Assessment and clinical implications of RANK/RANKL/OPG pathway as markers of bone tumor progression in patients with NET harboring bone metastases

Abstract

Introduction: The impact on the survival of bone metastases (BM) in patients with neuroendocrine tumor (NET) is a matter of debate. BM have a key role in causing symptoms and in decreasing patients’ quality of life. Although the mechanisms of the development of BM are not completely clear, it is now well understood that the Receptor Activator of Nuclear factor Kappa-B-/Ligand (RANK/RANKL)/osteoprotegerin (OPG) pathway plays a relevant role.

Aim: To characterize the RANK/RANKL/OPG pathway in patients affected with NET.

Patients and methods: Two cohorts of 15 patients each were enrolled in the study; one cohort was affected with NET without BM and the second cohort was affected with NET with BM. The serum RANK/RANKL/OPG pathway was assessed in both the groups.

Results: Serum OPG levels and RANKL/OPG ratio were lower and higher, respectively, in NET patients harboring BM than in those without BM. During the ROC analysis, a cut-off value of 1071?pg/ml for OPG and 0.62 for RANKL/OPG ratio were able to significantly distinguish between the two groups.

Conclusions: This study indicates that RANK/RANKL/OPG pathway is imbalanced in patients with NET harboring BM. Specific alterations of this pathway could predict an early development of BM.     

Role of whole brain radiotherapy in the management of infratentorial metastases from lung and breast cancer

BackgroundBrain metastases (BM) occur in almost one third of patients with systemic malignancies. Only a small number of studies focus on infratentorial location and whole brain radiotherapy (WBRT) as the main non-surgical management. The aim of the study was to compare the prognosis of patients treated with WBRT among patients with supra- or infratentorial lesions.Materials and methodsAt a single center, 263 patients with either breast (BC) or lung (LC) cancer, that had developed BM and received treatment with WBRT, were analyzed during an 8-year period.ResultsA total of 152 patients with BC and 111 with LC were analyzed, median age at the time of BM was 50.7 years, systemic activity other than BM was detected in 91%. Newly diagnosed BM were supratentorial in 40%, infratentorial in 10% and 51% in both locations. Median overall survival was 13 months (95% CI: 11.1–14.8 months), without significant difference between supra- or infratentorial location. WBRT alone was administered in 79% of patients, whereas WBRT with chemtoreapy was provided for 21%.ConclusionIn patients with BM from LC or BC that were not candidates for surgical resection, palliative WBRT appears to be equally effective in those with supra- or infratentorial locations.     

Relations between doses cumulated in bone marrow and dose delivery techniques during radiation therapy of cervical and endometrial cancer

PurposeTo compare normal tissue complication probability (NTCP) and average doses in the bone marrow (BM), obtained for five different radiotherapy delivery and planning strategies of cervical and endometrial cancer.Material/methods50 patients were taken to analysis. For each case, 3 different dose delivery techniques were used: 4-field, X15MV, 3DCRT; 7-field, X6MV, IMRT; and 2-arc, X6MV, VMAT. Two optimization scenarios were used for the IMRT and VMAT plans generation: with (+) and without (−) the inclusion of the BM as an optimized structure. Average doses and dose-volume histogram parameters for the PTV, BM, bladder, rectum, bowels and femoral heads were compared. In addition, the BM doses were analyzed with respect to the PTV and/or volume of the BM, and NTCP for the BM were computed.ResultsThe dose in PTV for evaluated plans was similar. The worst doses in organs at risk were obtained for 3DCRT. Using the BM during the optimization of IMRT and VMAT reduces an average dose in BM without increasing the doses in the bladder, rectum and bowels. Differences between doses in BM for IMRT(+) and VMAT(+) plans were similar while NTCP was lower for VMAT(+). A correlation between average dose in BM and the volume ratio of BM and PTV was found for each technique.ConclusionUsing the BM during the optimization of the IMRT and VMAT plans effectively reduces the dose in BM without increasing the dose in the bladder, rectum and bowels. The VMAT(+) plans were characterized by the lowest NTCP.     

Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First-passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64.64 ± 6.5 and 88.6 ± 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved independent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.     

