The BTLA-HVEM axis restricts CAR T cell efficacy in cancer.

The efficacy of T cell-based immunotherapies is limited by immunosuppressive pressures in the tumor microenvironment. Here we show a predominant role for the interaction between BTLA on effector T cells and HVEM (TNFRSF14) on immunosuppressive tumor microenvironment cells, namely regulatory T cells. High BTLA expression in chimeric antigen receptor (CAR) T cells correlated with poor clinical response to treatment. Therefore, we deleted BTLA in CAR T cells and show improved tumor control and persistence in models of lymphoma and solid malignancies. Mechanistically, BTLA inhibits CAR T cells via recruitment of tyrosine phosphatases SHP-1 and SHP-2, upon trans engagement with HVEM. BTLA knockout thus promotes CAR signaling and subsequently enhances effector function. Overall, these data indicate that the BTLA-HVEM axis is a crucial immune checkpoint in CAR T cell immunotherapy and warrants the use of strategies to overcome this barrier.

Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study.

BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.

Typhoid fever presenting with gastric ulcer bleeding.

BACKGROUND: Enteric fever is a systemic disease caused by Salmonella enterica serovar Typhi or Salmonella enterica serovar Paratyphi, characterized by high fever and abdominal pain. Most patients with enteric fever improve within a few days after antibiotic treatment. However, some patients do not recover as easily and develop fatal life-threatening complications, including intestinal hemorrhage. Lower gastrointestinal bleeding has been reported in 10% of cases. However, upper gastrointestinal bleeding has rarely been reported in patients with enteric fever. We present a case of gastric ulcer hemorrhage caused by enteric fever. CASE PRESENTATION: A 32-year-old woman, complaining of fever lasting four days and right upper quadrant pain and melena that started one day before admission, consulted our hospital. Abdominal computed tomography revealed mild hepatomegaly and gastroscopy revealed multiple active gastric ulcers with flat black hemorrhagic spots. The melena of the patient stopped on the third day. On the fifth admission day, she developed hematochezia. At that time, Salmonella enterica serovar Typhi was isolated from the blood culture. The antibiotic regimen was switched to ceftriaxone. Her hematochezia spontaneously resolved the following day. Finally, the patient was discharged on the 12th admission day without clinical symptoms. However, her fever recurred one month after discharge, and she was readmitted and Salmonella enterica serovar Typhi was confirmed again via blood culture. She was treated with ceftriaxone for one month, and was discharged without complications. CONCLUSION: Our case showed that although rare, active gastric ulcers can develop in patients with enteric fever. Therefore, upper and lower gastrointestinal bleeding should be suspected in patients with enteric fever, especially showing relapsing bacteremia.

Combination of Lacticaseibacillus paracasei BEPC22 and Lactiplantibacillus plantarum BELP53 attenuates fat accumulation and alters the metabolome and gut microbiota in mice with high-fat diet-induced obesity.

This study evaluated the effects of formulations with Lacticaseibacillus paracasei BEPC22 and Lactiplantibacillus plantarum BELP53 on adiposity, the alteration of microbiota, and the metabolome in high-fat diet-fed mice. The strains were selected based on their fat and glucose absorption inhibitory activities and potential metabolic interactions. The optimal ratio of the two strains in the probiotic formulation was determined based on their adipocyte differentiation inhibitory activities. Treatment of formulations with BEPC22 and BELP53 for 10 weeks decreased body weight gain at 6 weeks; it also decreased the food efficiency ratio, white adipose tissue volume, and adipocyte size. Moreover, it decreased the expression of the lipogenic gene Ppar-γ in the liver, while significantly increasing the expression of the fat oxidation gene Ppar-α in the white adipose tissue. Notably, treatment with a combination of the two strains significantly reduced the plasma levels of the obesity hormone leptin and altered the microbiota and metabolome. The omics data also indicated the alteration of anti-obesity microbes and metabolites such as Akkermansia and indolelactic acid, respectively. These findings suggest that treatment with a combination of BEPC22 and BELP53 exerts synergistic beneficial effects against obesity.

Does valgus femoral osteotomy improve femoral head roundness in severe Legg-Calvé-Perthes disease?

