Low concentrations of soybean lecithin nanoparticles had a positive impact on Holstein bulls' cryopreserved semen.

Spermatozoa can experience negative changes when subjected to freezing and thawing, including lowered motility, viability and acrosome response. Herein, the effects of different concentrations of soybean lecithin nanoparticles on cryopreserved Holstein bull semen were examined. Semen was collected, cryopreserved and utilized for sperm kinetic parameter analysis following dilution, equilibration and thawing with 0.5% soybean lecithin (E1), the control extender, and 0.75% (E2), 0.5% (E3), 0.25% (E4) and 0.125% (E5) of lecithin nanoparticles. Results revealed that following dilution, the progressive motility (PM) at E3, E4 and E5 of lecithin nanoparticles was higher (p < .05) than it was for E2. After equilibration, compared to the E1, E2, and E3 values, the PM, vitality, normal morphology, membrane integrity and intact acrosome values at the E5 were consistently greater (p < .05). Comparing the percentages of intact acrosome and membrane integrity at E2 and E3 to E4 and E5, a substantial decrease (p < .05) was seen. Following thawing, the percentage of PM improved at E2 and E5, even though their mean PM values were similar (p > .05) compared to E1, E3 and E4. Vigour and progression parameters of sperm (DAP, DCL, DSL, VAP, VCL, VSL and STR) at E5 were higher (p < .05) than those at E1, E2, E3 and E4. In conclusion, the cryopreserved sperm from Holstein bulls revealed outstanding properties both after equilibration and after thawing with 0.125% lecithin nanoparticles, and they were sensitive to high dosages.

Studying the Influence of Copper on the Growth Behavior, Antioxidative Status, and Histology of the Intestine and Liver of Striped Catfish (Pangasianodon hypophthalmus).

Microelements are well recognized as an essential approach in the field of aquaculture nutrition. Thus, this study aimed to evaluate copper (Cu) inclusion (0, 0.5, 1, and 2 mg/kg) on Striped catfish performances. Fish fed the Cu-incorporated diets for 60 days, then their growth behavior, antioxidative capacity, and intestinal and liver histological features were evaluated. The results showed a marked enhancement in Striped catfish's growth behavior fed 1-2 mg/kg of Cu, as shown by the final weight, weight gain, and specific growth rate. The feed and protein efficiency ratios were significantly affected by Cu in a dose-dependent manner. The highest level of Cu was accumulated in the whole body, muscle, liver, and gills of fish fed 2 mg/kg of Cu. The carcass composition of Striped catfish showed higher protein content in groups received 0.5, 1, and 2 mg/kg Cu in a linear and quadratic manner (p=0.001). The ash content was quadratically increased in Striped catfish fed 2 mg/kg Cu (p=0.001). However, no marked effects were observed on the moisture and lipid contents and the somatic indices (p>0.05). The incorporation of Cu showed meaningfully increased superoxide dismutase, catalase, and glutathione peroxidase but decreased malondialdehyde level in Striped catfish. The villous height exhibited visible growth and branching with increased doses of Cu without a significant increase in the goblet cells. No abnormal features were observed in the liver and hepatocytes of fish treated with Cu. It can be concluded that Cu is required at 1-2 mg/kg for better performances of Striped catfish.

Selenium nanoparticles are required for the optimum growth behavior, antioxidative capacity, and liver wellbeing of Striped catfish (Pangasianodon hypophthalmus).

Selenium (Se) is a multifunctional trace element required in specific amounts for the optimal growth of aquatic finfish species. For this reason, this study investigated the effect of Se nanoparticles on the growth behavior, antioxidative capacity, and liver wellbeing of Striped catfish (Pangasianodon hypophthalmus). Striped catfish fed varying Se nanoparticles levels (0. 0.5, 1, and 2 mg/kg) in triplicate units and kept for 60 days. Striped catfish delivered dietary Se nanoparticles had markedly increased growth performance, specific growth rate (SGR), consumed feed, and protein efficiency ratio but reduced feed conversion ratio (FCR). The whole body, liver, muscle, and gills have higher Se accumulation levels in fish that received Se nanoparticles than the control with the highest level in fish fed 2 mg/kg. The carcass composition showed higher protein content in fish fed 1 and 2 mg/kg (p = 0.001 and 0.001) and higher ash content (p = 0.001 and 0.002) in fish fed 2 mg/kg than the remaining groups. Superoxide dismutase was meaningfully activated in Striped catfish delivered 1 and 2 mg Se nanoparticles/kg compared with the control (p < 0.05). Also, catalase and glutathione peroxidase activities were higher, and malondialdehyde level was lower in Striped catfish fed Se nanoparticles at 0.5, 1, and 2 mg/kg than the control (p < 0.05). The villi exhibited a visible increase in both height and branching with an increased level of Se nanoparticles in addition to the increased number of goblet cells. The Se nanoparticles-treated fish revealed dose-dependent modifications fluctuated from diffuse fatty vacuolization in hepatocytes with eccentric pyknotic hepatocytes nuclei. In conclusion, Se nanoparticles are required for the optimum growth behavior, antioxidative capacity, and liver wellbeing of Striped catfish. Based on SGR and FCR data's regression analysis, Se nanoparticles are recommended at 1.02-1.11 mg/kg diet.

