BRD4 Inhibition Attenuates Inflammatory Pain by Ameliorating NLRP3 Inflammasome-Induced Pyroptosis.

Chronic pain, such as persistent inflammatory pain, remains a public health problem that has no effective treatment at present. Bromodomain-containing protein 4 (BRD4) inhibition, induced by JQ1 injection or BRD4 knockdown, has been used to attenuate inflammatory pain; However, it remains elusive whether BRD4 aggravates inflammatory pain by regulating inflammasome. Western blot and immunofluorescence staining showed that BRD4 expression increased after administration of complete Freund's adjuvant (CFA) and reached its peak on day 3. Immunofluorescence staining showed that BRD4 was mainly colocalized with NeuN-positive neurons in the spinal cord, which was accompanied by upregulation of inflammasome component proteins, such as NLRP3, gasdermin D, and caspase-1. JQ1 was intrathecally injected into mice 1 h before CFA administration, and the mechanical and thermal hyperalgesia levels were measured on days 1, 3, and 7 after CFA administration. CFA-induced inflammatory pain, paw inflammation, and swelling were attenuated by pre-treatment with JQ1. To our knowledge, this study was the first to prove that NLRP3 inflammasome-induced neuronal pyroptosis participates in inflammatory pain. BRD4 inhibition decreased the expression of pyroptosis-related proteins by inhibiting the activation of NF-κB signaling pathway, both in vivo and in vitro. Taken together, BRD4 inhibition exerted analgesic and anti-inflammatory effects against inflammatory pain by inhibiting NF-κB and inflammasome activation, which protected neural cells from pyroptosis.

Astrocyte-neuron lactate transport in the ACC contributes to the occurrence of long-lasting inflammatory pain in male mice.

Lactate transport is an important means of communication between astrocytes and neurons and is implicated in a variety of neurobiological processes. However, the connection between astrocyte-neuron lactate transport and nociceptive modulation has not been well established. Here, we found that Complete Freund's adjuvant (CFA)-induced inflammation pain leads to a significant increase in extracellular lactate levels in the anterior cingulate cortex (ACC). Inhibition of glycogenolysis and lactate release in the ACC disrupted the persistent, but not acute, inflammation pain induced by CFA, and this effect was reversed by exogenous L-lactate administration. Knocking down the expression of lactate transporters (MCT1, MCT4, or MCT2) also disrupted the long lasting inflammation pain induced by CFA. Moreover, glycogenolysis in the ACC is critical for the induction of molecular changes related to neuronal plasticity, including the induction of phospho- (p-) ERK, p-CREB, and Fos. Taken together, our findings indicate that astrocyte-neuron lactate transport in the ACC is critical for the occurrence of persistent inflammation pain, suggesting a novel mechanism underlying chronic pain.

Chronic presence of blood circulating anti-NMDAR1 autoantibodies impairs cognitive function in mice.

High titers of anti-NMDAR1 autoantibodies in brain cause anti-NMDAR1 encephalitis that displays psychiatric symptoms of schizophrenia and/or other psychiatric disorders in addition to neurological symptoms. Low titers of anti-NMDAR1 autoantibodies are reported in the blood of a subset of the general human population and psychiatric patients. Since ~0.1-0.2% of blood circulating antibodies cross the blood-brain barriers and antibodies can persist for months and years in human blood, it is important to investigate whether chronic presence of these blood circulating anti-NMDAR1 autoantibodies may impair human cognitive functions and contribute to the development of psychiatric symptoms. Here, we generated mice carrying low titers of anti-NMDAR1 autoantibodies in blood against a single antigenic epitope of mouse NMDAR1. Mice carrying the anti-NMDAR1 autoantibodies are healthy and display no differences in locomotion, sensorimotor gating, and contextual memory compared to controls. Chronic presence of the blood circulating anti-NMDAR1 autoantibodies, however, is sufficient to impair T-maze spontaneous alternation in the integrity of blood-brain barriers across all 3 independent mouse cohorts, indicating a robust cognitive deficit in spatial working memory and/or novelty detection. Our studies implicate that chronic presence of low titers of blood circulating anti-NMDAR1 autoantibodies may impair cognitive functions in both the general healthy human population and psychiatric patients.

