Recombinant Interferon-γ Research Articles

PRMT2 silencing regulates macrophage polarization through activation of STAT1 or inhibition of STAT6

BackgroundMacrophages play significant roles in innate immune responses and are heterogeneous cells that can be polarized into M1 or M2 phenotypes. PRMT2 is one of the type I protein arginine methyltransferases involved in inflammation. However, the role of PRMT2 in M1/M2 macrophage polarization remains unclear. Our study revealed the effect and mechanism of PRMT2 in macrophage polarization.MethodsBone marrow-derived macrophages (BMDMs) were polarized to M1 or M2 state by LPS plus murine recombinant interferon-γ (IFN-γ) or interleukin-4 (IL-4). Quantitative polymerase chain reaction (qPCR), western blot and flow cytometry (FCM) assay were performed and analyzed markers and signaling pathways of macrophage polarization.ResultsWe found that PRMT2 was obviously upregulated in LPS/IFN-γ-induced M1 macrophages, but it was little changed in IL-4-induced M2 macrophages. Furthermore, PRMT2 konckdown increased the expression of M1 macrophages markers through activation of STAT1 and decreased the expression of M2 macrophages markers through inhibition of STAT6.ConclusionsPRMT2 silencing modulates macrophage polarization by activating STAT1 to promote M1 and inhibiting STAT6 to attenuate the M2 state.

Specific Features of Pulmonary Tuberculosis Patients Treatment with Interferon-Gamma Depending on the Nature of the Tissue Inflammatory Reaction

The objective: to evaluate effectiveness of treatment of infiltrative pulmonary tuberculosis patients with exudative or productive types of the tissue inflammatory reaction when additional immunotherapy with recombinant IFN-γ is used.Subjects and Methods. 127 infiltrative tuberculosis patients were enrolled in the study, of them 66 had an exudative type of the inflammatory reaction (2 Groups OG-1 and GS-1 were formed), 61 had a proliferative type (2 Groups OG-2 and GS-2 were formed). Patients from OG-1 and OG-2 Groups additionally received different regimens with recombinant interferon-γ.Result: Comprehensive treatment of infiltrative tuberculosis including intramuscular administration (according to a certain regimen) of recombinant IFN-γ contributes to achievement of pronounced positive changes of clinical and radiological symptoms in patients with both exudative and productive types of the tissue inflammatory reaction. In the groups treated with recombinant IFN-γ, restoration of the cellular immune response, cellular cooperation, normalization of the cytokine profile and increased phagocytosis activity were confirmed by the results of laboratory tests.

Molecular identification and functional analysis of X-linked inhibitor of apoptosis -associated factor-1 (XAF1) in grass carp, Ctenopharyngodon idella

X-linked inhibitor of apoptosis protein (XIAP) -associated factor 1 (XAF1) is an interferon-stimulated gene which exhibits pro-apoptosis effect. In this study, XAF1 was characterized from grass carp Ctenopharyngodon idella and its expression pattern and function were analyzed. The open reading frame (orf) of XAF1 is 789 nucleotides (nt) encoding 262 amino acids. SMART online search results showed that a C2H2-type and six C2HC-type zinc-fingers were found in XAF1, however, the XAF1 of grass carp showed high sequence identity to zebrafish (71%), low sequence identity to tetrapods (21–22%). Rt-qPCR results showed that XAF1 was constitutively expressed in all tested organs/tissues with highest expression in blood. An inductive expression of XAF1 at mRNA level was observed in peripheral blood leucocytes (PBLs) and C. idellus kidney cells (CIKs) after treatment with C. idellus recombinant interferon-γ (rIFNg). Overexpressing XAF1 in CIKs exhibited resistance against grass carp reovirus (GCRV) and more sensitivity to cisplatin. These results implied a functional homologue of XAF1 in evolution, however the mechanism may require further investigation.

Antifungal chemotherapies and immunotherapies for the future.

