UNASSIGNED: Laminin is a major component of the basement membrane, containing multiple domains that bind integrin, collagen, nidogen, dystroglycan, and heparan sulfate. Laminin-221, expressed in skeletal and cardiac muscles, has strong affinity for the cell-surface receptor, integrin α7X2β1. The E8 domain of laminin-221, which is essential for cell integrin binding, is commercially available as a purified recombinant protein fragment. In this study, recombinant E8 fragment was used to purify primary rodent myoblasts. We established a facile and inexpensive method for primary myoblast culture exploiting the high affinity binding of integrin α7X2β1 to laminin-221.
UNASSIGNED: Total cell populations from dissociated muscle tissue were enzymatically digested and seeded onto laminin-221 E8 fragment-coated dishes. The culture medium containing non-adherent floating cells was removed after 2-hour culture at 37 °C. The adherent cells were subjected to immunofluorescence staining of desmin, differentiation experiments, and gene expression analysis.
UNASSIGNED: The cells obtained were 70.3 ± 5.49% (n = 5) desmin positive in mouse and 67.7 ± 1.65% (n = 3) in rat. Immunofluorescent staining and gene expression analyses of cultured cells showed phenotypic traits of myoblasts.
UNASSIGNED: This study reports a novel facile method for primary culture of myoblasts obtained from mouse and rat skeletal muscle by exploiting the high affinity of integrin α7X2β1 to laminin-221.

Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating in vitro has been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated morphology and exhibit gene expression profiles similar to adult PHHs, but importantly, they retain their phenotype over several passages. Further, unlike previous reports, where extensive manipulations of culture conditions are required to convert SHPCs to metabolically functional hepatocytes, SHPCs in our co-culture system maintain expression of xenobiotic metabolism-associated genes. We show that SHPCs in co-culture are able to perform xenobiotic metabolism at rates equal to their parent PHHs as evidenced by the metabolism of acetaminophen to all of its major metabolites. In summary, we present an improved co-culture system that allows generation of SHPCs from adult PHHs that maintain their differentiated phenotype over multiple passages. Our findings would be useful for expansion of limited PHHs for use in studies of drug metabolism and toxicity testing.

Monoclonal antibodies (mAbs) are widely used in many fields due to their high specificity and ability to recognize a broad range of antigens. IL-17A can induce a rapid inflammatory response both alone and synergistically with other proinflammatory cytokines. Accumulating evidence suggests that therapeutic intervention of IL-17A signaling offers an attractive treatment option for autoimmune diseases and cancer. Here, we present a combinatorial approach for optimizing the affinity and thermostability of a novel anti-hIL-17A antibody. From a large naïve phage-displayed library, we isolated the anti-IL-17A mAb 7H9 that can neutralize the effects of recombinant human IL-17A. However, the modest neutralization potency and poor thermostability limit its therapeutic applications. In vitro affinity optimization was then used to generate 8D3 by using yeast-displayed random mutagenesis libraries. This resulted in four key amino acid changes and provided an approximately 15-fold potency increase in a cell-based neutralization assay. Complementarity-determining regions (CDRs) of 8D3 were further grafted onto the stable framework of the huFv 4D5 to improve thermostability. The resulting hybrid antibody 9NT/S has superior stabilization and affinities beyond its original antibody. Human fibrosarcoma cell-based assays and in vivo analyses in mice indicated that the anti-IL-17A antibody 9NT/S efficiently inhibited the secretion of IL-17A-induced proinflammatory cytokines. Therefore, this lead anti-IL-17A mAb might be used as a potential best-in-class candidate for treating IL-17A related diseases.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Hypoxia is beneficial for the differentiation of stem cells transplanted for myocardial injury, but mechanisms underlying this benefit remain unsolved. Here, we report the impact of hypoxia-induced Jagged1 expression in cardiomyocytes (CMs) for driving the differentiation of cardiac stem cells (CSCs). Forced hypoxia-inducible factor 1α (HIF-1α) expression and physical hypoxia (5% O2) treatment could induce Jagged1 expression in neonatal rat CMs. Pharmacological inhibition of HIF-1α by YC-1 attenuated hypoxia-promoted Jagged1 expression in CMs. An ERK inhibitor (PD98059), but not inhibitors of JNK (SP600125), Notch (DAPT), NF-κB (PTDC), JAK (AG490), or STAT3 (Stattic) suppressed hypoxia-induced Jagged1 protein expression in CMs. c-Kit+ CSCs isolated from neonatal rat hearts using a magnetic-activated cell sorting method expressed GATA4, SM22α or vWF, but not Nkx2.5 and cTnI. Moreover, 87.3% of freshly isolated CSCs displayed Notch1 receptor expression. Direct co-culture of CMs with BrdU-labeled CSCs enhanced CSCs differentiation, as evidenced by an increased number of BrdU+/Nkx2.5+ cells, while intermittent hypoxia for 21 days promoted co-culture-triggered differentiation of CSCs into CM-like cells. Notably, YC-1 and DAPT attenuated hypoxia-induced differentiation. Our results suggest that hypoxia induces Jagged1 expression in CMs primarily through ERK signaling, and facilitates early cardiac lineage differentiation of CSCs in CM/CSC co-cultures via HIF-1α/Jagged1/Notch signaling.

