Impact of calcineurin inhibitors on bone metabolism in primary kidney transplant patients

OBJECTIVE: Posttransplant bone disease and bone metabolism markers were investigated in primary kidney transplant recipients receiving calcineurin inhibitor (CNI) based triple immunosuppression. We examined the safety profile and independent potential of CNIs on bone formation and bone resorption. The study also attempted to correct for modifiable and nonmodifiable factors that impact on posttransplantation bone metabolism, such as age, renal function, rejection, steroid dosage, and secondary hyperparathyroidism. MATERIALS AND METHODS: Serum alkaline phosphatase and osteocalcin were used as indices of bone formation and urinary deoxypyridinoline as a marker for bone resorption. Bone mineral density (BMD) data were assessed in all patients. Osteocalcin and deoxypyridinoline data were correlated with BMD scores to predict the clinical utility and sensitivity of these tests. Sixty-six patients among 300 kidney transplant recipients were enrolled as eligible candidates based upon more than 12 months' posttransplantation follow-up excellent graft function (GFR values >60 mL/min), and intact parathormone levels <100 pg/mL. RESULTS: Mean follow-up was 1395.3 +/- 179.3 days and 1488.9 +/- 225.1 days for cyclosporine (CsA) and FK506 groups, respectively. Mean values for alkaline phosphatase and osteocalcin were 108.8 +/- 6.0 versus 98.4 +/- 9.7 U/L and 10.1 +/- 1.2 versus 9.8 +/- 1.5 ng/mL for the CsA and FK506 groups, respectively. Both CsA and FK506 caused mild osteoblastic proliferation and matrix mineralization activity, as reflected by increased osteocalcin and alkaline phosphatase levels in 22.6% and 12.5% of patients, respectively. This bone formation activity was counterbalanced by a three-fold increase in urine deoxypyridinoline levels in both groups. Mean deoxypyridinoline levels were, respectively, 13.8 +/- 4.4 versus 11.3 +/- 2.1 nM/mMCr in the CsA and FK506 groups. Thirty-four (68%) patients in the CsA and 10 (62.5%) in the FK506 groups had elevated deoxypyridinoline levels. A strong correlation existed between deoxypyridinoline levels and BMD scores for the CsA group (P < .0001). Despite the presence of relatively greater elevations in deoxypyridinoline and BMD values among CsA-treated patients, there was no significant difference in terms of bone resorption potential of both groups. No correlation existed between iPTH values (<65 pg/mL or among 65 to 98.2 pg/mL) at any time versus osteocalcin, alkaline phosphatase, deoxypyridinoline, or BMD levels. The symptomatic bone disease and fracture rates were 0% in this series. CONCLUSION: Calcineurin inhibitor-based immunosuppression with low maintenance doses of glucocorticoids induces slight bone formation but relatively potent, clinically relevant bone resorption.     

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.     

Osteogenesis in xenogeneic bone transplantation, using an immunosuppressant: Rabbit-rat experiments

We investigated osteogenesis and lymphocyte subsets in xenogeneic bone transplantation, using the immunosuppressant FK506 (FK). Iliac bones of rabbits were transplanted as fresh and frozen xenogeneic bone grafts into an intramuscular pouch of rats. FK was injected intramuscularly in half of the rats in a dose of 3 mg/kg/day for 14 days after transplantation. At 2,4, and 8 weeks, transplanted grafts and the lymphocyte subsets of these rats were examined. in the group not given FK, the grafted bone became necrotic and infiltrated with small round cells around the trabeculae. in the FK group, at 2 and 4 weeks, new bone was formed in the fresh xenografts without infiltration of lymphocytes. At 8 weeks, the new bone became necrotic and lymphocytes were present. the percentage of T cells (CD 5), B cells and the ratio of CO 4 cells/CD 8 cells were smaller in the FK group. Using an immunosuppre-sant we concluded that xenogeneic bone has an osteogeneic potency.     

