Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.   Objective : Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods : Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl₂-treated non-irradiated mice, HgCl₂-treated non-irradiated mice infused with male BMCs 1 day after HgCl₂, and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results : Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl₂, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions : Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl₂, despite establishment of chimerism and cell proliferation in bone marrow and spleen.     

The repair processes and cell populations relationship in bone marrow of patients with acute radiation injury: morphological study

The results of bone marrow pathomorphologic investigation in patients who died of acute radiation sickness (3.7-13.7 Gy) after the Chernobyl accident are presented. Stratification into three consecutive periods was performed according to the length of postexposure survival (14-96 days) and the morphologic picture dynamics: 1) bone marrow aplasia (2-3 weeks after exposure) with a domination of stromal and plasma cell populations, exhibiting the morphologic characteristics of functional activity; 2) initial repair period (3-4 weeks after exposure) was characterized by the beginning of hematopoietic proliferation with simultaneous occurrence of cell death via apoptosis; 3) active repair period (4 and more weeks after exposure) was defined by increased marrow cellularity (trilineage regeneration), accompanied by decreased number of plasma cells and occurrence of large granular lymphocytes.     

Biochemical analysis of the bone of SHC rats with spontaneous hypercholesterolemia and renal dysfunction.

Spontaneously hypercholesterolemic (SHC) rats exhibit severe abnormalities in renal function and bone metabolism at old ages, in addition to hypercholesterolemia. SHC rats were also found to show endocrine abnormalities such as hyperthyroidism from young ages. In this study, biochemical and microdensitometric analyses were carried out using femurs to characterize further the abnormality in bone metabolism: whether biochemical markers of the bone may be affected by these abnormalities. At 6 weeks of age, the ashed weight and calcium content of the dried femurs were slightly lower in SHC rats than in age-matched Sprague-Dawley (SD) rats. None of the markers of microdensitometric analysis was changed. At 24 weeks of age, the ashed weight of dried femurs and the density of the marrow region of femurs were lower in the SHC rats. The results indicate that SHC rats exhibit severe abnormality in bone metabolism leading to biochemical changes in the bone at old ages whereas changes in bone markers were minimal at young ages before the onset of severe renal dysfunction.     

Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to bafilomycin A1 by a factor with restricted expression to bone and brain, neurochondrin

Bone, one of the favored sites for tumor metastasis, is a dynamic organ undergoing formation and resorption. We found bone metastasis with osteolytic lesion in the bone marrow of the femur by injecting BW5147 T-lymphoma cells into the tail vein of AKR mice. To understand this bone destruction, we constructed a cDNA library from BW5147 with a cloning vector that allowed in vitro synthesis of mRNAs, and then identified a particular cDNA clone by adding the conditioned medium from Xenopus oocytes following injection of the mRNA synthesized in vitro to primary bone marrow heterogeneous cell populations on hydroxyapatite thin films. By means of this method, we isolated a factor with 16% leucine residues, termed neurochondrin, that induces hydroxyapatite resorptive activity in bone marrow cells resistant to bafilomycin A1, an inhibitor of macrophage- and osteoclast-mediated resorption. Expression of the gene was localized to chondrocyte, osteoblast, and osteocyte in the bone and to the hippocampus and Purkinje cell layer of cerebellum in the brain. This may provide insights into the molecular mechanisms underlying bone resorption with potential implications for the activation of cells other than macrophages and osteoclasts in bone marrow cells.     

Immunocytochemical analysis of apoptotic bone marrow cells after treatment of mice with WR-2721 and chemotherapeutic drugs

The effects of the aminothiol WR-2721 (Amifostine) and the chemotherapeutic drugs, cyclophosphamide (CP) and cisplatin (CDDP) on induction of apoptosis in bone marrow cells of adult male Swiss mice were studied. The mice received intraperitoneal injections of WR-2721 (400 mg/kg), cyclophosphamide (200 mg/kg), and cisplatin (10 mg/kg). WR-2721 was administered alone, or 30 min before treatment of mice with CP or CDDP. The number of apoptotic bone marrow cells was determined at 7 h and 24 h after the agent(s) administration. The In Situ Cell Death Detection Kit, AP based on TUNEL technique, and Fast Red Substrate System were used for microscopic analysis of immunocytochemically stained apoptotic cells. Application of cyclophosphamide and cisplatin resulted in a distinct increase of the number of apoptotic cells in the mouse bone marrow. After treatment of mice with WR-2721 prior to administration of CP or CDDP, as compared to the chemotherapeutic treatment only, the tendency to a decrease--albeit statistically insignificant--in the number of apoptotic cells was observed. Application of WR-2721 alone, without subsequent administration of the chemotherapeutic agents caused an inconsiderable increase in the number of apoptotic cells. The degree of apoptotic DNA cleavage in cells of the mouse bone marrow varied depending on the agent(s) given and the time interval after the drug administration.     

