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Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost‐effective antivenom production based on plant‐made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin‐binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant‐made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice., The work carried out at IBMCP‐CSIC received financial support from MINECO and ERDF (project grants IPT‐2011‐0720‐010000, BIO2013‐42193‐R and BIO2016‐78601‐R). Research at IBV‐CSIC was supported by grant BFU2013‐42833‐P from the Spanish Ministry of Economy and Competitiveness, Madrid, Spain.

Lung transplantation is the ultimate treatment of end-stage lung disease. After transplantation, the 1-year survival rate is 80%. However, 5-year survival rates drop to 50% due to bronchiolitis obliterans syndrome (BOS). Ischemia/reperfusion injury, infections, and acute rejection are major risk factors contributing to the development of BOS. These risk factors are also associated with increased oxidative stress. Oxidative stress is a condition whereby prooxidants overwhelm the antioxidant defense system and may contribute to the pathogenesis of BOS by inducing more tissue injury and inflammation. This article reviews the current state of knowledge on oxidative stress in lung transplantation and BOS.

The airway epithelium is the primary target of inhaled pathogens such as human rhinovirus (HRV). Airway epithelial cells express ICAM-1, the major receptor for HRV. HRV binding to ICAM-1 mediates not only viral entry and replication but also a signaling cascade that leads to enhanced inflammatory mediator production. The specific signaling molecules and pathways activated by HRV-ICAM-1 interactions are not well characterized, although studies in human airway epithelia implicate a role for the p38 MAPK in HRV-induced cytokine production. In the current study, we report that Syk, an important immunoregulatory protein tyrosine kinase, is highly expressed by primary and cultured human airway epithelial cells and is activated in response to infection with HRV16. Biochemical studies revealed that ICAM-1 engagement by HRV and cross-linking Abs enhanced the coassociation of Syk with ICAM-1 and ezrin, a cytoskeletal linker protein. In polarized airway epithelial cells, Syk is diffusely distributed in the cytosol under basal conditions but, following engagement of ICAM-1 by cross-linking Abs, is recruited to the plasma membrane. The enhanced Syk-ICAM-1 association following HRV exposure is accompanied by Syk phosphorylation. ICAM-1 engagement by HRV and cross-linking Abs also induced phosphorylation of p38 in a Syk-dependent manner, and conversely, knockdown of Syk by short interfering (si)RNA substantially diminished p38 activation and IL-8 gene expression. Taken together, these observations identify Syk as an important mediator of the airway epithelial cell inflammatory response by modulating p38 phosphorylation and IL-8 gene expression following ICAM-1 engagement by HRV.

While current donor selection with clinical findings is generally effective, the imprecise nature of the assessment forces clinicians to remain on the conservative side. A reliable biological marker would assist donor selection and would improve donor organ utilization. We collected biopsies from 169 donor lungs before implantation. Expression levels of IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma and IL-1beta were measured by quantitative real-time RT-PCR (qRT-PCR). Seventeen cases died within 30 days after transplantation. No donor factor was significantly associated with 30-day mortality. Univariate analysis of the 84 cases for development of the prediction model showed that IL-6, IL-8, TNF-alpha and IL-1beta were risk factors for mortality and IL-10 and IFN-gamma were protective factors. We analyzed the cytokine expression ratios of risk to protective cytokines. A stepwise logistic regression for 30-day mortality demonstrated that a model containing the ratio of IL-6/IL-10 was the most predictive (p = 0.0013). When applied to the remaining 85 cases for validation, the test of model fit was significant (p = 0.039). Using the cytokine ratio, we were able to define three risk groups with striking differences in survival (p = 0.0003). Multi-cytokine analysis of the donor lung graft with qRT-PCR shows significant promise as a strategy to biologically evaluate the donor lung prior to implantation.

