Development of recombinant vaccine candidate molecule against Shigella infection.

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella's IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90-95% of mice whereas 80-85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60-70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe, efficacious and cost-effective recombinant vaccine against Shigella serotypes.

Lacrimal Cytokines Assessment in Subjects Exposed to Different Levels of Ambient Air Pollution in a Large Metropolitan Area.

BACKGROUND: Air pollution is one of the most environmental health concerns in the world and has serious impact on human health, particularly in the mucous membranes of the respiratory tract and eyes. However, ocular hazardous effects to air pollutants are scarcely found in the literature. DESIGN: Panel study to evaluate the effect of different levels of ambient air pollution on lacrimal film cytokine levels of outdoor workers from a large metropolitan area. METHODS: Thirty healthy male workers, among them nineteen professionals who work on streets (taxi drivers and traffic controllers, high pollutants exposure, Group 1) and eleven workers of a Forest Institute (Group 2, lower pollutants exposure compared to group 1) were evaluated twice, 15 days apart. Exposure to ambient PM2.5 (particulate matter equal or smaller than 2.5 µm) was 24 hour individually collected and the collection of tears was performed to measure interleukins (IL) 2, 4, 5 and 10 and interferon gamma (IFN-γ) levels. Data from both groups were compared using Student's t test or Mann- Whitney test for cytokines. Individual PM2.5 levels were categorized in tertiles (lower, middle and upper) and compared using one-way ANOVA. Relationship between PM2.5 and cytokine levels was evaluated using generalized estimating equations (GEE). RESULTS: PM2.5 levels in the three categories differed significantly (lower: ≤22 µg/m3; middle: 23-37.5 µg/m3; upper: >37.5 µg/m3; p<0.001). The subjects from the two groups were distributed unevenly in the lower category (Group 1 = 8%; Group 2 = 92%), the middle category (Group 1 = 89%; Group 2 = 11%) and the upper category (Group 1 = 100%). A significant relationship was found between IL-5 and IL-10 and PM2.5 levels of the group 1, with an average decrease of 1.65 pg/mL of IL-5 level and of 0.78 pg/mL of IL-10 level in tear samples for each increment of 50 µg/m3 of PM2.5 (p = 0.01 and p = 0.003, respectively). CONCLUSION: High levels of PM2.5 exposure is associated with decrease of IL-5 and IL-10 levels suggesting a possible modulatory action of ambient air pollution on ocular surface immune response.

Lung immunoreactivity and airway inflammation: their assessment after scorpion envenomation.

Release and activation of pro-inflammatory mediators are among the most important induced factors that are involved in the scorpion envenomation pathogenesis. Inflammatory response and lung reactivity were studied in mice following subcutaneous injection with Androctonus australis hector (Aah) venom. Venom immunodetection in lungs and sequestered cell population in the airways were determined. Cytokines, cellular peroxidase activities (eosinophil peroxidase, myeloperoxydase), and IgE antibodies were also assessed. Immunohistochemical study revealed a positive detection of the Aah venom in the alveolar wall, venule lumens, and inside inflammatory cells. Severe lung edema associated with rapid inflammatory response was observed after animal envenomation. Lung neutrophilia and eosinophilia were accompanied with IL-4, IL-5 release, and IgE synthesis. In conclusion, high cytokine levels, recruitment of inflammatory cells (eosinophils and neutrophils), and increased IgE concentration may contribute to the exacerbation and maintenance of the induced inflammatory response in lungs by scorpion venom. These results lead to the better understanding of this induced pathogenesis and could help the physicians to take care of envenomed patients.

Induction and maintenance of IL-4 expression are regulated differently by the 3' enhancer in CD4 T cells.

