β2-Microglobulin in postmenopausal osteoporosis

Summary The so-called bone-derived growth factor, or ₂-microglobulin, has a regulatory function in bone metabolism, stimulating osteoclastic activity. Osteoclastic activity is enhanced in postmenopausal osteoporosis, suggesting that ₂-microglobulin concentration may also be increased in this disease. ₂-microglobulin concentration was found to be raised (P < 0.001) in 30 women with postmenopausal osteoporosis as compared with 30 normal women of similar age; tartrate-resistant acid phosphatase concentration also was raised (P < 0.001), and total body bone mineral content was decreased (P < 0.001). Linear regression analysis revealed a highly negative correlation result between total body bone mineral content and ₂-microglobulin (r = 0.577,P < 0.001), and a positive correlation result between ₂-microglobulin and tartrate-resistant acid phosphatase concentration (r² = 0.806,P < 0.001). These findings, and the stimulatory effect of ₂-microglobulin on osteoclastic and osteoblastic activity, suggest that ₂-microglobulin may play an important role as a local regulatory factor in the pathogenesis of postmenopausal osteoporosis.     

Mutations in NOTCH2 in patients with Hajdu–Cheney syndrome

Summary

The Hajdu–Cheney syndrome is a very rare disease that affects several organ system, leading to severe osteoporosis and other abnormalities. We describe clinical and genetic findings of nine patients with this disease.

Introduction

The Hajdu–Cheney syndrome (HCS) is a rare autosomal dominant disorder characterized by severe osteoporosis, acroosteolysis of the distal phalanges, renal cysts, and other abnormalities. Recently, heterozygous mutations in NOTCH2 were identified as the cause of HCS.

Methods

Nine patients with typical presentations of HCS took part in this study: five affected patients from two small families and four sporadic cases. Peripheral blood DNA was obtained and exome sequencing performed in one affected individual per family and in all four sporadic cases. Sanger sequencing confirmed mutations in all patients.

Results

One of the identified mutations was introduced in a plasmid encoding NOTCH2. Wild-type and mutant NOTCH2 were transiently expressed in HEK293 cells to assess intracellular localization after ligand activation. Deleterious heterozygous mutations in the last NOTCH2 exon were identified in all patients; five of the six mutations were novel.

Conclusion

Consistent with previous reports, all mutations are predicted to result in a loss of the proline/glutamic acid/serine/threonine sequence, which harbors signals for degradation, therefore suggesting activating mutations. One of the six mutations furthermore predicted disruption of the second nuclear localization signal of NOTCH2, but the mutant revealed normal nuclear localization after transfection, which is consistent with the proposed gain-of-function mechanism as the cause of this autosomal dominant disease. Our findings confirm that heterozygous NOTCH2 mutations are the cause of HCS and expand the mutational spectrum of this disorder.     

β2-microglobulin in Paget's bone disease

On the basis of earlier findings of increased serum ₂-microglobulin concentration in women with postmenopausal osteoporosis, we decided to study serum ₂-microglobulin concentration in other bone diseases. In 28 patients with untreated Paget's bone disease, serum ₂-microglobulin concentration was normal (1.49±0.41 mg/liter versus 1.36±0.21 mg/liter in 42 control subjects, P= ns), a finding that contradicts reports in the literature. We found that serum ₂-microglobulin concentration was related negatively and significantly (r²=–0.154, P=0.0354) with serum total alkaline phosphatase concentration, but not with serum tartrate-resistant acid phosphatase concentration (p =ns). Urinary elimination of ₂-microglobulin was lower in the patients with Paget's disease than in the controls (34±28 versus 120±21 mg/liter, P<0.001). These findings suggest that ₂-microglobulin behaves similarly to osteocalcin (BGP) in Paget's bone disease and that its concentration remains within normal levels perhaps because of the rate of reuptake of ₂-microglobulin in bone neoformation.     