Fractalkine (CX3CL1) is of particular interest in atherogenesis because it can serve as an adhesion molecule and a chemokine. Fractalkine and its receptor CX3CR1 are expressed in atherosclerotic lesions of humans and mice. However, the effect of fractalkine deficiency on atherosclerosis susceptibility is unknown. Fractalkine-deficient mice on the C57BL/6 (B6) background were bred to the atherosclerosis-sensitizing B6.ApoE -/- and B6.LDLR -/- backgrounds. Compared with controls, aortic-root lesion area was unchanged in fractalkine-deficient male and female B6.ApoE -/- mice at 16 weeks of age and males at 12 weeks of age, but it was mildly reduced (30%, P = 0.005) in females at 12 weeks of age. In contrast, lesion area at the brachiocephalic artery (BCA) was reduced dramatically by ≈85% in fractalkine-deficient females [42,251 ± 26,136 μm 2 ( n = 15) vs. 6,538 ± 11,320 μm 2 ;( n = 24), P < 0.0001] and males [36,911 ± 32,504 μm 2 ( n = 24) vs. 6,768 ± 8,595 μm 2 ( n = 14); P = 0.001] at 16 weeks of age. Fractalkine-deficient B6.ApoE -/- mice were comparable with controls in body weight, plasma cholesterol, plasma high-density lipoprotein cholesterol and white blood cell counts. On the B6.LDLR -/- background, lesion areas were reduced by 35% at the aortic root ( P < 0.01) and by 50% at the BCA ( P < 0.05) in fractalkine-deficient females at 16 weeks of age. Lesions in fractalkine-deficient mice on the B6.ApoE -/- and B6.LDLR -/- backgrounds were less complex and contained significantly fewer macrophages than controls. In conclusion, the major reduction of atherosclerosis in fractalkine-deficient mice appears to be at the BCA rather than the aortic root.

Several autoimmune disease models depend on an imbalance in the activation of aggressor T(h)1 and CD4(+)CD25(+) regulatory T (T(reg)) cells. Here we compare the requirement for signals through the co-stimulatory molecules CD28 and inducible co-stimulator (ICOS) in chronic murine colitis, a model for inflammatory bowel disease. We used a colitis model in which disease-causing CD45RB(hi) T cells alone or in combination with CD4(+)CD25(+) T cells from either CD28-deficient or wild-type donors were transferred into T cell-deficient animals, half of which were treated with ICOS-blocking reagents. Blocking ICOS on the surface of CD28-deficient T(h)1 cells abrogated development of colitis, whereas blocking CD28 or ICOS alone had little to no effect on disease induction. In contrast to T(h)1 cells, regulatory T cell functioning depended mostly on CD28 signaling with only a minor contribution for ICOS. We conclude that CD28 and ICOS collaborate to development of murine colitis by aggressor T(h)1 cells, and that CD28 is required for T(reg) cells, which should caution against the use of CD28-blocking reagents in inflammatory bowel disease.

It is now fully appreciated that asthma is a disease of a chronic nature resulting from intermittent or continued aeroallergen exposure leading to airway inflammation. To investigate responses to continuous antigen exposure, mice were exposed to either house dust mite extract (HDM) or ovalbumin intranasally for five consecutive days, followed by 2 days of rest, for up to seven consecutive weeks. Continuous exposure to HDM, unlike ovalbumin, elicited severe and persistent eosinophilic airway inflammation. Flow cytometric analysis demonstrated an accumulation of CD4+ lymphocytes in the lung with elevated expression of inducible costimulator a marker of T cell activation, and of T1/ST2, a marker of helper T Type 2 effector cells. We also detected increased and sustained production of helper T cell Type 2-associated cytokines by splenocytes of HDM-exposed mice on in vitro HDM recall. Histologic analysis of the lung showed evidence of airway remodeling in mice exposed to HDM, with goblet cell hyperplasia, collagen deposition, and peribronchial accumulation of contractile tissue. In addition, HDM-exposed mice demonstrated severe airway hyperreactivity to methacholine. Finally, these responses were studied for up to 9 weeks after cessation of HDM exposure. We observed that whereas airway inflammation resolved fully, the remodeling changes did not resolve and airway hyperreactivity resolved only partly.

Background Mast cells (MCs) proliferate in response to T H 2 cytokines and express genes de novo after activation. Limited information is available concerning the interplay between these events. Objective We explored the potential for T H 2 cytokines to alter activation-dependent gene expression by MCs. Methods Cord blood–derived human (h)MCs maintained in stem cell factor (SCF) alone were compared with replicates treated with IL-4, IL-5, or IL-9, respectively, for their patterns of FceRI-dependent gene induction using microarray technology. Results Activation of SCF-treated hMCs upregulated their expression of roughly 140 transcripts at 2 hours, including genes involved in cell cycle progression and arrest. Each cytokine substantially modified this profile; ∼800 inducible genes apiece were controlled by IL-5 or IL-9, whereas 169 inducible genes were controlled by IL-4. IL-4 favored the induction of cytokines and of genes associated with cell growth arrest (GADD34, GAS-1, CIDE-A, INK4D, and BAX) and completely abolished the enhanced proliferation observed in the other 3 groups after activation. Conversely, IL-5 priming induced preferential upregulation of genes involved in cell proliferation and did not abolish thymidine incorporation. Conclusions T H 2 cytokines differentially modulate gene induction in hMCs after FceRI cross-linkage. IL-4 uniquely controls cytokine gene expression by hMCs and might also limit their activation-driven proliferation.

