NK cells mediate clearance of CD8+ T cell-resistant tumors in response to STING agonists.

Several immunotherapy approaches that mobilize CD8+ T cell responses stimulate tumor rejection, and some, such as checkpoint blockade, have been approved for several cancer indications and show impressive increases in patient survival. However, tumors may evade CD8+ T cell recognition via loss of MHC molecules or because they contain few or no neoantigens. Therefore, approaches are needed to combat CD8+ T cell-resistant cancers. STING-activating cyclic dinucleotides (CDNs) are a new class of immune-stimulating agents that elicit impressive CD8+ T cell-mediated tumor rejection in preclinical tumor models and are now being tested in clinical trials. Here, we demonstrate powerful CDN-induced, natural killer (NK) cell-mediated tumor rejection in numerous tumor models, independent of CD8+ T cells. CDNs enhanced NK cell activation, cytotoxicity, and antitumor effects in part by inducing type I interferon (IFN). IFN acted in part directly on NK cells in vivo and in part indirectly via the induction of IL-15 and IL-15 receptors, which were important for CDN-induced NK activation and tumor control. After in vivo administration of CDNs, dendritic cells (DCs) up-regulated IL-15Rα in an IFN-dependent manner. Mice lacking the type I IFN receptor specifically on DCs had reduced NK cell activation and tumor control. Therapeutics that activate NK cells, such as CDNs, checkpoint inhibitors, NK cell engagers, and cytokines, may represent next-generation approaches to cancer immunotherapy.

Amelioration of estrogen-deficiency-induced obesity by Ocimum gratissimum.

Objectives: Menopausal transition in women initiates with declining estrogen levels and is followed by significant changes in their physiological characteristics. These changes often lead to medical conditions, such as obesity, which is correlated with chronic low-grade/subclinical inflammation. Ocimum gratissimum L. is a food spice or traditional herb in many countries; the plant is rich in antioxidants, which possess anti-inflammation activities and multitude of other therapeutic functions. Methods: In this study, we evaluated effects of O. gratissimum extract (OGE) in preventing obesity by using ovariectomized (OVX) animal models to mimic menopausal women. Methods: OVX rats showed increase in body weight and in adipocyte size in perigonadal adipose tissue (p <0.05) and decrease in uterus weight. By contrast, OGE (0.2 mg/ml) significantly reduced body weight gain and adipocyte in OVX rats and showed insignificant changes in uterus weight. Further investigation indicated that OGE exerted no influence on levels of dorsal fat, serum total cholesterol, and serum triacylglycerol and on serum biochemical factors, calcium, phosphorus, and glucose. Conclusion: These findings suggested that OGE dietary supplements may be useful in controlling body weight of menopausal women.

Amelioration of estrogen deficiency-induced obesity by collagen hydrolysate.

Objectives: Menopausal transition with declining estrogen levels significantly affects the physiological properties of women and consequently contributes to a series of medical conditions, including obesity. Obesity is a crucial risk factor associated with cardiovascular diseases, diabetes mellitus, and breast cancer. Increasing dietary protein content improves satiety and energy expenditure. Thus, we hypothesize that supplementing with collagen, a common dietary protein, may alleviate menopause-induced obesity. Methods: We used ovariectomized (OVX) rats to mimic a menopausal human. The body weight of OVX rats significantly increased compared with that of sham-operated rats (P<0.05), but uterus weight was decreased. Adipocyte size in perigonadal adipose tissue also increased (P<0.05). Results: By contrast, OVX rats supplemented with aqueous collagen hydrolysate (2.5 mg/mL) exhibited significant attenuation in body weight gain and adipocyte enlargement (P<0.05), but insignificant change in uterus weight. Further investigation indicated that collagen hydrolysate supplementation insignificantly affected the levels of dorsal fat, serum total cholesterol, and serum triacylglycerol. Levels of serum biochemical factors, calcium, phosphorus, and glucose were also insignificantly altered by collagen hydrolysate supplementation. Conclusion: Collagen hydrolysate supplementation reduced body weight gain and adipocyte enlargement in response to ovariectomy but slightly affected blood lipids, calcium, and glucose in both sham-operated and OVX rats. Collagen hydrolysate supplementation is beneficial in ameliorating estrogen deficiency-induced obesity and its associated risk factors.

Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis.