B lymphocyte precursors in human bone marrow: an analysis of normal individuals and patients with antibody-deficiency states.

Lymphoid cells containing cytoplasmic IgM but lacking stable surface IgM are believed to be the direct precursors of B lymphocytes. We have characterized these pre-B cells in the bone marrow of normal individuals and patients with a variety of immunoglobulin deficiencies or hematologic disorders by using immunofluorescence and autoradiography. Pre-B cells comprised 5.8 +/- 5.7% of lymphoid cells in normal bone marrow. Eleven patients with infantile X-linked agammaglobulinemia (X-LA) lacked B lymphocytes but had a normal frequency (3.8 +/- 3.6%) of bone marrow pre-B cells. A smaller proportion of marrow pre-B cells from patients with X-LA were engaged in spontaneous DNA synthesis than was found for normal controls. In individuals other than the group with X-LA, the number of circulating B cells was positively correlated with the frequency of marrow pre-B cells. These results indicate that patients with X-LA have a defect in maturation of pre-B cells, and suggest that some patients with acquired B lymphocyte deficiency may have lost the capacity to generate pre-B cells from stem cells.     

The comparative analysis of homologous fresh frozen bone and autogenous bone graft,associated or not with autogenous bone marrow,in rabbit calvaria: a clinical and histomorphometric study

The aim of this study was to evaluate the potential of fresh frozen homologous and autogenous grafts, associated or not with autogenous bone marrow, to form bone. Sixty titanium cylinders were used, and were fixed to the skulls of 30 rabbits. These cylinders were filled with (A) autogenous bone (AM) autogenous bone associated with the bone marrow (H) fresh frozen homologous bone (HM) fresh frozen homologous bone associated with the bone marrow (M) pure autogenous bone marrow and (C) blood clot. The animals were sacrificed after 02 and 03 months. After clinical evaluation, the samples were stained with hematoxylin, eosin and Mallory Trichrome dyes for optical microscopy analysis and histomorphometric analysis. Experimental groups that received mineralized materials (A, AM, H, HM) showed the best bone formation results, presenting no statistical difference between them (P > 0.05). Groups that did not receive mineralized materials (M and C) showed the worst results (P < 0.05), but the M group showed better results than the C group. Most of the autogenous and homologous bone particles were resorbed and there was a larger amount of residual particles in the homologous graft (H, HM) when compared with the autogenous graft (A, AM; P < 0.05). These findings suggest that fresh frozen homologous grafts produced similar amounts of new bone when compared with the autogenous grafts. However, the amount of residual bone particles was larger in the homogenous groups, which may indicate a slower remodeling process. The homologous fresh frozen bone seems to be a good osteoconductive material. The use of only autogenous bone marrow showed better results when compared to the bood clot. However, this research indicates that association with mineralized materials is required.     

Global transcriptome analysis of human bone marrow stromal cells (BMSC) reveals proliferative, mobile and interactive cells that produce abundant extracellular matrix proteins, some of which may affect BMSC potency

Background aimsBone marrow stromal cells (BMSC) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality.MethodsEarly passage BMSC prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34⁺ cells from three healthy subjects and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35 000 probes.ResultsBMSC gene expression signatures of BMSC differed from those of hematopoietic stem cells (HSC), hESC and fibroblasts. Genes upregulated in BMSC were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The upregulated genes most probably belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor-2 (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/β catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (α5 and β5 integrin chains, fibronectin and collagen type IIIα1 and Vα1) and functional EMC proteins [connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and A disintegrin and metalloproteinase (ADAM12)].ConclusionsGlobal analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects.     

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.     