Application of autologous bone marrow mononuclear cells in six patients with advanced chronic critical limb ischemia as a result of diabetes: our experience

Background aimsPrevious clinical studies have reported that the injection of bone marrow (BM)-derived mononuclear cells (MNC) results in improvement in symptoms and healing of ulcers in patients with critical limb ischemia (CLI) up to stage IV of Fontaine's classification. However, most patients with Fontaine stage IV CLI limbs had to undergo amputation even after stem cell therapy. We report on six patients, who had poorly controlled diabetes with extensive ulceration and gangrene of limbs because of Fontaine stage IV CLI and had been advised amputation elsewhere, who underwent injection of autologous BM MNC.MethodsIn all six patients, BM was aspirated and the isolated MNC from the BM were injected intralesionally at various sites of the ulcer and its surroundings after necessary debridement. The patients were followed up at regular intervals for at least 6 months.ResultsAt the end of the 6-month follow-up, the lower limb pain and ulcers had improved significantly in all patients. The mean toe–brachial index had increased from 0.26 to 0.36. One patient died a month after therapy because of causes unrelated to the procedure. Limb salvage was possible in the remaining five patients and they had a pain-free walking distance of 100 m within 6 months.ConclusionsLimb salvage was possible in all six diabetic patients with Fontaine stage IV CLI following autologous BM MNC injection. The procedure was safe without any adverse outcomes.     

Investigating the role of T helper related cytokines in cerebrospinal fluid for the differential diagnosis of bacterial meningitis in pre-treated paediatric patients

Abstract

Purpose: Given the challenge in the diagnosis of bacterial meningitis (BM), we assessed different cytokines in the cerebrospinal fluid (CSF) of antibiotics pre-treated patients.

Materials and methods: Laboratory tests and polymerase chain reaction (PCR) were performed for 480 CSF samples from children (2 m to 14 y), suspicious to meningitis and pre-treated with antibiotics, to detect bacterial and viral aetiologies. Sixty-one CSF were included and the levels of 13 cytokines such as IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ and TNF-α were measured using flow-cytometry.

Results: All bacterial cultures were negative, but 29 and eight CSF were positive for bacterial and viral agents by PCR. IL-6, IL-10 and IFN-γ were significantly up-regulated in BM. T helper (Th) subset cytokines showed significant upregulation of Th1, Th2, Th17, Th22 and Tfh cytokines in BM. Common Th subsets cytokines (IL-6, IL-10 and TNF-α) were significantly different between the study groups. ROC curve analysis revealed good AUC for common Th related cytokines in discriminating BM.

Conclusions: In pre-treated BM patients with negative bacterial cultures, cytokines IL-6, IL-10 and IFN-γ can predict BM which could be beneficial for rapid diagnosis and treatment to decrease the sequela of the disease.     

Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer

Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic “shield” from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K_m for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.     

Validation plan of bone marrow collection,processing and distribution using the failure mode and effect analysis methodology: a technical report

Background aimsBone marrow (BM) is commonly used in the pediatric and adult setting as a source of hematopoietic stem cells (HSCs). The standards of the Joint Accreditation Committee of the International Society for Cell & Gene Therapy & European Society for Blood and Marrow Transplantation (JACIE) include specific requirements regarding BM collection, processing and distribution. To run this process, each transplant team develops a series of JACIE-compliant procedures, customizing them with regard to local settings and paths. Moreover, JACIE standards require that transplant teams validate and periodically revise their procedures to keep the entire process under control. In this article, the authors describe the methodology adopted in our center to fulfill the aforementioned JACIE requirements.MethodsThe authors developed a validation plan based on the failure mode and effect analysis (FMEA) methodology. According to the FMEA approach, the authors carefully revised activities and procedures connected to BM collection, processing and distribution at our institution. The entire process was initially divided into five main phases (assessment of donor eligibility, perioperative autologous blood donation, preparation of BM collection kit, BM harvesting and BM processing and distribution), comprising 17 subphases and 22 activities.ResultsFor each activity, one or more failure modes were identified, for a total of 28 failure modes, and a risk priority number (RPN) was then assigned to each failure mode. Although many procedures were validated, others were subjected to substantial changes according to the RPN rating. Moreover, specific indicators were identified for subsequent monitoring to contain the risk of failure of steps emerging as critical at FMEA.ConclusionsThis is the first study describing use of the FMEA methodology within an HSC transplant program. Shaping the risk analysis based on local experience may be a trustworthy tool for identifying critical issues, directing strict monitoring of critical steps or even amending connected procedures. Overall, the FMEA approach enabled the authors to improve our process, checking its consistency over time.     

