Natural History of Malignant Bone Disease in Renal Cancer: Final Results of an Italian Bone Metastasis Survey

Background

Bone metastasis represents an increasing clinical problem in advanced renal cell carcinoma (RCC) as disease-related survival improves. There are few data on the natural history of bone disease in RCC.

Patients and methods

Data on clinicopathology, survival, skeletal-related events (SREs), and bone-directed therapies for 398 deceased RCC patients (286 male, 112 female) with evidence of bone metastasis were statistically analyzed.

Results

Median time to bone metastasis was 25 months for patients without bone metastasis at diagnosis. Median time to diagnosis of bone metastasis by MSKCC risk was 24 months for good, 5 months for intermediate, and 0 months for poor risk. Median number of SREs/patient was one, and 71% of patients experienced at least one SRE. Median times to first, second, and third SRE were 2, 5, and 12 months, respectively. Median survival was 12 months after bone metastasis diagnosis and 10 months after first SRE. Among 181 patients who received zoledronic acid (ZOL), median time to first SRE was significantly prolonged versus control (n = 186) (3 months vs 1 month for control; P<0.05).

Conclusions

RCC patients with bone metastasis are at continuous risk of SREs, and in this survey ZOL effectively reduced this risk.     

Bone marrow support of the heart in pressure overload is lost with aging

Rationale

Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response.

Objective

To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging.

Methods and Results

Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice.

Conclusions

BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging.     

Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells

Background

VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions.

Methods

VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined.

Results

About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro.

Conclusions

Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration,but only a limited role in their protection by stromal cells.     

Alterations in the self-renewal and differentiation ability of bone marrow mesenchymal stem cells in a mouse model of rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease primarily involving the synovium. Evidence in recent years has suggested that the bone marrow (BM) may be involved, and may even be the initiating site of the disease. Abnormalities in haemopoietic stem cells'' (HSC) survival, proliferation and aging have been described in patients affected by RA and ascribed to abnormal support by the BM microenvironment. Mesenchymal stem cells (MSC) and their progeny constitute important components of the BM niche. In this study we test the hypothesis that the onset of inflammatory arthritis is associated with altered self-renewal and differentiation of bone marrow MSC, which alters the composition of the BM microenvironment.

Methods

We have used Balb/C Interleukin-1 receptor antagonist knock-out mice, which spontaneously develop RA-like disease in 100% of mice by 20 weeks of age to determine the number of mesenchymal progenitors and their differentiated progeny before, at the start and with progression of the disease.

Results

We showed a decrease in the number of mesenchymal progenitors with adipogenic potential and decreased bone marrow adipogenesis before disease onset. This is associated with a decrease in osteoclastogenesis. Moreover, at the onset of disease a significant increase in all mesenchymal progenitors is observed together with a block in their differentiation to osteoblasts. This is associated with accelerated bone loss.

Conclusions

Significant changes occur in the BM niche with the establishment and progression of RA-like disease. Those changes may be responsible for aspects of the disease, including the advance of osteoporosis. An understanding of the molecular mechanisms leading to those changes may lead to new strategies for therapeutic intervention.     

TNF-α Regulates the Effects of Irradiation in the Mouse Bone Marrow Microenvironment

Background

Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-α, known to regulate cell apoptosis, could modulate the onset of secondary MDS.

Principal Findings

We show that TNF-α is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-α deficient (TNF-α KO) mice or WT mice treated with a TNF-α-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-α KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (≥5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression.

Conclusion

Taken together, our data shows that TNF-α induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression.     

Non-Invasive Imaging Provides Spatiotemporal Information on Disease Progression and Response to Therapy in a Murine Model of Multiple Myeloma

Background

Multiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.

Material and Methods

A mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.

Results

Strong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase⁺ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase⁺ cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.

Conclusions

This study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.     

Dietary supplementation with probiotics improves hematopoiesis in malnourished mice

Background

Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation.

Objective

This study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice.