Effective Model of Food Allergy in Mice Sensitized with Ovalbumin and Freud's Adjuvant.

To better explore the pathophysiology of FA and its therapy, we aimed to establish a simple and practicable FA model with Freund's adjuvant and introduce an easy and reliable laboratory evaluation method for assessment of inflammation in intestinal segments at different anatomical locations. BALB/c mice were sensitized with ovalbumin combined with Freund's adjuvant. Complete Freund's adjuvant was chosen for the first sensitization and two weeks later incomplete Freund's adjuvant was used for a second sensitization. Two weeks later, the sensitized mice were challenged with 50 mg ovalbumin every other day. After the 6 challenge, all mice were assessed for systemic anaphylaxis, and then sacrificed for sample collection. All sensitized mice showed anaphylactic symptoms and markedly increased levels of serum ovalbumin-specific IgE and IgG1. The activity of mast cell protease-1 (mMCPT-1) was significantly increased in the serum and interstitial fluid of the duodenum, jejunum, ileum, and colon. A successful FA model was established, of which inflammation occurred in the duodenum, jejunum, ileum, and colon. This model provides a reliable and simple tool for analysis of the mechanism of FA and methods of immunotherapy. Moreover, combined detection of ovalbumin-specific antibody and local mMCPT-1 levels could potentially be used as the major indicator for assessment of food allergy.

Daphnetin inhibits spinal glial activation via Nrf2/HO-1/NF-κB signaling pathway and attenuates CFA-induced inflammatory pain.

Daphnetin (7, 8-dihydroxycoumarin, DAPH), a coumarin derivative isolated from Daphne odora var., recently draws much more attention as a promising drug candidate to treat neuroinflammatory diseases due to its protective effects against neuroinflammation. However, itscontribution to chronic inflammatory pain is largely unknown. In the current work, we investigated the effects of DAPH in a murine model of inflammatory pain induced by complete Freund's adjuvant (CFA) and its possible underlying mechanisms. Our results showed that DAPH treatment significantly attenuated mechanical allodynia provoked by CFA. A profound inhibition of spinal glial activation, followed by attenuated expression levels of spinal pro-inflammatory cytokines, was observed in DAPH-treated inflammatory pain mice. Further study demonstrated that DAPH mediated negative regulation of spinal NF-κB pathway, as well as its preferential activation of Nrf2/HO-1 signaling pathway in inflammatory pain mice. This study, for the first time, indicated that DAPH might preventthe development of mechanical allodynia in mice with inflammatory pain. And more importantly, these data provide evidence for the potential application of DAPH in the treatment of chronic inflammatory pain.

Immunogenicity in humans of a transdermal multipeptide melanoma vaccine administered with or without a TLR7 agonist.

BACKGROUND: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine. METHODS: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot. RESULTS: CD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%. CONCLUSIONS: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

Generation and characterization of a high-affinity chimeric anti-OX40 antibody with potent antitumor activity.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

Effects of Gliadin on Autoimmune Responses of Central Nervous System of C57BL/6 Mice.

