Immuno-phenotyping of stem cells isolated from human umbilical cord blood

Umbilical cord blood stem cells have gained notoriety in both clinical and research settings. The hallmark of hematopoietic stem cells is their potential regenerative therapy. This study aimed to assess the yield of umbilical cord blood CD34[+] cells as regard different obstetric and neonatal factors

Materials and Methods: Twenty umbilical cord blood samples from different clinical groups of newborns were included. Our research has concluded separation of momnonuclear blood cells umbilical cord blood, assay of viability and cell survival, magnetic labeling, enrichment and evaluation of CD34 cells by FACScan flow cytometer

Results and conclusion: our results for hematopoietic stem cells concentrations suggest that the most valuable UCB units for banking and thus transplantation come from full term, bigger babies who are born to younger mothers with few previous pregnancies. Further studying of these obstetric factors and other factors should be continued for confirmation or revealing more facts as regard our locality

Levels of Interleukin 17, Fas and eosinophil apoptosis in bronchial asthma patients

Bronchial asthma is a chronic inflammatory disorder of the airway in which many cells and cellular elements play a role. The chronic inflammation causes an associated increase in airway hyper responsiveness that leads to recurrent episodes of wheezing breathlessness, chest tightness and coughing particularly at night or in the early morning. These episodes are usually associated with widespread but variable airway obstruction that is often reversible either spontaneously or with treatment.IL-17 family members belong to a distinct category of cytokines that coordinate local tissue inflammation by inducing the release of pro-inflammatory and neutrophil-mobilizing cytokines. The importance of the IL-17 family in inflammatory and autoimmune disease is becoming increasingly apparent. In this prospective study, we measured sputum IL-17 and serum sFAS in bronchial asthma patients of various disease severities by ELISA technique and also detected sputum eosinophils apoptotic ratio [AR].This study was carried out at Mansoura University Hospitals, Medical Microbiology and Immunology and Thoracic Medicine Departments from August 2007 to August 2009. Fifty bronchial asthma patients and twenty healthy non-smoker subjects were enrolled in the study after informed consent. Sputum IL 17 and serum sFas were measured using ELISA technique and eosinophils apoptotic ratio was detected by identification the morphological features of apoptosis after staining by Giemsa stain. The levels of sputum IL 17 and serum sFAS were increased in bronchial asthma patients especially in severe asthma than control group with statistically significant difference, there is decrease in eosinophils apoptotic ratio in bronchial asthma patients than healthy controls. Bronchial asthma patients have higher levels of sputum IL 17, serum sFAS and decreased eosinophil apoptotic ratio, higher levels of IL 17 were associated with severity of the disease, high levels of serum sFAS inhibit the process of apoptosis and are associated with decrease in eosinophils apoptotic ratio

Detection of SHV-1 and SHV-2 in extended spectrum beta-lactamase producing klebsiella pneumoniae isolated from neonatal intensive care unit in Mansoura university children's hospital

Extended spectrum beta- Lactamase producing klebsiella [ESbetaL-KP] is an important cause of nosocomial infections in neonatal intensive care units [NICUs]. We conducted a prospective cohort study in the NICU, Mansoura University Children's Hospital, over a period of twelve months starting from June 2005 to May 2006, to assess the incidence of ESbetaL-KP, identify the frequency of SHV-1 and SHV-2 gene acquisition among ESbetaL-KP isolates, and the risk factors associated with ESbetaL-KP infection. Antimicrobial susceptibility was determined by, phenotypic confirmation of ESbetaL production was done by the double-disk synergy test [DDST] and Etest. Genetic detection of SHV-1 and SHV-2 genes in ESbetaL-KP isolates was done by polymerase chain reaction [PCR] and restriction fragment length polymorphisms [RFLP]. Risk factors associated with ESbetaL-KP infection were analyzed by both univariate and multiple logistic regression methods. Three hundred and ninety-eight neonates were enrolled in the study cohort. The overall nosocomial infection incidence rate was 36.6%. Klebsiella species was the commonest organism [27 among 138 bacterial isolates [19.6%]]. Eighteen klebsiella isolates [66.7%] exhibited phenotypic ESbetaL- resistance patterns. PCR amplicons from the 18 ESbetaL-KP isolates were subjected to RFLP analysis which revealed the presence of SHV-2 in all 18 isolates [100%], SHV-1 gene in 8 strains [44.4%]. Independent risk factors for ESbetaL-KP infection were: mechanical ventilation [odds ratio [OR]: 4.18, 95% confidence intervals [CI]: 1.57 -11.00]; birth weight < 1500 g [OR: 3.19, CI: 1.22 - 8.30]; duration of hospitalization > 15 days [OR: 4.09, CI: 1.17-14.40]; total parenteral nutrition [TPN] [OR: 4.93, CI: 1.12 - 21.70]; and prior use of oxyimino-antibiotics [OR: 4.87, CI: 1.10 -21.50]. Neonates infected with ESbetaL-KP higher mortality [27.8%] compared to other neonates [11%] [P=0.04]