Gene expression of bovine endometrial epithelial cells cultured in matrigel.

Glandular epithelial cells (GE) in the endometrium are thought to support the elongation and survival of ruminant embryos by secreting histotrophs. In the present study, the gene expression of bovine endometrial epithelial cells cultured in matrigel was analyzed and examined whether it could be an in vitro model of GE. Bovine endometrial epithelial cells (BEE) and stromal cells (BES) were isolated from the slaughterhouse uteri and cultured in DMEM/F12 + 10% FBS. BEE showed the gland-like structure morphological changes when cultured in 15% matrigel but could not be identified in higher concentrations of the matrigel (30% or 60%). The expression of typical genes expressed in GE, SERPINA14 and GRP, was substantially high in matrigel-cultured BEE than in monolayer (P  <  0.05). P4 and INFα have no significant effect on the SERPINA14 expression of BEE cultured in matrigel without co-culture with BES. On the other hand, when BEE were co-cultured with BES in matrigel culture, the expression of FGF13 was increased by the P4 treatment (P  <  0.05). Furthermore, SERPINA14 and TXN expressions were increased by P4 + IFNα treatment (P  <  0.05). These results demonstrate the appropriate conditions for BEE to form glandular structures in matrigel and the effect of co-culture with BES. The present study highlighted the possible use of matrigel for the culture of BEE to investigate the expression of cell-specific glandular epithelial genes as well as P4 and type-I IFN as factors controlling endometrial function during the implantation period.

The Role of Diffusion-Weighted MRI and Apparent Diffusion Coefficient in Assessment of Diabetic Kidney Disease: Preliminary Experience Study.

BACKGROUND: Diabetic kidney disease is the most common cause of ESRD. There is poor correlation between the degree of renal fibrosis and current screening markers. A noninvasive imaging technique is needed to assess the degree of structural changes in the kidney. The aim of this study was to assess the role of apparent diffusion coefficient (ADC) in the diagnosis of diabetic kidney disease. Forty adult diabetic patients with chronic kidney disease as well as 20 age- and sex-matched adult healthy controls were recruited from Nephrology Department of our University Hospital. All patients underwent renal MR-DWI and ADC mapping on a 1.5-T scanner (Philips Achieva) using phased array body coil. RESULTS: Among the studied 40 diabetic patients, five groups of patients were resulted 8 patients for each and the ADC values were inversely correlated with advancement in renal parenchymal affection, ie, in late stages of the disease the ADC values were lower than in early stages. The mean ADC values of renal parenchyma in patients with diabetic kidney disease were considerably lower than that of healthy controls with normal renal function (2.1±0.3x10-3 mm2/s vs 2.4±0.1x10-3 mm2/s with p<0.001). CONCLUSION: ADC value is a possible noninvasive technique in evaluating the stage of renal dysfunction with assessment of disease progression.

Copper Nanoparticles Mitigate the Growth, Immunity, and Oxidation Resistance in Common Carp (Cyprinus carpio).

