A Novel CD135+ Subset of Mouse Monocytes with a Distinct Differentiation Pathway and Antigen-Presenting Properties.

The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host defense.

Genetic architecture underlying IgG-RF production is distinct from that of IgM-RF.

OBJECTIVE: HLA-DRB1 alleles, particularly the shared epitope (SE) alleles, are strongly associated with RA. Different genetic structures underlie the production of the various autoantibodies in RA. While extensive genetic analyses have been conducted to generate a detailed profile of ACPA, a representative autoantibody in RA, the genetic architecture underlying subfractions of RF other than IgM-RF, namely IgG-RF, known to be associated with rheumatoid vasculitis, is not well understood. METHODS: We enrolled a total of 743 RA subjects whose detailed autoantibody (IgG-RF, IgM-RF, and ACPA) data were available. We evaluated co-presence and correlations of the levels of these autoantibodies. We analysed associations between the presence or levels of the autoantibodies and HLA-DRB1 alleles for the 743 RA patients and 2008 healthy controls. RESULTS: We found both IgG-RF(+) and IgG-RF(-) RA subjects showed comparable associations with SE alleles, which was not observed for the other autoantibodies. Furthermore, there was a clear difference in SE allele associations between IgG-RF(+) and (-) subsets: the association with the IgG-RF(+) subsets was solely driven by HLA-DRB1*04:05, the most frequent SE allele in the Japanese population, while not only HLA-DRB1*04:05 but also HLA-DRB1*04:01, less frequent in the Japanese population but the most frequent SE allele in Europeans, were main drivers of the association in the IgG-RF(-) subset. We confirmed that these associations were irrespective of ACPA presence. CONCLUSION: We found a unique genetic architecture for IgG-RF(-) RA, which showed a strong association with a SE allele not frequent in the Japanese population but the most frequent SE allele in Europeans. The findings could shed light on uncovered RA pathology.

C/EBPß is required for survival of Ly6C- monocytes.

The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) is highly expressed in monocytes/macrophages. However, its roles in monopoiesis are largely unknown. Here, we investigated the roles of C/EBPß in monopoiesis. Further subdivision of monocytes revealed that Cebpb messenger RNA was highly upregulated in Ly6C- monocytes in bone marrow. Accordingly, the number of Ly6C- monocytes was significantly reduced in Cebpb-/- mice. Bone marrow chimera experiments and Mx1-Cre-mediated deletion of Cebpb revealed a cell-intrinsic and monocyte-specific requirement for C/EBPß in monopoiesis. In Cebpb-/- mice, turnover of Ly6C- monocytes was highly accelerated and apoptosis of Ly6C- monocytes was increased. Expression of Csf1r, which encodes a receptor for macrophage colony-stimulating factor, was significantly reduced in Ly6C- monocytes of Cebpb-/- mice. C/EBPß bound to positive regulatory elements of Csf1r and promoted its transcription. Collectively, these results indicate that C/EBPß is a critical factor for Ly6C- monocyte survival, at least in part through upregulation of Csf1r.

Graft-Versus-Host Disease Amelioration by Human Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Is Associated with Peripheral Preservation of Naive T Cell Populations.

A substantial proportion of patients with acute graft-versus-host disease (aGVHD) respond to cell therapy with culture-expanded human bone marrow mesenchymal stromal/stem cells (BM-MSCs). However, the mechanisms by which these cells can ameliorate aGVHD-associated complications remain to be clarified. We show here that BM-MSC-derived extracellular vesicles (EVs) recapitulated the therapeutic effects of BM-MSCs against aGVHD. Systemic infusion of human BM-MSC-derived EVs prolonged the survival of mice with aGVHD and reduced the pathologic damage in multiple GVHD-targeted organs. In EV-treated GVHD mice, CD4+ and CD8+ T cells were suppressed. Importantly, the ratio of CD62L-CD44+ to CD62L + CD44- T cells was decreased, suggesting that BM-MSC-derived EVs suppressed the functional differentiation of T cells from a naive to an effector phenotype. BM-MSC-derived EVs also preserved CD4 + CD25 + Foxp3+ regulatory T cell populations. In a culture of CD3/CD28-stimulated human peripheral blood mononuclear cells with BM-MSC-derived EVs, CD3+ T cell activation was suppressed. However, these cells were not suppressed in cultures with EVs derived from normal human dermal fibroblasts (NHDFs). NHDF-derived EVs did not ameliorate the clinical or pathological characteristics of aGVHD in mice, suggesting an immunoregulatory function unique to BM-MSC-derived EVs. Microarray analysis of microRNAs in BM-MSC-derived EVs versus NHDF-derived EVs showed upregulation of miR-125a-3p and downregulation of cell proliferative processes, as identified by Gene Ontology enrichment analysis. Collectively, our findings provide the first evidence that amelioration of aGVHD by therapeutic infusion of BM-MSC-derived EVs is associated with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of BM-MSC-derived EVs. Stem Cells 2018;36:434-445.

Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O2). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O2). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-ß/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

Combination of panobinostat with ponatinib synergistically overcomes imatinib-resistant CML cells.

The major mechanism of imatinib (IM) resistance of CML is the reactivation of ABL kinase either through BCR-ABL gene amplification or mutation. We investigated the cytotoxicity of a pan-ABL tyrosine kinase inhibitor, ponatinib, and a pan-histone deacetylase inhibitor, panobinostat, against IM-resistant CML cells in vitro. Two different IM-resistant cell lines, K562/IM-R1 and Ba/F3/T315I were evaluated in comparison with their respective, parental cell lines, K562 and Ba/F3. K562/IM-R1 overexpressed BCR-ABL due to gene amplification. Ba/F3/T315I was transfected with a BCR-ABL gene encoding T315I-mutated BCR-ABL. Ponatinib inhibited the growth of both K562/IM-R1 and Ba/F3/T315I as potently as it inhibited their parental cells with an IC50 of 2-30 nM. Panobinostat also similarly inhibited the growth of all of the cell lines with an IC50 of 40-51 nM. This was accompanied by reduced histone deacetylase activity, induced histone H3 acetylation, and an increased protein level of heat shock protein 70, which suggested disruption of heat shock protein 90 chaperone function for BCR-ABL and its degradation. Importantly, the combination of ponatinib with panobinostat showed synergistic growth inhibition and induced a higher level of apoptosis than the sum of the apoptosis induced by each agent alone in all of the cell lines. Ponatinib inhibited phosphorylation not only of BCR-ABL but also of downstream signal transducer and activator of transcription 5, protein kinase B, and ERK1/2 in both K562/IM-R1 and Ba/F3/T315I, and the addition of panobinostat to ponatinib further inhibited these phosphorylations. In conclusion, panobinostat enhanced the cytotoxicity of ponatinib towards IM-resistant CML cells including those with T315I-mutated BCR-ABL.

Two susceptibility loci to Takayasu arteritis reveal a synergistic role of the IL12B and HLA-B regions in a Japanese population.

Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor.

Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles.

Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34(+) hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation.

Genotyping of relapsing polychondritis identified novel susceptibility HLA alleles and distinct genetic characteristics from other rheumatic diseases.

OBJECTIVE: To uncover the genetic background of relapsing polychondritis (RPC), a rare autoimmune disease with unknown mechanisms characterized by systemic inflammation of the cartilage, to deepen our understanding of the pathophysiology of RPC and show its distinct genetic characteristics from other rheumatic diseases. METHODS: A total of 102 patients with RPC and 1000 healthy subjects were recruited for a two-staged genetic association study and genotyped for six HLA classical loci. Haplotype association tests were also performed. The associations of amino acid (AA) residues and positions with susceptibility to RPC were analysed. Frequencies of representative susceptibility HLA alleles to other rheumatic diseases in RPC were also analysed. RESULTS: HLA-DRB1*16:02, HLA-DQB1*05:02 and HLA-B*67:01, which are in linkage disequilibrium with each other, were associated with RPC (P = 1.9 × 10(-6), 1.4 × 10(-5) and 0.00024, respectively). AA residue at position 57 in HLA-DQB1, the most significant position in type I diabetes mellitus, showed the strongest association among AA residues. HLA-DR4, a known susceptibility allele in Germans, showed a trend of susceptibility association without significance (P = 0.067). No associations were observed between the three alleles and clinical phenotypes. Representative susceptibility HLA alleles to RA, SLE, Behçet disease and Takayasu arteritis did not show enrichment in RPC in spite of sufficient statistical power. CONCLUSIONS: HLA-DRB1*16:02, HLA-DQB1*05:02 and HLA-B*67:01, in linkage disequilibrium with each other, are associated with susceptibility to RPC Importance of HLA-class II loci in RPC susceptibility is suggested. RPC is considered a genetically distinct disease from other rheumatic diseases.