Conclusion: This study confirms the high incidence of ESbetaL-KP in our NICU and further demonstrates the role of genes coding for SHV-1 and SHV-2 enzymes in clinical and environmental isolates. Independent risk factors for acquisition of ESbetaL-KP were mechanical ventilation; birth weight 15 days; and prior exposure to oxyimino antibiotics. Neonates infected with ESbetaL-KP experienced increased mortality compared to other neonates

Assessment of lannate toxicity in mice by comet assay

Lanate [methomyl], is a carbamate insecticide that is widely used. This study investigates the genotoxic effect of methomyl on mice. Fifty five mice were used. Mice were grouped into control group [15 animals] and two test groups [20 animals each]. Methomyl was given intraperitonealy to mice in two doses [2, 5 mg/kg and 5 mg/kg]. Animals were sacrificed after 4 and 24h. The comet assay was used for evaluation of DNA damage in lymphocytes. Methomyl was found to increase significantly DNA damage in peripheral lymphocytes. This effect did not improve after 24h. Increasing methomyl dose produced very high significant increase in DNA damage in the peripheral lymphocytes. So, it is recommended to investigate any pesticide for its drawbacks before marketing, calculate the most effective dose without side effects and generally decrease the use of pesticides. It is advised to use the comet assay as a monitoring method for high-risk groups to exclude genotoxicity. This test is rapid, simple and sensitive for measuring and analyzing DNA breakage

Relation between T lymphocytes activities and the intensity of schistosoma mansoni infection

This study was done on 60 schistosome patients and 12 cross matched healthy control persons. The schistosome patients were classified on the bases of intensity of infection into: 22 patients with light infection [one to 100 eggs/gm stool], 24 patients with moderate infection [101- 400 eggs/gm stool], 14 patients with heavy infection [>400 eggs/gm stool]. All the studied cases were submitted to flow cytometric analysis of peripheral blood mononuclear cells using monoclonal antibodies against CD3, CD4, CD8, CD28, HLA-DR. It was found that there was a significant decrease in CD3, CD4 and the expression of costimulatory molecule CD28 on CD8 T lymphocytes, while CD8 T lymphocytes and the activation marker HLA-DR expression on CD4 T lymphocytes were increased. These changes were more obvious with the increase in intensity of infection

Lymphocyte apoptosis and proliferation related gene products in childhood acute lymphoblastic leukemia

The aim of the study is to characterize markers of apoptosis in children with ALL in relation to treatment outcome of the disease. The study was performed on 34 children with ALL and 60 healthy children as a control group. Apoptosis was assessed by cell morphology; DNA fragmentation; ELISA and RT-PCR for CD95, CD95L, BcL2 and NF-KB; and flowcytometry for CD95, CD40, CD49d, and CD11a. Apoptosis was significantly lower in cases than controls. Apoptosis detected by CD95 ligand was significantly lower in cases with no remission after treatment than those with remission. Antiapoptotic factors: CD40, BcL2, and NF-KB were all found to be higher in cases than controls and in cases with no remission than those with remission, CD49d was significantly lower in cases than controls, and significantly lower in cases with no remission. CD11a levels were not different among various groups. Delayed apoptosis of ALL cells is genetically controlled either directly or indirectly by a network of oncogenes and tumor suppressor genes. CD40 appeared to stimulate both T and lineage and is considered the most potent influencer and predictor to resistance to therapy. Inhibitors for the activity of CD40, 8c/2 and NF-kB as well as stimulants to CD95 could have a potential therapeutic benefit

