This study examined the effect of hormonal environment on intranasal and subcutaneous routes of i... more This study examined the effect of hormonal environment on intranasal and subcutaneous routes of immunization in a genital herpes infection model. Ovariectomized mice were treated with estradiol (E(2)), progesterone (P(4)) or placebo hormone pellets and immunized intranasally (i.n.) or subcutaneously (s.c.) with attenuated HSV-2. Immunized mice were subsequently challenged, intravaginally, with wild-type HSV-2. Mice immunized under the influence of E(2) showed higher survival rates, reduced pathology and significantly lower viral shedding compared with those immunized under the influence of P(4) or placebo, by both i.n. and s.c. routes. Vaginal and serum anti-HSV-2 IgG, but not IgA, levels correlated with decreased pathology in E(2)-treated, i.n. immunized mice. We conclude that immunization under the influence of E(2) afforded better protection compared to placebo and P(4), by both routes of immunization. Female sex hormones can influence immune responses and outcome of viral challenge in the genital tract following systemic immunization.
Medicine and Science in Sports and Exercise, Feb 1, 2006
This study tested the hypothesis that exercise-induced perturbation and recovery of the immune sy... more This study tested the hypothesis that exercise-induced perturbation and recovery of the immune system would vary with age, puberty, and gender in healthy children and adolescents. Twelve-year-old girls (YG; N = 14) and boys (YB; N = 20), and 14-yr-old girls (OG; N = 11) and boys (OB; N = 13) cycled for 60 min at 70% VO2max. Blood was collected before, at 30 and 60 min of exercise, and at 30 and 60 min of recovery to measure total leukocytes, leukocyte and lymphocyte subsets, and cytokines. Age and pubertal (Tanner stage) effects within genders and gender effects within age and pubertal groups were determined. Exercise-induced increases in lymphocytes, CD3-CD16+CD56+ counts, and IL-6 were approximately 83, 90, and 390% greater in OG versus YG (P < 0.05). Recovery leukocytosis and neutrophilia were approximately 56 and 35% greater in OB versus YB (P < 0.05). Pubertal stage did not have a statistically significant influence on responses in girls, but the lowest pubertal stage consistently showed smaller changes in lymphocytes and CD3-CD16+CD56+ counts. Recovery neutrophilia was approximately 120% greater in postpubertal boys versus prepubertal or pubertal boys (P < 0.05). Responses of lymphocytes and CD3-CD16+CD56+ counts, respectively, were approximately 120 and 82% greater in OG versus OB (P < 0.05), with no differences between YG and YB. Exercise-induced increases in total leukocytes, lymphocytes, and CD3-CD16+CD56+ counts were at least 35% greater in girls versus boys of similar pubertal status (P < 0.05). Regardless of age, puberty, or gender, IL-8 levels were significantly higher during recovery versus rest (P < 0.05). These results highlight the need to control for age, puberty, and gender when interpreting immunologic responses to exercise in a pediatric population.
Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispec... more Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.
The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but m... more The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4 ؉ T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4 ؉ CD45RB high T cells developed colitis, and cotransferring DO11.10 CD45RB low CD4 ؉ T cells prevented CD4 ؉ CD45RB high T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4 ؉ T cell subsets were associated with an increase in endogenous TCR␣ chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4 ؉ CD45RB high attenuated the colitis in association with increased TGF- and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB high T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.
Recent investigations in our laboratory have shown that murine intestinal smooth muscle cells (IS... more Recent investigations in our laboratory have shown that murine intestinal smooth muscle cells (ISMCs) can exert an immunomodulatory effect on T-cells. Therefore, we examined the effects of substance P, calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) on the ability of ISMCs to modulate T-cell proliferation and lymphokine generation. T-cell proliferation was observed when these cells were co-cultured with IFN-pretreated C57/BL6 ISMCs which expressed major histocompatibility complex II (MHC II), but not during T-cell co-culture with C2D (MHC II -/-) ISMCs pretreated in the same manner. T-cell proliferation during co-culture with C57/BL6 ISMCs was also associated with significantly enhanced T-cell synthesis of IFN. When CGRP (at 10(-9) M), but not substance P or VIP, was added to C57/BL6 ISMCs during the IFN-pretreatment period. T-cell proliferation was significantly increased. However, increased T-cell proliferation was not observed if the concentration of CGRP was increased to 10(-6) M. At the higher concentration, addition of substance P or VIP during the pretreatment period significantly inhibited the subsequent T-cell proliferation. Pretreatment of C57/BL6 ISMCs with any of the three neuropeptides and IFN resulted in the diminished production of IL-4 and IFN by co-cultured T-cells. A similar pattern of cytokine secretion was observed during T-cell co-culture with IFN- and neuropeptide-pretreated C2D ISMCs except when 10(-6) M substance P was added; IFN secretion by co-cultured T-cells was increased 4-fold under these conditions. Taken together, these data show a direct modulatory role for neuropeptides in the interaction between ISMCs and T-cells and suggest that, in general, neuropeptides may dampen immune responses in the neuromuscular layers of the inflamed intestine.
Allergy, Asthma & Clinical Immunology, Oct 8, 2012
The immunological and clinical parameters that are associated with asthma remission are poorly un... more The immunological and clinical parameters that are associated with asthma remission are poorly understood. The cytokine and local mediator changes associated with the resolution of asthma symptoms were examined in three groups of subjects 12-18 years of age (n = 15 in each group): (a) continuing asthma group (CA) who had persistent symptoms since early childhood, (b) an age, sex and atopic status-matched group who had persistent symptoms in early childhood but in whom these had resolved (RA), and (c) a non-atopic, non-asthmatic control group. Clinical parameters, sputum cell counts, peripheral blood mononuclear cell (PBMC) cytokine production and activation marker expression were determined. All of the CA had methacholine airway hyperresponsiveness compared with only half of the RA subjects. The CA showed elevated numbers of eosinophils and increased ECP and IL-5 in sputum, which were not observed in the RA. PBMC cytokine studies revealed increased production of the type 1 cytokines IL-12, IFN-γ and TNF-α in the CA group compared with the RA group, under a range of activation conditions, however, the production of IL-4 and IL-5 were unchanged. These findings suggest that decreased type 1 cytokine expression as well as decreased eosinophilic inflammation is associated with the resolution of asthma symptoms.
