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VIP IMMUNOREACTIVITY IN THE RAT TRIGEMINAL GANGLION AFTER INFERIOR ALVEOLAR NERVE AXOTOMY (하치조신경 절단 후 흰쥐 삼차신경절에서 VIP-IR의 변화)

  • Kim, Heung-Joong;Park, Joo-Cheol;Kim, Hyun-Sub;Moon, Joo-Hoon
    • Restorative Dentistry and Endodontics
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    • v.25 no.2
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    • pp.225-234
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    • 2000
  • The purpose of this study was to investigate the distribution and fluorescene intensity of vasoactive intestinal polypeptide(VIP) immunoreactive cells in rat trigeminal ganglion after inferior alveolar nerve axotomy. The animals were divided into normal and two experimental groups. The experimental animals were sacrificed at 14th and 28th day after inferior alveolar nerve axotomy. The trigeminal ganglion was removed and immersed in the 4% paraformaldehyde-0.2% picric acid in 0.1M phosphate buffer. Serial frozon sections about $16{\mu}m$ in thickness were cut with a cryostat. The immunofluorescence staining was performed. The rabbit anti-VIP(1 : 8,000) was used as primary antibody and fluorescene isothiocynate(FITC)-conjugated anti-rabbit IgG(1 : 80) as secondary antibody. The slides were observed under confocal laser scanning microscope. Three-dimensional images were constructed from 9 serial images(each $1{\mu}m$ in thickness) made by automatic optical sectioning. Unprocessed optical sections were obtained and stored on a optical disk. Color picture were printed by a video copy processor. The results were as follows; 1. The appearance of VIP immunoreactive cells in the mandibular part of trigeminal ganglion was 8.79${\pm}$1.99% in normal group and 39.16${\pm}$5.62% in 14 days, 16.25${\pm}$2.39% in 28 days after inferior alveolar nerve axotomy groups. 2. The relative fluorescence intensity of VIP immunoreactive cell bodies in the mandibular part of trigeminal ganglion was 134.40${\pm}$10.39 in normal group and 192.88${\pm}$14.06 in 14 days, 143.10${\pm}$5.02 in 28 days after nerve axotomy groups. Therefore, the relative fluorescence intensity of 14 days after nerve axotomy group was 43.3% higher than intensity of normal group. 3. In optical single section analysis of VIP immunoreactive cell bodies, white cell bodies(moderate fluorescence intensity) were the most abundant in normal and 28 days after nerve axotomy groups. Whereas, in 14 days after nerve axotomy group, red cell bodies(high fluorescence intensity) were the most abundant. 4. In optical serial section analysis of VIP immunoreactive cell bodies, red cell bodies(high fluorescence intensity) were observed in a part of the 9 sections of normal and 24 days after nerve axotomy groups. Whereas, red cell bodies were observed in all of the 9 sections of 14 days after nerve axotomy group. 5. The results indicates that number and fluorescence intensity of VIP immunoreactive cells were increased in the mandibular part of trigeminal ganglion following inferior alveolar nerve axotomy.

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Beneficial Effects of Daebong Persimmon against Oxidative Stress, Inflammation, and Immunity in vivo (대봉감의 항산화, 항염증 및 면역증강 효과)

