Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100.

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvß3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

Inhibition of protein geranylgeranylation and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells.

Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without having a negative impact on immune reconstitution.

Primary bone lymphomas thought to be osteomyelitis urgently demand a rapid diagnosis in bone pathology.

We describe in detail three cases of primary bone lymphoma (PBL), initially treated as osteomyelitis of unknown etiology. These cases show which difficulties can occur with diagnosing this entity and highlight the urgent need for rapid diagnostic results in the field of bone pathology. Case 1: A 22-year-old man with the very rare diagnosis of precursor B-lymphoblastic lymphoma in the tibia (previously published). Case 2: A 13-year-old boy with an anaplastic large cell lymphoma of the "os pubis". With initial diagnosis pointing to multifocal osteomyelitis, histology of the left iliac crest revealed a florid/chronic osteomyelitis. During clinical regression with a new osteolytic lesion, he received treatment for chronic recurrent multifocal osteomyelitis. Case 3: A 60-year-old man with an anaplastic large cell lymphoma of the right sacrum, accompanied by a putrid, florid/chronic osteomyelitis. At first, an incisional biopsy revealed a florid/chronic osteomyelitis-only.

Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions.

BACKGROUND AIMS: Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees. METHODS: Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren-Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores. RESULTS: The expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05). CONCLUSIONS: These data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.

Immunohistological localization of BMP-2, BMP-7, and their receptors in knee joints with focal cartilage lesions.

INTRODUCTION: Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. METHODS: Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. RESULTS: BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P < 0.02), but there was no influence of these parameters on protein expression. CONCLUSIONS: BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

Deficiency of CD73/ecto-5'-nucleotidase in mice enhances acute graft-versus-host disease.

Extracellular ATP and adenosine have immunoregulatory roles during inflammation. Elevated extracellular ATP is known to exacerbate GVHD, and the pharmacologic activation of the adenosine A2A receptor is protective. However, the role of endogenous adenosine is unknown. We used gene-targeted mice and a pharmacologic inhibitor to test the role of adenosine generated by CD73/ecto-5'-nucleotidase in GVHD. In allogeneic transplants, both donor and recipient CD73 were protective, with recipient CD73 playing the dominant role. CD73 deficiency led to enhanced T-cell expansion and IFN-γ and IL-6 production, and the migratory capacity of Cd73-/- T cells in vitro was increased. However, the number of regulatory T cells and expression of costimulatory molecules on antigen-presenting cells were unchanged. A2A receptor deficiency led to increased numbers of allogeneic T cells, suggesting that signaling through the A2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GVHD. Pharmacologic blockade of CD73 also enhanced graft-versus-tumor activity. These data have clinical implications, as both the severity of GVHD and the strength of an alloimmune antitumor response could be manipulated by enhancing or blocking CD73 activity or adenosine receptor signaling depending on the clinical indication.

Pain perception in knees with circumscribed cartilage lesions is associated with intra-articular IGF-1 expression.

BACKGROUND: Circumscribed cartilage defects are considered as prearthritic lesions and lead to differential intra-articular cytokine expression. Mechanisms of associated pain development and influence of smoking behavior are not yet fully understood in humans. PURPOSE: This study aimed to reveal relations between synovial cytokine levels in knees with circumscribed cartilage defects and pain sensation. STUDY DESIGN: Descriptive laboratory study. METHODS: In a clinical trial, knee lavage fluids of 42 patients with circumscribed cartilage lesions treated by either microfracturing (n = 19) or by autologous chondrocyte implantation (n = 23) and fluids of 5 healthy control individuals were prospectively collected. Preoperative knee pain was evaluated according to frequency and strength; subjective knee function was assessed using a visual analog scale and the International Knee Documentation Committee (IKDC) score. Synovial concentrations of aggrecan, insulin-like growth factor (IGF)-I, basic fibroblast growth factor (bFGF), interleukin (IL)-1ß, bone morphogenetic protein (BMP)-2, and BMP-7 were determined by enzyme-linked immunosorbent assay. RESULTS: Pain strength showed a highly significant association with intra-articular IGF-1 levels (ρ = .48, P < .01), but no correlation with synovial concentrations of aggrecan, bFGF, IL-1ß, BMP-2, and BMP-7. Although pain strength and frequency demonstrated a statistically significant relationship, no substantial association between pain frequency and any of the examined cytokine levels was found. Intra-articular IGF-1 concentrations significantly correlated with the area of cartilage damage (ρ = .35, P < .02); the other investigated cytokines failed to show this association. Neither of the determined intra-articular mediators demonstrated statistically significant correlations with subjective knee function or IKDC score. Only intra-articular concentrations of IGF-1 and BMP-2 statistically significantly correlated with age; total protein content was negatively associated with body mass index (P < .05). In smokers, synovial expression of total protein content, IGF-1, and bFGF was significantly diminished compared to nonsmokers (P < .05). CONCLUSION: Insulin-like growth factor-I is present in knees with circumscribed cartilage lesions in a size-dependent manner. IGF-1 levels correlated with indicators of pain perception; smoking negatively influenced synovial cytokine expression related to cartilage metabolism, but pain perception was not altered.

CXCL12 mediates immunosuppression in the lymphoma microenvironment after allogeneic transplantation of hematopoietic cells.

Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen-presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)-based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12-dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT.

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

The sphingosine 1-phosphate receptor agonist FTY720 potently inhibits regulatory T cell proliferation in vitro and in vivo.

