• Title/Summary/Keyword: immunology

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Effect of Tumeric (Curcuma longa) on Bile Acid and UDP-glucuronyl Transferase Activity in Rats Fed a High-fat and -cholesterol Diet (울금(Curcuma longa L.)이 고지방·고콜레스테롤 식이 흰쥐에서의 담즙산 및 UDP-glucuronyl transferase 활성에 미치는 영향)

  • Kim, Min-Sun;Chun, Sung-Sik;Kim, Sang-Hun;Choi, Jung-Hwa
    • Journal of Life Science
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    • v.22 no.8
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    • pp.1064-1070
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    • 2012
  • The current study examined the effect of turmeric powder on bile acid and UDP-glucuronyl transferase activity in rats fed a high-fat and -cholesterol diet. Sprague-Dawley male rats weighing $120{\pm}10$ g were randomly assigned to a normal diet group (N group) and a high-fat and -cholesterol diet group (HF group), which was further divided into a high-fat and high-cholesterol with a 2.5% tumeric powder supplement group (TPA group) and 5% turmeric powder-supplemented group (TPB group). Body weight gain and food efficiency ratio were significantly increased in the N group as compared to the HF group, but they were significantly decreased in turmeric-supplemented groups as compared to the HF group. The total serum cholesterol and TG contents of the turmeric-supplemented groups were decreased as compared to those of the HF group. Especially, the TPB group was significantly decreased as compared to the HF group. The serum LDL-cholesterol and AI of the turmeric-supplemented groups were decreased as compared to the HF group. The hepatic triglyceride contents of all groups supplemented with the tumeric powder were significantly decreased as compared to the HF group. The hepatic UDP-glucuronyl transferase activity of the turmeric-supplemented groups was increased as compared to the HF group. In particular, the TPB group was significantly increased as compared to the HF group. The serum total bile acid contents of the turmeric-supplemented groups were increased as compared to the HF group. These results suggest that tumeric has powerful health benefits that are created via UDP-glucuronyl transferase activity, bile acid, and lipid metabolism.

Up-regulation of CD11c Expression on Human Acute Myelogenous Leukemia Cells by Flt-3 Ligand (인간 골수성 백혈병 세포에서 Flt-3 수용체 리간드에 의한 CD11c 발현의 증가)

  • Xu, Qi;Kwak, Jong-Young
    • Journal of Life Science
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    • v.19 no.12
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    • pp.1690-1697
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    • 2009
  • CD11c and costimulatory molecules such as CD80 and CD86 express mainly in dendritic cells (DCs). In this study, we investigated the biologic effects of recombinant Fms-like tyrosine kinase-3 (Flt-3) ligand on the expression of DC surface markers, including CD11c in leukemia cell lines, such as KG-1, HL-60, NB4, and THP-1 cells. The expression of the Flt-3 receptor was found in NB4 and HL-60 cells, as well as KG-1 cells, but not in THP-1 cells. When KG-1 cells were cultured in a medium containing Flt-3 ligand or granulocyte macrophage-colony stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-$\alpha$, cell proliferation was inhibited and the expression levels of CD11c, major histocompatibility complex (MHC)-I, and MHC-II were increased in the cells. Flt-3 ligand also increased the expression level of CD11c on HL-60 and NB4 cells, but not on THP-1 cells. In comparison with CD11c expression, the expression level of CD11b on KG-1 cells, but not on NB4 and HL-60 cells, was slightly increased by Flt-3 ligand. Flt-3 ligand induced phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and p38-mitogen-activated protein kinase (p38-MAPK) in KG-1 cells, and the up-regulation of CD11c expression by Flt-3 ligand in the cells was abrogated by PD98059, an inhibitor of MEK. The results suggest that Flt-3 ligand up-regulates DC surface markers on $CD34^+$ myelomonocytic KG-1 cells, as well as promyelocytic leukemia cells, and that the differentiation of the leukemia cells into DC-like cells by Flt-3 ligand is mediated by ERK-1/2 activity.

