Efficacy and safety of ReDuNing injection as a treatment for COVID-19 and its inhibitory effect against SARS-CoV-2.

BACKGROUND: Although the rapid emergence of coronavirus disease 2019 (COVID-19) poses a considerable threat to global public health, no specific treatment is available for COVID-19. ReDuNing injection (RDN) is a traditional Chinese medicine known to exert antibacterial, antiviral, antipyretic, and anti-inflammatory effects. In addition, RDN has been recommended in the diagnosis and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pneumonia by the National Health Council and the National Administration of Chinese Medicine. However, there is no information regarding its efficacy against COVID-19. AIM OF STUDY: This study was designed to determine the clinical efficacy of RDN in patients with COVID-19 and characterize its antiviral activity against SARS-CoV-2 in vitro. MATERIALS AND METHODS: A total of 50 adults with COVID-19 were included in this study, and the primary endpoint was recovery from clinical symptoms following 14 days of treatment. General improvements were defined as the disappearance of the major symptoms of infection including fever, fatigue, and cough. The secondary endpoints included the proportion of patients who achieved clinical symptom amelioration on days 7 and 10, time to clinical recovery, time to a negative nucleic acid test result, duration of hospitalization, and time to defervescence. Plaque reduction and cytopathic effect assays were also performed in vitro, and reverse-transcription quantitative PCR was performed to evaluate the expression of inflammatory cytokines (TNF-α, IP-10, MCP-1, IL-6, IFN-α, IFN-γ, IL-2 and CCL-5) during SARS-CoV-2 infection. RESULTS: The RDN group exhibited a shorter median time for the resolution of clinical symptoms (120 vs. 220 h, P < 0.0001), less time to a negative PCR test result (215 vs. 310 h, P = 0.0017), shorter hospitalization (14.8 vs. 18.5 days, P = 0.0002), and lower timeframe for defervescence (24.5 vs. 75 h, P = 0.0001) than the control group. In addition, time to improved imaging was also shorter in the RDN group than in the control group (6 vs.8.9 days, P = 0.0273); symptom resolution rates were higher in the RDN group than in the control group at 7 (96.30% vs. 39.13%, P < 0.0001) and 10 days (96.30% vs. 56.52%, P = 0.0008). No allergic reactions or anaphylactic responses were reported in this trial. RDN markedly inhibited SARS-CoV-2 proliferation and viral plaque formation in vitro. In addition, RDN significantly reduced inflammatory cytokine production in infected cells. CONCLUSIONS: RDN relieves clinical symptoms in patients with COVID-19 and reduces SARS-CoV-2 infection by regulating inflammatory cytokine-related disorders, suggestion that this medication might be a safe and effective treatment for COVID-19.

Betulinic Acid-Azaprostanoid Hybrids: Synthesis and Pharmacological Evaluation as Anti-inflammatory Agents.

BACKGROUND: Prevention and treatment of chronic inflammatory diseases require effective and low-toxic medicines. Molecular hybridization is an effective strategy to enhance the biological activity of new compounds. Triterpenoid scaffolds are in the focus of attention owing to their anti-inflammatory, antiviral, antiproliferative, and immunomodulatory activities. Heteroprostanoids have different pleiotropic effects in acute and chronic inflammatory processes. OBJECTIVE: The study aimed to develop structurally new and low toxic anti-inflammatory agents via hybridization of betulinic acid with azaprostanoic acids. METHODS: A series of betulinic acid-azaprostanoid hybrids was synthesized. The synthetic pathway included the transformation of betulin via Jones' oxidation into betulonic acid, reductive amination of the latter and coupling obtained by 3ß-amino-3-deoxybetulinic acid with the 7- or 13-azaprostanoic acids and their homo analogues. The hybrids 1-9 were investigated in vivo on histamine-, formalin- and concanavalin A-induced mouse paw edema models and two models of pain - the acetic acid-induced abdominal writhing and the hotplate test. The hybrids were in vitro evaluated for cytotoxic activity on cancer (MCF7, U- 87 MG) and non-cancer humane cell lines. RESULTS: In the immunogenic inflammation model, the substances showed a pronounced anti-inflammatory effect, which was comparable to that of indomethacin. In the models of the exudative inflammation, none of the compounds displayed a statistically significant effect. The hybrids produced weak or moderate analgesic effects. All the agents revealed low cytotoxicity on human immortalized fibroblasts and cancer cell lines compared with 3ß- amino-3-deoxybetulinic acid and doxorubicin. CONCLUSION: The results indicate that the principal anti-inflammatory effect of hybrids is substantially provided with the triterpenoid scaffold and in some cases with the azaprostanoid scaffold, but the latter makes a significant contribution to reducing the toxicity of hybrids. Hybrid 1 is of interest as a potent low toxic agent against immune-mediated inflammation.

