Aspirin 15cH has Different Effects on Morphology and Function of Lipopolysaccharide-Challenged RAW 264.7 Macrophages In Vitro Compared to a Pharmacological Dose of Aspirin.

INTRODUCTION: Aspirin is one of the most commonly used drugs worldwide. It is known to present antipyretic, anti-inflammatory and anti-thrombotic actions, making it extremely useful in a wide range of clinical contexts. Interestingly, homeopathically prepared Aspirin 15cH has been found to have a pro-thrombotic effect in rats, raising the hypothesis that Aspirin 15cH could also modulate the activity of inflammatory cells in different pathological processes. OBJECTIVE: Our objective was to assess what effect Aspirin 15cH has on RAW 264.7 macrophages in vitro. METHODS: The effects of Aspirin 15cH on biochemical and morphological activities of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were evaluated. These effects were compared with unchallenged macrophages (negative control), untreated LPS-stimulated macrophages, macrophages treated with succussed water (vehicle control), or aspirin 200 µg/mL (pharmacological inhibitor of LPS activity). Cell morphology (adhered cell area and cytoskeleton arrangements), cell viability, toll-like receptor-4 (TLR-4) expression, and the production of nitric oxide, cytokines and intracellular reactive oxygen species were assessed. RESULTS: Aspirin 15cH reduced the number of cells expressing TLR-4 on the surface (p = 0.03) and induced a "columnar" morphology of macrophage pseudopods, indicating changes in cytoskeleton arrangement. When cells were treated with both Aspirin 15cH and LPS, cell morphology became heterogeneous, suggesting that sub-populations of cells had differing sensitivities to LPS or Aspirin 15cH. Exposure of the cells to LPS alone, succussed water or aspirin 200 µg/mL produced effects consistent with the literature. CONCLUSION: Aspirin 15cH, aspirin 200 µg/mL, LPS and succussed water appear to act as independent stimuli able to induce different patterns of macrophage response. Aspirin 15cH induced changes suggestive of M2 polarization of the macrophages (i.e., toward a wound healing or tissue repair, rather than inflammatory, phenotype). These preliminary findings need to be confirmed in further specific studies.

The Clinical and Biological Effects of Homeopathically Prepared Signaling Molecules: A Scoping Review.

BACKGROUND: Signaling molecules such as cytokines and interleukins are key mediators for the immune response in responding to internal or external stimuli. Homeopathically prepared signaling molecules have been used therapeutically for about five decades. However, these types of products are not available in many countries and their usage by homoeopaths is also infrequent. The aim of this scoping review is to map the available pre-clinical and clinical data related to the therapeutic use of homeopathically prepared signaling molecules. METHODS: We conducted a scoping review of clinical and pre-clinical studies of therapeutically used signaling molecules that have been prepared in accordance with an officially recognized homeopathic pharmacopoeia. Articles in peer-reviewed journals reporting original clinical or pre-clinical research of homeopathically prepared signaling molecules such as interleukins, cytokines, antibodies, growth factors, neuropeptides and hormones, were eligible. Non-English language papers were excluded, unless we were able to obtain an English translation. An appraisal of eligible studies took place by rating the direction of the outcomes on a five-point scale. The quality of the papers was not systematically assessed. RESULTS: Twenty-eight eligible papers, reporting findings for four different manufacturers' products, were identified and reviewed. Seventeen papers reported pre-clinical studies, and 11 reported clinical studies (six experimental, five observational). A wide range of signaling molecules, as well as normal T-cell expressed specific nucleic acids, were used. A majority of the products (21 of 28) contained two or more signaling molecules. The most common clinical indications were psoriasis, vitiligo, rheumatoid arthritis, respiratory allergies, polycystic ovary syndrome, and herpes. The direction of the outcomes was positive in 26 papers and unclear in two papers. CONCLUSION: This scoping review found that there is a body of evidence on the use of homeopathically prepared signaling molecules. From a homeopathy perspective, these substances appear to have therapeutic potential. Further steps to explore this potential are warranted.