BACKGROUND: The up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. METHODS: In this study, we enrolled 107 women older than 45 years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. RESULTS: ERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (> 55 years), and low ERα mRNA levels in PBLs (≦ 250.39 copies/µg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. CONCLUSION: We conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. GENERAL SIGNIFICANCE: Our findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels.

Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells.

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

The metastatic tumor antigen 1-transglutaminase-2 pathway is involved in self-limitation of monosodium urate crystal-induced inflammation by upregulating TGF-ß1.

INTRODUCTION: Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied. METHODS: Immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with gouty arthritis. MSU crystal-exposed RAW264.7 mouse macrophages were analyzed for interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), transforming growth factor ß1 (TGF-ß1) and TG2 expression by RT-PCR and enzyme-linked immunosorbent assay (ELISA). TG2 small interfering (si)-RNA-mediated silencing and overexpression in RAW264.7 cells were used to evaluate the involvement of TG2 in resolving MSU crystal-induced inflammation. The role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1 si-RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model. RESULTS: TG2 expression was up-regulated in the synovium tissue and SFMCs from patients with gouty arthritis. The levels of MTA1, TG2, TGF-ß1, IL-1ß and TNF-α mRNAs were consistently increased in MSU crystal-stimulated RAW264.7 cells. si-MTA1 impaired the basal, as well as the MSU crystal-induced expression of TG2 and TGF-ß1, but increased that of IL-1ß and TNF-α. TG2 overexpression dramatically suppressed MSU crystal-induced IL-1ß and TNF-α, but significantly enhanced the TGF-ß1 production. Neutralizing TGF-ß antibodies or inhibition of the crosslinking activity of TG2 attenuated these effects. On the contrary, loss of TG2 resulted in a reduced TGF-ß, but in an increased IL-1ß and TNF-α production in MSU crystal-stimulated RAW264.7 cells and mouse embryonic fibroblasts (MEFs). MSU crystal-stimulated IL-1ß production was Janus kinase 2 (JAK2)-signaling dependent and TG2-induced TGF-ß suppressed the activity of it. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model. CONCLUSIONS: These findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-ß1 in macrophages that opposes the MSU crystal-induced JAK2-dependent pro-inflammatory cytokine formation.

Posterior instrumentation and simultaneous intertransverse approach using transforaminal cage fusion for thoracic pseudoarthrosis in ankylosing spondylitis: a case report.

BACKGROUND: Unrecognized or untreated injury in patients with ankylosing spondylitis (AS) may develop anterior column spinal pseudoarthrosis with an open wedge bone defect. The methods of surgical treatment are controversial. Combined anterior and posterior stabilizations or posterior instrumentation with osteoclasis are beneficial as shown in an existing literature review. CASE REPORT: A 36-year-old Asian man with AS sustained a motor vehicle accident 2 years before presentation. At that time, his immediate magnetic resonance imaging scan demonstrated T10-T11 bone edema and granulation tissue formation with fluid accumulation in T10-T11 disc space. He opted for conservative treatment. His back pain was then exacerbated 2 years after the accident, and he underwent three-dimensional (3D) computed tomography (CT) scan revealing a severe pseudoarthrosis with sclerotic margins across the T10 caudal end vertebra to the T11 upper end plate, with a maximal fracture gap of 15 mm. Spinal cord compression was not present. After selecting for an appropriate cage size with the aid of the preoperative 3D CT images, we used a single posterior approach to apply pedicle screws, removed pseudoarthrotic granulation tissue through an intertransverse posterior lateral approach without entering the spinal canal, and inserted a transforaminal lumbar interbody fusion (TLIF) cage with bone graft. There was radiographic evidence of spinal fusion at the 9-month follow-up, and the patient had resumed all normal daily activities. CONCLUSION: The authors found that a less invasive single posterior surgical approach using a TLIF cage and pedicle screws could be applied to AS patients with combined thoracic pseudoarthrosis and an anterior column defect. Using a TLIF cage may provide circumferential stability immediately, bone graft fusion, and sagittal plane correction simultaneously. An appropriate cage size and placement selected with preoperative 3D CT images are the keys to success.

Serum monocyte chemotactic protein-1 concentrations distinguish patients with ankylosing spondylitis from patients with mechanical low back pain.

