Enhancement of natural killer cell activity in vitro against human tumor cells by some plants from Jordan.

The effect of different concentrations (0, 0.01, 0.1, and 0.5 mg/ml) of plant aqueous extracts on the anti-tumor activity of natural killer (NK) cells isolated from human blood was examined. Plant extracts induced significant enhancement of (26.6-67.7%) of NK cell activity against K562 tumor cells. This increase in NK cell cytotoxicity was found to be due to the enhancement of NK cell production of interferon-gamma (87-337%), and on tumor necrosis factor-alpha (60-200%). Furthermore, the release of both granzyme A and N-acetyl-beta-D-glucosaminidase was increased significantly when compared with controls. Activation of granzyme A and N-acetyl-beta-D-glucosaminidase was clearly observed ranging from 24.2-106.4% to 26.8-110.7%, respectively. Lastly, in the absence of IL-2, plant extracts caused a significant increase in NK-cell-induced cytotoxicity (256%) against K562 tumor cells, and in the presence of IL-2 stimulated cells plant extracts caused an increase in NK cell-cytotoxicity (112%).

Effect of n-3 and n-6 polyunsaturated fatty acids on lymphocyte proliferation, interleukin production and phospholipid fatty acids composition in type 2 diabetic and healthy subjects in Jordan people.

Dietary lipid manipulation may affect a great number of immune parameters, such as lymphocyte proliferation, cytokine synthesis. In this study, lymphocytes of diabetic type 2 were incubated with different polyunsaturated fatty acid (docosahexaenoic, eicosapentaenoic, arachidonic acid) for investigated their effect on lymphoproliferation response, the concentration of interleukin 2 produced in each essay and phospholipid fatty acid composition of lymphocyte membrane. Our results found that the concanavalin A and insulin increase significantly the proliferative response while eicosapentaenoic, arachidonic and docosahexaenoic acid inhibited that by different degrees: 47%, 37% and 19%, respectively, for healthy subjects and 39%, 29% and 13% for diabetes. However, the concentration of IL-2 produced in presence of either docosahexaenoic, eicosapentaenoic or arachidonic acid was significantly reduced by 36%, 32% and 39%, respectively, in controls while 16%, 15% and 23%, respectively, in diabetics. On the other hand, the tested fatty acids demonstrated a major impact on the fatty acid composition of different phospholipid fractions of lymphocyte membrane but these fractions were different in their response to each fatty acid examined. For instance, the addition of docosahexaenoic acid to culture media was accompanied with a predominant composition of docosahexaenoic acid in phospholipid fractions. Also, our results showed a notable increased proportion of arachidonic, eicosapentaenoic and docosahexaenoic acids in control phospholipid fractions than those of diabetic.

Effects of sodium nitrite on natural killer cells isolated from human peripheral blood.

The effect of different concentrations (0, 0.1, 0.2, 0.4, and 0.8 mg/ml) of sodium nitrite on the anti-tumor activity of natural killer (NK) cells isolated from human peripheral blood was examined. Sodium nitrite induced significant inhibition (25.3-66.6%) of NK cell activity against WEHI-164 cells. This reduction in NK cell cytotoxicity was found to be partially due to inhibition of NK cell production of interferon-gamma (25.7-92.8%) and tumor necrosis factor-alpha (26.6 76.6%). In addition, exposure to sodium nitrite resulted in a significant decrease (17.5-81.1%) in proliferation of control and interleukin-2-stimulated NK cells. Furthermore, the release of granzyme A and N-acetyl-beta-D-glucosaminidase by NK cells was also decreased by 21.7-72.2% and 33.5-81.2%, respectively. Therefore, sodium nitrite is of environmental concern in view of its inhibitory effects on NK cell activity that might contribute to tumor promotion in vivo.

Augmentation of natural killer cell activity in vivo against tumour cells by some wild plants from Jordan.

