Dual Effect of Organogermanium Compound THGP on RIG-I-Mediated Viral Sensing and Viral Replication during Influenza a Virus Infection.

The interaction of viral nucleic acid with protein factors is a crucial process for initiating viral polymerase-mediated viral genome replication while activating pattern recognition receptor (PRR)-mediated innate immune responses. It has previously been reported that a hydrolysate of Ge-132, 3-(trihydroxygermyl) propanoic acid (THGP), shows a modulatory effect on microbial infections, inflammation, and immune responses. However, the detailed mechanism by which THGP can modify these processes during viral infections remained unknown. Here, we show that THGP can specifically downregulate type I interferon (IFN) production in response to stimulation with a cytosolic RNA sensor RIG-I ligand 5'-triphosphate RNA (3pRNA) but not double-stranded RNA, DNA, or lipopolysaccharide. Consistently, treatment with THGP resulted in the dose-dependent suppression of type I IFN induction upon infections with influenza virus (IAV) and vesicular stomatitis virus, which are known to be mainly sensed by RIG-I. Mechanistically, THGP directly binds to the 5'-triphosphate moiety of viral RNA and competes with RIG-I-mediated recognition. Furthermore, we found that THGP can directly counteract the replication of IAV but not EMCV (encephalitismyocarditis virus), by inhibiting the interaction of viral polymerase with RNA genome. Finally, IAV RNA levels were significantly reduced in the lung tissues of THGP-treated mice when compared with untreated mice. These results suggest a possible therapeutic implication of THGP and show direct antiviral action, together with the suppressive activity of innate inflammation.

Regulation of an adaptor protein STING by Hsp90ß to enhance innate immune responses against microbial infections.

Stimulator of interferon genes (STING) plays important roles in the DNA-mediated innate immune responses. However, the regulatory mechanism of STING in terms of stabilization is not fully understood. Here, we identified the chaperone protein Hsp90s as novel STING interacting proteins. Treatment with an Hsp90 inhibitor 17-AAG and knockdown of Hsp90ß but not Hsp90α reduced STING at protein level, resulted in the suppression of IFN induction in response to stimulation with cGAMP, and infections with HSV-1 and Listeria monocytogenes. Collectively, our results suggest that the control of STING protein by Hsp90ß is a critical biological process in the DNA sensing pathways.

Interleukin-34 expression in ovarian cancer: a possible correlation with disease progression.

Ovarian cancer is the second-most lethal gynecological malignancy and the seventh-commonest cause of cancer-related death in women around the world. Most of the ovarian cancer patients are diagnosed at advanced stages and suffer from recurrence after primary cytoreductive surgery and standard first-line chemotherapy. Thus, the successful management of ovarian cancer patients requires the identification of factors that contribute to progression and relapse. Interleukin-34 (IL-34) is a novel cytokine that acts as a tissue-specific ligand of colony-stimulating factor-1 receptor (CSF-1R). In cancer, IL-34 exerts pro-tumorigenic functions that promote tumor growth, metastasis, angiogenesis, immune suppression and therapeutic resistance. In this study, we evaluate the impact of IL-34 on progression and survival of ovarian cancer patients. First, IL-34 was found to be expressed in several human ovarian cancer cell lines and cancer tissues from patients. The expression of IL-34 was enhanced by cytotoxic chemotherapy in ovarian cancer cell lines and cancer tissues from chemotherapy-treated ovarian cancer patients. Importantly, high IL-34 expression correlated with worse progression-free survival (PFS) and overall survival in different cohorts. The assessment of PFS based on a combination between IL34 expression and other related genes such as CSF1R and CD163 helped further to reach more statistical significance compared with IL34 alone. Furthermore, in the murine ovarian cancer cell HM-1 in vivo model, it was suggested that IL-34-derived tumor cells was correlated with tumor progression and survival by modulating the immune environment. Collectively, these findings indicate a possible correlation between IL-34 expression and disease progression in ovarian cancer patients and the mouse model.