BACKGROUND: Many surgeons perform a varus femoral or Salter pelvic osteotomy in patients with Legg-Calvé-Perthes (LCP) disease. However, more severely deformed femoral heads show greater congruency in adduction rather than in abduction. Therefore, a valgus-(flexion) femoral osteotomy (VFO) seems preferable rather than a varus femoral or Salter pelvic osteotomy. QUESTIONS/PURPOSES: We evaluated whether the VFO improves (1) femoral head roundness, (2) radiographic parameters reflecting hip subluxation, and (3) function. METHODS: We treated 25 patients (25 hips; 18 lateral pillar C and seven B) in the late fragmentation stage by VFO. Seven patients had additional pelvic procedures. VFO was performed at a mean age of 9.8 years. Three hips were Stulberg Class II, 20 were Class III, and two were Class IV. The following components of femoral head roundness were calculated from preoperative MRI and final radiographs: lateral and medial head roundness (LHR and MHR); anterior and posterior head roundness (AHR and PHR); central head height; and the ratios MHR/LHR and PHR/AHR. Continuity of Shenton's line, medial gap ratio were evaluated. Function was determined with the Iowa hip score. Minimum followup was 3.1 years (mean, 6.3 years; range, 3.1-11.2 years). RESULTS: All femoral head roundness measurements improved, with greatest improvement in the lateral and anterior head. Pillar C hips showed greater relative improvement than pillar B hips. The continuity of Shenton's line improved and the mean medial gap ratio decreased. Mean Iowa hip score improved from 71 before surgery to 90 at the last followup. CONCLUSIONS: VFO appears to help the deformed femoral head in the fragmentation stage to remodel to fit the acetabulum. LEVEL OF EVIDENCE: Level III, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Disrupted-in-schizophrenia 1 (DISC1) plays essential roles in mitochondria in collaboration with Mitofilin.

Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophrenia-susceptibility gene affecting various neuronal functions. In this study, we characterized Mitofilin, a mitochondrial inner membrane protein, as a mediator of the mitochondrial function of DISC1. A fraction of DISC1 was localized to the inside of mitochondria and directly interacts with Mitofilin. A reduction in DISC1 function induced mitochondrial dysfunction, evidenced by decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP contents, and perturbed mitochondrial Ca(2+) dynamics. In addition, deficiencies in DISC1 and Mitofilin induced a reduction in mitochondrial monoamine oxidase-A activity. The mitochondrial dysfunctions evoked by the deficiency of DISC1 were partially phenocopied by an overexpression of truncated DISC1 that is associated with schizophrenia in human. DISC1 deficiencies induced the ubiquitination of Mitofilin, suggesting that DISC1 is critical for the stability of Mitofilin. Finally, the mitochondrial dysfunction induced by DISC1 deficiency was partially reversed by coexpression of Mitofilin, confirming a functional link between DISC1 and Mitofilin for the normal mitochondrial function. According to these results, we propose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin.

Construction of Rabbit Immune Antibody Libraries.

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II.

Platelet-derived growth factor (PDGF) is a potent mitogenic and migratory factor that regulates the tyrosine phosphorylation of a variety of signalling proteins via intracellular production of H2O2 (refs 1, 2-3). Mammalian 2-Cys peroxiredoxin type II (Prx II; gene symbol Prdx2) is a cellular peroxidase that eliminates endogenous H2O2 produced in response to growth factors such as PDGF and epidermal growth factor; however, its involvement in growth factor signalling is largely unknown. Here we show that Prx II is a negative regulator of PDGF signalling. Prx II deficiency results in increased production of H2O2, enhanced activation of PDGF receptor (PDGFR) and phospholipase Cgamma1, and subsequently increased cell proliferation and migration in response to PDGF. These responses are suppressed by expression of wild-type Prx II, but not an inactive mutant. Notably, Prx II is recruited to PDGFR upon PDGF stimulation, and suppresses protein tyrosine phosphatase inactivation. Prx II also leads to the suppression of PDGFR activation in primary culture and a murine restenosis model, including PDGF-dependent neointimal thickening of vascular smooth muscle cells. These results demonstrate a localized role for endogenous H2O2 in PDGF signalling, and indicate a biological function of Prx II in cardiovascular disease.

Impaired bone development and increased mesenchymal progenitor cells in calvaria of RB1-/- mice.

