Plasma proteins present in human cortical bone: Enrichment of theαHS-glycoprotein

The spectrum of plasma proteins present in human cortical bone and permanent dentine has been determined. One plasma glycoprotein, theHS-glycoprotein, was found to be present at a high concentration in both bone and dentine and was shown to be concentrated in the mineralized tissues with respect to the other plasma proteins by factors of between 30 and 100. In this respect theHS-glycoprotein is analogous to the G2B-glycoprotein and -glycoprotein of bovine and rabbit b one, respectively. The binding ofHS-glycoprotein and albumin to calcium phosphates generated within serum samples has been studied at different serum: precipitate ratios. In each case all theHS-glycoprotein was removed from the samples and theHS-glycoprotein was concentrated with respect to albumin by factors ranging from 370 at the highest serum: precipitate ratio to 25 at the lowest ratio. The plasmaHS-glycoprotein concentrations of patients with Paget's disease of bone were shown to be substantially lower than the normal range, with significant negative correlation between theHS-glycoprotein concentration and the plasma alkaline phosphatase activity.     

Role of endogenous interferon gamma in murine tumor growth and tumor necrosis factor alpha antitumor efficacy

Background: The anticancer role of tumor necrosis factor-alpha (TNF-) has been limited by toxicity. These experiments evaluate blocking endogenous interferon-gamma (IFN-) activity to abrogate TNF- toxicity. Methods: C57Bl/6 mice bearing MCA 105 tumor were treated with TNF- and anti-IFN- antibody (Ab) to evaluate the effect on the acute lethality of TNF- and their efficacy as evaluated by tumor growth rate, tumor histology, and survival. Results: Anti-IFN- Ab decreased TNF- lethality. Anti-IFN- Ab alone increased tumor growth significantly more than did nonimmune IgG (p₂<0.0001). Tumor-bearing mice that received nonimmune IgG and TNF- had slower tumor growth (p₂<0.02) and a trend toward improved survival (p=0.07) compared with saline-treated controls. Anti-IFN- Ab abrogated the antitumor effect of TNF-, prevented acute tumor necrosis histologically, and resulted in tumor growth rate and host survival similar to that of controls. The findings in mice that received anti-IFN- Ab and high-dose TNF- were comparable with those in mice that received a lower, equitoxic dose of TNF- alone. Conclusions: Blocking endogenous IFN- accelerates tumor growth in this model and partially abrogates the toxic and antitumor activity of exogenous TNF- equally. This suggests that blocking endogenous IFN- activity is not a useful strategy for limiting TNF- treatment toxicity.Presented in part at the 45th Annual Cancer Symposium of The Society of Surgical Oncology, New York, New York, March 15–18, 1992.     

Serum levels of alpha-1 microglobulin and beta-2 microglobulin in bone marrow transplant recipients treated with cyclosporin A

The levels of alpha-1 microglobulin ( ₁m) and beta-2 microglobulin ( ₂m) in serum were estimated in 77 bone marrow transplant recipients. In comparison to pretransplant levels, the highest levels of ₁m and ₂m were found during impairment of renal function, i.e., during cyclosporin-induced nephrotoxicity and during treatment with other nephrotoxic drugs (P<0.001). The ₁m levels were less elevated during infections and acute graft-versus-host disease (P<0.01), while ₂m levels were markedly elevated during the same conditions (P<0.001). The linear correlations between serum creatinine and ₁m and creatinine and ₂m were r=0.7 and 0.8, respectively (P<0.001). The overall correlation between ₁m and ₂m was 0.4 (P<0.001). It is concluded that ₁m might be a complement to serum creatinine levels in monitoring renal function after bone marrow transplantation.     

Integrin laminin receptors and breast carcinoma progression

This review explores the mechanistic basis of breast carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression not only for their ability to mediate physical interactions with extracellular matrices but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on the 6 integrins (61 and 64), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in breast cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease. Recent work by our group and others on mechanisms of breast carcinoma invasion and survival that are influenced by the 6 integrins are discussed.     