Gluten sensitivity contributes to various degrees of neurological manifestations and neurodegenerative immunological changes. We investigated the experimental features of anti-gliadin immune responses in the central nervous system (CNS) of mice. Female C57BL6 mice were divided into three groups. Mice immunized with complete Freund's adjuvant (CFA) or gliadin emulsified in CFA, and the control group received phosphate-buffered saline (PBS). Immunohistochemistry, hematoxylin-eosin, and Luxol fast blue staining were performed on the sections. The serum levels of interleukin (IL)-17 and interferon-gamma (IFN-γ) were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the mRNA levels of chemokine (C-X-C motif) ligand-2 (CXCL-2), C-C motif chemokine ligand-2 (CCL-2), and CXCL-10.  In gliadin+CFA immunized mice, the microscopic lesions included perivascular edema, focal-microgliosis, and acute neuronal necrosis in the cortex, subcortical, Purkinje cell layer, and ventral horn of the spinal cord. While extravasation of anti-IgG antibodies and selective targeting of Purkinje cells were observed in gliadin+CFA immunized mice. A significant increase in serum IL-17 and IFN-g levels (p<0.05), as well as expression of CXCL-2, CCL-2, and CXCL-10 in mice immunized with gliadin+CFA, were monitored versus controls. Our findings indicated that the immune responses directed against gliadin peptides might contribute to blood-brain barrier breakdown, extravasation of serum anti-IgG, gliosis, and acute neuronal necrosis in the cortex and cerebellar Purkinje cells. Anti-IgG antibodies may cause extravasation of blood-born anti-gliadin antibodies and selective targeting of Purkinje cells observed in mice immunized with peptide tryptic (pt) -gliadin in CFA.

Bioflavonoid Robinin from Astragalus falcatus Lam. Mildly Improves the Effect of Metothrexate in Rats with Adjuvant Arthritis.

Anti-inflammatory potential of orally administrated bioflavonoid-robinin, active sub-stance of original drug Flaroninum™ (FL), was investigated in the combination with methotrexate (MTX) and in monotherapy in rats suffering from adjuvant-induced arthritis (AA). Robinin (kaempferol-3-O-robinoside-7-O-rhamnoside) was isolated from the aerial parts of Astragalus falcatus Lam. The monotherapy with robinin was not efficient in alleviating symptoms of AA. The combination of MTX with robinin was similarly active as MTX alone in reducing the hind paw volume and change of body weight during the whole experiment. The combination, however, reduced plasma levels of Interleukin-17Aand activity of gamma-glutamyl transferase in joint more efficiently then MTX alone. Our results demonstrate that the novel combination of robinin and MTX mildly improved the reduction of inflammation in experimental arthritis.

Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice.

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund's adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Anticentromere antibody induced by immunization with centromere protein a and Freund's complete adjuvant may interfere with mouse oocyte meiosis.

BACKGROUND: Anticentromere antibody (ACA) is a member of the antinuclear antibody (ANA) family, and recent studies have found that ACA may be associated with oocyte maturation disorders; however, the possible mechanism behind this phenomenon remains unknown. We conducted this study to investigate whether ACA could penetrate into the living oocytes and interfere with oocyte meiosis in a mouse model. METHODS: We divided mice into three groups: human recombinant centromere protein-A (human CENP-A, HA) and complete Freund's adjuvant (CFA) were used to immunize mice for the study group (HA + CFA), and mice injected with CFA (CFA group) or saline (Saline group), respectively, served as controls. After immunization, serum anti-CENP-A antibody was detected by indirect immunofluorescence assay (IIFT) and enzyme-linked immunosorbent assay (ELISA). Chromosome alignment and intracellular IgG localization in MI- and MII-stage oocytes were investigated by immunofluorescence analysis. RESULTS: Positive ACAs were successfully induced by immunization with CENP-A and CFA, and results showed that the serum level of anti-CENP-A antibody was significantly higher in the HA + CFA group compared with the control groups. There was marked increase of chromosome misalignments in MI and MII oocytes in the HA + CFA group compared to the control groups. However, no oocytes from any of the three groups showed intracellular antibody immunofluorescence. CONCLUSIONS: The development and maturation of oocytes were impaired in peripheral ACA positive mice, which exhibited severe chromosomal misalignments in metaphase meiosis; however, no evidence of ACAs entering the oocytes was observed, thus the underlying mechanism needs further exploration.

Protective Effects of Chicken Egg Yolk Immunoglobulins (IgYs) against Vibrio vulnificus Infections.