IL-4 expression is known to be activated in CD4 T cells when they are differentiated to Th2 but not Th1 cells. However, CD4 T cells selected by MH class II-expressing thymocytes, named thymocyte-selected CD4 T cells (T-CD4 T cells), express IL-4 under both Th1 and Th2 conditions. In this study, we investigated molecular mechanisms by which IL-4 gene expression is regulated in T-CD4 T cells. We found that T-CD4 T cells express IL-4 soon after selection in the thymus. Deficiency of DNase I hypersensitive (HS) sites HS5a and HS5 at the 3'-enhancer region in the IL-4 gene decreased IL-4 production, but T-CD4 T cells were able to make IL-4 under the Th1-inducing condition. Consistent with this, IL-4 was expressed in Th1 differentiated T-CD4 T cells in the absence of recombination signal binding protein-J that interacts with HS5. When HS5 was examined separately from other endogenous regulatory elements using a reporter system, CD4 T cells that are selected by thymic epithelial cells cannot transcribe the IL-4 reporter gene with HS5 alone. However, HS5 was able to induce the expression of the IL-4 reporter gene in T-CD4 T cells. Interestingly, the Th1 differentiating signal led to deacetylation at HS5 of the IL-4 endogenous gene, whereas the Th2-inducing environment had no effect. Therefore, in T-CD4 T cells, HS5 plays an essential role during the induction phase of IL-4 expression, but the maintenance of IL-4 expression in Th1 cells requires additional regulatory elements.

Protective effects of Mentha haplocalyx ethanol extract (MH) in a mouse model of allergic asthma.

Mentha haplocalyx Briq., a commonly used herb in traditional Oriental medicine, has a variety of known pharmacological properties. However, neither the protective effects of Mentha haplocalyx ethanol extract (MH) against inflammation of the airway in an asthmatic model nor the mechanisms involved, have previously been reported. In the present study, an ovalbumin (OVA)-induced mouse model of allergic asthma was used to investigate whether MH was effective against the disease through regulation of airway inflammation. The MH treatment significantly inhibited increases in immunoglobulin (Ig) E and T-helper 2 (Th2)-type cytokines such as IL-4 and IL-5 in bronchoalveolar lavage fluid (BALF) and lung tissue. Inflammatory cell infiltration of the airway in mice treated with MH was effectively alleviated when compared with infiltration seen in the OVA-induced group. These data indicated that decreased cytokine levels are the result of the decreased number of invaded leukocytes. Also, the generation of reactive oxygen species (ROS) in BALF was diminished by MH treatment. Taken together, these findings indicate that the administration of MH may have potential therapeutic value in the treatment of inflammatory disease.

[Assessment of in vitro immune response in a young population exposed to atmospheric contamination of Guadalajara]. / Evaluación de la respuesta inmunológica in vitro en una población joven expuesta a la contaminación atmosférica de Guadalajara.

BACKGROUND: Atmospheric pollutants may cause alterations on health of persons exposed to urban environment. OBJECTIVE: To evaluate in vitro immunological response in young population exposed to different levels of atmospheric pollution. PARTICIPANTS AND METHODS: The study was performed in two groups of young men, one from Guadalajara, and the other from Tlajomulco. The volunteers had to be healthy and without precedents of atopia. The immunological responses studied on PBMC were: stimulation index by timidin incorporation, CD25 expression by flow citometry, and production of citokines IL-2 and IL-4 by ELISAtest. Atmospheric parameters monitored were: NO2, O3, SO2, CO and PM10. RESULTS: In Guadalajara the concentrations of NO2 and PM10 exceeded in 30% and 40%, respectively, the index established by WHO. Stimulation index of PBMC of the young men to Guadalajara was 18 +/- 4, whereas that of the volunteers from Tlajomulco was 23 +/- 3. Expression of CD25 did not show a significant difference between studied groups. IL-2 and IL-4 levels were similar between the young men of the city and those from the rural area. CONCLUSION: The environmental pollution in Guadalajara did not modify in a significant way proliferation, CD25 expression, nor secretion of IL-2 and IL-4 on PBMC. This demonstrates that healthy young men are less susceptible than other groups to the alterations caused by exposure to moderate levels of atmospheric pollutants.

Molecular assessment of the potential combination therapy of cytokines with biphalin and AZT for Friend leukemia virus infection in vitro.

