Proliferation Of Lymph Node Cells Research Articles

Effect of Rebamipide on the Colonic Barrier in Interleukin-10-Deficient Mice

Our aim was to study the effect of a mucosal protective agent, rebamipide, on the colonic barrier and the immune response in colitis-prone interleukin-10-deficient (IL-10-/-) C57BL/6 mice infected with Helicobacter hepaticus. After sacrifice, in all mice, control, or previously infected with H. hepaticus, or previously infected and treated with rebamipide enema, a histological examination of colonic samples was performed, intestinal permeability was studied in Ussing chamber, and mesenteric lymph node proliferation and cytokine secretion were measured. Mice treated with rebamipide presented a reinforcement of the distal colonic epithelial barrier, an increase of mesenteric lymph node cells proliferation, and of IFNgamma and IL-12 secretion. These results indicate that in IL-10-/- mice with mild colitis, rectally administered rebamipide reinforces the distal colonic barrier and has a slight Th1 immuno-stimulatory effect on mesenteric lymph node cells. These properties could be helpful in the management of some inflammatory bowel diseases.

FR180204, a novel and selective inhibitor of extracellular signal-regulated kinase, ameliorates collagen-induced arthritis in mice

Extracellular signal-regulated kinase (ERK), a serine/threonine protein kinase of the mitogen-activated protein kinase superfamily, is activated by various stimuli in inflammatory cells. We recently described FR180204 (5-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-amine), a novel selective ERK inhibitor. In this paper, we investigated the effect of FR180204 on collagen-induced arthritis (CIA) in DBA/1 mice, an animal model of rheumatoid arthritis (RA) mediated by type II collagen (CII)-reactive T cells and anti-CII antibodies. Preventive administration of FR180204 (100 mg/kg, i.p., b.i.d.) significantly ameliorated the clinical arthritis and body weight loss occurring in the CIA mice. Further, FR180204-treated mice showed a significant decrease in plasma anti-CII antibody levels (62%). FR180204 also attenuated delayed-type hypersensitivity in CII-immunized DBA/1 mice, an inflammatory response elicited by CII-reactive T cells, in a dose-dependent manner (52 and 62% inhibition at 32 and 100 mg/kg, respectively). Moreover, FR180204 inhibited in vitro CII-induced proliferation of lymph node cells prepared from CII-immunized mice, in which CII-specific T cells are known to undergo specific proliferation. In conclusion, our results suggest that ERK regulates both the cell-mediated and humoral immune responses in the development of CIA. ERK inhibitors may be useful as therapeutic reagents for the treatment of RA.

Human Prolactin Promotes Human Secondary Immunoglobulin Response in Human/SCID Mouse Chimeras

Recombinant human prolactin (rhPRL) was administered to huPBL-SCID mice to determine its effects on production of human immunoglobulin (Ig). The huPBL-SCID mice were injected intraperitoneally (i.p.) with 10 mug rhPRL every other day for a total of 10 injections. The results reconfirmed that rhPRL significantly increased the numbers of human CD3+ T cells and human CD19+ B cells in spleens, lymph nodes, and thymuses of huPBL-SCID mice. The huPBL-SCID mice were then concurrently given various doses of diphtheria-tetanus (DT) vaccine and 10-mug i.p. injections of rhPRL and were examined for the presence of human DT-specific proliferation of lymph node cells in vitro and antibody production in vivo. rhPRL greatly improved the engraftment of functional human lymphocytes (CD3+ T cells and CD19+ B cells) in DT-immunized huPBL-SCID mice. The rhPRL-treated, DT-immunized huPBL-SCID mice produced significantly larger amounts of DT-specific antibodies in response to the vaccine. The predominant Ig isotype induced after immunization was IgG. Thus, rhPRL stimulation promotes human secondary IgG responses in huPBL-SCID mice.