Vibrio (V.) vulnificus infection is a rare disease whose death rates exceed 50% despite aggressive antibiotic treatment and surgical debridement. The aim of this study was to assess the ability of specific anti-V. vulnificus immunoglobulins Y (IgYs) for preventing and treating V. vulnificus infections. IgYs were produced by immunizing egg laying hens with inactivated whole cell bacteria. Peritoneal cytokines, blood's bacterial load, and survival curves were obtained from both prophylactic and therapeutic mouse models. The results showed that the specific IgYs (i) inhibited the growth of V. vulnificus in vitro, (ii) dramatically reduced the inflammatory response and blood's bacterial load, and (iii) improved the survival rate of V. vulnificus-infected mice. These results prove that anti-V. vulnificus IgYs can be markedly effective means for the prophylaxis and the therapy of V. vulnificus infections.

Trial to evaluate the immunogenicity and safety of a melanoma helper peptide vaccine plus incomplete Freund's adjuvant, cyclophosphamide, and polyICLC (Mel63).

BACKGROUND: Peptide vaccines designed to stimulate melanoma-reactive CD4+ T cells can induce T cell and antibody (Ab) responses, associated with enhanced overall survival. We hypothesized that adding toll-like receptor 3 agonist polyICLC to an incomplete Freund's adjuvant (IFA) would be safe and would support strong, durable CD4+ T cell and Ab responses. We also hypothesized that oral low-dose metronomic cyclophosphamide (mCy) would be safe, would reduce circulating regulatory T cells (T-regs) and would further enhance immunogenicity. PARTICIPANTS AND METHODS: An adaptive design based on toxicity and durable CD4+ T cell immune response (dRsp) was used to assign participants with resected stage IIA-IV melanoma to one of four study regimens. The regimens included a vaccine comprising six melanoma peptides restricted by Class II MHC (6MHP) in an emulsion with IFA alone (Arm A), with IFA plus systemic mCy (Arm B), with IFA+ local polyICLC (Arm C), or with IFA+ polyICLC+ mCy (Arm D). Toxicities were recorded (CTCAE V.4.03). T cell responses were measured by interferon γ ELIspot assay ex vivo. Serum Ab responses to 6MHP were measured by ELISA. Circulating T-regs were assessed by flow cytometry. RESULTS: Forty-eight eligible participants were enrolled and treated. Early data on safety and dRsp favored enrollment on arm D. Total enrollment on Arms A-D were 3, 7, 6, and 32, respectively. Treatment-related dose-limiting toxicities (DLTs) were observed in 1/7 (14%) participants on arm B and 2/32 (6%) on arm D. None exceeded the 25% DLT threshold for early closure to enrollment for any arm. Strong durable T cell responses to 6MHP were detected ex vivo in 0%, 29%, 67%, and 47% of participants on arms A-D, respectively. IgG Ab responses were greatest for arms C and D. Circulating T-regs frequencies were not altered by mCy. CONCLUSIONS: 6MHP vaccines administered with IFA, polyICLC, and mCy were well tolerated. The dRsp rate for arm D of 47% (90% CI 32 to 63) exceeded the 18% (90% CI 11 to 26) rate previously observed with 6MHP in IFA alone. Vaccination with IFA+ polyICLC (arm C) also showed promise for enhancing T cell and Ab responses.

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

Identification of CTL Epitopes on Efflux Pumps of the ATP-Binding Cassette and the Major Facilitator Superfamily of Mycobacterium tuberculosis.

Tuberculosis is the world's most deadly infectious disease, with 10 million people falling ill and 1.5 million people dying from the disease every year. With the increasing number of drug-resistant Mycobacterium tuberculosis (MTB) strains and prevalence of coinfection of MTB with human immunodeficiency virus, many challenges remain in the prevention and treatment of tuberculosis. Therefore, the development of safe and effective tuberculosis vaccines is an urgent issue. In this study, we identified cytotoxic T lymphocyte epitopes on drug resistance-associated membrane protein efflux pumps of MTB, the ATP-binding cassette and the major facilitator superfamilies. First, three online software were used to predict HLA-A2-restricted epitopes. Then, the candidate epitopes were confirmed with the T2A2 cell binding affinity and peptide/MHC (pMHC) complex stability assays and in vitro immune activity experiments. Two drug-resistant T lymphocyte epitopes, designated Rv1218c-p24 and Rv2477c-p182, were selected, and their immunogenic activities studied in vivo in genetically engineered mice. The immune activities of these two epitopes were improved with the help of complete Freund's adjuvant (CFA). The epitopes identified here provide a foundation for the diagnosis and treatment of patients infected with drug resistant and the future development of a multiepitope vaccine.