The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of d... more The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of dendritic cells (DCs) during pulmonary viral infection was investigated by using a mouse model of GM-CSF transgene expression established with an adenoviral vector (AdGM-CSF). GM-CSF gene transfer resulted in increased levels of GM-CSF in the lung, which peaked at day 4 and remained increased up to day 19. A striking cellular response composed predominantly of macrophage-like cells was observed in the lung receiving AdGM-CSF but not control vector. By FACS analysis, the majority of these cells were identified at an early time point as macrophages and later as mature/activated myeloid DCs characterized by CD11b bright , CD11c bright , MHC class II bright , and B7.1 bright. In contrast, GM-CSF had a weak effect on a small DC population that was found present in normal lung and was characterized by CD11c bright and CD11b low. By immunohistochemistry staining for MHC II, the majority of activated antigenpresenting cells were localized to the airway epithelium and peribronchial/ perivascular areas in the lung. A concurrently enhanced Th1 immune response was observed under these conditions. The number of CD4 and CD8 T cells was markedly increased in the lung expressing GM-CSF, accompanied by increased release of interferon (IFN)␥ in the lung. Furthermore, lymphocytes isolated from either lung parenchyma or local draining lymph nodes of these mice but not the control mice released large amounts of IFN␥ on adenoviral antigen stimulation in vitro. These findings reveal that GM-CSF promotes the differentiation and activation of a myeloid DC population primarily by acting on macrophages during pulmonary immune responses.
Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses prote... more Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses protein Ags. We examined the Ab responses and allergic sensitization of several strains of mice to protein Ags, administered orally with CTX. The mice made strong IgA and IgG1 serum Ab responses, but little IgG2a Ab to Ags such as hen egg lysozyme (HEL) and OVA. However, when given a subsequent i.p. challenge with Ag alone, the same mice had immediate hypersensitivity reactions that included respiratory distress and death. Within 10 min of i.p. challenge, immunized mice had high levels of plasma histamine and extensive degranulation of mast cells in target tissues. These mice had detectable serum IgE Ab. Ag administered orally with the B subunit (CTB) of CTX did not sensitize mice. Intestinal tissues taken from these mice had Ag-specific ion-secretory responses in vitro, typical of intestinal anaphylaxis. Ag given s.c. without adjuvant could also sensitize for systemic and intestinal anaphylaxis. Sensitization with HEL given s.c. was dose dependent and correlated with a critical amount of HEL in the circulation. HEL was detected in the circulation after oral immunization, but CTX did not increase the uptake of HEL. Thus, oral immunization with a protein Ag in the presence of CTX can sensitize an animal for systemic and intestinal anaphylaxis. These results suggest a cautious approach to the use of CTX as an adjuvant in oral vaccines, and provide a new model to study immediate hypersensitivity reactions to intestinal Ag.
Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells i... more Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells in the intestine during the induced graft-vs-host reaction (GVHR). We investigated the activation and mucosal homing phenotype of the donor CD4 cells and the kinetics of cytokine responses within the intestine and associated lymphoid tissues during early GVHR. Significant frequencies of donor CD4 cells accumulated within recipient Peyer's patches (PP), mesenteric lymph nodes (MLN), lamina propria (LP), and spleen (SP), during the first 9 days of GVHR. Many donor CD4 cells in SP, MLN, and LP expressed CD44 and also expressed de novo the mucosal homing integrin ␣ 4  7 (LPAM-1). A large IFN-␥ response occurred by day 3 in cells from PP and MLN, but much later (day 9) in SP and LP cells. IL-10 production by SP and MLN cells was elevated initially but declined substantially by day 9. IL-4 production by SP, MLN, and PP cells was low on day 3 and showed gradual decline in LP by day 9. IL-5 production by LP cells gradually increased in direct contrast to IL-5 production by MLN cells. The MLN CD4 cells showed the most dynamic changes, with high numbers of activated/effector donor CD4 cells and altered cytokine production consistent with a developing Th1 response. The IFN-␥ responses in PP and MLN preceded that of the SP, suggesting an intestinal origin for some Th1 effector cells in GVHR. Donor CD4 T cells apparently acquire the ability to home to the LP during early GVHR.
The bacterial enterotoxins, cholera toxin and the heat labile toxin of E. coli, are well known ad... more The bacterial enterotoxins, cholera toxin and the heat labile toxin of E. coli, are well known adjuvants for mucosal immune response. Their common A chain mediates the toxigenic mechanism by causing ADP ribosylation of G proteins and subsequent elevation of cAMP in target cells. A large IgA and IgG antibody response to admixed protein antigen (Ag) is the hallmark of these adjuvants and is clearly associated with the A chain activity. Expansion of Ag-specific B and T cells, alteration of T cell cytokine production, and changes in regulatory T cells have been reported as adjuvant mechanisms. The B chain derivatives of these toxins can also weakly enhance immune response, especially if covalently associated with Ag and used for nasophyrangeal immunization. Importantly, these toxins or their B chain derivatives can alter the normal immune regulation that produces oral tolerance. This indicates that they modulate mechanisms operative between the mucosal and systemic immune systems. There are some discrepancies between in vitro models of CT or LT activity and in vivo manifestations of their adjuvant activities. Interpretation of current data regarding in vivo mechanism is hampered by an incomplete understanding of how mucosal B and T cells can interact with systemic lymphoid tissue and vice versa. More important, there is no clear understanding of the early effects of the toxins on the local (and draining) mucosal lymphoid tissues. This is especially true in the critical areas of antigen presentation, T and B cell activation, and cytokine production.