γδ T cells, a small subset of T cell population (5-10%), forms a bridge between innate and adaptive immunity. Although the role of γδ T cells in infectious diseases and antitumor immunity is well investigated, their role in bone biology needs to be explored. Aminobisphosphonates are used as a standard treatment modality for bone related disorders and are potent activators of γδ T cells. In the present study, we have compared the effect of \"activated\" and \"freshly isolated\" γδ T cells on osteoclast generation and function. We have shown that \"activated\" (αCD3/CD28 + rhIL2 or BrHPP + rhIL2 stimulated) γδ T cells inhibit osteoclastogenesis, while \"freshly isolated\" γδ T cells enhance osteoclast generation and function. Upon stimulation with phosphoantigen (BrHPP), \"freshly isolated\" γδ T cells were also able to suppress osteoclast generation and function. Cytokine profiles of these cells revealed that, \"freshly isolated\" γδ T cells secrete higher amounts of IL6 (pro-osteoclastogenic), while \"activated\" γδ T cells secrete high IFNγ levels (anti-osteoclastogenic). Neutralization of IFNγ and IL6 reversed the \"inhibitory\" or \"stimulatory\" effect of γδ T cells on osteoclastogenesis. In conclusion, we have shown that, activation status and dynamics of IL6 and IFNγ secretion dictate pro and anti-osteoclastogenic role of γδ T cells.

OBJECTIVE: Granulocyte macrophage colony-stimulating factor (GM-CSF) treatment induces clinical response in patients with active Crohn\'s disease. To explore whether monocytes mediate GM-CSF effects in vivo, we used a mouse model of chronic colitis induced by dextran sulfate sodium (DSS).
METHODS: Murine bone marrow-derived monocytes were activated with GM-CSF in vitro, and gene expression, phenotype, and function of GM-CSF-activated monocytes (GMaM) were analyzed. Therapeutic effects of GMaM were assessed in a model of chronic colitis induced by repeated cycles of DSS. Monocytes were administered intravenously and their immunomodulatory functions were evaluated in vivo by clinical monitoring, histology, endoscopy, immunohistochemistry, and expression of inflammatory markers in the colon. The distribution of injected monocytes in the intestine was measured by in vivo imaging.
RESULTS: GMaM expressed significantly higher levels of anti-inflammatory molecules. Production of reactive oxygen species was also increased while phagocytosis and adherence were decreased. GMaM up-regulated CD39 and CD73, which allows the conversion of adenosine triphosphate into adenosine and coincided with the induction of Foxp3+ (forkhead-box-protein P3 positive) regulatory T cells (Treg) in cocultures of GMaM and naive T cells. In chronic DSS-induced colitis, adoptive transfer of GMaM led to significant clinical improvement, as demonstrated by reduced weight loss, inflammatory infiltration, ulceration, and colon shrinkage. As GMaM migrated faster and persisted longer in the inflamed intestine compared with control monocytes, their presence induced Treg generation in vivo.
CONCLUSIONS: GM-CSF leads to specific monocyte activation that modulates experimental colitis via mechanisms that include the induction of Treg. We demonstrate a possible mechanism of Treg induction through CD39 and CD73 expression on monocytes.

