882 Primary Cutaneous T-Cell Lymphomas: Immunomodulation byPhotochemotherapy With Analysis of the Inflammatory-Expansive Cellular Dynamic

L. W. Bandeira1, A. M. Manhães2, E. A. Pagani2, A. L. Filgueira2;1Immunology / Dermatology, Santa Casa RJ / Federal University of Riode Janeiro, Rio de Janeiro, BRAZIL, 2Dermatology, Federal University ofRio de Janeiro, Rio de Janeiro, BRAZIL.RATIONALE: Primary cutaneous T-Cell lymphomas (CTCLs) are char-acterized by hyper-proliferation of malignant CD4+ T cells with primarylocalization of the lymphocytes on the skin. We evaluated theimmunomodulation effect of Photochemotherapy (PUVA) directly on thecellular infiltrate of patients suffering from CTCLs.METHODS: Pre and post-PUVA therapy punch skin biopsies of ninepatients were stained immunohistochemically for CD34+, CD8+, CD7+,CD16+, CD56+, and CD1a+ cells.RESULTS: The results showed a significant decrease in the mean ofcells/mm2 after PUVA therapy for the majority of the cellular markers:Epidermis: PrePUVACD34:10.097±3.920-Post:0±0;p=0.007; PrePU-VACD8: 94.037±28.889-Post: 8.032±4.377, p=0.013; PrePUVACD7:45.894±10.519- Post: 21.614±6.725, p = 0.073; PrePUVACD16:51.615±18.198-Post: 0±0, p=0; PrePUVACD56: 16.451±4.074-Post:0.862±0.862, p=0.003; PrePUVACD1a: 31.044±9.692-Post:38.236±11.686, p=0.628. Dermis: PrePUVACD34: 86.978±35.757-Post: 40.537±10.471, p=0.312; PrePUVACD8: 272.386±58.097-Post:29.885±9.042, p=0.004; PrePUVACD7: 165.391±42.949-Post:69.896±26.908, p=0.034; PrePUVACD16: 346.017±98.583-Post:39.162±14.621, p=0.001; PrePUVACD56: 271.583±116.852-Post:38.049±11.222, p=0.004; PrePUVACD1a: 298.061±68.937-Post:72.066±8.852, p=0.005.CONCLUSIONS: We could conclude that PUVA therapy immunomod-ulated the cells with an immunosuppressive effect on each inflammato-ry/expansive cell. These findings could represent an important step in thecomprehension of how PUVA acts, showing photochemotherapy as ahighly effective therapy for CTCL. The pre-photochemotherapy periodanalyses showed that each cell had no significant difference of density inthe infiltrate, independent of reactive or expansive pattern. These resultsshowed how cells take part in CTCL improving the possibilities of theunderstanding of the mechanism of disease and the control of this cancer.Funding: Conselho Nacional de Pesquisa-CNPq; Fundação José Bonifá-cio

883 Acidic Oligosaccharides Enhance Systemic Cellular ImmuneResponsiveness in Mice Via A Different Mechanism ThanGalacto- And Fructooligosaccharides

A. P. Vos1, A. Buco1, M. Haarman1, J. Knol1, M. Govers1, B. Stahl2, G.Boehm2, J. Garssen1, L. M’Rabet1; 1Numico Research, Wageningen, THENETHERLANDS, 2Numico Research, Friedrichsdorf, GERMANY.RATIONALE: Because it has been suggested that oligosaccharidesmight play a role in the development of the immune system in infants, westudied the effect of acidic oligosaccharides (AOS) on the systemicimmune system, as well as the interaction of AOS with a neutral prebiot-ic mixture containing galacto- and fructooligosaccharides (GOS/FOS)with a proven immunomodulatory capacity.METHODS: C57Bl/6 mice received control diet or supplemented dietscontaining 1%, to 5% (w/w%) AOS. For the interaction study, micereceived control and supplemented diets containing 2% AOS, 2%GOS/FOS or combinations of 1% + 1% or 0.2% + 1.8% of the respectiveingredients. Mice were subjected to a suboptimal vaccination protocolusing a protein-based influenza vaccine (Influvac) in combination with anadjuvant (Specol). Specific antibody serum levels and a (Th1 dependent)DTH response were measured.RESULTS: The mice that received AOS in the diet showed a dose-depen-dent enhancement of DTH responses. Moreover, both combinations ofGOS/FOS together with AOS showed at least an additive enhancement ofthe DTH response. No effects were detected of oligosaccharide supple-mentation on specific IgG antibody titers.