We have previously shown that the retinoblastoma protein (pRb) can activate expression of Runx2-dependent, bone-specific genes in cultured cells. We now show that pRb also plays a role early in osteogenesis, and that in primary RB1(-/-) calvarial cells there is an increased osteoprogenitor pool. To understand pRb's function in vivo, we generated a conditional RB1-KO mouse in which pRb expression is efficiently extinguished in osteoblasts. These animals display an apparent developmental defect in bones, most strikingly in the calvaria. Cultured RB1(-/-) calvarial osteoblasts fail to cease proliferation upon reaching confluence or following differentiation. Re-plating assays of primary RB1(-/-) calvarial cells after differentiation showed a clear adipogenic ability with increased multipotency. RB1(-/-) osteoblasts display a severe reduction in levels of mRNAs expressed late in differentiation. In this study, we present strong evidence that pRb has multiple regulatory roles in osteogenesis. Furthermore, in the absence of RB1(-/-) there is a larger pool of multipotent cells compared with the WT counterpart. This increased pool of osteoprogenitor cells may be susceptible to additional transforming events leading to osteosarcoma, and is therefore key to understanding RB1 as a target in malignancy.

MR imaging as a supplement to traditional decision-making in the treatment of LCP disease.

BACKGROUND: Heretofore, the general concept in treating Legg-Calve-Perthes (LCP) disease has been containment of the diseased femoral head into the acetabulum. However, surgery or bracing for containment of a deformed femoral head without accurate information on its dynamic relationship with the hip may aggravate hip congruity and lead to impingement between the femoral head and the acetabulum. We used magnetic resonance imaging on an outpatient clinic basis to evaluate the relationship between the deformed femoral head and the acetabulum in moderate-to-severe LCP disease, and applied these findings to management. METHODS: For 103 moderately and severely affected LCP patients (mean age 7.5 y), we made a total of 151 range of motion-magnetic resonance imagings (termed range of motion as each patient was scanned in 5 positions: neutral, abduction, abduction-internal rotation, abduction-internal rotation-flexion, and adduction). For each position, we calculated epiphyseal extrusion index (EEI), head coverage (HC), and medial gap ratio (MGR), and looked for differences between parameter values in neutral and the other positions. Disease severity was noted for each patient according to 3 classification systems (lateral pillar, Catterall, and Salter-Thompson), and differences in parameter values were examined for the various severity grades. The position of greatest congruity, and adjacent soft tissue changes, were also noted. Stulberg results were obtained for 54 patients who had reached skeletal maturity. RESULTS: For moderately affected (lateral pillar-B) patients, all 3 parameters (EEI, HC, and MGR) improved on abduction, supporting traditional containment theory. For severely affected (lateral pillar-C) patients, EEI and HC improved on abduction, but MGR did not, indicating hinge abduction by the deformed femoral head. The results do not seem to be greatly affected by 1 of the 3 classification systems which we use. In these patients, congruency was improved in adduction, and was aided by the surrounding soft tissues. Our pillar-B patients were treated conservatively and had mostly Stulberg I and II outcomes. Both conservative and operative treatment of our pillar-C patients resulted in mostly Stulberg III outcomes. CONCLUSIONS: For moderately affected patients, we support traditional treatment aimed at containment of the diseased femoral head into the acetabulum. For severely affected patients who show improved congruency in adduction, a valgus femoral osteotomy, aimed at achieving stable congruency rather than containment, may be used as a primary treatment to minimize acetabulofemoral impingement. LEVEL OF EVIDENCE: Therapeutic study, level II.

Preoperative plan for corrective osteotomy of the hip: a method using picture-editing software.

BACKGROUND: As the Picture Archiving and Communication System (PACS) has been adopted by many health centers for radiographic image storage and analysis, fewer and fewer physicians are using radiographic films and the traditional paper-drawing technique for creating preoperative surgical plans. Therefore, a new technique is required using the digital images in stored PACS. Any new method should not only be easy to perform while accurately reflecting the actual techniques of hip osteotomy, but also it should hopefully not require the purchase of expensive software. METHODS: We developed a method using widely available commercial image-editing software (Adobe Photoshop and Microsoft PowerPoint) that works with preoperative anteroposterior and lateral radiographs in stored PACS. To compare our technique with the traditional paper-drawing approach, we measured the time it took for 5 orthopaedic residents to prepare 1 surgical plan using each technique, for 6 different procedures (3 femoral osteotomy and 3 femoral plus pelvic osteotomy). RESULTS: The new method has been used in planning surgery on 133 hips. For femoral osteotomy, the average time required for the traditional and new techniques was 54 and 49 minutes, respectively; whereas for a combined femoral and pelvic osteotomy, the traditional technique took 71 minutes compared with 63 for the new approach. CONCLUSIONS: The new technique is not only cost effective and easy to learn, but also is more efficient and clearer than the conventional method using hand-made drawings, thus making the surgery itself easier to perform. LEVEL OF EVIDENCE: Level III, therapeutic study.