Regulation of bone metabolism in immunosuppressant (FK506)-treated rats

After organ transplantation, severe osteoporosis is occasionally seen, and the use of immunosuppressants is thought to be one of the causes of such osteoporosis. In the present study, we investigated the effects of FK506 monotherapy on bones and determined the mechanism of onset of osteoporosis, both by assessing chronological changes in bone metabolism and by identifying factors that facilitate bone resorption. In 8-week-old male Sprague-Dawley rats, FK506 (1mg/kg) was injected intraperitoneally every day for 5 weeks (FK506-treated group), and for comparison, physiological saline was administered in the same manner in a control group of rats. Serum and urine samples were collected at weeks 0, 1, 3, and 5 of administration. The femur and tibia were collected within 24h of the final administration. When compared to the control group, findings on three-dimensional micro-computed tomography of the femur for the FK506-treated group showed a significant decrease in trabecular bone volume. The level of serum osteocalcin in the FK506-treated group at week 1 of administration was significantly higher than the control. Throughout the administration period, the sum of urinary pyridinoline (PYD) and deoxypyridinoline (Dpd) was significantly higher in the FK506-treated group. Of the various bone resorption factors tested, the level of serum parathyroid hormone (PTH) in the FK506-treated group was significantly higher than the control at week 3 of administration. The results of the present study confirmed that FK506 monotherapy in rats induced high-turnover osteoporosis. Soon after the start of FK506 administration, bone formation and resorption were elevated, and PTH appeared to have been involved in the maintenance of the elevated bone resorption.     

Effect of DSG on xenogeneic immune reactivity with special emphasis on human anti-pig cellular reactions in vitro

Abstract: The effect of the immunosuppressive drug 15-deoxyspergualin (DSG) on xenogeneic human anti-porcine cellular reactivity in vitro, including MLR induced proliferation, interleukin-2 (II-2) production, generation of cytotoxic cells, and the effect on antibody-dependent cellular cytotoxicity (ADCC), were compared with the effects of cyclosporin A (CsA) and/or FK506. The cytotoxic response was evaluated for both direct and indirect pathways for antigen presentation. In addition, the effects of DSG and CsA on antibody production to pig peripheral blood lymphocytes (PBL) in mice was studied. The degree of immunosuppression of xenogeneic and allogeneic cellular responses was compared. CsA and FK506 effectively inhibited proliferation and II-2 production induced by allogeneic human PBL or xenogeneic porcine PBL, whereas DSG did not have any effect on these responses. However, DSG suppressed both the allogeneic and xenogeneic in vitro induced cytotoxic responses, to the same level whether induced via the direct or indirect pathways of immune activation. In contrast, CsA inhibited cytotoxicity induced by xenogeneic cells via the direct but not via the indirect pathway. No effect of FK506 and DSG on ADCC was demonstrated.
A 5-day treatment with DSG or CsA of mice immunized with pig PBL partly suppressed antibody production. In DSG treated mice anti-pig PBL antibodies were produced, but titers were lower than in nontreated or CsA treated mice. The results indicate that DSG may be more effective than CsA/FK506 in inhibiting cytotoxic responses and antibody production induced by xenogeneic pig cells. A possible explanation could be that cytotoxicity induced via the indirect activation pathway of xenoreactivity is mediated to a high degree by CD3^- CD16⁺ (natural killer) NK-like cells, and that stimulation of these cells may be more sensitive to DSG than to CsA/FK506.     