Effect of D-Fructose on Sugar Transport Systems in Trichoplusia ni Cells and Photolabeling of the Trichoplusia ni Cell-Expressed Human HepG2 Type Glucose Transport Protein (Trichoplusia ni 세포에 내재하는 당 수송체에 D-fructose가 미치는 효과와 Trichoplusia ni 세포에 발현된 사람 HepG2형 포도당 수송 단백질의 photolabelling)

  • Lee, Chong-Kee
    • Journal of Life Science
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    • v.24 no.1
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    • pp.86-91
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    • 2014
  • Trichoplusia ni cells are used as a host permissive cell line in the baculovirus expression system, which is useful for large-scale production of human sugar transport proteins. However, the activity of endogenous sugar transport systems in insect cells is extremely high. Therefore, the transport activity resulting from the expression of exogenous transporters is difficult to detect. Furthermore, very little is known about the nature of endogenous insect transporters. To exploit the expression system further, the effect of D-fructose on 2-deoxy-D-glucose (2dGlc) transport by T. ni cells was investigated, and T. ni cell-expressed human transporters were photolabeled with [$^3H$] cytochalasin B to develop a convenient method for measuring the biological activity of insect cell-expressed transporters. The uptake of 1 mM 2dGlc by uninfected- and recombinant AcMPV-GTL infected cells was examined in the presence and absence of 300 mM of D-fructose, with and without $20{\mu}M$ of cytochalasin B. The sugar uptake in the uninfected cells was strongly inhibited by fructose but only poorly inhibited by cytochalasin B. Interestingly, the AcMPV-GTL-infected cells showed an essentially identical pattern of transport inhibition, and the rate of 2dGlc uptake was somewhat less than that seen in the non-infected cells. In addition, a sharply labeled peak was produced only in the AcMPV-GTL-infected membranes labeled with [$^3H$] cytochalasin B in the presence of L-glucose. No peak of labeling was seen in the membranes prepared from the uninfected cells. Furthermore, photolabeling of the expressed protein was completely inhibited by the presence of D-glucose, demonstrating the stereoselectivity of labeling.

Components and Biological Activity of Aqueous Extract Isolated from Winged Stem of Euonymus alatus (화살나무 물 추출물의 구성성분과 생리활성)

  • Oh, Bong-Yun;Hwang, Soo-Kyung;Cheong, Mi-Young;Sin, Hong-Sig;Park, Bock-Hee;Lee, Jeong-Ho;Kim, Soo-Hyun
    • Korean Journal of Food Science and Technology
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    • v.37 no.6
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    • pp.898-904
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    • 2005
  • Although Euonymus alatus (EA) has been used as traditional medicine for cancer treatment, exact substances involved in curing of the disease are not yet known. Free radical scavenging and reactive oxygen species (ROS) removal activities of aqueous extract components isolated from winged stem of EA in animal cell line were investigated. Aqueous extract of EA (AEEA) was fractionated by ultrafiltration. All fractions mainly consisted of polysaccharide (44.8%), protein (2.1%), small amounts of phenol compounds and organic acids. Antioxidant activity of AEEA increased depending on concentration fractions, as determined by 1,1-diphenyl-2-picrylhydrazyl method. ROS removal activity was visualized in Chinese hamster ovary cell line using laser scanning confocal microscope, and AEEA activity increased in order of F IV>F III>F I>F II. These results suggest AETA has bioactive carbohydrates with potentials as functional foods and antioxidants.

Analysis of Clonorchis sinensis antigens and diagnosis of clonorchiasis using monoclonal antibodies (단세포군 항체를 이용한 간흡충 항원의 분석 및 간흡충증의 진단)

  • Yong, Tae-Sun;Im, Gyeong-Il;Jeong, Pyeong-Rim
    • Parasites, Hosts and Diseases
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    • v.29 no.3
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    • pp.293-310
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    • 1991
  • Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyse C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal anti- bodies were determined to be IgGl, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively, The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunoauorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity. The ELISA- inhibition test using monoclonal antibodies was found to have same sensitivity and definitely high specificity in comparison with conventional ELISA for serodiagnosis of human clonorchiasis.