Evidence for Reversal of Immunosuppression by Homeopathic Medicine to a Predominant Th1-type Immune Response in BALB/c Mice Infected with Leishmania donovani.

BACKGROUND: Visceral leishmaniasis (VL) is a neglected tropical disease that is fatal if treatment is not given. The available chemotherapeutic options are unsatisfactory, and so complementary therapies like homeopathy might be a promising approach. METHODS: A nosode from a pure axenic culture of Leishmania donovani was prepared and screened for its anti-leishmanial potential both in an in-vitro and an in-vivo experimental approach. RESULTS: Leishmania donovani amastigote promastigote nosode (LdAPN 30C) exhibited significant anti-leishmanial activity against the promastigote forms of Leishmania donovani and was found to be safe. A study conducted on VL-infected mice revealed that LdAPN 30C resolved the disease by modulating the host immune response toward the Th1 type through upregulating the pro-inflammatory cytokines (IFN-γ and IL-17) and inducing nitric oxide (NO) levels in the infected macrophages. The hepatic parasite load was also found to be significantly decreased. The nosode was found to be safe, as no histological alterations in the liver or kidney were observed in the animals treated with the LdAPN 30C. CONCLUSION: This is the first study in which an axenic culture of Leishmania donovani has been used for the preparation of a homeopathic medication. The study highlights the anti-leishmanial and immunomodulatory potential of a homeopathic nosode in experimental VL.

Phenotypic Changes in Mammary Adenocarcinoma (4T1) cells In Vitro after Treatment with Carcinosinum.

OBJECTIVE: The present study aimed to identify possible phenotypic changes in 4T1 (murine mammary adenocarcinoma) cells in vitro, including viability, HER-2 (human epidermal growth factor receptor-type 2) expression, and metastatic potential, after treatment with Carcinosinum in different homeopathic dilutions (12cH, 30cH, 200cH) shaken mechanically in pure, sterile, water from a commercial stock dilution. METHODS: Treated cells were cultured in R10 medium, using 24-well plates, 105 cells per well, and treated with vehicle, Carcinosinum 12cH, 30cH or 200cH; untreated cells were used as the baseline control. After 24 hours of treatment, the percentage of apoptotic cells was analyzed by annexin V. Cell morphology was evaluated by microscopy after hematoxylin-eosin and Giemsa staining, whilst HER-2 expression was assessed using immunocytochemistry. The metastatic potential was determined by the expression and activity of the enzyme matrix metalloproteinase 9 (MMP-9) using zymography. The cytokine profile was established using the cytometric bead array method. RESULT: Treatment of 4T1 cells in vitro with Carcinosinum 30cH produced an increase in the number of annexin V-positive cells (apoptosis) and decreased expression of proactivated MMP-9. Cells treated with Carcinosinum 200cH presented hyper-expression of HER-2 on the plasma membrane, identified by immunocytochemistry. There were no differences in cytokine production among treatments. CONCLUSION: The data show promising results for Carcinosinum 30cH in vitro, but in vivo studies are also required to evaluate the role of tumor microenvironment in its effects.

Preparation, Standardization and Anti-plasmodial Efficacy of Novel Malaria Nosodes.

