Recombinant human KAI1/CD82 attenuates M1 macrophage polarization on LPS-stimulated RAW264.7 cells via blocking TLR4/JNK/NF-κB signal pathway.

KAI1/CD82, a membrane tetraspanin protein, can prevent various cancers and retinal disorders through its anti-angiogenic and anti-metastatic capacity. However, little is known about its anti-inflammatory effect and molecular mechanism. Therefore, the present study aimed to inLPSvestigate effect of a recombinant protein of the large extracellular domain of human KAI1 (Gly 111-Leu 228, rhKAI1) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM) and to identify its underlying mechanism. Our data showed that rhKAI1 suppressed expression levels of classically macrophages (M1) phenotyperelated surface markers F4/80+CD86+ in LPS-stimulated BMDM and RAW264.7 cells. In addition, LPS markedly increased mRNA expression and release levels of pro-inflammatory cytokines and mediators such as interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2, whereas these increases were substantially down-regulated by rhKAI1. Furthermore, LPS strongly increased expression of NF-κB p65 in the nuclei and phosphorylation of ERK, JNK, and p38 MAPK. However, nuclear translocation of NF-κB p65 and phosphorylation of JNK were greatly reversed in the presence of rhKAI1. Especially, rhKAI1 markedly suppressed expression of toll-like receptor (TLR4) and prevented binding of LPS with TLR4 through molecular docking predict analysis. Importantly, Glu 214 of rhKAI1 residue strongly interacted with Lys 360 of TLR4 residue, with a binding distance of 2.9 Å. Taken together, these findings suggest that rhKAI1 has an anti-inflammatory effect on LPS-polarized macrophages by interacting with TLR4 and down-regulating the JNK/NF-κB signaling pathway. [BMB Reports 2023; 56(6): 359-364].

Effect of parasitic infection on muscular function of dystrophin gene (Dmd) deficient mouse.

BACKGROUND: Previous studies have reported many cases of Trichinella spiralis (T. spiralis) infection in normal skeletal muscle but there is little research on T. spiralis infection in abnormal muscle tissue. OBJECTIVE: To identify the effect of T. spiralis infection on muscular dystrophy, this study compared aspects of infection between normal (C57BL/10) and dystrophin-deficient Duchenne muscular dystrophy (DMD) mdx mice. METHOD: Infection rate was found to be lower in mdx mice than in C57BL/10 mice at early stages of infection; however, infection and inflammation in mdx mice persisted at later stages of infection while the infection rate and inflammation in C57BL/10 mice decreased gradually. The inflammation area was proportional to the degree of infection in both groups. Muscle strength was measured by the time of latency to fall in the wire-hanging test. Hanging time was shorter in the infected group than in the uninfected group in both C57BL/10 and mdx mice. RESULTS: Muscle strength was also reduced in mdx mice compared with C57BL/10 mice in both the un-infected and infected groups. The muscle intracellular cytokines TGF-ß and IL-6 were continuously expressed from early stage to late-stage infection. IL-10 was strongly expressed at the early stage of infection but decreased as the infection progressed. TNF-α expression remained stable from early to late-stage infection in mdx mice, while TNF-α was elevated only during early-stage infection in C57BL/10 mice. The degree of muscle damage was significantly higher in mdx mice than in C57BL/10 mice because of the high level of serum creatine kinase (CK). CONCLUSION: These results suggest that mdx mice continued in infection and inflammation until the late stages of disease, which was in contrast to the C57BL/10 mice that recovered to some extent in the late stage of infection. In addition, that dystrophin-deficient mice are not suitable for T. spiralis infection compared to normal mice, and the degree of inflammation may be worse in mdx mice.

CD82 attenuates TGF-ß1-mediated epithelial-mesenchymal transition by blocking smad-dependent signaling in ARPE-19 cells.