Reciprocal action between BMP-2 and BMP-3 in cultured fibroblast in vitro

Objective:To explore reciprocal action between BMP-2 (bone morphogenetic protein-2)and BMP-3 for better understanding of the mechanism of BMP during bone fracture union.Methods:rhBMB-2 was added into the cultured fibroblasts with the concentration of 1200 ng/ml.The expression of BMP-3 in fibroblasts was detected by immunohistochemistry.Eukaryotic expression vector pcDNA3-BMP-3 was transfected into the fibroblasts.After the effective expression of BMP-3 was identified,BMP-2 was also detected by immunohistochemistry in BMP-3 expression cells.The fibroblasts transfected with empty vector pcDNA3 were used as the control.Results:Exogenous rhBMP-2 could promote the expression of BMP-3 in fibroblasts. BMP-3 also could be detected in these cells.Conclusions:BMP-2 and BMP-3 could reciprocally adjust the expression in fibroblasts.     

Angiopoietin-2–Induced Arterial Stiffness in CKD

The mechanism of vascular calcification in CKD is not understood fully, but may involve collagen deposition in the arterial wall upon osteo/chondrocytic transformation of vascular smooth muscle cells (VSMCs). Increased levels of circulating angiopoietin-2 correlate with markers of CKD progression and angiopoietin-2 regulate inflammatory responses, including intercellular and vascular adhesion and recruitment of VSMCs. Here, we investigate the potential role of angiopoietin-2 in the pathogenesis of arterial stiffness associated with CKD. In a cohort of 416 patients with CKD, the plasma level of angiopoietin-2 correlated independently with the severity of arterial stiffness assessed by pulse wave velocity. In mice subjected to 5/6 subtotal nephrectomy or unilateral ureteral obstruction, plasma levels of angiopoietin-2 also increased. Angiopoietin-2 expression markedly increased in tubular epithelial cells of fibrotic kidneys but decreased in other tissues, including aorta and lung, after 5/6 subtotal nephrectomy. Expression of collagen and profibrotic genes in aortic VSMCs increased in mice after 5/6 subtotal nephrectomy and in mice producing human angiopoietin-2. Angiopoietin-2 stimulated endothelial expression of chemokines and adhesion molecules for monocytes, increased Ly6C^low macrophages in aorta, and increased the expression of the profibrotic cytokine TGF-β1 in aortic endothelial cells and Ly6C^low macrophages. Angiopoietin-2 blockade attenuated expression of monocyte chemokines, profibrotic cytokines, and collagen in aorta of mice after 5/6 subtotal nephrectomy. This study identifies angiopoietin-2 as a link between kidney fibrosis and arterial stiffness. Targeting angiopoietin-2 to attenuate inflammation and collagen expression may provide a novel therapy for cardiovascular disease in CKD.Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with CKD.^1–4 Arteriosclerosis characterized by arterial stiffness is the major vascular complication.^5,6 The independent predictive value of arterial stiffness for CVD has been well demonstrated in different populations.^7,8 Although arterial stiffness is a hallmark of the aging process, many other factors such as endothelial dysfunction, local or systemic inflammation, and genetic factors are also implicated in the pathogenesis of arterial stiffness.^9–11 Arterial stiffness in patients with CKD is characterized by arterial intima-media hypertrophy resulting from alterations of the intrinsic properties of the arterial wall.^3,12 Increased arterial stiffness is observed in the early stages of CKD.^6,9 Arterial stiffness is accelerated in patients with CKD compared with age-, sex-, and pressure-matched controls, suggesting unique CKD-related factors leading to such a complication.^6,9,12Traditional risk factors in patients with CKD, such as hypertension, diabetes, and hyperlipidemia, account for the increased CVD morbidity and mortality in part; however, actual mortality rates exceed expected rates even though these traditional risk factors are controlled.¹³ Much attention has been paid to the vascular calcification and functional alteration caused by disturbed mineral metabolism in patients with CKD.^14,15 Although the pathogenesis of vascular calcification has not yet been fully elucidated, it likely involves a transformation of vascular smooth muscle cells (VSMCs) into a synthetic phenotype, leading to arterial stiffness by deposits of collagen I– and collagen III–rich extracellular matrix (ECM) in the arterial wall.^12,16,17 Treatments correcting the mineral disturbance by phosphate binders, however, have not shown improvement in CVD mortality.¹⁸Cross-talk among endothelial cells, VSMCs, and other cells through humoral and mechanical mechanisms regulates vascular function in health and disease.^16,19 In these humoral factors, increased levels of circulating angiopoietin 2 (Angpt2) are correlated with scores of coronary, carotid, and peripheral artery diseases in dialysis patients.^20,21 Recent evidence further demonstrates that the levels of circulating Angpt2 have a reverse correlation with GFR, have a positive correlation with albuminuria, and predict long-term mortality in patients with CKD.^22–24 Angiopoietin-1 (Angpt1) and Angpt2 are ligands of the Tie-2 receptor, a family of growth factors specific for the vascular endothelium identified after discovery of vascular endothelial growth factor-A (VEGF-A).^25–28 In addition to angiogenesis, Angpt2 is an important regulator in numerous pathophysiologic processes, including inflammation.²⁹ Of note, circulating Angpt2 levels are correlated with systemic markers of microinflammation in patients with CKD.^23,24 Angpt1-mediated Tie-2 activation is required to maintain quiescent endothelium.³⁰ In contrast, Angpt2 destabilizes quiescent endothelium and primes it to respond to exogenous stimuli, thereby modulating activities of inflammatory (TNF-α) and angiogenic (VEGF-A) cytokines.^29,31 In mice undergoing femoral artery ligation, Angpt2 is shown to induce intercellular and vascular adhesion molecules, macrophage infiltration, and VSMC recruitment, thereby promoting arteriogenesis and blood flow recovery.³² Hence, it is reasonable to speculate that dysregulation of the Angpt/Tie-2 system in favor of Angpt2 may affect vascular remodeling through endothelial cells and VSMCs.     