Biphalin, a dimeric enkephalin analog, is under investigation as a potential, long-lasting medication of pain associated with chronic diseases, like cancer or AIDS. The role of cytokines, and splenocytes in anti-Friend leukemia virus (FLV) activity of biphalin, a synthetic opioid, and AZT was investigated in vitro. Mouse splenocytes inhibited FLV replication in Mus dunni (Dunni) cells when they were added to the cell culture. This inhibitory effect of splenocytes also was evident when cells were combined with biphalin and AZT as measured using a focus-forming assay. Under cell-free conditions, recombinant interferon gamma (IFNgamma), interleukin 2 (IL-2) and IL-4 directly inhibited the FLV reverse transcriptase (RT) activity by 27% to 36%. IFNgamma at 0.005 pg to 500 ng inhibited FLVRT activity by 61% to 80%. Acombination of 250 ng IFNgamma and 50 mug biphalin resulted in a 94% reduction of FLVRT activity, as compared with 61% inhibition by IFNgamma alone. The combination of AZT and IFNgamma, IL-2 or IL-4 also induced a stronger suppression of FLV RT activity than either cytokine or AZT used alone. In addition, cloned RT from Moloney murine leukemia virus (MMLV) was directly sensitive to inhibition by biphalin. Thus, the anti-FLV effects of splenocytes in combination with biphalin and AZT in cell culture are likely mediated to a large degree by the direct effect of cytokines. This antiviral activity of splenocytes or cytokines combined with chemotherapy, biphalin, and/or AZT, could be used as a complementary therapy to current approaches for retroviral infection and benefit acquired immunodeficiency syndrome (AIDS) patients. In conclusion, biphalin applied primarily as a new medicine for chronic pain treatment in AIDS patients may play a significant beneficial role as a component of antiviral HIV multidrug therapies.

Rapid qualitative and quantitative analysis of T-cell responses in HIV-1-infected individuals receiving successful HAART and HIV-1 sero-negative controls: concomitant assessment of perforin, IFN-gamma and IL-4 secretion.

The enzyme-linked immunospot (ELIspot) assay is a highly sensitive and valuable tool for determining the frequency of cytokine-secreting T cells. It is essential to determine both frequencies and functional capabilities of antigen-specific T cells, including cytokine secretion, degranulation, and cytotoxicity in order to obtain a fuller picture of the immune status of an individual. We describe here for the first time a perforin-release ELIspot assay which, when used in combination with IFN-gamma and IL-4 ELIspots, permits rapid assessment of these functional parameters for antigen-specific T cells. Whole antigen or peptides from HIV-1, recall and other viral antigens were used for in vitro stimulation. Anti-HIV-1 responses in treated chronically infected individuals were weak, both in terms of perforin and IFN-gamma production. Tetanus toxoid stimulation was associated with moderate perforin release and a predominantly type-2 IL-4 producing response, whilst herpes simplex virus antigen stimulation resulted in perforin release but only a weak type-1 IFN-gamma response. Anti-cytomegalovirus responses generated high levels of perforin in conjunction with IFN-gamma. Cytokines IL-2 and IL-12/IL-15 induced perforin release coupled with an IFN-gamma type-1 response. Perforin release strongly correlated with IFN-gamma production to individual influenza, Epstein-Barr virus or cytomegalovirus MHC class I restricted peptides, in an HIV-1 sero-negative cohort, indicating a cytolytic type-1 CD8+ T-cell response. Evaluation of immunogenicity and putative efficacy of candidate vaccines using IFN-gamma will not be as informative alone as when combined with perforin and IL-4 evaluations, which allow assessment of specific cytotoxic potential without extensive cell culture.

Assessment of selected co-stimulatory, adhesion and activatory molecules and cytokines of Th(1)/Th(2) balance in acute lymphoblastic leukemia in children.