The Largest Known Survival Analysis of Patients with Brain Metastasis from Thyroid Cancer Based on Prognostic Groups

PurposeTo analyze the clinical features and prognostic factors associated with the survival of patients with a very rare occurrence of brain metastasis (BM) from differentiated thyroid cancer (DTC).ResultsThe median age at BM was 63 years, and the median time from initial thyroid cancer diagnosis to BM was 3.8 years. The median survival and the 1-year actuarial survival rate after BM were 8.8 months and 47%, respectively. According to univariate and multivariate analyses, four good prognostic factors (GPFs) were identified including age ≤ 60 years, PS ≤ ECOG 2, ≤ 3 BM sites, and without extracranial metastasis prior to BM. Three prognostic groups were designed based on age and number of remaining GPFs: patients ≤ 60 years of age with at least 2 GPFs (Group A) had the most favorable prognosis with a median survival of 32.8 months; patients ≤ 60 years of age with fewer than 2 GPFs and those > 60 years of age with at least 2 GPFs (Group B) had an intermediate prognosis with a median survival of 9.4 months; and patients > 60 years of age with fewer than 2 GPFs (Group C) had the least favorable prognosis with a median survival of 1.5 months.ConclusionsThe survival of patients with BM form DTC differed among the prognostic groups based on the total number of good prognostic factors.     

Environmental and genetic influences on body mass and resting metabolic rates (RMR) in a natural population of weasel Mustela nivalis

Body mass (BM) and resting metabolic rates (RMR) are two inexorably linked traits strongly related to mammalian life histories. Yet, there have been no studies attempting to estimate heritable variation and covariation of BM and RMR in natural populations. We used a marker‐based approach to construct a pedigree and then the ‘animal model’ to estimate narrow sense heritability (h²) of these traits in a free‐living population of weasels Mustela nivalis—a small carnivore characterised by a wide range of BM and extremely high RMR. The most important factors affecting BM of weasels were sex and habitat type, whereas RMR was significantly affected only by seasonal variation of this trait. All environmental factors had only small effect on estimates of additive genetic variance of both BM and RMR. The amount of additive genetic variance associated with BM and estimates of heritability were high and significant in males (h² = 0.61), but low and not significant in females (h² = 0.32), probably due to small sample size for the latter sex. The results from the two‐trait model revealed significant phenotypic (r_P = 0.62) and genetic correlation (r_A = 0.89) between BM and whole body RMR. The estimate of heritability of whole body RMR (0.54) and BM corrected RMR (0.45) were lower than estimates of heritability for BM. Both phenotypic and genetic correlations between BM corrected RMR and BM had negative signals (r_P = ?0.42 and r_A = ?0.58). Our results indicate that total energy expenditures of individuals can quickly evolve through concerted changes in BM and RMR.     

Ultrafast Charge Generation Pathways in Photovoltaic Blends Based on Novel Star‐Shaped Conjugated Molecules

The quest for new materials is one of the main factors propelling recent advances in organic photovoltaics. Star‐shaped small molecules (SSMs) have been proven promising candidates as perspective donor material due to the increase in numbers of excitation pathways caused by the degeneracy of the lowest unoccupied molecular orbital (LUMO) level. In order to unravel the pathways of the initial photon‐to‐charge conversion, the photovoltaic blends based on three different SSMs with a generic structure of N(phenylene‐nthiophene‐dicyanovinyl‐alkyl)₃ (n = 1–3), and [6,6]‐phenyl‐C₇₁‐butyric acid methyl ester (PC₇₁BM) acceptor are investigated by ultrafast photoinduced absorption spectroscopy assisted by density functional theory calculations. It is shown that both electron transfer from SSMs to PC₇₁BM and hole transfer from PC₇₁BM to SSMs are equally significant for generation of long‐lived charges. In contrast, intramolecular (intra‐SSM) charge separation results in geminate recombination and therefore constitutes a loss channel. Overall, up to 60% of long‐lived separated charges are generated at the optimal PC₇₁BM concentrations. The obtained results suggest that further improvement of the SSM‐based solar cells is feasible via optimization of blend morphology and by suppressing the intra‐SSM recombination channel.     

Advantages of peripheral blood stem cells from unrelated donors versus bone marrow transplants in outcomes of adult acute myeloid leukemia patients