β2-Microglobulin in patients with urothelial cancer

β2-Microglobulin (β2-mi) is found on the surface of most nucleated cells, and in cancer patients its increased production and liberation into the blood has been described. We studied serum and urinary levels of β2-mi in 29 patients with urinary bladder cancer (UBC) and 38 patients with upper-tract urothelial cancer (UTUC). A statistically significant (P < 0.01) increase in serum β2-mi levels was demonstrated in patients with UTUC as compared with controls, whereas urinary excretion of β2-mi was increased (P < 0.05) in both UTUC and bladder cancer patients. β2-Mi production was studied in a 72-h culture of peripheral blood mononuclear cells (PBMC) under basal conditions and after stimulation with concanavalin A (Con A) or phytohemagglutinin (PHA). Basal production of β2-mi by control unstimulated PBMC was 89.3 ± 5; that of PHA-stimulated PBMC, 189 ± 22 μg/10⁶ cells; and that of Con A-stimulated PBMC, 210 ± 32 μg/10⁶ cells. β2-Mi production by PBMC of cancer patients was not statistically significantly different from the control value, ruling out the possibility of an increased production by lymphocytes in these patients. Increased production of β2-mi was demonstrated in 48-h ureteral and urinary bladder-cancer cell cultures, ranging from 440 to 2,600 μg. Serum β2-mi was found to be increased to values of over 2.7 mg/l in 4 UBC and 12 UTUC patients with normal serum creatinine levels. Tumor surgery in those patients with increased serum β2-mi resulted in normalization of the serum β2-mi levels; urinary β2-mi excretion was not significantly changed by surgery. This study establishes that increased serum levels of β2-mi found in patients with urothelial cancer and normal glomerular function might be due to the increased production by tumor cells. However, activation of the immune response by mononuclear cells to the neoplasm cannot be excluded.     