BACKGROUND: Resistance to artemisinin and its partner drugs has threatened the sustainability of continuing the global efforts to curb malaria, which urges the need to look for newer therapies to control the disease without any adverse side effects. In the present study, novel homeopathic nosodes were prepared from Plasmodium falciparum and also assessed for their in vitro and in vivo anti-plasmodial activity. METHODS: Three nosodes were prepared from P. falciparum (chloroquine [CQ]-sensitive [3D7] and CQ-resistant [RKL-9] strains) as per the Homeopathic Pharmacopoeia of India, viz. cell-free parasite nosode, infected RBCs nosode, mixture nosode. In vitro anti-malarial activity was assessed by schizont maturation inhibition assay. The in vitro cytotoxicity was evaluated by MTT assay. Knight and Peter's method was used to determine in vivo suppressive activity. Mice were inoculated with P. berghei-infected erythrocytes on day 1 and treatment was initiated on the same day. Biochemical, cytokine and histopathological analyses were carried out using standard methods. RESULTS: In vitro: the nosodes exhibited considerable activity against P. falciparum with maximum 71.42% (3D7) and 68.57% (RKL-9) inhibition by mixture nosode followed by cell-free parasite nosode (62.85% 3D7 and 60% RKL-9) and infected RBCs nosode (60.61% 3D7 and 57.14% RKL-9). The nosodes were non-toxic to RAW macrophage cell line with >70% cell viability. In vivo: Considerable suppressive efficacy was observed in mixture nosode-treated mice, with 0.005 ± 0.001% parasitemia on day 35. Levels of liver and kidney function biomarkers were within the normal range in the mixture nosode-treated groups. Cytokine analysis revealed increased levels of IL-4 and IL-10, whilst a decline in IL-17 and IFN-γ was evident in the mixture nosode-treated mice. CONCLUSION: The mixture nosode exhibited promising anti-malarial activity against P. falciparum and P. berghei. Biochemical and histopathological studies also highlighted the safety of the nosode for the rodent host. The study provides valuable insight into a novel medicament that has potential for use in the treatment of malaria.

Biological Actions, Electrical Conductance and Silicon-Containing Microparticles of Arsenicum Album Prepared in Plastic and Glass Vials.

INTRODUCTION: According to the "silica hypothesis" formulated to explain homeopathy, the information of starting materials would be transferred to cells by silica nanoparticles detached from the glassware walls by serial dilution and agitation through epitaxy. We compared the biological activity, electrical current and silicon microparticle content (by means of scanning electron microscopy/energy-dispersive X-ray spectroscopy) of high dilutions (HDs) of arsenic prepared in plastic and glass vials to investigate the role of silica in their biological effects in vitro. MATERIALS AND METHODS: Co-cultures of macrophages and yeast (Saccharomyces cerevisiae) were treated with different HDs of arsenic prepared in plastic and glass vials. Macrophage morphology, phagocytosis index, nitric oxide (NO), and cytokine production were evaluated. RESULTS: Measurable amounts of silicon microparticles were detected only in the HDs prepared in glass vials, but ultra-centrifugation eliminated them. Specific and non-specific results were observed. Non-specific pro-inflammatory effects were seen in all dilutions prepared in plastic vials, including elevation of pro-inflammatory cytokines, NO and macrophage phagocytic index. Only the 200th centesimal dilution of arsenic produced specific decrease in interleukin-6 production in macrophages, and it was independent of the vial type or the presence of microparticles of silica in the medicine samples. The nature of the vials had an impact on the electric flow in the respective fluids. CONCLUSION: The non-specific, pro-inflammatory effects might be attributed to organic residuals detached from the vials' plastic walls during manipulation. Instead, specific silica-independent effects of the homeopathic medicine can be attributed to the decrease of interleukin-6 after treatment with the 200th centesimal dilution of arsenic.

Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum.

The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n=138) infected with Leishmania infantum, and treated with Leishmania infantum 30× (10-30) biotherapy - BioLi30×. We performed controlled experiments using 8-to-12-week-old mice, infected with 5×107L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30× pre-treated), G4 (BioLi30× pre/post-treated), G5 (BioLi30× post-treated), G6 (Water 30× post-treated), G7 (Antimonium crudum 30× post-treated) and G8 (Glucantime® post-treated). G3-G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received Glucantime® (0.6mg/100µl of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals' serum was measured after 49days of infection, and cytokines (IFN-γ, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-γ/IL-4 and IFN-γ/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3-G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2-G8), and intense disruption (G2-G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-γ/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance.