HLA class I antigens of the human major histocompatibility complex play an important role in immu... more HLA class I antigens of the human major histocompatibility complex play an important role in immune response. These molecules present foreign antigenic peptides to cytotoxic T lymphocytes and thereby play a role in the immune surveillance of cells infected with virus or other intracellular pathogens or altered by malignant transformation. A marked deficiency or lack of expression of these antigens has been reported in a variety of human neoplasms. In the present study, we examined the expression of class I a chain, P,-microglobulin, TAP (TAP1 and TAP2) and LMP (LMP2 and LMP7) genes in a number of human tumor cell lines including small-cell lung carcinoma, hepatocellular carcinoma, colon adenocarcinoma and bdsophilic leukaemia. These cell lines were deficient in expression of both class I CI chain and /Y,-microglobulin gene products. In addition, these cell lines lacked the products of MHC-encoded proteasome subunit LMP2 as well as the putative peptide transporter TAP1 genes. In contrast, TAP2 and LMP7 genes were expressed in these cell lines. Treatment of cells with y-IFN markedly enhanced the expression of class I c(chain, a,-microglobulin, TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. The upregulation of TAP1 and LMP2 expression is associated with increased class I expression, suggesting that endogenous antigens, e.g. tumor antigens, could be presented by class I molecules following treatment of tumor cells with >I-IFN.
Recognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immun... more Recognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immune responses has led to increasing interest in methods for the identification of DC within the circulation. We sought to develop a flow cytometric method that would allow the reliable enumeration of absolute myeloid DC counts in minimally manipulated blood samples. Myeloid DC were identified by three-color staining of whole blood leukocytes as a discrete population of mononuclear cells expressing high levels of HLA-DR and CD33, yet having little or no expression of CD14 and CD16. This method was analyzed for reproducibility and variation in blood DC number during typical clinical day hours and after exercise. The new method was compared to an established commercial kit method. FACS sorting of the CD33(+) DC showed that they morphologically resembled immature DC, and developed cytoplasmic projections typical of mature DC following overnight culture in granulocyte macrophage-colony stimulating factor (GM-CSF). Within peripheral blood, these DC were found at a mean concentration of 17. 4 +/- 5.4 x 10(6) per liter, corresponding to 0.93 +/- 0.27% of mononuclear cells. Comparison of duplicate samples stained and analyzed in parallel showed that the intrasample variability was very low, with an intraclass correlation coefficient of 0.95. The frequency of CD33(+) myeloid DC and their light scatter characteristics were similar to that of CD11c(+) myeloid cells. Four-color FACS analysis revealed complete identity of CD11c(hi), HLA-DR(+) DC with CD33(+), HLA-DR(+) DC. Only rare CD33(+) DC coexpressed CD123 and HLA-DR. Numbers of blood myeloid DC, identified by CD33 staining, showed no significant variation during standard laboratory hours. However, their numbers rose significantly during vigorous exercise, in parallel to other blood cells. The method described herein is rapid, reproducible, requires only small volumes of blood, can be readily used by a clinical immunology laboratory, and requires fewer antibodies than a currently available commercial method.
We analyzed the appearance and level of Giardia muris-specific antibody of immunoglobulin A (IgA)... more We analyzed the appearance and level of Giardia muris-specific antibody of immunoglobulin A (IgA), IgG, and IgM isotypes, at weekly intervals, over the course of a 7-week infection in BALB/c and C57BL/6 mice. Using sensitive immunoradiometric assays, we observed that IgA antibody was the only detectable anti-G. muris antibody in intestinal secretions throughout the course of infection. No secreted IgG or IgM anti-G. muris antibody was detected even in concentrated intestinal secretions. The expulsion of G. muris by the mice was associated closely with the appearance and increasing levels of secreted anti-G. muds IgA antibody. Both IgG and IgA serum antibody to G. muds were detected, but no serum IgM antibody was detected. Serum IgA and IgG anti-G. muris antibody remained at high levels up to 10 weeks following clearance of the parasite. An interesting observation indicated that serum IgA antibody to G. muds developed more slowly in response to infection than secreted IgA antibody. An analysis of the molecular weight distribution of total serum IgA in infected mice determined that infection produced a transient but significant shift in serum IgA to highmolecular-weight (.dimeric IgA) forms. The results indicate that a substantial IgA antibody response occurs in sera and in gut secretions of G. muds-resistant mice and that IgA antibody is the dominant and possibly the only effector antibody active in intestinal secretions during G. murs infection in mice.
The Journal of Allergy and Clinical Immunology, Jul 1, 2004
Dendritic cells are important antigen-presenting cells. After an allergen inhalation, their numbe... more Dendritic cells are important antigen-presenting cells. After an allergen inhalation, their numbers rapidly decrease in circulation and increase in the airway mucosa. To investigate whether allergen-induced changes in the number of circulating dendritic cells are mediated by cysteinyl leukotrienes. In a randomized, double-blind, crossover study, we examined the effects of 2 weeks of treatment with pranlukast (a cysteinyl leukotriene 1 [CysLT1] receptor antagonist) 300 mg twice daily and placebo on allergen-induced changes in airway responses and circulating dendritic cells in 15 subjects with mild asthma. We examined by flow cytometry, before and at 3 hours and 24 hours after allergen inhalation, the proportion of myeloid (CD33+) and plasmacytoid (CD123+) dendritic cells (HLA-DR+, CD14-, CD16-) among all peripheral blood mononuclear cells. The fraction of dendritic cells expressing CysLT1 receptor was also determined. Compared with placebo, pranlukast significantly attenuated both the maximum early (by 55%) and the late (by 39%) asthma responses, the allergen-induced methacholine airway hyperresponsiveness, and the increase in macrophage inflammatory protein 1alpha and 3alpha in induced sputum. A significantly greater proportion of CD33+ cells (55%) expressed CysLT1 receptor compared with CD123+ cells (11%). Consistent with this, pranlukast prevented the allergen-induced decrease in CD33+ dendritic cells at 3 hours postallergen (mean Delta from baseline, +4.4%) compared with placebo (mean Delta, -8.4; P <.05), but not CD123+ cells. Pretreatment with pranlukast attenuated allergen-induced airway responses and the decrease in circulating myeloid dendritic cells, demonstrating a novel role of cysteinyl leukotrienes in dendritic cell trafficking.