Human fungal pathogens cause a broad plethora of infections, spanning cutaneous dermatophytoses to invasive infections in immunocompromised hosts. As eukaryotic pathogens are capable of morphotype switching, they present unique challenges both for drug development and the immunological response. Whilst current antifungal therapies are limited to the orally available triazoles, intravenous echonocandins and polyenes, and flucytosine and terbinafine, there has been recent significant progress in the antifungal armamentorium with ibrexafungerp, a novel orally available terpanoid that inhibits 1,3-beta-D-glucan-approved by Food and Drug Administration in 2021, and fosmanogepix, an orally available pro-drug of manogepix, which targets glycosylphosphatidylinositol-anchored protein maturation entering Phase 3 studies for candidaemia. A number of further candidates are in development. There has been significant use of existing immunotherapies such as recombinant interferon-γ and G-CSF for fungal disease in immunocompromised patients, and there are emerging opportunities for monoclonal antibodies targeting TH2 inflammation. Omalizumab, an anti-IgE monoclonal antibody in asthma, is now used routinely for the treatment of allergic bronchopulmonary aspergillosis, and further agents targeting IL-4 and IL-5 are being evaluated. In addition, T-cell CAR therapy is showing early promise for fungal disease. Thus, we are likely to see rapid advances to our approach to the management of fungal disease in the near future.

P212 Fatal invasive aspergillosis in a child with Chronic Granulomatous disease; a case report

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMIn this article, we have reported a case of invasive aspergillosis in an 8-year-old boy with chronic granulomatous disease, who presented with pleural effusion and pneumonia, cerebral venous sinus thrombosis, and unusual skin lesions (Fig. 1) caused by Aspergillus fumigatus (Fig. 2). The patient received recombinant human interferon-γ subcutaneously (1-2 doses in even days) and antimicrobial prophylaxis, including itraconazole (100 mg/d), cotrimoxazole (5 mg trimethoprim/kg body weight/d), and ofloxacin (3 times a week, 200 mg), during hospitalization. It is noteworthy that treatment was immediately started according to the direct examination results. Due to the MRI findings suggestive of brain abscesses and brain edema, empirical amphotericin and voriconazole were started. The in vitro antifungal susceptibility tests (AFST) of A. fumigatus utilized the microbroth dilution method of the Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol for five antifungal agents including; voriconazole, itraconazole, fluconazole, caspofungin, and amphotericin B, that the minimum inhibitory concentrations (MIC) of antifungal agents were 0.25, 1, 16, 0.125, and 4 mg/L, respectively. Voriconazole and caspofungin were shown to be the most potent antifungal drugs against this A. fumigatus strain. On the other hand, the IDSA protocols recommend voriconazole as the initial therapy of invasive aspergillosis in most patients. So, in response to the results of antifungal susceptibility testing, treatment was switched to voriconazole at the dose of 100 mg twice a day (taken every 12 h), and interferon-γ subcutaneously (1-3 doses in even days) was introduced. Unfortunately, the patient passed away due to cerebral venous sinus thrombosis (CVST), and intracerebral hemorrhage (ICH) following increased intracranial pressure (ICP) after 1 month. In conclusion, we have reported a case in which a patient who came with pleural effusion and pneumonia in the chest x-ray, CVST, and unusual skin lesion caused by A. fumigatus expired after one month. Our report suggests the importance of early diagnosis in children presenting with invasive fungal infections particularly, those involving the central nervous system.

Expression of recombinant human IFN-γ protein in soybean (Glycine max L.)

In recent years, the diagnostic and therapeutic application of recombinant proteins is increasing rapidly. One of these proteins is human interferon-γ (hIFN-γ), which shows great value in therapy against the viruses. The different biological drugs, such as cytokines, vaccines, growth factors, and antibodies are produced extensively through plant-based expression systems. In the present investigation, we report the successful expression of recombinant human interferon-γ (hIFN-γ) protein in soybean by Agrobacterium-mediated. For this purpose, the recombinant construct (pCAMBIA1304-hIFN-γ) was transferred in the GV3101 strain of Agrobacterium tumefaciens and applied for stable transformation of soybean explants. The results have confirmed the presence of the hIFN-γ gene in transgenic soybean by PCR, and also, the integration and copy number of the hIFN-γ gene was confirmed by southern blot analysis. The expression of the hIFN-γ transcription was confirmed by RT-PCR. Moreover, the total protein of transgenic soybean was extracted, and the expression of the hIFN-γ proteins was determined by western blot. Finally, bioactivity assay demonstrates that recombinant hIFN-γ protein was active biologically and protects the Vero cells versus infection that produced by the VSV. This investigation can be used as a basis for further researches in the large scale production of hIFN-γ and other recombinant proteins in soybean. For the first time, the successful transformation and expression of human gamma interferon gene in soybean plant by Agrobacterium tumefaciens were reported and the antiviral activity of recombinant hIFN-γ protein produced in soybean was confirmed.