Types of “H2O” in human enamel and in precipitated apatites

Summary Types of H₂O in human enamel and in precipitated apatites are characterized using X-ray diffraction, infrared (IR) absorption spectroscopic and thermogravimetric analyses. Changes in lattice parameters (principally in the -axis dimensions) and in the character of the IR absorption bands are correlated with weight losses at pyrolysis temperatures of 100° to 400°C and with effect of rehydration and reignition of previously ignited samples.This study demonstrated that the loss of H₂O below 200°C is reversible and causes no significant change in the lattice parameter of these apatites, whereas loss of H₂O between 200° and 400°C is irreversible and causes a contraction in the -axis dimension. It is proposed that two general types of H₂O are present in these apatites: (a)adsorbed H ₂O—characterized by reversibility, thermal instability below 200°C, and lack of effect on lattice parameters; and (b)lattice H ₂O—characterized by irreversibility, thermal instability between 200 and 400°C, and induction of expansion in the -axis dimensions of human enamel and precipitated apatites. Lattice H₂O is assumed to be due to H₂O-for-OH and/or HPO₄-for-PO₄ substitutions in these apatites. Loss of adsorbed H₂O caused sharpening of the OH absorption bands in the spectra of these apatites. Loss of lattice H₂O caused the appearance of P–O–P absorption bands (due to the presence of P₂O₇ ^4– group) in precipitated apatites containing small amounts of CO₃ ^2–.The observed larger -axis of human enamel apatite, i.e., 9.445±0.003A, compared to that of the mineral or synthetic (prepared at 1000°C) OH-apatite, i.e., 9.442A, may be attributed to the presence of lattice H₂O, Cl-for-OH, and concerted substitutions of larger cations (e.g., Sr, Ba, Pb, K) for Ca in this apatite.This paper was presented in part at the International Association for Dental Research Meeting in Copenhagen, March, 1977 (abstract no. 48).     

Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes

Podocyte structural changes have been suggested to be involved in the pathogenesis of albuminuria in diabetes. We evaluated podocytes density, number, and structure in 67 white patients with type 2 diabetes: 21 normoalbuminuric (NA), 23 microalbuminuric (MA), and 23 proteinuric (P). Kidney function and biopsy studies were performed; 20 kidney donors served as control subjects. Electron microscopic morphometric analysis was used to estimate numerical density of podocytes per glomerulus [Nv(epi/glom)], filtration slit length density per glomerulus (FSLv/glom), and foot process width (FPW). The number of podocytes per glomerulus (Epi N/glom) was obtained by multiplying Nv(epi/glom) by mean glomerular volume. Nv(epi/glom) was significantly decreased in all type 2 diabetic groups compared with control subjects and was lower in MA and P than in NA (P < 0.0001, ANOVA). Epi N/glom was lower in MA and P patients compared with control subjects (P < 0.002, ANOVA); however, there were no significant differences among the type 2 diabetic groups. In addition, MA and P had decreased FSLv/glom and increased FPW compared with NA (P < 0.005 for both, ANOVA). The albumin excretion rate was inversely related to Nv(epi/glom) and FSLv/glom and directly to FPW (P < 0.0005 for all), whereas there was no correlation with Epi N/glom. In conclusion, changes in podocyte structure and density occur since the early stages of diabetic nephropathy and might contribute to increasing albuminuria in type 2 diabetic patients. These findings also suggest that in white type 2 diabetic patients, the density of podocytes may be functionally more relevant than the absolute number.     

Erweiterte kardiopulmonale Reanimation in besonderen Situationen: Teil 2

Based on the 2005 International Consensus Conference on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations of the International Liaison Committee on Resuscitation (ILCOR), guidelines were published for managing basic and advanced life-saving procedures in the event of cardiac arrest. The fact that special circumstances for cardiac arrest must be considered resulted in a separate chapter. This two-part article reviews essential information as well as necessary modifications of the standard advanced life support algorithm in cases of electrolyte disorders, hyperthermia and hypothermia, cardiac arrest in pregnancy, trauma, electrical emergencies and cardiac surgery. Part 1 has already dealt with life-threatening drowning, asthma, anaphylaxis and poisoning.     