A role for IL-34 in osteolytic disease of multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that grows in multiple sites of the axial skeleton and causes debilitating osteolytic disease. Interleukin-34 (IL-34) is a newly discovered cytokine that acts as a ligand of colony-stimulating factor-1 (CSF-1) receptor and can replace CSF-1 for osteoclast differentiation. In this study, we identify IL-34 as an osteoclastogenic cytokine that accelerates osteolytic disease in MM. IL-34 was found to be expressed in the murine MM cell line MOPC315.BM, and the expression of IL-34 was enhanced by stimulation with proinflammatory cytokines or by bone marrow (BM) stromal cells. MM-cell-derived IL-34 promoted osteoclast formation from mouse BM cells in vitro. Targeting Il34 by specific small interfering RNA impaired osteoclast formation in vitro and attenuated osteolytic disease in vivo. In BM aspirates from MM patients, the expression levels of IL-34 in CD138+ populations vary among patients from high to weak to absent. MM cell-derived IL-34 promoted osteoclast formation from human CD14+ monocytes, which was reduced by a neutralizing antibody against IL-34. Taken together, this study describes for the first time the expression of IL-34 in MM cells, indicating that it may enhance osteolysis and suggesting IL-34 as a potential therapeutic target to control pathological osteoclastogenesis in MM patients.

Potential anti-lymphoma effect of M-CSFR inhibitor in adult T-cell leukemia/lymphoma.

The c-fms proto-oncogene is also known as macrophage colony stimulating factor receptor (M-CSFR) or colony-stimulating factor-1 receptor (CSF-1R), and is expressed on several types of malignant tumor cells and myeloid cells. In the present study, we found that overexpression of M-CSFR was present in adult T-cell leukemia/lymphoma (ATLL) cases. M-CSFR signaling was associated with lymphoma cell proliferation, and M-CSFR inhibition induced apoptosis in lymphoma cells. The ATLL cell line ATL-T expressed M-CSF/CSF-1 and interleukin (IL)-34, which are both M-CSFR ligands. M-CSF and IL-34 expression was seen in ATLL cases, and co-expression of these ligands was detected in 11 of 13 ATLL cases. M-CSFR inhibition suppressed programmed death-1 and -2 ligand in ATL-T cells and macrophages stimulated with conditioned medium from ATL-T cells. Thus, an M-CSFR inhibitor may be useful as additional therapy against ATLL due to direct and indirect mechanisms.

Enhanced expression of IL-34 in an inflammatory cyst of the submandibular gland: a case report.

BACKGROUND: Cysts of the salivary glands are common lesions that occur in the context of various etiologies. Although the diagnostic importance of cysts in salivary gland diseases has been well studied, molecular mechanisms that control the related pathological process remain largely unknown. IL-34 is a novel cytokine that was discovered recently as a tissue-specific ligand of colony stimulating factor-1 receptor. Since its discovery, accumulating evidence has revealed emerging roles of IL-34 in various pathological conditions and has been suggested to correlate remarkably with inflammation. In this study, we report a medical case of an inflammatory cyst within the submandibular gland, through which evaluating the possible involvement of IL-34 in salivary gland disorders. CASE PRESENTATION: A 37-year-old male patient suffered from a sudden swelling in the right submandibular region, started initially small and had gradually increased in size to reach 3-4 cm in 1 week, accompanied by pain and local fever. Ultrasonography and MRI imaging revealed the existence of a well-defined cystic lesion with sharp borders measuring 39.8 mm × 19.7 mm within the right submandibular gland. The cyst was removed surgically, and the diagnostic decision was determined based on histopathological observations as an inflammatory cyst in the submandibular gland. Sections were generated from different regions of the surgically resected inflammatory cyst and used to examine IL-34 expression by immunohistochemistry compared to normal salivary gland tissues. Immunohistochemical staining showed enhanced expression of IL-34 in the ductal epithelial cells and endothelial cells of blood vessels, with a tendency to be accompanied with high infiltration of immune cells, which suggests a possible involvement of IL-34 in the pathogenesis of salivary gland inflammation. CONCLUSIONS: In this report, we introduce interesting findings of enhanced IL-34 expression in a case of an inflamed submandibular gland. Our findings emphasize the pathological roles of IL-34 as an inflammation amplifier and angiogenic enhancer in inflammatory conditions, such as in salivary gland disorders.