Screening of medicinal herbs for cytotoxic activity to leukemia cells.

PURPOSE: This study aimed to find a new source of anti-leukemia agents from Vietnamese medicinal plants. METHODS: The human leukemia cell lines TCCY, KU-812, TCC-S, KOPB-26, and HL60 were used. The crude ethanol extracts of 17 medicinal plants were collected and evaluated for their cytotoxicity against these leukemia cell lines by the trypan blue dye exclusion test. Morphological changes of cells were observed under phase-contrast inverted microscope. Bioactive compounds were evaluated using the method described by Ciulei. 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was carried out for evaluating the antioxidant effect. RESULTS: Among the tested samples, Artemisia vulgaris (A.vulgaris) crude ethanol extract effectively inhibited the viability of leukemia cell in both dose and time-dependent manner. The IC50 value was different for cell lines and ranged from 18.07±1.64 µg/ml to 45.87±3.49 µg/ml. Moreover, the phytoconstituents analysis results showed coumarin, flavonoid, anthocyanin, cardiac glycoside, tannins, reduced sugar compounds were present in the A.vulgaris extract. The total polyphenol and flavonoid contents of the dry extract were calculated as 3.81 mg GAE/g dry weight and 11.64 mg RUE/g dry weight of A.vulgaris. A.vulgaris exhibited antioxidant activity with IC50 is 145.10 ± 6.34 µg/ml. CONCLUSION: Among the 17 Vietnamese plants used to treat a variety of cancer-related diseases, A.vulgaris has been able to suppress the growth of leukemia cells.

MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities.

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (ß-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and ß-arrestin endpoints and a specificity of 93% (ß-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.

Cross-company evaluation of the human lymphocyte activation assay.

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.

Assessment of IFN-γ and granzyme-B production by in "sitro" technology.

Tumor neantigens (TNAs) and tumor-associated antigens (TAAs) are crucial triggers of anticancer immune responses. Through major histocompatibility complex, such antigens activate T cells, which, by releasing interferon gamma (IFN-γ) and granzyme B (GRZB), act as crucial effectors against tumor onset and progression. However, in response to immune pressure, cancer cells use different strategies to favor the establishment of an immunosuppressive tumor microenvironment (TME). Elucidating the dynamics of tumor-host co-evolution provides novel opportunities for personalized cancer immunotherapies. The in sitro (in vitro+in situ) technology is an experimental approach involving the preparation of heterocellular cell suspensions from fresh tumors and their use in vitro. The in sitro experimental setup offers the possibility to (1) analyze immune-related parameters (e.g., quantification of cytokines released in the TME), (2) reveal the mechanism of action of drugs, and (3) unveil crucial cell-intrinsic and cell-extrinsic processes boosting anticancer immune responses. Nonetheless, the in sitro technology does not fully recapitulate the complexity of the tissue "in situ" nor models the patterns of infiltrating immune cell localization, and hence parallel experimentation should be scheduled. In this chapter we discuss in sitro technology to analyze and quantify IFN-γ and GRZB production by T cells either co-cultured with cancer cells in the presence of exogenous adjuvant stimuli (i.e., an antibody targeting the immune checkpoint programmed cell death protein 1, and recombinant calreticulin) and boosting with TAAs (i.e., the model SIINFEKL ovalbumin antigen). Specifically, we describe IFN-γ and GRZB quantification by flow cytometry, ELISA and ELISpot technologies.

Psychoneuroimmunology and Natural Killer Cells: The Chromium-Release Whole-Blood Assay.

Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter describes a chromium (51Cr)-release bioassay designed to measure to the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 erythroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4-h in vitro assay.

Comparison between xCELLigence biosensor technology and conventional cell culture system for real-time monitoring human tenocytes proliferation and drugs cytotoxicity screening.