OBJECTIVES: This study aimed to identify potential blood-derived biomarkers distinguishing patients with ankylosing spondylitis from those with mechanical low back pain. METHODS: Serum and synovial fluid samples from our cohorts were assayed by using enzyme-linked immunosorbent assay for the following inflammatory biomarkers: interleukin (IL)-1α, IL-6, IL-8, IL-17, IL-23, monocyte chemotactic protein (MCP)-1, macrophage inflammatory proteins (MIP)-1α, MIP-1ß, tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), IFN-ß, metalloproteinase (MMP-3), and bone morphogenetic protein 7 (BMP-7). RESULTS: After screening, a panel of serum and synovial fluid samples with a series of potential biomarkers, cytokines including IL-6, IL-8, MMP-3, and MCP-1 were selected for additional testing because they exhibited higher concentrations than paired serum samples in the synovial fluid. Sera obtained from 50 patients with ankylosing spondylitis and 27 patients with mechanical low back pain were measured for these biomarkers. CONCLUSIONS: The MCP-1 serum was identified as a biomarker candidate, distinguishing ankylosing spondylitis from mechanical low back pain with a sensitivity of 96% and a specificity of 83.3%.

Platelet-rich fibrin modulates the expression of extracellular signal-regulated protein kinase and osteoprotegerin in human osteoblasts.

Platelet-rich fibrin (PRF) by Choukroun's technique is produced in a totally natural manner, without using anticoagulant during blood harvest nor bovine thrombin and calcium chloride for platelet activation and fibrin polymerization. When delicately pressed between two gauzes, the PRF clot becomes a strong membrane with high potential in clinical application and tissue engineering. In this study, blood collection was carried out from healthy volunteers. Osteoblast cell line U2OS was used to evaluate the cell proliferation resulting from PRF by using colorimetric assay. Western blot was employed to evaluate the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK), receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in U2OS cells. PRF was found to increase osteoblast proliferation during 5 day incubation period (p < 0.05). PRF was found to increase ERK phosphorylation in U2OS cells (p < 0.05). OPG was significantly elevated by the stimulation with PRF (p < 0.05). However, there was no significant change in RANKL expression (p > 0.05). Taken together, PRF can stimulate osteoblasts proliferation. The activation of p-ERK and OPG expression by PRF suggests a potential role for new bone formation. The application of PRF may provide the benefit for the bone regeneration.

Shikonin induces apoptosis through reactive oxygen species/extracellular signal-regulated kinase pathway in osteosarcoma cells.

Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose- and time-dependent manner. The IC(50) at 24 h and 48 h for 143B cells was 4.55 and 2.01microM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8microM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.

Surgical versus pharmacologic treatment of intraspinal gout.

UNLABELLED: A controversy between pharmacologic and surgical treatment of intraspinal gout exists in the literature. If gout is diagnosed timely, pharmacologic therapy may avert the need of surgery. The lack of readily available synovial fluid makes the diagnosis particularly difficult. The purpose of this study was to evaluate the clinical pictures and magnetic resonance imaging features in rapid differentiations of intraspinal gout. I retrospectively evaluated lumbar intraspinal tophaceous gout without the classic radiographic punched-out lesions. Four patients (average age, 65 years) had a history of hyperuricemia with multiple tophaceous deposits in the joints or visceral organs or both. The common presentations were low back pain with or without inflammatory reaction (fever, elevated C-reactive protein level, and mild leukocytosis). The patients also presented with intermittent claudication or radiculopathy of variable duration or both. The gouty tophi yielded homogeneous and hypointense masses on T1- and T2-weighted images, with multiple hypointense speckles. The masses were located in bilateral lumbar facet joints in all patients, with additional midline extension along the ligamentum flavum in three. All patients had uneventful outcomes after surgical decompression and pharmacologic treatment. Rapid deposition of tophi may aggravate nerve compression. If neurologic deficits are found, surgical decompression can provide a satisfactory outcome. LEVEL OF EVIDENCE: Therapeutic study, Level IV. See the Guidelines for Authors for a complete description of levels of evidence.

Salmonella spondylodiscitis in patients without sickle cell disease.