Natural killer-T [NKT] cells and human leukocyte antigen-G [HLA-G] in children with insulin dependent diabetes mellitus

Background: natural killer T [NKT] Cells are unique immunoregulatory cells reported to be deficient in many autoimmune disorders as systemic sclerosis with controversy as regards their role in IDDM. HLA-G is a non-classical HL4 class I molecule with immunotolerant function through T and NK cells inhibition but its role in IDDM is not defined. Bur aim was to evaluate NKT cells frequency and activity as well as the level and expression of HLA-G in children with IDDM to highlight their role in disease pathogenesis

Study design: this study included 28 children with IDDM who attended Mansoura University Children's Hospital consecutively from June, 2003 to March, 2004. A group of 18 healthy children with matched age and sex having no family history of diabetes mellitus served as control. All subjects were exposed to thorough history and clinical examination beside routine investigations that included random blood sugar and glycosylated hemoglobin. NKT cell receptors [Valfa24-JalfaQ] were assessed by ELISA before and after mitogen stimulation. NKT cell receptor expression was also assessed by quantitative reverse transcriptase polymerase chain reaction [RT-PCR]. Soluble HLA-G was also determined by ELISA and its expression was assessed by RT-PCR

Results: NKT cell receptors were significantly lower in patients before and after mitogen stimulation compared to control [p<0.001]. Similarly, NKT cell receptor expression was significantly lower in patients than control [P<0.001]. On the other hand, sHLA-G and HLA-G expression were significantly higher in patients than control [p<0.001] A. highly significant negative correlation was found be between NKT receptors and HLA-G [p<0.001]. But no significant correlation was observed between NKT cell receptors or HLA-G and either age, duration of illness, random blood sugar, dose of insulin or level of glycosylated hemoglobin

Conclusion: NXT cells frequency and activity are decreased in children with IDDM which may contribute to disease development. HLA-G level and expression are increased in diabetic children suggesting a role for this molecule in DDM pathogenesis

Herpes simplex virus type-1 and apoptosis in pemphigus: a causal relationship

The objective of this study was to evaluate the possible role of herpes simplex virus type-I [HSV-I] infection and apoptosis in the etiopathogenesis of pemphigus vulgaris [PV]. The study included 29 patients with PV and 7 apparently healthy subjects as controls. The patients were grouped into three groups: Group I [recent acute onset [6 cases]], group II [chronic exacerbated under steroid therapy [16 cases]] and group III [chronic controlled with steroid therapy [7 cases]]. Apoptosis was detected in peripheral blood monocytes [PBMC] using flow cytometry and that in the skin by TUNEL. HSV-I DNA was detected in PBMC and the skin using PCR. The antibody titer of serum IgG for HSV-I was detected using indirect immunofluorescence technique [IF]. The study concluded that apoptosis is increased in PV. Both HSV-I and apoptosis have to be considered in the immune-pathogenesis and can be used for immuno-modulation and suggestion of strategy of therapy or even to prevent exacerbation

Some biological and immunological studies in vitiligo

The present study comprised 48 vitiligo patients; 31 of them were not treated and the remaining 17 patients were on PUVA treatment for 6 months. They were subjected to thorough history taking and clinical examination. Vitiligo was associated with other autoimmune diseases in six patients in the form of alopecia areata [three patients], diabetes mellitus [two patients] and thyroid disease [one patient]. The melanocyte cultures were used as an antigen in the immunofluorescence [IF] and ELISA test using sera from the vitiligo patients and 20 control subjects. IF was positive in 54.2% in vitiligo sera in comparison with 5% positivity in the control sera. ELISA was positive in 77.1% in the vitiligo sera and in 20% of the control sera. Patients who were treated with PUVA had lower positive results to both IF and ELISA with a significant decrease in the optical density [OD] than the untreated patients. Thus, the IF is more specific for the detection of antibody in vitiligo serum against melanocytes whereas ELISA is more sensitive and more accurate

