A novel adjuvant, the general opioid antagonist naloxone, elicits a robust cellular immune response for a DNA vaccine.

While many adjuvants have been discovered and used in research, only a few adjuvants have been permitted for use with human vaccination. We have previously shown that the administration of naloxone (NLX), a general opioid antagonist, during infection with a non-virulent strain of herpes simplex virus type 1 (HSV-1) could enhance protection against HSV-1 challenge. Here, the adjuvant activity of NLX has been evaluated using a DNA vaccine for HSV-1 as a model. BALB/c mice were divided into four groups; for experimental groups, mice received the glycoprotein D1 (gD1) DNA vaccine alone or in combination with the adjuvant NLX. A positive control group received the KOS strain of HSV-1, and a negative control group received PBS. All mice were immunized three times on days 0, 21 and 42. Three weeks after the last immunization, immune responses against HSV-1 were assessed. Our results indicate that the administration of NLX as an adjuvant increased the ability of the gD1 DNA vaccine to enhance cytolytic T lymphocyte activity, lymphocyte proliferation, delayed-type hypersensitivity and shifting the immune response toward a T helper (Th)1 pattern and improved protective immunity against HSV-1. NLX also increased the IgG2a/IgG1 ratio, though it did not affect the production of HSV-1 antiserum. In conclusion, administration of NLX as an adjuvant in combination with the gD1 DNA vaccine can enhance cell-mediated immunity and shift the immune responses to Th1.

Invasive aspergillosis promotes tumor growth and severity in a tumor-bearing mouse model.

Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.

Capecitabine-loaded anti-cancer nanocomposite hydrogel drug delivery systems: in vitro and in vivo efficacy against the 4T1 murine breast cancer cells.

In this work, nanocomposite hydrogel drug delivery systems based on polyvinyl alcohol and montmorillonite loaded with the capecitabine, as an anti-cancer drug, were developed for oral administration. The gel fraction and swelling ability of the prepared nanocomposite hydrogels were experimentally measured. In vitro release kinetics of capecitabine in nanocomposite hydrogel drug delivery systems were studied. In vitro flow cytometry assay was utilized to exhibit the anti-cancer activity of the prepared nanocomposite hydrogel drug delivery systems against 4T1 cancer cell line. The anti-tumor efficacy of the nanocomposite hydrogel drug delivery systems was also studied in vivo on animal models. The results showed that the amount of montmorillonite incorporated into the nanocomposite hydrogel drug delivery systems could be recognized as a key parameter to adjust the values of the gel fraction, swelling and capecitabine release rate in a manner which by increasing the montmorillonite content, the gel fraction is increased while the swelling and drug release rate are decreased. The flow cytometry results demonstrated the better anti-cancer activity of the capecitabine-loaded nanocomposite hydrogel drug delivery systems as compared with the pure capecitabine. The in vivo assays indicated that the administration of nanocomposite hydrogel drug delivery systems had a significant effect on the reduction of the tumor growth in animal models as compared with pure capecitabine administration. In general, the prepared nanocomposite hydrogel drug delivery systems exhibited a suitable efficacy against 4T1 cancer cell line both in vitro and in vivo and they could be considered as promising candidates for controlled release of anti-cancer drugs in chemotherapy with enhanced therapeutic effects.

The efficient generation of immunocompetent dendritic cells from leukemic blasts in acute myeloid leukemia: a local experience.

