Influence of 1-MCP on texture, related enzymes, quality and their relative gene expression in 'Amrapali' mango (Mangifera indica L.) fruits.

The mango fruits remain biologically active even after harvest as they continue respiration, transpiration and other bio-chemical processes. Being highly perishable, the fruit quality deteriorates fast under ambient conditions (30 ± 5 °C and 50 ± 5% RH), rendering them unmarketable within 5-6 days. In order to extend the shelf-life of 'Amrapali' mango fruits, we have treated them with three different concentrations (500, 750 and 1000 ppb) of 1-Methylcyclopropene (1-MCP) @ 20 °C and stored at ambient conditions. Among all the treatments, 1000 ppb was found to be an effective in extending shelf-life till twelfth day with minimum physiological loss in weight (19.24%), maximum firmness (10.43 N), highest retention of quality parameters such as soluble solid concentrates (27.88 °B), ascorbic acid (28.49 mg 100 g-1 FW) and total antioxidant activity (675.41 µmol Trolox g-1 FW) compared to untreated mango fruits (21.79%, 5.45 N, 23.17 °B, 19.55 mg 100 g-1 FW and 265.41 µmol Trolox g-1 FW, respectively). Gene expression studies have revealed that the texture related gene expansin was significantly repressed till fifth day of storage with increasing concentrations of 1-MCP.

Outcome and complications of percutaneous nephrolithotomy as primary versus secondary procedure for renal calculi.

PURPOSE: To compare the efficacy of percutaneous nephrolithotomy (PCNL) as a primary procedure of patients following previous open surgery or post percutaneous nephrolithotomy (PCNL) for renal calculi. MATERIALS AND METHODS: The medical records of 367 patients who underwent PCNL by a single surgeon from January 2008 to December 2013 were reviewed retrospectively. All patients were divided into 3 Groups. Group-1 (n=232) included patients with no history of ipsilateral open stone surgery. Group 2 (n=86) patients had undergone one or more open stone surgeries before PCNL, patients with failed or recurrence following PCNL were placed in Group-3 (n=49). The demographic data, operation duration, stone free rate (SFR), number of attempts to access the collecting system and intra operative and postoperative complications between the three Groups were compared. RESULTS: There was no difference in sex, Body Mass Index (BMI), stone burden and laterality among the three Groups. Operation time was significantly less in first Group, while there was a statistically significant difference in operation duration between second and third Groups (p<0.05). The number of attempts to enter the collecting system was lower in the first Group in comparison to other two Groups (p<0.5). There was no significant differences among three groups in stone free rate. Intra operative and postoperative complications were slightly more frequent in Groups 2 and 3. Mortality occurred in 1 patient with colon perforation in Group-2. CONCLUSION: Our study demonstrated that PCNL can be performed in patients even as secondary procedure without further complications.

Ischemia-modified albumin levels in overt and subclinical hypothyroidism.

BACKGROUND: Free radical-mediated oxidative stress (OS) has been implicated in the pathogenesis of thyroid disorders. The ischemia-modified albumin (IMA) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. AIM: Our aim was to evaluate IMA, malondialdehyde (MDA), and reduced glutathione (GSH) levels in patients with overt hypothyroidism (OHT) and subclinical hypothyroidism (SHT) in comparison to euthyroid controls. SUBJECTS AND METHODS: Albumin, IMA, IMA/albumin ratio, MDA, GSH, total cholesterol (TC), triglycerides (TG), HDL-Cholesterol were assessed in 105 subjects grouped into OHT, SHT patients, and euthyroid controls with 35 subjects in each group. RESULTS: MDA and IMA levels were significantly elevated while the GSH concentrations were significantly lower in OHT and SHT patients compared to controls (p < 0.01). When IMA values were normalized for albumin concentrations, the IMA/albumin ratio was also significantly elevated in both patient groups compared to controls (p < 0.01). These changes were more pronounced in the OHT group when compared to SHT group. In OHT group, thyroid-stimulating hormone (TSH) levels showed significant positive correlation with MDA (r = 0.470, p = 0.004), IMA (r = 0.530, p = 0.001), and IMA/albumin ratio (r = 0.525, p = 0.001). Both IMA (r = -0.342, p = 0.041), IMA/albumin ratio (r = -0.378, p = 0.023) showed significant negative correlation with GSH in OHT patients. No significant correlation between variables was, however, observed in SHT group. CONCLUSIONS: Increase of MDA and IMA levels with decreased antioxidant status indicate the presence of OS in hypothyroid patients, which was more pronounced in OHT patients. Elevated levels of IMA can be a clinically useful marker of protein oxidative damage and OS in hypothyroidism.

