Abstract: This study was conducted to support the investigations of probable immunological changes in asthmatic patients and to study the relationship between some immunological markers and the clinically estimated asthma severity in order to find the more correlated immunological marker with asthma severity. This study involved 61 patients of both genders ( ٤٦ female and 15 male) of age varied between 13 to 53 years from recently diagnosed cases that arrived to the allergy institute of Baghdad or from steroids and other antiinflammatory therapy ceased patients and for a period that allow to carry out study tests without effecting the results, In addition to control group which consisted of 23 apparently healthy individuals.Skin test analysis of ten allergens showed a significant differences between asthma patients and healthy controls in rate of positive reactivity for at least one tested molds or grass allergens where they attained 43.1 % and 35.3% in patients’ group respectively, results also revealed that there was a significant differences in rate of reactivity for M4 allergen only with difference in asthma severity.Total and specific serum IgE were assessed by the Enzyme Linked Immunosorbent assay (ELISA). The geometric mean of serum total IgE was significantly higher in asthmatics than healthy controls, Another Significant elevation was revealed in asthmatics as compared to control in the rate of 100 IU/ml overlapped serum total IgE individuals, this rate also showed significant difference among the different categories of asthma severity. 83 IU/ml was suggested as a typical cut off value that differentiates asthmatic patients from healthy individuals.From other side, estimation of serum specific IgE for 10 allergens demonstrated significant differences in I203 allergen specific IgE levels in addition to specific IgE for food allergens (F25, F27, F33, F35 and F81) between asthmatics and healthy controls. Its ought to mention that specific IgE for food allergens (F25, F27 and F33) also recorded significant differences by difference in asthma severity which may refer actual effect for some food allergens in asthma pathogenesis .The results didn’t document any significant difference in levels of other immunoglobulins IgM, IgG, and IgA and complement component C4 in serum between asthmatic patients and control group, Whereas a significant elevation was documented in serum C3 level of asthmatics as compared to healthy controls which supports the studies that refered some complement component role in asthma pathogenesis. From other side, this study didn’t observe any significant differences in the levels of those immunoglobulins with difference in asthma severity. Total and differential white blood cells were estimated during the study, the results showed a significant elevation in mean of total white blood cells, eosinophils and neutrophils numbers in asthmatics’ blood as compared to control, while there were no significant differences in association with remaining white blood cells. The relationship between asthma severity and white blood cells was also assessed, the results showed a significant difference in geometric mean of total white blood cells and also lymphocytes numbers in asthmatics’ blood and asthma severity.The data demonstrated that there was a significant elevation in level of eosinophil cationic protein (ECP) in asthmatics’ serum (median = 40 ng/ml) as compared with control group (median = 15.5 ng/ml) , it was also observed that elevation in serum ECP level proportionate significantly increasing of asthma severity. This study suggested that 29 ng/ml of serum ECP as a typical cut off value of statistically significant denotation for differentiation between asthmatic patients and healthy individuals. The ratio of serum eosinophil cationic protein to blood eosinophils was calculated in this study, Despite not attaining the significance level between patients’ group and controls as regards this ratio, results showed a significant elevation in ECP/Eo associated with increasing ofasthma severity among different categories. During the study of correlation among some immunological markers that adapted in many allergy centers as preliminary diagnostic tests for asthmatic patients especially the determination of total serum IgE level and blood eosinophils number, there was no significant correlation between total serum IgE level and asthma severity, correlation between blood eosinophils and asthma severity was also not significant too (r = 0.16 and 0.01 respectively). Contrary to the previous results, Statistical analysis showed a significant positive linear correlation between ECP level and also ECP/Eo ratio and clinically estimated asthma severity (r = 0.41 and 0.43 respectively, P<0.01).According to results the ratio 0.38 Pg/Cell suggested as a typical cut off value for diagnosing severe persistent asthma and 137.5 ng/ml of serum ECP concentration also suggested as a typical cut off value for differentiating severe persistent asthma cases from other less severe categories. According to the results, Determination of serum ECP can be considered as a diagnostic tool for differentiating asthma cases from not, besides it reflects asthma severity significantly, whereas calculation of ECP/Eo ratio may be an effective marker in reflecting asthma severity which may be useful in monitoring asthma cases and in determining the recovery of disease severity during therapy stages but it has no benefit for diagnosing asthma cases where no significant differences were recorded between asthmatics and healthy controls

دراسة التاثيرات الوراثية لمتعدد السكريد خارج خلوي لبكتريا Enterococcus faecium في الخلايا السرطانية == Study of the Genetic Effects of Exopolysaccharide (EPS) from Enterococcus faecium in the Cancer Cells

Author name: هويدة كريم عبد الجنابي

Supervisor name: هيفاء هادي حساني

General topic: Biology

Specific topic: Microbiology - Bacteria

Degree: Master

University: University of Baghdad

Language: English

University location: Baghdad

First pages:

