Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions.

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.

Enhancing ex vivo expansion of cord blood-derived unrestricted somatic stem cells for clinical applications.

OBJECTIVES: To study effects of serum-containing medium (SCM) versus serum-free medium (SFM) and influence of seeding density, on rate of expansion of cord blood (CB) unrestricted somatic stem cells (USSCs), as a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. MATERIAL AND METHODS: Isolation, propagation and characterization of USSCs from CB samples were performed and followed by their passage 3 culture in SCM and SFM, at cell densities of 5, 50, 500 and 5000 cells/cm(2) . RESULTS: The cells were CD44(+) , CD90(+) , CD73(+) , CD105(+) , CD34(-) , CD45(-) , and HLA-DR, with Oct4 & Sox2 gene expression; they were differentiated into osteoblasts and adipocytes. USSCs cultured in SCM had significantly higher population doubling levels (P < 0.01) than those cultured in SFM. Those cultured in SCM at 5 cells/cm(2) and those cultured in SFM at 50 cells/cm(2) had significantly higher population doubling (P < 0.01) levels than those cultured at higher cell densities. CONCLUSIONS: For scaling up of USSCs from 106 (?) to 1012 (?) in 6 weeks, culturing of CB-derived cells of early passage (≤P3) in SCM at low cell seeding density (5 cells/cm(2) ) is suggested for increasing cell count with lower passaging frequency, followed by culture of expanded USSCs at 50 cells/cm(2) in SFM, to avoid undesirable effects of bovine serum in clinical applications.

The challenge of achieving measles elimination in the eastern Mediterranean region by 2010.

In 1997, the Eastern Mediterranean Region (EMR) of the World Health Organization adopted a resolution to eliminate measles by 2010. Of the 23 EMR member countries, 18 are polio-free and are building on this success to eliminate measles. The 5 countries where polio remains endemic are prioritizing polio eradication and working to improve measles control. Measles incidence has been reduced from 193/100,000 in 1981 to 6.8/100,000 in 2001. Supplemental vaccination campaigns for measles have been conducted since 1994 in 14 of the 18 polio-free countries. More than 50 million children have been immunized in these supplemental activities. However, in Afghanistan, Sudan, Somalia, Djibouti, and Pakistan, where 34% of the EMR population live, routine vaccination coverage for measles remains below 60% and measles deaths are estimated at 81,000 annually among children <5 years old. Significant resources must be allocated to these last 5 countries to achieve regional measles elimination by 2010.

Progress toward interruption of endemic measles transmission in Tunisia.

Measles was the second leading cause of infant mortality in Tunisia prior to introduction of measles vaccination in 1979. The number of reported measles cases has decreased from 3007 in 1981 to 47 cases in 2000 due in part to the high coverage rates achieved after 1992. During 1998, a measles catch-up campaign vaccinated 1,846,657 children (95%) aged 6-16 years, and a follow-up campaign for children aged 9 months to 5 years in 2001 reached 547,766 (94%). During 1999-2001, 1717 cases of rash and fever illness were tested for measles; only 3 (0.2%) were positive for measles. From February to July 2002, an outbreak of measles involving 87 cases occurred in Tunisia in a health care setting and 56 (64%) patients were aged 15-30 years. The low number of laboratory-confirmed measles cases during 1999-2001 suggests endemic measles transmission may have been interrupted.

Mass psychogenic illness following tetanus-diphtheria toxoid vaccination in Jordan.

In September 1998, more than 800 young people in Jordan believed they had suffered from the side-effects of tetanus-diphtheria toxoid vaccine administered at school; 122 of them were admitted to hospital. For the vast majority, their symptoms did not result from the vaccine but arose from mass psychogenic illness. The role played by the media, the children's parents, and the medical profession in the escalation of this mass reaction appeared, at first sight, to be unusual and even unique to the circumstances in Jordan at the time. A review of the literature showed, however, that this mass reaction was similar in many ways to previous outbreaks, even though the underlying causes varied. There are about 200 published accounts of mass responses to situations involving suspected poisoning or other events. Because such mass reactions are relatively rare and the triggers so diverse, individuals faced with responding to them are unlikely to have prior experience in how to handle them and are unlikely to take bold steps to prevent their escalation. Indeed they may be unaware that such events have been recorded before. The lessons learned from this incident in Jordan may help other immunization programme managers to handle crisis situations elsewhere.

Introduction of Hib conjugate vaccines in the non-industrialized world: experience in four 'newly adopting' countries.