Amentoflavone inhibits M1 polarization of THP-1-derived foam cells by activating PPAR-α/γ

To investigate the mechanism by which amentoflavone inhibits polarization of THP-1-derived foam cells to M1 phenotype. Human monocyte cell line THP-1 was stimulated to differentiate into M1-type macrophages using phorbol 12-myrislate13-acetate (PMA) combined with lipopolysaccharide (LPS) and recombinant human interferon-γ (rhlFN-γ). M1 polarization of THP-1-derived macrophages was confirmed by observing morphological changes of the cells and detecting the mRNA expression of L-6 and TNF-α with RT-qPCR. THP-1-derived foam cells treated with 5 or 10 μmol/L amentoflavone for 24 h were examined for cytokines using ELISA. The mRNA and protein expressions of IL-6, IL-10, TNF-α, TGF-β, PPAR-α/γ, Arg-1 and Fizz1 in the cells were detected using RT-qPCR and Western blotting. Amentoflavone prevented induced M1 polarization of THP-1 cells. Amentoflavone down-regulated the mRNA expressions of IL-6 and TNF-α, up-regulated mRNA expressions of IL-8 and TGF-β mRNA (P < 0.05), and increased the protein expressions of PPAR-α/γ, Arg-1 and Fizz1. Molecular docking simulation showed that amentoflavone could bind to the surface of PPARα/γ. Amentoflavone can inhibit the differentiation of macrophages into M1 type by activating PPAR-α/γ and restoring the expressions of the gene Arg-1 and Fizz1.

Cloning, Expression, and Purification of Recombinant Mouse Interferon-γ

Background: Interferon-gamma [IFN-γ) is the most important cytokine in the immune system. This protein has been expressed in bacterial cells. However, bacterial cloning is not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ and overcome problems in favor of commercial purposes. Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method. Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting. Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.

The inhibitory effect of canine interferon gamma on the growth of canine tumors

Recombinant canine interferon-γ (rc-IFNγ; InterdogⓇ) was exclusively approved as a therapeutic for canine atopic dermatitis. However, it has been used off-label for the treatment of canine cancer. We examined the inhibitory effect of rc-IFNγ on the growth of canine tumor cell lines and analyzed its mechanism of action. Three (CTB-p, CTB-m, and CNM-m) out of seven mammary gland tumor cell lines and two (VIMC and CoMS) out of four mast cell tumor cell lines showed remarkable growth inhibition after treatment with rc-IFNγ. However, one (CLBL-1) out of nine lymphoma cell lines showed a significant amount of cell death. Using CTB-p and CTB-m cell lines, we showed that STAT1 was essential for inducing the growth inhibitory effect of rc-IFNγ. Although rc-IFNγ induced G1 growth arrest in CTB-p cell line, treatment with rc-IFNγ did not alter the expression of cell cycle regulatory proteins. In this study, we observed direct cytotoxicity or cytostatic effects of rc-IFNγ in canine tumor cell lines. However, the detailed mechanisms responsible for these effects need to be elucidated in the future.

Interleukin-35 Suppresses CD8+ T Cell Activity in Patients with Viral Hepatitis-Induced Acute-on-Chronic Liver Failure.

Interleukin (IL)-35 is a newly indentified cytokine and induces immunotolerance via suppression of CD8+ T cell activity in chronic viral hepatitis. To investigate the modulatory function of IL-35 to CD8+ T cells in viral hepatitis-induced acute-on-chronic liver failure (ACLF). Fifty-five ACLF patients and 21 healthy controls were enrolled. Serum IL-35 concentration was measured by ELISA. Absolute accounts for T cells, immune checkpoint molecules, and cytotoxic molecules in CD8+ T cells were measured by flow cytometry and real-time PCR, respectively. Direct and indirect contact co-culture systems between CD8+ T cells and HepG2 cells were set up. The regulatory function of IL-35 to CD8+ T cells was assessed by measuring lactate dehydrogenase expression and cytokine production. Serum IL-35 concentration was elevated in ACLF patients and positively correlated with total bilirubin, but negatively correlated with prothrombin time activity. Peripheral CD8+ T cells showed exhausted phenotype in ACLF patients, which manifested as up-regulation of programmed death-1 (PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), and lymphocyte activation gene-3 (LAG-3) but down-regulation of perforin, granzyme B, and FasL. Recombinant IL-35 stimulation dampened cytotoxicity and interferon-γ production in both direct and indirect contact co-culture systems. This process was accompanied by elevation of PD-1, CTLA-4, and LAG3, as well as reduction of perforin, granzyme B, and FasL in CD8+ T cells. Elevated IL-35 suppressed both cytolytic and non-cytolytic activity of CD8+ T cells in ACLF patients.