M1 homeopathic complex trigger effective responses against Leishmania (L) amazonensis in vivo and in vitro.

Leishmaniasis is a term referring to a range of clinical conditions caused by protozoan parasites of the genus Leishmania, Trypanosomatidae family, Kinetoplastida order that is transmitted by the bite of certain species of mosquitoes Phlebotominae subfamily. These parasites infect hosts wild and domestic mammals, considered as natural reservoirs and can also infect humans. Leishmania are obligate intramacrophage protozoa that have exclusively intracellular life style. This suggests that the amastigotes possess mechanisms to avoid killing by host cells. Cutaneous leishmaniasis, the most common form of the disease, causes ulcers on exposed parts of the body, leading to disfigurement, permanent scars, and stigma and in some cases disability. Many studies concluded that the cytokines profile and immune system of host have fundamental role in humans and animals natural self-healing. Conventional treatments are far from ideals and the search for new therapeutic alternatives is considered a strategic priority line of research by the World Health Organization. A promising approach in the field of basic research in homeopathy is the treatment of experimental infections with homeopathic drugs prepared from natural substances associations highly diluted, which comprise a combination of several different compounds considered as useful for a symptom or disease. Therefore, this study aimed to evaluate the effect of M1, a complex homeopathic product, in macrophage-Leishmania interaction in vitro and in vivo. It was used RAW cells lineage and BALB/c mice as a host for the promastigotes of L. amazonensis (WHOM/BR/75/Josefa). Several biochemical and morphological parameters were determined. Together, the harmonic results obtained in this study indicate that, in general, the highly diluted products trigger rapid and effective responses by living organisms, cells and mice, against Leishmania, by altering cytokines profile, by NO increasing (p<0.05), by decreasing parasitic load (p<0.001), and modifying classical maturation and biogenesis of parasitophorous vacuoles (p<0.001). M1 complex decreased endocytic index (p<0.001), and the % of infected macrophages (p<0.05), preventing the development of lesions (p<0.05) caused by L. amazonensis by increasing Th1 response (p<0.05). Therefore the M1complex can be a good candidate for a complementary therapy to conventional treatments, since all the parameters observed in vitro and in vivo improved. It could be an interesting clinical tool in association to a classical anti-parasitic treatment, maybe resulting in better quality of life to the patients, with less toxicity.

Homeopathic medicine Cantharis modulates uropathogenic E. coli (UPEC)-induced cystitis in susceptible mice.

OBJECTIVE: This is a random blinded placebo controlled murine experimental model to study the effects of Cantharis 6 CH, a homeopathic medicine, on E coli-induced cystitis. METHODS: 24 adult susceptible female BALB/c mice were inoculated with E coli - UPEC O4:K-:H5 by a transurethral catheter. Cantharis 6cH or vehicle (placebo) was offered to mice by free access into the drinking water (1:100), during 24 h after infection. Spleen, bladder and kidneys were processed for quantitative histopathology after immunohistochemistry, using anti-CD3, CD79, MIF, NK and VEGF antibodies; the cytokines present in the bladder washing fluid were measured using a LUMINEX-Magpix KIT. Mann-Whitney and Fisher exact test were used as statistical analysis. RESULTS: Cantharis 6 CH increased IL12p40, IFN-γ and decreased IL10 concentrations in the bladder fluid (p⩽0.05); in the bladder mucosa, it increased the ratio between B and T lymphocytes (31%) and between B lymphocytes and MIF+ macrophages (57%, p⩽0.05). In the pelvis, instead, it decreased the B/T cells ratio (41%, p⩽0.05) and increased the M1/M2 macrophage ratio (42%, p⩽0.05). No differences were seen in the kidney and spleen analysis. CONCLUSION: The inverted balance of inflammatory cells and cytokines in bladder and pelvis mucosa shows specific local immune modulation induced by Cantharis 6cH.