BACKGROUND: Local injections of anesthetics, NSAIDs, and corticosteroids for tendinopathies are empirically used. They are believed to have some cytotoxicity toward tenocytes. The maximal efficacy dosages of local injections should be determined. A commercial 2D microfluidic xCELLigence system had been developed to detect real-time cellular proliferation and their responses to different stimuli and had been used in several biomedical applications. The purpose of this study is to determine if human tenocytes can successfully proliferate inside xCELLigence system and the result has high correlation with conventional cell culture methods in the same condition. METHODS: First passage of human tenocytes was seeded in xCELLigence and conventional 24-well plates. Ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone with different concentrations (100, 50, and 10% diluted of clinical usage) were exposed in both systems. Gene expression of type I collagen, type III collagen, tenascin-C, decorin, and scleraxis were compared between two systems. RESULTS: Human tenocytes could proliferate both in xCELLigence and conventional cell culture systems. Cytotoxicity of each drug revealed dose-dependency when exposed to tenocytes in both systems. Significance was found between groups. All the four drugs had comparable cytotoxicity in their 100% concentration. When 50% concentration was used, betamethasone had a relatively decreased cytotoxicity among them in xCELLigence but not in conventional culture. When 10% concentration was used, betamethasone had the least cytotoxicity. Strong and positive correlation was found between cell index of xCELLigence and result of WST-1 assay (Pearson's correlation [r] = 0.914). Positive correlation of gene expression between tenocytes in xCELLigence and conventional culture was also observed. Type I collagen: [r] = 0.823; type III collagen: [r] = 0.899; tenascin-C: [r] = 0.917; decorin: [r] = 0.874; and scleraxis: [r] = 0.965. CONCLUSIONS: Human tenocytes could proliferate inside xCELLigence system. These responses varied when tenocytes were exposed to different concentrations of ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone. The result of cell proliferation and gene expression of tenocytes in both xCELLigence and conventional culture system is strongly correlated. CLINICAL RELEVANCE: xCELLigence culture system may replace conventional cell culture, which made real-time tenocyte proliferation monitoring possible.

Investigation of the Mechanism of Anti-Inflammatory Action and Cytotoxicity of a Semipurified Fraction and Isolated Compounds From the Leaf of Peltophorum africanum (Fabaceae).

Peltophorum africanum extracts have been shown to possess many important medicinal benefits, including anti-inflammatory and antiviral activities. However, the mechanism of action is poorly understood. The mechanism of anti-inflammatory action was determined by measuring the synthesis of cytokines in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage cells in vitro. Compound 1 (CP1), compound 2 (CP2), and fraction F3.3.0 (F3.3.0) significantly reduced the synthesis of interleukin 1ß (IL-1ß) from RAW 264.7 cells (1.18, 1.32, and 0.92 ng/mL), respectively. Similarly, CP1, CP2, and F3.3.0 inhibited the production of IL-2 and tumor necrosis factor-α (TNF-α) by RAW 264.7 cells (0.41, 0.60, 0.74 and 0.11, 0.27, 0.24 ng/mL, respectively. In addition, CP1 and CP2 had lower cytotoxicity toward RAW 264.7 cells, with CP2 indicating the lowest cytotoxicity (LD50 = 207.88 µg/mL). The mechanism of action was found to be via the inhibition of pro-inflammation cytokines (IL-1 ß and TNF-α). This observation may support the use of P africanum to treat pain-related conditions.

Differential Requirements for Tcf1 Long Isoforms in CD8+ and CD4+ T Cell Responses to Acute Viral Infection.

In response to acute viral infection, activated naive T cells give rise to effector T cells that clear the pathogen and memory T cells that persist long-term and provide heightened protection. T cell factor 1 (Tcf1) is essential for several of these differentiation processes. Tcf1 is expressed in multiple isoforms, with all isoforms sharing the same HDAC and DNA-binding domains and the long isoforms containing a unique N-terminal ß-catenin-interacting domain. In this study, we specifically ablated Tcf1 long isoforms in mice, while retaining expression of Tcf1 short isoforms. During CD8+ T cell responses, Tcf1 long isoforms were dispensable for generating cytotoxic CD8+ effector T cells and maintaining memory CD8+ T cell pool size, but they contributed to optimal maturation of central memory CD8+ T cells and their optimal secondary expansion in a recall response. In contrast, Tcf1 long isoforms were required for differentiation of T follicular helper (TFH) cells, but not TH1 effectors, elicited by viral infection. Although Tcf1 short isoforms adequately supported Bcl6 and ICOS expression in TFH cells, Tcf1 long isoforms remained important for suppressing the expression of Blimp1 and TH1-associated genes and for positively regulating Id3 to restrain germinal center TFH cell differentiation. Furthermore, formation of memory TH1 and memory TFH cells strongly depended on Tcf1 long isoforms. These data reveal that Tcf1 long and short isoforms have distinct, yet complementary, functions and may represent an evolutionarily conserved means to ensure proper programming of CD8+ and CD4+ T cell responses to viral infection.