INTRODUCTION: Recent years have seen a rise in the importance of cytokine production and co-stimulatory/activatory molecule expression in the immune response in leukemia. The aim of our study was to assess the function of T lymphocytes in children with acute lymphoblastic leukemia (ALL) during remission induction based on selected cytokine and co-stimulatory/activatory molecule expression. MATERIAL/METHODS: The study group consisted of 50 children with ALL (B cell precursor). Peripheral blood samples were taken before treatment (day 0), after the prednisone prophase (day 8), and during (day 15) and after (day 33) remission induction. The percentages of T cells with interferon (IFN)-gamma (Th(1)), interleukin (IL)-4 (Th(2)) and IL-2 receptor (IL-2R), CD28, CTLA-4, CD38, ICAM-1, and HLA-DR expression were assessed by tricolor flow cytometry. RESULTS: At the time of diagnosis we noted higher percentages of T cells with adhesion molecule ICAM-1, activation molecule CD38 expression, and an increased population of Th(2 )cells (IL-4) compared with the control group. During and after remission induction we observed a decreased population of CD38(+) T cells, elevated percentages of helper T lymphocytes with IL-2R expression, and a rise in helper T lymphocytes producing IFN-gamma (Th(1)). During fever/infection, higher levels of activated T lymphocytes (CD4(+)HLA-DR(+), CD8(+)HLA-DR(+)), a rise in Th1, and no change in Th(2 )populations were observed. CONCLUSIONS: The results suggest T cell activation and Th(2 )predominance at the time of diagnosis and during remission induction in ALL in children. These results confirm the involvement of cellular immunity in the leukemic process and can be used in immune therapy in leukemia.

Assessment of preferential Th1 or Th2 induction by low-molecular-weight compounds using a reverse transcription-polymerase chain reaction method: comparison of two mouse strains, C57BL/6 and BALB/c.

The aim of this study was to examine whether the RT-PCR method for various Th1/Th2 cytokines is appropriate for determination of response to allergens using C57BL/6 and Balb/c mice, which are known to preferentially demonstrate Th1 and Th2 responses, respectively. To this end, both strains of mice were sensitized by skin painting with the contact allergen dinitrochlorobenzene (DNCB) or the respiratory allergen trimellitic anhydride (TMA). We used the sensitizing protocol adopted by Kimber and coworkers (Toxicology 103, 63-73, 1995). At various time points after the last application, the levels of mRNA expression for Th1-type cytokines IFN-gamma, IL-18, and IL-12p40, as well as receptor IL-18R, and the Th2-type cytokine IL-4 and the receptor ST2L, in lymph nodes were measured. The results suggest that differential expression of IL-12p40 and IL-4 mRNA after 24 h allows clear discrimination between DNCB and TMA in C57BL/6 mice, more obviously than in Balb/c mice. Furthermore, to examine this method, C57BL/6 mice were exposed to OXA, DNFB, and TNCB (Th1-predominant allergens) or PA, TDI, and MDI (Th2-predominant allergens). Elevation of IL-12p40 expression was significant with the Th1 inducers, while the level of IL-4 was higher with Th2-predominant allergens. The results of the present study demonstrate, for the first time, that differential expression of IL-12p40 and IL-4 in C57BL/6 mice may be useful as a parameter for assessing influence of contact and respiratory allergens.

An assessment of the immunological status of patients with renal cell carcinoma based on the relative abundance of T-helper 1- and -2 cytokine-producing CD4+ cells in peripheral blood.

OBJECTIVE: To assess the immunological status of patients with renal cell carcinoma (RCC), by analysing the proportion of cluster-of-differentiation 4-positive (CD4+) cells showing intracellular cytokine production, i.e. interferon-gamma derived from T-helper (Th) 1 and interleukin-4 derived from Th2 cells, among peripheral blood lymphocytes from these patients Patients, subjects and methods Peripheral blood samples (5 mL) were collected from 36 patients (mean age 61 years, range 44-78) with RCC before and after they underwent nephrectomy. The proportion of cytokine-producing CD4+ cells was determined by flow cytometric analysis after stimulating the cells with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A, and staining the cells with fluorescein isothiocyanate-labelled anti-interferon-gamma, anti-interleukin-4 and anti-immunoglubulin-2b antibodies. The results were expressed as the percentage of cytokine-producing cells in the CD4+ population. As a control, peripheral blood obtained from 35 healthy volunteers (mean age 34 years, range 22-49) was also analysed. RESULTS: The proportion of CD4+ cells producing interferon-gamma and interleukin-4 was significantly higher (P < 0.04 and P < 0.001, respectively) in patients with RCC than in controls. The Th1/Th2 ratio (i.e. the ratio of CD4+ cells producing each cytokine) was significantly lower in patients with RCC (P < 0.001). There was a significant correlation in the controls between interferon-gamma and interleukin-4 production (r = 0.489, P < 0.01) but not in patients with RCC. The proportion of CD4+ cells producing interleukin-4 was significantly higher and the Th1/Th2 ratio significantly lower in patients with high-stage than in those with low-stage RCC (P < 0.05). The percentage of CD4+ cells producing interleukin-4 was significantly less after nephrectomy in those with low-stage RCC (P < 0.01) and the Th1/Th2 ratio significantly greater (P < 0.05) than before nephrectomy; there was no such trend in patients with high-stage RCC. Conclusion An evaluation of the production of interferon-gamma and interleukin-4 in CD4+ peripheral blood lymphocytes is useful for assessing the immunological status of patients with RCC; there is a change in the predominant response from Th1 to Th2 with increasing stage of RCC.