Although T helper (T(H)) cell-mediated immunity is required to effectively eliminate pathogens, unrestrained T(H) activity also contributes to tissue injury in many inflammatory and autoimmune diseases. We report here that the T(H) type 1 (T(H)1)-specific Tim-3 (T cell immunoglobulin domain, mucin domain) protein functions to inhibit aggressive T(H)1-mediated auto- and alloimmune responses. Tim-3 pathway blockade accelerated diabetes in nonobese diabetic mice and prevented acquisition of transplantation tolerance induced by costimulation blockade. These effects were mediated, at least in part, by dampening of the antigen-specific immunosuppressive function of CD4(+)CD25(+) regulatory T cell populations. Our data indicate that the Tim-3 pathway provides an important mechanism to down-regulate T(H)1-dependent immune responses and to facilitate the development of immunological tolerance.

Remarkable advances in our knowledge of the genome sequence have led to the creation of databases that afford the opportunity to discover genes based on the presence of specific sequence motifs. The application of this strategy to the identification of proteins in families of immunological interest has had a visible impact on the sizes of the interleukin 1/18 and the CD28/B7 costimulatory molecule families, among many others. This accelerated pace of discovery presents a new challenge to match the rate of discovery with biological understanding of the functions of these extended protein families.

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9+ lymphocytes in blood from normal donors. A subset of CCR9+ T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9−, CCR9+CD4+ peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-γ and IL-10. IL-10-producing cells were exclusively detected within the CCR9+ subset of CD4+ T cells by intracellular staining and were distinct from IL-2- and IFN-γ-producing cells. Moreover, memory CCR9+CD4+ lymphocytes respond to CD2 stimulation with proliferation and IFN-γ/IL-10 production, whereas memory CCR9−CD4+ cells were unresponsive. In addition, memory CCR9+CD4+ T cells support Ig production by cocultured CD19+ B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9+CD4+ T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.

Background: Conventional models of allergic airway inflammation involve intraperitoneal administration of ovalbumin in conjunction with a chemical adjuvant (generally aluminum hydroxide) to generate allergic airways inflammation. Here we have investigated the effect of respiratory mucosal exposure to a ragweed extract in the absence of chemical adjuvant on the generation of allergic responses. Objectives: We sought to develop a mouse model of ragweed-induced allergic airway inflammation through mucosal sensitization and to investigate the role of GM-CSF in this process. Methods: Ragweed was delivered intranasally to an airway microenvironment enriched with GM-CSF, which was achieved by means of either multiple coadministrations of recombinant GM-CSF or a single delivery of an adenoviral vector carrying the GM-CSF transgene. Results: Administration of a purified ragweed extract leads to TH2 sensitization (and not inhalation tolerance) accompanied by mild airway inflammation, modest clinical symptoms, and moderate production of TH2 cytokines by splenocytes on ragweed restimulation. The administration of anti-GM-CSF antibodies in conjunction with ragweed diminished TH2-associated cytokine production. These responses were amplified by enriching the airway microenvironment with GM-CSF. Under these conditions, all TH2-associated immune-inflammatory responses, as well as the clinical responses, were considerably enhanced. To investigate the mechanism underlying these effects, we examined lung mononuclear cells by means of flow cytometry and detected a substantial expansion of antigen-presenting cells, particularly dendritic cells, as well as a substantially increased activation of these antigen-presenting cells, as demonstrated by the expression of B7 molecules, particularly B7.2. Conclusion: GM-CSF plays an important role in the generation of allergic immune-inflammatory responses to ragweed. (J Allergy Clin Immunol 2003;111:1076-86.)