The most limiting factor regarding the use of TNF␣ in serum concentrations of mTNF␣ (approximatel... more The most limiting factor regarding the use of TNF␣ in serum concentrations of mTNF␣ (approximately 1 ng/ml) tumour therapy is systemic toxicity. The expression of were detected only in Ad-mTNF-wt-treated mice, while membrane-bound (nonsecreted) TNF␣ within a tumour both vectors induced substantial disruption of tumour pathmay serve to reduce systemic toxicity while retaining anti-ology. The wt TNF vector was highly toxic, killing 12 of 16 tumour activity. Two adenovirus (Ad) vectors were con-mice at a dose of 5 × 10 8 p.f.u., whereas the Ad-mTNFstructed: (1) Ad-mTNF-wt expressing wild-type murine MEM vector showed low toxicity killing three of 27 at the TNF␣; and (2) Ad-mTNF-MEM expressing a mutant non-same dose. Both vectors induced partial, and in some secreted (membrane-bound) form. Only the Ad-mTNF-wt cases, permanent tumour regressions, with cured mice disvector induced high levels of TNF␣ secretion in transduced playing protective immunity and specific CTL activity cells (approximately 400 ng/10 6 cells), however, both vec-against the tumour. These results indicate that the use of tors induced efficient cell surface expression as detected a nonsecreted form of TNF␣ can result in a relatively large by FACS. These vectors were used in tumour immunother-reduction in systemic toxicity with little or no reduction in apy trials in a murine transgenic breast cancer model. High antitumour activity.
The objective of this study was to investigate the contribution of the interaction between CD40 a... more The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG 1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNF ␣ in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNF ␣ in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and-challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNF ␣ by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNF ␣ or Ad/IL-4 into OVA-sensitized and-challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNF ␣ and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.
B-cell depletion therapy may impair vaccine responses and increase infection risk in patients wit... more B-cell depletion therapy may impair vaccine responses and increase infection risk in patients with immune thrombocytopenia (ITP). Capitalizing on a multicenter randomized placebo-controlled trial, we investigated the effects of rituximab on the antibody and cellular responses to Streptococcus pneumoniae polysaccharide vaccine and Haemophilus influenzae type b (Hib) conjugate vaccine in ITP patients. Of 60 patients in the main trial, 24 patients received both vaccines 6 months after rituximab (n=17) or placebo (n=7). Among 20 evaluable patients, 3/14 (21%) in the rituximab group and 4/6 (67%) in the placebo group achieved a 4-fold increase in anti-pneumococcal antibodies (p=0.12). For anti-Hib antibodies, 4/14 (29%) and 5/6 (83%), respectively, achieved a 4-fold increase (p<0.05). Fewer patients in the rituximab group demonstrated functional Hib killing (2/14 [14%] versus 5/6 [83%], p<0.05). Three of 14
Systemic immunization can elicit a significant response of IgG producing activated B cell subsets... more Systemic immunization can elicit a significant response of IgG producing activated B cell subsets in human blood, part of which is not toward the vaccine. However, the effect of vaccination on IgA antibody secreting B cell subsets has had limited investigation. We immunized healthy, adult volunteers with a tetanus/diphtheria vaccine and observed a significant burst of IgA-secreting pre-plasma cells (PPC). Isolated PPC produced IgG, but not IgA antibody to tetanus antigen, and produced IgA and IgG antibody specific for poliovirus and herpes simplex virus. Thus, a vaccine that generates a systemic recall response to tetanus also induces blood PPC secreting IgA and IgG antibody relevant to mucosal protection.
Mice deficient in MHC class II expression (C2d mice) do not make antibody to protein antigens adm... more Mice deficient in MHC class II expression (C2d mice) do not make antibody to protein antigens administered systemically, but their ability to produce IgA antibody to antigen administered at mucosal sites has not been described. We investigated IgA production by C2d mice and their IgA antibody response to antigen given orally. Young C2d mice had normal amounts of serum IgA, intestinal-secreted IgA and normal numbers of intestinal IgA plasma cells, compared to control C57BL/6 mice. IgA production by C2d mice increased with age. Following oral immunization with cholera toxin, C57BL/6 mice responded with IgA and IgG antibody, and had increased numbers of IgA plasma cells, but C2d mice gave no response. The Peyer's patch and mesenteric lymph node tissues of C2d mice contained very few CD4-expressing T cells. Thus, C2d mice have no typical mucosal CD4 T h cells and cannot respond to a strong oral immunogen, yet they still produced and secreted IgA. We hypothesized that B-1 lymphocytes could provide a source of IgA independent of antigen-specific T cell help. Young C2d mice had normal numbers of peritoneal B-1a cells and their frequency increased with age. To test the role of these B-1a cells, we bred C2d mice to obtain mice that had no MHC class II expression and expressed the Xid gene that confers deficiency in B-1a cells. These double-deficient mice had 10-fold less serum and secreted IgA than all other F 2 littermates. We conclude that B-1a cells are essential for the majority of IgA production in C2d mice. Thus, the C2d mouse may provide a useful tool for analysis of the role of intestinal IgA provided by B-1a cells.