  • Lee, Hee Jae;Lim, So Young;Kang, Min-Gyung;Park, Jeongjin;Chung, Hyun-Jung;Yang, Soo Jin
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.44 no.4
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    • pp.491-496
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    • 2015
  • The purpose of this study was to assess the antioxidant, anti-inflammatory, and immuno-enhancing effects of Daebong persimmon (DP) and Bansi (BS) in vivo. Two types of astringent persimmons (DP and BS) were used for this experiment. C57BL/6J mice were assigned to the following groups: 1) lean control, 2) high-fat diet control (HF), 3) A region DP (3% wt/wt) with HF diet (A-DP), 4) B region DP with HF diet (B-DP), 5) C region DP with HF diet (C-DP), 6) D region BS with HF diet (D-BS), and 7) E region BS with HF diet (E-BS). All mice were sacrificed after 4 weeks of treatment, after which blood and tissues were collected. Antioxidant enzyme activities, inflammatory markers, and immune factors were evaluated. DP and BS treatments did not alter food intake or body weight, compared with HF. Administration of B-DP increased catalase activities in serum. Hepatic levels of malondialdehyde, a product of lipid peroxidation, were significantly lower in A-DP mice than in the HF group. A-DP had down-regulatory effects against inflammation induced by high-fat diet feeding, as shown by significant reduction of interleukin (IL)-$1{\beta}$, IL-6, and tumor necrosis factor-${\alpha}$. Additionally, A-DP treatment exerted an immuno-stimulatory effect, as shown by increasing levels of immunoglobulin G. DP treatment improved the level of insulin-like growth factor-1. These results indicate that DP has beneficial health effects on oxidative stress, inflammation, and immunity in vivo.

Clinical Characteristics, Histology and Prognosis of Autoimmune Hepatitis in Korean Children (소아 자가면역성 간염: 임상적 특성, 조직 소견 및 예후)

  • Chung, Dae-Lim;Seo, Jeong Kee;Yang, Hye Ran;Ko, Jae Sung;Park, Sung Hye
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.7 no.2
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    • pp.186-196
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    • 2004
  • Purpose: Autoimmune hepatitis is a chronic inflammatory liver disease with unknown cause that is characterized by liver histology, circulating autoantibodies and increased levels of immunoglobulin G. Only sporadic reports are available on autoimmune hepatitis in children. The aim of this study was to evaluate the clinical, biochemical, and histological features, and the long-term outcome of autoimmune hepatitis in Korean children. Methods: We reviewed the medical records of 14 children diagnosed as having autoimmune hepatitis at Seoul National University Children's Hospital from 1990 to 2004, and analyzed clinical, biochemical, and histological features, and clinical outcomes. Results: Mean age at diagnosis was 9 years and 11 of the 14 children were female. Six children presented with acute hepatitis-like manifestations. Jaundice and fatigue were the most common symptoms. Other autoimmune diseases accompanied in 6 children. Anti-nuclear antibody was detected in 13 patients and anti-smooth muscle antibody was positive in 8. All 14 patients were type 1 autoimmune hepatitis. The main histologic findings were interface hepatitis, rosette formation, and cirrhosis. Clinical and biochemical features were improved in six patients treated with ursodeoxycholic acid. Eight patients were treated with corticosteroid alone or in combination with azathioprine and five of them are in biochemical remission. Conclusion: Autoimmune hepatitis is an inflammatory liver disease, which has a favorable long-term outcome if it is diagnosed and treated promptly. Therefore, autoimmune hepatitis should be suspected in children with chronic hepatitis of unknown etiology, especially in female patients who show hypergammaglobulinemia or some clinical features of autoimmune disease.

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Preservation Conditions of Aqueous Samples Containing silver Nanomaterials (은나노물질을 포함한 수질시료의 보관조건)

  • Kang, Mun Hee;Park, Sol;Lee, Sang-Woo;Kim, Hyun-A;Lee, Byung-Tae;Eom, Ig-Chun;Kim, Soon-Oh
    • Journal of Korean Society of Environmental Engineers
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    • v.37 no.4
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    • pp.218-227
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    • 2015
  • A prerequisite for precise quantification of nanomaterials contained in environmental samples is to prepare suitable preservation conditions of samples. This study was initiated to suggest preservation conditions of aqueous samples for analyses of metal nanomaterials. Variation in the size of silver nanomaterial (cit-AgNP) was observed according to change in various conditions, such as pH, electrolyte concentration, temperature, nanomaterial concentration, and time. Aggregation of AgNP was characterized for each environmental condition, and finally proper preservation conditions of samples were proposed based on experimental results on AgNP aggregation. In addition, the preservation period of sample was computed by the doublet time of AgNP. The results indicate that the aggregation rate of cit-AgNP was close to 0 at the conditions of pH of ${\geq}7$, electrolyte ($Ca(NO_3)_2$) concentration of ${\leq}3mM$, temperature of $4^{\circ}C$, and cit-AgNP concentration of ${\leq}2mg/L$. Furthermore, the experimental results on doublet time of cit-AgNP suggest that maximum preservation period was evaluated to be 15.79~17.53 days when the concentration of 100 nm cit-AgNP is assumed to be $1{\mu}g/L$ which is considered as an environmentally-relevant concentration of engineered nanomaterials. Our results suggest that samples should be preserved at $4^{\circ}C$ and analyzed within 2 weeks.