CD4(+)CD25(+) regulatory T cell (Treg) entry into secondary lymphoid organs and local expansion is critical for their immunosuppressive function. Long-term application of the sphingosine-1 phosphate receptor agonist FTY720 exerts pleiotropic anti-inflammatory effects, whereas short-term FTY720 boosts antiviral immunity. In this study, we provide evidence that FTY720 potently inhibits Treg proliferation in vitro and in vivo without affecting their viability, phenotype, or in vitro immunosuppression. In contrast, adoptively transferred Treg exposed ex vivo to FTY720 lost their protective effects in murine models of acute glomerulonephritis and acute graft-vs-host disease. On a cellular level, FTY720 inhibits IL-2-induced STAT-5 phosphorylation, paralleled by a loss of FoxP3 expression during Treg expansion in vitro. Notably, loss of in vivo immunosuppression is not due to impaired migration to or localization within secondary lymphoid organs. We could even show a selective trapping of adoptively transferred Treg in inflammatory lymph nodes by FTY720. Finally, Treg isolated from animals systemically exposed to FTY720 also exhibit a significantly impaired proliferative response upon restimulation when compared with Treg isolated from solvent-treated animals. In summary, our data suggest that sphingosine-1 phosphate receptor-mediated signals induced by FTY720 abrogate their in vivo immunosuppressive potential by blocking IL-2 induced expansion, which is indispensable for their in vivo immunosuppressive activity.

Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation.

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-gammaR-mediated endocytosis. Locally injected luc(+) DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.

Centrosome-, chromosomal-passenger- and cell-cycle-associated mRNAs are differentially regulated in the development of sporadic colorectal cancer.

Dysregulation of the centrosome complex and chromosomal segregation has been associated with aneuploid cells and aggressive solid tumours, but the relevance of this mechanism to the adenoma-carcinoma sequence of sporadic colorectal cancer (sCRC), especially tumours showing chromosomal instability (CIN), is still unknown. In a series of matching normal epithelial cells (n = 41), dysplastic cells (n = 18), and invasive carcinoma cells (n = 41) from cases with sCRC, mRNA levels of the centrosomal kinase Aurora-A/STK15 and the chromosomal passenger- and cell cycle-associated molecules Incenp, Survivin, Mad-2, and Cyclin-D1 were therefore measured with specific reference to the type of genetic instability. Compared with normal epithelium, significant up-regulation of mRNAs was already present for Aurora-A/STK15 (p = 0.0313) in dysplastic cells and for all investigated markers in invasive carcinoma. Whereas Aurora-A/STK15 mRNA levels were similarly up-regulated in dysplastic and invasive carcinoma cells (p = 0.0797), Survivin (p = 0.0046) and Cyclin-D1 (p = 0.0017) mRNA levels increased from dysplastic to invasive carcinoma cells. In carcinomas, Incenp mRNA correlated with T category (p = 0.0149), and Survivin (p = 0.0382) and Cyclin-D1 (p = 0.0185) were associated with tumour differentiation. Importantly, a significantly higher (p = 0.0419) fold-change of Aurora-A/STK15 mRNA (p = 0.0419), but not Incenp, Survivin, Mad-2 or Cyclin-D1, was observed in sCRC cases with CIN (n = 29) when compared with tumours showing microsatellite instability (MIN, n = 10). The present data are the first to show an early increase of the centrosomal kinase Aurora-A/STK15 in the adenoma-carcinoma sequence of sCRC. The regulation of this kinase differs in CIN- and MIN-type sCRCs and the pattern of changes is different from those of the cell-cycle-associated markers Survivin, Mad-2, and Cyclin-D1. This reinforces the concept of preferential dysregulation of the centrosome complex in CIN-type (aneuploid), compared with MIN-type, sporadic colorectal cancers and may influence the response to and efficiency of novel therapeutics targeting Aurora kinases.

Numerical and structural centrosome aberrations are an early and stable event in the adenoma-carcinoma sequence of colorectal carcinomas.

AIMS: Numerical and structural centrosome changes have been described for and linked with genetic instability in solid tumors. Here, we specifically address centrosome aberrations in the adenoma-carcinoma sequence of colorectal cancer by detailed evaluation of gamma-tubulin staining patterns. METHODS: Formalin-fixed and paraffin-embedded specimens (normal colonic epithelium n=21; low-grade intraepithelial neoplasia n=27, high-grade intraepithelial neoplasia n=16 and invasive adenocarcinomas n=33) were stained by an anti-gamma-tubulin antibody using standard immunofluorescence. Three-dimensional image stacks of the stainings were recorded (Zeiss LSM510 confocal microscope), followed by numerical and structural data analysis (DIAS software package) and statistical evaluation (NCSS-software). RESULTS: The mean centrosome signal per cell differed significantly (P<0.0001) between normal colonic epithelium (0.8775) and each low-grade intraepithelial neoplasia (1.787), high-grade intraepithelial neoplasia (2.259) and invasive carcinomas (2.267). Similarly, both the centrosomes' structural entropy (SE) and minimal spanning tree (MST) differed significantly (P<0.001) between normal (SE=3.956, MST=38.78) and each low- (SE=6.39, MST=26) and high-grade intraepithelial neoplasia (SE=5.75, MST=26.97) and invasive carcinoma (SE=6.86, MST=28.08). CONCLUSION: Numerical and structural centrosome dysregulation is seen as early as in low-grade dysplastic lesions of the adenoma-carcinoma sequence of colorectal carcinomas and may, as such, play an initial role in the carcinogenic process.