This study examined the effect of hormonal environment on intranasal and subcutaneous routes of i... more This study examined the effect of hormonal environment on intranasal and subcutaneous routes of immunization in a genital herpes infection model. Ovariectomized mice were treated with estradiol (E(2)), progesterone (P(4)) or placebo hormone pellets and immunized intranasally (i.n.) or subcutaneously (s.c.) with attenuated HSV-2. Immunized mice were subsequently challenged, intravaginally, with wild-type HSV-2. Mice immunized under the influence of E(2) showed higher survival rates, reduced pathology and significantly lower viral shedding compared with those immunized under the influence of P(4) or placebo, by both i.n. and s.c. routes. Vaginal and serum anti-HSV-2 IgG, but not IgA, levels correlated with decreased pathology in E(2)-treated, i.n. immunized mice. We conclude that immunization under the influence of E(2) afforded better protection compared to placebo and P(4), by both routes of immunization. Female sex hormones can influence immune responses and outcome of viral challenge in the genital tract following systemic immunization.
Medicine and Science in Sports and Exercise, Feb 1, 2006
This study tested the hypothesis that exercise-induced perturbation and recovery of the immune sy... more This study tested the hypothesis that exercise-induced perturbation and recovery of the immune system would vary with age, puberty, and gender in healthy children and adolescents. Twelve-year-old girls (YG; N = 14) and boys (YB; N = 20), and 14-yr-old girls (OG; N = 11) and boys (OB; N = 13) cycled for 60 min at 70% VO2max. Blood was collected before, at 30 and 60 min of exercise, and at 30 and 60 min of recovery to measure total leukocytes, leukocyte and lymphocyte subsets, and cytokines. Age and pubertal (Tanner stage) effects within genders and gender effects within age and pubertal groups were determined. Exercise-induced increases in lymphocytes, CD3-CD16+CD56+ counts, and IL-6 were approximately 83, 90, and 390% greater in OG versus YG (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Recovery leukocytosis and neutrophilia were approximately 56 and 35% greater in OB versus YB (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Pubertal stage did not have a statistically significant influence on responses in girls, but the lowest pubertal stage consistently showed smaller changes in lymphocytes and CD3-CD16+CD56+ counts. Recovery neutrophilia was approximately 120% greater in postpubertal boys versus prepubertal or pubertal boys (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Responses of lymphocytes and CD3-CD16+CD56+ counts, respectively, were approximately 120 and 82% greater in OG versus OB (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), with no differences between YG and YB. Exercise-induced increases in total leukocytes, lymphocytes, and CD3-CD16+CD56+ counts were at least 35% greater in girls versus boys of similar pubertal status (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Regardless of age, puberty, or gender, IL-8 levels were significantly higher during recovery versus rest (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). These results highlight the need to control for age, puberty, and gender when interpreting immunologic responses to exercise in a pediatric population.
Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispec... more Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab&#39;2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.
The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but m... more The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4 ؉ T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4 ؉ CD45RB high T cells developed colitis, and cotransferring DO11.10 CD45RB low CD4 ؉ T cells prevented CD4 ؉ CD45RB high T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4 ؉ T cell subsets were associated with an increase in endogenous TCR␣ chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4 ؉ CD45RB high attenuated the colitis in association with increased TGF- and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB high T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.
Recent investigations in our laboratory have shown that murine intestinal smooth muscle cells (IS... more Recent investigations in our laboratory have shown that murine intestinal smooth muscle cells (ISMCs) can exert an immunomodulatory effect on T-cells. Therefore, we examined the effects of substance P, calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) on the ability of ISMCs to modulate T-cell proliferation and lymphokine generation. T-cell proliferation was observed when these cells were co-cultured with IFN-pretreated C57/BL6 ISMCs which expressed major histocompatibility complex II (MHC II), but not during T-cell co-culture with C2D (MHC II -/-) ISMCs pretreated in the same manner. T-cell proliferation during co-culture with C57/BL6 ISMCs was also associated with significantly enhanced T-cell synthesis of IFN. When CGRP (at 10(-9) M), but not substance P or VIP, was added to C57/BL6 ISMCs during the IFN-pretreatment period. T-cell proliferation was significantly increased. However, increased T-cell proliferation was not observed if the concentration of CGRP was increased to 10(-6) M. At the higher concentration, addition of substance P or VIP during the pretreatment period significantly inhibited the subsequent T-cell proliferation. Pretreatment of C57/BL6 ISMCs with any of the three neuropeptides and IFN resulted in the diminished production of IL-4 and IFN by co-cultured T-cells. A similar pattern of cytokine secretion was observed during T-cell co-culture with IFN- and neuropeptide-pretreated C2D ISMCs except when 10(-6) M substance P was added; IFN secretion by co-cultured T-cells was increased 4-fold under these conditions. Taken together, these data show a direct modulatory role for neuropeptides in the interaction between ISMCs and T-cells and suggest that, in general, neuropeptides may dampen immune responses in the neuromuscular layers of the inflamed intestine.
Allergy, Asthma & Clinical Immunology, Oct 8, 2012
The immunological and clinical parameters that are associated with asthma remission are poorly un... more The immunological and clinical parameters that are associated with asthma remission are poorly understood. The cytokine and local mediator changes associated with the resolution of asthma symptoms were examined in three groups of subjects 12-18 years of age (n = 15 in each group): (a) continuing asthma group (CA) who had persistent symptoms since early childhood, (b) an age, sex and atopic status-matched group who had persistent symptoms in early childhood but in whom these had resolved (RA), and (c) a non-atopic, non-asthmatic control group. Clinical parameters, sputum cell counts, peripheral blood mononuclear cell (PBMC) cytokine production and activation marker expression were determined. All of the CA had methacholine airway hyperresponsiveness compared with only half of the RA subjects. The CA showed elevated numbers of eosinophils and increased ECP and IL-5 in sputum, which were not observed in the RA. PBMC cytokine studies revealed increased production of the type 1 cytokines IL-12, IFN-γ and TNF-α in the CA group compared with the RA group, under a range of activation conditions, however, the production of IL-4 and IL-5 were unchanged. These findings suggest that decreased type 1 cytokine expression as well as decreased eosinophilic inflammation is associated with the resolution of asthma symptoms.