Immunoelectron Microscopic Study on the Paneth Cell of Rabbit after the Common Bile Duct Ligation (총담관결찰후 집토끼 Paneth세포의 변화에 대한 면역전자현미경적 연구)

  • Park, Kyung-Ho;Cho, Hwee-Dong;Yang, Nam-Gil;Ahn, E-Tay;Ko, Jeong-Sik;Kim, Jin-Gook
    • Applied Microscopy
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    • v.24 no.2
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    • pp.78-92
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    • 1994
  • Lysozyme has been reported to be present in the secretory granules of the Paneth cell, and lysozyme immunoreactivity has been detected by immunogold method in Paneth cells of the intestine of human, mouse and rat. The present study was aimed at clarifying the intracellular distribution and changes of the lysozyme immunoreactivity in rabbit Paneth cell after common bile duct ligation of rabbit, using the electron microscope immunogold technique. Healthy adult rabbits weighing about 2kg body weight were divided into normal and bile duct ligated groups. Common bile duct ligation was performed aseptically under ether anesthesia. Experimental animals were sacrificed on the 1st, the 3rd, the 5th, the 7th and the 14th day after the operation. Mucosal specimens from the intestinal gland of ileum were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde, followed by 1% osmium tetroxide, embedded in araldite mixture, cut with LKB-V ultratome. Ultrathin sections were placed on parlodion coated nickel grids (200mesh). The section-bearing grids were floated upside down on the added substance in a moist chamber at room temperature except for the primary antibody step, which was at $4^{\circ}C$. Sections were etched with a saturated solution of sodium m-periodate for 60min. After etching, sections were pretreated with 0.02M tris buffered saline (TBS), pH 8.4, with 1% bovine serum albumin (BSA, Sigma) for 60min, then treated polyclonal rabbit anti-human lysozyme (Dakipatts) diluted 1 : 50 in TBS with 0.1% BSA for 20hr. Subsequently, grids were incubated 60min in biotinylated goat anti rabbit IgG (Amersham) diluted 1 : 100 in TBS with 0.1% BSA. After this, sections were incubated 60min on streptavidin gold G10 (Amersham) diluted 1 : 50 in TBS with 0.1% BSA. After each step, the grids were briefly rinsed with TBS with 0.1% BSA. After the strepavidin gold step, the sections were jet washed with distilled water. Counterstain of the sections performed by uranyl acetate and lead citrate, and observed with JEM 100 CX II electron microscope. Observed results were as follow; 1. Secretory granules of mouse Paneth cells have a lysozyme immunoreactivity and also eosinophil leucocyte of rabbit applied for the positive-control stain, are well labeld with gold particles. 2. Normal rabbit Paneth cells have a lysozyme immunoreactivity restricted on the secretory granules. 3. Amount lysosomes containing myelin figures in the Paneth cells were significantly increased from 5th day after the common bile duct ligation. 4. Immunoreactivity of Paneth cell secretory granules were more activated on the 3rd day after the common bile duct ligation as compared with those of the normal animal. But the lysozyme immunoreactivity were decreased from the 5th day after the common bile duct ligation. 5. Considering the above finding, lysozyme contained Paneth cell are affected following of common bile duct ligation, whereas lysosomes containing myelin-figure do not exhibit any immunoreactive relationship with those of secretory granules.