WR-2721 reduces bone loss after hindlimb tenotomy in rats

WR-2721 is a thiophosphate analog of cysteamine that produces hypocalcemia in vivo. Previous studies suggest that WR-2721 produces hypocalcemia by independent inhibitory effects on parathyroid hormone (PTH) secretion, osteoclastic bone resorption, and tubular reabsorption of calcium. We sought to determine if WR-2721 would decrease bone loss in an animal model of disuse osteoporosis produced by unilateral knee tenotomy in the rat. Tenotomy significantly increased osteoclast number in tibias on the side of the procedure compared with tibias on the opposite side which had not undergone the procedure at 3 and 14 days. Femoral weight of tenotomized limbs were also reduced significantly compared with the contralateral limb at 3 and 14 days. WR-2721 treatment (240 mg/kg daily) prevented 26% of the loss of femoral dry weight and 29% of the loss of femoral ashed weight produced 14 days after tenotomy. In addition, WR-2721 treated (240 mg/kg daily) animals had fewer osteoclasts in tenotomized tibias than control animals at 3 days (6.6 +/- 0.7/mm versus 10.3 +/- 0.9/mm, p less than 0.02) and at 14 days (5.8 +/- 0.3/mm versus 8.7 +/- 0.4/mm, p less than 0.02). These data suggest that WR-2721 decreases bone loss in this model by decreasing osteoclastic bone resorption.     

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.     

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption

Summary

The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506.

Introduction

Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling.

Methods

FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments.

Results

Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA.

Conclusion

These results show that DHNA has some effects for improving bone mass reduction caused by FK506.     

Heterotopic new bone formation causes resorption of the inductive bone matrix

The bone matrix of growing rats was labeled by multiple injections of 3H-proline, and demineralized bone matrix (DBM) was prepared. The DBM was allotransplanted heterotopically into growing rats. New bone formation was induced in and around the implants. The new bone formation was accompanied by a decrease in the content of 3H; 20 and 30 days after implantation, 72% and 46%, respectively, of the activity remained in the implants. Daily injections of indomethacin (2 mg/kg) inhibited calcium uptake by about 20% at 20 and 30 days and inhibited the release of 3H from the DBM to a similar degree. Heterotopic bone induction by DBM is accompanied by matrix resorption, and inhibition of the new bone formation decreases the resorption of DBM.     

他克莫司对肝移植受者外周血T淋巴细胞亚群及共刺激分子表达的影响

目的 探讨他克莫司（FK506）对肝移植受者外周血T淋巴细胞亚群及其表面共刺激分子表达的影响。方法 采用荧光标记单克隆抗体和流式细胞技术，测定术后采用FK506治疗的肝移植受者（FK506治疗组）在用FK506后1、2、3、4周时的外周血T淋巴细胞亚群及其表面共刺激分子CD28、CD152和ICOS分子的表达情况，以健康志愿者（健康对照组）和患终末期肝脏疾病拟行肝移植者（肝病对照组）为对照。结果 CD3^＋T淋巴细胞在各组间的差异均无统计学意义（P〉0．05）。经FK506治疗后，肝移植患者的CD4^＋T淋巴细胞逐渐减少，CD8^＋T淋巴细胞逐渐增加，并恢复至健康对照组水平（P〉0．05）。FK506治疗组T淋巴细胞亚群表面CD28分子和ICOS分子表达逐渐下降，并明显低于健康对照组（P〈0．05），而CD152分子表达增加，且明显高于健康对照组（P〈0．05）；其ICOS分子表达水平的下降晚于CD28分子，CD4^＋CD28^＋T淋巴细胞、CD8^＋CD28^＋T淋巴细胞和CD4^＋ICOS^＋T淋巴细胞均呈现相近的变化规律。结论 FK506能迅速纠正移植受者T淋巴细胞亚群紊乱，并抑制正性共刺激分子CD28和ICOS的表达，促进负性共刺激分子CD152的表达。     

Application of immune cell functional assay in monitoring immune status in renal transplant recipients

