Serum levels of C-terminal telopeptide of type I collagen: a useful new marker of cortical bone loss in hemodialysis patients

Renal osteodystrophy is a major complication in hemodialysis patients. Measurement of serum peptide derived from the degradation of bone collagen could potentially provide an indirect estimate of bone resorption. The present study estimated the significance of the C-terminal telopeptide of type I collagen (-CTx) as a serum bone resorption marker in male hemodialysis patients. The mean age and hemodialysis duration of the 160 patients were 59.7 years (26–86 years) and 67.2 months (17–142 months), respectively. Bone mineral density (BMD) in the distal third of the radius was measured using dual-energy X-ray absorptiometry twice with a 2-year interval. A blood sample was collected immediately before the hemodialysis session at the time of the second BMD measurement. Other serum bone markers determined were bone-specific alkaline phosphatase (BAP) and intact and N-terminal midfragment (N-Mid) osteocalcin (OC) as bone-formation markers and serum pyridinoline (PYD) and deoxypyridinoline (DPD) as bone resorption markers. Serum -CTx correlated significantly in a positive manner with serum PYD, DPD, BAP, intact OC, and N-Mid OC. Serum -CTx, as well as PYD, DPD, BAP, intact OC, and N-Mid OC, correlated significantly with BMD in the distal third of the radius at the second measurement and with the rate of BMD reduction during the preceding 2 years. The highest quartile of serum -CTx was positively associated with rapid bone loss, defined as a change in the value for BMD in the distal third of the radius falling within the upper tertile of patients, in 55% of cases, and each quartile progress in serum -CTx increased the odds ratio of rapid bone loss by a factor of 1.73. Since the Youden index was twice as accurate for -CTx, BAP and N-Mid OC as for intact PTH, these bone-remodeling markers may be better risk markers of cortical bone loss than intact PTH. Inclusion in the highest quartile of PTH (above 288 pg/ml) predicted rapid bone loss with a sensitivity of only 26%. This means that the upper limit for serum PTH level recommended by K/DOQI may be too high, since 74% of cases with rapid bone loss showed serum PTH levels of below 288 pg/ml. In conclusion, serum measurement of -CTx may provide a new commercially viable and relevant serum assay to reflect cortical bone resorption in hemodialysis patients.     

Potentiation of transforming growth factor (TGF-β1) by natural coral and fibrin in a rabbit cranioplasty model

The association of a biodegradable material and a growth factor could be of clinical value for treating bone defects. We therefore tested the association of transforming growth factor (TGF-1) in fibrin glue and coral granules to heal skull defects in rabbits. Adult rabbits underwent a double trepanation symmetrically in both parietal bones. Using histomorphometry, we compared bone repair after 1 month in control animals (n=5) and in animals treated with either TGF-1 as a single injection of 1 g in methylcellulose (n=5) or in fibrin glue (n=5), or with coral granules in fibrin glue (n=4) or with coral granules and TGF-1 1 g in fibrin glue (n=5). We measured the diameter of the remaining defect and the surface of the bone growth. TGF-1 without coral in either methyl cellulose or fibrin induced a partial closure of the defect as assessed by a significant decrease in the defect diameter, compared with the control group. However, the association of TGF-1 in fibrin and coral induced an area of the bone growth higher than in any other groups (P<0.05). Two months after surgery, this triple association induced a better healing of the defect than coral alone or control group. In each group treated with TGF-1, the mineralization rate was increased not only at the treated side but also in the contralateral defect which was untreated, suggesting a diffusion of the growth factor. Indeed, when pooled together, the diameter of the defect at the contralateral side of 14 animals that had received TGF-1 was reduced compared with the control group. Significant coral granules resorption occurred between month 1 and 2 and was unchanged by the addition of TGF-1. In conclusion, the triple association of coral granules and TGF-1 in fibrin could be of interest for treating bone defects.     