Chronic intramedullary infusion of interleukin-1α increases bone mineral content in rats

Interleukin-1 (IL-1) is known to have a potent bone-resorbing activity in vitro, however, results from in vivo studies are conflicting. We performed experiments with the continuous infusion of recombinant IL-1 directly into the femoral bone marrow of rats for 2 weeks and examined its effects on bone by soft X-ray photographs, bone mineral assessment, and histological examination. Infusion of IL-1 at doses greater than 1 g/ml (0.6 ng/hour) caused sclerosis around the infusion site on soft X-ray photographs, and the bone mineral content of IL-1 infused femora was increased significantly. Histologically, extensive periosteal bone formation was observed around the infusion site and small mononuclear cells replaced the normal fat tissue. Infusion of prostaglandin E₂ alone produced intramedullary bone formation more extensively. Simultaneous infusion of IL-1 and indomethacin (10^-3 M; 179 ng/hour) inhibited the increase of bone mineral content (BMC) induced by IL-1. Thus, the chronic intramedullary administration of IL-1 stimulates bone formation, especially in the periosteum, and increases BMC in intact rats.     

Renal α-adrenergic receptors and genetic hypertension

The hypothesis has been proposed that an increase in the number of renal -adrenergic receptors may contribute to the pathogenesis of genetic hypertension. Herein we review recent findings regarding expression of renal ₁ (_{1 a},_{1 b})- and ₂ (_{2 a},_{2 b})-adregenergic subtypes and we provide an updated revision of the above-stated hypothesis. Enhancement in receptor number or in post-receptor components responsible for ₁- and ₂-adregenergic mediated sodium reabsorption in proximal tubule may contribute to sodium retention and an elevation in blood pressure. Perhaps such changes contribute to the increase in blood pressure in genetically determined hypertension in humans, although direct tests of this notion have not yet been performed.     

Mechanisms and factors involved in development of hypertrophic scars

Fibroblasts in granulation tissue (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle cells, such as the presence of microfilament bundles and the expression of -smooth muscle actin. The mechanisms leading to the development of such myofibroblastic features remain unclear. Both in vivo and in vitro investigations suggest the participation of growth factors, cytokines, and extracellular matrix in the regulation of -smooth muscle actin expression. During normal wound healing, myofibroblasts disappear when the wound is fully re-epithelialized. In contrast, the expression of -smooth muscle actin is a permanent feature of myofibroblasts present in fibrocontractive diseases. In hypertrophic scars, the presence of these cells may represent an important element in the pathogenesis of retraction. Different therapeutic modalities available for the treatment of hypertrophic scars cause a decrease in the number of -smooth muscle actin expressing myofibroblasts. Further studies regarding the expression of cytoskeleton markers in fibroblastic cells are needed to understand the mechanisms of scarring and fibrosis.     

Cytotoxic effect of diphtheria toxin used alone or in combination with other agents on human renal cell carcinoma cell lines

Treatment of renal cell carcinoma (RCC) by conventional chemotherapy and immunotherapy has resulted in minimal remissions. Alternative forms of therapy are therefore being sought. The present study investigated the sensitivity of RCC cell lines to several toxins used alone and in combination with other agents. RCC lines were relatively sensitive to the direct cytotoxic effect of diphtheria toxin (DTX), Pseudomonas aeruginosa exotosin A (PEA) and ricin. Furthermore, DTX in combination with tumor necrosis factor- (TNF-) resulted in synergistic cytotoxic activity. The mechanism of synergy was examined. A possible mechanism of resistance to TNF- in tumor cells is the expression of TNF- mRNA or protein. R11 cells did not constitutively express mRNA for TNF-, however, treatment of R11 cells with TNF- induced the expression of TNF- mRNA. When DTX was used in combination with TNF-, the level of TNF- mRNA induced by TNF- was markedly reduced. These studies suggest that DTX in combination with TNF- can overcome the resistance of RCC lines and that the marked downregulation of TNF- mRNA by DTX may play a role in the enhanced cytotoxicity seen with the combination of DTX and TNF-. Furthermore, the combination treatment might also potentiate the antitumor host responses. The implications of these findings in clinical therapy are discussed.     