A Brief Review on Advanced Sandwich Structures with Customized Design Core and Composite Face Sheet.

Sandwich structures are a class of multifunctional high-performance structural composites that have the advantages of being lightweight, of a high strength-to-weight ratio, and of high specific energy absorption capabilities. The creative design of the core along with the apposite material selection for the fabrication of the face sheet and core are the two prerequisites with encouraging areas for further expedition towards the fabrication of advanced composite sandwich structures. The current review work focused on different types of core designs, such as truss, foam, corrugated, honeycomb, derivative, hybrid, hollow, hierarchical, gradient, folded, and smart core along with different composite materials accessible for face sheet fabrication, including fiber-reinforced composite, metal matrix composite, and polymer matrix composite are considered. The joining method plays a major role for the performance evolution of sandwich structures, which were also investigated. Further discussions are aligned to address major challenges in the fabrication of sandwich structures and further enlighten the future direction of the advanced composite sandwich structure. Finally, the work is summarized with a brief conclusion. This review article provides wider guidelines for researchers in designing and manufacturing next-generation lightweight multilayer core sandwich structures.

A Study of Morphological Characteristics of Lung Fissures and Trachea in the Indian Population.

Tracheal length and lung anatomy have been rarely studied; however, the anatomy of the lung has been shown to vary significantly. Moreover, the surgery regarding trachea are few, and hence the surgeons do not have extensive experience in the trachea. OBJECTIVE: We aimed to study the variations of the lung anatomy and the relation between tracheal length and body height in the Indian population. MATERIALS AND METHODS: This is an observational study to observe the tracheal length in relation to body height and sex and gross morphological anatomy of the lung in 70 cadavers. The data was collected from the forensic department of Bangalore Medical College and Research Institute (BMCRI), and further analysis was done at Kidwai Memorial Institute of Oncology. RESULTS: Deviation from normal lung morphology was seen in 37.86% of the specimens studied. The tracheal length (average, 9.97 cm) correlated with the body length (average, 147.02 cm) with a Pearson coefficient of 0.806 (p value=0.001) Conclusion: The study of lung fissure morphology guides clinicians in understanding and planning lung disease treatment, especially lobectomy/segmentectomy surgeries. The information of the average length of the trachea with respect to body height in a given ethnicity will help during endotracheal intubation and tracheal surgical planning.

Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation.

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell-cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell-cell contact caused evident morphological changes accompanied with tight cell-cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow-derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.

Non-invasive evaluation of bladder outlet obstruction in benign prostatic hyperplasia: a clinical correlation study.