The protease inhibitor, Bowman-Birk Inhibitor, suppresses experimental autoimmune encephalomyelitis: a potential oral therapy for multiple sclerosis

Available treatments for multiple sclerosis (MS) require frequent injections and have significant side effects. Proteases generated during inflammation are involved in the induction of tissue damage during inflammatory demyelination in the central nervous system (CNS). The Bowman-Birk Inhibitor (BBI), a soy-derived protease inhibitor with anti-carcinogenic and anti-inflammatory properties, has been shown to be well tolerated in clinical trials for pre-cancerous conditions, such as oral leukoplakia and the inflammatory disease, ulcerative colitis. We hypothesized that BBI may modulate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The BBI concentrate (BBIC), a soybean extract enriched in BBI, was administered to myelin basic protein (MBP)-immunized Lewis rats by gastric gavage in different treatment regimens, during the induction or the effector phase of disease. BBIC significantly delayed disease onset and suppressed disease severity, clinically and pathologically, in all treatment protocols. Both in vitro and ex vivo, BBIC inhibited MBP-specific proliferation of lymph node cells. BBIC reduced the activity of matrix metalloproteinase (MMP)-2 and -9 in spleen cell supernatants and was detected in the CNS of treated rats. BBIC suppresses EAE, it can be administered orally, and it is safe and relatively inexpensive. It may have a therapeutic role in patients with MS.

Screening for immunomodulatory proteins of the intestinal parasitic nematode Heligmosomoides polygyrus

Parasitic nematodes are constantly exposed to the immune effector mechanisms of their hosts. One strategy of the worms to cope with these defence reactions is the secretion of modulatory proteins that down-regulate cell-mediated immune responses. We analysed the proliferation of mesenteric lymph node cells of mice infected with Heligmosomoides polygyrus and showed that cellular proliferation was strongly suppressed in the chronic phase of infection. To identify proteins of H. polygyrus that are involved in parasite-induced immunomodulation, worm extract and culture supernatant of adult H. polygyrus were fractionated by gel chromatography and activity of each fraction was determined. One of the fractions (fraction 9) of worm extract as well as worm secretory products inhibited the antigen-specific cellular proliferation by about 40%. This reduced cellular reactivity coincided with a down-regulation of inducible nitric oxide production of mouse macrophages by 57%. Furthermore, fraction 9 contained antigens that were recognized by IgE antibodies of H. polygyrus-infected mice and induced degranulation of an IgE-sensitized basophil cell line. Single proteins of fraction 9 were analysed by mass spectrometry. These data suggest that antigens that are recognised by IgE antibodies might play an important role in immunomodulation exerted by nematode infections.

Evaluation of immunomodulation by Lactobacillus casei Shirota: Immune function, autoimmunity and gene expression

Lactic acid bacteria are claimed to have immunomodulating effects. Stimulation as well as suppression of T helper (Th)1 mediated immune responses, have been described for various strains. Experiments involving Lactobacillus casei Shirota (LcS) detected mainly enhancement of innate immune responses and promotion of Th1 mediated immune reactivity. To confirm and further investigate modulation of Th1 responses and development of autoimmune disease by LcS, the consequences of oral administration of LcS were assessed in several experiments. The effect of LcS varied between the different models. No modulation was found in the mitogen-induced cell proliferation and cytokine release assays in mesenteric lymph nodes of Wistar rats. LcS inhibited the Th1 mediated immune response in an adapted murine Local Lymph Node Assay (LLNA) in BALB/c mice, whereas experimental autoimmune encephalomyelitis (EAE) in Lewis rats was aggravated. These varying effects on Th1 responses indicate that beneficial as well as harmful effects on immune related disorders could occur after LcS consumption. Since microarray analysis is suggested to be more sensitive and predictive than functional tests, gene expression profiling was included as an alternative endpoint in the testing of immunomodulation. The detected gene expression profiles did not reflect the effects of LcS on the immune system. Microarray analysis may therefore have no more predictive value than immune function assays when investigating immunomodulation by probiotics. To gain further insight into effects of probiotics on immune function, experiments including cytokine assays and gene expression analysis combined with disease models could be useful.