The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of d... more The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of dendritic cells (DCs) during pulmonary viral infection was investigated by using a mouse model of GM-CSF transgene expression established with an adenoviral vector (AdGM-CSF). GM-CSF gene transfer resulted in increased levels of GM-CSF in the lung, which peaked at day 4 and remained increased up to day 19. A striking cellular response composed predominantly of macrophage-like cells was observed in the lung receiving AdGM-CSF but not control vector. By FACS analysis, the majority of these cells were identified at an early time point as macrophages and later as mature/activated myeloid DCs characterized by CD11b bright , CD11c bright , MHC class II bright , and B7.1 bright. In contrast, GM-CSF had a weak effect on a small DC population that was found present in normal lung and was characterized by CD11c bright and CD11b low. By immunohistochemistry staining for MHC II, the majority of activated antigenpresenting cells were localized to the airway epithelium and peribronchial/ perivascular areas in the lung. A concurrently enhanced Th1 immune response was observed under these conditions. The number of CD4 and CD8 T cells was markedly increased in the lung expressing GM-CSF, accompanied by increased release of interferon (IFN)␥ in the lung. Furthermore, lymphocytes isolated from either lung parenchyma or local draining lymph nodes of these mice but not the control mice released large amounts of IFN␥ on adenoviral antigen stimulation in vitro. These findings reveal that GM-CSF promotes the differentiation and activation of a myeloid DC population primarily by acting on macrophages during pulmonary immune responses.
Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses prote... more Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses protein Ags. We examined the Ab responses and allergic sensitization of several strains of mice to protein Ags, administered orally with CTX. The mice made strong IgA and IgG1 serum Ab responses, but little IgG2a Ab to Ags such as hen egg lysozyme (HEL) and OVA. However, when given a subsequent i.p. challenge with Ag alone, the same mice had immediate hypersensitivity reactions that included respiratory distress and death. Within 10 min of i.p. challenge, immunized mice had high levels of plasma histamine and extensive degranulation of mast cells in target tissues. These mice had detectable serum IgE Ab. Ag administered orally with the B subunit (CTB) of CTX did not sensitize mice. Intestinal tissues taken from these mice had Ag-specific ion-secretory responses in vitro, typical of intestinal anaphylaxis. Ag given s.c. without adjuvant could also sensitize for systemic and intestinal anaphylaxis. Sensitization with HEL given s.c. was dose dependent and correlated with a critical amount of HEL in the circulation. HEL was detected in the circulation after oral immunization, but CTX did not increase the uptake of HEL. Thus, oral immunization with a protein Ag in the presence of CTX can sensitize an animal for systemic and intestinal anaphylaxis. These results suggest a cautious approach to the use of CTX as an adjuvant in oral vaccines, and provide a new model to study immediate hypersensitivity reactions to intestinal Ag.
Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells i... more Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells in the intestine during the induced graft-vs-host reaction (GVHR). We investigated the activation and mucosal homing phenotype of the donor CD4 cells and the kinetics of cytokine responses within the intestine and associated lymphoid tissues during early GVHR. Significant frequencies of donor CD4 cells accumulated within recipient Peyer's patches (PP), mesenteric lymph nodes (MLN), lamina propria (LP), and spleen (SP), during the first 9 days of GVHR. Many donor CD4 cells in SP, MLN, and LP expressed CD44 and also expressed de novo the mucosal homing integrin ␣ 4  7 (LPAM-1). A large IFN-␥ response occurred by day 3 in cells from PP and MLN, but much later (day 9) in SP and LP cells. IL-10 production by SP and MLN cells was elevated initially but declined substantially by day 9. IL-4 production by SP, MLN, and PP cells was low on day 3 and showed gradual decline in LP by day 9. IL-5 production by LP cells gradually increased in direct contrast to IL-5 production by MLN cells. The MLN CD4 cells showed the most dynamic changes, with high numbers of activated/effector donor CD4 cells and altered cytokine production consistent with a developing Th1 response. The IFN-␥ responses in PP and MLN preceded that of the SP, suggesting an intestinal origin for some Th1 effector cells in GVHR. Donor CD4 T cells apparently acquire the ability to home to the LP during early GVHR.
The bacterial enterotoxins, cholera toxin and the heat labile toxin of E. coli, are well known ad... more The bacterial enterotoxins, cholera toxin and the heat labile toxin of E. coli, are well known adjuvants for mucosal immune response. Their common A chain mediates the toxigenic mechanism by causing ADP ribosylation of G proteins and subsequent elevation of cAMP in target cells. A large IgA and IgG antibody response to admixed protein antigen (Ag) is the hallmark of these adjuvants and is clearly associated with the A chain activity. Expansion of Ag-specific B and T cells, alteration of T cell cytokine production, and changes in regulatory T cells have been reported as adjuvant mechanisms. The B chain derivatives of these toxins can also weakly enhance immune response, especially if covalently associated with Ag and used for nasophyrangeal immunization. Importantly, these toxins or their B chain derivatives can alter the normal immune regulation that produces oral tolerance. This indicates that they modulate mechanisms operative between the mucosal and systemic immune systems. There are some discrepancies between in vitro models of CT or LT activity and in vivo manifestations of their adjuvant activities. Interpretation of current data regarding in vivo mechanism is hampered by an incomplete understanding of how mucosal B and T cells can interact with systemic lymphoid tissue and vice versa. More important, there is no clear understanding of the early effects of the toxins on the local (and draining) mucosal lymphoid tissues. This is especially true in the critical areas of antigen presentation, T and B cell activation, and cytokine production.