Ethics for clinical research.

In order to translate research results obtained with technical innovations into the novel therapeutic approaches expeditiously, it is mandatory that both researchers and clinicians collaborate with pharmaceutical companies. In other words, investigators, pharmaceutical companies, and patients should work together to make newly innovative drugs more patient-friendly and more effective, with fewer adverse side effects. Patient cooperation is also indispensable for carrying out investigator-initiated clinical trials providing informative evidence for the development of clinical practice guidelines. In order to achieve these goals, it is necessary that the general public allow the implementation of investigatorinitiated clinical trials. For this purpose, it is mandatory to ripen the "social soil", while at the same time building a "system" including such essentials as regulations. Enhancing the transparency of conflicts of interest is one aspect of such a "system", and it is impossible to ripen the "social soil" in the settings of scientific misconduct and fraudulent medical research or clinical trials. In order to foster the "social soil" concept, it is clearly important to cultivate firm insights and lofty ethics of individual physicians and researchers.

Targeting the canonical Wnt/ß-catenin pathway in hematological malignancies.

The canonical Wnt/ß-catenin pathway plays an important role in different developmental processes through the regulation of stem cell functions. In the activation of the canonical Wnt/ß-catenin pathway, ß-catenin protein is imported into the nucleus and activates transcription of target genes including cyclin D1 and c-myc. Aberrant activation of the Wnt/ß-catenin pathway contributes to carcinogenesis and malignant behaviors, and Wnt signaling is essential for the maintenance of cancer stem cells. The canonical Wnt/ß-catenin pathway has been investigated extensively as a target in cancer treatment and several specific inhibitors of this signaling pathway have been identified through high-throughput screening. In this review, the significance of the canonical Wnt/ß-catenin pathway in hematological carcinogenesis and screening methods for specific inhibitors are discussed.

Non-steady-state hematopoiesis regulated by the C/EBPß transcription factor.

Steady-state hematopoiesis responds to extracellular stimuli to meet changing demands and also to pathologically altered intracellular signaling. Granulocyte production increases following infection or in response to cytokine stimulation, and activation of the CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for such stress-induced granulopoiesis, whereas C/EBPα plays a critical role in maintaining steady-state granulopoiesis. Different roles of these C/EBP transcription factors in different modes of hematopoiesis are evolutionally conserved from zebrafish to humans. In addition to reactions against infections, C/EBPß is responsible for cancer-driven myelopoiesis, which promotes cancer progression, at least in part, by abrogating the immune response in the cancer microenvironment. The BCR-ABL fusion protein activates emergency-specific pathway of granulopoiesis by upregulating C/EBPß. This in turn causes chronic phase chronic myeloid leukemia, which is characterized by myeloid expansion. The C/EBPß transcription factor also plays a role in other hematological malignancies of both myeloid and lymphoid lineage origin. Thus, elucidation of the upstream and downstream networks surrounding C/EBPß will lead to the development of novel therapeutic strategies for diseases mediated by non-steady-state hematopoiesis.

Accelerated apoptosis of peripheral blood monocytes in Cebpb-deficient mice.

The CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for granulopoiesis under stress conditions. However, little is known about its roles in steady state hematopoiesis. Here, we analyzed the peripheral blood and bone marrow of Cebpb(-/-) mice at steady state by flow cytometry and unexpectedly found that the number of peripheral blood monocytes was severely reduced, while the number of bone marrow monocytes was maintained. The ability of Cebpb(-/-) bone marrow cells to give rise to macrophages/monocytes in vitro was comparable to that of wild-type bone marrow cells. Apoptosis of monocytes was enhanced in the peripheral blood, but not in the bone marrow of Cebpb(-/-) mice. These results indicate that C/EBPß is required for the survival of monocytes in peripheral blood.

Isopentenyl pyrophosphate secreted from Zoledronate-stimulated myeloma cells, activates the chemotaxis of γδT cells.