CONCLUSIONS: These results suggest that the mechanism by whichAOS induces the DTH response is different from the mechanism by whichGOS/FOS induces the DTH response. Moreover, this shows that dietaryAOS supplementation can modulate systemic cellular immune responsesthat are Th1 dependent, as was previously shown for neutral oligosaccha-rides. Whether this is a direct effect or that the intestinal microflora isinvolved remains to be elucidated.Funding: Numico Research

884 Timing of Acute Stress Hormone Exposure Produces VariableEffect on Human Peripheral Blood Mononuclear Cells

A. N. Salicru1, G. D. Marshall, Jr.2; 1Department of Pathology, Universi-ty of Texas Health Science Center, Houston, TX, 2Department of ClinicalImmunology and Allergy, University of Mississippi Medical Center, Mis-sissippi, MS.RATIONALE: Acutely stressful events have been associated with wors-ening of various immune-based disorders including allergic asthma andatopic dermatitis. We examined the timing of acute stress exposure rela-tive to antigen stimulation on human peripheral blood mononuclear cells(PBMC) in vitro.METHODS: Human PBMC are incubated with stress levels of epineph-rine (EPI) at various time intervals from tetanus stimulation. Culturesupernatants are collected on the 11th day and are analyzed for cytokineproduction (IFN-�, IL-10, IL-4).RESULTS: Here we show that exposure of PBMC to EPI in vitro shiftscytokine profiles from Th1 to Th2 and that this effect diminishes withincreasing intervals of time after initial antigen exposure. In particular,EPI decreases IFN-� production from percent baseline control (58 ± 15,p<0.01) and increases IL-10 (261 ± 73, p<0.01) and IL-4 (372 ± 193,p<0.01) production most significantly at time zero. Additionally, EPIlooses the ability to significantly impact IFN-� production at 48 hours (91± 10). Similar results are observed for IL-10 at 24 hours (151 ± 36, IL-10)and IL-4 at 12 hours (255 ± 135, IL-4).SUMMARY: These data indicate that cytokine production in response toantigen can be influenced by the timing of sympathetic exposure.Funding: NASA GSRP/University of Texas Health Science Center Hous-ton

S222 Abstracts J ALLERGY CLIN IMMUNOL

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885 Gantrez® AN Nanoparticles Do Not Alter Allergenicity ofLollium Extract

M. Ferrer1, S. Gomez2, M. L. Sanz1, E. Luquin1, A. Grau3, A. Molina3,S. Beatriz2, C. Gamazo4, J. M. Irache2; 1Allergy and Clinical Immunolo-gy, Universidad de Navarra, Pamplona, SPAIN, 2Pharmaceutical Technol-ogy, Universidad de Navarra, Pamplona, SPAIN, 3Applied MolecularDevelopement, Madrid, SPAIN, 4Microbiology, Universidad de Navarra,Pamplona, SPAIN.RATIONALE: We previously demonstrated that Gantrez® AN nanopar-ticles act as an adjuvant in an OVA allergic animal model. We wanted toexamine whether Lollium extract encapsulated in these nanoparticles doesmaintain the ability to activate sensitised basophils.METHODS: Lollium perenne pollen was purified and the protein extract(Lol p1: 6.61% and Lol p5: 3.08%) used to load in Gantrez® AN nanopar-ticles (NP). Some batches of these nanoparticles also included roughlipopolysaccharide (RLPS) as immunomodulator. Both nanoparticles andcommercial pollen were incubated with basophils from patients allergic toLollium and histamine release was measured.RESULTS: All nanoparticles displayed homogeneous spherical surface,with a size of about 200 nm. The presence of R-LPS did not influence thephysicochemical characteristics. These carriers were able to encapsulate38 �g Lollium perenne extract per mg nanoparticles. Basophils frompatients allergic to Lollium were induced to release histamine with encap-sulated allergen in a similar way than with the sole Lollium extract. Thepresence of R-LPS associated to nanoparticles appears to slightly increasethe release of histamine by basophils. On the contrary, blank nanoparticlesdo not stimulate those basophils.CONCLUSIONS: The encapsulation method to obtain Lollium -Gantrez® AN nanoparticles do not alter the recognition and activation of

