Clinical characteristics and outcomes of Epstein-Barr virus viral load after allogeneic hematopoietic stem cell transplantation.

Epstein-Barr virus (EBV) reactivation can occur following allogenic hematopoietic stem cell transplantation (allo-HSCT). However, the clinical characteristics and outcomes of EBV-viral load are not well known. Thus, we retrospectively analyzed the clinical features and prognostic impact of the EBV viral load in 121 allo-HSCT recipients from our hospital. EBV DNA quantification was performed in whole blood after transplantation. Patients were grouped based on whether EBV DNA quantification reached > 1000 copies/mL during follow-up (N = 50) or not (N = 71). Patients with EBV > 1000 EBV copies/mL were relatively more common in the groups with graft versus host disease (GVHD) prophylaxis including ATG, haploidentical donor type, peripheral blood as a donor source, and acute GVHD II-IV. The 20-month OS and DFS were not significantly different between patients with < 1000 EBV copies/mL and patients with > 1000 EBV copies/mL (20-month OS, 56.0% vs. 60.6%; p = 0.503, 20-month DFS, 50.0% vs. 57.7%; p = 0.179). Immunosuppressant (ISS) dose reduction was achieved after the maximum increase in EBV in 41/50 (82%) patients. Additionally, 30/50 (60%) patients achieved a 50% dose reduction or no restarting of ISS within 3 months of the maximum EBV increase. Among cases wherein EBV DNA quantification reached > 1000 copies/mL, those that achieved rapid dose reduction of ISS tended to have longer overall survival ("not reached" vs 5.4 months, p < 0.001) and disease-free survival (88.4 months vs 5.3 months, p < 0.001) than those in patients who did not. Our data highlight the importance of rapid ISS reduction in post-transplant EBV reactivation.

Clinical and pathological analysis of giant cell tumor of bone with denosumab treatment and local recurrence.

BACKGROUND: Giant cell tumor of bone (GCTB) is a primary bone tumor which comprises giant cells and two types of stromal cells. Recent studies have suggested therapeutic risks of denosumab. No previous studies have reported changes in serum TRACP-5b and SUVmax of 18F-FDG-PET/CT in recurred GCTB after denosumab treatment. Therefore, we assessed the relationship between clinical and pathological features of GCTB which recurred after denosumab treatment. METHODS: We retrospectively reviewed the medical records of 26 patients with GCTB who underwent curettage between 2010 and 2018. Fourteen patients treated with denosumab were defined as the denosumab group. We evaluated TRACP-5b and SUVmax values in the denosumab group. H&E staining and immunohistochemistry for H3.3 G34W were performed for pathological assessment. Twelve patients treated without denosumab were defined as the non-denosumab group and compared with denosumab group. RESULTS: The local recurrence rate in the denosumab group was 57.4%. The mean TRACP-5b and SUVmax values were significantly decreased after denosumab therapy (P < 0.001, 1077 ± 161 to 74 ± 9 mU/dL and 8.88 ± 0.40 to 3.79 ± 0.56, respectively). Both parameters significantly increased with local recurrence. H&E staining after denosumab treatment revealed the disappearance of giant cells and histological changes in stromal cells. Specimens of local recurrence subjected to H&E staining and immunohistochemistry for H3.3 G34W demonstrated almost identical features to those in the first biopsy. CONCLUSION: Although denosumab can prevent GCTB from osteolysis, local recurrence cannot be reduced by denosumab treatment. The clinical and pathological results were almost the same as those before denosumab treatment, suggesting that the changes of GCTB by denosumab are reversible.

Quantitative Measurements of Intercellular Adhesion Strengths between Cancer Cells with Different Malignancies Using Atomic Force Microscopy.

Intercellular adhesion strengths between two kinds of murine breast cancer cells with different malignancies were measured quantitatively using a metal cup-attached chip with atomic force microscopy (AFM). The cup-attached chip was used to approach a cell, pick it up, and then approach another cell, and the adhesion strengths were measured according to the contact time of the cells between 0 to 60 s. Separation work was used as a parameter for quantitative comparisons of the strengths. As a result, the work of a highly metastatic cancer cell (FP10SC2) was greater than a low metastatic cancer cell (4T1-LM) throughout all contact times examined. Adhesion was analyzed from a point of a view of binding kinetics of receptors on cells, and two possibilities were found: one was the number of cell adhesive receptors increased, and the other was the work to separate single molecular binding increased with increasing cancer cell malignancy. These results indicated quantitative measurements of intercellular adhesion strengths using AFM yielded information to understand the mechanism of the cancer progression from a new perspective.

Giant cell tumours of bone treated with denosumab: histological, immunohistochemical and H3F3A mutation analyses.