Enhanced IL-34 expression in Nivolumab-resistant metastatic melanoma.

BACKGROUND: Immunotherapies that target immune-checkpoint molecules such PD-1 have helped to achieve durable responses in melanoma treatment. However, 25% of melanoma patients who showed objective responses to PD-1 blockade develop resistance and suffer from disease progression and ultimately death, which necessitates the identification of related resistance mechanisms.IL-34 is a cytokine that controls the biology of myeloid cell lineage through binding to CSF-1R. IL-34 is importantly involved in the pathogenesis of various diseases. In cancer, the expression of IL-34 has been suggested to associate with tumor growth, metastasis, angiogenesis, and therapeutic resistance such as in lung cancers and malignant pleural mesotheliomas. In this study, we evaluate the possible involvement of IL-34 in immunotherapeutic resistance. CASE PRESENTATION: Melanoma resection species were obtained from a patient who developed a refractory melanoma against immunotherapy with Nivolumab, and stained with anti-IL-34, anti-melanoma antigens and anti-CD163 antibody. Staining of these markers was compared between primary or metastatic refractory melanoma tissues. Immunohistochemistry staining of melanoma tissues showed an enhanced expression of IL-34 in metastatic refractory melanoma compared to primary melanoma tissues, which correlates with increased frequencies of CD163+ macrophages. CONCLUSION: We introduce for the first time a clinical case of a patient with metastatic refractory melanoma that acquired resistance to anti-PD-1 immunotherapy, showing an enhanced expression of IL-34 in refractory melanoma tissues.

High co-expression of IL-34 and M-CSF correlates with tumor progression and poor survival in lung cancers.

Despite recent advances in diagnosis and treatment of lung cancers, the 5-year survival rate remains unsatisfactory, which necessitates the identification of novel factors that associates with disease progression and malignant degree for improving diagnostic and therapeutic strategies. Recent progress in cancer immunology research has unveiled critical roles for colony stimulating factor 1 receptor (CSF1R) in multiple aspects of the tumor microenvironment. CSF1R is expressed on tumor-associated macrophages (TAMs), and mediates important pro-tumorigenic functions. CSF1R also provides critical autocrine signals that promote cancer cell survival and proliferation. Activation of CSF1R can be achieved by two independent ligands; macrophage colony-stimulating factor (M-CSF) and interleukin 34 (IL-34). Accordingly, the expression of these ligands in cancer is expected to result in poor prognosis. In this study, we show that IL-34 and M-CSF expression correlates with poor survival in a cohort of lung cancer patients. Importantly, high co-expression of IL-34 and M-CSF associates with the poorest survival compared to cancers that show weak or absent expression of the two ligands. Furthermore, high expression of IL-34 and M-CSF associates with advanced stages of lung cancers. Together, these results indicate a correlation between IL-34/M-CSF expression with poor survival and disease progression in lung cancer patients.

Imipramine ameliorates pain-related negative emotion via induction of brain-derived neurotrophic factor.

Depression-like behavior is often complicated by chronic pain. Antidepressants including imipramine (IMI) are widely used to treat chronic pain, but the mechanisms are not fully understood. Brain-derived neurotrophic factor (BDNF) is a neuromodulator that reduces depression by regulating synaptic transmission. We aimed to characterize the antidepressant effects of IMI without analgesia based on BDNF (trkB)-mediated signaling and gene expression in chronic pain. A chronic constriction injury (CCI) model was constructed in Sprague-Dawley (SD) rats. IMI (5 mg/kg, i.p.) was administered from day 10 after CCI. The pain response was assessed using the paw withdrawal latency (PWL) and depression was judged from the immobility time in a forced swim test. Anti-BDNF antibody, K252a, or 5,7-dihydroxytryptamine (5,7-DHT) were used to examine the antidepressant effects of imipramine. Changes in pERK1/2 (immunohistochemistry), 5-HT and BDNF (ELISA), and BDNF mRNA (RT-PCR) were measured in the anterior cingulate cortex (ACC), rostral ventromedial medulla (RVM), and spinal cord. After CCI, rats showed decreased PWL and increased immobility time. A low dose of IMI reduced the immobility time without having analgesic effects. This antidepressant effect was reversed by anti-BDNF antibody, K252a, and 5,7-DHT. IMI reduced excessive activation of pERK1/2 associated with decreased pCREB and BDNF mRNA, and these changes were reversed by 5,7-DHT. These results show that IMI reduces pain-related negative emotion without influencing pain and that this effect is diminished by denervation of 5-HT neurons and by anti-BDNF treatment. IMI also normalizes derangement of ERK/CREB coupling, which leads to induction of BDNF. This suggests a possible interaction between 5-HT and BDNF.