HLA class I antigens of the human major histocompatibility complex play an important role in immu... more HLA class I antigens of the human major histocompatibility complex play an important role in immune response. These molecules present foreign antigenic peptides to cytotoxic T lymphocytes and thereby play a role in the immune surveillance of cells infected with virus or other intracellular pathogens or altered by malignant transformation. A marked deficiency or lack of expression of these antigens has been reported in a variety of human neoplasms. In the present study, we examined the expression of class I a chain, P,-microglobulin, TAP (TAP1 and TAP2) and LMP (LMP2 and LMP7) genes in a number of human tumor cell lines including small-cell lung carcinoma, hepatocellular carcinoma, colon adenocarcinoma and bdsophilic leukaemia. These cell lines were deficient in expression of both class I CI chain and /Y,-microglobulin gene products. In addition, these cell lines lacked the products of MHC-encoded proteasome subunit LMP2 as well as the putative peptide transporter TAP1 genes. In contrast, TAP2 and LMP7 genes were expressed in these cell lines. Treatment of cells with y-IFN markedly enhanced the expression of class I c(chain, a,-microglobulin, TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. The upregulation of TAP1 and LMP2 expression is associated with increased class I expression, suggesting that endogenous antigens, e.g. tumor antigens, could be presented by class I molecules following treatment of tumor cells with >I-IFN.
Recognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immun... more Recognition of the importance of dendritic cells (DC) in the initiation of T-cell-dependent immune responses has led to increasing interest in methods for the identification of DC within the circulation. We sought to develop a flow cytometric method that would allow the reliable enumeration of absolute myeloid DC counts in minimally manipulated blood samples. Myeloid DC were identified by three-color staining of whole blood leukocytes as a discrete population of mononuclear cells expressing high levels of HLA-DR and CD33, yet having little or no expression of CD14 and CD16. This method was analyzed for reproducibility and variation in blood DC number during typical clinical day hours and after exercise. The new method was compared to an established commercial kit method. FACS sorting of the CD33(+) DC showed that they morphologically resembled immature DC, and developed cytoplasmic projections typical of mature DC following overnight culture in granulocyte macrophage-colony stimulating factor (GM-CSF). Within peripheral blood, these DC were found at a mean concentration of 17. 4 +/- 5.4 x 10(6) per liter, corresponding to 0.93 +/- 0.27% of mononuclear cells. Comparison of duplicate samples stained and analyzed in parallel showed that the intrasample variability was very low, with an intraclass correlation coefficient of 0.95. The frequency of CD33(+) myeloid DC and their light scatter characteristics were similar to that of CD11c(+) myeloid cells. Four-color FACS analysis revealed complete identity of CD11c(hi), HLA-DR(+) DC with CD33(+), HLA-DR(+) DC. Only rare CD33(+) DC coexpressed CD123 and HLA-DR. Numbers of blood myeloid DC, identified by CD33 staining, showed no significant variation during standard laboratory hours. However, their numbers rose significantly during vigorous exercise, in parallel to other blood cells. The method described herein is rapid, reproducible, requires only small volumes of blood, can be readily used by a clinical immunology laboratory, and requires fewer antibodies than a currently available commercial method.
We analyzed the appearance and level of Giardia muris-specific antibody of immunoglobulin A (IgA)... more We analyzed the appearance and level of Giardia muris-specific antibody of immunoglobulin A (IgA), IgG, and IgM isotypes, at weekly intervals, over the course of a 7-week infection in BALB/c and C57BL/6 mice. Using sensitive immunoradiometric assays, we observed that IgA antibody was the only detectable anti-G. muris antibody in intestinal secretions throughout the course of infection. No secreted IgG or IgM anti-G. muris antibody was detected even in concentrated intestinal secretions. The expulsion of G. muris by the mice was associated closely with the appearance and increasing levels of secreted anti-G. muds IgA antibody. Both IgG and IgA serum antibody to G. muds were detected, but no serum IgM antibody was detected. Serum IgA and IgG anti-G. muris antibody remained at high levels up to 10 weeks following clearance of the parasite. An interesting observation indicated that serum IgA antibody to G. muds developed more slowly in response to infection than secreted IgA antibody. An analysis of the molecular weight distribution of total serum IgA in infected mice determined that infection produced a transient but significant shift in serum IgA to highmolecular-weight (.dimeric IgA) forms. The results indicate that a substantial IgA antibody response occurs in sera and in gut secretions of G. muds-resistant mice and that IgA antibody is the dominant and possibly the only effector antibody active in intestinal secretions during G. murs infection in mice.
The Journal of Allergy and Clinical Immunology, Jul 1, 2004
Dendritic cells are important antigen-presenting cells. After an allergen inhalation, their numbe... more Dendritic cells are important antigen-presenting cells. After an allergen inhalation, their numbers rapidly decrease in circulation and increase in the airway mucosa. To investigate whether allergen-induced changes in the number of circulating dendritic cells are mediated by cysteinyl leukotrienes. In a randomized, double-blind, crossover study, we examined the effects of 2 weeks of treatment with pranlukast (a cysteinyl leukotriene 1 [CysLT1] receptor antagonist) 300 mg twice daily and placebo on allergen-induced changes in airway responses and circulating dendritic cells in 15 subjects with mild asthma. We examined by flow cytometry, before and at 3 hours and 24 hours after allergen inhalation, the proportion of myeloid (CD33+) and plasmacytoid (CD123+) dendritic cells (HLA-DR+, CD14-, CD16-) among all peripheral blood mononuclear cells. The fraction of dendritic cells expressing CysLT1 receptor was also determined. Compared with placebo, pranlukast significantly attenuated both the maximum early (by 55%) and the late (by 39%) asthma responses, the allergen-induced methacholine airway hyperresponsiveness, and the increase in macrophage inflammatory protein 1alpha and 3alpha in induced sputum. A significantly greater proportion of CD33+ cells (55%) expressed CysLT1 receptor compared with CD123+ cells (11%). Consistent with this, pranlukast prevented the allergen-induced decrease in CD33+ dendritic cells at 3 hours postallergen (mean Delta from baseline, +4.4%) compared with placebo (mean Delta, -8.4; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.05), but not CD123+ cells. Pretreatment with pranlukast attenuated allergen-induced airway responses and the decrease in circulating myeloid dendritic cells, demonstrating a novel role of cysteinyl leukotrienes in dendritic cell trafficking.