Objective To evaluate the value of immune cell functional assay (ImmuKnow CD4+ T cell ATP assay) in monitoring immune status in renal recipients. Methods A total of 131 adult renal transplant recipients who received transplantation for the first time were under investigation. According to the dynamic monitoring ATP concentration before operation, 2 week, 1, 3, 6 months after operation and during infect or rejection, samples were divided into the following groups: health control group (HC), pretransplant (Pre-Tx) group, stable (Tx) group, infect group, acute rejection (AR) group, acute kidney injury (AKI) group. Immune cell functions were detected by ImmuKnow CD4+ T cell ATP assay. Lymphocyte subsets (CD4+/CD8+) were analysed and serum concentrations of FK506 were tested. Mixed lymphocyte reaction(MLR) was analysed. Results The ATP concentration was no significant difference between Pre-Tx and HC group. The ATP concentration of 2 weeks, 1 months after operation were significantly higher than Pre-Tx group (P＜0.01). After 3 months, 6 months follow-up, the ATP concentration stabilized with time. The ATP concentration of AR group was significantly higher than other three groups (Tx, infect and AKI group, all P＜0.05). The correlation coefficient between the ATP concentration and MLR, CD4+/CD8+, FK506 level were R2＝0.0072, R2＝10-6, R2＝0.004 respectively (all P﹥0.05). Conclusions The cell-mediated immunity of recipients is relatively strongger during the first month after transplantation. The ATP concentration is not related to the levels of MLR, CD4+/ CD8+, FK506. ImmuKnow ATP assay is a valuable predictor in acute rejection diagnosis.     

Cyclosporine A and FK506 induce osteoclast apoptosis in mouse bone marrow cell cultures

Studies were carried out to characterize the effects of cyclosporines and FK506 on the formation and survival of osteoclasts deriving from mouse bone marrow cultures. Cyclosporin A (CsA), cyclosporin B (CsB), cyclosporin H (CsH), and FK506 all inhibited receptor activator of NFkappaB ligand (RANKL)-stimulated tartrate-resistant acid phosphatase (TRAP) activity and generation of TRAP+ multinucleated cells in the cultures. CsA and CsG were approximately equipotent, CsH was approximately one order of magnitude less potent than the other cyclosporines, and FK506 was approximately two orders of magnitude more potent than CsA and CsG. All of the inhibitors demonstrated greater potency and efficacy on decreasing the number of TRAP+ multinucleated cells than on decreasing total TRAP activity. Further evidence that late stages were more sensitive to inhibition was obtained in experiments in which CsA was present for different segments of the RANKL-stimulated culture period. CsA was as efficacious when added for the final 2 days of a 4-day culture as when added for the entire culture period, whereas it was less effective if added for only the first 2 days of the culture. When CsA or FK506 were added for 1 day to cultures in which osteoclasts had already formed, the numbers of TRAP+ osteoclasts decreased. Treatment with CsA or FK506 produced nuclear fragmentation and disruption of the multinucleated osteoclasts and an increase in caspase-3 activity. The apoptosis inhibitor z-VAD partially prevented the inhibitory effects of CsA and FK506 on the survival of TRAP+ multinucleated cells in the cultures and also preserved the normal osteoclast morphology. The data indicate that an important component of the inhibitory effects of CsA and FK506 on marrow-derived osteoclasts is the induction of apoptosis.     

Inducing host acceptance to encapsulated xenogeneic myoblasts

BACKGROUND: Cell encapsulation holds promise for the chronic delivery of recombinant proteins such as erythropoietin. Encapsulated xenogeneic mouse C2C12 myoblasts display long-term survival in the central nervous system whereas they do not in the subcutaneous tissue, suggesting that encapsulation only partially prevents affector and effector mechanisms of the host immune response. Transient immunosuppression with FK506 at the time of subcutaneous implantation leads, however, to their long-term survival. The nature of this acceptance was further investigated in this report. METHODS: Fischer rats were rendered unresponsive to encapsulated murine C2C12 myoblasts secreting mouse erythropoietin by either a 1- or 4-week initial treatment of FK506. To examine the extent of xenograft acceptance, animal were challenged with a second implant 9 weeks after the initial implantation. RESULTS: Challenging animals treated only 1 week with FK506 led to rejection of both primary and secondary implants. Animals administered FK506 for 4 weeks accepted both implants over the period investigated. However, these animals rejected unencapsulated xenogeneic cells injected at a later time, highlighting the requirement of the polymer membrane for immune protection. Developed unresponsiveness to encapsulated xenogeneic myoblasts lasted over extended periods (at least 7 months), in the absence of both immunosuppression and stimulating xenoantigens. CONCLUSIONS: These findings reveal that host acceptance of encapsulated but not unencapsulated xenogeneic myoblasts can be developed in the subcutaneous tissue after transient FK506 immunosuppression. This may have direct clinical relevance as it enables capsules to be replaced without additional immunosuppression, facilitating long-term cell-based therapies.     