4-Methylcatechol prevents derangements of brain-derived neurotrophic factor and TrkB-related signaling in anterior cingulate cortex in chronic pain with depression-like behavior.

Chronic pain with mood disorder, resulting from a peripheral nerve injury, is a serious clinical problem affecting the quality of life. A lack of brain-derived neurotrophic factor (BDNF) and abnormal intercellular signaling in the brain can mediate this symptom. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but little is known about its role in pain-emotion. Thus, we characterized the actions of 4-MC on TrkB receptor-related pERK and BDNF mRNA in discreet brain regions related to pain-emotion after chronic pain in rat. Rats implanted with a stainless steel cannula into the lateral ventricular were subjected to chronic constriction injury (CCI). Pain was assessed by changes in paw withdrawal latency (PWL) to heat stimuli after CCI. Immobility time during the forced swimming testing was measured for depression-like behavior. Analgesic and antidepression modulations with 4-MC were examined by an anti-BDNF antibody (K252a, a TrkB receptor inhibitor). The animals were perfused and fixed (4% paraformaldehyde) for immunohistochemistry analysis (c-FOS/pERK). BDNF mRNA expression (anterior cingulate cortex) was determined using reverse transcription-PCR. Rats showed a sustained decrease in PWL, associated with a prolonged immobility time after CCI. 4-MC reduced decreases in PWL and increased immobility time. 4-MC reduced increases in pERK immunoreactivity and decreases in BDNF mRNA expression in regions related to pain and the limbic system. Anti-BDNF blocked effects induced by 4-MC. We suggest that a lack of BDNF associated with activated extracellular signal-regulated kinase in the pain-emotion network may be involved in depression-like behavior during chronic pain. 4-MC ameliorates pain-emotion symptoms by inducing BDNF and normalizing pERK activities.

Free arterialised flow-through venous flap with venous anastomosis as the outflow (A-A-V flap) for reconstruction after severe finger injuries.

Abstract For reconstruction of volar defects, an arterialised flow-through venous flap (A-A flap) can be used to restore the soft tissues and the digital artery at the same time. However, there have been reports that the circulation of this flap is inadequate. This study used a venous flap with only one venous anastomosis for the outflow of an A-A flap (A-A-V flap) to solve this problem. Six patients with defects of the finger soft tissues and digital artery after severe finger injuries were performed. The venous flap with a Y-shaped vein was harvested. The digital artery was reconstructed, after which the other proximal vein of this flap was anastomosed to the dorsal subcutaneous vein. The flap survived in all patients and histological examination of flap tissue showed a nearly normal architecture. This study describes the good results obtained with an A-A-V flap, and discusses the utility of our flap in comparison with previously reported venous flaps.

Activation of different signals identified with glia cells contribute to the progression of hyperalgesia.