Twelve aqueous extracts prepared from Jordanian plants that are currently used in traditional medicine to treat various types of cancer were tested in mice for their augmentation of natural killer cells in vivo in generating cytotoxicity against YAC tumour targets. After 1 week of oral administration of aqueous extracts of fresh Nigella sativum (N.s.) seeds and Allium sativum (A.s.) bulbs significant augmentation of splenic natural killer (NK) cells (62.3% +/- 6.4% and 52.6% +/-5.4% cytotoxicity, respectively), was obtained in comparison with spontaneous activity (24.5% +/- 1.6%) at 200:1 effector:target ratio. Onopordum acanthium (O.a.) stem and leaves and Allium cepa (A.c.) bulbs showed intermediate augmentation (38.6% +/- 3.8% and 30.6% +/- 3.4% cytotoxicity, respectively) while the rest showed insignificant augmentation activity. The fresh aqueous extract of a mixture of the plants with high and intermediate activity showed little insignificant augmentation activity (72.7% +/- 6.7% cytotoxicity) of NK cells compared with that obtained with each plant alone.

The effect of sodium nitrite on some parameters of the immune system.

The effect of orally administered sublethal doses of 25, 50 and 100 mg/kg of sodium nitrite in drinking water ad lib. for 21 days on the immune response of Balb/c mice was investigated. The immunological parameters were examined at three phases: 1 day (phase A), 1 week (phase B) and 3 weeks (phase C) after the end of exposure to sodium nitrite. A significant decrease in dose-dependent manner was obtained in the following tests: lymphocyte percentages, concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced lymphocyte proliferation assessed by the colorimetric MTT method, natural killer (NK) cell activity against WEHI-164 target cells, as well as IgM and IgG titers against injected sheep erythrocytes. Maximum suppressions were obtained in phase A after treatment with sodium nitrite at 100 mg/kg including lymphocyte count (17.5%), Con A-induced lymphocyte proliferation (40.1%), LPS-induced lymphocyte proliferation (31.4%), IL-2-stimulated NK cell activity (59.2%), unstimulated NK cell activity (59.6%), IgM titer (57.5%) and IgG titer (61.1%). On the other hand, a significant dose-dependent increase in neutrophil count (71.3%) in phase A and phagocytic activation (133%) in the first two phases was obtained using the nitroblue tetrazolium (NBT) assay in the presence of phorbol myristate acetate (PMA). It was found that the immunosuppressive effect of sodium nitrite is reversible after cessation of exposure.

Seroepidemiologic study of herpes simplex virus type 2 and cytomegalovirus among young adults in northern Jordan.

Blood samples were randomly collected from 360 males and 390 females among apparently healthy university students aged 18-24 years and tested for herpes virus type 2 (HSV-2) and cytomegalovirus (CMV) antibodies. The prevalence of HSV-2 seropositivity was 52.8% for males and 41.5% for females as detected by ELISA. On the other hand, the prevalence of CMV seropositivity was 75.6% in males and 77.2% in females. The high percentage of seropositivity in our study is most probably due to the crowded living and low socioeconomic status of the Jordan population. The higher prevalence in males could be due to the way of life in the Middle East that gives males the freedom to play outdoors more than females.

Augmentation of natural killer cell activity in vitro against tumor cells by wild plants from Jordan.

Thirteen aqueous extracts prepared from Jordanian plants, that are currently used in traditional medicine to treat various types of cancer, were tested in mice for their ability to augment natural killer (NK) cell function in vitro in generating cytotoxicity against YAC tumor targets. Lymphoid cells at a concentration of 5x10(6)/ml were incubated in medium alone or in medium containing different dilutions of either plant extract or purified interferon alpha for 20 h and tested for NK activity. Maximum NK activity (62. 3%) was obtained at 1:50 dilution of Nigella sativum fresh aqueous extract, 48.5% at 1:100 dilution for Allium sativum (and 38.3% at 1:50 dilution for Onopordum acanthium. Fresh aqueous plant extracts appeared to be more potent than old dried aqueous extract or ethanolic extracts. NK augmentation by plant extracts using nylon wool non-adherent spleen cells was slightly higher than the whole spleen cells.

Respiratory syncytial virus infection in infants hospitalized with respiratory illness in northern Jordan.