The present investigation aimed to evaluate the influence of copper nanoparticles (Cu-NPs) on the growth, immunity, and oxidation resistance of common carp (3.02 ± 0.01 g, initial mean weight ± S.E.). Five groups of fish fed diets with Cu-NPs at 0, 0.5, 1, 2, and 4 mg/kg for 8 weeks. The results suggested that Cu-NPs in diets increased the growth performance and reduced FCR with linear and quadratic model (P < 0.05). Also, common carp fed Cu-NPs showed increased carcass protein, lipid, and ash contents in a dose-dependent manner (P < 0.05). The Cu accumulation in the carcass, liver, muscle, and gills increased by Cu-NPs and showed the maximum at 4 mg Cu-NPs/kg (P < 0.05). No significant alterations were found in the blood variables due to Cu-NP supplementation except for the Hb, RBCs, total protein, albumin, and globulin levels which showed the highest level in 2 mg/kg (P < 0.05). IgM level, phagocytic, lysozyme, SOD, CAT, and GPX activities were boosted by Cu-NPs with decreased malondialdehyde (MDA) content (P < 0.05). Based on regression analysis, the requirement of dietary Cu-NPs for common carp was estimated to be 2.19 to 2.91 mg/kg diet.

Effects of fruit drinks on surface roughness of two esthetic restorative materials.

OBJECTIVES: Restorative materials may be exposed in the oral cavity to chemical agents found in beverages, which may lead to their biodegradation. The purpose of this in vitro study was to evaluate the effect of two fruit drinks commonly used by children on surface roughness of two esthetic restorative materials. MATERIALS AND METHODS: One resin composite (RC), one resin-modified glass ionomer (RMGI) and two fruit drinks (orange and cocktail) were used in this study. Specimens (n=20) of each material were fabricated against Mylar strip. Baseline measurements of surface roughness were recorded for each group using noncontact surface profilometer. Each specimen was placed in the tested fruit drinks for 24 hours and then surface roughness was recorded. RESULTS: The mean (±SD) surface roughness of RC before and after immersion in orange and cocktail were 0.04±0.02, 0.12±0.05, 0.06±0.03 and 0.11±0.06, respectively and for RMGI were 0.72±0.14, 0.60±0.19, 0.56±0.11, and 0.52±0.15. For RC there was significant difference between surface roughness (Sa) before and after immersion in orange and cocktail (P<0.05). For RMGI, there was significant difference between surface roughness before and after immersion in orange (P<0.05), but no significant difference before and after immersion in cocktail (P>0.05). CONCLUSIONS: The surface roughness of the RC and RMGI examined showed a significant change in the surface roughness after immersion for 24 hours in the tested fruit drinks.

Premature ovarian failure of autoimmune etiology in 46XX patients: is there a hope?

OBJECTIVES: To evaluate the efficacy of live bee stings at fertility points and acupuncture in treating symptoms and managing infertility in premature ovarian failure (POF) of autoimmune etiology. PATIENTS AND METHODS: Patients with primary POF were allocated randomly into two groups: group I: subjected to acupuncture at specific fertility points and group II: subjected to live bee stings at sites of fertility points. RESULTS: A total of 24 cases show significant reduction of Follicle stimulating hormone (FSH) level to normal range with gradual decline over the study duration: 13 cases in group I and 11 cases in group II. Eight cases got pregnant while the other 13 cases regained normal menses but still infertile. CONCLUSIONS: Both bee sting therapy and acupuncture were effective in reduction of FSH levels with restoration of regular menstrual patterns and restoration of fertility. The bee sting therapy was superior in the pregnancy rate, while acupuncture was superior in alleviation of symptoms.

Heat generation during removal of an abutment screw fragment from dental implants.

STATEMENT OF PROBLEM: Little information is available on the effect of drilling speed on surrounding bone during the removal of an abutment screw fragment. PURPOSE: The purpose of this in vitro study was to compare, in vitro, the peak temperature increase during the removal of fractured abutment screws from implants placed in a porcine mandible, using drilling speeds of 600 or 2000 rpm. MATERIAL AND METHODS: Twenty 4.3×13-mm dental implants were placed in 10 dissected porcine mandibles: 2 implants per mandible, 1 on each side. Localized defects were created in 20 surface-treated abutment screws, which were then tightened into each implant until a reproducible fracture occurred in each screw. The fractured screws were removed with a handpiece removal kit and irrigated with room-temperature water at either 600 or 2000 rpm. The temperature rise at the implant surface was measured at 3 levels with 3 type-K thermocouples. Repeated measure ANOVA was performed with the Tukey-Kramer post hoc test for mean pair-wise comparisons (α=.05 for all tests). RESULTS: Mean peak temperatures were significantly higher at 2000 rpm than at 600 rpm in the mid-body (P<.001) and crestal (P=.003) regions but not in the apical (P=.225) implant locations. No significant differences in mean peak temperatures were found among the 3 locations using 600 rpm (P=.179). In the 2000-rpm group, mean peak temperature in the mid-body area was consistently higher than that in the apical (P<.001) area, and more instances of temperature rise above 56°C and 60°C were observed. In 1 implant from this group, the estimated peak temperature exceeded the bone damage threshold value (50°C for 30 seconds). CONCLUSIONS: A drilling speed of 2000 rpm during the removal of abutment screw fragments caused overheating of the outer surface of the implant which may damage the surrounding bone; a speed of 600 rpm appears to be safe.