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The Significance of Acetylcholine Receptor Autoantibody Test (아세틸콜린 수용체 항체(Acetylcholine receptor autoantibody) 검사의 의의)

  • Yoo, Soh-Yeon;Lim, Soo-Yeon;Pack, Song-Ran;Seo, Mi-Hye;Moon, Hyung-Ho;You, Sun-Hee
    • The Korean Journal of Nuclear Medicine Technology
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    • v.15 no.1
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    • pp.113-116
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    • 2011
  • Purpose: Acetylcholine receptor antibodies cause acetylcholine receptor loss, which is responsible for failure of the neuromuscular junction in the acetylcholine receptor autoantibody. The disease characterized by muscle weakness and fatigue, myasthenia gravis(MG) occurs when the body inappropriately produces antibodies against acetylcholine receptors, and thus inhibits proper acetylcholine signal transmission. And this reason, the measurement of acetylcholine receptor antibodies can be of considerable value in disease diagnosis. Methods: From 2010. August to September, we tested orderd AchRAb 19 samples to get the results. 1. Pipette $5{\mu}{\ell}$ undiluted patient sera and kit control and add 125I AChR $50{\mu}{\ell}$ and incubate at R.T for 2 hours. 2. Pipette $50{\mu}{\ell}$ of anti-human IgG into each tube, and incubate at $2{\sim}8^{\circ}C$ for 2 hours. 3. Pipette $25{\mu}{\ell}$ precipitation enhancer into each tube and add 1mL washing solution into all tubes. 4. Centrifuge each tube for 20minutes at $2{\sim}8^{\circ}C$ at 1500g. 5. Aspirate or decant the supernatant. 6. Pipette 1 mL washing solution into all tubes and resuspend the pellet and repeat centrifugation. 7. Aspirate or decant the supernatant and count all tubes on a gamma counter. Results: Cut off value is 0.2 nmol/L and the results taken below 0.2 nmol/L are negative, the results above that identified as being positive values. We assayed the 19 patients samples and got 7 positive results. Of which, 6 patients were diagnosed as MG.(85.7%). Conclusions: Acetylcholine Receptor autoantibody test is intended for use by persons only for the quantitative determination of it in human serum. Even if measurement of the antibodies is not a routine test, it can be of considerable value in disease diagnosis.

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Growth Performance, Immune Response and Carcass Characteristics of Finishing Pigs by Feeding Stevia and Charcoal (스테비아와 숯이 급여된 비육돈의 사양성적, 면역력 및 도체특성)

  • Choi, Jung-Soek;Jung, Dong-Soon;Lee, Ju-Ho;Choi, Yang-Il;Lee, Jae-Joon
    • Food Science of Animal Resources
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    • v.32 no.2
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    • pp.228-233
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    • 2012
  • This study was conducted to determine the effects of stevia (Stevia rebaudiana bertoni) and charcoal supplementation on growth performance, immune response and carcass characteristics of finishing pigs. A total of 420 pigs (LYD) were randomly allocated into 7 treatments with 3 replications. Dietary treatments were 1) T1 (basal diet), 2) T2 (basal diet+0.3% stevia), 3) T3 (basal diet+0.6% stevia), 4) T4 (basal diet+0.3% charcoal), 5) T5 (basal diet+0.6% charcoal), 6) T6 (basal diet+0.3% stevia+0.3% charcoal) and 7) T7 (basal diet+0.6% stevia+0.6% charcoal). During the experimental period, average daily gain (ADG) was higher in T2 and T6 groups than the other treatments (p<0.05). Feed conversion ratio (FCR) was higher in T6 group compared to the others (p<0.05). There were no significant differences in total cholesterol level and glutamic pyruvic transaminase (GPT) activity of blood among treatments. In glutamic oxaloacetic transaminase (GOT) activity, T3, T5, T6 and T7 groups showed lower values (p<0.05) compared to T1. Insulin-like growth factor-1 concentration was higher in T2 and T6 groups than the others (p<0.05), but there were no significant differences in immunoglobulin G, lymphocyte, eosinophil, basophil and atypical lymph levels among treatments. In neutrophil, T6 showed higher level compared to the others (p<0.05). In the carcass characteristics, T6 showed higher level of a carcass grade compared to the other treatments. However, carcass length did not show any significant difference among treatments. As a result, dietary supplementation of 0.3% stevia and 0.3% charcoal showed higher ADG, higher FCR and better immune response resulting in better growth performance and carcass characteristics of finishing pigs.