Quantitative assessment of mast cells and expression of IgE protein and mRNA for IgE and interleukin 4 in the gastrointestinal tract of healthy dogs and dogs with inflammatory bowel disease.

OBJECTIVE: To characterize the mucosal IgE network in dogs affected with inflammatory bowel disease (IBD) and compare it with that for healthy dogs. ANIMALS: 9 healthy dogs and 20 dogs with IBD. PROCEDURE: In situ hybridization of mRNA specific for IgE and interleukin 4 (IL-4) and immunohistochemical analysis for IgE protein and 2 markers of mast cells (ie, tryptase and chymase) were performed on tissue sections obtained from the gastrointestinal (GI) tract and lymph nodes of dogs. RESULTS: Dogs with IBD had significantly more cells positive for IgE protein and more mast cells in the GI mucosa than healthy dogs. Despite this significant increase in number of cells positive for IgE, cells positive for IgE mRNA were rarely detected in the GI mucosa; most cells positive for IgE mRNA were found in mesenteric lymph nodes. Signal pattern of IL-4 mRNA was similar to that of IgE mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: The increased numbers of cells positive for IgE and mast cells in dogs with IBD suggest hypersensitivity such as hypersensitivity to bacterial or dietary-derived antigens in the intestinal lumen. Future studies need to elucidate whether this represents a cause of inflammation or is a result of the inflammatory process of IBD.

[Does the changed Th1/Th2 activity in children with the assessment of body water in children with nephrotic syndrome: initial results]. / Czy zmieniona aktywnosc limfocytów Th1/Th2 u dzieci z zespolem nerczycowym moze byc wskaznikiem reaktywnosci na leczenie?

T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity(SS) or resistance(SR). Activity of Th1 and Th2 cells was defined by the production of IL-2, IFN-gamma and IL-4, IL-10 (ELISA), respectively, in the supernatants of the culture of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double or triple colour flow cytometry. In SS children with NS we found the cytokine synthesis indicating the predominance of Th2 activity. We conclude that prior to treatment the Th1 and Th2 cell activity provides a useful tool to evaluate the probability of steroid sensitivity in patients with primary NS.

Assessment of preferential T-helper 1 or T-helper 2 induction by low molecular weight compounds using the local lymph node assay in conjunction with RT-PCR and ELISA for interferon-gamma and interleukin-4.