The most limiting factor regarding the use of TNF␣ in serum concentrations of mTNF␣ (approximatel... more The most limiting factor regarding the use of TNF␣ in serum concentrations of mTNF␣ (approximately 1 ng/ml) tumour therapy is systemic toxicity. The expression of were detected only in Ad-mTNF-wt-treated mice, while membrane-bound (nonsecreted) TNF␣ within a tumour both vectors induced substantial disruption of tumour pathmay serve to reduce systemic toxicity while retaining anti-ology. The wt TNF vector was highly toxic, killing 12 of 16 tumour activity. Two adenovirus (Ad) vectors were con-mice at a dose of 5 × 10 8 p.f.u., whereas the Ad-mTNFstructed: (1) Ad-mTNF-wt expressing wild-type murine MEM vector showed low toxicity killing three of 27 at the TNF␣; and (2) Ad-mTNF-MEM expressing a mutant non-same dose. Both vectors induced partial, and in some secreted (membrane-bound) form. Only the Ad-mTNF-wt cases, permanent tumour regressions, with cured mice disvector induced high levels of TNF␣ secretion in transduced playing protective immunity and specific CTL activity cells (approximately 400 ng/10 6 cells), however, both vec-against the tumour. These results indicate that the use of tors induced efficient cell surface expression as detected a nonsecreted form of TNF␣ can result in a relatively large by FACS. These vectors were used in tumour immunother-reduction in systemic toxicity with little or no reduction in apy trials in a murine transgenic breast cancer model. High antitumour activity.
The objective of this study was to investigate the contribution of the interaction between CD40 a... more The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG 1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNF ␣ in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNF ␣ in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and-challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNF ␣ by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNF ␣ or Ad/IL-4 into OVA-sensitized and-challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNF ␣ and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.
B-cell depletion therapy may impair vaccine responses and increase infection risk in patients wit... more B-cell depletion therapy may impair vaccine responses and increase infection risk in patients with immune thrombocytopenia (ITP). Capitalizing on a multicenter randomized placebo-controlled trial, we investigated the effects of rituximab on the antibody and cellular responses to Streptococcus pneumoniae polysaccharide vaccine and Haemophilus influenzae type b (Hib) conjugate vaccine in ITP patients. Of 60 patients in the main trial, 24 patients received both vaccines 6 months after rituximab (n=17) or placebo (n=7). Among 20 evaluable patients, 3/14 (21%) in the rituximab group and 4/6 (67%) in the placebo group achieved a 4-fold increase in anti-pneumococcal antibodies (p=0.12). For anti-Hib antibodies, 4/14 (29%) and 5/6 (83%), respectively, achieved a 4-fold increase (p<0.05). Fewer patients in the rituximab group demonstrated functional Hib killing (2/14 [14%] versus 5/6 [83%], p<0.05). Three of 14
Systemic immunization can elicit a significant response of IgG producing activated B cell subsets... more Systemic immunization can elicit a significant response of IgG producing activated B cell subsets in human blood, part of which is not toward the vaccine. However, the effect of vaccination on IgA antibody secreting B cell subsets has had limited investigation. We immunized healthy, adult volunteers with a tetanus/diphtheria vaccine and observed a significant burst of IgA-secreting pre-plasma cells (PPC). Isolated PPC produced IgG, but not IgA antibody to tetanus antigen, and produced IgA and IgG antibody specific for poliovirus and herpes simplex virus. Thus, a vaccine that generates a systemic recall response to tetanus also induces blood PPC secreting IgA and IgG antibody relevant to mucosal protection.
Mice deficient in MHC class II expression (C2d mice) do not make antibody to protein antigens adm... more Mice deficient in MHC class II expression (C2d mice) do not make antibody to protein antigens administered systemically, but their ability to produce IgA antibody to antigen administered at mucosal sites has not been described. We investigated IgA production by C2d mice and their IgA antibody response to antigen given orally. Young C2d mice had normal amounts of serum IgA, intestinal-secreted IgA and normal numbers of intestinal IgA plasma cells, compared to control C57BL/6 mice. IgA production by C2d mice increased with age. Following oral immunization with cholera toxin, C57BL/6 mice responded with IgA and IgG antibody, and had increased numbers of IgA plasma cells, but C2d mice gave no response. The Peyer's patch and mesenteric lymph node tissues of C2d mice contained very few CD4-expressing T cells. Thus, C2d mice have no typical mucosal CD4 T h cells and cannot respond to a strong oral immunogen, yet they still produced and secreted IgA. We hypothesized that B-1 lymphocytes could provide a source of IgA independent of antigen-specific T cell help. Young C2d mice had normal numbers of peritoneal B-1a cells and their frequency increased with age. To test the role of these B-1a cells, we bred C2d mice to obtain mice that had no MHC class II expression and expressed the Xid gene that confers deficiency in B-1a cells. These double-deficient mice had 10-fold less serum and secreted IgA than all other F 2 littermates. We conclude that B-1a cells are essential for the majority of IgA production in C2d mice. Thus, the C2d mouse may provide a useful tool for analysis of the role of intestinal IgA provided by B-1a cells.
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Papers by Denis Snider