Dendritic cells (DCs) are the most important antigen presenting cells with potentially useful applications in cancer immunotherapy. Leukemic cells of patients with acute myeloid leukemia (AML) could be differentiated to DC-like cells possessing the ability of stimulating anti-leukemic immune response. Despite obvious progress in DC-based immunotherapy, some discrepancies were reported in differentiation potential of AML blasts from all patients toward DC like cells. The present study, as a local experience, was set up to generate DCs from AML blasts of various subtypes. Leukemic Blasts from 16 Iranian AML patients were differentiated into functional DCs by culturing in the presence of rhGM-CSF, rhIL-4 and TNF-alpha for 8 days. The morphology, expression of key surface molecules and allostimulatory activity of resultant DCs were compared with primary blasts and cultured but cytokine untreated control groups. The pattern of angiotensin-converting enzyme (ACE) expression was used to approve the leukemic origin of generated DCs. Neo-expression or upregulation of DC-associated markers were occurred during culturing period in cytokine treated cells compared with primary blasts and cultured but cytokine untreated control groups: CD1a (63.22% vs. 3.22% and 11.79%), CD83 (41.27% vs. 0.11% and 0.70%), CD40 (15.17% vs. 0.00% and 0.04%), CD80 (49.96 vs. 0.02% and 0.32%), CD86 (56.49% vs. 0.50% and 5.71%) and HLA-DR (52.52% vs. 14.32% and 2.49%) respectively. The potency of generated DCs to induce allogeneic T cell proliferation increased significantly compared to pre and post culture control groups (27,533.4 +/- 2,548.3, 8,820.4 +/- 1,639.4 and 3,200.35 +/- 976 respectively). The expression pattern of ACE in AML-DCs, blast cells and DCs derived from normal monocytes (7.93%, 1.28% and 74.97% respectively) confirmed the leukemic origin of DCs. Our data confirmed the generation of sufficient AML-derived cells with the properties of DCs in all cases. This potency of AML blasts, offers a useful route for active immunotherapy of AML patients.

Hot and Cold natures and some parameters of neuroendocrine and immune systems in traditional Iranian medicine: a preliminary study.

AIM: The purpose of this study was to assess differences in persons of a Hot or Cold nature (according to traditional Iranian medicine), in terms of changes in their neuroendocrine and immune systems. MATERIALS AND METHODS: Thirty-seven (37) male volunteers (20-40 years old) were divided into two groups, by whether they had a Hot or Cold nature. In addition, the Warmth/Coldness ratio of all the volunteers was assessed. Plasma concentrations of epinephrine, norepinephrine and cortisol, and also the concentrations of interferon (IFN)-gamma and interleukin (IL)-4 produced by peripheral blood mononuclear cells stimulated by mitogen were measured. RESULTS: The results showed that norepinephrine/epinephrine and norepinephrine/cortisol ratios were significantly higher, and that there was a borderline significantly increased IL-4/IFN-gamma ratio in the Hot nature group compared with those in the Cold nature group. In addition, there was a significant linear positive correlation between the norepinephrine/epinephrine and Warmth/Coldness ratios and a significant nonlinear association between the IL-4/IFN-gamma and Warmth/Coldness ratios. CONCLUSIONS: It can be deduced that the persons of a Hot nature had more sympathetic nervous system activity, less adrenal sympathetic, adrenal corticosteroid, and parasympathetic nervous system activities and more deviation of the immune system toward T-helper (Th)2 responses than the persons of a Cold nature. Moreover, the activity of the sympathetic nervous system was increased and adrenal sympathetic was decreased with an increasing Warmth/Coldness ratio. Furthermore, when the person's nature veered toward extreme Warmth or extreme Coldness, the deviation of the immune system toward Th2-like responses was greater, but this increased deviation was much more marked when veering toward extreme Warmth than toward extreme Coldness.

Naturally occurring self-reactive CD4+CD25+ regulatory T cells: universal immune code.

Naturally occurring thymus-arisen CD4(+)CD25(+) regulatory T (Treg) cells are considered to play a central role in self-tolerance. Precise signals that promote the development of Treg cells remain elusive, but considerable evidence suggests that costimulatory molecules, cytokines, the nature of the TCR and the niche or the context in which the T cell encounters antigen in the thymus play important roles. Analysis of TCR from Treg cells has demonstrated that a large proportion of this population has a higher avidity to self-antigen in comparison with TCR from CD4(+)CD25(-) cells and that peripheral antigen is required for their development, maintenance, or expansion. Treg cells have been shown to undergo expansion in the periphery, likely regulated by the presence of self-antigen. Many studies have shown that the involvement of Treg cells in the tolerance induction is antigen-specific, even with MHC-mismatched, in transplantation/graft versus host disease (GVHD), autoimmunity, cancer, and pregnancy. Theses studies concluded a vital role for self-reactive Treg cells in maintenance of the body integrity. Based on those studies, we hypothesize that self-reactive Treg cells are shared among all healthy individuals and recognize same self-antigens and their TCR encodes for few dominant antigens of each organ which defines the healthy self. These dominant self antigens can be regarded as "universal immune code".

