Effect Of Local Hyperthermia On Mice Sporotrichosis Lesions And Lesional IFN-γ Expression

IntroductionSporotrichosis is one of the most common deep mycosis.Potassium iodide and itraconazole orally are both effective.Local hyperthermia is often a useful adjunct.For pregnant women and patients with severe adverse reaction,local hyperthermia is the treatment of first choice.Further more,local hyperthermia is simple,economical,and less adverse.It is believed that the mechanism of local hyperthermia for sporotrichosis involves not only it's effect on the causative agent but also the immunity.Enhancing antigen presentation is one effect of hyperthermia on immunity.Cell-mediated immunity to S. schenckii antigen is a key immunologic defense mechanism that controls the infection. The function of DCs from monocytes is antigen presentation.S.schenckii can activate them to induce th1-prone immune response.Cellular immunity is mediated by th1 cells which can produce cytokines such as IFN—_γactivating macrophage to kill the pathogens.Heat can effect the upper phase of the immune response so it may contribute to the host defending against the pathogens.In this study we observe the curative effect of local hyperemia on mice model of sporotrichosis and we also study the changes of cytokine in the lesions after heat treatment.The purpose of the current study was to find the probable mechanism of the heat.Materials and methodsPart one 1,materialsStrain:The S.schenckii strain(12608) isolated from a patent with sporotrichisis of lymphatic form was used throughout the study.Mice:Female BALB/c mice aged 8-10 weeks(mean weight 20-22g) were purchased from the Laboratory Animal Centre of China Medical University(Shenyang, China).Thirty mice were divided into two groups of fifteen mice.All the mice were maintained in a specific pathogen-free environment.The mice received food and filtered water ad libitum.For experimental infection,thirty mice were injected subcutaneously at two points on their tails with 0.05 ml of the yeast form suspension (10~8/ml).Two mice injected with sterile phosphate-buffered saline served as the negative controls.2,methodsLocal hyperthmia:The lesions appeared about two weeks after inoculation then the tails of infected mice were locally heated by warm water.The temperature is 43℃, three times weekly,fifteen minutes per time.The control group wasn't heated.As the therapy beginning,the changes of lesions and strength of inflammatory reaction were observed.It is effective when the lesions fully receded and the skin of affected area returned to normal.3,Statistical analysisSoftware SPSS13 was used and the data was analyzed by corrected chi square test. P Values less than 0.05 were considered to represent statistically significant differences.Part two1,materialsThe animal models and the use of local hyperthermia are same as elucidated above in the part one.2,methods After the second treatment,animals from each group were submitted to euthanasia to obtain material from the lesions of the tail to be processed for histopathology analyses(buffered formaline at 10%was used as the fixative agent).Histology sections were stained with HE and PAS(Schiff' s period acid).IFN-_γImmunohistochemical staining was also performed.Interpretation of the immunohistochemistry slides:dark red cells with AEC staining are the IFN-_γpositive cells.In the cutaneous lesions:read the positive infiltrated cells in continuous 3 high power lens in dermis.Positive cells less than 10% is negative,positive cells has four levels:10%to 25%is weak;25%to 50%is moderate;50%to 75%is strong;more than 75%is extremely strong.3,Statistical analysisThe data was analyzed by Mann-Whitney U test in software SPSS 13.Statistical significance was established if the two sided P value of a test was less than 0.05.ResultsPart oneLesions appeared about one week after inoculation.The lesions were significant from week two to week three.The characteristic lesions may be red papules,nodules and pustules then evolved to ulcers,suppuration and crust.There were no changes about the activities and diets of the mice.The lesions of the treatment group receded gradually comparable with the control group.After six times of treatment the rate of regression was about 60%comparable with only 13.3%in the control group.When twelve times treatment were performed,the rate of regression increases to 80% comparable with 20%in the control group.They were significant differences in statistics.Part twoIFN-_γexpresses extensively in the epidermis.The basal layer is stronger and it fades gradually in the upper layers.The follicles in the dermis were also stained lightly. The staining in epidermis was no different in the normal skin,treatment group and control group.IFN-_γpositive cell were present in the granulomatous lesions in dermis. They were detected in in all samples.The pattern of IFN-gamma staining appeared to be combination of intracellular staining in mononuclear lymphoid cells and extracellular deposition in the surrounding tissue.Statistical significance was established between the dermis of treatment group and the control group.Conclusions1,The animal models of mice with sporotrichosis present tendency of self cure.2,The efficacy of local hyperthermia on mice with sporotrichosis was confirmed.3,The expression of IFN-_γincreased in granulomatous lesion of dermis after thermotherapy.