Inducing T cell responses requires at least two distinct signals: 1) TCR engagement of MHC-peptide and 2) binding of CD28 to B7.1/2. However, the recent avalanche of newly described costimulatory molecules may represent additional signals which can modify events after the initial two-signal activation. Inducible costimulatory molecule (ICOS) is a CD28 family member expressed on T cells rapidly following activation that augments both Th1 and Th2 T cell responses and has been implicated in sustaining rather than initiating T cell responses. Although it is known that blockade of ICOS-B7-related protein 1 (B7RP-1) in vivo dramatically reduces germinal center formation and Ab production, the mechanism(s) remains unclear. An optimal T cell-dependent Ab response requires T and B cell activation, expansion, differentiation, survival, and migration, and the ICOS-B7RP-1 interaction could be involved in any or all of these processes. Understanding this will have important implications for targeting ICOS-B7RP-1 therapeutically. We have therefore used a double-adoptive transfer system, in which all of the above events can be analyzed, to assess the role of ICOS-B7RP-1 in T cell help for B cell responses. We have shown that ICOS signaling is involved in the initial clonal expansion of primary and primed Th1 and Th2 cells in response to immunization. Furthermore, while ICOS-B7RP-1 interactions have no effect on the migration of T cells into B cell follicles, it is essential for their ability to support B cell responses.

A characteristic feature of rheumatoid arthritis is the abundance of inflammatory cells in the diseased joint. Two major components of this infiltrate are neutrophils in the synovial fluid and macrophages in the synovial tissue. These cells produce cytokines including tumor necrosis factor α and other proinflammatory mediators that likely drive the disease through its effector phases. To investigate what mechanisms underlie the recruitment of these cells into the synovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a related family that includes the anaphylatoxin receptors and the formyl-MetLeuPhe receptor. We then examined the effect of targeted disruption of two abundantly expressed chemoattractant receptors, the receptors for C3a and C5a, on arthritogenesis in a mouse model of disease. We report that genetic ablation of C5a receptor expression completely protects mice from arthritis.

Notch signaling plays a critical role in cell fate determination in many developmental systems, including the hematopoietic system. We and others have recently cloned a novel Notch ligand called Delta4. In this study, we show the effect of retrovirus-mediated ectopic expression of Delta4 in hematopoietic cells. Lethally irradiated mice transplanted with bone marrow cells expressing Delta4 initially suffered from leukopenia and thrombocytopenia. Although all lineages were affected, the deficit in B cells and platelets was the most durable and profound. A rapid expansion of CD4+CD8+ cells occurred shortly after transplantation. CD4+CD8+ cells progressively invaded all tissues analyzed except the thymus, which surprisingly was atrophic. CD4+CD8+cells were mainly non–Delta4-transduced cells, strongly suggesting that the disease was not cell autonomous. Around 15 weeks after transplantation, mice died from this severe lymphoproliferative disorder, which was not transplantable in late-stage disease into secondary recipients. Mice transduced with a soluble form of Delta4 behaved like control mice. Characterization of early hematopoietic development revealed that Delta4 expression impaired formation of day-12 spleen colony-forming units (CFU-Ss) and, to a greater extent, pre–CFU-Ss. No effect was observed on myeloid colony-forming cells (CFU-Cs), indicating that Delta4 specifically acted on the earliest hematopoietic stem cell compartment. These results show that constitutive expression of Delta4 in hematopoietic cells impairs the development of B cells, platelets, and early stem cells and induces a lethal lymphoproliferative disease.

LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT−/− mice by homologous recombination. The mean survival of fully major histocompatibility complex–mismatched vascularized cardiac allografts in LIGHT−/− mice (10 days, P < 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P < 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT−/− allograft recipients (30 days) was considerably enhanced (P < 0.001) compared with the 10 days of mean survival in either untreated LIGHT−/− mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-γ, plus IFN-γ–induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P < 0.001). Our data suggest that T cell to T cell–mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.