Hib conjugate vaccines are widely used in the industrialized world, but are just now beginning to be introduced into other countries. To identify factors facilitating rapid global introduction, we evaluated the decision-making process, mode of introduction, effectiveness, and impact on the immunization program of Hib conjugate vaccine introduction in four non- industrialized countries through site visits and use of a standardized questionnaire. The key promoters of Hib introduction were the pediatric community and ministries of health. Local surveillance and severity data were critical in the decision to adopt Hib vaccine. Assistance with surveillance, introduction guidelines, educational material, tenders, and funding is needed to accelerate wider adoption.

Differential responsiveness of humans with early-stage schistosomiasis haematobium to Schistosoma haematobium soluble adult-worm and egg antigens.

Schoolchildren (7-8 years old) infected with Schistosoma haematobium were tested for lymphocyte proliferative responses, in vitro granuloma formation (IVGF), and cytokine release in T-cell Western assays and for serum antibody reactivity by enzyme-linked immunosorbent assay (ELISA) and immunoblotting against S. haematobium soluble adult-worm (SAWA) and egg (SEA) antigens. The lymphoproliferative response rate of individual subjects against 10 SAWA and 15 SEA electroseparated bands ranged from 0 to 33% and from 11 to 66%, respectively. The SAWA bands essentially failed to elicit significant IVGF, in contrast to the SEA bands, all of which were capable of inducing IVGF from peripheral blood mononuclear cells (PBMC) of 30-80% of individual donors. The exclusive ability of SEA bands to induce IVGF could not be attributed to selective release of interleukin 2 (IL-2), IL-4, or interferon-gamma, as SEA and SAWA bands were capable of eliciting release of a similar array of cytokines in supernatants of 4-day PBMC cultures. The antibody response to SEA was stronger than that to SAWA, yet the proportion of SAWA bands binding humoral antibodies of individual donors was significantly larger than that observed for SEA. The study thus suggests that humans with early-stage S. haematobium infection respond poorly to SAWA but mount strong cellular immune responses to SEA that result in granuloma and antibody formation.

Effect of cold storage on the haemostatic cascade systems.

This study dealt with the effect of citrate phosphate dextrose adenine (CPDA) whole blood stored at 4 degrees C for 5 weeks and fresh frozen plasma at -30 degrees C for 12 months on coagulation, fibrinolytic and kallikrein system activation. Stored whole blood showed a significant decrease in ATIII activity by the second week with a significant decrease in thrombin-antithrombin complex by the fourth week. alpha 2-antiplasmin and plasminogen decreased significantly by the first and second week, respectively, accompanied by a significant increase in D-dimer level by the fourth week. A significant decrease in C1-inhibitor activity occurred by the first week associated with a significant increase in kallikrein activity by the third week. However, all measured parameters were minimally affected in fresh frozen plasma. Therefore, fresh frozen plasma supplemented with packed RBCs are preferred to whole blood stored over 3 weeks especially in patients with proteolytic enzyme system activation.

Campylobacter diarrhea in Alexandria, Egypt.

The role of Campylobacter as a cause of bacterial diarrhea in young children in Alexandria, Egypt was investigated. Stools or rectal swabs were collected from 880 children (mean age 9.8 months) presenting to a hospital with the primary complaint of diarrhea and from 1,079 well children (mean age 8.8 months) attending a vaccination clinic. Isolation of Campylobacter was significantly (p<0.0002) more frequent from cases (17.2%) than from controls (6.4%). Campylobacter was isolated from children presenting with diarrhea more frequently than Salmonella (3% isolation rate), Shigella (2% isolation rate), or other bacterial pathogens (1% isolatoin rate). Isolation of Campylobacter was significantly more frequent during the rainy season (p<0.0012). These results implicate Campylobacter as a major bacterial cause of diarrhea for which young children are brought for medical attention in Alexandria, Egypt.

Identification of Schistosoma haematobium soluble egg antigens that elicit human granuloma formation in vitro.