Estrogen receptor alpha gene analysis in osteoporosis and familial osteoporosis

Estrogens are important determinants of bone mineral density (BMD) mediating their effects via estrogen receptor (ER) and (ER). The strong genetic predisposition to osteoporosis, and the fact that alterations in the aminoterminal region of ER have been linked to bone disturbances, prompted us to identify genetic alterations in exon 1 and exon 2 of ER in osteoporotic individuals. Sixty-two unrelated normal subjects (age 46.1±9.5 years) and 72 unrelated osteoporotic subjects (age 52.3±7.9 years) were studied. Their menopausal status was pre- and perimenopausal. We also included 30 related osteoporotic individuals (mother-daughter or sister-sister relationship) (age 46.2±12.8 years) belonging to 14 families who where also pre- and perimenopausal. DNA was extracted from peripheral blood, exons 1 and 2 were amplified by polymerase chain reaction (PCR) and were further submitted to denaturing gradient gel electrophoresis (DGGE), single stranded conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and sequence analysis. Bone turnover markers were also determined. Two polymorphisms were identified in exon 1 (codons 10 and 87) in both normal and osteoporotic women. Statistical analysis revealed no difference (P>0.05) in the ER genotype frequencies within osteoporotic families as compared with the same genotypes in the unrelated normal or osteoporotic subjects. Codon 10, codon 87 polymorphisms were not related to BMD or bone turnover markers. No other mutations were found in exons 1 and 2 in all subjects studied. Genetic alterations in exons 1 and 2 of ER are not associated to osteoporosis and familial osteoporosis. Moreover, the codon 10 and codon 87 polymorphisms do not seem to be correlated with BMD and bone turnover markers.     

Combined interferon alpha with levamisole in patients with metastatic renal cell carcinoma

To evaluate the efficacy and toxicity of interferon alpha-2b (IFN-2b) and levamisole treatment regimen in patients with metastatic renal cell carcinoma (RCC). Seventeen patients with metastatic RCC were treated using recombinant IFN-2b at a dose of 10 MU/m2 body surface subcutaneously three times in a week, for 3 months, with levamisole 50 mg t.d.s orally on days 1–3on alternate weeks. The mean follow-up period was 10.7 (range 2–23) months. We achieved 1 complete response (lasting for 12+months) and 1 partial response (lasting for 15 months), for an objective response rate of 11.7%. A further 7 patients (41%) had a stabilization of disease. The overall toxicity was moderate, with mainly grade I or II side effects. Grade III toxicities reported among 3 patients including vomiting (2 patients) and anorexia (1 patient). There was no treatment related death. Although additions of levamisole to IFN- do not result in any significant increase in treatment toxicity, the response rate appears to be no better than IFN- monotherapy reported in the literature.     

Growth factors and kidney development

The formation of the metanephric kidney is dependent upon the timed and sequential expression of a number of polypeptide growth factors. To shed light on the participation of members of the insulin-like growth factor (IGF) and epidermal growth factor/transforming growth factor- (EGF/TGF-) families, we measured the synthesis of IGF-I, IGF-II, EGF and TGF- by developing rat metanephroi in organ culture and determined the effect of anti-growth factor antibodies on growth and development. IGF-I, IGF-II and TGF- were produced by metanephroi and released into culture media. We could detect no EGF. Inclusion of anti-IGF-I, anti-IGF-II, anti-IGF-II receptor or anti-TGF- antibodies in organ cultures inhibited growth and development of metanephroi. Our findings suggest that both members of the IGF family and TGF- are produced within the developing metanephros and promote renal organogenesis.     

Denervation supersensitivity of the urethra to α-adrenergics in the chronic neurogenic bladder

Summary The response of the urethral pressure profile to the administration of various autonomic drugs was compared between a group of eight patients with chronic neurogenic bladder as evidenced by denervation supersensitivity to besacholine^R and a group of 10 control subjects. A supersensitive response to the administration of an -stimulant with a rise of maximum urethral pressure of 10 mmHg or more above the control urethral pressure was uniformly observed in the urethra of patients with chronically denervated bladders. Mechanisms of supersensitivity are postulated and the significance of -adrenergic innervation of the urethra are stressed. These results appear to add pharmacological evidence of -adrenergic predominance in the urethra which is now believed to be dually innervated.     

Immunoreactive low-molecular-weight epidermal growth factor in urine of patients with renal cell carcinoma

Summary A specific heterogeneous enzyme-linked immunosorbent assay (ELISA) has been established in order to determine levels of low-molecular-weight epidermal growth factor (EGF) in the urine of patients with renal cell carcinoma who had undergone unilateral radical nephrectomy. Urine specimens, i.e., 20 pre- and postsurgical specimens from a group of patients and 22 from a control group, were assaved after the urine had been freed from high-molecular-weight proteins (>30kDa) and salts. EGF levels were expressed as urinary EGF/creatinine ratios, and a highly significant decrease (=0.0005 by Student's t-test) of urinary EGF was found in the patient group prior to surgery. The cancer patients also showed an additional loss of urinary EGF after unilateral nephrectomy (=0.0005 by Student's t-test). These data correlate with our previous findings that pro-EGF gene expression is decreased in human renal carcinoma and support the concept that low-molecular-weight urinary EGF is derived from high-molecular-weight kidney pro-EGF.Supported by a grant from Deutsche Krebshife, Bonn, FRG (W70/87 Pe-2).     