γδT cell receptor (TCR)-positive T cells, which control the innate immune system, display anti-tumor immunity as well as other non-immune-mediated anti-cancer effects. γδT cells expanded ex vivo by nitrogen-containing bisphosphonate (N-BP) treatment can kill tumor cells. N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for γδT cells. We have previously observed that as they get closer, migrating γδT cells increase in speed toward target multiple myeloma (MM) cells. In the present study, we investigated the γδT cell chemotactic factors involving using a micro total analysis system-based microfluidic cellular analysis device. The addition of supernatant from RPMI8226 MM cells treated with the N-BP zoledronic acid (ZOL) or the addition of IPP to the device induced chemotaxis of γδT cells and increased the speed of migration compared to controls. Analysis of the ZOL-treated RPMI8226 cell supernatant revealed that it contained IPP secreted in a ZOL-dose-dependent manner. These observations indicate that IPP activates the chemotaxis of γδT cells toward target MM cells treated with ZOL.

CCAAT/enhancer-binding protein ß expressed by bone marrow mesenchymal stromal cells regulates early B-cell lymphopoiesis.

The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) regulates the differentiation of a variety of cell types. Here, the role of C/EBPß expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B-cell lymphopoiesis was examined. The size of the precursor B-cell population in bone marrow was reduced in C/EBPß-knockout (KO) mice. When bone marrow cells from C/EBPß-KO mice were transplanted into lethally irradiated wild-type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B-cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPß-KO mice did not support the differentiation of WT c-Kit(+) Sca-1(+) Lineage(-) hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell-derived factor-1 by the cocultured C/EBPß-deficient BMMSCs. The ability of C/EBPß-deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPß-deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPß expressed by BMMSCs plays a crucial role in early B-cell lymphopoiesis.

Parathyroid hormone enhances hematopoietic expansion via upregulation of cadherin-11 in bone marrow mesenchymal stromal cells.

Parathyroid hormone (PTH) stimulates hematopoiesis in mouse models. The involvement of osteoblasts in this process has been well investigated; however, the effects of PTH on human hematopoiesis and bone marrow mesenchymal stromal cells (BM-MSCs) are unclear. Here, we show that BM-MSCs contribute to the hematopoiesis-stimulating effects of PTH via upregulation of cadherin-11 (CDH11). When culture-expanded human BM-MSCs were stimulated with PTH, their ability to expand cocultured CD34(+) hematopoietic progenitor cells (HPCs) was enhanced. Furthermore, when PTH-treated BM-MSCs were subcutaneously implanted into NOD/SCID mice, the induction of hematopoietic cells was enhanced. Culture-expanded human BM-MSCs expressed CDH11, and the level of CDH11 expression increased following PTH stimulation. Depletion of CDH11 expression in BM-MSCs using small interfering RNA abolished the enhancement of HPC expansion by PTH-treated BM-MSCs. In lethally irradiated mice that underwent BM transplantation, CDH11 expression in BM-MSCs was higher and survival was better in PTH-treated mice than in control mice. The number of hematopoietic cells in BM and the number of red blood cells in peripheral blood were higher in PTH-treated mice than in control mice. Our results demonstrate that PTH stimulates hematopoiesis through promoting the upregulation of CDH11 expression in BM-MSCs, at least in part. PTH treatment may be an effective strategy to enhance the ability of BM-MSCs to support hematopoiesis.

Myeloid-derived suppressor cells play crucial roles in the regulation of mouse collagen-induced arthritis.

Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. The role of MDSCs in autoimmune diseases remains controversial, and little is known about the function of MDSCs in autoimmune arthritis. In this study, we clarify that MDSCs play crucial roles in the regulation of proinflammatory immune response in a collagen-induced arthritis (CIA) mouse model. MDSCs accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited the proliferation of CD4(+) T cells and their differentiation into Th17 cells in vitro. Moreover, MDSCs inhibited the production of IFN-γ, IL-2, TNF-α, and IL-6 by CD4(+) T cells in vitro, whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced the severity of CIA in vivo, which was accompanied by a decrease in the number of CD4(+) T cells and Th17 cells in the draining lymph nodes. However, depletion of MDSCs abrogated the spontaneous improvement of CIA. In conclusion, MDSCs in CIA suppress the progression of CIA by inhibiting the proinflammatory immune response of CD4(+) T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis.

[Research ethics and conflicts of interest].