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection.

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine, and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL, or sulfadiazine, and the following parameters analyzed: Cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non-treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model.

Triagem fitoquímica, análise antimicrobiana e citotóxica e dos extratos das plantas: schinus terebinthifolia, maytenus ilicifolia reissek, tabebuia avellanedae, anadenanthera colubrina (Bell. ) Brenan / Phy tochemical selection, antimicrobial and cy totoxic analysis of extracts from plants: schinusterebinthifolia, maytenusilicifolia reissek, tabebuia avellanedae, anadenanthera colubrina ( Vell. ) Brenan

O reino vegetal oferece maior variedade de substâncias potencialmente úteis nas enfermidades humanas, especialmente aquelas causadas por micro-organismos. Um dos maiores problemas de Saúde Pública enfrentado nos últimos anos é o agravamento da resistência bacteriana e isto se tornou mais grave com a dificuldade para a descoberta de novos antibióticos. O objetivo deste estudo foi avaliar o perfil fitoquímico, analisando a atividade antimicrobiana e citotóxica dos extratos das plantas Schinus terebinthifolia, Maytenus ilicifolia Reissek, Tabebuia avellanedae, Anadenanthera colubrina (Vell.) Brenan. Os extratos das plantas estudadas apresentaram taninos e alcalóides em sua composição fitoquímica. T. avellanedae não teve atividade inibitória frente aos micro-organismos testados, enquanto que os outros extratos testados apresentaram atividade inibitória em diferentes concentrações. No teste de citotoxicidade pode-se observar que T. avellanedae apresentou alta toxicidade. A atividade antimicrobiana desejada pode ser encontrada em espécies de plantas medicinais, para isso deve-se verificar cientificamente o uso popular de plantas medicinais, caracterizando seus princípios ativos e o mecanismo de ação destes.

The vegetal kingdom provides a great variety of compounds which are highly useful in human illnesses, especially those caused by microorganisms. One of the most relevant issues during the last decades is bacterial resistance, which is extremely serious due to difficulties in discovering new antibiotic agents. Current study evaluates the phytochemical profile by analyzing the antimicrobial and cytotoxic activity of the extracts of the plants Schinusterebinthifolia, Maytenusilicifolia Reissek, Tabebuia avellanedae, Anadenanthera colubrina (Vell.) Brenan. Plants´ extracts included tannins and alkaloids in their phytochemical compositions. T. avellanedae did not have any inhibitory activity to the microorganisms tested, whereas the other extracts tested presented inhibitory activity at different concentrations. The cytotoxicity test showed that T. avellanedae had high toxicity. The required antimicrobial activity may be found in several species of medicinal plants. However, the use of folk medicine plants should be scientifically assessed so that their main active agents and mechanisms may be characterized.

Use of S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol as an adjuvant improved protective immunity associated with a DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus in mice.

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.

Effect of aged garlic extract on immune responses to experimental fibrosarcoma tumor in BALB/c mice.

BACKGROUND: Aged garlic extract (AGE) has many biological activities including radical scavenging, antioxidative and immunomodulative effects. AIM: In this research work, the antitumor and immunomodulatory effects of AGE against fibrosarcoma implanted tumor were studied. MATERIALS AND METHODS: WEHI-164 fibrosarcoma cells were implanted subcutaneously on day 0 into the right flank of 40 BALB/c mice at age of 8 weeks. Mice were randomly categorized in two separate groups: First received AGE (100 mg/kg, IP), second group as the control group received phosphate buffered saline. Treatments were carried out 3 times/week. Tumor growth was measured and morbidity was recorded. Subpopulations of CD4+/CD8+ T cells were determined using flow cytometry. WEHI-164 cell specific cytotoxicity of splenocytes and in vitro production of interferon gamma (IFN-γ) and interleukin-4 cytokines were measured. RESULTS: The mice received AGE had significantly longer survival time compared with the control mice. The inhibitory effect on tumor growth was seen in AGE treated mice. The CD4+/CD8+ ratio and in vitro IFN-γ production of splenocytes were significantly increased in AGE group. WEHI-164 specific cytotoxicity of splenocytes from AGE mice was also significantly increased at 25:1 E: T ratio. CONCLUSION: Administration of AGE resulted in improved immune responses against experimentally implanted fibrosarcoma tumors in BALB/c mice. AGE showed significant effects on inhibition of tumor growth and longevity of survival times.