Augmentation of surgical angiogenesis in vascularized bone allotransplants with host‐derived a/v bundle implantation,fibroblast growth factor‐2, and vascular endothelial growth factor administration

We have previously shown experimental transplantation of living allogeneic bone to be feasible without long‐term immunosuppression by development of a recipient‐derived neoangiogenic circulation within bone. In this study, we examine the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(D,L‐lactide‐co‐glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both, were inserted intramedullarly along with a recipient‐derived arteriovenous (a/v) bundle. FK‐506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic, and histomorphometric analyses were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p < 0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p < 0.005). Bone formation rates were greater with VEGF (p < 0.01) and FGF+VEGF (p < 0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation, and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1015–1021, 2010     

Functionally improved bone in calbindin-D28k knockout mice

In vitro studies indicate that Calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization of bone extracellular matrix. The recent creation of a Calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the Calbindin-D28k protein on bone remodeling in vivo. In this experiment, histomorphometry, microCT, and bend testing were used to characterize bones in Calbindin-D28k KO (knockout) mice. The femora of Calbindin-D28k KO mice had significantly increased cortical bone volume (60.4% +/- 3.1) compared to wild-type (WT) mice (45.4% +/- 4.6). The increased bone volume was due to a decrease in marrow cavity area, and significantly decreased endosteal perimeters (3.397 mm +/- 0.278 in Calbindin-D28k KO mice, and 4.046 mm +/- 0.450 in WT mice). Similar changes were noted in the analysis of the tibias in both mice. The bone formation rates were similar in the femoral and tibial cortical bones of both mice. microCT analysis of the trabecular bone in the tibial plateau indicated that Calbindin-D28k KO mice had an increased bone volume (35.2% +/- 3.1) compared to WT mice (24.7% +/- 4.9) which was primarily due to increased trabecular number (8.99 mm(-1) +/- 0.94 in Calbindin-D28k KO mice compared to 6.75 mm(-1) +/- 0.85 in WT mice). Bone mineral content analysis of the tibias indicated that there is no difference in the calcium or phosphorus content between the Calbindin-D28k KO and WT mice. Cantilever bend testing of the femora demonstrated significantly lower strains in the bones of Calbindin-D28k KO mice (4135 micro strain/kg +/- 1266) compared to WT mice (6973 micro strain/kg +/- 998) indicating that the KO mice had stiffer bones. Three-point bending demonstrated increased failure loads in bones of Calbindin-D28k KO mice (31.6 N +/- 2.1) compared to WT mice (15.0 N +/- 1.7). In conclusion, Calbindin-D28k KO mice had increased bone volume and stiffness indicating that Calbindin-D28k plays an important role in bone remodeling.     

Baohuoside-1, a novel immunosuppressive molecule, inhibits lymphocyte activation in vitro and in vivo