In retinal pigment epithelial (RPE) cells, transforming growth factor-beta (TGF-ß) plays a critical role in epithelial-mesenchymal transition (EMT), which contributes to various fibrotic retinal disorders. In the present study, we investigated the effect of recombinant human cluster of differentiation 82 (rhCD82), a tumor metastasis suppressor, on TGF-ß-induced EMT in the human RPE cell line APRE-19. The results show that TGF-ß1 significantly enhanced cell migration, invasion and the expression of EMT-mediate factors in ARPE-19 cells. However, rhCD82 markedly inhibited cell mobility and the expression of epithelial marker, zonula occludens-1, as well as increased the expression of mesenchymal markers, such as vimentin and α-smooth muscle actin in TGF-ß1-treated APRE-19 cells. In addition, TGF-ß1 upregulated the phosphorylation of Smad, extracellular signal regulated kinase (ERK) and glycogen synthase kinase-3ß (GSK-3ß), but only phosphorylation of Smad was suppressed by rhCD82. Noteworthy, rhCD82 greatly suppressed the expression of TGF-ß receptor I (TGFRI), TGFRII and integrins in TGF-ß1-treated APRE-19 cells. In particular, the result of molecular docking analysis and structural modeling show that rhCD82 partially interacts with the TGF-ß1 binding sites of TGFRI, TGFRII, integrin ß1 and integrin αv. Taken together, this finding suggested that rhCD82 suppressed TGF-ß1-induced EMT of RPE by blocking of Smad-dependent pathway, which is caused by rhCD82 interaction with TGFRs and integrins, suggesting new insight into CD82 as a potential therapeutic strategy in fibrotic retinal disorders.

Exercise training reduces the risk of opportunistic infections after acute exercise and improves cytokine antigen recognition.

In general, acute exercise is thought to inhibit immune function and increase the risk of opportunistic infections, but there is some opposition to this due to a lack of quantitative evaluation. Therefore, we quantified the effect of exercise on immune function and observed the interaction between antigens and cytokines using an intramuscular infection with Trichinella spiralis (T. spiralis), a common parasitic infection model. C57BL/6 mice were used for a non-infection experiment and an infection (Inf) experiment. Each experiment was divided further into three groups: one control (CON) group, and an exercise pre-infection (PIE)-only group and exercise-sustained (ES) group, each of which was subjected to exercise for 7 weeks. All animals in the infection experiment were infected with T. spiralis 30 min after acute exercise. After infection, the ES and Inf-ES groups continued exercise for 7 additional weeks. The number of T. spiralis nurse cells remaining in skeletal muscles was fewer in the infected exercise groups compared with the infected control. Expression of interleukin-6 (IL-6) and interleukin-10 (IL-10) was higher in the Inf-CON group and transforming growth factor beta (TGF-ß) expression was lower in the Inf-CON group than in the CON group, as measured by RT-PCR. In the infection experiment, only IL-10 had significant differences between the groups. Immunofluorescence revealed that most cytokines were specifically expressed around the antigenic nurse cells following exercise. In conclusion, exercise training does not increase the risk of opportunistic infections even after acute exercise, but rather reduces it. These results may be due to antigen-specific immune responses.

Induction of Angiogenesis by Malarial Infection through Hypoxia Dependent Manner.

Malarial infection induces tissue hypoxia in the host through destruction of red blood cells. Tissue hypoxia in malarial infection may increase the activity of HIF1α through an intracellular oxygen-sensing pathway. Activation of HIF1α may also induce vascular endothelial growth factor (VEGF) to trigger angiogenesis. To investigate whether malarial infection actually generates hypoxia-induced angiogenesis, we analyzed severity of hypoxia, the expression of hypoxia-related angiogenic factors, and numbers of blood vessels in various tissues infected with Plasmodium berghei. Infection in mice was performed by intraperitoneal injection of 2×106 parasitized red blood cells. After infection, we studied parasitemia and survival. We analyzed hypoxia, numbers of blood vessels, and expression of hypoxia-related angiogenic factors including VEGF and HIF1α. We used Western blot, immunofluorescence, and immunohistochemistry to analyze various tissues from Plasmodium berghei-infected mice. In malaria-infected mice, parasitemia was increased over the duration of infection and directly associated with mortality rate. Expression of VEGF and HIF1α increased with the parasitemia in various tissues. Additionally, numbers of blood vessels significantly increased in each tissue type of the malaria-infected group compared to the uninfected control group. These results suggest that malarial infection in mice activates hypoxia-induced angiogenesis by stimulation of HIF1α and VEGF in various tissues.