Methods

Protein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat''s milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells.

Results

Malnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups.

Conclusions

Protein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised-malnourished hosts.     

Natural History of Malignant Bone Disease in Gastric Cancer: Final Results of a Multicenter Bone Metastasis Survey

Background

Bone metastasis represents an increasing clinical problem in advanced gastric cancer (GC) as disease-related survival improves. In literature, few data on the natural history of bone disease in GC are available.

Patients and Methods

Data on clinicopathology, skeletal outcomes, skeletal-related events (SREs), and bone-directed therapies for 208 deceased GC patients with evidence of bone metastasis were statistically analyzed.

Results

Median time to bone metastasis was 8 months (CI 95%, 6.125–9.875 months) considering all included patients. Median number of SREs/patient was one. Less than half of the patients (31%) experienced at least one and only 4 and 2% experienced at least two and three events, respectively. Median times to first and second SRE were 2 and 4 months, respectively. Median survival was 6 months after bone metastasis diagnosis and 3 months after first SRE. Median survival in patients who did not experience SREs was 5 months. Among patients who received zoledronic acid before the first SRE, the median time to appearance of first SRE was significantly prolonged compared to control (7 months vs 4 months for control; P: 0.0005).

Conclusions

To our knowledge, this retrospective analysis is the largest multicenter study to demonstrate that bone metastases from GC are not so rare, are commonly aggressive and result in relatively early onset of SREs in the majority of patients. Indeed, our large study, which included 90 patients treated with ZOL, showed, for the first time in literature, a significant extension of time to first SRE and increase in the median survival time after diagnosis of bone metastasis. Taken together, these data may support the beneficial effects of ZOL in GC patients.     

Catabolic and anabolic periarticular bone changes in patients with rheumatoid arthritis: a computed tomography study on the role of age,disease duration and bone markers

Introduction

The aim of this study was to determine the factors, including markers of bone resorption and bone formation, which determine catabolic and anabolic periarticular bone changes in patients with rheumatoid arthritis (RA).

Methods

Forty RA patients received high-resolution peripheral quantitative computed tomography (HR-pQCT) analysis of the metacarpophalangeal joints II and III of the dominantly affected hand at two sequential time points (baseline, one year follow-up). Erosion counts and scores as well as osteophyte counts and scores were recorded. Simultaneously, serum markers of bone resorption (C-terminal telopeptide of type I collagen (CTX I), tartrate-resistant acid phosphatase 5b (TRAP5b)), bone formation (bone alkaline phosphatase (BAP), osteocalcin (OC)) and calcium homeostasis (parathyroid hormone (PTH), 25-hydroxyvitamin D3 (Vit D)) were assessed. Bone biomarkers were correlated to imaging data by partial correlation adjusting for various demographic and disease-specific parameters. Additionally, imaging data were analyzed by mixed linear model regression.

Results

Partial correlation analysis showed that TRAP5b levels correlate significantly with bone erosions, whereas BAP levels correlate with osteophytes at both time points. In the mixed linear model with erosions as the dependent variable, disease duration (P <0.001) was the key determinant for these catabolic bone changes. In contrast, BAP (P = 0.001) as well as age (P = 0.018), but not disease duration (P = 0.762), were the main determinants for the anabolic changes (osteophytes) of the periarticular bone in patients with RA.

Conclusions

This study shows that structural bone changes assessed with HR-pQCT are accompanied by alterations in systemic markers of bone resorption and bone formation. Besides, it can be shown that bone erosions in RA patients depend on disease duration, whereas osteophytes are associated with age as well as serum level of BAP. Therefore, these data not only suggest that different variables are involved in formation of bone erosions and osteophytes in RA patients, but also that periarticular bone changes correlate with alterations in systemic markers of bone metabolism, pointing out BAP as an important parameter.     

Effects of whole-body vibration on acute bone turnover marker responses to resistance exercise in young men

Objective:

We investigated acute bone turnover marker (BTM) responses to high-intensity resistance exercise with and without whole-body vibration (WBV) in young men (n=10).