Extracellular matrix and integrin composition of the normal bladder wall

Summary We performed an immunohistochemistry study of the normal human bladder so as to understand the interactions of extracellular matrix (ECM) components and the integrins of cell adhesion that accommodate the volume changes and maintain an impermeable barrier to reabsorption of urine in the bladder. The normal human urothelial cell and/or its plasma membrane contained integrins 3, V, 1, and 4 but did not contain integrin 3. The urothelial basement membrane (UBM) contained collagen type IV and laminin. Fibronectin and integrins 3 and 4 were found in or near the UBM area, with types I and III collagen and tenascin abutting the area. The patterns of collagen, laminin, tenascin, vitronectin, fobronectin, and the 3, V, 1, and 3 integrins in the lamina propria, vessels, nerves, and smooth-muscle layers are described. These findings detail the normal anatomical ECM/integrin relationship that provides the cellular basis for bladder-wall relationships responsible for its impermeable state and other functions.     

Comparison of adrenoceptor subtype expression in porcine and human bladder and prostate

We have quantified and characterized ₁-, ₂-and -adrenoceptor subtypes in porcine bladder detrusor and bladder neck, human bladder detrusor, and porcine and human prostate. ₁-, ₂- and -adrenoceptor were identified in radioligand binding studies using [³H]prazosin, [³H]RX 821002 and [¹²⁵I]iodocyanopindolol, respectively, as the radioligands. In porcine male and female detrusor and bladder neck and male prostate, adrenoceptors were detected in the order of abundance > _{2 1} (not detectable), with no major differences between the sexes or between detrusor and bladder neck. In human detrusor and prostate the order of abundance was > _{2 1} (not detectable) and ₁ > ₂. respectively. The ₂-adrenoceptors in all tissues were homogeneously of the _2A-subtype as evidenced by competition binding studies with yohimbine, prazosin, ARC 239 and oxymetazoline. The -adrenoceptors represented a mixed population with a dominance of the ₂-subtype in all tissues as demonstrated by competition binding with ICI 118,551 and CGP 20,712A. We conclude that pigs may be a suitable model for studies of detrusor function with respect to adrenoceptor expression. They may be less suitable for studies of bladder neck or prostate function.     

Methylated β-cyclodextrins are able to improve the nasal absorption of salmon calcitonin

The absorption enhancing effect of methylated -cyclodextrins on the nasal absorption of salmon calcitonin (sCT) was studied in rats and rabbits. The nasal absorption of sCT following administration without additives was low in both species. The absorption in rats could be largely improved by coadministration of cyclodextrins as apparent from the effect on serum calcium concentrations. Trimethyl--cyclodextrin (TMCD), at a concentration of 5% (w/v), was the least potent enhancer. Randomly methylated--cyclodextrin (RMCD) and dimethyl--cyclodextrin (DMCD), all at a concentration of 5% (w/v), were almost equally effective in decreasing serum calcium levels, and the hypocalcemic responses were similar to those of i.v. and s.c. injected sCT. Absorption enhancement was already achieved with 1% DMCD added to the nasal formulations. In rabbits, only the effect of DMCD on the nasal sCT absorption was investigated. A total serum calcium decrement in 4 hours of 9.4±3.9% (mean±SD) was observed following nasal administration of 12.6 IU/kg sCT with 5% DMCD, comparable to that of i.v.-injected sCT. In conclusion, the methylated cyclodextrins DMCD and RMCD are suitable absorption enhancers for nasal sCT administration, which is expected to have a clinical impact on the therapy with calcitonin.     

Anterior lumbar interbody fusion with carbon fiber cage loaded with bioceramics and platelet-rich plasma. An experimental study on pigs