Bispecific Antibody Molecule Inhibits Tumor Cell Proliferation More Efficiently Than the Two-Molecule Combination.

BACKGROUND: Monoclonal antibodies (mAbs) have proved to be a valuable tool for the treatment of different cancer types. However, clinical use of an increasing number of mAbs, have also highlighted limitations with monotherapy for cancers, in particular for such with more complex mechanisms, requiring action on additional molecules or pathways, or for cancers quickly acquiring resistance following monotherapy. An example for the latter is the mAb trastuzumab, FDA approved for treatment of metastatic gastric carcinoma. To circumvent this, researchers have reported synergistic, anti-proliferative effects by combination targeting of HER2 and EGFR by trastuzumab and the EGFR-targeting mAb Cetuximab overcoming trastuzumab resistance. METHODS: Maintaining the proven functionality of trastuzumab, we have designed bi-specific antibody molecules, called AffiMabs, by fusing an EGFR-targeting Affibody molecule to trastuzumab's heavy or light chains. Having confirmed binding to EGFR and Her2 and cytotoxicity of our AffiMabs, we analyzed apoptosis rate, receptor surface levels, phosphorylation levels of receptors and associated signaling pathways as well as differentially expressed genes on transcriptome level with the aim to elucidate the mode of action of our AffiMabs. RESULTS: The AffiMabs are able to simultaneously bind HER2 and EGFR and show increased cytotoxic effect compared to the original trastuzumab therapeutic molecule and, more importantly, even to the combination of trastuzumab and EGFR-targeting Affibody molecule. Analyzing the mode of action, we could show that bi-specific AffiMabs lead to reduced surface receptor levels and a downregulation of cell cycle associated genes on transcriptome level. CONCLUSION: Our study shows that transcriptome analysis can be used to validate the choice of receptor targets and guide the design of novel multi-specific molecules. The inherent modularity of the AffiMab format renders it readily applicable to other receptor targets.

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

Construction of Rabbit Immune Antibody Libraries.

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Association of CACNA1C with bipolar disorder among the Pakistani population.

Many single nucleotide polymorphisms (SNPs) have been identified for Bipolar disorder (BD), but association between SNPs and BD can vary depending on the population tested. SNPs rs10994336 and rs9804190 in ANK3 and rs1006737 in CACNA1C have emerged as the most highly replicated SNPs significantly associated with BD. The aim of the present study was to assess the association of these SNPs with BD in the Pakistani population, which has never before been examined. A total of 120 BD and 120 control individuals from Pakistan were examined in this analysis. Genotyping results indicated that rs1006737 in CACNA1C was significantly associated with BD, while rs10994336 or rs9804190 in ANK3 was not significant when examined individually. However, risk score assessment found that the presence of two or more risk alleles was significantly associated with disease, indicating that risk alleles from ANK3 and CACNA1C may additively contribute to BD. A protein-protein interaction network was generated using STRING to probe the relationship between ANK3 and CACNA1C interactors and their associations with BD. While none of the interactors are directly linked to BD, they play a role in pathways linked to BD, including oxytocin and dopamine signaling pathways. Collectively, these results reveal a significant association of CACNA1C with BD among the Pakistani population, extending results from other ethnic groups to the Pakistani population for the first time.

Establishment of a Patient-derived Xenograft for Development of Personalized HER2-targeting Therapy in Gastric Cancer.

BACKGROUND/AIM: To maximize success rate for development of HER2-targeted therapeutics, patient-derived xenograft (PDX) models reflecting HER2-positive gastric cancer (HER2+ GC) patients were established. MATERIALS AND METHODS: GC tissues obtained from surgery of GC patients were implanted into immune-deficient mice, and tumor tissue of HER2+ PDXs were verified of the patient-mimic HER2 expression by immunohistochemistry and explored for the feasibility by testing with Herceptin, the approved therapeutics and novel HER2 antibody therapeutics being developed. RESULTS: We obtained 5 cases of HER2+ GC PDX models reflecting patient's GC tumor, consisting of 2 cases of HER2 3+ and 2 cases of HER2 2+. Novel HER2 antibody displayed significantly improved anti-cancer efficacy in combination with Herceptin. CONCLUSION: The HER2+ GC PDX models were successfully established to be utilized for preclinical evaluation of HER2-targeting drugs and combined therapies for GC treatment, as an ideal platform of personalized tools for precision therapy.

Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference.

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylation in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphorylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.

HES1 cooperates with pRb to activate RUNX2-dependent transcription.

UNLABELLED: The retinoblastoma protein, pRb, can activate the transcription factor RUNX2, an essential regulator of osteogenic differentiation, but the mechanism of this activation is unknown. Here we studied the interaction of pRb and RUNX2 with HES1, previously reported to augment RUNX2 activity. PRb can act to promote RUNX2/HES1 association with concomitant promoter occupancy and transcriptional activation in bone cells. INTRODUCTION: RUNX2 (also known as OSF2/CBFA1) is a transcription factor required for osteoblast differentiation and bone formation. We have reported that RUNX2 can associate with the retinoblastoma protein pRb, a common tumor suppressor in bone, and the resultant complex can bind and activate transcription from bone-specific promoters. This activity of the pRb/RUNX2 complex may thus link differentiation control with tumor suppressor activity. However, the mechanism through which pRb can activate RUNX2 is unknown. HES1 is a reported co-activator of RUNX2 that shares a binding site on RUNX2 with pRb. Thus, we have tested the cooperativity among these factors in activating transcription from bone specific promoters. MATERIALS AND METHODS: Coimmunoprecipitation, chromatin immunoprecipitation, and EMSA experiments were used to study the interaction of RUNX2, HES1, and pRb in cell lysates and on DNA. Transcriptional reporter assays were used to analyze the activity of RUNX2 in the presence and absence of HES1 and pRb. RESULTS: We showed that pRb can associate with HES1, a previously described RUNX2 interactor that can itself augment RUNX2-dependent transcription. The association of HES1 with RUNX2 is augmented by pRb. Furthermore, both pRb and HES1 increase the amount of RUNX2 bound to promoter sites in vivo, pRb and HES1 synergistically activate a RUNX2-dependent reporter gene, and depletion of HES1 reduces RUNX2/pRb activity. CONCLUSIONS: These data indicate that pRb acts as a RUNX2 co-activator at least in part by recruiting HES1 into the pRb/RUNX2 complex and further elucidate a novel role for pRb as a transcriptional co-activator in osteogenesis.

Alterations in the differentiation ability of mesenchymal stem cells in patients with nontraumatic osteonecrosis of the femoral head: comparative analysis according to the risk factor.

It has been suggested that decreased replication capacity of mesenchymal stem cells (MSCs) or decreased MSCs activity in the bone marrow is related to nontraumatic osteonecrosis (ON). However, little is known about differentiation ability of MSCs according to the risk factor of nontraumatic ON. We hypothesize that differentiation abnormalities in MSCs of the bone marrow of the proximal femurs might be related to nontraumatic ON of the femoral head. The purpose of this study was to investigate the osteogenic and adipogenic differentiation ability of MSCs in patients with nontraumatic ON of the femoral head. We examined the differentiation ability of MSCs in cultures derived from the bone marrow of the proximal femurs obtained from 10 patients with hip osteoarthritis (OA) and 37 patients with nontraumatic ON of the femoral head undergoing hip replacement surgery. We analyzed the osteogenic and adipogenic differentiation ability of MSCs according to the risk factor [alcohol-induced (15 patients), idiopathic (12 patients) and steroid-induced (10 patients)] of nontraumatic ON of the femoral head separately and compared it with patients with hip OA. The osteogenic activity was measured as the extracellular matrix calcification by alizarin red S staining and the alkaline phosphatase activity, and the adipogenic activity was measured as the accumulation of Oil red O-positive lipid vacuoles. The osteogenic differentiation ability of MSCs in patients with alcohol-induced and idiopathic ON was significantly reduced compared with that in patients with OA (p < 0.05 and p < 0.05, respectively). In patients with steroid-induced ON, the osteogenic differentiation ability was found to be increased, but the difference was not statistically significant. The adipogenic differentiation ability of MSCs was not significantly changed in patients with alcohol-induced, idiopathic, and steroid-induced ON compared to patients with OA. Our results indicate that altered osteogenic differentiation ability in MSCs is related to nontraumatic ON of the femoral head and the differentiation potential of MSCs in patients with nontraumatic ON differs according to its risk factor.