anti-TGFβ Antibody Research Articles

Abstract LB-331: Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide on a mouse model of breast cancer.

Abstract Elimination of CD4+FoxP3+ regulatory T cells (Tregs) has become an important strategy of cancer immunotherapy. It was reported that TGFβ is responsible for accumulation of Tregs in tumor. Thus, we treated mouse 4T1 mammary carcinoma model with 1D11 (0.1 mg/mouse), a neutralizing antibody of TGFβ(1,2,3). The treatment delayed growth of primary tumor, however, unexpectedly increased the proportion of FoxP3+ Tregs present in the intratumoral CD4 cells. In order to enhance the anti-tumor effect, 1D11 was administered with a single dose of cyclophosphamide (4 mg/mouse), a chemotherapeutic agent which was previously shown to selectively eliminate intratumoral TNFR2+ Tregs. This combination, but not the therapeutics by themselves, resulted in tumor-free long term survival of up to 80% of mice, and those tumor free mice were more resistant to re-challenge with 4T1 tumor. In order to examine the phenotype of tumor infiltrating immune cells, 4T1 tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide (2 mg/mouse). This treatment markedly inhibited tumor growth, accompanied by a massive infiltration by IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic MDSCs, and increased their expression levels of MHC II and CD80. Taken together, our data show that anti-TGFβ Ab and cyclophosphamide represents a novel chemoimmunotherapeutic combination which may prove useful in the treatment of human cancer patients.(Funded by NCI Contract HHSN261200800001E) Citation Format: Xin Chen, Yuan Yang, Qiong Zhou, O.M.Zack Howard, John McPherson, Lalage Wakefield, Joost J. Oppenheim. Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide on a mouse model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-331. doi:10.1158/1538-7445.AM2013-LB-331 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2143: Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models.

Abstract Anthracyclines (doxorubicin) are one of the most effective and widely used chemotherapeutic agent for the treatment of several cancer types, including early stage and metastatic breast cancer. However, doxorubicin has been known to cause long-term bone damage and growth defects in patients with childhood cancers, which was also confirmed by several preclinical studies. This raises concern regarding the usage of doxorubicin for the treatment of patients with metastatic breast cancer who are already at increased risk of osteolytic bone damage leading to severe bone loss due to. A number of clinical and preclinical studies have indicated that doxorubicin causes both long term and short term bone damages by lowering bone mineral density, increasing bone loss and may disrupt bone homeostasis by directly affecting bone marrow stem cells, however, the underlying mechanism is poorly understood. We have previously reported that treatment with doxorubicin increases circulating levels of TGFβ, a cytokine which has been implicated in the increase in osteoclast differentiation and survival, suppression of osteoblast differentiation and has been associated with advancement of breast cancer to bone metastasis. We hypothesize, breast cancer patients undergoing doxorubicin treatment will experience accelerated bone loss due to an increase in serum TGFβ levels and blocking TGFβ will rescue this bone loss, at least partially. We have tested the efficacy of anti-TGFβ antibody 1D11, in blocking doxorubicin-mediated bone damage in cardiac injection and orthotopic models of breast cancer to bone metastasis. X-ray image analysis revealed that doxorubicin treatment resulted in an eightfold increase in osteolytic lesion areas (P=0.002) and microCT analysis has shown almost 50% decrease in BV/TV (P=0.0005), in a preclinical murine model metastatic breast cancer. Doxorubicin treatment has also decreased BV/TV in non-tumor bearing animals, suggesting a direct damaging effect of this agent to bone at the sites which are not affected by cancer infestation. As anticipated, treatment with anti-TGFβ antibody 1D11 was able to rescue doxorubicin-mediated bone loss in all three preclinical models tested. At the cellular level, direct effect of doxorubicin treatment on the bone marrow microenvironment included decreased osteoblast survival and reduced mineralized matrix formation and increased osteoclast formation, thereby affecting both bone formation and bone resorption. Taken together, we conclude that, doxorubicin therapy decreases bone formation and increases bone resorption causing additional bone loss in patients with osteolytic bone disease and an anti-TGFβ therapy may be affective in controlling that bone loss. Citation Format: Tapasi Rana, Anwesa Chakrabarti, Michael Freeman, Swati Biswas. Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2013-2143

Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1

BackgroundChronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor β1 (TGFβ1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFβ1 may become activated in the alveolar space of the alcoholic lung.MethodsPrimary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague–Dawley rats. Expression of TGFβ1 and the epithelial integrin αvβ6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran.ResultsTGFβ1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFβ1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFβ1 antibody treatment, indicating the presence of bioactive TGFβ1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFβ1 and anti-αvβ6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFβ1 in vitro, increased the expression of αvβ6 integrin, which is known to activate TGFβ1, in alveolar epithelial cells.ConclusionsTaken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFβ1 at points of cell-cell contact.

Neutrophil cannibalism triggers transforming growth factor β1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystal–induced inflammation in mice

To identify macrophage-independent sources of transforming growth factor β1 (TGFβ1) production during monosodium urate monohydrate (MSU) crystal-induced inflammation and to determine how TGFβ1 alters MSU crystal-recruited neutrophil functions. C57BL/6J mice were injected intraperitoneally with MSU crystals with or without TGFβ1-neutralizing antibody. MSU crystal-recruited peritoneal and blood neutrophils were purified and cultured ex vivo. Peritoneal neutrophils were treated with the caspase inhibitor Q-VD-OPh, anti-TGFβ1 antibody, or fluorochrome-labeled apoptotic neutrophils. Neutrophils were analyzed for expression of annexin V, caspase 3, and TGFβ1 by flow cytometry or fluorescence microscopy, for superoxide production using the redox-sensitive dye water-soluble tetrazolium 1, and for TGFβ1 and interleukin-1β (IL-1β) production by enzyme-linked immunosorbent assay. Eighteen hours after MSU crystal administration in vivo, TGFβ1 levels were elevated in peritoneal lavage fluids, and a significant number of peritoneal neutrophils were TGFβ1+. Purified blood or peritoneal neutrophils cultured ex vivo showed TGFβ1+ neutrophils coexpressing the apoptosis marker caspase 3 and increased TGFβ1 production, both of which dropped following inhibition of apoptosis. Live neutrophils that had phagocytosed apoptotic neutrophils showed greatest TGFβ1 expression. Superoxide production by purified MSU crystal-recruited neutrophils ex vivo was enhanced by anti-TGFβ1 antibody treatment. Neutrophils purified from the peritoneum of MSU crystal-challenged mice treated with anti-TGFβ1 antibody produced elevated levels of superoxide, but neutrophil IL-1β production was unaffected. Neutrophil cannibalism and TGFβ1 production have the potential to make a significant contribution to the controlled resolution of neutrophil-driven inflammatory diseases such as gout.

Abrogation of Myeloid Derived Suppressor Cell-Like Inhibitory Activity of K562 Restores Antigen Presenting Cell Functions

AbstractAbstract 4118The human leukemia cell line K562 represents an attractive platform for creating an artificial antigen presenting cell (AAPC): it is readily expandable, does not express HLA class I and II and can be stably transduced with various genes. To generate an AAPC to expand CMV antigen-specific T cells for adoptive immunotherapy, we stably transduced K562 with HLA-A2 in combination with 4-1BB ligand, or CD64 or HLA-DR15. In preliminary experiments, irradiated K562 cells expressing HLA-A2 and 4-1BB ligand pulsed with CMV pp65 and IE-1 HLA-A2 specific peptides failed to elicit antigen-specific CD8+ T cells in HLA-A2+ peripheral blood mononuclear cells (PBMC) or isolated T cells. Since CMV peptides added directly to the PBMC readily expanded antigen-specific CD8+ T cells, we concluded that K562 AAPC inhibited the T cell response. We found that both parental K562 cells and AAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcus enterotoxin B (SEB), anti-CD3 stimulation with OKT3, and in MLR. The inhibitory effect of K562 appeared to be T cell specific since K562 cells did not suppress EBV-transformed B cells.Transwell experiments demonstrated preservation of the inhibition, suggesting that suppression was mediated by a soluble factor. MLR inhibition was not reversed by neutralizing anti-TGFβ antibody or PGE2 inhibitors. Finally, the full abrogation of the suppressive activity of K562 was achieved by a brief fixation of cells with formaldehyde at concentrations as low as 0.1%: The MLR was restored when K562 was fixed, and donor T cell response to SEB- and OKT3-loaded K562 AAPC was significantly higher when using fixed K562 cells compared to unfixed cells. Moreover, fixed pp65 and IE-1 peptide-loaded HLA A2+ AAPC expressing 4-1BB ligand induced robust (3–5 fold improved) expansion of CMV-specific T cells from all tested HLA-A2+ donors when compared with irradiated AAPC control. Thus, fixed K562 cell constructs efficiently presented antigen and stimulated T cells.Overall, we demonstrate that K562 line can serve as a source of AAPC for cell therapy approaches after abrogation of their suppressive activity using formaldehyde. However, our results also revealed a previously unappreciated feature of K562 biology, clearly indicating that these commonly used cells are potent inhibitors of peptide antigen-, superantigen-, and OKT3- driven T cell proliferation. Thus K562 line displays a myeloid-derived suppressor cell-like functionality. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemias and other tumors. Disclosures:No relevant conflicts of interest to declare.

Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti-TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation invitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis.

Dermal Melanocortin Receptor Rebound in Diffuse Systemic Sclerosis after Anti‐TGFβ1 Antibody Therapy

Disturbed transforming growth factor beta (TGFβ) signalling leads to enhanced synthesis of extracellular matrix (ECM), which is manifested as systemic sclerosis (SSc), but this may be attenuated by the melanocortin system. Here, we report of rebound reaction in the gene expression of melanocortin receptor (MCR) subtypes and of the precursor of these receptors' ligands, the pro-opio-melanocortin protein (POMC), in the acute skin lesion of diffuse systemic sclerosis (dSSc) after treatment with a recombinant human anti-TGFβ1 antibody. Biopsies, taken from the leading edge of the skin lesion, before and after treatment of a patient with recent onset dSSc, were examined. Before treatment, increased levels of TGFβ mRNA and suppressed levels of POMC mRNA and MCR subtypes MC(1-3, 5) R mRNAs were seen in the lesion, compared with healthy controls. After treatment, there was a rebound expression of POMC, MC(2, 3, 5) R mRNAs. As the melanocortin system regulates collagen and melanin production, our findings add a new understanding to the pathogenetic mechanisms involved in the acute skin lesion of dSSc, which is characterized by enhanced ECM formation and changes in skin pigmentation.

Abstract 660: Inducible Ablation Of Emilin1 In Smooth Muscle Cells Increases Blood Pressure And Myogenic Tone By Enhancing Tgf-beta/alk5 Signaling.

Emilin1 (E1), a protein of the elastic extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, precursor to the LAP/TGFβ complex, that releases the growth factor. E1 null mice are hypertensives, with an increased activation of TGFβ. As E1 is expressed in blood vessels starting from early embryonic life to adulthood, is still unknown whether the E1 null phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER T2 [a tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain ( Smmhc ) promoter. TAM administration induced Cre activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1 flox/flox mice carrying the Smmhc -CreER T2 ( Smmhc -CreER T2 E1 flox/flox ) were given TAM for 7 days, E1 completely disappeared in 10 days from start of treatment. After that, blood pressure significantly increased in Smmhc -CreER T2 E1 flox/flox as compared to E1 flox/flox (124±1 vs 106±1 mmHg, n=10), whereas no structural changes of both large and resistance arteries were detected. Interestingly, a treatment with anti-TGFβ antibodies rescued the hypertensive phenotype of Smmhc -CreER T2 E1 flox/flox mice. Then, we found that isolated resistance arteries of Smmhc -CreER T2 E1 flox/flox displayed and increased myogenic response to perfusion pressure as compared to E1 flox/flox (7±1 vs 16±2 %, n=10). Moreover, we further clarified that the increased myogenic response was rescued by resistance arteries exposure to TGFβ neutralizing antibody. Finally, we found that the inhibition of TGFβ receptor ALK5, obtained transfecting isolated vessels with an adenovirus carrying a dominant negative ALK5mutant, rescued as well the increased myogenic response of Smmhc -CreER T2 E1 flox/flox. . Our results demonstrate that E1 exerts a homeostatic control of blood pressure by regulating myogenic tone of resistance arteries, through TGFβ/ALK5 in SMCs, thus suggesting that this pathway should be carefully investigated in essential hypertension.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Repetitive mechanical stretching modulates transforming growth factor-β induced collagen synthesis and apoptosis in human patellar tendon fibroblasts