888 A Comparison of Lymphocyte Responses in PBMC, by Humanand Chimpanzees in Response to PMA/I Stimulation, or Anti-CD3/Anti-CD28 Costimulation, Using Microarray Analysis

J. A. DeVoti; Allergy/Immunology, North Shore-LIJ Health System, New

J ALLERGY CLIN IMMUNOL Abstracts S223VOLUME 115, NUMBER 2

basophils from patients allergic to Lollium. This observation allows fur-ther research in this new way to carry the allergen.Funding: National Dept Science and Technology # SAF 2001-0690-C03-01

886 New Adjuvant for Immunotherapy: Gantrez® AN Nanoparti-cles

S. Gomez1, C. Carlos2, E. Luquin3, B. San Roman1, S. Espuelas1, M. L.Sanz3, M. Ferrer3, J. M. Irache1; 1Pharmaceutical Technology, Universi-dad de Navarra, Pamplona, SPAIN, 2Microbiology, Universidad deNavarra, Pamplona, SPAIN, 3Allergy and Clinical Immunology, Univer-sidad de Navarra, Pamplona, SPAIN.RATIONALE: Gantrez® AN were examined as immunoadjuvant study-ing their protective effect in sensitized mice with ovalbumin.METHODS: Ovalbumin-loaded nanoparticles were prepared by a sol-vent displacement method. In some cases, nanoparticles were prepared inthe presence of rough lipopolysacharide (R-LPS) as immunomodulator(R-LPS-NP). BALB/c mice were sensitized by treatment with 50 �g oval-bumin adsorbed in alhydrogel on days 0 and 7th. On day 13th , 16th and19th the animals received i.d. doses of different nanoparticle formulations.On day 34th, mice were challenged by i.p. administration of 1 mg ovalbu-min in PBS. The intensity of the anaphylaxis was evaluated determiningmortality or symptomatology, and by quantification of the histamineblood level and body temperature.RESULTS: Gantrez® AN nanoparticles displayed a size of about 200 nmand were able to carry about 30 �g OVA/mg nanoparticle. The presenceof R-LPS did not influence the physicochemical characteristics. In vivo,mice treated with nanoparticles containing R-LPS (R-LPS-NP) displayedthe most favorable symptomatology. In fact, this group of mice showed asignificantly lower capacity to rise the histamine blood level after thechallenge. Similarly, the decrease on the body temperature was almostinappreciable. In addition, all the animals that treated were protectedagainst death by anaphylactic shock.CONCLUSIONS: Gantrez® AN nanoparticles containing R-LPS protectagainst the anaphylactic shock in experimentally sensitized mice withovalbumin, which encourages the application of these nanoparticles inimmunotherapy.Funding: Natl Dept Science and Technology # SAF 2001-0690-C03-01

887 Effects of Oral Ingestion of Nigella sativa Seeds (BlackCumin) on Murine Immune System

A. K. Y. Daoud; Internal Medicine, Jordan University Science and Tech-nology, Irbid, JORDAN.RATIONALE: We wanted to study the effects on the murine immunesystem for oral ingestion of Nigella sativa seeds, which is a common herbfrom the family Raununuculaceae used as a food spice and thought tohave many medicinal values.METHODS: 3 groups of 18 BALB/c mice were given daily either 0,40gms or 100gms of the seeds and complete consumption was assured.The following tests were done after 2 and 3 weeks of ingestion and at 1week after stopping the intake: - (1) White Blood Cell Count and differ-ential,(2) NBT test on peritoneal macrophages stimulated by PMA, (3)IgM and IgG Plaque Forming Cell assays on splenocytes using sRBC asantigen and (4) Mitogenic response of splenocytes to Lipopolysaccaride(LPS) and Concanavalin A (Con A). Appropriate statistical analysis wasapplied.RESULTS: There was no difference between the groups for the WBC’sor differential at all times. NBT test and IgG PFC assay were significant-ly higher at 3 weeks and after stopping the intake with increasing doses.IgM PFC assay was significantly higher from week 2 and on with increas-ing doses. LPS and Con A induced proliferations were significantly high-er at all times and doses.CONCLUSIONS: Nigella sativa seeds have diffuse murine immune sys-tem stimulant effects and further studies are needed on the human immunesystem with clarification and isolation of the active components fromthem.