Increased of the hepatocytes and splenocytes apoptosis accompanies clinical improvement and higher survival in mice infected with Trypanosoma cruzi and treated with highly diluted Lycopodium clavatum.

Recent evidence includes apoptosis as a defense against Trypanosoma cruzi infection, which promotes an immune response in the host induced by T cells, type 1, 2 and 17. Currently, there is no medicine completely preventing the progression of this disease. We investigated the immunological and apoptotic effects, morbidity and survival of mice infected with T. cruzi and treated with dynamized homeopathic compounds 13c: Kalium causticum (GCaus), Conium maculatum, (GCon), Lycopodium clavatum (GLy) and 7% alcohol solution (control, vehicle compounds, GCI). There was significant difference in the increase of apoptosis in the treated groups, compared with GCI, which might indicate action of the compounds in these cells. Infected animals treated with Lycopodium clavatum presented better performance compared with other groups. GLy showed a higher amount of hepatocytes and splenocytes undergoing apoptosis, higher number of apoptotic bodies in the liver, predominance of Th1 response, increased TNF-α and decreased IL-6, higher survival, lower morbidity, higher water consumption, body temperature, tendency to higher feed intake and weight gain compared with GCI. Conium maculatum had worse results with increased Th2 response with increased IL-4, worsening of the infection with early mortality of the animals. Together, these data suggest that highly diluted medicines modulate the immune response and apoptosis, affecting the morbidity of animals infected with a highly virulent strain of T. cruzi, being able to minimize the course of infection, providing more alternative approaches in the treatment of Chagas disease.

Beneficial immunomodulatory and neuro digestive effect in Trypanosoma cruzi infection after Lycopodium clavatum 13c treatment.

Studies show that highly diluted medications demonstrate benefits in treating infections, constituting an alternative for their treatment. The present study evaluated the effects of Lycopodium clavatum, dynamization 13c, in Wistar rats infected with T. cruzi. In this study 42 male rats were intraperitoneally inoculated with T. cruzi - Y strain and allocated into groups: IC (infected control group) and Ly (treated with L. clavatum 13c). The cytokines dosage (IFN-γ, IL-12, IL-10, IL-4), quantification and morphometry of myenteric neurons were evaluated. The treatment with L. clavatum modifies the immune response, with increase of IFN-γ on day 10 a.i. and IL-12 on day 24 a.i., decrease of IL-10 concentration on day 10 a.i. and subsequent increase of this cytokine and IL-4 on day 24 a.i., affording a bigger number of myenteric neurons compared to IC group. Thus, L. clavatum 13c promoted on rats infected with T. cruzi a beneficial immunomodulatory action reducing the pathogenic progression of digestive Chagas disease.

Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative Real-Time PCR.

BACKGROUND: Arnica montana is a popular traditional remedy widely used in complementary medicine, also for its wound healing properties. Despite its acknowledged action in clinical settings at various doses, the molecular aspects relating to how A. montana promotes wound healing remain to be elucidated. To fill this gap, we evaluated the whole plant extract, in a wide range of dilutions, in THP-1 human cells, differentiated into mature macrophages and into an alternative IL-4-activated phenotype involved in tissue remodelling and healing. METHODS: Real-time quantitative Reverse Transcription Polymerase Chain Reaction (PCR) analysis was used to study the changes in the expression of a customized panel of key genes, mainly cytokines, receptors and transcription factors. RESULTS: On macrophages differentiated towards the wound healing phenotype, A. montana affected the expression of several genes. In particular CXC chemokine ligand 1 (CXCL1), coding for an chief chemokine, exhibited the most consistent increase of expression, while also CXC chemokine ligand 2 (CXCL2), Interleukin8 (IL8) and bone morphogenetic protein (BMP2) were slightly up-regulated, suggesting a positive influence of A. montana on neutrophil recruitment and on angiogenesis. MMP1, coding for a metalloproteinase capable of cleaving extracellular matrix substrates, was down-regulated. Most results showed non-linearity of the dose-effect relationship. CONCLUSIONS: This exploratory study provides new insights into the cellular and molecular mechanisms of action of A. montana as a promoter of healing, since some of the genes it modifies are key regulators of tissue remodelling, inflammation and chemotaxis.