Assessment of the relative skin sensitization potency of siloranes and bis‐GMA using the local lymph node assay and QSAR predicted potency

Siloranes are silicon and oxirane (epoxy) containing monomers used for new dental composite development. The siloranes 3,4-epoxycyclohexylethyl-cyclopolymethylsiloxane (Tet-Sil) and bis-3,4-epoxycyclohexylethyl-phenyl-methylsilane (Ph-Sil) have in common cycloaliphatic epoxy moieties. The epoxy group is of concern in their biocompatibility since most epoxy compounds are known skin sensitizers. The objective of this study was to determine the in vivo skin sensitization potency of the siloranes in the local lymph node assay. A comparison was made with well-known chemical allergens, bis-GMA and DNCB. Female mice (CBA/CaJ) were exposed topically (dorsum of both ears) to several doses of acetone:olive oil in the ratio of 4:1 v/v. Doses were defined by a predictive structure-activity model (QSAR) for contact sensitization. Lymph node cell (LNC) proliferation was measured on the sixth day by incorporation of radioactive thymidine into DNA of lymph node cells. The effective concentration (EC3) that produced a 3-fold stimulation in LNC proliferation relative to controls was extrapolated from dose-response curves. DNCB was a strong sensitizer (EC3 = 0.06%). The EC3 values of Ph-Sil and bis-GMA were 19% and 45%, respectively, making these weak contact sensitizers. Tet-Sil did not increase lymph node proliferation when compared with controls. In contrast to Tet-Sil, the unpolymerized monomers Ph-Sil and bis-GMA have the capacity to induce LNC proliferation, characteristic of a T-cell mediated skin contact sensitization.

Nested N-Terminal Megalin Fragments Induce High-Titer Autoantibody and Attenuated Heymann Nephritis

It was shown previously that an N-terminal fragment (nM60) that encompasses amino acid residues 1 to 563 of megalin could induce active Heymann nephritis (AHN) as efficiently as the native protein. For delineation of a minimal structure within this fragment that is sufficient to induce AHN, smaller protein fragments that encompass residues 1 to 236 (L6), 1 to 195 (L5), 1 to 156 (L4), and 1 to 120 (L3), representing successive C-terminal truncations within ligand-binding repeats of nM60, were cloned and produced in a baculovirus insect cell expression system. Protein fragments L4, L5, and L6 clearly were glycosylated. All four fragments stimulated proliferation of megalin-sensitized lymph node cells and induced high-titer anti-megalin autoantibodies in Lewis rats. A full-blown disease, as assessed by severity of proteinuria, was observed in rats that were immunized with L6 and L5, whereas animals that were immunized with L4 and L3 developed only mild disease. The proteinuria levels correlated with staining for complement (C3, C5b-9) and IgG1 isotype in glomerular immune deposits. The results suggest that one or more molecular determinants on the region that comprises amino acid residues 157 to 236 contribute to the induction of a full-blown form of AHN. Study of the structure, conformation, and posttranslational modifications of these determinants could provide greater insight into the molecular correlates of immunopathogenesis in this disease model.

Development of a Modified Local Lymph Node Assay using ATP Measurement as an Endpoint

Several proposed modified local lymph node assays (LLNAs) use a non-RI method, but such methods tend to yield lower stimulation indices (SIs) than those obtained with the standard LLNA when used to assess moderate sensitizers. To address this issue, we propose a modified LLNA in which mice are exposed to the test material four times, instead of three times, and adenosine triphosphate (ATP) content is used as a measure of proliferation of lymph node cells (LNCs). The chemicals tested were 2.4dinitrochlorobenzene (DNCB), eugenol, and -hexyl cinnamic aldehyde (HCA), which are classified as strong-to-moderate sensitizers in the LLNA. Methyl salicylate (MS) was used as an example of a non-sensitizer. Using an SI of 3 as a cut-off, as recommended in the standard LLNA, DNCB, eugenol and HCA were classified as positive, while MS was classified as negative using the ATP-based non-RI method. Furthermore, there were no marked differences in SI levels for 10% eugenol and 10% HCA (a moderate sensitizer) between the non-RI method and the standard LLNA. Thus, it is reasonable to conclude that the non-RI method proposed in the present study could be a valid alternative to the standard LLNA.