Abstract: تم الحصول على ثلاث عزلات لبكتريا Enterococcus faecium من 120 عينة منتجات الالبان المحلية والمستوردة، وقد شخصت هذه العزلات باستخدام الفحوصات البكتريولوجية والكيموحيوية، استخلص متعدد السكريد خارج خلوي Exopoly Saccharide من العزلة رقم (1) لبكتريا E. faecium وذلك لكون هذه العزلة اعطت اعلى معدل انتاجية من المستخلص وبلغ 300 ملغرام/لتر من الوزن الجاف.درس التاثير السمي الخلوي لمستخلص EPS في اثنين من الخطوط الخلوية السرطانية هما AMN - 3 وHeLa، واظهر المستخلص فعالية تثبيطية في الخلايا السرطانية اعتمادا على التركيز المستخدم ومدة التعريض خصوصا عند التركيزين 2500 و5000 مايكروغرام/مللتر حيث وصلت نسب التثبيط الى 59.3% و86.2% على التوالي في خلايا AMN - 3، ثم بدات هذه النسب بالانخفاض مع تقليل التركيز المستخدم الى 1250 مايكروغرام/مللتر لتصل الى (8.3)% بينما استمرت الخلايا بالنمو والتضاعف خلال مدد التعريض الثلاث 24 ، 48 و72 ساعة عند التركيز 625 مايكروغرام/مللتر وكانت النسبة المئوية لتضاعفها (157.1%) مقارنة بعينة السيطرة (100%).اما تاثير المستخلص في خلايا HeLa فقد اظهر تاثيرا تثبيطيا على هذه الخلايا ازدادت نسبته مع زيادة التركيز ومدة التعريض وان نسب التثبيط في خلايا HeLa كانت اقل بفارق معنوي عن نسب التثبيط في خلايا AMN - 3 حيث بلغت نسب التثبيط في خلايا HeLa عند التركيزين 2500 و5000 مايكروغرام/مللتر 28.0% و31.1% على التوالي ولمدة تعرض 72 ساعة واستمر انخفاض الفعالية التثبيطية للمستخلص في خلايا HeLa في التراكيز الواطئة 625 و1250 مايكروغرام/ مللتر حيث بلغت 10.7% و14.4% على التوالي ولمدة تعرض 72 ساعة مقارنة بعينة السيطرة.درس تاثير مستخلص (EPS) وراثيا في الخلايا اللمفاوية للانسان من خلال حساب معامل التحول الارومي Blast index (BI) ومعامل الانقسام الخيطي (MI) Mitotic index وفحصت التغايرات الكروموسومية والفعالية التحفيزية لانقسام الخلايا. وجد ان هذا المستخلص قد سبب انخفاضا معنويا في معدلات معامل الانقسام ومعامل التحول الارومي يتناسب مع زيادة التركيز المستعمل ومدة التعريض لكنه لم يحدث اية تاثيرات سمية وراثية في كروموسومات الخلايا اللمفاوية المعاملة. | Three isolates of Enterococcus faecium were obtained from 120 of dairy products. These isolates were identified using bacteriological and biochemical assays.Exopolysarccharide (EPS) was extracted by (TCA 10%) method from isolate E. faecium no.1 for high production. One gram of dry weight produced 300 mg/ml of EPS.Cytotoxicity effect of EPS on cancer cell lines AMN - 3 and HeLa. An inhibitory activity of EPS against cancer cells was noticed, it was depended on EPS concentration and time exposure. Both concentrations 2500 and 5000 µg/ml were inhibited cells of AMN - 3; the percentage of inhibition was 59.3 and 86.2% respectively. Whereas, the percentage of inhibition was reduced when EPS concentration was low, 1250 µg/ml of EPS was showed 8.3% of inhibition rate. Notable, the low concentration (625µg/ml) of EPS was induced proliferation of AMN - 3 cancer cells of AMN - 3 for proliferation; the viability rate of cancer cells was 157.1% in comparison with untreated cells (control). In addition, the cytotoxic activity of EPS on cancer cell line HeLa was also studied. Significantly, inhibition rate on HeLa cells was less than that against AMN - 3 cells. The inhibition rate of HeLa cells at 2500 and 5000 µg/ml of EPS after 72 hr. was 28.06 and 31.1 % respectively. While the inhibitory activity of EPS at low concentrations (625 and 1250 µg/ml) on HeLa cells were 10.7% and 14.4% respectively.Genetically, the effect of EPS on normal human lymphocytes using different parameters such as mitotic index (MI), blastogenic index (BI), rearrangement of chromosomes, and mitogenic activity was examined. The results showed that EPS was significantly reduced MI and BI. Moreover, no genotoxic effect on chromosomes of healthy human lymphocytes was noticed