Homeopathic Rhus toxicodendron has dual effects on the inflammatory response in the mouse preosteoblastic cell line MC3T3-e1.

BACKGROUND: Homeopathic remedy Rhus toxicodendron (Rhus tox) is used for several symptoms including skin irritations, rheumatic pains, mucous membrane afflictions, and typhoid type fever. Previously, we reported that Rhus tox treatment increased the cyclooxygenase-2 (COX-2) mRNA expression in primary cultured mouse chondrocytes. METHODS: A preosteoblastic mouse cell line, MC3T3-e1, was treated with different homeopathic dilutions of Rhus tox and the COX-2 mRNA and protein expression was examined using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting. Additionally, nitric oxide (NO) generation was examined in LPS-induced MC3T3-e1 cells using a Griess reaction assay. RESULTS: Stimulation with different concentrations of Rhus tox increased the expression of Cox2 mRNA, with 30X Rhus tox showing the most prominent increase in mRNA expression. In addition, treatment with 30X Rhus tox significantly increased prostaglandin E2 (PGE2) release compared with other homeopathic dilutions. However, the COX-2 protein expression level differed slightly from its mRNA expression, because the 30C Rhus tox treatment increased COX-2 protein to a greater extent compared with other dilutions. NO generation was dramatically decreased in MC3T3-e1 cells after Rhus tox treatment co-stimulated with lipopolysaccharide. CONCLUSION: Homeopathic dilution of Rhus tox has a dual activity that increases COX-2 expression and decreases NO generation, thus modulating inflammation. Further study is needed to examine the cellular signaling mechanisms that are associated with inflammatory regulation by Rhus tox treatment in greater detail.

Identification of an active fraction of Kangfuxin in the treatment of periodontitis in a rat model.

INTRODUCTION: Periodontitis is a serious gum infection that disrupts the soft tissue around teeth. This study aimed to identify the most effective fraction of the Chinese medicine Kangfuxin for periodontitis treatment in a rat model. MATERIAL AND METHODS: Kangfuxin solution was subjected to sequential extraction using chloroform, ethyl acetate, n-butanol, and water. The extracts were evaporated, dissolved in DMSO, diluted in water, and administered to rats via gavage (0.5 mL/day) for 2 weeks. The n-butanol extract was further fractionated using macroporous resin chromatography with 10%, 30%, 50%, 70%, and 90% ethanol elution. Levels of inflammatory cytokines IL-6, IL-1ß, and TNF-α in periodontitis samples were examined by ELISA. Leukocyte infiltration in the cementum was analysed by haematoxylin and eosin (H&E) staining. RESULTS: The n-butanol extract showed the best anti-inflammatory effect, reducing IL-6, IL-1ß, and TNF-α levels in periodontitis samples and alleviating tissue damage and leukocyte infiltration in the cementum. Further fractionation revealed that the 50% ethanol fraction of the n-butanol extract had the most potent action in attenuating inflammation. This fraction suppressed the activation of the PI3K-AKT-mTOR signalling pathway in periodontitis samples. Application of a PI3K activator counteracted the anti-inflammatory effect of the 50% ethanol fraction. CONCLUSIONS: We identified a potent anti-inflammatory fraction (50% ethanol fraction of the n-butanol extract) of Kangfuxin for periodontitis treatment. This fraction suppressed the activity of the PI3K-AKT-mTOR signalling pathway in periodontitis samples. Further research is needed to isolate and characterise the specific bioactive compounds within this fraction.

Increased hepcidin expression in multibacillary leprosy.

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

Association of miR-155, miR-187 and Inflammatory Cytokines IL-6, IL-10 and TNF-α in Chronic Opium Abusers.