The optimal treatment of salmonella spondylodiscitis is controversial. The cases of eight patients who had salmonella spondylitis without sickle cell disease were reviewed. Back pain (100%), fever (75%), and elevated C-reactive protein levels (100%) were common, but gastrointestinal symptoms were not (0%). Six patients had positive blood cultures, and the other two had positive tissue cultures. Group C1 salmonella was the most common serotype. Two patients with coexisting aortic mycotic aneurysms had immediate aneurysm resection. Three others responded favorably to appropriate antibiotics, and three required subsequent surgical reconstruction because of neurologic impairment or osseous instability. Clinical outcomes were significantly better than those of 46 previously reported patients. Salmonella spondylodiscitis usually responds favorably to appropriate antibiotics; consequently, a tissue diagnosis is important. Operative interventions are necessary only for patients with coexisting aneurysms or ongoing osseous instability. A ruptured aortic aneurysm with pseudoaneurysm may mimic a paravertebral abscess, and surgery at the site of an unsuspected aneurysm may precipitate life-threatening hemorrhage. Satisfactory results may be depend on early surgical intervention for a mycotic aneurysm and also are related to host immunity.

Surgical experience in symptomatic congenital intraspinal cysts.

Symptomatic congenital intraspinal cysts are uncommon but not rare. Since these cysts may have various manifestations, a careful differential diagnosis is needed. We retrospectively reviewed findings in 3 women and 2 men (age range, 10-50 years) with 6 symptomatic cysts of the thoracic or thoracolumbar spine. In addition, we statistically analyzed the patients' Nurick myelopathy grades before and after treatment. Pain and spastic paraparesis were the most frequent manifestations. Radiographs showed the widening of the spinal bony canal in 3 patients with extradural arachnoid cysts. All patients received limited laminectomy and appropriate surgical procedures. Pathology reports indicated neurenteric (n = 1), arachnoid (n = 4), and epidermoid (n = 1) cysts. Their functional status significantly improved by a mean of 2.6 Nurick points (p = 0.0002). Our findings confirm that these cysts have various manifestations. Congenital intraspinal cyst should be included in the differential diagnosis for patients with back pain, radiculopathy, cauda equina syndrome, Brown-Sequard syndrome, or myelopathy. The advent of MR imaging and increased knowledge about the pathogenesis of these cysts have improved the ease and accuracy of their early diagnosis; in addition, postoperative prognoses are excellent if surgery is performed early.

Spinal cord compression caused by extradural arachnoid cysts. Clinical examples and review.

Most spinal arachnoid cysts are asymptomatic and detected incidentally during magnetic resonance imaging or myelography. The etiology of intraspinal arachnoid cyst is not yet clear. We present two children with three spinal extradural arachnoid cysts and each cyst protruded from a separate dura defect. In both patients, plain radiographs demonstrated widening of the interpedicular distance, which suggested progressive widening of the spinal bony canal. Limited laminectomy was performed to remove the intraspinal cysts. Separate dura defects, the apparent predisposing factors, were also found and repaired. The patients completely recovered neurologically. Radical cyst removal and dura defect closure are the surgical intervention of choice in patients with symptomatic extradural arachnoid cyst.

Thoracic neurenteric cyst in a middle aged adult presenting with Brown-Sequard syndrome.

STUDY DESIGN: To report an unusual presentation of a thoracic neurenteric cyst. OBJECTIVES: To increase knowledge about the pathogenesis and treatment of intraspinal neurenteric cyst. SUMMARY OF BACKGROUND DATA: Intraspinal neurenteric cysts (enterogenous cysts) are very rare congenital cysts of endodermal origin. The diagnosis usually is established during the first or second decade of life. Those cysts are frequently associated with vertebral or spinal cord anomalies and dual malformation with mediastinal or abdominal cysts. METHODS: A 50-year-old man presented with 1 year of left midthoracic intercostal pain after chest compression injury. Several months before admission, he felt left lower extremity weakness with right-side numbness. Plain radiography of thoracic spine was normal while MRI showed a cystic mass at T7, T8 level ventral to the spinal cord with cord compression. The spinal cord was displaced to the posterior more to the right side, mimicking hemisection of the left side of the spinal cord. RESULTS: Thoracic laminectomy was performed and the intraspinal cyst was removed. The pathology report indicated neuroenteric cyst. The postoperative course was uneventful and the signs of myelopathy improved immediately. The patient appeared well after 2 years of follow-up. CONCLUSIONS: Intraspinal neuroenteric cyst without plain vertebral anomaly may occur after trauma in middle aged adult life with Brown-Sequard syndrome. Successful treatment requires early recognition of those cysts and their associated abnormalities.