Development of an in vitro model to study uterine functions and early implantation using rat uterine explants.

This study was conducted to develop an in vitro model using rat uterine explants to explore complex uterine functions. Rat uterine explants (1-2 mm) were isolated, cultured and further characterized. Steroid hormone treatment of cultured explants showed that both Muc1 and Pr were significantly up-regulated (P < 0.05) by E2. Areg was significantly up-regulated (P < 0.05) by P4 and Igfbp1 was significantly up-regulated (P < 0.05) by the combination of E2 and P4, although, in rat, Igfbp1 is E2-dependent. In vitro decidualization of cultured explants was induced and two potential markers of decidualization, Prl8a2 and Bmp2, were examined. Real-time quantitative PCR data revealed that both Prl8a2 and Bmp2 were significantly up-regulated (P < 0.05) in MPA- and db-cAMP-treated explants compared to the control group of explants. Then, an individual hatched blastocyst and cultured explant was placed in a 96-well (round-bottom U-shaped) plate. Co-culture results showed that stable attachments were observed after 48 h, where embryos were stably attached to the explants and could not be dislodged after mild shaking and/or pipetting. The rates of attachment of embryos to the explants were increased significantly in the P4-treated group (63.6%) compared to the control group (35.5%), after steroid hormone treatment. The rates of attachment were reduced significantly in the E2-treated group (0.0%), where no stable attachments were observed. Despite the necessity of comprehensive investigation, our results suggest that the cultured rat uterine explants can be a useful in vitro model to study uterine functions and early implantation.

Characterization of primary osteocyte-like cells from rat mandibles.

OBJECTIVE: The mandible is continuously undergoing remodeling as a result of mechanobiologic factors, such as chewing forces, tooth loss, orthodontic forces, and periodontitis. The effects of mechanical stress and biologic signals in bone homeostasis have been the focus of many investigations. However, much of this research utilized osteocytes derived from long bones, but little is known about the mandible-derived osteocytes. This study tests a protocol to isolate and grow osteocytes from rat mandible. STUDY DESIGN: Rat mandibles were harvested, sectioned into small pieces, and subjected to a sequence chemical treatment and enzymatic digestion. The treated tissues were cultured for a few weeks while cells emerged. Cells were sorted by using the osteocyte marker podoplanin, an early marker for osteocyte differentiation. The cells were then characterized according to morphology, biochemical markers (osteocalcin, podoplanin, and sclerostin), and alkaline phosphatase activity and compared with an isotype cell line MLO-Y4 cells. RESULTS: The mandibular osteocytic cells had stellate shape and were positive for osteocalcin, podoplanin, and sclerostin and lower alkaline phosphatase activity compared with MLO-Y4 osteocyte-like cells. CONCLUSIONS: The protocol to isolate osteocyte-like cells will allow the investigators to investigate the mechanobiologic differences in biomechanical response between these mandibular and long bone osteocyte-like cells under various conditions.

Effect of organic and inorganic selenium supplementation on semen quality and blood enzymes in buffalo bulls.

The present study aimed to evaluate the effect of organic and inorganic selenium (Se) supplementation on semen quality and blood serum profiles of buffalo bulls. Nine mature buffalo bulls were divided into three groups: control (non-supplemented); organic Se (10 mg Sel-Plex®/head twice weekly) and inorganic Se (10 mg sodium selenite/head twice weekly). Semen was collected twice a week for 3 months during Se supplementation. Semen properties were evaluated from fresh ejaculate. Moreover, fructose concentration, aspartate and alanine transaminase (AST and ALT) activities, total protein and total cholesterol were assayed in seminal plasma. Additionally AST, ALT, testosterone and Se levels were determined in the blood serum. Results showed that Se supplementation significantly (P < 0.05) influences the semen parameters during 3 months of treatment. Organic Se significantly (P < 0.05) increased the percentage of viable sperms compared to inorganic Se and the control group. Fructose concentration was significantly higher (P < 0.05) in the seminal plasma of organic Se-treated bulls. Serum testosterone and Se concentrations were significantly (P < 0.05) increased in the Se supplemented groups than the control group. In conclusion, Se supplementation improved the parameters of buffalo bull semen and more precisely, organic Se was more effective for the improvement of semen quality and some blood components than inorganic Se.