Hyde Park, NY.RATIONALE: Although the AIDS epidemic continues to claim millionsof lives annually, there is a paucity of novel therapeutic approaches beingexplored. The completion of the genomic sequence of the chimpanzee hasprovided a unique opportunity to explore the genetic basis for the resis-tance of our closest evolutionary relative to develop AIDS. METHODS: We used the Affymetrix microarray gene chip to explore thedifferences in gene expression between human and chimpanzee PBMCfollowing in-vitro stimulation with Phorbol ester and Ionomycin or mon-oclonal antibodies specific for CD3 and CD28 which activate T-cellsthrough the CD3-TCR complex and the co-stimulatory molecule CD28. RESULTS: We found a high degree of concordance between the humanand chimpanzee gene expression profiles regardless of the type of stimu-lation used. In addition, the levels of induction for individual genes werevery similar for the two species, with very little intra-, and inter-speciesvariation. CONCLUSIONS: The remarkably similar profiles of human and chim-panzee gene expression suggest that the overall program of immunocytegene regulation has been highly conserved between these species, despitethe larger than expected differences in DNA sequences of the chimpanzeeand human genomes. Thus, relatively few key differences in HIV-inducedgene regulation may be responsible for the drastic differences in clinicaloutcome in these two genetically similar species following HIV-1 infec-tion. Identifying novel regulatory differences in chimpanzees may providea rationale for new therapeutic approaches in humans.Funding: NIH

889 Effects of Sleep Disorders and Fatigue on HIV Disease Pro-gression in Older Children and Adolescents

M. E. Paul1, P. Y. Brouwers1, D. G. Glaze1, J. M. Reuben2, B. Lee2, L. L.Harris1, C. A. Kozinetz1, M. W. Kline1, H. L. Schwarzwald1, S. B. Fos-ter1, C. D. Jackson1, W. T. Shearer1; 1Pediatrics, Baylor College of Med-icine, Houston, TX, 2Molecular Biology, The University of Texas, M.D.Anderson Cancer Center, Houston, TX.RATIONALE: Children with chronic illnesses have sleep disruption, day-time attention deficits, and impulsivity, and altered blood leukocyte sub-types, sleep-regulating hormones, and pro- and anti-inflammatory cytokines.Children with HIV infection exhibit similar sleep disorders and fatigue, buttheir effects upon HIV disease progression and immunity are unknown.METHODS: A cohort of 30 HIV+ and 30 HIV-exposed children (8-18yr.) with detailed clinical database was secured for study. Previously val-idated questionnaires and test instruments (sleep habits, daytime sleepi-ness, neurocognition) were administered to HIV+ and control children.Blood from these children was activated with phorbol myristate acetate(PMA) or lipopolysaccharide (LPS) to assess tumor necrosis factor-alpha(TNF-a, pro-inflammatory, sleep-inducing cytokine) and interleukin-10(IL-10, anti-inflammatory, sleep-inhibiting cytokine) expression in acti-vated T cells to determine the relationship between sleep disturbance,fatigue, and cytokines levels.RESULTS: Psychological testing revealed that there was a positive rela-tionship between plasma IL-10 and neurocognitive functioning in 30HIV+ children (r = 0.40, p < 0.05). A study of a subgroup of 11 HIV+ chil-dren and 10 controls showed that TNF-a was elevated in PMA-stimulatedCD3+ T cells (p = 0.049), and IL-10 was lower in LPS-stimulated CD3+

T cells (p = 0.008).CONCLUSIONS: Children with HIV infection demonstrate higherblood levels of the sleep-inducing cytokine, TNF-a; reduced blood levelsof the sleep-inhibiting cytokine, IL-10; and neurocognitive deficits. Theseearly results of this clinical study indicate that it may be possible to deter-mine if sleep disorders and fatigue are related with disease progression inHIV+ children.Funding: NIH, NHLBI HL079533 T

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