Plasma neutrophil gelatinase-associated lipocalin as a potential predictor of adverse renal outcomes in immunoglobulin A nephropathy.

BACKGROUND/AIMS: Neutrophil gelatinase-associated lipocalin (NGAL) is a well-known biomarker of acute kidney injury. We evaluated the value of plasma NGAL (pNGAL) as an independent predictor of prognosis in immunoglobulin A nephropathy (IgAN). METHODS: In total, 91 patients with biopsy-proven IgAN at a single center were evaluated. pNGAL was measured using a commercial enzyme-linked immunosorbent assay kit (R&D Systems). Adverse renal outcome was defined as chronic kidney disease (CKD) stage 3 or above at the last follow-up. Pearson correlation coefficient and Cox regression were used for analyses. RESULTS: The mean age of all patients (male:female, 48:43) was 35 years (range, 18 to 77). pNGAL ranged between 21.68 and 446.40 ng/mL (median, 123.97) and showed a correlation with age (r = 0.332, p = 0.001), creatinine (r = 0.336, p = 0.001), estimated glomerular filtration rate (r = -0.397, p < 0.001), uric acid (r = 0.289, p = 0.006), and the protein-to-creatinine ratio (r = 0.288, p = 0.006). During a mean follow-up period of 37.6 months, 11 patients (12.1%) had CKD stage 3 or above. In a multivariate Cox regression model, hypertension (hazard ratio [HR], 8.779; 95% confidence interval [CI], 1.526 to 50.496; p = 0.015), proteinuria > 1 g/day (HR, 5.184; 95% CI, 1.124 to 23.921; p = 0.035), and pNGAL (HR, 1.012; 95% CI, 1.003 to 1.022; p = 0.013) were independent predictors associated with adverse renal outcome. CONCLUSIONS: pNGAL showed strong correlations with other clinical prognostic factors and was also an independent predictor of adverse renal outcome. We suggest pNGAL as a potential predictor for prognosis in IgAN, while further studies are needed to confirm the clinical value.

Expression of human endogenous retrovirus env genes in the blood of breast cancer patients.

Human endogenous retroviruses (HERV) env proteins have been recently reported to be significantly up-regulated in certain cancers. Specifically, mRNA and protein levels of HERV-K (HML-2) are up-regulated in the blood plasma or serum of breast cancer patients. Here, we collected blood samples of 49 breast cancer patients and analyzed mRNA expressions of various HERVs env genes including HERV-R, HERV-H, HERV-K, and HERV-P by real-time PCR. The expression of env genes were significantly increased in the blood of primary breast cancer patients but were decreased in patients undergoing chemotherapy to a similar level with benign patients. When we compared the group currently undergoing chemotherapy and those patients undergoing chemotherapy simultaneously with radiotherapy, HERVs env genes were reduced more in the chemotherapy only group, suggesting that chemotherapy is more effective in reducing HERV env gene expression than is radiotherapy. Among chemotherapy groups, HERV env gene expression was the lowest in the taxotere- or taxol-treated group, suggesting that taxotere and taxol can reduce HERVs env expression. These data suggest the potential to use HERVs env genes as a diagnosis marker for primary breast cancer, and further studies are needed to identify the mechanism and physiological significance of the reduction of HERV env gene expression during chemotherapy.

DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers.

DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

Over-expression of thymosin ß4 in granulomatous lung tissue with active pulmonary tuberculosis.