Translating research results to clinical settings requires that both researchers and clinicians work together with pharmaceutical companies. At the same time, however, it must be recognized that pharmaceutical companies are basically profit-motivated organizations. When pharmaceutical companies develop deeper doctor-patient relationships, the benefits of individual doctors and organizations inevitably conflict with the benefits or interests of patients. To promote industry-university cooperative activities, research quality and research ethics should be of major importance and we must ensure the reliability of the scientific nature and ethics of the research for the sake of the public. In other words, "confirming the quality and reliability of research", "ensuring transparency", and "accountability of research activity" are all clearly warranted. For this purpose, it is mandatory that each individual involved has the ethical sophistication and professionalism expected of doctors and researchers. Each physician and researcher must bear in mind that firm insights and the highest ethical judgments regarding the problems of conflict of interest, by themselves, are the only and the final barrier preserving the fairness and transparency of medical research.

Loss of SMAD4 from colorectal cancer cells promotes CCL15 expression to recruit CCR1+ myeloid cells and facilitate liver metastasis.

BACKGROUND & AIMS: Loss of the tumor suppressor SMAD4 correlates with progression of colorectal cancer (CRC). In mice, colon tumors that express CCL9 recruit CCR1(+) myeloid cells, which facilitate tumor invasion and metastasis by secreting matrix metalloproteinase 9. METHODS: We used human CRC cell lines to investigate the ability of SMAD4 to regulate expression of CCL15, a human ortholog of mouse CCL9. We used immunohistochemistry to compare levels of CCL15 and other proteins in 141 samples of human liver metastases. RESULTS: In human CRC cell lines, knockdown of SMAD4 increased CCL15 expression, and overexpression of SMAD4 decreased it. SMAD4 bound directly to the promoter region of the CCL15 gene to negatively regulate its expression; transforming growth factor-ß increased binding of SMAD4 to the CCL15 promoter and transcriptional repression. In livers of nude mice, SMAD4-deficient human CRC cells up-regulated CCL15 to recruit CCR1(+) cells and promote metastasis. In human tumor samples, there was a strong inverse correlation between levels of CCL15 and SMAD4; metastases that expressed CCL15 contained 3-fold more CCR1(+) cells than those without CCL15. Patients with CCL15-expressing metastases had significantly shorter times of disease-free survival than those with CCL15-negative metastases. CCR1(+) cells in the metastases expressed the myeloid cell markers CD11b and myeloperoxidase, and also matrix metalloproteinase 9. CONCLUSIONS: In human CRC cells, loss of SMAD4 leads to up-regulation of CCL15 expression. Human liver metastases that express CCL15 contain higher numbers CCR1(+) cells; patients with these metastases have shorter times of disease-free survival. Reagents designed to block CCL15 recruitment of CCR1(+) cells could prevent metastasis of CRC to liver.

Application of complement component 4d immunohistochemistry to ABO-compatible and ABO-incompatible liver transplantation.

Antibody-mediated rejection (AMR) is difficult to diagnose after ABO-compatible or ABO-identical (ABO-C) liver transplantation. To determine whether complement component 4d (C4d) immunostaining would be useful for diagnosing AMR, we compared the results of C4d immunohistochemistry for allograft biopsy samples with assays for anti-donor antibodies performed at the time of biopsy. One hundred fourteen patients with ABO-C grafts and 29 patients with ABO-incompatible (ABO-I) grafts were included. Linear C4d endothelial staining (identifiable with a 4× objective lens) or staining seen in 50% or more of the portal tracts was considered positive. Five of the 114 patients (4%) with ABO-C grafts and 15 of the 29 patients (52%) with ABO-I grafts showed C4d positivity. In the ABO-C cases, C4d positivity in late biopsy samples (≥30 days after transplantation) was associated with stage 2 or higher fibrosis (METAVIR score; P = 0.01) and with the presence of donor-specific anti-human leukocyte antigen DR antibodies (HLA-DR DSAs) with a mean fluorescence intensity > 5000 according to the Luminex single-antigen bead assay (P = 0.04). Conversely, the presence of HLA-DR DSAs was associated with the presence of stage 2 or higher fibrosis, acute cellular rejection, and C4d positivity. During the 2-year follow-up, neither C4d positivity nor HLA-DR DSAs were related to graft loss. Among ABO-I patients, C4d positivity was not associated with allograft dysfunction or fibrosis. Only 3 of the 15 C4d-positive patients (20%) showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO-I grafts, and they were the only cases associated with elevations in anti-donor A/B antibody titers. In conclusion, C4d endothelial positivity among ABO-C patients is an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common among ABO-I patients and may not be associated with allograft dysfunction if alloantibody titers are not elevated.