Inherent physical characteristics and gene expression differences between alveolar and basal bones.

OBJECTIVES: The objective of this study was to evaluate the baseline differences between alveolar and basal areas of the rat mandible. STUDY DESIGN: Rat mandibular alveolar and basal bones were evaluated using histology and micro-computed tomography to compare osteocyte number as well as bone density and architecture and polymerase chain reaction to measure gene expression levels. RESULTS: Micro-computed tomography data indicated that basal bone is denser and less porous than alveolar bone. Histologic analysis showed that alveolar bone has more osteocytes per unit area compared with basal bone. Real-time polymerase chain reaction results showed higher levels of expression of the following genes in basal bone than in alveolar bone: SOST, E-11, DMP-1, and MEPE. CONCLUSIONS: Three of these gene products are associated with mature osteocytes, and this suggests that basal bone has more mature osteocyte phenotypes compared with alveolar bone. These findings are suggestive of fewer bone mineralization units and therefore a slower remodeling rate.

Kid depletion in mouse oocytes associated with multinucleated blastomere formation and inferior embryo development.

This study investigated the knockdown (KD) of Kid on maturation developmental competence and multinucleation of mouse germinal vesicle (GV) oocytes after parthenogenetic activation. Data revealed that Kid messenger RNA (mRNA) was expressed in GV and MII stage oocyte and 1- and 2-cell embryos. Additionally, Kid mRNA expression in the Kid KD group decreased by nearly 46% compared to the control small interfering RNA (siRNA) groups. The rate of multinucleated embryos in the Kid KD group (52.4%) was significantly higher (P < 0.05) than the control siRNA group (4.7%). Finally, the developmental rates were significantly lower in the Kid siRNA group at > 4-cell stage (28.6% vs. 53.5%) and the blastocyst stage (2.4% vs. 23.3%) compared to the control siRNA groups. Suppression of Kid using siRNA caused multinucleation in early embryos with high frequency and it may increase 2- to 4-cell arrested embryos and reduce the developmental competence to blastocyst.

Histologic Evaluation of Osseous Regeneration Following Combination Therapy With Platelet-Rich Plasma and Bio-Oss in a Rat Calvarial Critical-Size Defect Model.

Platelet-rich plasma (PRP) is an autogenous source of growth factors shown to facilitate human bone growth. Bio-Oss, an osteoconductive xenograft, is used clinically to regenerate periodontal defects, restore dental alveolar ridges, and facilitate sinus-lift procedures. The purpose of this study was to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration better than either material alone. PRP and/or Bio-Oss were administered in an 8-mm critical-size defect (CSD) rat calvarial model of bone defect between 2 polytetrafluoroethylene membranes to prevent soft tissue incursion. Eight weeks after the induction of the CSD, histologic sections were stained with hematoxylin and eosin stain and analyzed via light microscopy. Qualitative analyses revealed new bone regeneration in all 4 groups. The Bio-Oss and PRP plus Bio-Oss groups demonstrated greater areas of closure in the defects than the control or PRP-only groups because of the space-maintaining ability of Bio-Oss. The groups grafted with Bio-Oss showed close contact with new bone growth throughout the defects, suggesting a stronger graft. The use of PRP alone or in combination with Bio-Oss, however, did not appear to enhance osseous regeneration at 8 weeks. Areas grafted with Bio-Oss demonstrated greater space-maintaining capacity than controls, and PRP was an effective vehicle for placement of the Bio-Oss. However, at 8 weeks this study was unable to demonstrate a significant advantage of using PRP plus Bio-Oss over using Bio-Oss alone.

Human periodontal ligament fibroblast responses to compression in chronic periodontitis.