Background aimsIn allogeneic stem cell transplantation, unrelated donors are chosen in cases where appropriate related donors are not available. Peripheral blood stem cells (PBSCs) are more often selected as a graft source than bone marrow (BM). However, the prognostic benefits of PBSCs versus BM transplants from unrelated donors have not been carefully examined in patients with acute myeloid leukemia (AML). This study compared outcomes of adult AML patients who underwent unrelated PBSC and BM transplantation, evaluating post-transplant complications, including engraftment, graft-versus-host disease (GVHD) and infections, and determined subgroups of patients who are most likely to benefit from unrelated PBSCs compared with BM transplants.MethodsThe authors analyzed 2962 adult AML patients who underwent unrelated PBSC or BM transplants between 2011 and 2018 (221 PBSC and 2741 BM) using the Japanese nationwide registry database, in which graft source selection is not skewed toward PBSCs.ResultsIn 49.7% of patients, disease status at transplantation was first complete remission (CR1). In 57.1% of cases, HLA-matched donors were selected. Myeloablative conditioning was performed in 75.1% of cases, and anti-thymocyte globulin (ATG) was added to conditioning in 10.5%. Multivariate analyses showed a trend toward favorable non-relapse mortality (NRM) in PBSC recipients compared with BM recipients (hazard ratio [HR], 0.731, P = 0.096), whereas overall survival (OS) (HR, 0.959, P = 0.230) and disease-free survival (DFS) (HR, 0.868, P = 0.221) were comparable between PBSC and BM recipients. Although the rate of chronic GVHD (cGVHD) was significantly higher in PBSC patients (HR, 1.367, P = 0.016), NRM was not increased, mainly as a result of significantly reduced risk of bacterial infections (HR, 0.618, P = 0.010), reflecting more prompt engraftments in PBSC recipients. Subgroup analyses revealed that PBSC transplantation was advantageous in patients transplanted at CR1 and in those without ATG use. PBSC recipients experienced significantly better OS and/or DFS compared with BM recipients in this patient group.ConclusionsThe authors' results confirmed the overall safety of unrelated PBSC transplantation for adult AML patients and suggested an advantage of PBSCs, especially for those in CR1. Further optimization of the prophylactic strategy for cGVHD is required to improve the overall outcome in transplantation from unrelated PBSC donors.     

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides

BackgroundCytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure.MethodsThe metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures.ResultsLower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F–G loop regions and active site cavity volumes for the F87A mutated structures.ConclusionsAnalysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site.General significanceThe metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.     

Changes in egg and body temperature indicate triggering of egg desertion at a body mass threshold in fasting incubating blue petrels (Halobaena caerulea)

The triggering of transitory egg desertion in fasting and incubating blue petrels (Halobaena caerulea, nocturnal burrowing seabirds living in the subantarctic region) was investigated by continuously monitoring both body temperature (T _sto) and egg temperature (T _egg) with a telemetry system, and by measuring body mass (BM) loss. The birds were kept captive in their burrow and incubated day and night without any interruption; there was no day-night cycle in T _sto and T _egg, which averaged 39.9 °C and 32.0 °C, respectively. There was no evidence of hypothermia as a way to save energy in this fasting situation. Egg desertion occurred at night and was an abrupt and definitive phenomenon reflected by a simultaneous fall in T _egg and a peak in T _sto. After egg desertion, a distinct day-night cycle of body temperature was observed, T _sto being 0.6 °C higher during night-time (P < 0.05), probably reflecting increased nocturnal activity. BM at egg desertion averaged 166.7 ± 3.8 g in telemetered birds and 164.4 ± 1.6 g in␣a group of free-living birds. Throughout fasting, the␣specific daily BM loss remained at 46 ± 1 g · kg⁻¹ · day⁻¹, but increased sharply below a critical BM of 160.0 ± 2.5 g. Thus, fasting incubating blue petrels spontaneously desert their egg when reaching a BM threshold. This BM is very close to a critical value in fasting birds and mammals that corresponds to a critical depletion of fat stores and to a shift from lipid to protein utilization. This strongly suggests that such a metabolic shift triggers behavioural changes leading to egg desertion and refeeding, which is of great relevance to the understanding of the long-term control of food intake and BM. Accepted: 16 July 1998     

Selective local irradiation improves islet engraftment and survival in intra–bone marrow islet transplantation

BackgroundBone marrow (BM) is as an alternative site for islet transplantation, but it is not an immunoprotected microenvironment and allogeneic islets are rejected. However, the BM, for its structure and anatomic position, offers the possibility to modulate microenvironment by local interventions. We here investigate whether local irradiation is able to improve islet engraftment and prevent rejection in BM in the absence of immunosuppression.MethodsA model of BM local irradiation was set up. Islets were transplanted in syngeneic and fully major histocompatibility complex–mismatched recipients in control and locally irradiated BM; gain of normoglycemia and time to rejection were evaluated.ResultsBM local irradiation proved to be a selective and safe procedure. Syngeneic islet transplantation into locally irradiated BM had better outcome compared with not irradiated recipients in terms of capacity to gain normoglycemia (100% versus 56% in irradiated versus not irradiated mice). In the allogenic setting, glycemia was significantly lower in the first days after transplantation in the group of irradiated mice and local irradiation also delayed time to graft rejection (from 4 ± 1 days for not irradiated to 11 ± 1 days for locally irradiated mice).DiscussionThese data indicate that local immunosuppression by irradiation before islet transplantation in BM favors islet engraftment and delays time to rejection.     