Platelet-rich plasma (PRP) is an autogenous source of growth factor and has been shown to enhance bone healing both in clinical and experimental studies. PRP in combination with porous hydroxyapatite has been shown to increase the bone ingrowth in a bone chamber rat model. The present study investigated whether the combination of beta tricalcium phosphate (-TCP) and PRP may enhance spinal fusion in a controlled animal study. Ten Danish Landrace pigs were used as a spinal fusion model. Immediately prior to the surgery, 55 ml blood was collected from each pig for processing PRP. Three-level anterior lumbar interbody fusion was performed with carbon fiber cages and staples on each pig. Autogenous bone graft, -TCP, and -TCP loaded with PRP were randomly assigned to each level. Pigs were killed at the end of the third month. Fusion was evaluated by radiographs, CT scanning, and histomorphometric analysis. All ten pigs survived the surgery. Platelet concentration increased 4.4-fold after processing. Radiograph examination showed 70% (7/10) fusion rate in the autograft level. All the levels with -TCP+PRP showed partial fusion, while -TCP alone levels had six partial fusions and four non-fusions (P=0.08). CT evaluation of fusion rate demonstrated fusion in 50% (5/10) of the autograft levels. Only partial fusion was seen at -TCP levels and -TCP+PRP levels. Histomorphometric evaluation found no difference between -TCP and -TCP+PRP levels on new bone volume, remaining -TCP particles, and bone marrow and fibrous tissue volume, while the same parameters differ significantly when compared with autogenous bone graft levels. We concluded from our results in pigs that the PRP of the concentration we used did not improve the bone-forming capacity of -TCP biomaterial in anterior spine fusion. Both -TCP and -TCP+PRP had poorer radiological and histological outcomes than that of autograft after 3 months.     

Dexamethasone regulates IL-1β and TNF-α-induced interleukin-8 production in human bone marrow stromal and osteoblast-like cells

We have investigated both constitutive- and cytokine-induced secretion of interleukin-8 (IL-8) and its regulation by dexamethasone and 17-estradiol in normal human bone marrow stromal (HBMS), osteoblast-like cells (hOB), and osteosarcoma MG-63 cells. Although HBMS cells secrete low levels of IL-8 constitutively, treatment with IL-1 and tumor necrosis factor- (TNF-) induced IL-8 secretion. Their effects were synergistic but IL-8 production was not affected by 17-estradiol. Human osteosarcoma MG-63 cells also secreted low levels of IL-8 constitutively; the production was induced by IL-1 and TNF- and was also not affected by 17-estradiol. The magnitude of the response to cytokine stimulation of IL-8 in MG-63 cells was much lower than that of HBMS and hOB cells, indicating differences in response in normal and osteoblastic osteosarcoma cells. Dexamethasone (10^-7 M) significantly inhibited IL-1 plus TNF- stimulated IL-8 production in HBMS, MG-63, and hOB cells. The accumulated results demonstrate that IL-8 is secreted by HBMS, MG-63, and hOB cells, suggesting that IL-8 may play a role in the regulation of bone cell function. These data also emphasize the importance of glucocorticoids in controlling cytokine secretion in HBMS, hOB, and MG-63 cells.     

Experimental study of bone formation around a titanium rod with β-tricalcium phosphate and prostaglandin E2 receptor agonists

-Tricalcium phosphate (-TCP) is an excellent bone-filling material that is completely absorbed by the body and replaced by autologous bone. Unfortunately, its mechanical strength is low, rendering its application at loaded regions difficult. The purpose of this study is to evaluate the histological and mechanical effects of single and combined use of -TCP and EP4 agonist on bone formation around a titanium rod. -TCP was loaded into the femoral bone marrow from the distal end of the femur, where the titanium implants were inserted, and the animals received twice-daily subcutaneous injections of EP4 agonist. Group I received the rod only and was designated the control group; group II received EP4 agonist only; group III received -TCP only; and group IV received both -TCP and EP4 agonist. Examination of decalcified specimens revealed favorable bone formation in all treatment groups compared with that in group I, with the most active bone formation seen in group IV. Mechanical evaluation revealed significant differences in maximum pull-out force compared with group I at weeks 4 and 8. There were no differences between groups II and III at either week 4 or 8, but the values seen in group IV at weeks 4 and 8 were significantly higher compared with the other groups. Combined use of -TCP and EP4 agonist is expected to compensate for bone defects resulting from revision total joint arthroplasty and to achieve stability at an early stage.     