AIM: Test whether human periodontal ligament fibroblasts (PDLFs) retain homeostatic responses to a physiological compressive force during chronic periodontitis. MATERIAL AND METHODS: Six cell lines were established from periodontally healthy individuals (H-PDLFs) and another six were cultured from patients diagnosed with chronic periodontitis (D-PDLFs). Compressive force at 150 psi was applied to H-and D-PDLFs for 3 h on 2 consecutive days. After compression, comparisons between H-and D-PDLFs were performed by gene expression analysis of IL-6, proteases and 84 inflammation-related targets using real-time PCR. RESULTS: Compression of H-PDLFs resulted in a significant increase only in MMP-1 mRNA. In contrast, the same compressive force on D-PDLFs produced significant increases in the expression of MMPs-1,-7,-9 and -16. Moreover, compression of H-PDLFs resulted in down-regulation of IL-6, while IL-6 was significantly up-regulated in compressed D-PDLFs. Compression of H-PDLFs slightly up-regulated 3 and significantly down-regulated 15 inflammation-related genes, while the same treatment strongly up-regulated 21 inflammation-related genes in D-PDLFs. CONCLUSION: These results suggest a fundamental difference in the inflammatory response of healthy versus diseased PDLFs under physiological compression. Maintenance of these characteristics in vitro suggests that these cells may be at least partly responsible for the persistence of inflammation and localized susceptibility in chronic periodontitis.

Delayed versus immediate reconstruction of mandibular segmental defects using recombinant human bone morphogenetic protein 2/absorbable collagen sponge.

PURPOSE: To compare the efficiency of recombinant human bone morphogenetic protein 2 (rhBMP2)/absorbable collagen sponge (ACS) in the delayed versus immediate reconstruction of mandibular segmental defects in a canine model. METHODS: We randomized 11 dogs into 2 groups: immediate reconstruction (group 1, n = 6) and delayed reconstruction (group 2, n = 5). A 35-mm osteoperiosteal segmental defect was created on the left side of the mandible. Reconstruction with rhBMP2/ACS was carried out in the same setting in group 1 or at 4 weeks postoperatively in group 2. The contralateral side acted as an internal control. Animals were monitored both clinically and radiographically throughout the experiment. Twelve weeks after the application of rhBMP2/ACS, the quantity of bone formation was evaluated using regenerate mapping and histomorphometric analysis. Qualitative evaluation was performed based on bone mineral density and Vickers microhardness (µHV) testing. RESULTS: Postoperative seromas were observed in 83.3% of group 1 dogs only. Group 1 showed significantly larger physical dimensions than group 2 in most regenerate zones. Successful regeneration was achieved in 83.3% of group 1 dogs (discontinuity defect was seen in 1 of 6 dogs in group 1). Meanwhile, none of the 5 dogs in group 2 could be considered to have undergone successful regeneration (3 dogs had discontinuity defects, bony union occurred only in the basal third in the fourth dog, and the last dog showed union with only a shell of bone). The percent bone area and percent defect filling were significantly higher in group 1 than in group 2 (percent bone area, 52.4% ± 5.6% in group 1 and 36.6% ± 11.2% in group 2 [P = .02]; percent defect filling, 56.3% ± 5.5% in group 1 and 38.5% ± 10.8% in group 2 [P = .01]). Group 1 showed higher bone mineral density (0.7 ± 0.3 mg/cm(3) in group 1 and 0.4 ± 0.1 mg/cm(3) in group 2, P = .1). Finally, µHV was significantly higher in group 1 (20.3 ± 2.6 µHV) than in group 2 (13.2 ± 2.4 µHV) (P = .01). CONCLUSIONS: Delaying the application of rhBMP2/ACS for 4 weeks attenuated the quantity and quality of regenerated bone in mandibular segmental defects.

NF-κB2 mutation targets survival, proliferation and differentiation pathways in the pathogenesis of plasma cell tumors.

BACKGROUND: Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines. METHODS: We conducted a detailed histopathological characterization of lymphomas developed in p80HT transgenic mice and microarray gene expression profiling of p80HT B cells with the goal of identifying genes that drive plasma cell tumor development. We further verified the significance of our findings in human multiple myeloma cell lines. RESULTS: Approximately 40% of p80HT mice showed elevated levels of monoclonal immunoglobulin (M-protein) in the serum and developed plasma cell tumors. Some of these mice displayed key features of human multiple myeloma with accumulation of plasma cells in the bone marrow, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of B cells from M-protein-positive p80HT mice revealed aberrant expression of genes known to be important in the pathogenesis of multiple myeloma, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. In vitro assays demonstrated a critical role of Stat3, a key downstream component of IL-10 signaling, in the survival of human multiple myeloma cells. CONCLUSIONS: These findings provide a mouse model for human multiple myeloma with aberrant NF-κB2 activation and suggest a molecular mechanism for NF-κB2 signaling in the pathogenesis of plasma cell tumors by coordinated regulation of plasma cell generation, proliferation and survival.