Hyperalgesia results from a decreased pain threshold, often subsequent to peripheral tissue damage. Recent reports revealed several promising mechanisms of hyperalgesia, but many issues remain unclear. The glial activation accompanying inflammation of neurotransmission in the spinal cord might be related to the initiation and maintenance of hyperalgesia. The present study investigated the pharmacological pain-modifying effects of mitogen-associated protein kinase (MAPK)-related inhibitors identified with glia cells over time during inflammatory pain. A model of inflammatory pain was produced by injecting mustard oil (MO) into the hind paws of rats. Following MO injection, the changes in paws flinching as the early onset of pain and paw withdrawal latency (PWL) in response to thermal stimulation were measured as delayed-onset hyperalgesia. Before and after the MO injection, one of the inhibitors, a p38-MAPK (SB), nuclear factor (NF)-κB (PDTC), BDNF-trk-B (K252a), or JNK-1 (SP), was administered and flinching and PWL were measured. In the SB, PDTC, and k252a groups, early flinching following MO injection was moderately suppressed. Hyperalgesia was significantly suppressed in the left-right difference of PWL in animals receiving SB, k252a, or PDTC pre-treatment. In animals receiving post-treatment, the suppressive effects were most potent in the SP group. The present results revealed that microglial activation resulting from the release of the phosphatase p38-MAPK, the transcription factor NF-κB, and BDNF contributes to the early stage of inflammatory pain. Astrocyte activation accompanying JNK activation contributes to subsequent hyperalgesia. Activation of different signals identified with glia cells is thought to contribute to the progression of hyperalgesia, which represents an applicable finding for the treatment of hyperalgesia.

Intracerebroventricular 4-methylcatechol (4-MC) ameliorates chronic pain associated with depression-like behavior via induction of brain-derived neurotrophic factor (BDNF).

Neuropathic pain concurrent with mood disorder from peripheral nerve injury is a serious clinical problem that significantly affects quality of life. Recent studies have suggested that a lack of brain-derived neurotrophic factor (BDNF) in the limbic system may cause this pain-emotion. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but the role of 4-MC-induced BDNF in pain-emotion is poorly understood. Thus, we assessed the possible involvement of BDNF in brain in depression-like behavior during chronic pain following peripheral nerve injury. In addition, we examined whether intracerebroventricular (i.c.v.) 4-MC prevents chronic pain in rats and produces an antidepressant effect. Sprague-Dawley rats implanted intracerebroventricularly with a PE-10 tube were subjected to chronic constriction injury (CCI). Pain was assessed by a reduction in paw withdrawal latency (PWL) to heat stimuli after CCI. We also used a forced swimming testing (FST; time of immobility, in seconds) from day 14 to day 21 after CCI. Modulation of pain and emotional behavior was performed by injection of PD0325901 (a MEK1/2 inhibitor). 4-MC (100 nM) was continuously administered i.c.v. for 3 days during the period from day 14 to day 21 after CCI. To block analgesic and antidepressant effects, anti-BDNF antibody or K252a (a TrkB receptor inhibitor) was injected in combination with 4-MC. Naloxone was also coadministered to confirm the analgesic effect of 4-MC. During the chronic stage after CCI, the rats showed a sustained decrease in PWL (thermal hyperalgesia) associated with extension of the time of immobility (depression-like behavior). PD0325901 significantly reduced the decrease in PWL and the increased time of immobility after CCI. The decreased PWL and increased time of immobility were also reduced by 4-MC and by treatment with an ERK1/2 inhibitor. These effects of 4-MC i.c.v. were reversed by anti-BDNF and K252a. The analgesic effect of 4-MC i.c.v. was also antagonized by naloxone. Based on these results, we suggest that a lack of BDNF and activation of ERK1/2 in the pain-emotion network in the CNS may be involved in depression-like behavior during chronic pain. 4-MC i.c.v. ameliorates chronic pain and depression-like behavior by producing of BDNF and normalization of ERK1/2 activation. Therefore, enhancement of BDNF may be a new treatment strategy for chronic pain associated with depression.

Cutaneous magnetic stimulation reduces rat chronic pain via activation of the supra-spinal descending pathway.