The cellular and molecular mechanisms underlying the development of tendinopathy are not clear, but inflammatory mediators produced by tendon fibroblasts in response to repetitive mechanical loading may be an important factor for this illness. In this study, we explored the effect of cyclic mechanical stretching on collagen synthesis and apoptosis of human patellar tendon fibroblasts (HPTFs). The role of a candidate inflammatory mediator, transforming growth factor-β1 (TGFβ1), which we identified in a cytokine antibody array, in collagen synthesis and apoptosis during repetitive mechanical stretching was also investigated. Our results showed that there was a significant increase in collagen type I synthesis at 4% and 8% stretch. Significantly, enhancement of apoptosis may account for the observed decrease in fibroblast numbers after 8% stretching. Furthermore, the exogenous addition of an anti-TGFβ1 antibody or gene silencing by si-TGFβ1 eliminated the increase in collagen type I production and activities of caspases during apoptosis under cyclic uniaxial stretching conditions. These results suggest that TGFβ1 may take part in the increase of cellular production of collagen type I and apoptosis during the development of tendinopathy. Furthermore, caspase 8 mediates activation of caspase 3 and poly ADP-ribose polymerase (PARP) cleavage during TGFβ1-induced apoptosis in stretching HPTFs.

TGFβ inhibition during expansion phase increases the chondrogenic re-differentiation capacity of human articular chondrocytes

Autologous chondrocyte implantation is a cell-based treatment to repair articular cartilage defects, relying on the availability of expanded (de-differentiated) chondrocytes. Unfortunately, the expansion process causes several phenotypical changes, requiring re-establishment of the native chondrogenic phenotype to sustain proper repair. Among other proteins, transforming growth factor-β (TGFβ) is known to influence the chondrogenic re-differentiation of human articular chondrocytes (HACs) and their matrix deposition. Thus we investigated the effects of TGFβ-depletion during the expansion phase. HACs were isolated from articular cartilage and expanded in the canonical serum-supplemented medium [fetal calf serum (FCS)] or in a chemically-defined (CD) medium, with or without anti-TGFβ antibody administration. The re-differentiation potential of the cells was assessed by pellet cultures, gene expression analysis and histology. Cell proliferation proceeded more rapidly in CD-medium than in FCS-medium; it was not affected by the use of anti-TGFβ antibody but was further increased by addition of exogenous TGFβ1, via increased p-Smad1/5/8. Conversely, in FCS-medium, addition of anti-TGFβ antibody decreased both proliferation and p-Smad1/5/8 level. Challenging either FCS- or CD-medium with anti-TGFβ antibody during expansion enhanced chondrogenesis in the subsequent pellet cultures. Moreover, TGFβ-depletion during expansion in CD-medium inhibited mRNA expression of hypertrophic markers, collagen type-X (COL10) and matrix metalloproteinase-13 (MMP-13). Interestingly, the TGFβ1 level detected by enzyme-linked immunosorbent sandwich assay (ELISA) during cell expansion was correlated with COL10 mRNA expression after re-differentiation. TGFβ-depletion during expansion improves the re-differentiation capacity of chondrocytes and inhibits hypertrophy. These results indicate the importance of the expansion medium composition to improve chondrogenic re-differentiation and to inhibit hypertrophy.