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Potential Contamination Sources on Fresh Produce Associated with Food Safety

  • Choi, Jungmin;Lee, Sang In;Rackerby, Bryna;Moppert, Ian;McGorrin, Robert;Ha, Sang-Do;Park, Si Hong
    • Journal of Food Hygiene and Safety
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    • v.34 no.1
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    • pp.1-12
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    • 2019
  • The health benefits associated with consumption of fresh produce have been clearly demonstrated and encouraged by international nutrition and health authorities. However, since fresh produce is usually minimally processed, increased consumption of fresh fruits and vegetables has also led to a simultaneous escalation of foodborne illness cases. According to the report by the World Health Organization (WHO), 1 in 10 people suffer from foodborne diseases and 420,000 die every year globally. In comparison to other processed foods, fresh produce can be easily contaminated by various routes at different points in the supply chain from farm to fork. This review is focused on the identification and characterization of possible sources of foodborne illnesses from chemical, biological, and physical hazards and the applicable methodologies to detect potential contaminants. Agro-chemicals (pesticides, fungicides and herbicides), natural toxins (mycotoxins and plant toxins), and heavy metals (mercury and cadmium) are the main sources of chemical hazards, which can be detected by several methods including chromatography and nano-techniques based on nanostructured materials such as noble metal nanoparticles (NMPs), quantum dots (QDs) and magnetic nanoparticles or nanotube. However, the diversity of chemical structures complicates the establishment of one standard method to differentiate the variety of chemical compounds. In addition, fresh fruits and vegetables contain high nutrient contents and moisture, which promote the growth of unwanted microorganisms including bacterial pathogens (Salmonella, E. coli O157: H7, Shigella, Listeria monocytogenes, and Bacillus cereus) and non-bacterial pathogens (norovirus and parasites). In order to detect specific pathogens in fresh produce, methods based on molecular biology such as PCR and immunology are commonly used. Finally, physical hazards including contamination by glass, metal, and gravel in food can cause serious injuries to customers. In order to decrease physical hazards, vision systems such as X-ray inspection have been adopted to detect physical contaminants in food, while exceptional handling skills by food production employees are required to prevent additional contamination.

Antimicrobial effect of toothbrush with light emitting diode on dental biofilm attached to zirconia surface: an in vitro study (지르코니아 표면에 부착된 바이오필름에 대한 LED 치솔의 항균효과)

  • Park, Jong Hew;Kim, Yong-Gun;Um, Heung-Sik;Lee, Si Young;Lee, Jae-Kwan;Chang, Beom-Seok
    • Journal of Dental Rehabilitation and Applied Science
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    • v.35 no.3
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    • pp.160-169
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    • 2019
  • Purpose: The purpose of this study was to evaluate the antimicrobial effects of a toothbrush with light-emitting diodes (LEDs) on periodontitis-associated dental biofilm attached to a zirconia surface by static and dynamic methods. Materials and Methods: Zirconia disks (12 mm diameter, 2.5 mm thickness) were inserted into a 24-well plate (static method) or inside a Center for Disease Control and Prevention (CDC) biofilm reactor (dynamic method) to form dental biofilms using Streptococcus gordonii and Fusobacterium nucleatum. The disks with biofilm were subdivided into five treatment groups-control, commercial photodynamic therapy (PDT), toothbrush alone (B), brush with LED (BL), and brush with LED+erythrosine (BLE). After treatment, the disks were agitated to detach the bacteria, and the resulting solutions were spread directly on selective agar. The number of viable bacteria and percentage of bacterial reduction were determined from colony counts. Scanning electron microscopy (SEM) was performed to visualize alterations in bacterial morphology. Results: No significant difference in biofilm formation was observed between dynamic and static methods. A significant difference was observed in the number of viable bacteria between the control and all experimental groups (P < 0.05). The percentage of bacterial reduction in the BLE group was significantly higher than in the other treated groups (P < 0.05). SEM revealed damaged bacterial cell walls in the PDT, BL, and BLE groups, but intact cell walls in the control and B groups. Conclusion: The findings suggest that an LED toothbrush with erythrosine is more effective than other treatments in reducing the viability of periodontitis-associated bacteria attached to zirconia in vitro.