AIMS: Denosumab, a human monoclonal antibody directed against the receptor activator of nuclear factor-κB ligand (RANKL), is a therapeutic agent for giant cell tumour of bone (GCTB). Although some studies have reported that denosumab shrinks tumours and induces bone formation, the actual effects of RANKL suppression on GCTB remain unclear. A mutation in the H3 histone family member 3A gene (H3F3A) was recently identified as a genetic signature for GCTB. The aim of this study was to investigate the histopathological features and H3F3A mutation status of GCTBs treated with denosumab. METHODS AND RESULTS: Nine biopsy-diagnosed patients with GCTB, who underwent curettage after neoadjuvant denosumab therapy, were reviewed. Immunohistochemistry for NFATc1 (an osteoclast marker), RUNX2 (an osteoblast marker) and histone H3.3 G34W (G34W, a GCTB marker) was performed; furthermore, H3F3A mutation status was examined with direct sequencing. Before therapy, GCTBs comprised NFATc1+ and RUNX2+ cells. All cases were G34W+ and contained H3F3A mutations. After therapy, the osteoclast-like giant cells disappeared. Areas of slender spindle cell proliferation and reticular woven bone that were NFATc1- and RUNX2+ replaced the lesions in various proportions. However, all post-therapy lesions still contained many G34W+ cells and harboured H3F3A mutations. Immunofluorescence double staining revealed that RUNX2+ mononuclear cells coexpressed G34W in pre-therapy and post-therapy lesions. Two patients experienced radiologically detected local recurrence within 2 years. CONCLUSIONS: Denosumab therapy effectively decreases the number of osteoclastic cells in GCTBs. However, the neoplastic cells with H3F3A mutation survive denosumab treatment and undergo dramatic histological changes in response to this agent.

Phosphoproteome analysis of synoviocytes from patients with rheumatoid arthritis.

AIM: To explore disease-associated molecules in rheumatoid arthritis (RA), we comprehensively analyzed phosphoproteins purified from RA synoviocytes. METHOD: Synoviocytes were obtained from three patients with RA and three patients with osteoarthritis (OA). Profiles of phosphoproteins purified from the synoviocytes were compared by two-dimensional differential gel electrophoresis (2D-DIGE) between the RA and OA groups. Protein spots with significantly different phosphorylation levels were identified by mass spectrometry. Recombinant protein of annexin A4 (ANXA4), one of the identified phosphoproteins, was transfected into synoviocytes from an OA patient to mimic RA synoviocytes and humoral factor secretion was compared between rANXA4-transfected and non-transfected synoviocytes under a tumor necrosis factor-α (TNFα)-stimulated condition. RESULTS: In 2D-DIGE, 318 phosphoprotein spots were detected, of which 94 spots showed significantly different intensities between the two groups (P < 0.05). Among the 94 spots, 22 spots showed two-fold or higher intensity and one spot showed less than 1/2-fold intensity in the RA group compared to the OA group. From the 22 spots, 11 phosphoproteins were identified, which included kinases, carrier and chaperone proteins, cytoskeletal proteins, proteases and calcium-binding proteins. One of the identified calcium-binding proteins was ANXA4, an exocytosis-regulating protein. The transfected rANXA4 was found to be phosphorylated intracellularly, and secretion of chemokine (C-X-C motif) ligand 1 and interleukin-8 induced by TNFα stimulation was significantly suppressed by the transfection (P < 0.01). CONCLUSION: The phosphoprotein profile of RA synoviocytes was different from that of OA synoviocytes. This difference would reflect the different pathophysiologies of the diseases. ANXA4 may be one of therapeutic targets in RA.

Pyoderma gangrenosum with wrist joint destruction: case report.

Pyoderma gangrenosum (PG) is a rare, noninfectious, neurotrophic dermatosis. We observed a case of PG mimicking cutaneous and osteoarticular infections that presented with a prolonged ulcer on the forearm, severe wrist pain, anemia, substantial local and systemic inflammation as evaluated by serum laboratory data, and carpal osteolysis. Although PG rarely damages joints, the ulcer extended to the joint and destroyed the osteochondral tissues. Advanced ulcerative colitis, which is a most common comorbidity of PG, proved to be an underlying disease. Antibiotic and surgical treatment did not heal the ulcer, which was successfully treated with corticosteroids. This intractable ulcer is often misdiagnosed. Hence when a patient presents with an enlarged, painful, unusual skin lesion, PG should always be considered.

Arthritogenicity of annexin VII revealed by phosphoproteomics of rheumatoid synoviocytes.