The local lymph node assay (LLNA) is a new and promising test in mice used to identify contact allergens by means of dermal exposure. Experimentally this assay, which comprises a sensitizing phase only, is also used to identify respiratory allergens. Another, experimentally used test in mice to identify allergens is also based on dermal exposure, but comprises both a sensitizing and effector phase. In this latter test, it has been shown that contact allergens preferentially induce a T-helper 1 (TH1) response, whereas respiratory allergens preferentially induce a T-helper 2 (TH2) response. These responses can be discriminated on the basis of cytokine production, such as IFN-gamma, which is produced by TH1 cells, and IL-4, which is produced by TH2 cells. The aim of the study was to establish whether the LLNA was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen. To this end, LLNA responses to the contact allergen dinitrochlorobenzene (DNCB) and the respiratory allergen trimellitic anhydride (TMA) were determined using IFN-gamma and IL-4 mRNA expression and production as parameters. Topical application of TMA resulted in a threefold higher lymphocyte proliferation compared to DNCB 3 and 5 days after the first application, while a similar proliferation was found from Day 7 and onward. RT-PCR showed a similar induction of IFN-gamma and IL-4 mRNA expression. While both DNCB and TMA induced IFN-gamma production, TMA but not DNCB induced IL-4 production. Thus, only IL-4 production seemed a suitable parameter to discriminate between the two compounds. In a second study, the respiratory allergens toluene-2,4-diisocyanate (TDI) and phthalic anhydride (PA) were also assayed 7 days after the first application. Topical application of DNCB and PA resulted in a similar lymphocyte proliferation, while application of TMA and TDI resulted in a 1.8-fold higher proliferation. IFN-gamma production was similar for DNCB, TMA, and TDI, and fourfold lower for PA, while IL-4 production was similar for TMA, TDI, and PA, and 24-fold lower for DNCB. In summary, both studies showed induction of IL-4 production by respiratory allergens, with little or no induction by the contact allergen, holding promise for the possibility of identifying respiratory allergens within the LLNA by measuring IL-4 production 7 days after the first application.

Assessment of chemokine receptor expression by human Th1 and Th2 cells in vitro and in vivo.

The preferential association of some chemokine receptors with human Th1 or Th2 cells has recently been reported. In this study, the expression of CCR3, CCR5, CXCR3, and CXCR4 were analyzed by flow cytometry in three distinct in vitro models of Th1/Th2 polarization, activated naive and memory T cells, and T-cell clones, in which the intracellular synthesis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) and the surface expression of CD30 and LAG-3 were also assessed. Moreover, by using immunohistochemistry the in vivo expression of CCR3, CCR5, CXCR3, and CXCR4 was examined in the gut of patients suffering from Crohn's disease, a Th1-dominated disorder, and in the skin of patients suffering from systemic sclerosis, a Th2-dominated disorder. CCR5 and LAG-3 exhibited the same pathway of Th1 association, whereas CXCR3 did not discriminate between Th1- and Th2-dominated responses. On the other hand, CCR3 was found only occasionally in a small proportion of allergen-specific memory T cells with Th2/ThO profile of cytokine production in vitro. However, it was neither seen in Th2-polarized activated naive T cells nor in established Th2 clones and could be detected in vivo only on non-T cells. Finally, whereas CXCR4 expression was not limited to Th2 cells in vivo, it was markedly up-regulated by IL-4 and down-regulated by IFN-gamma in vitro. Thus, the results of this study confirm the existence of flexible programs of chemokine receptor expression during the development of Th1 and Th2 cells. However, caution is advised in interpreting these receptors as surrogate markers of a given type of effector response.

Characterization of antigens and allergens of date palm (Pheonix dactylifera) pollen. Immunologic assessment of atopic patients by whole extract and its fractions [corrected].

Antigenic and allergenic components of date palm (Phoenix dactylifera) pollen were investigated to observe their effects on the skin test reactivity, lymphocyte blastogenesis and cytokine production in atopic and healthy individuals. Date pollen extracts were fractionated using SDS-PAGE and Sephacryl S-200 gel filtration. Western blotting of SDS-PAGE separated components with antiserum raised against whole pollen extract in rabbits revealed at least 22 immunoreactive bands ranging in molecular weight between 12 and 94 kD. The immunogenicity of the pollen extract was further confirmed by strong positive reactions in ELISA and Ouchterlony's double diffusion tests. Immunoblot analyses revealed IgG and IgE reactive components (14-94 kD for IgG and 12-90 kD for IgE) in the skin test-positive patients' sera against whole pollen extract. Fifteen of 60 atopics reacted positively to either whole or some fractions of date pollen extract when skin tested. In response to whole or components of date pollen extract atopic patients showed differential peripheral blood lymphocyte (PBL) proliferative response and cytokine (IL-2, IL-4) production when compared with PBL of normal subjects. Our findings strongly suggest that date palm pollen should be considered a reaginic component and should be included in the battery of allergens for determining the allergic status of atopic patients, particularly in those parts of the world where the date palm is grown commercially.