Serum auto-antibodies in juvenile rheumatoid arthritis

We investigated the relationship between the serum levels of autoantibodies in juvenile rheumatoid arthritis [JRA] to evaluate the diagnostic value of each serological marker and of associations of markers with disease activity. The study was conducted on sixty JRA patients [42 girls, and 18 boys] and 60 controls matching in age and sex. The patients were classified as pauciarticular [24, 40%], polyarticular [27, 45%], and systemic onset [9,15%]. Nine autoantibodies [AFA, AKA, ANA, ANCA, ANTIKU, APF, dsDNA, ssDNA, PDA] were detected and correlated with disease onset and activity markers as well as HLA-DR typing, alleles being done with PCR. To find out correlations between autoantibodies and disease character, we categorized the patients either active [41,68.3%] or in remission [19,31.7%]. The levels of autoantibodies was significantly higher in JRA active group than in remission, [p<0.0001]. No significant correlation was found between autoantibodies and HLA-DR typing. The frequencies of HLA-DR8, DR6, and DR5 were significantly higher among JRA group as compared to normal controls. There was a significant correlation between dsDNA and morning stiffness, ssDNA, EAM, ESR and MS. The level of autoantibodies differed between active JRA and patients in remission. These markers were prognostic in predicting disease outcome

IL-10 gene expression as a marker for detection of protective immunity levels with irradiated cercarial vaccine against experimental Schistosoma mansoni infection

Cercariae were obtained in a large number from the maintained life cycle of S. mansoni. They were attenuated at different doses [20 Kr, 50 Kr,60Kr, 70 Kr and 80 Kr] of gamma radiation. Laboratory bred Swiss Albino mice were classified into 7 groups. Five groups were immunized with +/- 500 S. mansoni cercariae. Two groups were used as positive and negative controls. All animals were sacrificed after 8 weeks. Spleen cell proliferative responses to Phytohaemagglutinin [PHA] were assessed in all groups. IL-10 was measured by ELISA in serum and splenic cells secretion in vitro. RNA extracted from freshly isolated liver cells was analyzed for detection of m RNA of IL-10 by reverse transcriptase polymerase chain reaction [RT-PCR]. The results showed augmentation of proliferative cell from the spleen in all vaccinated groups except with 80 Kr. irradiated cercariae group. The highest percentage of lymphocytes transformation was recorded among the mice immunized with 60 Kr. irradiated cercariae. After challenge, splenic reasponses in all groups declined progressively to the control level. IL-10 secretion from spleen cells of all vaccinated groups increased after challenge with the least level in 60 Kr. immunized challenged group IL-10 m RNA expression was higher among 60 Kr. immunized with irradiated cercariae group than 70 Kr. one, but with no expression among 80 Kr. cercariae immunized ones

Immunogenetic study of diabetic patients with periodontal disease

This study compared the periodontal status of diabetic patients [whether insulin-dependent or non-insulin-dependent] with non-diabetic patients, in addition to the determination of the level of IL-8, and the frequency of HLA class I and class II antigens on lymphocytes among these patients and comparing them with the control group. The study groups consisted of 28 male patients with an age from 30 to 60 years and 10 healthy control subjects of similar age and sex. Clinical periodontal evaluation were performed for all teeth in each patient using Russel periodontal index. Blood was obtained from all subjects for the determination of the level of protein concentration of IL-8 using ELISA technique. Class I and class II HLA antigens were determined by the microlymphocytotoxicity assay. Analysis of the data demonstrated no statistically significant difference in the overall means for all diseased groups as regards the periodontal index [PI]. However, insulin-dependent diabetic patients [IDDP] showed highest mean, while non-diabetic patients had the lowest mean [P >/0.05]. The mean blood levels of IL-8 in all the diseased groups were significantly higher than the mean blood level of IL-8 in the control group, i.e. the difference among the four groups were highly significant [P <0.001]. There were no significant difference between the distribution of HLA antigens between the diseased groups and the control group in any class except in HLA [B37 + W67] which was significantly higher in NIDD than the control group [P <0.05]