Immunization With WT1-derived Peptides by Tattooing Inhibits the Growth of TRAMP-C2 Prostate Tumor in Mice

The expression of the transcription factor encoded by the Wilms tumor gene 1 (WT1) is associated with a variety of human cancers. WT1 protein has been reported to serve as a target antigen for tumor-specific immune responses. We observed that the immunization of mice with peptide vaccines derived from WT1 in a mixture with the CpG adjuvant (ODN 1826) by tattoo administration was superior to subcutaneous delivery of the peptides in combination with CpG formulated with the mineral oil adjuvant or a DNA vaccine or a recombinant vaccinia virus vaccine expressing the truncated WT1 protein. Tattooing with the WT1122-140 and WT1126-134 peptide elicited the response of WT1-specific interferon-γ-producing T cells. Peptide vaccine administered with a tattoo device had an antitumor effect on the growth of the prostate tumor cell line TRAMP-C2, provided that the transforming growth factor-β produced by tumor cells was neutralized by anti-TGFβ monoclonal antibody. The treatment of the tumor-bearing mice with 5-azadeoxycytidine or poly IC did not work in synergy with the peptide vaccine.

Abstract 16: Downregulation of CYLD leads to acquisition of mesenchymal state and increased migration via ligand-independent TGFβR1 activation in human oral squamous cell carcinoma cells

Abstract The 5-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA transfection on OSCC progression using 5 OSCC cell lines (SAS, Tu4, HSC3, Ca9-22, and SCC-NA) and HaCaT keratinocytes. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with the correlation with the increased tumor size, advanced clinical stage, and poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in all the OSCC cell lines as well as HaCaT keratinocytes. Notably, such EMT-like changes were completely blocked by a TGFβR1 inhibitor in all the OSCC cell lines, but not in HaCaT keratinocytes. Furthermore, treatment with excess amount of anti-TGFβ antibody (1D11) did not inhibit the EMT-like changes induced by CYLD repression. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ligand-independent TGFβR1 activation in OSCC cells in mechanisms distinct from normal epithelium. Further studies for these mechanisms probably provide new insights into the biology including “TGFβ switch” and development of a novel therapy in OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 16. doi:1538-7445.AM2012-16

Echinacoside Stimulates Cell Proliferation and Prevents Cell Apoptosis in Intestinal Epithelial MODE-K Cells by Up-Regulation of Transforming Growth Factor-β1 Expression

Cistanche deserticola MA (C. deserticola) has been widely used as a laxative herbal in herbal medicine for the treatment of irritable bowel syndrome or constipation, and echinacoside (ECH) is one of the major bioactive ingredients in this herbal. Our aim was to investigate the effect of ECH on intestinal epithelial cell growth and death. MODE-K, an intestinal epithelial cell line, was used as an in vitro model of the intestine. Cell proliferation was measured by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was determined with Annexin-V staining. Here we showed that in cultured MODE-K cells, ECH significantly stimulated cell proliferation and enhanced cell survival by reducing cell apoptosis in the presence of H2O2 or the mixture of pro-inflammatory cytokines, while transforming growth factor (TGF)-β1 expression was up-regulated in a dose-dependent manner. Knockdown of TGF-β1 expression disrupted both the proliferative and cytoprotective activities of ECH, which was further confirmed by neutralization of TGF-β1 activity using anti–TGF-β1 antibody. These data suggest that ECH as one of bioactive ingredients in herbal C. deserticola and others may improve mucosal tissue repair by stimulating intestinal epithelial cell proliferation and preventing cell death via up-regulation of TGF-β.

Anti-Transforming Growth Factor ß Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.

Dysregulation of macrophage activation by decidual regulatory T cells in unexplained recurrent miscarriage patients

CD4(+)CD25(+) T cells (Treg cells) and macrophages play roles in the maintenance of maternal-fetal immunological tolerance. Treg cells suppress the function of macrophages via mechanisms mediated by cell-cell contact and production of soluble factors. The purpose of this study was to investigate regulation of macrophages by Treg cells within decidua from patients with unexplained recurrent miscarriage (RM) and normal control women during early pregnancy. Treg cells and macrophages were isolated from deciduas of unexplained RM (n=15) and control women (n=15) by magnetic cell separation and co-cultured for six days. Regulation of macrophages by Treg cells was assessed in the presence and absence of neutralizing anti-TGFβ antibodies and in transwell experiments. Expression of CD80, CD86, IL10, and IFNγ by macrophages was measured by flow cytometry or ELISA. Macrophage expression of CD80 and CD86 was higher in deciduas of unexplained RM patients compared with controls whereas the expression of IL10 was lower. There was no difference in the expression of IFNγ by macrophages between the two groups. Treg cells inhibited macrophage expression of CD80, CD86 and IFNγ and increased the expression of IL10. The regulatory effects of Treg cells were abrogated in the presence of neutralizing anti-TGFβ antibodies or by transwell culture. The phenotype of macrophages therefore differed in unexplained RM patients compared with normal early pregnant subjects. Macrophage regulation by Treg cells was shown to be mediated by cell-cell contact and TGFβ and this capacity was decreased in unexplained RM patients.

