Comparative cytomorphology of skin, lymph node, liver and bone marrow in patients with lepromatous leprosy1

OBJECTIVE: The aim of this study was to compare the cytological changes in skin, lymph nodes, liver and bone marrow in patients with lepromatous leprosy. METHODS: Skin lesion, lymph node, liver and bone marrow aspirates were analysed. May-Grunwald-Giemsa (MGG) and Ziehl-Neelsen (Z-N) stains were employed. Comparative cytomorphology was studied. RESULTS: Twenty patients with lepromatous leprosy were studied. Lepra cells (LC) predominated in the skin aspirates of 12 patients with lepromatous leprosy (LL), lymphocytes accompanied LC in eight patients with borderline-lepromatous (BL) leprosy. Three patients of LL leprosy and two of BL leprosy in type 2 reaction additionally had numerous neutrophils. Two patterns of lymph node aspirates were seen: partial replacement with few LC in a reactive lymphoid background (10), complete replacement with either only LC or LC in a background of degenerating neutrophils (10), the latter a feature of type 2 reaction. Liver aspiration was performed in seven patients and of bone marrow in eight patients. Occasional LC were present in five liver-aspirated patients, steatosis and Kupffer cell hyperplasia in four patients, and myelopoiesis in two patients. Bone marrow smears invariably had occasional LC and a relative increase in mature plasma cells; sea-blue histiocytes were seen in six patients. CONCLUSION: Lepra cells predominated in skin and lymph node aspirates with complete replacement. In comparison, liver, bone marrow and lymph node aspirates with partial replacement were dominated by a preponderance of cells native to these organs with only few or occasional LC.     

The effect of resorption cavities on bone stiffness is site dependent

Resorption cavities formed during the bone remodelling cycle change the structure and thus the mechanical properties of trabecular bone. We tested the hypotheses that bone stiffness loss due to resorption cavities depends on anatomical location, and that for identical eroded bone volumes, cavities would cause more stiffness loss than homogeneous erosion. For this purpose, we used beam–shell finite element models. This new approach was validated against voxel-based FE models. We found an excellent agreement for the elastic stiffness behaviour of individual trabeculae in axial compression (R² = 1.00) and in bending (R²>0.98), as well as for entire trabecular bone samples to which resorption cavities were digitally added (R² = 0.96, RMSE = 5.2%). After validation, this new method was used to model discrete cavities, with dimensions taken from a statistical distribution, on a dataset of 120 trabecular bone samples from three anatomical sites (4th lumbar vertebra, femoral head, iliac crest). Resorption cavities led to significant reductions in bone stiffness. The largest stiffness loss was found for samples from the 4th lumbar vertebra, the lowest for femoral head samples. For all anatomical sites, resorption cavities caused significantly more stiffness loss than homogeneous erosion did. This novel technique can be used further to evaluate the impact of resorption cavities, which are known to change in several metabolic bone diseases and due to treatment, on bone competence.     

Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Hematopoietic growth factors in autologous transplantation

Hematopoietic growth factors (HGFs) sustain the survival, proliferation and differentiation of hematopoietic stem cells and some functions of mature blood cells. In man several HGFs have been characterised and cloned so far, and this has allowed investigators to confer the rationale for the clinical application of these molecules in hematology and oncology. In particular G-CSF and GM-CSF are currently utilised to abrogate the hematological toxicity of chemotherapy for standard and dose-intensified therapy, neutropenia following bone marrow and peripheral blood stem cell transplantation. Moreover there has recently been great interest in the ex vivo expansion of hematopoietic stem and progenitor cells for a variety of applications, such as in vitro tumor cell purging or for reducing the volume of blood processed by the leukapheresis. Several combinations of HGFs have been described to sustain the ex vivo survival and proliferation of these cells disclosing new opportunities in the field of stem cells transplants.     

围手术期综合护理对脊柱损伤并四肢骨折疗效的影响

目的：探讨固手术期系统综合护理对脊柱损伤并四肢骨折疗效的影响。方法：收集50例脊柱损伤并四肢骨折患者,按手术编号随机分为观察组和对照组,每组各25例,两组患者均采用内固定手术及常规治疗,但观察组围手术期配合综合系统护理,对照组不配合,连续观察至出院并随访6个月,比较护理对比两组患者临床疗效、并发症情况及疾病恢复时间的影响。结果：经治疗,两组患者均较前好转,且观察组临床有效率92％明显高于对照组84％（P〈0．05）;观察组并发症发生率28％明显低于对照组44％（P〈0．05）;术后疾病恢复时间观察组明显短于对照组（P〈0．05）。结论：围手术期综合系统护理有助于脊柱损伤并四肢骨折术后恢复,能显著提高疗效、减少并发症,缩短疾病恢复时间,临床值得应推广实施。     

Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

Objectives

Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2.

Materials and methods

Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR.

Results

BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA.

Conclusions

These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation.     

The TβR-I pre-helix extension is structurally ordered in the unbound form and its flanking prolines are essential for binding

Transforming growth factor β isoforms (TGF-β) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-β play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-β type I receptor (TβR-I) and TGF-β type II receptor (TβR-II). TGF-β's specificity for TβR-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-β and TβR-II. The structure and backbone dynamics of the unbound form of the TβR-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the β-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 3₁₀ helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-β signaling complex with its unique specificity, departing from the ancestral promiscuity of the bone morphogenetic protein subfamily, where the binding interface of the type I receptor is highly flexible.