Antibodies directed at mouse IL-2-R α and β chains act in synergy to abolish T-cell proliferation in vitro and delayed type hypersensitivity reaction in vivo

The anti-mouse IL-2-R chain mAb TM-1 which, by itself, does not affect IL-2-dependent proliferation throught the high affinity mouse IL-2 receptor, was shown to cooperate in a synergistic way with a set of anti-IL-2-R chain mAbs both in vitro and in vivo. In vitro, when associated at equimolar concentrations, the TM-1/anti- mAb association was four to ten times more efficient at inhibiting the proliferation of the CTL-L2 cell line than was a similar concentration of anti- mAb alone. In addition, a bispecific antibody in which a Fab' fragment of TM-1 was covalently linked to a Fab' fragment of one of the anti- mAb (5A2) was shown to be as efficient as the TM-1/5A2 association. The association of TM-1 with 5A2 was also tested in vivo in a sheep red blood cell-induced delayed type hypersensitivity (DTH) model. TM-1 which, by itself, had no effect on DTH, induced a two- to threefold decrease in the doses of 5A2 required to suppress this cell-mediated immune reaction.     

Latency and activation in the control of TGF-β

The biological activity of the transforming growth factor-'s (TGF-)³ is tightly controlled by their persistance in the extracellular compartment as latent complexes. Each of the three mammalian isoform genes encodes a product that is cleaved intracellularly to form two polypeptides, each of which dimerizes. Mature TGF-, a 24 kD homodimer, is noncovalently associated with the 80 kD latency-associated peptide (LAP). LAP is a fundamental component of TGF- that is required for its efficient secretion, prevents it from binding to ubiquitous cell surface receptors, and maintains its availability in a large extracellular reservoir that is readily accessed by activation. This latent TGF- complex (LTGF-) is secreted by all cells and is abundant both in circulating forms and bound to the extracellular matrix. Activation describes the collective events leading to the release of TGF-. Despite the importance of TGF- regulation of growth and differentiation in physiological and malignant tissue processes, remarkably little is known about the mechanisms of activationin situ. Recent studies of irradiated mammary gland reveal certain features of TGF-1 activation that may shed light on its regulation and potential roles in the normal and neoplastic mammary gland.     

The Normal and Malignant Mammary Gland: A Fresh Look with ERβ Onboard

Estrogens are important for the development and function of the normal mammary gland as well as for development of mammary cancer. The frontline therapy for treatment of estrogen receptor (ER)⁴ positive breast cancer is antiestrogens. A second estrogen receptor (ER) is also expressed in the breast but it has not been measured because it is not detected by the immunoassays used to detect ER. In many cell systems ER has actions which are opposite to those of ER and this finding has raised questions about the role of ER in the development and treatment of breast cancer.     

Differentiation of intercalated cells in culture

The renal collecting duct is a heterogenous epithelium consisting of intercalated cells (ICCs) and principal cells (PCs). To test the hypothesis that the two cell types might originate from one another and to determine which one of the two is a stem cell, -ICCs and PCs were isolated by fluorescence-activated cell sorting and grown on permeable supports. Cultures of sorted PCs maintained their PC phenotype [electrogenic sodium (Na⁺) reabsorption and potassium (K⁺) secretion and expression of PC-specific antigens]. In contrast, cultures of sorted -ICCs acquired -ICC-specific functions (e.g. proton secretion) and gradually expressed functions specific for PCs (amiloride-blockable Na⁺ current and K⁺ secretion). Most cells in cultures of sorted -ICCs also acquired a central cilium, a characteristic feature of PCs. Dual-staining of -ICC cultures with cell-specific antibodies against surface antigens revealed that approximately 45% of the cells expressed only ICC-specific antigens and approximately 20% expressed only PC antigens. The remainder of the cells were ICC/PC hybrids and stained for both markers. Such hybrid cells were also observed in situ, albeit with a lower frequency, on kidney sections dualstained with cell type-specific markers. The proliferation rate of the two cell types, assessed by pulse labeling cells in S-phase with bromodeoxyuridine or staining with an antibody against a proliferation antigen (KI-67), revealed a significantly higher proliferation rate among -ICCs than PCs. In aggregate, these data suggest that -ICCs in culture are capable of differentiating into -ICCs and PCs and raise the possibility that -ICC is the stem cell of the collecting duct.     