The possible role of TNF-α and IL-2 in inducing tumor-associated metabolic alterations

This study was conducted to investigate the role of tumor necrosis factor- (TNF-) and interleukin-2 (IL-2) in inducing cancer cachexia, and the results were compared with those obtained from our previous study on Fisher 344 rats with methylcholanthrene-induced sarcoma. Three groups of male Fisher 344 rats received one of the following regimens: 4×10⁴ IU of human recombinant TNF- per rat per day subcutaneously (sc) for 5 consecutive days (n=5), 3.5×10⁵ U human recombinant IL-2 per rat per day sc for 14 consecutive days (n=5), or normal saline (n=5). The activities of both phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) were increased slightly in the IL-2 group. Furthermore, LPL activity was significantly increased in the adipose tissue of the TNF group and in the cardiac muscle of the IL-2 group, but not in that of the TNF group. These results show that there is a significant difference between the metabolic alterations seen in the tumor-bearing state and those induced by either TNF- or IL-2 alone. Thus, it is unlikely that IL-2 or TNF- is the sole mediator of cancer cachexia in this tumor and rat model.     

Expression of nephrin,podocin, α-actinin,and WT1 in children with nephrotic syndrome

Recently, nephrin, podocin, -actinin, and WT1, which are located at the slit diaphragm and expressed by the podocyte, were found to be causative in congenital/familial nephrotic syndrome (NS), but their role in acquired NS remains unclear. We studied their expression in NS with the aim of disclosing their possible role in the development of proteinuria. Immunofluorescence, confocal microscopy, and image analysis were used to study the expression and the distribution in 19 children with primary NS, 9 with isolated hematuria, and 9 controls. All the children with NS presented with heavy proteinuria and foot process effacement was identified by electron microscopy. No proteinuria and foot process effacement was seen in the group with hematuria. A dramatic decrease of podocin expression was found in NS (86.66±22.74) compared with control groups (P=0.014). Furthermore, we also found the pattern of distribution of nephrin, podocin, and -actinin changed in children with NS. In conclusion, a dramatic decrease of podocin expression and abnormal distribution of nephrin, podocin, and -actinin were found in children with NS. No differences were found in children with isolated hematuria, suggesting involvement of these molecules in the development of proteinuria in primary NS.     

Postnatal renal development of rats from mothers that received increased sodium intake

The newborn rat kidney is not fully developed until approximately 12 days after birth. Several lines of evidence suggest that angiotensin II (AII) participates in the postnatal development of the kidney. The aim of the present study was to analyze proliferating cell nuclear antigen (PCNA), fibronectin, -smooth muscle-actin (-SM-actin), and AII expression in renal cortex during development in rats born to mothers that received a normal (control) or increased (experimental) sodium intake during pregnancy. Ninety Wistar rats aged 1, 7, 15, and 30 days from the control and experimental groups were killed and the kidneys removed for histological and immunohistochemical studies. The results showed higher fibronectin, -SM-actin, PCNA, and AII expression in the glomerular and tubulointerstitial areas of the renal cortex of 1- and 7-day-old animals, which decreased with renal development. The animals from the experimental group showed at 1 day of age a decrease in -SM-actin, fibronectin, PCNA, and AII expression compared with controls of the same age ( P <0.05). In conclusion, our data show that increased sodium intake during pregnancy induces a reduction of -SM-actin, fibronectin, and PCNA expression in the renal cortex tubulointerstitium and glomeruli of neonatal rats. These alterations may be related to the decrease of AII expression also observed in the renal cortex from these animals.     