Recent studies have shown that thymosin ß4 (Tß4) stimulates angiogenesis by inducing vascular endothelial growth factor (VEGF) expression and stabilizing hypoxia inducible factor-1α (HIF-1α) protein. Pulmonary tuberculosis (TB), a type of granulomatous disease, is accompanied by intense angiogenesis and VEGF levels have been reported to be elevated in serum or tissue inflamed by pulmonary tuberculosis. We investigated the expression of Tß4 in granulomatous lung tissues at various stages of active pulmonary tuberculosis, and we also examined the expression patterns of VEGF and HIF-1α to compare their Tß4 expression patterns in patients' tissues and in the tissue microarray of TB patients. Tß4 was highly expressed in both granulomas and surrounding lymphocytes in nascent granulomatous lung tissue, but was expressed only surrounding tissues of necrotic or caseous necrotic regions. The expression pattern of HIF-1α was similar to that of Tß4. VEGF was expressed in both granulomas and blood vessels surrounding granulomas. The expression pattern of VEGF co-localized with CD31 (platelet endothelial cell adhesion molecule, PECAM-1), a blood endothelial cell marker, and partially co-localized with Tß4. However, the expression of Tß4 did not co-localize with alveolar macrophages. Stained alveolar macrophages were present surrounding regions of granuloma highly expressing Tß4. We also analyzed mRNA expression in the sputum of 10 normal and 19 pulmonary TB patients. Expression of Tß4 was significantly higher in patients with pulmonary tuberculosis than in normal controls. These data suggest that Tß4 is highly expressed in granulomatous lung tissue with active pulmonary TB and is associated with HIF-1α- and VEGF-mediated inflammation and angiogenesis. Furthermore, the expression of Tß4 in the sputum of pulmonary tuberculosis patients can be used as a potential marker for diagnosis.

Trichinella spiralis infection reduces tumor growth and metastasis of B16-F10 melanoma cells.

Recently, attempts have been made to use parasites as novel candidates for live vaccine vectors against solid tumors. In this study, we examined the effects of Trichinella spiralis (T. spiralis) infection on solid tumor growth and metastasis. After oral infection with T. spiralis larvae, B16-F10 cells were injected subcutaneously and intravenously into C57BL/6 mice to evaluate tumor growth and metastatic potential, respectively. Tumor growth and lung metastases in T. spiralis infected mice were significantly reduced compared with control mice. To elucidate the mechanism of tumor reduction by parasitic infection, we conducted cytokine arrays using mouse serum. CXCL9 and CXCL10 were increased in the infection group and decreased in the infection-tumor group. However, the expression level was not changed in the infection-metastasis group compared to the infection or control-metastasis groups. Although SDF-1 and IL-4 were increased in the infection group, there was no significant change in expression in the infection-tumor group or the infection-metastasis group. Additionally, IL-4 and KC were increased in the infection-tumor group compared to the control-tumor group, but there was no difference in expression between the control-metastasis group and the infection-metastasis group. CXCL13 was significantly increased in the infection-metastasis group only. These results suggest that T. spiralis infection reduced tumor growth and metastasis through a complex transition in cytokine regulation profiles including CXCL9, CXCL10, and CXCL13.

Expression patterns of Thymosin ß4 and cancer stem cell marker CD133 in ovarian cancers.

Thymosin ß4 (Tß4), a small acidic actin binding peptide, is overexpressed in a side population of cancer stem cells and CD133-positive colorectal cancer stem cells. In order to understand the relationship between Tß4 and CD133, we studied the expression patterns of Tß4 and CD133 in ovarian cancers. The expression patterns of Tß4 and CD133 were studied in normal ovaries, primary ovarian cancers, metastatic ovarian cancers, primary stomach cancers, and normal stomachs by Western blot and immunohistochemistry. Expression patterns and co-localization of Tß4 and CD133 were examined by immunofluorescence and confocal laser-scanning microscopy. Tß4 is overexpressed in primary ovarian cancers, but not in primary stomach cancers, when compared with normal controls. However, Tß4 levels in metastatic stomach cancers to the ovary are significantly upregulated compared with levels in normal stomachs and primary stomach cancers. These results suggest that Tß4 levels are related to tumorigenesis in ovarian cancers and metastasis in stomach cancers. The expression of Tß4 in normal ovaries and normal stomachs was weak, but was co-localized with CD133 expression. Tß4 expression was also co-localized with CD133 expression in primary ovarian carcinomas, metastatic ovarian cancers from stomach cancers and primary stomach cancers. These data suggest that Tß4 expression is strongly related to CD133 expression and is a characteristic of stem cells or cancer stem cells.