Cytotoxicity of erythrosine (E-127), brilliant blue (E-133) and red 40 (E-129) food dyes in a plant test system / Citotoxicidade dos corantes alimentares erythrosine (E-127), azul brilhante (E-133) e red 40 (E-129) em sistema-teste vegetal

The objective of this work was to evaluate the cytotoxic effect of the food dyes erythrosine, brilliant blue and red 40 on the cell cycle of Allium cepa L. Each dye was evaluated at doses of 0.4 and 4.0 ml, at exposure times of 24 and 48 hours, in onion root tip cells. Cells and the presence of chromosomal aberrations were analyzed throughout the whole cell cycle, totaling 5,000 cells for each group of bulbs. The mitotic index was calculated and the statistical analysis was conducted through the Chi-square test (p < 0.05). From the obtained results, it was verified that the food additives erythrosine and brilliant blue were not cytotoxic to the cells of the test system. However, the red 40 dye, at the two evaluated doses and the two exposure times used in this bioassay have promoted a significant reduction in cell division and induced the emergence of anaphasic and telophasic bridge aberrations and micronucleated cells. Additional cytotoxicity studies should be conducted to add information to these and other previously obtained results in order to evaluate, with property, the action of these three dyes on a cellular level.

Este trabalho teve por objetivo avaliar o efeito citotóxico dos corantes alimentares eritrosina, azul brilhante e vermelho 40 sobre o ciclo celular de Allium cepa L. Cada corante foi avaliado nas doses de 0,4 e 4,0mL, nos tempos de exposição de 24 e 48h, em células meristemáticas de raízes de cebolas. Foram analisadas células em todo o ciclo celular e a presença de aberrações cromossômicas, totalizando 5.000 células para cada grupo de bulbos. Calculou-se o índice mitótico e a análise estatística foi feita por meio do teste Qui-quadrado (p < 0,05). A partir dos resultados obtidos, verificou-se que os aditivos alimentares eritrosina e azul brilhante não foram citotóxicos às células do sistema-teste em questão. Já o corante vermelho 40, nas duas doses avaliadas e nos dois tempos de exposição estipulados para este bioensaio, promoveu redução significativa da divisão celular e induziu o aparecimento de aberrações dos tipos ponte anafásica, ponte telofásica e célula micronucleada. Estudos adicionais de citotoxicidade devem ser conduzidos para se somar a estes para assim avaliar, com propriedade, a ação destes três corantes em nível celular.

Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines.

Preclinical characterization of novel nanotechnology-based formulations is often challenged by physicochemical characteristics, sterility/sterilization issues, safety and efficacy. Such challenges are not unique to nanomedicine, as they are common in the development of small and macromolecular drugs. However, due to the lack of a general consensus on critical characterization parameters, a shortage of harmonized protocols to support testing, and the vast variety of engineered nanomaterials, the translation of nanomedicines into clinic is particularly complex. Understanding the immune compatibility of nanoformulations has been identified as one of the important factors in (pre)clinical development and requires reliable in vitro and in vivo immunotoxicity tests. The generally low sensitivity of standard in vivo toxicity tests to immunotoxicities, inter-species variability in the structure and function of the immune system, high costs and relatively low throughput of in vivo tests, and ethical concerns about animal use underscore the need for trustworthy in vitro assays. Here, we consider the correlation (or lack thereof) between in vitro and in vivo immunotoxicity tests as a mean to identify useful in vitro assays. We review literature examples and case studies from the experience of the NCI Nanotechnology Characterization Lab, and highlight assays where predictability has been demonstrated for a variety of nanomaterials and assays with high potential for predictability in vivo.

Thai herbal formulas used for wound treatment: a study of their antibacterial potency, anti-inflammatory, antioxidant, and cytotoxicity effects.