Glioblastoma cells produce and release high amounts of glutamate into the extracellular milieu and subsequently can trigger seizure in patients. Tumor-associated microglia/macrophages (TAMs), consisting of both parenchymal microglia and monocytes-derived macrophages (MDMs) recruited from the blood, are known to populate up to 1/3 of the glioblastoma tumor environment and exhibit an alternative, tumor-promoting and supporting phenotype. However, it is unknown how TAMs respond to the excess extracellular glutamate in the glioblastoma microenvironment. We investigated the expressions of genes related to glutamate transport and metabolism in human TAMs freshly isolated from glioblastoma resections. Quantitative real-time PCR analysis showed (i) significant increases in the expressions of GRIA2 (GluA2 or AMPA receptor 2), SLC1A2 (EAAT2), SLC1A3 (EAAT1), (ii) a near-significant decrease in the expression of SLC7A11 (cystine-glutamate antiporter xCT) and (iii) a remarkable increase in GLUL expression (glutamine synthetase) in these cells compared to adult primary human microglia. TAMs co-cultured with glioblastoma cells also exhibited a similar glutamatergic profile as freshly isolated TAMs except for a slight increase in SLC7A11 expression. We next analyzed these genes expressions in cultured human MDMs derived from peripheral blood monocytes for comparison. In contrast, MDMs co-cultured with glioblastoma cells compared to MDMs co-cultured with normal astrocytes exhibited decreased expressions in the tested genes except for GLUL. This is the first study to demonstrate transcriptional changes in glutamatergic signaling of TAMs in a glioblastoma microenvironment, and the findings here suggest that TAMs and MDMs might potentially elicit different cellular responses in the presence of excess extracellular glutamate.

A promising vaccine strategy for the treatment of cancer involves the use of vaccines incorporating tumor antigen-derived synthetic peptides that can be coordinately recognized by specific CD4+ and CD8+ T-cells. Previously, we reported that a MAGE-A6-derived peptide (MAGE-A6172-187) and its highly-immunogenic and cross-reactive homolog derived from Mycoplasma penetrans HF-2 permease (HF-2216-229) are promiscuously presented by multiple HLA-DR alleles to responder CD4+ T-cells obtained from healthy donors and melanoma patients. Here, we investigated whether these same peptides could concomitantly stimulate cross-reactive MAGE-A6-specific CD8+ T-cell responses in vitro using cells isolated from HLA-A*0201 (HLA-A2)+ healthy individuals and patients with melanoma. We now show that MAGE-A6172-187 and, even more so, HF-2216-229, induce memory CD8+ T cells that recognize HLA-A2+ MAGE-A6+ tumor target cells. The immunogenicity of these peptides was at least partially attributed to their embedded MAGE-A6176-185 and HF-2220-229 \"homologous\" sequences. The functional avidity of HF-2216-229 peptide-primed CD8+ T cells for the MAGE-A6172-187 peptide was more than 100-fold greater than that of CD8+ T cells primed with the corresponding MAGE-A6 peptide. Additionally, these 2 peptides were recognized in interferon γ (IFNγ) and granzyme B ELISPOT assays by CD8+ T-cell clones displaying variable T-cell receptor (TCR) Vβ usage. These data suggest that the immune cross-reactivity of the MAGE-A6172-187 and HF-2216-229 peptides extends to CD8+ T cells, at least in HLA-A2+ donors, and supports the potential translational utility of these epitopes in clinical vaccine formulations and for immunomonitoring of cancer patients.

Tumor associated macrophages and tumor infiltrating regulatory T cells greatly hamper host-protective antitumor responses. Therefore, we utilized a novel immunomodulator, heat-killed Mycobacterium indicus pranii (Mw), to repolarize TAM and an agonistic GITR antibody (DTA-1) to reduce intratumoral regulatory T cell frequency for generation of a host-protective antitumor response. Although, the combination of Mw and DTA-1was found to be effective against advanced stage tumors, however, Mw or DTA-1 failed to do so when administered individually. The presence of high level of regulatory T cells abrogated the only Mw induced antitumor functions, whereas only DTA-1 treatment was found to be ineffective due to its inability to induce TAM repolarization in vivo. The combination therapy was found to be effective since DTA-1 treatment reduced the frequency of regulatory T cells to such an extent where they could not attenuate Mw induced TAM repolarization in vivo. Therefore, the combination therapy involving Mw and DTA-1 may be utilized to the success of advanced stage solid tumor immunotherapies.