MT1-MMP Cooperates with KrasG12D to Promote Pancreatic Fibrosis through Increased TGF-β Signaling

Pancreatic cancer is associated with a pronounced fibrotic reaction that was recently shown to limit delivery of chemotherapy. To identify potential therapeutic targets to overcome this fibrosis, we examined the interplay between fibrosis and the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14), which is required for growth and invasion in the collagen-rich microenvironment. In this article, we show that compared with control mice (Kras(+)/MT1-MMP(-)) that express an activating Kras(G12D) mutation necessary for pancreatic cancer development, littermate mice that express both MT1-MMP and Kras(G12D) (Kras(+)/MT1-MMP(+)) developed a greater number of large, dysplastic mucin-containing papillary lesions. These lesions were associated with a significant amount of surrounding fibrosis, increased α-smooth muscle actin (+) cells in the stroma, indicative of activated myofibroblasts, and increased Smad2 phosphorylation. To further understand how MT1-MMP promotes fibrosis, we established an in vitro model to examine the effect of expressing MT1-MMP in pancreatic ductal adenocarcinoma (PDAC) cells on stellate cell collagen deposition. Conditioned media from MT1-MMP-expressing PDAC cells grown in three-dimensional collagen enhanced Smad2 nuclear translocation, promoted Smad2 phosphorylation, and increased collagen production by stellate cells. Inhibiting the activity or expression of the TGF-β type I receptor in stellate cells attenuated MT1-MMP conditioned medium-induced collagen expression by stellate cells. In addition, a function-blocking anti-TGF-β antibody also inhibited MT1-MMP conditioned medium-induced collagen expression in stellate cells. Overall, we show that the bona fide collagenase MT1-MMP paradoxically contributes to fibrosis by increasing TGF-β signaling and that targeting MT1-MMP may thus help to mitigate fibrosis.

Are non‐traditional CD4+CD69+CD25– regulatory T cells involved in disease progression of human hepatocellular carcinoma?

See article in J. Gastroenterol. Hepatol. 2011; 26: 1519–1526.

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors

AbstractCD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34+ progenitors. Transforming growth factor-β1 (TGF-β1) and anti–TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti–TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133+ progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.

Role of CD200 expression in regulation of metastasis of EMT6 tumor cells in mice

Previous studies have confirmed that levels of CD200 expression on the cells of the transplantable EMT6 mouse breast cancer line are increased markedly during growth in immunocompetent mice, unlike the persistent low levels of expression observed in NOD-SCID.IL-2(γr-/-) mice or mice with generalized over-expression of a CD200 transgene (CD200(tg) mice). Faster tumor growth occurs in both of these latter mice, with decreased evidence for a host immune reaction in lymph nodes draining the tumor (DLN). We now report evidence for a role for CD200 expression (by the host and/or tumor cells) in increased seeding of tumor cells to DLN in immunocompromised (CD200(tg) or NOD-SCID.IL-2(γr-/-)) vs immunocompetent mice, by limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN), using control and GFP-tagged EMT6 cells. Neutralization of expressed CD200 by anti-CD200mAbs decreased the tumor metastasis at the same time as increasing detection of cytotoxic anti-tumor immune cells in DLN. Infusion of either anti-CD4 to deplete T-effector cells, or anti-TGFβ antibody, increased metastasis to DLN, as did indeed the infusion of EMT6 cells selected for the loss of TGFβRII expression. It is concluded that the increased CD200 expression by breast cancer cells (and/or host tissue) may be an important variable involved in determining the risk of metastasis.