Clinical Features, Diagnosis, Management, and Outcomes of Idiopathic Pulmonary Fibrosis in Korea: Analysis of the Korea IPF Cohort (KICO) Registry

  • Jegal, Yangjin;Park, Jong Sun;Kim, Song Yee;Yoo, Hongseok;Jeong, Sung Hwan;Song, Jin Woo;Lee, Jae Ha;Lee, Hong Lyeol;Choi, Sun Mi;Kim, Young Whan;Kim, Yong Hyun;Choi, Hye Sook;Lee, Jongmin;Uh, Soo-Taek;Kim, Tae-Hyung;Kim, Sang-Heon;Lee, Won-Yeon;Kim, Yee Hyung;Lee, Hyun-kyung;Lee, Eun Joo;Heo, Eun Young;Yang, Sei Hoon;Kang, Hyung Koo;Chung, Man Pyo;Korea ILD Study Group,
    • Tuberculosis and Respiratory Diseases
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    • v.85 no.2
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    • pp.185-194
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    • 2022
  • Background: The Korea Interstitial Lung Disease Study Group has made a new nationwide idiopathic pulmonary fibrosis (IPF) registry because the routine clinical practice has changed due to new guidelines and newly developed antifibrotic agents in the recent decade. The aim of this study was to describe recent clinical characteristics of Korean IPF patients. Methods: Both newly diagnosed and following IPF patients diagnosed after the previous registry in 2008 were enrolled. Survival analysis was only conducted for patients diagnosed with IPF after 2016 because antifibrotic agents started to be covered by medical insurance of Korea in October 2015. Results: A total of 2,139 patients were analyzed. Their mean age at diagnosis was 67.4±9.3 years. Of these patients, 76.1% were males, 71.0% were ever-smokers, 14.4% were asymptomatic at the time of diagnosis, and 56.9% were at gender-age-physiology stage I. Occupational toxic material exposure was reported in 534 patients. The mean forced vital capacity was 74.6% and the diffusing capacity for carbon monoxide was 63.6%. Treatment with pirfenidone was increased over time: 62.4% of IPF patients were treated with pirfenidone initially. And 79.2% of patients were treated with antifiboritics for more than three months during the course of the disease since 2016. Old age, acute exacerbation, treatment without antifibrotics, and exposure to wood and stone dust were associated with higher mortality. Conclusion: In the recent Korean IPF registry, the percentage of IPF patients treated with antifibrotics was increased compared to that in the previous IPF registry. Old age, acute exacerbation, treatment without antifibrotics, and exposure to wood and stone dust were associated with higher mortality.

Evaluation of zinc oxide and copper oxide nanoparticles as potential alternatives to antibiotics for managing fowl typhoid in broilers