Methods:

In this randomized crossover study, subjects performed 2 protocols separated by 2-week wash out periods: 1) resistance exercise only (RE) (3 sets 10 repetitions 80% 1RM for 9 exercises); and 2) WBV + RE (side-alternating vibration platform 5 intermittent, 1-minute bouts 20 Hz, 3.38 mm peak-to-peak displacement followed by RE). Fasting morning blood draws were taken before RE or WBV (PRE), immediately post RE (IP), and 30 minutes post RE (30P). WBV + RE also had a blood draw after the WBV exposure (POST WBV). Blood samples were analyzed for lactate, hematocrit, bone-specific alkaline phosphatase (Bone ALP, U/L), C-terminal telopeptide of type I collagen (CTX-I, ng/mL) and tartrate-resistant acid phosphatase 5b (TRAP5b, U/L).

Results:

Lactate, hematocrit, and Bone ALP significantly increased (p<0.05) IP for both protocols. Bone resorption markers did not change during RE only. CTX-I significantly decreased POST WBV. TRAP5b increased POST WBV, then significantly decreased at 30P.

Conclusions:

Generally, BTM changes to RE only were not significant when adjusted for hemoconcentration. The WBV stimulus altered bone resorption marker but not bone formation marker responses.     

Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading

Background

Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading.

Methodology/Principal Findings

We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP_8–37, for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP_8–37 was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP_8–37 both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP_8–37 administration.

Conclusions

CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton.     

Quantitative Analysis of Protein and Gene Expression in Salivary Glands of Sjogren’s-Like Disease NOD Mice Treated by Bone Marrow Soup

Background

Bone marrow cell extract (termed as BM Soup) has been demonstrated to repair irradiated salivary glands (SGs) and restore saliva secretion in our previous study. In the present study, we aim to investigate if the function of damaged SGs in non-obese diabetic (NOD) mice can be restored by BM Soup treatment and the molecular alterations associated with the treatment.

Methods

Whole BM cells were lysed and soluble intracellular contents (“BM Soup”) were injected I.V. into NOD mice. Tandem mass tagging with 2-D liquid chromatography-mass spectrometry was used to quantify proteins in the submandibular glands (SMGs) between untreated and BM Soup-treated mice. Quantitative PCR was used to identify genes with altered expression in the treated mice.

Results BM Soup

restored salivary flow rates to normal levels and significantly reduced the focus scores of SMGs in NOD mice. More than 1800 proteins in SMG cells were quantified by the proteomic approach. Many SMG proteins involved in inflammation and apoptosis were found to be down-regulated whereas those involved in salivary gland biology and development/regeneration were up-regulated in the BM Soup-treated mice. qPCR analysis also revealed expression changes of growth factors and cytokines in the SMGs of the treated NOD mice.

Conclusion

BM Soup treatment is effective to restore the function of damaged SGs in NOD mice. Through gene/protein expression analysis, we have found that BM Soup treatment might effectuate via inhibiting apoptosis, focal adhesion and inflammation whereas promoting development, regeneration and differentiation of the SG cells in NOD mice. These findings provide important insights on the potential mechanisms underlying the BM Soup treatment for functional restoration of damaged SGs in NOD mice. Additional studies are needed to further confirm the identified target genes and their related signaling pathways that are responsible for the BM Soup treatment.     

Paracrine Effects of Bone Marrow Soup Restore Organ Function,Regeneration, and Repair in Salivary Glands Damaged by Irradiation

Background

There are reports that bone marrow cell (BM) transplants repaired irradiated salivary glands (SGs) and re-established saliva secretion. However, the mechanisms of action behind these reports have not been elucidated.