In vitro study of matrix metalloproteinase/tissue inhibitor of metalloproteinase production by mesenchymal stromal cells in response to inflammatory cytokines: the role of their migration in injured tissues

Background aimsThe transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production.MethodsThe migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel^® in the presence and absence of stromal cell-derived (SDF)-1α, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels.ResultsWe have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli.ConclusionsOur observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.     

Digestibility of amino acids in fish meal and blood-derived protein sources fed to pigs

ObjectiveAn experiment was conducted to determine the standardized ileal digestibility (SID) of amino acids (AA) in fish meal (FM) and blood-derived protein sources including spray-dried porcine plasma (SDPP), porcine red blood cells (PRBC), and blood meal (BM) fed to growing pigs.MethodsTen barrows (mean initial body weight of 22.1±1.54 kg) surgically fitted with T-cannulas at the distal ileum were allotted to a duplicated 5×4 incomplete Latin square design with 5 experimental diets and 4 periods. Four experimental diets were prepared to contain FM, SDPP, PRBC, or BM as the sole source of nitrogen. A nitrogen-free diet was prepared and included to estimate the basal ileal endogenous losses of AA. For the 7-day experimental period, pigs were fed for 5 days as adaptation, and ileal digesta samples were collected for 9 hours on days 6 and 7.ResultsThe SID of crude protein in BM (48.0%) was less (p<0.05) than in FM, SDPP, and PRBC (83.4%, 83.9%, and 87.3%, respectively). Pigs fed the diet containing BM had less (p<0.05) SID of AA, except isoleucine and proline, than those fed the diet containing FM, SDPP, or PRBC. Among FM, SDPP, and PRBC, there was no difference in the SID of crude protein and all AA, except isoleucine. The SID of isoleucine in PRBC and BM (62.7% and 48.3%, respectively) was less (p<0.05) than in FM and SDPP (88.0% and 84.9%, respectively). The SID of lysine in FM, SDPP, PRBC, and BM was 85.4%, 84.9%, 89.7%, and 51.9%, respectively.ConclusionThe SID of most AA was not different among FM, SDPP, and PRBC, but BM had lower SID of most AA than FM, SDPP, and PRBC.     

Mesenchymal stromal/stem cells markers in the human bone marrow

Background aimsMesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation.MethodsTo characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression.ResultsMicroscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers.ConclusionsTargeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.     

Bone marrow angiogenesis and angiogenic factors in multiple myeloma treated with novel agents

Introduction. An increased bone marrow (BM) angiogenesis is associated with poor outcome in multiple myeloma (MM). Objective. Angiogenesis study in MM treated with novel antimyeloma agents: thalidomide, lenalidomide, bortezomib, and with dexamethasone. Patients and methods. Forty-four patients with MM (14 newly diagnosed, 30 refractory/relapsed) were treated with novel agents at our institution. A BM biopsy was obtained before the initiation of therapy in 19. Angiogenesis was assessed by microvessel density (MVD) estimation in BM biopsies stained with the monoclonal anti-CD34 antibody, and by serum levels of angiogenic factors (VEGF, bFGF, and HGF) and cytokines (IL-6 and TNF-α). Results. A positive correlation was found between BM plasma cell involvement and MVD estimation (p = 0.01). However, MVD was not significantly correlated with either disease phase (p = 0.065) or response to therapy (p = 0.79). Neither baseline serum levels of angiogenic cytokines correlated to response to treatment. No significant correlation was found between BM MVD and serum levels of angiogenic cytokines. Serum levels of angiogenic cytokines before and after therapy showed a significant increase of bFGF (p = 0.008). Conclusion. There is no relationship between MVD estimation and baseline serum levels of angiogenic cytokines, neither between each of them and response to therapy.     

Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro

Background aimsThe ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood.MethodsC57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU.ResultsAt a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105⁺). A 3-fold reduction in the percentage of CD105⁺ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105⁺ cells coincided with a 10–20% increase in the frequency of proliferating CD105^? cells. Removal of CD105^? stroma caused increased proliferation in CD105⁺ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105⁺ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105^? cells.ConclusionsThis work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.