Immunostimulatory activities of a high molecular weight fraction of Cynanchum auriculatum royle ex wight root obtained by ultrafiltration

Background: The root of Cynanchum auriculatum Royle ex Wight has been traditionally used as a folk medicine in the eastern Asia. Objectives: In this study, we explored the immune-enhancing effect of the high-molecular weight fraction of C. auriculatum (CAHF) root extracts in RAW 264.7 macrophages and cyclophosphamide (CYP)-induced immune-suppressed mice.Materials and Methods: To obtain the CAHF, ultrafiltration was performed using 30-kDa molecular weight cutoff polyethersulfone membranes in a cross-flow microfiltration system. In vitro and in vivo experiments were performed to evaluate the immunostimulatory effects of CAHF. Results: CAHF increased the production of nitric oxide and immunostimulatory cytokines (interleukin-6 and tumor necrosis factor-α) in recombinant interferon-γ primed macrophages. In addition, the phagocytic activity of macrophage cells was enhanced by CAHF. Furthermore, CAHF treatment markedly restored the decreased body weight, immune organs (spleen and thymus) weight, natural killer cell activity, and T- and B-lymphocyte proliferation in CYP-induced immune-suppressed mice. Conclusion: These results suggest that CAHF is an effective immunostimulatory agent.

The extended understanding of chronic granulomatous disease.

We briefly address the advances in genetics, pathophysiology, and phenotypes of chronic granulomatous disease (CGD). This is one of the most studied primary immunodeficiencies, which comprise mutations in genes encoding the different subunits of the NADPH oxidase system. Those mutations lead to defective reactive oxygen species production, and consequently a failure to eliminate pathogens. Patients with CGD are susceptible to fungal, bacterial, and parasitic infections. Other symptoms, as systemic adverse effects to BCG vaccine and hyperinflammation, are also important clinical conditions in this disease. This wide-ranging clinical spectrum of CGD comes from heterogeneity of mutations, X-linked-CGD or autosomal recessive inheritance, and diverse environmental pressure factors. Early accurate diagnosis and prompt treatment are necessary to diminish the consequences of the disease. The most used diagnostic tests are dihydrorhodamine, cytochrome c reduction, and luminol-enhanced chemiluminescence assay. The determination of mutations is essential for diagnosis confirmation and genetic counseling. CGD treatment usually includes prophylactic antibiotics and antifungals. Prophylactic recombinant human interferon-γ, immunosuppressors or immune modulators may be, respectively, indicated for preventing infections or inflammatory manifestations. Hematopoietic stem cell transplantation and gene therapy are currently the available options for curative treatment of CGD.

A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease

Reactive microglia are a major pathological feature of Alzheimer's disease (AD). However, the exact role of microglia in AD pathogenesis is still unclear. Here, using metabolic profiling, we found that exposure to amyloid-β triggers acute microglial inflammation accompanied by metabolic reprogramming from oxidative phosphorylation to glycolysis. It was dependent on the mTOR-HIF-1α pathway. However, once activated, microglia reached a chronic tolerant phase as a result of broad defects in energy metabolisms and subsequently diminished immune responses, including cytokine secretion and phagocytosis. Using genome-wide RNA sequencing and multiphoton microscopy techniques, we further identified metabolically defective microglia in 5XFAD mice, an AD mouse model. Finally, we showed that metabolic boosting with recombinant interferon-γ treatment reversed the defective glycolytic metabolism and inflammatory functions of microglia, thereby mitigating the AD pathology of 5XFAD mice. Collectively, metabolic reprogramming is crucial for microglial functions in AD, and modulating metabolism might be a new therapeutic strategy for AD.

Characterization of anti-interferon-γ antibodies in HIV-negative immunodeficient patients infected with unusual intracellular microorganisms.