Objectives: To determine the utility of ultrasonography (US)-derived parameters (e.g. prostate volume [PV], bladder wall thickness [BWT], post-void residual urine volume [PVR], and intravesical prostatic protrusion [IPP]) and uroflowmetry for identifying bladder outlet obstruction (BOO) by correlating them with the results of pressure-flow urodynamic studies (UDS). Patients and methods: In all, 164 patients presenting with lower urinary tract symptoms suggestive of benign prostatic hyperplasia (BPH), from May 2016 to December 2018, were included in this study. All had International Prostate Symptoms Score (IPSS), Quality-of-Life (QOL) index, uroflowmetry (including maximum urinary flow rate [Qmax]) and PVR measured by transabdominal US. Pressure-flow UDS were performed on all men and BOO was defined by a BOO Index (BOOI) >40. Men with a Qmax of ≥12.0 mL/s were considered to have 'good' flow. Results: Amongst the 164 men, the mean (SD) age, PV, BWT and Qmax were 66.72 (9.88) years, 51.91 (13.24) mm, 5.07 (0.91) mm, and 8.46 (3.59) mL/s, respectively. In all, 91 (55.49%) patients had BOO with a BOOI >40 and nine (5.49%) had equivocal BOO with a BOOI of 20-40. The IPP was a statistically significant predictor (P < 0.001) of BOO compared with other variables in the initial evaluation. In patients with BOO confirmed by the pressure-flow UDS, IPP Grade III was associated with a higher BOOI than was Grade I and II (P < 0.001). Conclusion: BWT, PV and PVR in conjunction with IPP are good predictors of clinically significant BOO due to BPH. Abbreviations: AUC: area under the curve; BOOI: BOO Index; BPO, benign prostatic obstruction; BWT, bladder wall thickness; IPP: intravesical prostatic protrusion; Pdet: detrusor pressure; PV: prostate volume; PVR: post-void residual urine volume; Qmax: maximum urinary flow rate; QOL: quality of life; ROC: receiver operating characteristic; (TA)US: (transabdominal) ultrasonography; UDS: urodynamic studies.

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype.

Osteoclasts (OCLs) in Paget's disease are markedly increased in number and size, have increased numbers of nuclei per multinucleated cell, and demonstrate increased resorption capacity and increased sensitivity to 1,25-(OH)(2)D(3), the active form of vitamin D. These cells also contain nuclear inclusions, reminiscent of those seen in paramyxovirus-infected cells, which cross-react with antibodies to measles virus nucleocapsid (MVNP) antigen. To elucidate the role of MV in the abnormal OCL phenotype of Paget's disease, we transduced normal OCL precursors with retroviral vectors expressing MVNP and the MV matrix (MVM) genes. The transduced cells were then cultured with 1,25-(OH)(2)D(3) for14 or 21 days to induce formation of OCL-like multinucleated cells. The MVNP-transduced cells formed increased numbers of multinucleated cells, which contained many more nuclei and had increased resorption capacity compared with multinucleated cells derived from empty vector-transduced (EV-transduced) and MVM-transduced or normal bone marrow cells. Furthermore, MVNP-transduced cells showed increased sensitivity to 1, 25-(OH)(2)D(3), and formed OCLs at concentrations of 1, 25-(OH)(2)D(3) that were 1 log lower than that required for normal, EV-transduced, or MVM-transduced cells. These results demonstrate that expression of the MVNP gene in normal OCL precursors stimulates OCL formation and induces OCLs that express a phenotype similar to that of pagetic OCLs. These results support a potential pathophysiologic role for MV infection in the abnormal OCL activity and morphology that are characteristic of pagetic OCLs.

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption.

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures.

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget's disease of bone.

Paget's disease is characterized by highly localized areas of increased osteoclast (OCL) activity. This suggests that the microenvironment in pagetic lesions is highly osteoclastogenic, or that OCL precursors in these lesions are hyperresponsive to osteoclastogenic factors (or both). To examine these possibilities, we compared RANK ligand (RANKL) mRNA expression in a marrow stromal cell line developed from a pagetic lesion (PSV10) with that in a normal stromal cell line (Saka), and expression in marrow samples from affected bones of Paget's patients with that in normal marrow. RANKL mRNA was increased in PSV10 cells and pagetic marrow compared with Saka cells and normal marrow, and was also increased in marrow from affected bones compared with uninvolved bones from Paget's patients. Furthermore, pagetic marrow cells formed OCLs at much lower RANKL concentrations than did normal marrow. Anti-IL-6 decreased the RANKL responsivity of pagetic marrow to normal levels, whereas addition of IL-6 to normal marrow enhanced RANKL responsivity. Thus, RANKL expression and responsivity is increased in pagetic lesions, in part mediated by IL-6. These data suggest that the combination of enhanced expression of RANKL in affected bones and increased RANKL sensitivity of pagetic OCL precursors may contribute to the elevated numbers of OCLs in Paget's disease.

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.

Measles virus nucleocapsid transcript expression is not restricted to the osteoclast lineage in patients with Paget's disease of bone.