Any beneficial effects of mycobacteria on multiple sclerosis and experimental autoimmune encephalitis may include stimulation of the sympathetic nervous system.

UNLABELLED: The inhibitory effects of mycobacterial infection and mycobacterium components on multiple sclerosis (MS) and experimental autoimmune encephalitis (EAE; an animal model for MS) have been known for years. However, this effect seems like a paradox that both mycobacterial infection and MS induce type I immune responses. Some mechanisms have been proposed or even proven for this effect in different studies, but among them there is no hint of a possible role for the nervous system (NS). Regarding the close relations between sympathetic nervous system (SNS) and MS disease course, it can be hypothesized that SNS may have a role in the effects of mycobacterium on MS. HYPOTHESIS: SNS can be stimulated by pro-inflammatory cytokines such as TNF-alpha and IL1-beta, production of which are induced by mycobacterial infection or mycobacterium components. Although these cytokines can inhibit SNS in the site of inflammation caused by mycobacterium, they increase sympathetic tone in other places. The beneficial role of SNS in inhibiting or attenuating the course of MS and EAE has been suggested. Inhibitory effects of stimulated SNS on MS may occur via different ways such as inhibiting the production of pro-inflammatory cytokines and inducing the synthesis of anti-inflammatory cytokines, in other words, shifting the immune responses from type 1 toward type 2, as well as, induction of suppressor/regulator T lymphocytes, induction of heat shock proteins in brain and increasing the expression of Fas and Fas-ligand. Therefore, it seems that stimulation of SNS by mycobacterial infection or mycobacterium components is a key step in the mechanism of beneficial effects of mycobacterium on MS.

Sympathetic nervous system plays an important role in the relationship between immune mediated diseases.

UNLABELLED: T helper (Th) lymphocytes have been classified into distinct subsets, Th1 and Th2 on the basis of the cytokines they produce. According to the cross-regulatory properties of Th1 and Th2 cells, one would assume that to be affected by a Th1 type disease increases susceptibility to a Th1 type disease and inhibits a Th2 type disease and vice versa about being affected by a Th2 type disease. However, the pattern of related diseases does not necessarily follow the conventional pattern of inhibitory effects of Th1 and Th2 immune responses on each other. For example, Mycobacteria including BCG, that induce Th1 immune responses; can modulate some Th1 type autoimmune diseases including MS, experimental autoimmune encephalomyelitis (EAE; an animal model for Multiple Sclerosis) and insulin-dependent diabetes mellitus (IDDM) thereby leading to an alleviation of their symptoms. Also BCG precipitates a syndrome similar to systemic lupus erythematosus (SLE), a Th2 type disease; in NOD mice. The coexistence of the major Th2-mediated atopic diseases such as asthma, eczema and allergic rhinitis with the Th1-mediated autoimmune conditions including; coeliac disease (CD), IDDM, rheumatoid arthritis (RA) and psoriasis is another example that is in apparent disagreement with counter-regulatory effects of Th1/Th2 phenotypes. HYPOTHESIS: SNS can be stimulated by pro-inflammatory cytokines, production of which is induced by mycobacteria including BCG. Although these cytokines can inhibit SNS activity in the site of inflammation and secondary lymphoid organs, they increase sympathetic tone in other places. Increased sympathetic tone can induce an anti-inflammatory and Th2 type milieu. This milieu can inhibit MS and IDDM and provide a susceptible environment for starting of SLE. Atopic diseases are Th2 type immune mediated diseases; therefore, they increase the production of Th2 type cytokine and decrease production of pro-inflammatory cytokines in the site of allergic reaction and also in secondary lymphoid organs. Therefore, atopic diseases decrease sympathetic tone in all tissues except in the sites of allergic reaction and secondary lymphoid organs. Decreased sympathetic tone results in a pro-inflammatory milieu and in such an environment, Th1 type autoimmune diseases can affect tissues.

In vitro and in vivo assays on egg white/polyvinyl alcohol/clay nanocomposite hydrogel wound dressings.