Recent studies have demonstrated that magnetic stimulation (MS) can induce cellular responses such as Ca(2+) influx into the cultured neurons and glia, leading to increased intracellular phosphorylation. We have demonstrated previously that MS reduces rat neuropathic pain associated with the prevention of neuronal degeneration. Thus, we aimed to elucidate the actions of MS in relation to modulation of spinal neuron-glia and the descending inhibitory system in chronic pain. The male SD rats intrathecally implanted with catheters were subjected to sciatic nerve ligation (CCI). MS is a low power apparatus characterized by two different frequencies, 2 KHz and 83 MHz. Rats were given MS to the skin (injured sciatic nerve) for 10 min from the seventh day after CCI. The paw withdrawal latency (PWL) evoked by thermal stimuli was measured for 14 days after CCI. Immunohistochemistry for Iba-1 or GFAP was performed after 4% paraformaldehyde fixation (microscopic analysis). We employed microdialysis for measuring CSF 5-HIAA as a reflection of 5-HT release by MS stimulation. Following CCI, rats showed a decrease in PWL after CCI, and the decrease continued until the 14th day. With MS treatment, the decrease in PWL was reduced during the 10-14 day after CCI. Injection of JNK-1 inhibitors on the 14th day antagonized the analgesic effect of MS. MS also eliminated the CCI-induced decrease in GFAP immunoreactivity. Moreover, MS evoked spinal 5-HT release reflected by increase in spinal 5-HIAA level. Thus, we demonstrate that a novel magnetic stimulator used cutaneously can ameliorate chronic pain by not only preventing abnormal spinal neuron-glia interaction, but also through the activation of the supra-spinal descending inhibitory system.

Importance of the reconstruction of the extensor retinaculum after injury.

We reconstructed an extensive soft tissue defect of the dorsum of the left wrist after an injury using a free anterolateral thigh flap and a tendon graft, and intending to substitute the extensor retinaculum for the fascia in this flap. As a result, maximum function of the extension mechanism was restored to the reconstructed hand.

Distraction arthrolysis using an external fixator and flexor tenolysis for proximal interphalangeal joint extension contracture after severe crush injury.

PURPOSE: To introduce a technique for distraction arthrolysis with an external fixator followed by flexor tendon tenolysis for extension contracture of the proximal interphalangeal (PIP) joint after severe crush injury. We also assessed the results of this method in all patients treated. METHODS: Five fingers of 4 men with extension contracture of the PIP joint after severe injury underwent distraction arthrolysis using an external fixator, followed by flexor tenolysis. On the day of attaching the external fixator, moderate distraction was applied to the joint and the gap was widened to approximately 2 mm. From the following day onward, the PIP joint was gradually widened for 10 days until a gap of about 5 mm was attained. After sustaining this amount of distraction for 3 or 4 days, the fixator was removed. Passive range of motion was performed for about one week until swelling of the affected digit subsided. Then, flexor tenolysis was performed. Patients were follow-up for an average of 31 months after surgery. RESULTS: After tenolysis, the average improvement of active range of motion was 20 degrees, average gain of active flexion was 41 degrees, and average loss of active extension was 21 degrees. The average range of active motion was from 6 degrees to 38 degrees preoperatively, and from 27 degrees to 79 degrees postoperatively. The average median of active motion was 22 degrees preoperatively, and 52 degrees postoperatively. In all fingers, there was no significant difference in the total arc of active motion preoperatively and postoperatively, but there was a significant difference between preoperative and postoperative maximum active flexion. In all patients, painless motion was maintained and arthritic changes of the PIP joint did not worsen during the follow-up period. CONCLUSIONS: Distraction arthrolysis with an external fixator followed by flexor tenolysis was a useful treatment for our patients with extension contracture of the PIP joint and tendon adhesions after severe crush injury. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Free latissimus dorsi musclocutaneous flap transfer for chronic osteomyelitis of the tibia: 16-year follow-up.

Free latissimus dorsi musculocutaneous flap transfer is one of the surgical methods for treating the difficult problem of chronic osteomyelitis of the tibia. We evaluated the viability of the flap and the influence on the bone in a patient at 16 years after surgery by using X-ray films, computed tomography and magnetic resonance imaging.

Dorsal digital perforator flap for reconstruction of distal dorsal finger defects.

Three patients are presented in whom defects of the distal part of the dorsum of the finger were covered with a rotation flap or V-Y advancement flap based on a single perforating branch of the digital artery running from the volar to the dorsal side. This method is useful for the reconstruction of the distal dorsal region of the fingers, because the flap is more mobile, has a smaller skin island and is less invasive compared to the previous flaps. This type of flap conforms to the concept of a perforator flap arising from the main artery.