Protective effect of Corynoline on the CFA induced Rheumatoid arthritis via attenuation of oxidative and inflammatory mediators.

Rheumatoid arthritis (RA) is a long-standing and growing autoimmune disease. Therefore, the present study was intended to investigate the effect of Corynoline (COR) on CFA induced rheumatoid arthritis in a rat model. Results suggested that COR causes significant reduction in paw swelling, edema, arthritis score, thymus and spleen indexes and neutrophil infiltration (p < 0.01). Moreover, the levels of inflammatory cytokines (interleukin- 1ß, -6, and -17, and TNF-α) and anti-collagen II-specific immunoglobulins (IgG1 and IgG2a) were decreased significantly (p < 0.01) together with increase in antioxidant enzymes (SOD, CAT, and GSH) (p < 0.01) in COR-treated group in dose-dependent manner. In western blot analysis, COR-treated group showed concentration-dependent reduction of expression of COX-2, 5-LOX and NF-p65 as compared to CFA rats. Moreover, COR-treated group showed mild inflammation of cartilage with fewer cartilage erosion and synovititis with most significant reversal of arthritic features in the rats treated with 30 mg/kg. It has been concluded that, COR alleviates oxidative stress and inflammation in arthritic rats, thus verifying its anti-rheumatoid arthritis property.

Deficit in voluntary wheel running in chronic inflammatory and neuropathic pain models in mice: Impact of sex and genotype.

Patients with chronic pain report decreased general activity and emotional distress. Therefore, the development of various animal models that encompass different aspects of pain are crucial for the discovery of genetic differences and the assessment of novel analgesics to improve quality of life. C57BL/6J and DBA/2J mice received unilateral intraplantar injections of 100 % CFA, paclitaxel, or CCI surgery to compare their distance traveled in a voluntary wheel running assay, paw edema diameter, and mechanical sensitivity. Mechanical withdrawal thresholds were lower in both strains of mice that received CFA when compared to their vehicle. However, a decrease in distance traveled was observed in CFA-treated C57BL/6J but not DBA/2J mice. In a separate group, chemotherapy agent paclitaxel 8 mg/kg, i.p. was administered to both strains of mice to induce CIPN which was confirmed by lower mechanical thresholds in paclitaxel-treated mice compared to vehicle-treated mice. Only female C57BL/6J mice showed attenuation of distance traveled following treatment, whereas male C57BL/6J and DBA/2J mice did not. Lastly, C57BL/6J mice underwent chronic constriction injury (CCI) or sham surgery to observe the impact of another chronic neuropathic pain model in wheel running assay. CCI mice showed a gradual decrease in mechanical withdrawal threshold and a decrease in distance traveled compared to sham 5 days following the procedure. Comparing these chronic inflammatory and neuropathic pain models in different mouse strains may help us better understand genetic differences underlying pain perception and its impact on reflexive and nonreflexive outcome measures.

Ceria-based nanotheranostic agent for rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that affects 1-2% of the human population worldwide, and effective therapies with targeted delivery for local immune suppression have not been described. We address this problem by developing a novel theranostic nanoparticle for RA and assessed its therapeutic and targeting effects under image-guidance. Methods: Albumin-cerium oxide nanoparticles were synthesized by the biomineralization process and further conjugated with near-infrared, indocyanine green (ICG) dye. Enzymatic-like properties and reactive oxygen species (ROS) scavenging activities, as well as the ability to reprogram macrophages, were determined on a monocyte cell line in culture. The therapeutic effect and systemic targeting potential were evaluated in collagen-induced arthritis (CIA) mouse model using optical/optoacoustic tomographic imaging. Results: Small nanotheranostics with narrow size distribution and high colloidal stability were fabricated and displayed high ROS scavenging and enzymatic-like activity, as well as advanced efficacy in a converting pro-inflammatory macrophage phenotype into anti-inflammatory phenotype. When administrated into affected animals, these nanoparticles accumulated in inflamed joints and revealed a therapeutic effect similar to the gold-standard therapy for RA, methotrexate. Conclusions: The inflammation-targeting, inherent contrast and therapeutic activity of this new albumin-cerium oxide nanoparticle may make it a relevant agent for assessing severity in RA, and other inflammatory diseases, and controlling inflammation with image-guidance. The design of these nanotheranostics will enable potential clinical translation as systemic therapy for RA.