Initial Ecological Risk Assessment of 1,2-Benzisothiazol-3-one in Environment (환경 중 1,2-Benzisothiazol-3-one에 대한 초기 생태위해성 평가)

  • Han, Hye-Jin;Kim, EunJu;Yoo, SunKyoung;Ro, Hi-Young;Baek, Yong-Wook;Shim, IlSeob;Eom, Ig-Chun;Kim, Hyun-Mi;Kim, PilJe;Choi, Kyunghee
    • Journal of Korean Society of Environmental Engineers
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    • v.35 no.3
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    • pp.165-170
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    • 2013
  • In this study, physico-chemical properties and environmental fate were investigated and ecotoxicity tests using fish, daphnia and algae were conducted for an initial ecological risk assessment of 1,2-Benzisothiazol-3-one. Due to low volatility of the test substance under environmental conditions, it is likely to distributed in soil and water environment. The compound has low adsorption in the soil, with low bioconcentration potential. Acute toxicity results showed that 96 h-$LC_{50}$ for Oryzias laties was 4.7 mg/L (measured) and 48h-$EC_{50}$ for Daphnia magna was 3.3 mg/L (measured). In a growth inhibition test with Pseudokirchneriella subcapitata, 72 h-$EC_{50}$ was 0.456 mg/L (growth rate, nominal) and 0.262 mg/L (yield, nominal). Using the acute toxicity value of algae, predicted no-effect concentration (PNEC) in the aquatic environment was determined to be 2.62 ${\mu}g/L$ using an factor of 100. According to globally harmonized system (GHS), the compound was categorized as aquatic acute 1 for algae, while it was categorized as aquatic acute 2 for fish and daphnia. This screening assessment suggests that the test substance may pose ecological risks in the aquatic environment.

Histopathological and Serological Observations on Experimental Anisakiasis of Rabbits (실험적 토끼 아니사키스증에서의 조직학적 병변과 혈청 항체가의 경시적 변동 양상)

  • 홍성태;이손형
    • Parasites, Hosts and Diseases
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    • v.25 no.2
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    • pp.168-180
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    • 1987
  • This study was performed to observe histopathological changes and serological reactions in chronic anisakiasis of rabbits. Each rabbit was infected per os with 30 larvae of Anisakis type I. Their sera were collected chronologically and the rabbits were killed for histopathological examination, 3, 13, 20, 30, 60, 90 and 150 days after the infection. The results were summarized as below. 1. Most of the larvae were recovered from the stomach, but a few from the omentum, intestine, mesentery and abdominal wall. The recovery rates and distribution of worms by organ were not differed by duration of infection. 2. Histologically the lesion was abscess type on 13 days, i.e., the dead worms were surrounded by fibrinous exudate, histiocytes and thick zone of numerous inflammatory cells. After 30 days, histiocytes were found to invade the worms and the lesion was changing into abscessgranulomatous type. Also a calcified worm was found on the 30th day. After then the worms were observed to be dissolved slowly until 90 days. On 150 day, only one calcified worm was observed. 3. The levels of serum IgG antibody by ELISA reached their maximum 30 days after the infection. After then, it decreased slowly until 150 days after the infection. Above serological and histopathological findings indicated that antigenic stimulation from degenerating Anisakis larvae was the greatest during the first 30 days after infection. This period was corresponding with the beginning of worm resolution or calcification. Serologic test by ELISA would be a valuable tool for confirming chronic anisakiasis.

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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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