A major characteristic of immunodeficiency associated with life-threatening intracellular infection in adults is the presence of anti-interferon-γ antibodies. Although little is known about the mechanism underlying this syndrome, it is believed that the antibodies inhibit the activity of downstream signaling pathway of interferon-γ. In this study, the characteristics of these antibodies in patients who presented, or have a history of, intracellular infection and were positive to anti-interferon-γ antibodies were investigated. The antibodies exhibited mainly the IgG1 and the IgG4 subtypes and recognized the C-terminal of the interferon-γ linear epitope containing the KRKR motif, which is required for the biological activity of interferon-γ. The antibodies bound to recombinant interferon-γ with significantly lower avidity than antibodies to a recall antigen, tetanus toxoid, suggesting that the antibodies might have not undergone affinity maturation. The data from this study may provide fundamental information to better understand the properties of anti-interferon-γ antibodies, which can be useful for future studies. Impact statement An increase in the number of immunodeficient patients related to autoantibodies to interferon (IFN)-γ has been observed particularly in East Asian adults. These patients are often presented with opportunistic infections caused by intracellular pathogens, including non-tuberculous mycobacteria (NTM), Cryptococcus neoformans, Penicillium marneffei (now called Talaromyces marneffei), and non-typhoidal Salmonella spp. The mortality rate for this syndrome is relatively high with 32% patients dying at the median time of 25 months after diagnosis. Characterization of these autoantibodies may promote better understanding of the syndrome, an emerging health problem affecting East Asia populations and impeding their welfare and economic development.

Effects of recombinant canine interferon-γ injected before general anesthesia with propofol and isoflurane on natural killer cytotoxic activity during anesthesia in dogs

We aimed to determine whether subcutaneous injection of recombinant canine interferon-γ (rCaIFN-γ) 1h before general anesthesia with a combination of propofol and isoflurane (P-I) changes the cytotoxic activity of natural killer (NK) cells during anesthesia in dogs. Nine clinically healthy beagles were assigned to one of two groups: rCaIFN-γ–treated group (n=5) and control group (no treatment, n=4). One hour before P-I anesthesia, rCaIFN-γ was subcutaneously injected at a dose of 1×104U/kg body weight for the test group and physiologic saline was subcutaneously injected for the control group. Blood samples were collected 1h before P-I anesthesia (immediately before rCaIFN-γ injection) and 1 and 3h after anesthesia induction. NK cytotoxic activity was measured using the Rose Bengal assay, using canine thyroid adenocarcinoma cells as target cells. In the control group, NK cytotoxic activity decreased significantly over time (P<0.05). On the other hand, in the rCaIFN-γ group, the activity increased for the first hour after anesthesia induction without a significant difference from the level before anesthesia; furthermore, the activity at 3h after anesthesia induction was comparable with that before anesthesia. NK cytotoxic activity at 1 and 3h after anesthesia induction was significantly higher in the rCaIFNγ group than in the control group (P<0.05). Subcutaneous injection of rCaIFN-γ 1h before P-I general anesthesia not only maintained but also enhanced NK cytotoxic activity up to 3h after anesthesia induction in dogs. In conclusion, subcutaneous injection of rCaIFN-γ before cancer resection in cancer-bearing dogs might be effective for preventing intraoperative dissemination of cancer cells and postoperative cancer recurrence and metastasis.

Probucol reverses homocysteine induced inflammatory monocytes differentiation and oxidative stress

Reactive oxygen species have been demonstrated to involve in homocysteine-induced Ly-6Chi monocytes differentiation. Probucol is an anti-oxidant agent that has been used to treat atherosclerosis. We sought to evaluate the effect and potential mechanism of probucol on homocysteine-induced inflammatory monocytes differentiation. The primary mouse splenocytes suspensions were initiated by recombinant interferon-γ and cultured with L-homocysteine in the presence or absence of probucol. The cells were co-incubated with monoclonal antibodies to CD11b–PE and Ly-6C FITC. Flow cytometry analysis was performed on BD FACS caliber. Data were analyzed using the FlowJo software. Mononuclear cells were gated according to the lower granular and larger size, distinguished with granulocytes and lymphocytes. Monocytes were defined as CD11b+ mononuclear cells and further divided into three groups based on their Ly-6C expressions, Ly-6Chi, Ly-6Cmid and Ly-6Clow subsets. The productions of reactive oxygen species in monocytes subsets were detected by 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) containing monocytes were marked as DCFH-DA+ cells in both Ly-6C+ and Ly-6C- subsets. The activity of nicotinamide adenine dinucleotide phosphate oxidase in THP-1 cells was measured by assay kit on enzyme-labelling instrument. L-homocysteine promoted inflammatory monocytes differentiation and its reactive oxygen species productions in dose-dependent manner. Probucol dose-dependently suppressed the differentiation and reactive oxygen species productions of inflammatory monocytes induced by L-homocysteine. Furthermore, the increased NADPH oxidase activity induced by L-homocysteine was significantly reversed by probucol in THP-1 cells. Probucol prevented L-homocysteine-induced inflammatory monocytes differentiation and its reactive oxygen species generation probably through inhibiting NADPH oxidase activity.