OBJECTIVE: To identify novel proteins involved in the pathogenesis of rheumatoid arthritis (RA) and to characterise the identified proteins based on pathogenic and therapeutic aspects. METHODS: The authors applied differential phosphoproteomic analysis to articular synoviocytes between RA and osteoarthritis (OA) to identify proteins differently phosphorylated between RA and OA. Focusing on annexin VII (Anx7), one of the highly phosphorylated proteins in RA, the authors prepared Anx7-transgenic C57BL/6 (Anx7-Tg-B6) mice to evaluate their susceptibility to collagen-induced arthritis (CIA). In addition, the authors examined the effect of anti-Anx7 antibodies (Abs) on CIA and serum levels of cytokines in wild-type DBA/1J mice, which are known to be susceptible to CIA, and in Anx7-Tg-B6 mice. In vitro, the authors examined the effect of the Anx7 knockdown by small interfering RNA on the secretion of cytokines in rheumatoid synoviocytes and the human synovial sarcoma cell line SW982. RESULTS: The Anx7 transgene altered the CIA-resistant B6 mice to CIA-susceptible ones. The Abs treatment suppressed CIA even in the wild-type DBA/1J mice. The serum levels of cytokines including interleukin 6 (IL-6) and TNFα were not altered by the Abs treatment in vivo. On the other hand, the knockdown of Anx7 by small interfering RNA caused downregulation of IL-8 secretion in vitro. CONCLUSIONS: These results indicate that Anx7 participates in the pathogenesis of RA partly through the secretion of IL-8. The study data have demonstrated the pathogenic roles and therapeutic significance of Anx7 in RA for the first time.

Adipose-specific deletion of Src homology phosphatase 2 does not significantly alter systemic glucose homeostasis.

The SH2 domain-containing protein-tyrosine phosphatase Src homology phosphatase 2 (Shp2) has been implicated in a variety of growth factor signaling pathways, but its metabolic role in some peripheral insulin-responsive tissues remains unknown. To address the metabolic function of Shp2 in adipose tissue, we generated mice with adipose-specific Shp2 deletion using adiponectin-Cre transgenic mice. We then analyzed insulin sensitivity, glucose tolerance, and body mass in adipose-specific Shp2-deficient and control mice on regular chow and high-fat diet (HFD). Control mice on HFD exhibited increased Shp2 expression in various adipose depots compared with those on regular chow. Adiponectin-Cre mice enabled efficient and specific deletion of Shp2 in adipose tissue. However, adipose Shp2 deletion did not significantly alter body mass in mice on chow or HFD. In addition, mice with adipose Shp2 deletion exhibited comparable insulin sensitivity and glucose tolerance compared with controls. Consistent with this, basal and insulin-stimulated Erk and Akt phosphorylations were comparable in adipose tissue of Shp2-deficient and control mice. Our findings indicate that adipose-specific Shp2 deletion does not significantly alter systemic insulin sensitivity and glucose homeostasis.

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase.

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma ß cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic ß cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.

Altered glucose homeostasis in mice with liver-specific deletion of Src homology phosphatase 2.

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser(612)) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.

Comprehensive investigation of disease-specific short peptides in sera from patients with systemic sclerosis: complement C3f-des-arginine, detected predominantly in systemic sclerosis sera, enhances proliferation of vascular endothelial cells.

OBJECTIVE: To identify pathogenic and/or disease-specific short peptides in sera from patients with systemic sclerosis (SSc). METHODS: Serum samples from 40 patients with SSc, 30 patients with systemic lupus erythematosus, 21 patients with rheumatoid arthritis, 30 patients with osteoarthritis, and 26 healthy donors were tested. Short peptides with molecular weights of smaller than approximately 3 kd, purified from the sera by magnetic bead-based hydrophobic interaction chromatography 18, were detected and their amino acid sequences determined using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Effects of the identified peptides on fibroblasts and microvascular endothelial cells were tested using synthesized peptides and sera containing the peptides. RESULTS: A group of peptides with mass/charge (m/z) values of 1,865, 1,778, 1,691, 1,563, and 1,450 were detected predominantly in the SSc sera. These peptides were identified as family members of complement C3f-des-arginine (DRC3f) derived from C3b. The level of DRC3f (m/z 1,865) was related to vascular involvement in SSc and to SSc disease activity. The synthesized peptides of DRC3f and C3f, as well as the filtrated sera containing DRC3f, enhanced proliferation of microvascular endothelial cells, but not fibroblasts. Both DRC3f and C3f increased production of transforming growth factor beta1 by dermal microvascular endothelial cells. CONCLUSION: This comprehensive peptidomics analysis revealed the predominance of DRC3f in the sera of patients with SSc. Investigation of DRC3f may be a useful tool for the diagnosis and evaluation of disease activity in SSc. Moreover, its demonstrated effects on endothelial cells suggest a potential role for DRC3f in the pathophysiologic mechanisms of SSc.