Methods

To test if a paracrine mechanism was the main effect behind this reported improvement in salivary organ function, whole BM cells were lysed and its soluble intracellular contents (termed as “BM Soup”) injected into mice with irradiation-injured SGs. The hypothesis was that BM Soup would protect salivary cells, increase tissue neovascularization, function, and regeneration. Two minor aims were also tested a) comparing two routes of delivering BM Soup, intravenous (I.V.) versus intra-glandular injections, and b) comparing the age of the BM Soup’s donors. The treatment-comparison group consisted of irradiated mice receiving injections of living whole BM cells. Control mice received irradiation and injections of saline or sham-irradiation. All mice were followed for 8 weeks post-irradiation.

Results

BM Soup restored salivary flow rates to normal levels, protected salivary acinar, ductal, myoepithelial, and progenitor cells, increased cell proliferation and blood vessels, and up-regulated expression of tissue remodeling/repair/regenerative genes (MMP2, CyclinD1, BMP7, EGF, NGF). BM Soup was as an efficient therapeutic agent as injections of live BM cells. Both intra-glandular or I.V. injections of BM Soup, and from both young and older mouse donors were as effective in repairing irradiated SGs. The intra-glandular route reduced injection frequency/dosage by four-fold.

Conclusion

BM Soup, which contains only the cell by-products, can be advantageously used to repair irradiation-damaged SGs rather than transplanting whole live BM cells which carry the risk of differentiating into unwanted/tumorigenic cell types in SGs.     

Evidence that prefibrotic myelofibrosis is aligned along a clinical and biological continuum featuring primary myelofibrosis

Purpose

In the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.

Patients and Methods

We investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.

Results

As compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.

Conclusion

Pre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.     

Calcitonin inhibits SDCP-induced osteoclast apoptosis and increases its efficacy in a rat model of osteoporosis

Introduction

Treatment for osteoporosis commonly includes the use of bisphosphonates. Serious side effects of these drugs are caused by the inhibition of bone resorption as a result of osteoclast apoptosis. Treatment using calcitonin along with bisphosphonates overcomes these side-effects in some patients. Calcitonin is known to inhibit bone resorption without reducing the number of osteoclasts and is thought to prolong osteoclast survival through the inhibition of apoptosis. Further understanding of how calcitonin inhibits apoptosis could prove useful to the development of alternative treatment regimens for osteoporosis. This study aimed to analyze the mechanism by which calcitonin influences osteoclast apoptosis induced by a bisphosphate analog, sintered dicalcium pyrophosphate (SDCP), and to determine the effects of co-treatment with calcitonin and SDCP on apoptotic signaling in osteoclasts.

Methods

Isolated osteoclasts were treated with CT, SDCP or both for 48 h. Osteoclast apoptosis assays, pit formation assays, and tartrate-resistant acid phosphatase (TRAP) staining were performed. Using an osteoporosis rat model, ovariectomized (OVX) rats received calcitonin, SDCP, or calcitonin + SDCP. The microarchitecture of the fifth lumbar trabecular bone was investigated, and histomorphometric and biochemical analyses were performed.

Results

Calcitonin inhibited SDCP-induced apoptosis in primary osteoclast cultures, increased Bcl-2 and Erk activity, and decreased Mcl-1 activity. Calcitonin prevented decreased osteoclast survival but not resorption induced by SDCP. Histomorphometric analysis of the tibia revealed increased bone formation, and microcomputed tomography of the fifth lumbar vertebrate showed an additive effect of calcitonin and SDCP on bone volume. Finally, analysis of the serum bone markers CTX-I and P1NP suggests that the increased bone volume induced by co-treatment with calcitonin and SDCP may be due to decreased bone resorption and increased bone formation.

Conclusions

Calcitonin reduces SDCP-induced osteoclast apoptosis and increases its efficacy in an in vivo model of osteoporosis.     

Sequestration and homing of bone marrow-derived lineage negative progenitor cells in the lung during pneumococcal pneumonia

Background

Bone marrow (BM)-derived progenitor cells have been shown to have the potential to differentiate into a diversity of cell types involved in tissue repair. The characteristics of these progenitor cells in pneumonia lung is unknown. We have previously shown that Streptococcus pneumoniae induces a strong stimulus for the release of leukocytes from the BM and these leukocytes preferentially sequester in the lung capillaries. Here we report the behavior of BM-derived lineage negative progenitor cells (Lin- PCs) during pneumococcal pneumonia using quantum dots (QDs), nanocrystal fluorescent probes as a cell-tracking technique.