Abundant evidence supports a viral etiology for Paget's disease of bone (PD), however, an infectious virus has not been isolated from PD patients. Thus, it is unclear how the virus is maintained for the many years that the disease persists in patients. We considered if a primitive multipotential hematopoietic stem cell (HSC), which is self-renewing, passes the virus to its differentiated progeny and serves as a reservoir for the pathogen. If a primitive stem cell harbored measles virus (MV), then other hematopoietic lineages derived from this stem cell in PD patients should also express MV transcripts. Therefore, because the human hematopoietic stem cell has not been clearly identified or isolated in large numbers, we isolated RNA from highly purified erythroid and multipotential hematopoietic progenitors that are the precursors for erythroid, granulocyte, megakaryocyte and macrophages (CFU-GEMM), and used RT-PCR to determine if MV nucleocapsid transcripts were present. MV transcripts were detected in PD patients in early erythroid (BFU-E) and more primitive multipotential myeloid progenitors (CFU-GEMM). Nonhematopoietic stromal cells from PD patients did not express MV transcripts. The expression of MV transcripts in erythroid progenitors was further confirmed by in situ hybridization using antisense riboprobes to MV nucleocapsid transcripts. Thus, our findings suggest that the pluripotent HSCs may be a potential reservoir for the virus. We propose that when HSCs, which contain MV, divide they produce a second HSC that serves as a reservoir for the virus and also transmit the virus to their more differentiated progeny in the erythroid and myeloid lineages. This mechanism would permit a defective virus to persist in HSCs of PD patients for many years, since HSCs are usually in G0 phase, and then be transmitted to more differentiated cells. This model further suggests that a mature complete virus that affects cell function could only act pathogenetically in the osteoclast lineage, which offers a permissive milieu.

Molecular docking and dynamic simulation studies evidenced plausible immunotherapeutic anticancer property by Withaferin A targeting indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic drug target for the treatment of cancer characterized by pathological immune suppression. IDO catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway. Reduction in local tryptophan concentration and the production of immunomodulatory tryptophan metabolites contribute to the immunosuppressive effects of IDO. Presence of IDO on dentritic cells in tumor-draining lymph nodes leading to the activation of T cells toward forming immunosuppressive microenvironment for the survival of tumor cells has confirmed the importance of IDO as a promising novel anticancer immunotherapy drug target. On the other hand, Withaferin A (WA) - active constituent of Withania Somnifera ayurvedic herb has shown to be having a wide range of targeted anticancer properties. In the present study conducted here is an attempt to explore the potential of WA in attenuating IDO for immunotherapeutic tumor arresting activity and to elucidate the underlying mode of action in a computational approach. Our docking and molecular dynamic simulation results predict high binding affinity of the ligand to the receptor with up to -11.51 kcal/mol of energy and 3.63 nM of IC50 value. Further, de novo molecular dynamic simulations predicted stable ligand interactions with critically important residues SER167; ARG231; LYS377, and heme moiety involved in IDO's activity. Conclusively, our results strongly suggest WA as a valuable small ligand molecule with strong binding affinity toward IDO.

Detection of measles virus nucleocapsid transcripts in circulating blood cells from patients with Paget disease.