BACKGROUND: We evaluated the in vitro and in vivo immunosuppressive effects of baohuoside-1 (B1), a novel flavonoid isolated from Epimedium davidii. METHODS: Proliferation assay was used to determine the antiproliferative properties on T-cell and B-cell proliferation. Flow cytometry analysis was applied to detect changes of phenotypes on activated cells. RESULTS: B1 inhibits the lymphocyte proliferation activated by polyclonal mitogens and mixed lymphocyte reaction with a 50% inhibitory concentration of low micromolar concentration. Also, B1 suppressed T-cell activation in T cell receptor/CD3-mediated signaling pathways in a dose- and time-dependent manner. The suppression of B1 was not simply a result of a toxic effect and was recovered by withdrawing the drug. B1 down-regulated the expression of some phenotype molecules. In Ca(2+)-independent or -dependent antigen stimulation, although B1 had different inhibitive patterns on CD69 expression stimulated by phorbol 12-myristate 13-acetate (PMA) or Ca2+ ionophore, it inhibited T-cell proliferation induced by CD3/CD28 or PMA/ionomycin and partially blocked that induced by PMA/CD28. Interestingly, an additive inhibition between B1 and tacrolimus (FK506) was found in the CD69 expression stimulated by PMA/CD28 and PMA/ionomycin. Similarly, this immunosuppression by combination therapy was observed in a heart transplantation model in vivo and might act through an immunosuppressive mechanism different from FK506. CONCLUSIONS: B1, whose mechanism of action is not similar to that of FK506, has selectively immunosuppressive effects on T-cell and B-cell activation in vitro and effectively prevents rat heart allograft rejection in vivo.     

Mechanism of FK 506/520 action on rat renal proximal tubular Na+, K+-ATPase activity.

BACKGROUND: The neurotransmitter in renal sympathetic nerves, norepinephrine (NE), regulates the activity of proximal tubule (PT) Na+,K+-ATPase in a bidirectional manner via stimulation of alpha- and beta-adrenoceptors. The stimulatory alpha-adrenergic pathway is mediated by calcineurin, the target molecule for FK 506 and related compounds. We examined whether the FK 506 analogue FK 520, by interrupting the calcineurin-mediated alpha-adrenergic signaling pathway, enhance the inhibitory beta-adrenergic effect of NE on PT Na+,K+-ATPase activity. METHODS: The effects of three days of treatment with FK 520 were examined on rat renal PT Na+,K+-ATPase activity, measured as ouabain-sensitive ATP hydrolysis in single, microdissected PT segments. Renal function studies, including glomerular filtration rate (GFR) and urinary excretion of N-acetyl-3-D-glucoseaminidase (NAG), were examined using conventional clearance techniques after three days of treatment with FK 506. RESULTS: FK 520 treatment induced a pronounced and dose-dependent decrease in PT Na+,K+-ATPase activity. This effect was completely reversed by the competitive FK 520 antagonist, L 685 818, indicating that the effect was dependent on inhibition of calcineurin. To test whether the FK 520-induced decrease in Na+, K+-ATPase activity was mediated by enhanced beta-adrenoceptor signaling, the FK 520 effect was examined in rats pretreated with a beta-adrenoceptor antagonist (propranolol) or rats subjected to renal denervation. Both of these procedures prevented the FK 520-induced decrease in Na+,K+-ATPase activity. Thus, during FK 520 treatment, renal sympathetic nerves mediate an inhibitory effect on PT Na+,K+-ATPase activity via beta-adrenoceptors. Propranolol pretreatment also prevented FK 506-induced decreases in GFR and urinary excretion of NAG, an index of PT dysfunction. CONCLUSIONS: The results support the hypothesis that the net effect of the neurotransmitter NE on Na+,K+-ATPase activity is dependent on the balance between the alpha- and beta-adrenergic signaling pathways and suggest that agents that interfere with these pathways may, by altering the activity of tubular Na+,K+-ATPase, also alter the function of the renal tubular epithelial cell.     