Methods

Whole BM cells or purified Lin- PCs, harvested from C57/BL6 mice, were labeled with QDs and intravenously transfused into pneumonia mice infected by intratracheal instillation of Streptococcus pneumoniae. Saline was instilled for control. The recipients were sacrificed 2 and 24 hours following infusion and QD-positive cells retained in the circulation, BM and lungs were quantified.

Results

Pneumonia prolonged the clearance of Lin- PCs from the circulation compared with control (21.7 ± 2.7% vs. 7.7 ± 0.9%, at 2 hours, P < 0.01), caused preferential sequestration of Lin- PCs in the lung microvessels (43.3 ± 8.6% vs. 11.2 ± 3.9%, at 2 hours, P < 0.05), and homing of these cells to both the lung (15.1 ± 3.6% vs. 2.4 ± 1.2%, at 24 hours, P < 0.05) and BM as compared to control (18.5 ± 0.8% vs. 9.5 ± 0.4%, at 24 hours, P < 0.01). Very few Lin- PCs migrated into air spaces.

Conclusion

In this study, we demonstrated that BM-derived progenitor cells are preferentially sequestered and retained in pneumonic mouse lungs. These cells potentially contribute to the repair of damaged lung tissue.     

TNF inhibits production of stromal cell-derived factor 1 by bone stromal cells and increases osteoclast precursor mobilization from bone marrow to peripheral blood

Introduction

The objective of the present study was to investigate the role of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis in TNF-induced mobilization of osteoclast precursors (OCPs) from bone marrow.

Methods

OCPs were generated from bone marrow cells of TNF-transgenic mice or wild-type mice treated with TNF or PBS. The percentage of CD11b⁺/Gr-1^-/lo OCPs was assessed by fluorescence-activated cell sorting. OCP migration to the SDF-1 gradient and the osteoclast forming potency were assessed in chemotaxis/osteoclastogenic assays. SDF-1 expression was assessed by real-time RT-PCR, ELISA and immunostaining in primary bone marrow stromal cells, in the ST2 bone marrow stromal cell line, and in bones from TNF-injected mice.

Results

OCPs generated in vitro from wild-type mice migrated to SDF-1 gradients and subsequently gave rise to osteoclasts in response to RANKL and macrophage colony-stimulating factor. TNF reduced SDF-1 expression by ST2 cells. Bone marrow stromal cells from TNF-transgenic mice produced low levels of SDF-1. TNF treatment of wild-type mice decreased the SDF-1 concentration in bone marrow extracts and decreased the SDF-1 immunostaining of bone marrow stromal cells, and it also increased the circulating OCP numbers. The percentage of bone marrow CXCR4⁺ OCPs was similar in TNF-transgenic mice and wild-type littermates and in TNF-treated and PBS-treated wild-type mice.

Conclusion

Systemically elevated TNF levels inhibit bone marrow stromal cell production of SDF-1 and increase the release of bone marrow OCPs to the peripheral blood. Disruption of the SDF-1/CXCR4 axis by TNF may play an important role in mediating OCP mobilization from the bone marrow cavity in chronic inflammatory arthritis.     

Granulocyte Colony-Stimulating Factor Reduces Fibrosis in a Mouse Model of Chronic Pancreatitis

Background

Chronic pancreatitis (CP) is a necroinflammatory process resulting in extensive pancreatic fibrosis. Granulocyte colony-stimulating factor (G-CSF), a hematopoietic stem cell mobilizer, has been shown to exert an anti-fibrotic effect partly through the enrichment of bone marrow (BM) cells in fibrotic organ. We aimed to test the effect of G-CSF on fibrosis in a mouse model of CP.