Novel nanocomposite hydrogel wound dressings on the basis of egg white and polyvinyl alcohol, as matrix, and natural Na-montmorillonite clay, as reinforcing agent, were prepared and their performances on wound healing investigated in vitro and in vivo. In vitro cytotoxicity assay revealed non-cytotoxic activity and excellent biocompatibility level of prepared nanocomposite hydrogel wound dressings. The bacterial penetration assay showed the prepared nanocomposite hydrogel wound dressings are excellent barriers against microorganisms and could protect the wound from infection during the wound healing. In vivo animal study showed that the wound healing process was considerably faster in wounds covered with nanocomposite hydrogel wound dressings compared to the conventional wound dressing, i.e. sterile gauze, due to creation of a moist environment on the wound surface and faster migration rate of the epidermal cells. The mechanical properties of healed wounds with nanocomposite hydrogel wound dressings were better than those control wounds covered with sterile gauze due to their better collagen formation ability as a result of created moist healing condition as well as the presence of egg white, as a source of proteins, in their structures.

Tumor Associated Mesenchymal Stromal Cells Show Higher Immunosuppressive and Angiogenic Properties Compared to Adipose Derived MSCs.

BACKGROUND: Differentiation, migratory properties and availability of Mesenchymal Stromal Cells (MSC) have become an important part of biomedical research. However, the functional heterogeneity of cells derived from different tissues has hampered providing definitive phenotypic markers for these cells. OBJECTIVE: To characterize and compare the phenotype and cytokines of adipose derived MSCs (AD-MSCs) and tumoral-MSCs (T-MSCs) isolated from mammary tumors of BALB/c mice. METHODS: Immunophenotyping and in vitro differentiation tests were used for MSC characterization. Cytokine and enzyme profiles were assessed using ELISA and Real-time PCR, respectively. RESULTS: T-MSCs expressed significantly higher levels of HLA-DR (p=0.04). Higher levels of PGE2 and COX-2 enzyme were also observed in T-MSCs (p=0.07 and p=0.00, respectively). Additionally, T-MSCs expressed higher levels of iNOS and MMP9 (p=0.01 and p=0.01, respectively). T-MSCs were also able to induce higher levels of proliferation and migration of HUVEC endothelial cells in wound scratch assay compared to AD-MSCs (p=0.015). CONCLUSION: Functional differences showed by the surface markers of MSCs, cytokine and enzyme production indicate the effect of different microenvironments on MSCs phenotype and function.

Study of immunomodulatory effects of arteether administrated intratumorally.

Recent studies have indicated the profound anti-tumor activity of artemisinin's compounds, among which; arteether is an oil-soluble derivative of artemisinin with an endoperoxide bridge that can induce apoptosis in tumor cells but not in the normal cells. An experiment was carried out on tumor-bearing Balb/c mice to estimate the effects of Arteether on tumor growth and antitumor immune responses. Briefly, 6mg/kg/day of Arteether and diluents were administered to two groups of mice. Tumor sizes were measured using digital verniercallipers. Mice were sacrificed and splenocytes were harvested for lymphocyte proliferation assay, the level of IL-4 and IFN-γ cytokines, and the percentage of splenic T regulatory cells were measured. According to the findings, there were no statistical differences between the groups with respect to the level of IFN-γ, IL-4 and proliferation assay; while our results showed that Arteether is effective in the reduction of tumor growth rate. In general, intra-tumoral injection of Arteether as an oil-soluble derivative of artemisinin brings to light some antitumor properties that may aid in development of more effective antitumor agents.

Long acting propranolol and HSP-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in Balb/c mice.