During the winter seasons of 1993 and 1994, a total of 256 nasopharyngeal aspirates (NPA) from infants aged less than 1 year old admitted to the pediatric ward of Princess Rahma Hospital, northern Jordan, with bronchiolitis and/or pneumonia, were tested for the presence of respiratory syncytial virus (RSV) using direct immunofluorescence assay (DFA) and cell culture (CC). Of the 256 specimens, 129 (50 per cent) were found positive by both DFA and CC, whereas 24 specimens (9 per cent) and 16 specimens (6 per cent) were found positive by DFA and CC, respectively. In an evaluation of the collected NPA specimens detected by DFA, a sensitivity of 89 per cent and a specificity of 78 per cent were demonstrated. These data suggest that virus isolation in CC is still important for the diagnosis of RSV, although DFA is a valuable, rapid diagnostic assay.

Seroprevalence of brucellosis among high risk people in northern Jordan.

BACKGROUND: Brucellosis is considered the most important zoonosis in Jordan with high prevalence among man and livestock. METHODS: This study was carried out on high risk people in 1992 in order to assess the seroprevalence of brucellosis in northern Jordan. The sera of 1236 individuals (636 at high risk and 600 controls) were evaluated using the Rose Bengal plate agglutination test (RBPT) and enzyme-linked immunosorbent assay (ELISA-IgG) tests. RESULTS: A significantly (P < 0.05) higher seroprevalence of brucellosis among high risk people (8.2 percent) compared to the control sample (0.5 percent) was found. The overall seroprevalence was significantly higher among sheep farmers and meat handlers than in other occupations tested. The seroprevalence increased with age and years at work, but was not influenced by sex or locality. The results indicated a higher seroprevalence among veterinarians in northern Jordan, compared to central Jordan but the difference was not significant. Seroprevalence was present only among veterinarians working in clinics especially in the working age group (34-43 years). CONCLUSION: The results of this study emphasized the importance of contact infections, namely contact with infected animals and their products, as a method of transmission of brucellosis compared to ingestion of contaminated animal products.

Electroblotting of proteins on agarose gel containing specific antibodies.

A method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3 complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.

Seroprevalence of Toxocara canis antibodies in humans in northern Jordan.

Sera of 699 individuals, aged between 5-24 years, from the Irbid area, Jordan, were tested for Toxocara canis antibodies using an ELISA-IgG test. Crude prevalence was 10.9% (76 of 699) but age-adjusted prevalence was 14.3%. The highest prevalence was observed in females aged 5-9 years, 23.3% (7 of 30), and males of 15-19 years of age, 19.5% (16 of 82). The lowest prevalence was observed in females aged 20-25 years, 5.2% (8 of 155). Significant differences (P < 0.05) between the prevalences of the toxocaral antibodies in males and females were observed in the age groups 5-9, 15-19 and 20-24 years. The trend of prevalence in relation to age was different according to sex.

Detection of proteins after immunoblotting on nitrocellulose using fluorescent antibodies.

A Western blot method is described utilizing 5-dimethylaminonaphthalene-1-sulfonyl (dansyl chloride)-conjugated antibodies to detect proteins transferred onto nitrocellulose. Four proteins of different molecular weight, including human albumin, gamma heavy chain, kappa and lambda light chains of immunoglobulins, were tested. Labeling with the fluorochrome was compared with the method of enzyme-conjugated antibodies and was found to have similar sensitivity, specificity, and safety. Moreover, this method is less expensive, and the labeled antibodies can be prepared fresh in a short time and are free from the disadvantages of enzyme label whose activity is affected by the presence of activators or inhibitors in the tested specimens.

The immune status of young adults to rubella virus in Northern Jordan.

Serum samples from 1252 young adults were screened for rubella antibodies using an enzyme-linked immunosorbent assay (ELISA) method and hemagglutination ihibition test (HIT) in an attempt to establish the immune status for rubella among northern Jordanians. The overall percentage of susceptible adult males was 8.2%, while in females it was 8.6%. This implies that, in spite of the high prevalence of rubella-specific antibodies in the adult Jordanian female population, protection against rubella through natural infection appears inadequate. Therefore, it is recommended that a nationwide vaccination program be instituted for adolescent and adult females who are not pregnant and will not become pregnant within 3 months of vaccination, and who show no history of rubella vaccination or clinically diagnosed disease. The two techniques correlated well, though ELISA exhibited a higher sensitivity than did HIT.