Effect of Se-Yeon-Eum on nitric oxide and tumor necrosis factor-α production in C6 glial cells induced by recombinant interferon-γ and cigarette extract

Even though Se-Yeon-Eum (SYE) has been used to treat symptoms induced by cigarette smoking in genuine Korean medicine, scientific evidence for this traditional use has not been provided. It is well known that nitric oxide (NO) and tumor necrosis factor-α (TNF-α) are well known to be inflammatory mediators of cerebral ischemia due to cigarette smoking. The objective of this study was toe detemrine the effect of SYE on NO and TNF-α production in C6 glial cells induced by recombinant interferon-γ (rIFN-γ) plus cigarette extract (CE). Results showed that SYE (500, 1000 μg/ml) inhibited NO production induced by rIFN-γ (50 U/ml) and CE (1/100, 1/50 dilution). SYE (1000 μg/ml) also suppressed TNF-α secretion induced by rIFN-γ (50 U/ml) and CE (1/100 dilution). Moreover, inducible nitric oxide synthase expression and cytoplasmic TNF-α level stimulated by rIFN-γ and CE were inhibited by SYE (1000 μg/ml). These results suggest that SYE might have anti-inflammatory effect under cerebral ischemic condition.

Chemical Compounds in Natural Medicines That Affect Macropharges and Adipocyte Cells.

Macrophages play major roles in inflammation, immunity and host defense mechanisms. Once activated they produce and release cytokines, oxygen and nitrogen species, and eicosanoids. The best characterized stimuli to induce the transcription of genes encoding pro-inflammatory proteins in macrophages in vitro is bacterial lipopolysaccharide (LPS). LPS could be used alone or in combination with recombinant mouse interferon-γ (IFN-γ). Such stimulation results in cytokine release and the synthesis of enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The nitric oxide (NO) radical is known to play a central role in inflammatory and immune reactions for self-protection. However, the excessive production of NO may lead to tissue damage. In inflammatory diseases such as rheumatoid arthritis, excessive NO production by activated macrophages has been observed. Adipose tissue is composed of various cell types such as mature adipocytes, preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages. Recent studies indicate that obesity is associated with low-grade chronic inflammation of adipose tissues, and that such inflammation is one of the potential mechanisms leading to the insulin resistance. It has been demonstrated that obese adipose tissue is characterized by the increased infiltration of macrophages. Therefore, we attempted to identify natural anti-inflammatory compounds that not only inhibit the secretion of NO from RAW 264.7 cells, but also inhibit triglyceride accumulation in 3T3-L1 adipocytes. This review describes the NO prpduction inhibitory activity or the TG accumulation inhibitory activity of the compounds obtained from 18 plants and a fungi that have been used as traditional medicines.

Cucurbita moschata Duch. and its active component, β-carotene effectively promote the immune responses through the activation of splenocytes and macrophages

Cucurbita moschata Duch. has long been used for traditional health food in many countries. However, to enhance the immune system of Cucurbita moschata Duch. and its major component, β-carotene is not clear. Here, we determined the immune enhancement effect of Cucurbita moschata Duch. and β-carotene in mouse splenocytes and RAW 264.7 macrophage cell line. We prepared baked Cucurbita moschata Duch. (Sweetme Sweet PumpkinTM, SSP) and steamed Cucurbita moschata Duch. (SC). Splenocytes isolated from the spleen of BALB/c mice were treated with SSP, SC, and β-carotene for 24 h. RAW 264.7 cells were stimulated with recombinant interferon-γ (rIFN-γ) for 6 h before treatment with SSP, SC, or β-carotene. SSP, SC and β-carotene significantly up-regulated the proliferation of splenocyte and mRNA expression of KI-67. The levels of interleukin-2 and IFN-γ were up-regulated by SSP, SC, or β-carotene in the splenocytes. SC and β-carotene also increased the levels of tumor necrosis factor-α (TNF-α) in the splenocytes. In addition, SSP, SC, or β-carotene significantly increased the levels of TNF-α through the nuclear translocation of the nuclear factor-κB and phosphorylation of IκBα in the rIFN-γ-primed RAW 264.7 cells. These data indicate that Cucurbita moschata Duch. and β-carotene may have an immune-enhancing effect through the production of Th1 cytokines by activation of splenocytes and macrophages.

Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis.

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.