Trichinella spiralis infection induces angiogenic factor thymosin ß4 expression.

Trichinella spiralis (T. spiralis) has been reported to up-regulate the expression of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. In order to analyze the induction of angiogenesis by T. spiralis, the expression patterns of angiogenesis-related proteins were investigated by immunohistochemical analysis. VEGF expression was induced in the infected muscles at an early stage of infection (10 days after infection) and diminished after 3 weeks. Thymosin ß4, a major factor which induces VEGF, showed increased expression in muscle fibers 10 days after infection, and the expression remained high in the nurse cells for 6 weeks, when the formation of the nurse cell complex was completed. The hypoxia inducible factor (HIF)-1α showed a diffuse expression pattern around the infected muscle fibers and was strongly expressed in inflammatory cells but was not related to the hypoxic condition caused by nurse cell formation. Localization of the hypoxic regions by the hypoxia marker, pimonidazole showed T. spiralis infection does not induce a hypoxic condition in nurse cells. These results suggest that the expression of VEGF and thymosin ß4 induce angiogenesis and the expression of thymosin ß4 remained elevated to potentially regulate nurse cell formation and maintenance.

Elevated expression of thymosin ß4, vascular endothelial growth factor (VEGF), and hypoxia inducible factor (HIF)-1α in early-stage cervical cancers.

Recent studies have shown that thymosin ß4 (TB-4) is highly related with tumor metastasis and angiogenesis. In addition, TB-4 induced the expression of VEGF in melanoma cells. We investigated the expression patterns of TB-4 and related angiogenic proteins, VEGF, and HIF-1α, at various stages of cervical cancers and also identified the expression pattern of these proteins in metastatic cervical cancers. Expression patterns of TB-4, VEGF, and HIF-1α were studied with tissue microarray containing 42 samples of cervical cancers. In addition, 15 cervical cancers and metastatic tumors in lymph nodes from patients who have metastatic tumors were also analyzed to confirm the role of TB-4, VEGF, and HIF -1α in cervical cancer metastasis. The expression levels of TB-4, VEGF, and HIF-1α were very weak at early cancer stages (stages 0 to 1A) but significantly increased at stage 1B. The numbers of blood vessels in tumors were also increased at stage 1B. The expression patterns of TB-4, VEGF, and HIF-1α were compared in tumors without lymph node metastasis, primary tumors with lymph node metastasis, and metastatic tumors in lymph nodes. The expression levels of TB-4, VEGF, and HIF-1α in primary tumors with lymph node metastasis and their metastatic tumors in lymph node were less than in tumors without lymph node metastasis. These data suggest that TB-4, VEGF, and HIF-1α triggered angiogensis and tumor invasiveness to surrounding tissues at early stage of cervical carcinoma but have a negative or no effect on the metastatic potential.

Thymosin ß4 expression in human tissues and in tumors using tissue microarrays.