Cytokines in human milk: Properties and potential effects upon the mammary gland and the neonate

Epidemiologic and immunologic studies of breastfed and nonbreastfed infants and investigations of certain biologic activities in human milk led to the identification of immunomodulating agents in human milk. Among them were the cytokines interleukin-1 (IL-1); IL-6, IL-8, IL-10, granulocyte-colony stimulating factor, macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-, interferon-, epithelial growth factor (EGF), transforming growth factor- (TGF-), and TGF-2. Inteferon- may originate from T cells in milk; EGF, TGF-, TGF-, M-CSF, IL-6, and IL-8 may be produced by mammary gland epithelium. Based upon their known functions, we hypothesize that cytokines influence the development and immunologic function of the mammary gland and the neonate. Thosein vivo functions remain to be defined by future investigations.     

The reducing end of αGal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl--Gal gave weak inhibition, Gal1-3Gal substantially inhibited the reaction (400 M), and Gal1-3Gal2-4GlcNAc was ten times more efficient (30 M). The modification from to anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-Gal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal1-3Gal1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.     

Role of p38 MAP kinase pathway in a toxin-induced model of hemolytic uremic syndrome

The role of proinflammatory cytokines in a rat model of toxin-induced hemolytic uremic syndrome (HUS) was studied. Male Sprague-Dawley rats underwent continuous saline infusion (6 ml/h) via a tail vein and received a bolus injection of saline (control), lipopolysaccharide (LPS, 10 g/100 g body weight), ricin (6.7 g/100 g body weight), or ricin with LPS (ricin+LPS). They were then observed for 8 h. Blood samples and kidney tissues were obtained at the end of the experiment. The effects of FR 167653, a potent inhibitor of interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) production, were also examined in ricin+LPS-treated rats. Only ricin+LPS-treated rats developed significant thrombocytopenia, hemolysis, and oliguric acute renal failure with extensive glomerular thrombotic microangiopathy, which was characterized by glomerular microthrombi and apoptosis of glomerular endothelial cells. Thrombotic microangiopathy was not detected in other organs, including the brain, liver, spleen, pancreas, lung, colon, and intestine. Significantly elevated levels of serum IL-1 and TNF- were detected only in ricin+LPS-treated rats. Treatment of ricin+LPS-treated rats with FR 167653 significantly reduced the serum levels of IL-1 and TNF-, accompanied by improvement of the oliguric renal failure and glomerular thrombotic microangiopathy. These findings indicate that the increased serum levels of IL-1 and TNF-, which probably result in the apoptosis of glomerular endothelial cells, play a pivotal role in the development of this rat model of toxin-induced HUS. The findings also suggest that inhibition of these proinflammatory cytokines may prevent the development of HUS.     

TGF-β and functional differentiation

A review of the pertinent literature suggests that TGF-1 may play a multifaceted role in functional differentiation of mammary epithelium. Evidence for the expression of TGF-1 RNA and the presence of functional TGF-1 protein in differentiating mammary epithelial cells from a pregnant mouse has been recently reported. The specific role of mammary-epithelial-cell-produced TGF-1 in the differentiating mammary gland is presently unclear. However, several possible functions are suggested from the following observations. Milk protein production is negatively regulated by exogenous TGF-1 during gestational development of the gland but not during lactation. Consistent with reports linking TGF-1 gene expression with mammary gland involution following lactation, overexpression of TGF-1 in the differentiating secretory epithelium leads to premature programmed cell death in the absence of a negative effect on secretory epithelial cell proliferation. A role for TGF-1 in cell cycle control and suppression of malignant progression independent from its inhibitory effect on epithelial cell growth has been demonstrated in keratinocytes. A similar function could provide protection against malignancy in proliferating mammary epithelium and account for TGF-1 suppression of mammary tumorigenesis in transgenic mice overexpressing transforming growth factor alpha (TGF-).³     