Substance use disorders are known to be associated with inflammation. However, the dynamics of inflammatory cytokines and microRNA in chronic opium use is yet unexplored. The current study determined the levels of inflammatory cytokines TNF-α, IL-6, IL-10 and immune-regulatory miR-155 and miR-187 expressions in chronic opioid use disorder. Adults (n = 48) meeting the 5th Edition of the DSM criteria regarding opioid use disorder and healthy controls (n = 46) were included in the study. Inflammatory cytokines IL-10, IL-6, and TNF-α were analyzed from serum samples, and peripheral blood mononuclear cells processed for miRNA expression. Cases showed significantly raised IL-10 and TNF-α and reduced IL-6. Dose-dependent upregulation of miR-155-5p and miR-187-5p was evident at opium dose >1500 g/month, with a corresponding increase of TNF-α and IL-10. MiR-155 showed a significant positive correlation with IL-6 and TNF-α levels, while miR-187 showed a significant negative association with TNF-α at ≥1000 g/month consumption. Therefore, increasing consumption of opium probably enhances inflammation leading to immunomodulation and aberrant expression of hsa-miR-155-5p and hsa-miR-187-5p in opioid use disorder.

[Effects of Cordyceps extract on cytokines and transcription factors in peripheral blood mononuclear cells of asthmatic children during remission stage].

OBJECTIVE: To explore the effects of Cordyceps extract in regulating the imbalance of Th1/Th2 ratio and inhibiting the inflammatory reaction, and to find the theoretical basis of Cordyceps extract for treating asthma in remission stage. METHODS: A total of 20 peripheral venous blood samples (3 mL) were collected from 20 asthmatic children during remission stage, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll method. PBMCs were separated into three groups (blank group, low-dose group and high-dose group). The PBMCs were incubated in vitro for 48 hours in the absence (blank group) or presence (low-dose group and high-dose group) of Cordyceps extract at different concentrations (10, 20 microg/mL). The expressions of interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-10, T-box expressed in T cells (T-bet), GATA-binding protein-3 (GATA-3) and forkhead/winged-helix transcription factor-3 (Foxp3) mRNAs in PBMCs were measured by real-time fluorescent quantitative polymerase chain reaction, and the contents of IFN-gamma, IL-4 and IL-10 in supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: The expressions of IFN-gamma mRNA showed no significant differences among the three groups. The expressions of the IL-4 mRNA in the high-dose group and the low-dose group were lower than that in the blank group (P=0.014, P=0.011). The expression of IL-10 mRNA in the high-dose group was higher than that in the blank group (P=0.034). And the differences of the IFN-gamma mRNA/IL-4 mRNA ratio presented no statistic significance among the three groups. The level of IL-4 content in the high-dose group was lower than that in the blank group (P=0.018), but the level of IL-10 content, and ratio of IFN-gamma/IL-4 in the high-dose group were higher than those in the blank group (P=0.011, P=0.045). The differences of the IFN-gamma presented no statistic significance among the three groups. The T-bet/GATA-3 ratio and Foxp3 mRNA expression in the high-dose group were higher than those in the blank group (P=0.001, P=0.015). There was significant difference in expression of GATA-3 mRNA between the high-dose group and the blank group (P=0.028), and between the low-dose group and the blank group (P=0.019). The expression differences of T-bet mRNA were insignificant between any two groups. CONCLUSION: Cordyceps extract can inhibit the proliferation and differentiation of Th2 cells and reduce the expression of related cytokines by down-regulating the expression of GATA-3 mRNA and up-regulating the expression of Foxp3 mRNA in PBMCs. Meanwhile, it can alleviate the chronic allergic inflammation by increasing the content of IL-10.

[The effect of the type II collagen protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis].