AIM: This present work was aimed to investigate wound healing-related biologic activities of traditional herbal formulas used for wound treatment in southern Thailand. METHODS: Water and ethanol extracts of the formulas (THR-SK004, THR-SK010, and THR-SK011) were tested for their antibacterial potency against methicillin-resistant Staphylococcus aureus (MRSA) and -susceptible S. aureus. Anti-inflammatory activities of the extracts were assessed by detection of the inhibition of lipopolysaccharide-induced nitric oxide production. Anti-oxidant activities and cytotoxicity of the extracts were also measured. RESULTS: Among the tested formulas, ethanol extract of THR-SK010 consisting of four herbs: Curcuma longa L., Areca catechu L., Oryza sativa L., and Garcinia mangostana L., was found to possess promising antibacterial activities with MIC90 of 4 µg mL(-1) against MRSA isolates. This ethanol extract offered the highest anti-inflammatory activity as well as DPPH and hydroxyl radical scavenging activities. CONCLUSIONS: Remarkable antibacterial, anti-inflammatory, and antioxidant activities as well as low toxicity on Vero cells of THR-SK010 ethanol extract provide scientific information to support the topical use of the formula for wound treatment. This information proposes the potential to develop a new generation of phytopharmaceuticals based on traditional knowledge.

After TGN1412: recent developments in cytokine release assays.

The failure of regulatory science to keep pace with and support the development of new biological medicines was very publically highlighted in March 2006 when the first-in-man Phase I clinical trial of the immunomodulatory CD28-specific monoclonal antibody (mAb) TGN1412 ended in disaster when all six volunteers suffered a life-threatening adverse reaction termed a 'Cytokine Storm'. The poor predictive value of standard pre-clinical safety tests and animal models applied to TGN1412 demonstrated the need for a new generation of immunotoxicity assays and animal models that are both sensitive and predictive of clinical outcome in man. The non-predictive result obtained from pre-clinical safety testing in cynomolgus macaques has now been attributed to a lack of CD28 expression on CD4+ effector memory T-cells that therefore cannot be stimulated by TGN1412. In contrast, high levels of CD28 are expressed on human CD4+ effector memory T-cells, the source of most TGN1412-stimulated pro-inflammatory cytokines. Standard in vitro safety tests with human cells were also non-predictive as they did not replicate in vivo presentation of TGN1412. It was subsequently shown that, if an immobilized therapeutic mAb-based assay or endothelial cell co-culture assay was used to evaluate TGN1412, then these would have predicted a pro-inflammatory response in man. New in vitro assays based on these approaches are now being applied to emerging therapeutics to hopefully prevent a repeat of the TGN1412 incident. It has emerged that the mechanism of pro-inflammatory cytokine release stimulated by TGN1412 is different to that of other therapeutic mAbs, such that standard pro-inflammatory markers such as TNFα and IL-8 are not discriminatory. Rather, IL-2 release and lymphoproliferation are optimal readouts of a TGN1412-like pro-inflammatory response.

A randomized placebo-controlled trial of massage therapy on the immune system of preterm infants.

OBJECTIVES: The aim of this study was to investigate the effects of massage therapy (MT) on the immune system of preterm infants. The primary hypothesis was that MT compared with sham therapy (control) will enhance the immune system of stable premature infants by increasing the proportion of their natural killer (NK) cell numbers. METHODS: A randomized placebo-controlled trial of MT versus sham therapy (control) was conducted among stable premature infants in the NICU. Study intervention was provided 5 days per week until hospital discharge for a maximum of 4 weeks. Immunologic evaluations (absolute NK cells, T and B cells, T cell subsets, and NK cytotoxicity), weight, number of infections, and length of hospital stay were also evaluated. RESULTS: The study enrolled 120 infants (58 massage; 62 control). At the end of the study, absolute NK cells were not different between the 2 groups; however, NK cytotoxicity was higher in the massage group, particularly among those who received ≥5 consecutive days of study intervention compared with control (13.79 vs 10 lytic units, respectively; P = .04). Infants in the massage group were heavier at end of study and had greater daily weight gain compared with those in the control group; other immunologic parameters, number of infections, and length of stay were not different between the 2 groups. CONCLUSIONS: In this study, MT administered to stable preterm infants was associated with higher NK cytotoxicity and more daily weight gain. MT may improve the overall outcome of these infants. Larger studies are needed.

Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases.

BACKGROUND: Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses. METHODS: To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell™ cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT. RESULTS: Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway. CONCLUSIONS: The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.