Immune status of young adults to mumps virus infection in northern Jordan.

Eight hundred and seventy-five sera, collected randomly from university students aged 18-24 years, were tested for mumps virus antibodies. The prevalence of seropositivity was 85.3% for males and 87.1% for females using the enzyme linked immunosorbent assay method (ELISA) and 83.4% for males and 77.6% for females using the micro-complement fixation test (CFT). The remaining 14.7%-16.7% of males and 12.9%-22.3% of females, who had non-reactive sera in ELISA and CFT tests respectively indicated the presence of a sizeable population with a high risk of contracting the infection and this recommends the implementation of routine vaccination against mumps in Jordan. The two techniques correlated well, though ELISA exhibited a higher sensitivity than CFT.

Two CEA and three NCA species, although distinguishable by monoclonal antibodies, have nearly identical peptide patterns.

In perchloric acid extracts of normal lung and colonic tumors, 3 NCA molecules were identified by monoclonal antibodies that cross-reacted with CEA, which itself gave 2 bands in SDS-PAGE. The proteins had molecular weights of 50, 75 and 97 kd, while the 2 CEA molecules banded at 180 and 160 kd in SDS-PAGE. No MAb recognized only one molecule, with the exception of MAb 3/13 which precipitated solely the upper CEA band. Analysis of the biochemical relationship of the cross-reactive antigens showed that none of them contained any internal methionine. Furthermore, after digestion by thermolysin, the peptide maps of the immunoprecipitated molecules showed very close similarities, if not identity. When the cross-reactive and the CEA were compared, the only differences found were in the upper CEA band, which apparently lacked one hydrophobic peptide, while the 97 kd cross-reacting protein showed one extra peptide. We conclude from our results that CEA and the cross-reacting molecules are composed of nearly identical, small (i.e. less than 50 kd) polypeptide chains.

Non-specific crossreacting antigen (NCA) does not contain methionine.

The presence of methionine in non-specific cross-reacting antigen (NCA) is one of the properties distinguishing it from carcinoembryonic antigen (CEA). In this paper we show that the appearance of methionine in NCA is due to regularly copurified materials, which were immunologically identified as alpha-1-antitrypsin and alpha-1-antichymotrypsin-like proteins. NCA is itself not cleavable by cyanogen bromide, which means that the molecule is devoid of any internal methionine. This proved to be true for biochemically-purified NCA as well as for NCA purified by immunoprecipitation with anti-serum followed by perchloric acid extraction.

Isolation and characterization of two proteins copurifying with carcinoembryonic antigen.

Using common purification procedures CEA was eluted as a symmetrical peak after gel-chromatography with a molecular weight (mw) of 180,000. The purity was assessed by the Ouchterlony test and immunoelectrophoresis, and by SDS-PAGE, where only one precipitation line and one band were obtained. However, two weak bands with a mw of 45,000 and 58,000 appeared, when iodinated CEA preparations were analyzed on SDS-PAGE. It was impossible to separate these proteins from CEA by a great variety of purification procedures. Out of several different immune sera, only two, anti-alpha-antitrypsin and anti-alpha-1-antichymotrypsin, reacted with the proteins. Furthermore, antisera against these protease inhibitors also immunoprecipitated the typical 180,000 mw band of CEA. This was also true for CEA prepared by other laboratories. We have purified and partially characterized both proteins. Although reacting with antisera against alpha-1-antitrypsin and alpha-1-antichymotrypsin they were not identical to the protease inhibitors, because they possess a different N-terminal amino acid sequence than published for them. However, the comparison of their sequences to 1900 total protein sequences made by computer search revealed a strong homology of the N-terminal sequence of the 45,000 mw protein with an internal sequence of alpha-1-antitrypsin. For the 58,000 mw protein no significant homology was found.