Difference in soft tissue response between immediate and delayed delivery suggests a new mechanism for recombinant human bone morphogenetic protein 2 action in large segmental bone defects.

The ability of recombinant human bone morphogenetic protein 2 on absorbable collagen sponge (rhBMP2/ACS) to regenerate bone in segmental defect has been well characterized. However, clinical results of rhBMP2/ACS constructs in secondary reconstruction of large mandibular and craniofacial defects have not been consistent. We hypothesized that rhBMP2 delivery triggers an endogenous response in the soft tissues surrounding the defect, in the form of expression of BMP2 and vascular endothelial growth factor (VEGF). Such osteogenic response will occur only after immediate, as opposed to delayed, rhBMP2 delivery, suggesting a new explanation to the difference in bone regeneration between the two settings. A 35-mm segmental bone and periosteum defect was created on one side of the mandible in 16 dogs divided in three groups. Group 1 (Gp1, n=6) ACS was loaded with 8 mL of rhBMP2 (0.2 mg/mL). In Gp2 (n=5) the same dose of rhBMP2/ACS was delivered into the defect 4 weeks after surgery. In Gp3 (control; n=5) the defect was reconstructed using ACS loaded with 8 mL of buffer only (devoid of rhBMP2). Tissues were collected after 12 weeks of reconstruction in all groups. Direct measurement of physical dimensions of regenerates and bone morphometry was performed to evaluate bone regeneration. The mRNA expression of both BMP2 and VEGF in the soft tissue surrounding the defect was evaluated using real-time quantitative PCR. Both BMP2 and VEGF proteins were quantified in immunostained sections. Immunoflurescence colocalization of BMP2 and acetylated low density lipoprotein (AcLDL) was done to detect the source of BMP2. Immediate delivery yielded better bone regeneration. Both BMP2 and VEGF mRNA expression was upregulated only in Gp1 (+7.3, p=0.001; +1.53, p=0.001, respectively). BMP2 protein was significantly higher in the immediate reconstruction group; however, VEGF protein was undetected in the examined sections. Immediate delivery of rhBMP2 seemed to induce endogenous release of BMP2 from the surrounding soft tissues, an effect that was lacking in delayed delivery and may explain the variability of clinical results associated with BMP2 use. Colocalization of BMP2 and endothelial cells (ECs) suggested that ECs could be the source of endogenous BMP2.

Recombinant bone morphogenetic protein-2 induces up-regulation of vascular endothelial growth factor and interleukin 6 in human pre-osteoblasts: role of reactive oxygen species.

OBJECTIVE: Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) have been reported in many studies to play a major role in the communication between endothelial cells and osteoblasts. The inflammatory reaction and relative hypoxia at the site of bone injury are the first stages of the fracture repair. rhBMP-2 has been used extensively in spinal fusion and reconstruction of maxillofacial bone defects with main complication is the formation of seroma. The aim of this study was to test whether rhBMP-2 regulates the expression of the angiogenic and inflammatory mediators in pre-osteoblasts via generating reactive oxygen species (ROS). METHODS: rhBMP-2 effect on angiogenesis and inflammatory genes was assessed using normal human osteoblasts (NHOst). Angiogenesis genes were measured using angiogenic PCR array. VEGF and IL6 production were analysed using ELISA kit and real-time PCR. ROS production was assessed using dihydroethidine and dichlorofluorescein staining and lipid peroxidation. HIF-1α immunoreactivity was performed using immunofluorescence staining. RESULTS: There was an increase in the pro-angiogenic and -inflammatory genes as well as VEGF and IL6 protein expression in NHOst by rhBMP-2. This increase in VEGF and IL6 was blocked by the ROS scavenger N-acetyl cysteine (NAC). CONCLUSION: The regulatory effect of rhBMP-2 on angiogenesis and inflammation is mediated through a ROS-dependent mechanism, which involves upregulation of crucial angiogenic and inflammatory mediators such as VEGF and IL6. These findings highlight the need for future studies to identify new therapeutic targets downstream from rhBMP-2 to potentiate its beneficial effect or limit its complications such as seroma formation.