  • Muhammad Atif Raza;Eungyung Kim;Muhammad Shakeel;Muhammad Fiaz;Lei Ma;Hyeonjin Kim;Chae Yeon Kim;Zhibin Liu;Ke Huang;Kanghyun Park;Muhammad Tariq Javed;Myoung Ok Kim
    • Journal of Animal Science and Technology
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    • v.66 no.5
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    • pp.962-980
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    • 2024
  • Antimicrobial resistance poses challenges to humans and animals, especially to the poultry sector in control of fowl typhoid with antibiotics, leading to increased mortality and food insecurity. Therefore, it is essential to develop more effective medications as alternatives to antibiotics. Currently, zinc oxide and copper oxide nanoparticles are of such significant interest due to their antibacterial properties. This study aimed to evaluate antimicrobial activity of zinc oxide and copper oxide nanoparticles against fowl typhoid in broilers. Ninety broiler chicks were raised under suitable management conditions. On day 10 of age, chicks were divided into six groups: control negative, control positive, T1, T2, T3, and T4. On day 19 of age, chicks in all groups except control negative were infected with Salmonella gallinarum (0.2 mL, 108 CFU/mL). After appearance of clinical signs, the treatments (Florfenicol; 50 mg/L drinking water [T1], and zinc oxide + copper oxide nanoparticles; 25 + 10 mg/kg/d [T2], 37.5 + 15 mg/kg/d [T3], and 50 + 20 mg/kg/d [T4]) were administered to chicks. Chicks were sacrificed on 26th and 30th day of age, and samples of blood and tissue were obtained. Hematological analysis with gross and histopathological examination of spleen, thymus and bursa of Fabricius was performed. Results revealed that there was no visible congestion in spleen and thymus of T3 and T4 at 11th day post infection. Antibody level against new castle's disease and lymphoproliferative response showed no significant difference in all groups. However, phagocytic response in nanoparticles treated groups exhibited a notable (p < 0.01) distinction compared to control positive. Notably, T3 demonstrated the highest level of phagocytic activity. Hematological parameters, including lymphocytes, heterophils, eosinophils, and heterophils/lymphocytes ratio in groups T2, T3, and T4, indicated significant (p < 0.01) difference compared to control positive. However, lymphocytes, heterophils, and heterophils/lymphocytes ratio in groups T2, T3, and T4 showed no significant difference when compared to T1. Nanoparticle treated groups showed decreased (p < 0.01) congestion of spleen and thymus as compared to control positive. Overall, zinc oxide and copper oxide nanoparticles have potential to serve as an alternative to florfenicol in treatment of fowl typhoid.

A clinical study of allergic rhinitis (알레르기 비염에 관한 임상적 연구)