Transforming growth factor-β and prostate cancer

Summary This review focuses on the possible role of transforming growth factor- isoforms 1–3 (TGF) in prostate cancer. TGF1 appears to inhibit the cellular proliferation of normal prostate cells. Surprisingly, TGF1 is overexpressed in prostate cancer. To help explain this apparent paradox, it has been revealed that with tumor progression, prostate cancer cells acquire reduced sensitivity to the growth-inhibitory effects of TGF1. Aberrations of the TGF1 signaling pathway at the prereceptor, receptor, or postreceptor level may lead to prostate cancer cell resistance to TGF1 growth inhibition. Indirectly, elevated levels of TGF1 may induce host effects that may be beneficial to prostate tumor growth by suppressing the immune system, promoting angiogenesis and extracellular matrix formation, and enhancing metastatic potential. Consequently, TGF1 appears to be important in prostate carcinogenesis and tumorigenicity. TGF2 and TGF3 are only briefly presented as very little is known about their role in prostate cancer.     

Complex role of tumor cell transforming growth factor (TGF)-βs on breast carcinoma progression

Growth inhibition by the TGF-s has been extensively studied in both normal and transformed mammary epithelial cells. It has been proposed that loss of autocrine TGF- mediated growth regulation is a critical event in breast tumorigenesis and several lines ofin vitro andin vivo data support this hypothesis. However, a positive association between the expression of TGF-s by tumor cells and the progression or maintenance of breast cancinoma cells has been observed in many studies inin vivo tumor models. Possible mechanisms for these growth enhancing effects of TGF- include immunosuppression mediated by tumor TGF-s, enhanced angiogenesis, increased peritumoral stroma formation, and cell adhesion. The net effect of tumor cell TGF- on the biology of breast carcinogenesis would depend on the balance between autocrine growth inhibition of mammary epithelial cells and these growth enhancing effects.     

Elevated concentrations of the β-subunit of S100 protein in renal cell tumors in rats

Concentrations of and -subunits of S100 protein (S100- and S100-) in rat kidney neoplasms, including renal cell and mesenchymal tumors, were determined using a highly sensitive enzyme immunoassay, and both types immunohistochemically localized in tissue sections. Concentration of S100- in each histological type of rat tumor were lower than in normal kidney, whereas levels of S100- (mean±SE: 29.7±14.2 ng/mg protein, n=15) in renal cell tumors were significantly higher than in normal kidneys (0.55±0.06 ng/mg protein, n=7), or mesenchymal tumors (1.21±0.43 ng/mg protein, n=9). In normal rat kidney tissues S100- was immunohistochemically positive in epithelial cells of the distal tubules, the thin limbs of loops of Henle, and the collecting ducts. No appreciable immunostaining for S100- was found in any nephron segment. Both S100- and S100- were positive for renal cell tumors, indicating new appearance of the latter during renal carcinogenesis in rats.     

TGF-β1 impairs homocysteine metabolism in human renal cells: possible implications for transplantation

We hypothesized that TGF-1 influences the metabolism of homocysteine (Hcy) and increases its cellular export, which may lead to hyperhomocysteinemia in patients with renal transplants. We exposed human renal proximal tubule epithelial cells (huRPTECs) to different concentrations of TGF-1, IL-1, IL-10, or methionine and measured total Hcy (tHcy) in culture supernatants. We then examined the relationship between plasma levels of tHcy and TGF-1 in renal graft recipients. In multivariate analysis, the factors mediator (TGF-1, IL-1, IL-10), mediator concentration, methionine concentration, and "mediator × concentration" interaction independently influenced tHcy concentrations in culture supernatants. A 31% increase in tHcy was observed after exposure of huRPTECs to TGF-1 compared to medium alone. However, TGF-1 plasma levels in kidney graft recipients showed no independent association with tHcy plasma concentrations. We demonstrated that the release of Hcy from huRPTECs is enhanced by TGF-1, but that TGF-1 plasma levels in renal graft recipients show no independent relationship with hyperhomocysteinemia.