Epitope Spreading and Autoimmune Glomerulonephritis in Rats Induced by a T Cell Epitope of Goodpasture’s Antigen

An amino-terminal region of alpha3 chain of type IV collagen noncollagenous domain [alpha3(IV)NC1] that induces experimental autoimmune glomerulonephritis (EAG) in rats has been identified. Only recombinant antigens that contain a nine-amino acid (AA) span of alpha3(IV)NC1, consistent with a T cell epitope, could induce EAG. It was hypothesized that synthetic peptides of this region should induce EAG. Human and rat peptides of this region were synthesized and rats were immunized to define the nephritogenic epitope. A 13-AA rat peptide induced EAG with proteinuria, decreased renal function, and glomerular basement membrane (GBM)-bound deposits in half of the rats. This peptide induces lymph node cell proliferation and development of antibodies to epitopes of alpha3(IV)NC1 external to the peptide immunogen. Carboxy-terminal extension to 21 amino acids results in all rats' demonstrating anti-GBM antibody and severe EAG. Asparagine at position 19 is critical for EAG induction. None of the 50 rats that were immunized with peptide that contained human sequence with isoleucine at position 19 developed EAG, whereas rat sequence with asparagine 19 induced EAG. Truncation of amino terminal AA of the peptide aborts EAG induction. These studies demonstrate that a T cell epitope of alpha3(IV)NC1 induces lymph node cell proliferation, EAG, and intramolecular epitope spreading; that the length of this peptide influences the formation of anti-GBM antibody; and that the presence of asparagine at position 19 of the peptide is critical to disease induction.

The local lymph node assay being too sensitive?

The local lymph node assay (LLNA) and modifications thereof were recently recognized by the OECD as stand-alone methods for the detection of skin-sensitizing potential. However, although the validity of the LLNA was acknowledged by the ICCVAM, attention was drawn to one major problem, i.e., the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). This is based on the fact that inflammatory processes in the skin may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions. In this paper, we have compared both endpoints: (1) cell proliferation alone and (2) cell proliferation in combination with inflammatory (irritating) processes. It turned out that a considerable number of tests were "false positive" to the definition mentioned above. By excluding such false positive results the LLNA seems not to be more sensitive than relevant guinea pig assays. These various methods and results are described here.

Well-defined and potent liposomal meningococcal B vaccines adjuvated with LPS derivatives

Potent liposomal PorA formulations containing various lipopolysaccharide (LPS) derivatives were developed. The following adjuvants were compared: the commonly used aluminum phosphate (AlPO 4), and three LPS like adjuvants: monophosphoryl lipid A (MPL), lipopolysaccharide ( galE LPS) and the less toxic LPS mutant lpxL1. The immunogenicity in mice was evaluated and compared with that against an outer membrane vesicle (OMV) vaccine. The IgG isotype distribution and bactericidal activity were determined. Furthermore, PorA specific proliferation of lymph node cells after immunization and restimulation in vitro was studied with selected formulations. Both AlPO 4 and MPL were unable to improve the functional immunogenicity (i.e. bactericidal response) of liposomal PorA. Besides, when these adjuvants were used, the percentage of responders in the groups did not reach 100%. This was also observed with non adjuvated PorA-liposomes or OMV. Of the adjuvants studied, only galE LPS and lpxL1 LPS were capable of increasing the immunogenicity and avoid non responsiveness against PorA-liposomes. Importantly, the adjuvant activity of lpxL1 LPS was accompanied by an improved PorA specific proliferation of lymph node cells and a concomitant increase in IL-2 production. In conclusion and considering its lower toxicity, lpxL1 LPS adjuvated liposomes are superior to other formulations tested.