Schistosoma haematobium soluble egg antigens (SH SEAs) induce intense granulomas in human hosts that often culminate in severe disease. In an attempt to identify the SH SEA fractions that are responsible for pathology, we combined T-cell Western blotting and an in vitro model of granuloma formation. Whole SH SEAs were dotted onto nitrocellulose pieces or were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose paper. Horizontal strips bearing the separated antigens were solubilized in dimethylsulfoxide and precipitated in carbonate/bicarbonate buffer. Antigen-free and antigen-bearing particles were used to stimulate peripheral blood mononuclear cells (PBMCs) obtained from S. haematobium-infected patients and sex- and age-matched healthy controls to form granulomas in vitro. Whole SH SEA-bearing nitrocellulose particles elicited in vitro formation of granulomas by PBMCs from infected donors. The response was similar in sensitivity, specificity, and reproducibility to that evoked by SH SEA-bound polyacrylamide beads. The results obtained in samples form 30 patients and 10 controls tested with SH SEA-separated fractions revealed that SEA bands of 84,000, 63,000, 57,000, 55,000, 40,000, 30,000, and 28,000 Da elicited in vitro granuloma reactions by PBMCs of almost all infected patients. Conversely, separated soluble adult-worm antigens failed to stimulate PBMCs of infected patients to form granulomas. This study is the first to identify the SH SEA fractions that evoke in vitro granuloma formation and represents an initial step toward the development of an anti-urinary schistosomiasis pathology vaccine.

Identification of the Schistosoma haematobium soluble egg antigens inducing antibody production and/or T cell proliferation in humans.

Soluble antigens were prepared from Schistosoma haematobium eggs collected from urine of 6-16 year-old children with urinary schistosomiasis. The electrophoretic profile of the soluble egg antigen (SEAH) preparation was almost identical to that (SEAh) obtained from UNDP/World Bank/WHO, Switzerland and prepared from S. haematobium eggs retrieved from intestines of infected hamsters. Reactivity of 50 individual patients with S. haematobium in Western blots led to the identification of the SEA protein bands carrying human B cell epitopes. Some, but not all, of these SEA proteins initiated peripheral blood T lymphocyte proliferation in T cell Western assays. These antigens are probably the ones inducing granulomatous response in vivo, and that are responsible for the immunopathology of the disease.

Peripheral blood lymphocyte subsets in urinary bladder carcinoma patients.

The percentages of pan T (CD3+), T helper (CD4+), T cytotoxic/suppressor (CD8+), B (CD22+) and natural killer (CD57+) cells in peripheral blood lymphocytes of 15 urinary bladder carcinoma patients and in parallel, 10 healthy donors were estimated, using monoclonal antibodies in indirect membrane immunofluorescence. A significant decrease in the percentage of CD3+ lymphocytes and a highly significant decrease in the proportion of CD8+ cells was revealed in urinary bladder cancer patients. This change was accompanied by a significant increase in the CD4/CD8 ratio and in the frequency of CD57+ (HNK-1+) cells. Our data document, for the first time, the complete lymphocyte profile of patients with advanced (T3) urinary bladder carcinoma. The reason and significance of the decline in CD8+ lymphocyte percentage and the increase of CD57+ cells are discussed.

Assessment of IgG & IgM, ELISA and IFAT in diagnosis of toxoplasmosis in pregnancies at risk.

This study was carried out on 100 pregnant cases, divided into 5 groups of patients having congenitally malformed newborns, suffering from stillbirths, premature labours, repeated abortions and control group. The incidence of positive Toxoplasma antibodies was higher in the congenital malformation and stillbirths groups than other groups. Analysis of the results showed that an excellent correlation exists between ELISA and IFAT with a minimal differences (less than 5%).

Dose-response aggregometry in maternal/neonatal platelets.

The aggregation of platelets collected from maternal/neonatal pairs (n = 240) at the time of childbirth, was studied in response to multiple doses of ADP, collagen, arachidonic acid and ristocetin. Similar responses were obtained from healthy nonpregnant adult controls for comparison. The lag phase, slope of the aggregation curves as well as maximum aggregation (MA%) were recorded and analysed. Neonatal and adult platelets exhibited more enhanced responses to decreasing doses of ADP, arachidonic acid and ristocetin, than maternal platelets. These enhanced responses were exhibited more consistently in the slopes of the aggregation curves than in MA%. Although neonatal platelets have shown longer lag phase in their responses to collagen, the rate of the aggregation reaction was significantly faster than maternal platelets, with no differences in MA%. These results contradict many previous reports suggesting impaired aggregation responses of neonatal platelets to these agonist. The possible reasons for these contradictions were discussed.

Relation of maternal and cord blood serum ferritin.

Serum ferritin was measured in 51 term normal pregnant mothers and the corresponding cord blood samples. All of the mothers had received prophylactic oral iron and folate during pregnancy. The mean (+/-SD) maternal serum ferritin at the end of pregnancy was 58 +/- 42.9 microgram/l (range 16-201 microgram/l), compared to a mean of 183.2 +/- 61.2 microgram/l (range 62-313 microgram/l) in these newborns. No correlation was found between the serum ferritin of mothers and babies, nor between the serum ferritin and serum iron of mothers at the end of pregnancy or between these parameters in the newborn.