Thymosin ß4 has been reported to play the key roles in tumor growth, metastasis, and angiogenesis. Although the importance of thymosin ß4 in angiogenesis and metastasis is known, few studies to show the expression patterns of thymosin ß4 in human tissues including tumors have been conducted. The comparisons of the expression of thymosin ß4 between the normal and tumor tissues are also needed to study the role of thymosin ß4 in tumor formation. Using tissue microarray analysis, we compared the expression patterns of thymosin ß4 in the normal human tissues and in the tumors to screen certain tumors and upregulated the expression of thymosin ß4 by tumorigenesis. Thymosin ß4 was highly expressed in the hepatic cells in the normal adult liver, duct, and acinar cells in pancreas, and muscle cells in the heart and also expressed highly in certain tumor cells, including osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. Comparing the thymosin ß4 expression between normal and tumors, thymosin ß4 was upregulated specifically in osteosarcoma, colorectal carcinoma, and esophageal cancer. To confirm the over-expression of thymosin ß4 in these tumors, we analyzed expression of thymosin ß4 with each additional microarray of osteosarcoma, colorectal carcinoma, and esophageal cancer. The significant increased expression of thymosin ß4 was observed in osteosarcoma and in colorectal cancer. These results suggest that the expression of thymosin ß4 is highly related with tumorigenesis of certain tumors including the osteosarcoma and colorectal cancers.

Thymosin ß4 induces the expression of vascular endothelial growth factor (VEGF) in a hypoxia-inducible factor (HIF)-1α-dependent manner.

Thymosin ß4 has multi-functional roles in cell physiology, but little is known about its mechanism(s) of action. We previously reported that thymosin ß4 stimulated angiogenesis through the induction of vascular endothelial growth factor (VEGF). To identify the mechanism of VEGF induction by thymosin ß4, we have used a luciferase assay system with VEGF in the 5' promoter region. We also analyzed the effect of thymosin ß4 on VEGF mRNA stability and on the expression and stability of hypoxia-inducible factor (HIF)-1α. We found that thymosin ß4 induces VEGF expression by an increase in the stability of HIF-1α protein. Analysis of the expression patterns of thymosin ß4 and HIF-1α in colon cancer tissue microarray showed that thymosin ß4 and HIF-1α co-localized in these biopsies. These data show that thymosin ß4 induces the expression of VEGF indirectly by increasing the protein stability of HIF-1α.

Primary cardiac lymphoma presenting with atrioventricular block.

Primary cardiac lymphomas (PCL) are extremely rare. Clinical manifestations may be variable and are attributed to location. Here, we report on a case of PCL presenting with atrioventricular (AV) block. A 55 year-old male had experienced chest discomfort with unexplained dyspnea and night sweating. His initial electrocardiogram (ECG) revealed a first degree AV block. Along with worsening chest discomfort and dyspnea, his ECG changed to show second degree AV block (Mobitz type I). Computed tomography (CT) scan showed a cardiac mass (about 7 cm) and biopsy was performed. Pathologic finding confirmed diffuse large B-cell lymphoma. The patient was treated with multi-drug combination chemotherapy (R-CHOP: Rituximab, cyclophoshamide, anthracycline, vincristine, and prednisone). After treatment, ECG changed to show normal sinus rhythm with complete remission on follow-up CT scan.

Inhibition of microsomal triglyceride transfer protein expression and atherogenic risk factor apolipoprotein B100 secretion by tanshinone IIA in HepG2 cells.

Salvia miltiorrhiza Bunge is known to be effective for the treatment of cardiovascular diseases. Here, we have isolated tanshinone IIA (T-IIA) from S. miltiorrhiza Bunge. The aim of this study is to address the mechanisms where apolipoprotein B-100 (ApoB) regulation is associated with T-IIA, since T-IIA regulates the lipoprotein metabolism in liver cells. Human HepG2 cells treated with T-IIA for 24 h exerted a dose-dependent inhibitory effect on ApoB secretion together with triglyceride. However, another secretory protein, albumin, was unaffected by T-IIA treatment, indicating that the effect of T-IIA is specific for ApoB secretion. T-IIA decreased the transcription level of microsomal triglyceride transfer protein gene, suggesting that lipoprotein assembly is likely to be involved in the inhibited ApoB secretion. Interestingly, T-IIA inhibited ApoB secretion via a proteasome-dependent pathway. Our results suggest that T-IIA is an influential inhibitor of ApoB secretion and triglyceride secretion in liver cells.