BACKGROUND: Noradrenaline (NA), the principal neurotransmitter released from sympathetic nerve terminals, influences T-cell maturation, not only directly in developing T cells, but also indirectly, by acting on the thymic nonlymphoid cells. In vitro and in vivo studies have demonstrated the anti-proliferative, anti-migratory, anti-angiogenic and cytotoxic properties of propranolol, ß-AR blocker, against various cancers. OBJECTIVES: To evaluate the effect of propranolol on efficacy of HSP-70 rich lysate vaccine in immunotherapy of fibrosarcoma. METHODS: Mouse fibrosarcoma WEHI-164 cells were used to immunize tumor-bearing mice with or without propranolol and HSP-70. Splenocytes proliferation, cytotoxicity activity of the splenocytes, naturally occurring CD4+ CD25high T-reg cells and IFN-γ and IL-4 secretion as well as tumor size, were assessed to describe the anti-tumor immune response. RESULTS: A significant increase in the level of IFN-γ in the mice vaccinated with WEHI-164 cells enriched with HSP-70 and co-treated with propranolol was observed compared to controls. However, HSP enrichment or propranolol treatment alone did not enhance the immune response as measured by the level of IFN-γ. Likewise, a decrease in tumor growth in the test group (p<0.01) and a significant increase in CTL activity (p<0.05) was observed. CONCLUSION: HSP enriched vaccine shows anti-tumor activity, probably due to the modulation of immune responses.

In vivo and cytotoxic assays of a poly(vinyl alcohol)/clay nanocomposite hydrogel wound dressing.

In the present work, a nanocomposite hydrogel wound dressing was prepared on the basis of poly(vinyl alcohol) using organically-modified montmorillonite as nanoclay by the freezing-thawing cyclic method. In vivo assays were performed to evaluate its performance as an applicable wound dressing on animals. It showed an improved healing process for wounds covered by the prepared nanocomposite hydrogel compared with control wounds covered by sterile gauze. Significant improvements, such as better creation of moist surfaces on the wound with less scar formation, shorter duration of healing operation and better closing of the wound edges with enhanced tensile properties of the healed wound, i.e., tensile strength and elongation-at-break, were observed using the prepared nanocomposite hydrogel in comparison to the sterile gauze. An in vitro cytotoxic assay was also utilized to determine the biocompatibility of the prepared nanocomposite hydrogel. It showed that the prepared nanocomposite hydrogel is non-toxic and can be used as a biocompatible wound dressing in practical wound management.

Antitumor and immunomodulatory properties of artemether and its ability to reduce CD4+ CD25+ FoxP3+ T reg cells in vivo.

BACKGROUND: Development of agents that specifically kill cancer cells and simultaneously elicit antitumor immune response is a step forward in cancer therapy. In the present study, we investigated whether the administration of artemether contributes to the augmentation of antitumor immunity and the regression of tumor tissues in a mouse model of breast cancer. METHODS: An optimal immunostimulatory dose of artemether (ART) was defined by DTH reaction and antibody production in sRBC-challenged mice. Subsequent experiments were carried out on tumor-bearing BALB/c mice. In the first group of tumor-bearing mice, the dose of 10 mg/kg/day of artemether were intraperitoneally administered to each animal for six times. The second group was treated with 20 mg/kg/day of cyclophosphamide as a positive control, and the last group (negative control) received the ART diluents. Tumor size was measured during the 10-day experiment; on the last day, mice were sacrificed and their splenocytes and tumor infiltrating lymphocytes were harvested. The concentration of IL-4 and IFN-γ cytokines (using ELISA assay) and the percentage of splenic and tumor Treg cells (using Flowcytometry analysis) were measured. RESULTS: Artemether could increase both DTH reaction and the production of hemagglutinating antibody in normal mice. Administration of ART profoundly suppressed the progression of tumor tissues. As well, it was significantly effective in the depletion of splenic CD4+ CD25+ Foxp3+ Treg cells (p-value>0.05). ART also increased the production of IL-4 (p-value<0.05) and IFN-γ (p-value>0.05). As a conclusion, the cytotoxic and immunomodulatory properties of artemether were acknowledged in vivo.

Evaluation of the adjuvant activity of naloxone, an opioid receptor antagonist, in combination with heat-killed Listeria monocytogenes vaccine.