  • 채병윤
    • The Journal of Korean Medicine
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    • v.21 no.3
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    • pp.149-165
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    • 2000
  • As recent developments of Immunology and Nuclear medicine, serum IgE and IgG values are helpful in the diagnosis and evaluation of the therapeutic effects of nasal allergies. But in Korea, air pollution and the increased use of food additives have become leading factors in nasal allergies, It seems to be induced by environmental change, especially industrialization and urbanization, so allergic rhinitis in our environment has changed in accordance with the changes made in the living environment. Therefore this study is attempted in order to observe a clinical analysis which places more importance on allergic rhinitis. We studied 200 patients who had visited Kyunghee Oriental Medical Center with allergic rhinitis from January 1, 1999 to December 31, 1999 The results were as follows: 1. The sex distribution was 114 males(57%) and 86 females(43%). In age distribution, the average age was 25. In males, ages ranged from 3 to 66 years old and the average was 23.81. In females, ages ranged from 4 to 67 years old and the average was 28.57. The peak age was 30~39 years old(24%); under 9 years old and 10~19 years old were each 18%; 20~29 years old was 22%; 40~49 years old was 11 %; over 50 years old 6.5%. The gulf between males and females showed a statistically significant difference(P<0.025). 2. In the age of onset, male' s maximum was 62.5, minimum was 0.25; female s maximum was 59.5, minimum was 0.2. Under 9 years old was the most with 34%(male 24%, female 10%), 10~19 years old was 18%, 20~29 years old was 22.5%, 30~39 years old was 13.50%, over 40 years old was 12%, The gulf between males and females were showed statistically significant difference.(p<0.014) 3. The average duration of the disease was 5.67 years. In male and female, the maximum was 30, the minimum was 0.05; under 5 years old was the most with 62%(male 34.50%, female 27.50%); 6~10 years old was 23%. So, under 10 years old was 85%. There was no statistically significant difference in the duration of disease. 4. Regarding type of residence, 47.50% of patients with allergic rhinitis lived in apartments, 52.50% lived in houses. In males, 29.50% lived in apartments, 27.50% lived in houses. In females, 18% lived in apartments, 25% lived in houses. There was no statistically significant difference in the residence by T-test and chi-test. 5. In the distribution of season, spring is the most with 29.5% of patients, winter 28%, fall 25.5%, and summer 17%. But there was no statistically significant difference. 6. After observing 200 patients with allergic rhinitis, classifying main symptoms into 5 types, sneezing was the main symptom in 177cases(88.50%), nasal obstruction in 176cases(88%), rhinorrhea in 169cases(84.5%), post nasal discharge in 87cases(43.50%), and itching in I04cases(52%). The Cumulus ration is 98.50% and symptoms overlapped with an average 3.57±0.1 times but in an analysis of variance of these symptoms, the gulf between males and females was not recognized as statistically significant by T-test and ANOVA. 7. Patients whose families have allergic diseases account for 90 cases(45%) : 49cases(24.50%) male and 41cases(20.50%) female. There were 4 cases (71.11 %) whose families have allergic rhinitis, 9cases(10%) of asthma, and 7.78% with allergic dermatitis. There were 61 (67.80%) cases of patients whose parents have allergic diseases; cases wherein the patient s child had allergic diseases numbered 13 (14.45%); and cases with a sibling with allergic diseases totalled 16cases (17.80%). There was no statistically significant difference in allergic disease regarding sex, parents, or siblings by chi-test. 8. Blood type: For males, type A is the most common, with 37cases(18.5%), followed by type B with 32cases(16%), type O 28cases(l4%) and type AB 13cases(6.5%). For females, type B is the most common, with 30cases(15%), followed by type O with 23cases(l1.5%), type A with 18cases(9%) and type AB with 13cases(6.5%). There was no statistically significant difference in blood type by chi-test. 9. In the selection of prefered food, most patients prefer cool food, with 98 such cases(49%), tepid food in 54cases(27%) and warm food in 48cases(24%). These showed a statistically significant difference in the selection of prefered food between males and females by chi-test(p<0.009). 10. The state of Past History was classified into II types. chronic hypertrophic rhinitis is the most common with 11cases (18.64%), tonsil and adenoid hypertrophy is 8cases(l3.56%), sinusitis is 6cases(10.17%), nasal septum deviation is 4cases, nasal polyp is 2cases, others are 10cases(l6.95%). No statistically significant difference in past history between males and females was shown, but a statistically significant difference was shown when males and females were compared with total cases by T-test(p<0.002, P<0.0008). 11. Regarding complications, 37 patients (28.91%) had sinusitis: 22cases(17.19%) in male, 15cases(11.72%) in female. Chronic hypertrophic rhinitis was found in 15cases(11.72%). Others are under 10%. There was no statistically significant difference in the type of complications between males and females, but a statistically significant difference was shown when males and females were compared with total cases by T-test(P<0.00l, P<0.007). 12. In the treatment, medication was used 1691 times, an average of 2.58 times. No.34 was used 370 times for 124 cases, an average of 2.98 times. No. 152 was used 318 times for 106 cases, an average of 3.00 times. No.151 was used 307 times for 97cases, an average of 3.16 times. No. 31 was used 117 times for 33 cases, an average of 3.55 times. No 25 was used 116 times for 33 cases, an average of 3.52 times. 13. In the duration of treatment, the most frequent is 1 week(69cases, 34.50%), the maximum is 20weeks, and the minimum is 1week. A treatment period of 2~3 weeks accounted for 32% of cases, a period of 4~5weeks accounted for 13.5%. The gulf between males and females showed a statistically significant difference in the duration of treatment.(p<0.01). There was a statistical significance when the males were compared with total cases by ANOVA(P<0.03). 14. A comparison between before-treatment and after-treatment showed a statistically significant difference in treatment by T-test (p<0.01) and F-test (p<0.0058).

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