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round

The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8–10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A–C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies.

Ectopic mesothelial cell proliferation in cervical lymph nodes after severe ovarian hyperstimulation syndrome

To report a case of ectopic mesothelial proliferation in cervical lymph nodes after severe ovarian hyperstimulation syndrome. Case report. University-affiliated teaching hospital. A 42-year-old woman underwent a successful IVF attempt and developed severe ovarian hyperstimulation syndrome and pathologically enlarged cervical lymph nodes. Paracentesis, cervical lymph node biopsy followed by cytology, histology, and immunohistochemistry. Resolution of symptoms, pregnancy outcome, correct pathological diagnosis. Paracentesis resulted in resolution of symptoms of ovarian hyperstimulation. The diagnosis of ectopic mesothelial proliferation in cervical lymph nodes was made after immunohistochemical examination of cervical lymph nodes. The pregnancy progressed normally, and at 40 weeks the patient spontaneously delivered a healthy son weighing 3,060 g. This case describes ectopic mesothelial cell proliferation localized in and around cervical lymph nodes occurring at 9 weeks' gestation in a patient who earlier developed severe ovarian hyperstimulation syndrome 10 days after ET. Ectopic mesothelial cell proliferation in lymph nodes is an extremely rare event, and this is the first case described after ovarian hyperstimulation. Familiarity with this event is important for the clinician as well as for the pathologist in preventing the misdiagnosis of malignancy.

TLR2 modulates inflammation in zymosan-induced arthritis in mice

The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dectin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dectin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro, it had no effect on ZIA in vivo. These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

Intratracheal Instillation of Cytoplasmic Granules from Phleum pratense Pollen Induces IgE- and Cell-Mediated Responses in the Brown Norway Rat

Background: Release of cytoplasmic granules from grass pollen upon contact with water is thought to be an important source of airborne allergens. Objectives: To investigate the humoral and cellular responses to intratracheal instillation of Phleum pratense (timothy grass) pollen cytoplasmic granules (PCG) in the Brown Norway rat. Methods: PCG were purified from timothy grass pollen by filtration through 5-µm-mesh filters. Rats were sensitized (day 0) and challenged (day 21) intratracheally with purified PCG suspended in saline (6 × 10⁶ PCG/rat). Rats were then challenged 4 weeks later (1.5 × 10⁶ PCG/rat). Blood samples, bronchial lymph nodes and lungs were collected from the rats 4 days after the second challenge. PCG-specific IgE and IgG1 levels and specificity were determined by ELISA and Western blotting. Pollen, pollen extract and PCG-induced proliferation of lymph node cells were monitored by [³H]-thymidine incorporation in a lymph node assay. Histopathological examination was carried out on the lungs. Results: Specific IgE and IgG1 were present in the sera. Cultured lymph node cells proliferated in the presence of pollen, pollen extract and PCG. Western blots showed that all major pollen allergens are recognized by IgE and IgG1 from PCG-treated rats. Histopathological examination revealed features of a mild allergic reaction. Conclusions: In our rat model of allergy, purified timothy grass PCG instillation induced specific antibodies and lymph node cell responses, comparable to those obtained with intact pollen.