Methods

CP was induced in C57Bl/6J mice by consecutive cerulein injection (50 µg/kg/day, 2 days a week) for 6 weeks. Mice were then treated with G-CSF (200 µg/kg/day, 5 day a week) or normal saline for 1 week, and sacrificed at week 7 or week 9 after first cerulein injection. Pancreatic histology, pancreatic matrix metallopeptidase 9 (MMP-9), MMP-13 and collagen expression were examined. Pancreatic myofibroblasts were isolated and cultured with G-CSF. Collagen, MMP-9 and MMP-13 expression by myofibroblasts was examined. The BM-mismatched mice model was used to examine the change of BM-derived myofibroblasts and non-myofibroblastic BM cells by G-CSF in the pancreas.

Results

The pancreatic collagen expression were significantly decreased in the G-CSF-treated group sacrificed at week 9. While collagen produced from myofibroblasts was not affected by G-CSF, the increase of MMP13 expression was observed in vitro. There were no effect of G-CSF in the number of myofibroblasts and BM-derived myofibroblasts. However, the number of non-myofibroblastic BM cells and macrophages were significantly increased in the pancreata of cerulein- and G-CSF-treated mice, suggesting a potential anti-fibrotic role of non-myofibroblastic BM cells and macrophages stimulated by G-CSF.

Conclusions

Our data indicated that G-CSF contributed to the regression of pancreatic fibrosis. The anti-fibrotic effects were possibly through the stimulation of MMP-13 from myofibroblasts, and the enhanced accumulation of non-myofibroblastic BM cells and macrophages in the pancreas.     

The Inhibition of Subchondral Bone Lesions Significantly Reversed the Weight-Bearing Deficit and the Overexpression of CGRP in DRG Neurons,GFAP and Iba-1 in the Spinal Dorsal Horn in the Monosodium Iodoacetate Induced Model of Osteoarthritis Pain

Background

Chronic pain is the most prominent and disabling symptom of osteoarthritis (OA). Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain.

Methods

Osteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA) into the rat knee joint. Zoledronic acid (ZOL), a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG), and spinal glial activation status using glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling.

Results

MIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected.

Conclusions

The inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.     

Bone marrow cells in murine colitis: multi-signal analysis confirms pericryptal myofibroblast engraftment without epithelial involvement

Background

The contribution of bone marrow-derived cells to epithelial tissues in the inflamed gut remains controversial. Recent reports have suggested that cell fusion between bone marrow-derived cells and the intestinal epithelium takes place in inflammatory conditions.

Methods

In attempts to confirm this, we have undertaken gender mis-matched bone marrow (BM) transplants from male Swiss Webster (SWR) mice to B and T cell-deficient female Rag2 KO mice which, 4 weeks later, were given 5% dextran sodium sulphate in drinking water to induce acute colitis. A further BM-treated group of animals with a graft versus host-like condition was also studied. We developed a new method to combine up to three brightfield or fluorescent lectin- or immuno-histochemical signals with fluorescent in situ hybridisation for the Y and X chromosomes to enable us unequivocally to identify BM-derived male cells which presented as different cell types in the gastrointestinal tract.

Principal Findings

In rolled preparations of whole intestines we scanned around 1.5 million crypts at many tissue levels. In no instance did we see a Y chromosome-positive cell in the epithelial compartment, which was not also CD45-positive. We saw no evidence of cell fusion, based on combined X and Y chromosome analysis. Levels of CD45-positive stromal and lymphoid cells and pericryptal myfibroblasts (positive for α-smooth muscle actin) increased with time up to a plateau, which resembled the level seen in untreated control grafted animals. We saw very few Y chromosome-positive endothelial cells in intestinal stromal vessels.

Conclusions

We conclude that whole BM transplantation does not result in intestinal epithelial engraftment in this model. Our new methods can usefully assist in multi-signal analyses of cell phenotypes following BM transplant and in models of chimaerism and regenerative medicine.