Detailed analysis of intrahepatic CD8 T cells in the normal and hepatitis C-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes

In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7-, L-selectin-) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin- cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin- intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.     

Multilocus phylogeographical analysis of Trypanosoma (Megatrypanum) genotypes from sympatric cattle and water buffalo populations supports evolutionary host constraint and close phylogenetic relationships with genotypes found in other ruminants

Species of the subgenus Trypanosoma (Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle (Bos taurus), but only one in water buffalo (Bubalus bubalis). However, the small number of isolates examined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associations and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate locations in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochondrial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all isolates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymorphisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, antelope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corresponds to the subgenus T. (Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host-parasite co-evolution, indicating that host switches have occurred across ruminants followed by divergences, giving rise to new trypanosome genotypes adapted exclusively to one host species.     

Follow-up analysis of translocation and dicentric frequencies, measured by FISH-chromosome painting in breast cancer patients after partial-body radiotherapy with little bone marrow exposure

Fanconi anemia (FA) is one of several genetic diseases with characteristic cellular hypersensitivity to DNA crosslinking agents which suggest that FA proteins may function as part of DNA repair processes. At the clinical level, FA is characterized by bone marrow failure that affects children at an early age. The clinical phenotype is heterogeneous and includes various congenital malformations as well as cancer predisposition. FA patients are distributed into eight complementation groups suggesting a complex molecular pathway. Three of the eight possible FA genes have been cloned, although their function(s) have not been identified. FA cells are highly sensitive to DNA crosslinking agents (mitomycin C (MMC) and diepoxybutane), with some variability between cell lines. Sensitivity to monofunctional alkylating agents has been reported in some cases, although these studies were performed with genetically unclassified FA cells. To further analyse and characterize the newly identified FA complementation groups, we tested their sensitivity to UV radiation, monofunctional and bifunctional alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA complementation groups D to H show increased sensitivity to the X-ray mimetic drug bleomycin. Furthermore, the single known FA-H cell line shows increased sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in addition to the characteristic sensitivity to crosslinking agents, suggesting a broader spectrum of drug sensitivities in FA cells.     

International cohort study of 73 anti-Ku-positive patients: association of p70/p80 anti-Ku antibodies with joint/bone features and differentiation of disease populations by using principal-components analysis

Introduction

An international cohort study of 73 anti-Ku-positive patients with different connective tissue diseases was conducted to differentiate the anti-Ku-positive populations of patients based on their autoantibody profile and clinical signs/symptoms and to establish possible correlations between antibodies against Ku p70 and Ku p80 with autoimmune diseases.

Methods

Sera of anti-Ku-positive patients were collected from six European centers and were all secondarily tested (in the reference center); 73 were confirmed as positive. Anti-Ku antibodies were detected with counter-immunoelectrophoresis (CIE), line immunoassay (LIA), and immunoblot analyses. All clinical and laboratory data were follow-up cumulative data, except for anti-Ku antibodies. Statistical analyses were performed by using R (V 2.12.1). The Fisher Exact test was used to evaluate the association between anti-Ku antibodies and diagnosis, gender, clinical signs, and other observed antibodies. The P values were adjusted for multiple testing. Separation of disease populations based on the presence of antibodies and clinical signs was investigated by principal-components analysis, which was performed by using thr// R's prcomp function with standard parameters.

Results

A 16% higher prevalence of anti-Ku p70 was found over anti-Ku p80 antibodies. In 41 (57%) patients, a combination of both was detected. Five (7%) patients, who were CIE and/or LIA anti-Ku positive, were negative for both subsets, as detected with the immunoblot; 31% of the patients had undifferentiated connective tissue disease (UCTD); 29% had systemic sclerosis (SSc); 18% had systemic lupus erythematosus (SLE); 11% had rheumatoid arthritis; 7% had polymyositis; and 3% had Sjögren syndrome.

Conclusions

A significant positive association was found between female patients with anti-Ku p70 and joint/bone features, and a significant negative association was found between female patients with anti-Ku p80 only and joint/bone features (P = 0.05, respectively). By using the first and the third components of the principal-component analysis (PCA) with 29 parameters evaluated, we observed that the anti-Ku-positive population of UCTD patients had overlapping parameters, especially with SLE, as opposed to SSc, which could be helpful in delineating UCTD patients.