterogeneity after metabolic labelling with [3H] glucosamine. Arterial basement membrane-like material was isolated from cultures of aortic myomedial cells by a sonication-differential centrifugation technique. Glycopeptides were then obtained by proteinase digestion and Sephadex G-50 chromatography. Alkaline borohydride treatment of the glycopeptides followed by Sephadex G-25 chromatography revealed that 10-14% of the oligosaccharides were released by a beta-eliminative reaction. The molecular weight of the alkali labile carbohydrate units was estimated to be approximately 750. Alkali stable oligosaccharides were released from the glycopeptides by hydrazinolysis. The liberated oligosaccharides were retarded by Sephadex G-50 chromatography corresponding to a molecular weight of 2700 using unit B thyroglobulin oligosaccharides as standard. The chromatographic pattern obtained by anion exchange chromatography of the glycopeptides after neuraminidase treatment showed that a major part of the glycopeptides contain one or more sialic acid residues, but also other negatively charged groups--possibly phosphomannosyl residues were present as suggested by [32PO4(3-)] labelling and concanavalin-A-Sepharose chromatography. Concanavalin-A-Sepharose chromatography combined with alpha- mannosidase treatment and gel filtration suggested that a minor part of the glycopeptides were of high-mannose- type.

Proteolysis of arterial basement membrane containing different amounts of carbohydrates.

The arterial basement-membrane-like material was isolated from cultures of rabbit aortic myomedial cells by a sonication/differential-centrifugation technique. Using exoglycosidases the enzymic removal of fucose, glucose, mannose, N-acetylglucosamine and sialic acid resulted in a statistically significant increased susceptibility against the tryptic degradation (2 P less than 0.02), whereas the removal of galactose did not have any effect on the degradation of the basement-membrane-like material. Application of endo-beta-N-acetylglucosaminidase H caused a statistically significant increased sensitivity against trypsin digestion (2 P less than 0.02), whereas the proteolytic degradation of the basement-membrane-like material was unchanged when endo-beta-N-acetylglucosaminidase D was applied.

Atmospheric air vs. normal middle ear gas: effects on in vitro growth and collagen synthesis in normal middle ear fibroblasts.

The present study was undertaken to quantitate the effects of atmospheric air and normal middle ear gas on cultured fibroblasts obtained from normal rabbit middle ear mucosa. The cells were exposed to three different gas compositions: 7% O2:5% CO2:88% N2, 21% O2:5% CO2:74% N2, and 75% O2:5% CO2:20% N2. The growth was monitored by measuring the total content of cell protein, the amount of DNA, and the cell division activity. The activity of the synthetic apparatus was determined by the collagen synthesis. For comparison, rabbit skin fibroblasts were grown under identical conditions. The results demonstrated significantly higher replication rate of middle ear fibroblasts at 7% oxygen than at atmospheric air whereas the collagen synthesis was significantly lower at 7%. Furthermore, the responses varied significantly between rabbit middle ear and rabbit skin fibroblasts. Thus the present study substantiates the hypothesis of an influence of atmospheric air on the middle ear mucosa which might be of importance, e.g., in relation to insertion of ventilation tubes or longstanding perforations of the tympanic membrane in otitis media.

Keratocyte migration and peptide growth factors: the effect of PDGF, bFGF, EGF, IGF-I, aFGF and TGF-beta on human keratocyte migration in a collagen gel.

PURPOSE: Peptide growth factors are known accelerators of corneal wound healing, probably mediated through increased proliferation of the cells; however, information about their effect on keratocyte motility is lacking. The influence of peptide growth factors on keratocyte migratory activity was investigated, using the following growth factors: platelet derived growth factor (PDGF-BB), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-beta 1). METHODS: Keratocytes were seeded on gels of type 1 collagen, growth factor added, and the cells left to migrate for 72 hours. Subsequently, the number of keratocytes at the different levels in the collagen gel was evaluated by optically sectioning the gel at 20 microns, intervals, with an inverted phase contrast microscope. RESULTS: PDGF, EGF and bFGF at 10 ng/ml, all increased the number of keratocytes at the different levels of the gel as compared to a non-stimulated control (p < 0.05 or p < 0.01, students t-test). TGF-beta proved to be a strong inhibitor of keratocyte migration, decreasing the number of keratocytes observed at every level in the gel (p < 0.05 and p < 0.01, students t-test), whereas no effect of IGF-I and aFGF was found. During the 72 hours of migration, no contraction of the collagen gels was observed. Autoradiography of histological sections of the gels showed that during the 72-hour period only TGF-beta and 10% fetal bovine serum induced an increase in keratocyte proliferation. CONCLUSION: PDGF, EGF and bFGF increase keratocyte migration, independent of proliferation in a collagen gel invasion assay and might promote corneal wound healing, not only by increasing cell proliferation, but also through increased motility.

The keratocyte density of human donor corneas.

The cellularity of the human corneal stroma has not been described in the literature. In the present study we calculated the density of keratocytes in human donor corneas using a new method for biochemical measurement of the stromal DNA content (sDNA). The DNA measurements were compared to morphological counts of the number of keratocyte nuclei per area (KNPA) obtained from histological sections. A significant correlation was found between the data achieved by the two methods (r = +0.52, p < 0.001, n = 46). No significant change in either sDNA or KNPA was found during 28 days of organ culture, and no influence of donor age, sex, or post mortem time was found on either sDNA or KNPA. Both sDNA and KNPA approximated a normal distribution with a mean sDNA of 1.10 +/- 0.25 micrograms DNA/mg dry tissue weight and an average KNPA of 200 +/- 53 nuclei/mm2 (n = 35). Between paired corneas the sDNA were closely correlated (r = +0.83, p < 0.005, n = 11 pairs) with an intra-individual variation of only 0.5%. Using the sDNA data, the keratocyte density in the central region of human donor corneas was calculated to be 129,000 +/- 29.000 per mg dry tissue weight (n = 35). Thus, when corneal grafting is performed (using a 7 mm trephine) an average of 818,000 +/- 186,000 donor keratocytes are transplanted. Assuming a uniform cellularity throughout the stroma, the average number of keratocytes was calculated to be 2,430.000 +/- 551,000 per human donor cornea.(ABSTRACT TRUNCATED AT 250 WORDS)