Paget disease of bone is characterized by abnormalities in all phases of bone remodeling, but the fundamental cellular abnormality resides in the osteoclast (OCL). Osteoclasts in bone involved by Paget disease contain viral-like nuclear and cytoplasmic inclusions that react with antibodies directed against paramyxovirus nucleocapsid proteins, such as measles virus, respiratory syncytial virus, or canine distemper virus. However, the identity of the virus or the mechanisms responsible for its persistence or pathologic role in Paget disease is unclear. Furthermore, although Paget disease persists for many years, it remains a highly localized process with new lesions rarely if ever developing in previously unaffected bones. Since osteoclasts are formed by fusion of mononuclear precursors derived from colony forming unit-granulocyte macrophage (CFU-GM), the granulocyte-macrophage progenitor, we used reverse transcriptase polymerase chain reaction (RT-PCR) analysis to determine if CFU-GM, more differentiated osteoclast precursors, and peripheral blood cells derived from CFU-GM express measles virus nucleocapsid (MV-N) transcripts. We found that osteoclast precursors, as well as peripheral blood mononuclear cells, express MV transcripts in 9 of 13 patients. Sequence analysis of the PCR amplified products confirmed nucleotide identity of MV-N transcripts expressed in peripheral blood and bone marrow-derived cells from the same patient. In contrast, MV-N transcripts were not detected in OCL precursors or the peripheral blood from 10 normal subjects. In situ hybridization studies using 35S-labeled antisense riboprobes to MV-N transcripts further confirmed the expression of MV transcripts in these cells. Sequence analysis of the PCR amplified product from one of these patients also identified a novel mutation that converted lysine441 to glutamic acid441 in the MV-N transcript. These data demonstrate that OCL precursors and circulating peripheral blood cells also express MV transcripts in patients with Paget disease and suggest that the pagetic marrow microenvironment plays a critical role in maintaining the highly localized nature of the lesions in Paget disease.

Interleukin-6 antisense deoxyoligonucleotides inhibit bone resorption by giant cells from human giant cell tumors of bone.

The effects of antisense constructs to IL-6 on the bone-resorbing capacity of purified giant cells from giant cell tumors of bone were examined to further define the role of IL-6 in human osteoclastic bone resorption. In addition, we wanted to determine the utility of antisense constructs to cytokines produced by osteoclasts as probes to identify the molecular events responsible for the bone-resorptive process. Giant cells were cultured on sperm whale dentin for 24 h in the presence of fluoresceinated antisense or scrambled antisense deoxyoligonucleotides complementary to IL-6 mRNA. The giant cells actively incorporated these oligonucleotides, as evidenced by their intense fluorescence. The number of resorptive lacunae formed and the area of the dentin resorbed were significantly decreased in cultures of giant cells treated with antisense IL-6 constructs compared with control cultures treated with scrambled antisense constructs to IL-6 (60 +/- 13 versus 12 +/- 6 lacunae and 1.2 +/- 0.3 versus 0.26 +/- 0.1 x 10(5) microns2). IL-6 levels in conditioned media from giant cell cultures treated with IL-6 antisense constructs were fourfold lower than those in control media obtained from giant cells treated with scrambled antisense constructs to IL-6. These data confirm the capacity of IL-6 antisense oligomers to block IL-6 production by these cells. These observations show that IL-6 plays an important role in the bone-resorptive process of human osteoclasts and suggest that antisense constructs to cytokines produced by bone cells may be useful for determining the molecular events occurring during bone resorption.

IL-6 mediates the effects of IL-1 or TNF, but not PTHrP or 1,25(OH)2D3, on osteoclast-like cell formation in normal human bone marrow cultures.

A potent interleukin-6 (IL-6) antagonist (Sant 5), which binds tightly to the IL-6alpha receptor but has impaired gp130 heterodimerization, has been developed recently by site-directed mutagenesis of human IL-6. We report here that Sant 5 inhibits IL-6-stimulated osteoclast-like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL-1 or tumor necrosis factor alpha (TNF-alpha in these cultures. We further show that a neutralizing antibody to IL-6 also inhibits the stimulatory effects of IL-1 or TNF-alpha in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25-dihydroxyvitamin D3 stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL-6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast-like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL-6 mediates the effects of IL-1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL-6 antagonist and inhibit osteoclast formation in vitro.

1,25-Dihydroxyvitamin D3 hypersensitivity of osteoclast precursors from patients with Paget's disease.