Urinary ?2-microglobulin in very preterm neonates with chorioamnionitis

It is important to identify premature infants with prenatal inflammation as it contributes to short- and long-term complications. Our object was to study how prenatal inflammation affects the urinary β₂-microglobulin (β₂-MG) level. Preterm neonates were divided based on the presence of chorioamnionitis (CAM) into the CAM (n = 100) and non-CAM groups (n = 117). These were further subdivided into five groups each: 30 preterm neonates of 23–26; 42 neonates of 27–28; 54 neonates of 29–30; 51 neonates of 31–32; and 40 neonates of 33–34 weeks’ gestation. The urinary β₂-MG level within 48 h of birth was significantly higher in the CAM group than in the non-CAM group among the neonates of 23–26 weeks’ gestation (18.3 ± 6.9 vs 10.0 ± 5.6 × 10⁴ μg/gCr, p = 0.0018) and the neonates of 27–28 weeks’ gestation (16.2 ± 10.8 vs 8.8 ± 3.3 × 10⁴ μg/gCr, p = 0.0101). However, there was no difference in urinary β₂-MG level between the CAM and the non-CAM group among the neonates ≥ 29 weeks ’gestation. Moreover, the elevated urinary β₂-MG level in the neonates ≤ 28 weeks ’ gestation with CAM had disappeared by 1 week after birth. The reasons for the increase in urinary β₂-MG level within 48 h of birth in very preterm neonates (≤ 28 weeks’ gestation) with CAM are believed to be not only prematurity, but also prenatal inflammation. It is suggested that the urinary β₂-MG level during the early postnatal period can identify prenatal inflammation.     

Mueller reinforcement rings in acetabular revision Outcome in 164 hips followed for 2–17 years

Background?In revision hip arthroplasty, cages are commonly used for acetabular reconstruction in cases with loss of bone stock. It is important to follow this patient group closely, in order to better understand failure mechanisms and the chance of long-term success.

Patients and methods?We followed our first 164 acetabular revisions with the Mueller reinforcement ring (ARR) in 164 patients, with an average follow-up period of 6 (2–17) years. Mean age at surgery was 69 (29–92) years. 39 patients died (39 hips) during follow-up, but only 3 patients (3 hips) were lost to follow-up.

Results?In the observation period, 13 of the hips had to undergo acetabular re-revision for aseptic and septic loosening. Overall survival at 5 years was 95% (CI: 89%–98%) and 90% (CI: 76%–95%) at 8 years using acetabular re-revision or implant removal for all reasons as endpoint. In addition, mechanical and clinical failure was seen in 2 cases. The mean Harris hip score was 70 points, whilst the Merle d'Aubigné score averaged 7 points. Radiolucent lines according to DeLee and Charnley were observed twice in zone I, 6 times in zone II and 14 times in zone III.

Interpretation?We found that mid- to long-term survival of the ARR is acceptable. However, failure of the implant due to allograft collapse/resorption or deep infection, and also poor clinical outcome, remain major concerns in acetabular revision arthroplasty. This should be recognized when advising patients.     

Interferon-α inhibits cyclooxygenase-1 and stimulates cyclooxygenase-2 expression in bladder cancer cells in vitro

The enzymes cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2) catalyze the initial step in the formation of prostaglandins (PGs). PGs are known to be involved in numerous processes, for example inflammation, immune responses, carcinogenesis, and tumor angiogenesis. The formation of PGs is stimulated in various cancers since the expression of Cox-2 is upregulated. Interferon (IFN)-α is used in the treatment of bladder cancer, although not all of the effects of such treatment are thoroughly known. Therefore, we investigated the expression of cyclooxygenases in two bladder cancer cell lines, 5637 and T24, under basal conditions and in the presence of human recombinant IFN-α (100, 1,000, and 10,000 U/ml). The mRNA of Cox-1 and Cox-2 was expressed in both cultured bladder carcinoma cell lines. The level of Cox-1 expression was low in 5637 cells and higher in T24 cells. In contrast, Cox-2 expression was prominent in 5637 cells and low in T24 cancer cells. The highest IFN-α concentration (10,000 U/ml) decreased the expression of Cox-1 to 47 and 28% of the control levels in 5637 and T24 cells, respectively. In contrast, Cox-2 expression increased in both cell lines. In 5,637 cells, Cox-2 expression increased 1.3-fold with 10,000 U/ml of IFN-α. In T24 cells, the maximum effect was achieved by 1,000 U/ml of IFN-α, which increased the expression of Cox-2 up to 2.4-fold. These findings may have relevance in the outcome of patients treated with IFN-α because upregulated Cox-2 expression may suppress the cell-mediated defense system. On the other hand, the inhibition of Cox-1 could be beneficial because Cox-1 is known to stimulate angiogenesis. Received: 5 August 1999 / Accepted: 8 September 2000