Allogeneic core amino acids of an immunodominant allopeptide are important for MHC binding and TCR recognition

The indirect alloimmune response seems to be restricted to a few dominant major histocompatibility complex (MHC)-derived peptides responsible for T-cell activation in allograft rejection. The molecular mechanisms of indirect T-cell activation have been studied using peptide analogues derived from the dominant allopeptide in vitro, whereas the in vivo effects of peptide analogues have not been well characterized yet. In the present study, we generated allochimeric peptide analogues by replacing the three allogeneic amino acids 5L, 9L, and 10T in the sequence of the dominant MHC class I allopeptide P1. These allochimeric peptide analogues were used to define the allogeneic amino acids critical for the MHC binding and TCR recognition. We found that position 5 (5L) of the dominant allopeptide acts as an MHC-binding residue, while the other two allogeneic positions, 9 and 10, are important for the T-cell receptor (TCR) recognition. A peptide containing the MHC-binding residue 5L, as the only different amino acid between donor (RT1.A u) and recipient (RT1.A l) sequences, did not induce proliferation of lymph node cells primed with the dominant peptide and prevented dominant peptide-induced acceleration of allograft rejection. Identification of MHC and TCR contact residues should facilitate the development of antigen-specific therapies to inhibit or regulate the indirect alloimmune response.

Gammadelta T cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and IL-12 production.

Using an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-reactive lymph node cells (LNC), we have shown that depletion of gammadelta T cells from LNC resulted in diminished severity of EAE in recipient mice, both clinically and histopathologically. The reduced potency of gammadelta T cell-depleted LNC to induce EAE correlated with decreased cell proliferation in response to MBP. The gammadelta T cell effect upon the threshold of MBP-induced LNC proliferation and EAE transfer was restored by reconstitution of gammadelta T cells derived from either MBP-immunized or naive mice, indicating that this effect was not Ag specific. The enhancing effect of gammadelta T cells on MBP-induced proliferation and EAE transfer required direct cell-to-cell contact with LNC. The gammadelta T cell effect upon the LNC response to MBP did not involve a change in expression of the costimulatory molecules CD28, CD40L, and CTLA-4 on TCRalphabeta(+) cells, and CD40, CD80, and CD86 on CD19(+) and CD11b(+) cells. However, depletion of gammadelta T cells resulted in significant reduction in IL-12 production by LNC. That gammadelta T cells enhanced the MBP response and severity of adoptive EAE by stimulating IL-12 production was supported by experiments showing that reconstitution of the gammadelta T cell population restored IL-12 production, and that gammadelta T cell depletion-induced effects were reversed by the addition of IL-12. These results suggest a role for gammadelta T cells in the early effector phase of the immune response in EAE.

Arsenic-induced alterations in the contact hypersensitivity response in Balb/c mice

Previous studies in our laboratory indicate that arsenic alters secretion of growth promoting and inflammatory cytokines in the skin that can regulate the migration and maturation of Langerhans cells (LC) during allergic contact dermatitis. Therefore, we hypothesized that arsenic may modulate hypersensitivity responses to cutaneous sensitizing agents by altering cytokine production, LC migration, and T-cell proliferation. To investigate this hypothesis, we examined the induction and elicitation phases of dermal sensitization. Mice exposed to 50 mg/l arsenic in the drinking water for 4 weeks demonstrated a reduction in lymph node cell (LNC) proliferation and ear swelling following sensitization with 2,4-dinitrofluorobenzene (DNFB), compared to control mice. LC and T-cell populations in the draining lymph nodes of DNFB-sensitized mice were evaluated by fluorescence-activated cell sorting; activated LC were reduced in cervical lymph nodes, suggesting that LC migration may be altered following arsenic exposure. Lymphocytes from arsenic-treated animals sensitized with fluorescein isothiocyanate (FITC) exhibited reduced proliferative responses following T-cell mitogen stimulation in vitro; however, lymphocyte proliferation from nonsensitized, arsenic-treated mice was comparable to controls. Arsenic exposure also reduced the number of thioglycollate-induced peritoneal macrophages and circulating neutrophils. These studies demonstrate that repeated, prolonged exposure to nontoxic concentrations of sodium arsenite alters immune cell populations and results in functional changes in immune responses, specifically attenuation of contact hypersensitivity.

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy.

Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.