Our previous studies suggested that increased osteoclast formation and activity in Paget's disease may be related in part to increased responsiveness of highly purified osteoclast precursors to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. However, the basis for this enhanced sensitivity to 1,25-(OH)2D3 is unclear. To address this question, we examined 24-hydroxylase and 1,25-(OH)2D3 receptor (VDR) messenger RNA (mRNA) expression during human osteoclast differentiation from normal subjects and patients with Paget's disease in response to 1,25-(OH)2D3 as well as VDR content and affinity. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of granulocyte-macrophage colony-forming unit (GM-CFU), the earliest identifiable osteoclast precursor, derived from patients with Paget's disease demonstrated 24-hydroxylase mRNA expression in response to 1,25-(OH)2D3 was induced at concentrations of 1,25-(OH)2D3 that were at least one log less than that required for normal GM-CFU. VDR mRNA and VDR protein were detected in both immature and more differentiated osteoclast precursors, as well as in osteoclast-like multinucleated cells (MNCs). However, VDR expression was lower in MNCs than the mononuclear precursor cells. Osteoclast precursors and MNCs from patients with Paget's disease had levels of VDR expression similar to those of normal subjects but showed increased VDR affinity for 1,25-(OH)2D3. Because the effects of 1,25-(OH)2D3 are in part mediated by induction of expression of RANK ligand on marrow stromal cells, which in turn stimulates osteoclast formation, we examined expression of RANK ligand mRNA by marrow stromal cell lines derived from patients with Paget's disease and normal subjects in response to 1,25-(OH)2D3. RT-PCR analysis showed no difference in sensitivity of marrow stromal cells to 1,25-(OH)2D3 from normal subjects or patients with Paget's disease although the Paget's stromal cells expressed increased basal levels of RANK ligand mRNA. These results show that VDR protein is expressed in early and more differentiated osteoclast precursors, that expression levels of VDR decline with osteoclast differentiation, and that 1,25-(OH)2D3 has direct effects on osteoclast precursors. The enhanced sensitivity to 1,25-(OH)2D3 is an intrinsic property of osteoclast precursors from patients with Paget's disease that distinguishes them from normal osteoclast precursors. Furthermore, our results suggest that an increased affinity of VDR for 1,25-(OH)2D3 may be responsible for the enhanced 1,25-(OH)2D3 sensitivity of osteoclast precursors in patients with Paget's disease compared with normal subjects.

Cell biology of Paget's disease.

Paget's disease is characterized by markedly increased osteoclast formation and bone resorption followed by excessive new bone formation. Osteoclasts in Paget's disease are increased both in number and size, contain paramyxoviral-like nuclear inclusions, and can have up to 100 nuclei per cell. Marrow culture studies have identified several abnormalities in osteoclast formation in Paget's disease. Osteoclast-like multinucleated cells formed more rapidly in marrow cultures from patients with Paget's disease, produced increased levels of interleukin-6 (IL-6), and expressed high levels of IL-6 receptors compared to normals. IL-6 levels were also increased in bone marrow and peripheral blood of patients with Paget's disease. In addition, osteoclast precursors from patients with Paget's disease are hyperresponsive to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and calcitonin. The increased sensitivity of osteoclast precursors to 1,25(OH)2D3 is mediated through the vitamin D receptor (VDR), since 24-hydroxylase activity is also up-regulated at concentrations of 1,25(OH)2D3 that are one log less than that needed to induce 24-hydroxylase activity in osteoclast precursors from normals. However, VDR numbers and affinity for 1,25(OH)2D3 do not differ in osteoclast precursors from Paget's patients compared to those from normals. Synergistic interactions between cytokines such as IL-6 and 1,25(OH)2D3 also cannot explain the enhanced sensitivity of osteoclast precursors from patients with Paget's disease to 1,25(OH)2D3. Interestingly, coculture studies of osteoclast precursors and cells from the marrow microenvironment of patients with Paget's disease and normals have demonstrated that the marrow microenvironment is more osteoclastogenic than normal. Thus, studies of the cell biology of osteoclasts in Paget's disease have demonstrated an increased rate of osteoclast formation and abnormalities in both osteoclast precursors and the marrow microenvironment. Enhanced IL-6 production by osteoclasts in Paget's disease may further amplify the increased osteoclast formation already ongoing in the pagetic lesion, and may explain the increased bone turnover at uninvolved sites distant from the pagetic lesion.

Cloning and characterization of the 5'-flanking region of the mouse tartrate-resistant acid phosphatase gene.

Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb SmaI fragment, containing the 5'-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the SmaI fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for AP1 and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at -1 to -393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at -552 bp from the ATG. The region from -1846 to 2bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.