OBJECTIVE: To investigate the effect of the type II collagen (C II) protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis (AA). METHODS: Type II collagen protein was abstracted and purificated from Zaocys. Adjuvant arthritis (AA) was induced by a single intradermal injection of 0.1 mL of complete Freund's adjuvant into the left hind paw. Synoviocytes' supernatants were harvested and synoviocytes-Peyer's Patches (PP) cells coculture system were developed. Tumor necrosis factor-alpha (TNF-alpha) activity was measured by L929 cytotoxicity bioassay and Interleukin (IL)-1beta activity was measured by MTT dye reduction. The synoviocytes' supernatants cytokines' levels were detected by ELISA. RESULTS: Each concentration of C II from Zaocys had no effect on IL-1beta and TNF-alpha production by synoviocytes in vitro. Middle concentration of C II suppressed the activity of IL-1beta and TNF-alpha production by synoviocytes-PP cells coculture system (P < 0.05). Treating with low and high dose of C II suppressed the activity of TNF-alpha and IL-1beta producing by synoviocyte (P < 0.05), significantly suppressed in the group of AA rats treated with middle dose of C II (P < 0.01). Treating with middle and high dose of C II decreased the level of synoviocytes' supernatants TNF-alpha (P < 0.05), the level of synoviocytes' supernatants IL-1beta decreased in all treating groups (P < 0.05). Treating with middle dose of C II increased the level of serum TGF-beta (P < 0.05). Middle concentration of C II suppressed the activity of IL-1 and TNF production by synoviocytes-PP cells coculture system (P < 0.05). CONCLUSIONS: C II from Zaocys has no direct effect on the activity of IL-1beta and TNF production by synoviocytes in vitro. Oral administration of type II collagen protein from Zaocys can effectively suppressed the activity and level of the cytokines production by synoviocytes in rats with adjuvant arthritis (AA).

Gene expression profiling of macrophages following mice treatment with an immunomodulator medication.

Canova (CA) is a complex homeopathic medication used in diseases where the immune system is depressed. Previous studies demonstrated that it is neither toxic nor mutagenic and activates macrophages. We now evaluate CA effects on cytokine production and gene expression from mice macrophages. The global view of changes in expression of genes with known functions can provide a vivid picture of the way in which cell adapts to a changing environment or a challenge. We found a decrease in IL-2 and IL-4 production and a differential expression in 147 genes from CA group. These genes are mainly involved in transcription/translation, cell structure and dynamics, immune response, cytoprotection, enzymatic process, and receptors/ligands. With gene expression analysis we state that this medication provokes a reaction that involves alterations in gene expression profile mainly in the ones involved with macrophages activation, corroborating the laboratorial research and the clinical data.

Differentiation of human monocytes in vitro following exposure to Canova in the absence of cytokines.

Canova is an immunomodulatory, homeopathic preparation that has been shown to activate macrophages in vitro and in vivo, with resultant enhanced spreading of the cells and formation of microvillus extensions from the cell body. Since monocytes are the precursor cells of macrophages and dendritic cells, the objective of the current study was to investigate the effects of Canova on the differentiation of human blood monocytes in vitro. Monocytes were isolated, grown in culture, and exposed to 10 and 20% Canova without the addition of cytokines. After 48 h, monocytes were prepared for analysis by scanning electron microscopy, while cells kept in culture for 7 days and exposed to Canova on days 1, 3, and 4 were analyzed by flow cytometry for alterations in the levels of expression of CD1a, CD11c, CD14, CD80, CD83, CD86, and HLA-DR. SEM revealed that monocytes exposed to 10% Canova had a morphological appearance similar to that of macrophages. Various cytoplasmic projections were observed with pseudopodia formation. Flow cytometric analysis after exposure of monocytes to 10 and 20% Canova indicated high cell viability and upregulation of CD80, compatible with differentiation into either macrophages or dendritic cells. Exposure to Canova per se causes activation of monocytes with resultant differentiation into large macrophage-like cells of indeterminate phenotype that have increased expression of CD80. Like cytokines, Canova induces differentiation of monocytes, an activity that may underpin the immunomodulatory activity of this product.