Prolonged survival of donor-specific rat intestinal allograft by administration of bone-marrow-derived immature dendritic cells

It has been reported that intraportal administration of donor antigens induced donor-specific hyporesponsiveness. We studied here the effects of transplantation of BM-derived immature dendritic cells (imDCs) and mature DCs (mDCs) via portal vein on rat small intestinal allograft survival. This study comprised four treatment groups: 1) untreated controls; 2) FK506 alone; 3) intraportal donor-specific BM-derived imDCs transplantation+FK506; 4) mDCs/Tx+FK506. Allograft survival was minimal in control group (5.2+/-0.8 days) and maximal in imDC+FK506 group (28.4+/-3.0 days). The rats in mDC+FK506 group showed systemic inflammatory reaction due to GVHR, and died within 10 days after transplantation. The in vitro MLR reaction using imDCs was also strongly inhibited both in direct and indirect recognition pathways. The impact of imDCs for the specific induction of transplant tolerance may suggest that immunization with donor-specific imDCs has therapeutic potential in organ transplantation.     

Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury

PURPOSE: We investigated the long-term effect of immunophilin ligands on erection physiology and cavernous tissue histology using rat models of unilateral and bilateral cavernous nerve (CN) injury. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were administered the immunophilin ligand FK506 (5 mg/kg subcutaneously daily for 5 days), the nonimmunosuppressant FK506 derivative GPI1046 (3 to 3-pyridyl)-1-propyl(2 seconds)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate) (40 mg/kg subcutaneously daily for 5 or 28 days) or saline immediately upon induction of 2 paradigms of cavernous nerve injury, namely focal transection of the CN unilaterally, and a combination of focal transection of the CN unilaterally and excision of a 5 mm segment of contralateral CN (BIL treated). At 28 days following CN injury electrical stimulation of the transected CN and penile erection monitoring were performed. Whole penes were removed and evaluated immunohistochemically for the nitric oxide generator neuronal nitric oxide synthase, the neuronal marker synaptophysin, the endothelial marker CD31 and the smooth muscle marker alpha-actin. RESULTS: In unilaterally treated groups erection recovery was significantly greater in FK506 treated than in saline treated animals ((83.5% +/- 9.2% vs 24.3% +/- 8.1%, p <0.001) and similarly greater in GPI1046 treated animals than in the respective saline treated controls for this group (57.9% +/- 12.6% vs 23.8% +/- 7.0%, p <0.05). In BIL treated groups erection recovery for FK506 and GPI1046 treated rats exceeded saline treated values by approximately 70% (p <0.05) and 40% (p = 0.14), respectively. After 28 days of continuous treatment in BIL treated groups erection recovery for GPI1046 treated rats exceeded saline treated values by 140% (p <0.05). Neuronal and endothelial staining was preserved after immunophilin ligand treatment. CONCLUSIONS: Immunophilin ligands exert neurotrophic effects on the penile innervation that preserve cavernous tissue structure and promote erectile function recovery in rats after extensive CN injury.     

Immunosuppression with FK506 has no influence on fracture healing in the rat

Immunosuppressant drugs like cyclosporine A and FK506 are widely used for solid organ transplantation. They are accelerating bone remodeling but cause net bone loss. The aim of this study was to investigate the effect of FK506 on fracture healing in the rat. Eighty Lewis rats were divided into four groups, which received FK506 (1 mg/kg BW) or no treatment for 2 or 4 weeks, beginning after production of a closed, nondisplaced unilateral tibial fracture. Radiographic, histological, and biomechanical studies were used to evaluate fracture healing and histomorphometric analysis of the tibial metaphysis of the intact contralateral side was performed. Radiographs revealed no difference of the healing of the control fractures compared with the fractures in the FK506-treated group at 2 and 4 weeks. The mechanical parameters of the tested contralateral intact tibiae and of the fracture callus demonstrated no difference between control and immunosuppressed animals. Tibial bone histomorphometry revealed increased measures of bone formation and bone resorption, accompanied by a significant reduction of percent trabecular area. At 4 weeks, the fractures showed osseous healing with woven bone at the fracture site and only minimal amounts of cartilage. Histological grading was not different between the control and the FK506 group at both time points. We conclude that systemic application of FK506 has no biomechanical and histological effects of experimental fracture healing in the rat. However, resorption far in excess of formation leads to a net bone loss in the trabecular bone of the tibia that has no effect on the stability of the intact bone.