We have previously demonstrated the adjuvant activity of naloxone (NLX), a general opioid antagonist, using a DNA vaccine for herpes simplex virus type 1. Here, the adjuvant activity of NLX has been evaluated using a heat-killed Listeria monocytogenes (HKLM) vaccine as a model for general immunization against intracellular bacteria. BALB/c mice were divided into three groups: the Vac group received the HKLM vaccine alone; the NLX-Vac group received the HKLM vaccine in combination with the adjuvant NLX; and the control group received phosphate buffered saline (PBS). Our results indicate that the administration of NLX as an adjuvant enhances the ability of the HKLM vaccine to increase lymphocyte proliferation, delayed type hypersensitivity, and skewing of the immune response toward a T-helper 1 (Th1) pattern. Additionally, combination of NLX with the HKLM vaccine improves protective immunity against L. monocytogenes. In conclusion, administration of NLX as an adjuvant for the HKLM vaccine can enhance cell-mediated immunity and shift the immune response to Th1.

Protective effects of Helicobacter pylori against gastroesophageal reflux disease may be due to a neuroimmunological anti-inflammatory mechanism.

There is some evidence that Helicobacter pylori infection has a protective effect against gastroesophageal reflux disease (GORD) and its complications such as Barrett's oesophagus and oesophageal adenocarcinoma. In this paper, we propose that a neuroimmunological mechanism is responsible for the protective effect of H. pylori on GORD. H. pylori infection of the gastric mucosa induces a T helper1-like immune response and production of pro-inflammatory cytokines. These cytokines can inhibit local sympathetic tone, whereas they increase systemic sympathetic tone. Increased sympathetic tone can induce an anti-inflammatory milieu, which in turn can inhibit inflammation in the oesophagus and lower oesophageal sphincter (LOS). Furthermore, H. pylori infection may stimulate the cholinergic anti-inflammatory pathway. It has been suggested that reflux-induced oesophageal inflammation plays an important role in the pathogenesis of reflux oesophagitis. Reduction of oesophageal inflammation by increased systemic sympathetic tone and vagal activity may lead to a decrease in reflux-induced oesophageal injury and LOS dysfunction in GORD.

Purification of astaxanthin from mutant of Phaffia rhodozyma JH-82 which isolated from forests trees of Iran.

Astaxanthin have been extracted and purified from mutant isolate of Phaffia rhodozyma JH-82. Purified astaxanthin was identified by spectrophotometric, TLC and HPLC analysis and were compared with synthetic astaxanthin. Results of TLC analysis indicated that isolate of P. rhodozyma JH-82 were able to produce nine different carotenoids and high level of carotenoids was belong to astaxanthin. Results of this study for pure astaxanthin production indicated that mutant of JH-82 of P. rhodozyma (230 microg g(-1)(dried yeast)) produced more astaxanthin than natural isolate JH-80 (140 microg g(-1)(dried yeast)). The HPLC spectrum showed retention time 11 min for both purified and synthetic astaxanthin and solvent was CDCl3.

The effect of post-burn local hyperthermia on the reducing burn injury: the possible role of opioids.

PURPOSE: This paper studied the effect of post-burn local hyperthermia on burn induced injury. METHODS: A second-degree burn injury was induced on the right and left flanks of Balb/c mice. Thirty-two burn wounds were divided into four groups. Opioid receptor blocking was done for groups 3 and 4 by intra-peritoneal administration of Naloxone (NLX) 30 min before the thermal injury. Local hyperthermia (45 degrees C, 30 s) was applied only for the burn wounds of groups 2 and 4. Twenty-four hours after burn injury, the burned wounds were assessed for the level of iNOS (by immunohistochemistry) and the number of hair follicles (as an indicator of tissue injury). RESULTS: The wounds that received hyperthermia (group 2) had significantly more hair follicles (p < 0.001) compared to the control wounds (group 1). There was no significant difference between the number of hair follicles and acute inflammation of group 1 and group 3 (NLX + burn). Group 4 (NLX + burn + hyperthermia) had significantly fewer hair follicles compared to group 1 (p < 0.001), group 2 (p < 0.001) and group 3 (p < 0.001). The level of iNOS in groups 1, 3 and 4 was not significantly different but significantly more than group 2 (p < 0.001, p < 0.001 and p < 0.001, respectively). CONCLUSIONS: The results showed that local hyperthermia after second degree burn decreased the tissue injury and iNOS expression. It is also concluded that endogenous opioid response may have a key role in the above mentioned effects of post-burn local hyperthermia.