A case of renal sarcoidosis: a special reference to calcium metabolism as a diagnostic and the therapeutic implications

Sarcoidosis is a systemic granulomatous disease of unknown etiology and is associated with a wide variety of renal disorders including nephrolithiasis, hypercalciuria, hypercalcemia, nephrocalcinosis, tubular defect, glomerulonephritis, and granulomatous interstitial nephritis. We report a case of renal sarcoidosis in which we could not detect any evidence of extrarenal involvements that was diagnosed by renal biopsy and abnormal calcium metabolism incompatible with chronic renal insufficiency. On laboratory findings, decreased creatinine clearance, proteinuria, hypercalcemia, hypercalciuria, and mildly elevated serum angiotensin-converting enzyme (ACE) were seen. Serum intact parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,-25 vit D) were lower and higher than normal range, respectively, whereas the patient was already in chronic renal insufficiency. He was treated with oral corticosteroid. Serum ACE tended to fall, and 1,-25 vit D level decreased with substantial fall of serum calcium and daily calcium excretion. In contrast, intact PTH increased slowly in accordance with a fall of serum calcium compatible with the level of renal impairment. Creatinine clearance and daily excretion of protein improved. The case reported here may propose that serial measurement of serum level of 1,-25 vit D, calcium level, and magnitude of daily calcium excretion into urine is a simple and meaningful tool to detect the therapeutic response in sarcoidosis with abnormal calcium metabolism.This case was reported at the 38th Kanagawa nephritis research meeting in 2002 and the 32nd eastern regional meeting, Japanese Society of Nephrology, in 2002.     

Multiple endocrine neoplasia type 1 in end-stage renal failure

A 59-year-old woman with chronic renal failure due to type 2 diabetes mellitus (DM) is presented. Her father and a brother had a history of brain tumor. Her blood urea nitrogen and serum creatinine levels were 102mg/dl and 4.5mg/dl, respectively. Her serum Ca²⁺ and Pi were within the normal range (9.4mg/dl and 5.4mg/dl, respectively). Her intact parathyroid hormone (PTH) level was 1730000pg/ml. A ^99mTc-methoxy-isobutylisonitrile scintigraphy showed high uptake in three parathyroid glands. A magnetic resonance image showed microadenoma in the pituitary gland. The serum gastrin level was high. Genetic examination revealed a mutation of the MEN1 gene (894–9 G A). From these findings, she was diagnosed with multiple endocrine neoplasia (MEN) type 1. Subsequently, a parathyroidectomy was performed successfully, a parathyroid gland was transplanted to her right forearm, and her serum Ca²⁺ level was controlled at 8.5–9.0mg/dl. It is very important to identify MEN1 if an end-stage renal disease (ESRD) patient has hyperparathyroidism with multigland involvement. Examination of the MEN1 gene may be valuable to make an accurate diagnosis and choose the appropriate therapy in some ESRD patients with hyperparathyroidism.     

Characterization of protein components of human urinary crystal surface binding substance

We previously extracted crystal surface binding substance (CSBS) from human urine and showed that it appeared to constitute a substantial proportion of urinary macromolecular inhibitors of calcium oxalate crystallization. CSBS was isolated from human urine and fractionated by three consecutive chromatography procedures in order to characterize protein inhibitors of calcium oxalate crystallization. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and NH₂-terminal amino acid sequencing revealed that inhibitory fractions eluted from a final, hydroxyapatite column contained prothrombin and osteopontin. Hydroxyapatite column fractions also contained other, unidentified protein inhibitors of calcium oxalate crystallization. CSBS contained also human serum albumin, ₁-acid glycoprotein, ₂-microglobulin, ₂-HS glycoprotein, retinol-binding protein, transferrin, and Tamm-Horsfall protein, but these proteins seemed to play no direct role in inhibitory activity.     

Estrogen Receptor Alpha in Human Breast Cancer: Occurrence and Significance

Estrogens have long been recognized as being important for stimulating the growth of a large proportion of breast cancers. Now it is recognized that estrogen action is mediated by two receptors, and the presence of estrogen receptor (ER)³ correlates with better prognosis and the likelihood of response to hormonal therapy. Over half of all breast cancers overexpress ER and around 70% of these respond to anti-estrogen (for example tamoxifen) therapy. In addition, the presence of elevated levels of ER in benign breast epithelium appears to indicate an increased risk of breast cancer, suggesting a role for ER in breast cancer initiation, as well as progression. However, a proportion of ER-positive tumors does not respond to endocrine therapy and the majority of those that do respond eventually become resistant. Most resistant tumors remain ER-positive and frequently respond to alternative endocrine treatment, indicative of a continued role for ER in breast cancer cell proliferation. The problem of resistance has resulted in the search for and the development of diverse hormonal therapies designed to inhibit ER action, while research on the mechanisms which underlie resistance has shed light on the cellular mechanisms, other than ligand binding, which control ER function.