Determination of in vitro and in vivo immune response to recombinant cholesterol oxidase from Mycobacterium tuberculosis.

Cholesterol oxidase (ChoD) is an enzyme that is involved but is dispensable in the process of cholesterol degradation by Mycobacterium tuberculosis (Mtb). Interestingly, ChoD is a virulence factor of Mtb, and it strongly modulates the function of human macrophages in vitro, allowing the intracellular survival of bacteria. Here, we determined the immunogenic activity of recombinant ChoD from Mtb in a mouse model. We found that peritoneal exudate cells obtained from mice injected i.p. with ChoD but not those from mice injected with PBS responded in vitro with highly spontaneous, as well as phorbol 12-myristate 13-acetate (PMA)-stimulated, production of reactive oxygen species (ROS). However, ChoD significantly reduced the ROS response to PMA in re-stimulated cells in vitro. The cytokine secretion pattern in mice immunized s.c. with ChoD emulsified with incomplete Freund's adjuvant (IFA) showed evidence of Th2-induced or proinflammatory immune responses. The main cytokines detected in sera were interleukin (IL) 6 and 5, tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein 1, while IL-2 and IL-12 as well as interferon γ were undetectable. Similarly, ChoD protein alone activated THP-1-derived macrophages to release proinflammatory IL-6, IL-8 and TNF-α, in vitro. Moreover, a statistically significant predominance of the IgG1 isotype over that of IgG2a in the sera of mice immunized with ChoD/IFA was observed. In conclusion, we demonstrated here that ChoD of Mtb is an active protein, which is able to induce the immune response both in vivo and in vitro.

The recombinant anti-TNF-α fusion protein ameliorates rheumatoid arthritis by the protective role of autophagy.

The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signaling that cause collateral damage to protective signaling cascades carrying the potential for unwanted side effects. The variable domains of heavy-chain only antibodies (HCAbs) discovered in Camelidae are stable and display to be fully functional in antigen-binding against variable targets, which seem to be attractive candidates for the next-generation biologic drug study. The purpose of our study was to establish a simple prokaryotic expression system for large-scale expression, purification, and refolding of the recombinant anti-tumor necrosis factor α (TNF-α) fusion protein (FVH1-1) from inclusion bodies. Over 95% purity of the recombinant anti-TNF-α fusion proteins was obtained by just one purification step in our developed prokaryotic expression system, while the results of surface plasmon resonance (SPR) established the high-efficiency potent binding ability of FVH1-1 to human TNF-α. The counteraction of TNF-α cytotoxic effect experiment on the mouse fibroblast fibrosarcoma cell line (L929) confirmed that the expressed FVH1-1 were able to selectively and highly combine with human recombinant TNF-α (hTNF-α) in vitro. Western blot results showed that FVH1-1 can inhibit the activation of caspase-9 and PARP, which are the apoptotic signaling pathway proteins activated by hTNF-α. Meanwhile, lysosome autophagy signaling pathways stimulated by hTNF-α were inhibited by FVH1-1, which down-regulated the expression of LC3II/LC3I and up-regulated the expression of P62, indicating that the autophagy linked with TNF-α-induced apoptosis in response to rheumatoid arthritis. The results of the AIA rat model experiment presented that FVH1-1 can reduce the degree of joint swelling and inflammatory factors to a certain extent in vivo.