Background Exposure to aerosolized harmless antigen such as ovalbumin (OVA) has previously been shown to induce inhalation tolerance, a state characterized by inhibition of IgE synthesis and airway inflammation, upon secondary immunogenic antigen encounter. Immune events associated with this phenomenon are still poorly understood. Objective The aim of this study was to investigate cellular and molecular mechanisms underlying this state of ‘unresponsiveness’. Methods; After initial repeated OVA exposure, mice were subjected to a protocol of antigen-induced airway inflammation, encompassing two intraperitoneal injections of OVA adsorbed to aluminium hydroxide followed by airway challenge. We assessed immune events in the draining lymph nodes after sensitization, and in the lungs after challenge. Results In animals initially exposed to OVA, we observed, at the time of sensitization, considerable expansion of T cells, many of which expressed the activation markers CD69 and CD25, as well as increased numbers of antigen-presenting cells, particularly B cells. While these animals produced low levels of IgE, the observed elevated levels of IgG1 signified isotype switching. Splenocytes and lymph node cells from OVA-exposed mice produced low levels of IL-4, IL-5, IL-13 and IFN-γ, indicating aborted effector function of both T helper (Th)2- and Th1-associated cytokines. Real time quantitative polymerase chain reaction (PCR) (TaqMan) analysis of costimulatory molecules in the lungs after in vivo challenge showed that B7.1, B7.2, CD28 and CTLA-4 mRNA expression was low in animals initially exposed to OVA. Ultimately, these events were associated with abrogated airway inflammation and attenuated airway hyper-responsiveness. The decreased inflammation was antigen-specific and independent of IL-10 or IFN-γ. Conclusion Initial exposure to OVA establishes a programme that prevents the generation of intact, fully functional inflammatory responses upon secondary antigen encounter. The absence of inflammation, however, is not associated with categorical immune unresponsiveness.

The SLAM family of human genes currently consists of seven related members of the immunoglobulin superfamily, membrane-associated proteins, including CD150 (SLAM), CD244 (2B4), CD84, CD229 ( Ly-9), BLAME, CD48, and 19A. These genes are expressed to varying degrees in subsets of immune cells (T, B, natural killer, and myeloid cells) and may function as ligands or receptors. This set of genes, related to CD2 and CD58 on Chromosome (Chr) 1p98, are found clustered close together in the human genome on Chr 1q22. Four of these family members (CD150, CD244, CD84, CD229) contain conserved tyrosine motifs in their cytoplasmic tails that enable them to bind intracellular signaling molecules SAP and EAT-2. SAP is mutated in human X-linked lymphoproliferative disease (XLP), and studies in XLP patients have shown that improper signaling via molecules that bind SAP contributes to the disease. We have identified two new members of the SLAM family (SF), which we term SF2000 and SF2001, which are expressed in immune cells and map in the SLAM gene cluster. SF2001 does not contain SAP-binding motifs in its short cytoplasmic tail. SF2000, which is co-expressed with SAP in T cells, binds both SAP and EAT-2. The data suggest that signaling through SF2000, together with CD150, CD244, CD84, and CD229, is controlled by SAP and therefore contributes to the pathogenesis of XLP.

The objective of this study was to define phenotypic changes of antigen-presenting cells (APCs) and T cells in a murine model of antigen-induced airways inflammation that involves intraperitoneal sensitization with ovalbumin (OVA)/adjuvant followed by antigen aerosolization. We investigated the APC and T-cell compartments both after sensitization (primary immune response) and after challenge (secondary immune response) at the thoracic lymph nodes (initiation site) and the lung (effector site). Our findings document a major cellular expansion in the lymph nodes after both sensitization and challenge. After sensitization, this expansion was comprised mainly of B cells, a considerable proportion of which expressed B7.2. At this time, T cells were markedly expanded and activated as assessed by CD69 expression; further, although GATA-3 and signal transducer and activator of transcription-6 were expressed at this time point, expression of interleukin (IL)-4, IL-5, and IL-13 messenger RNA (mRNA) levels were marginal. However, in vitro stimulation of lymph-node cells with OVA led to cytokine production. In contrast, 24 h after challenge, but not after sensitization, there was a major expansion of dendritic cells and macrophages in the lungs. This expansion was associated with enhanced expression of both B7.1 and B7.2. We also observed expansion of activated CD3(+)/CD4(+) T cells expressing the T helper-2-associated marker T1/ST2 in the lung, most notably 5 d after challenge. Further, IL-4, IL-5, and IL-13, but not interferon-gamma mRNA were expressed at high levels 3 h after challenge. This study helps to elucidate the "geography" of immune responses generated in a conventional murine model of allergic airways inflammation.

The inducible costimulatory molecule (ICOS) is expressed on activated T cells and participates in a variety of important immunoregulatory functions. After the induction of experimental allergic encephalomyelitis in SJL mice with proteolipid protein (PLP), brain ICOS mRNA and protein were up-regulated on infiltrating CD3+ T cells before disease onset. ICOS blockade during the efferent immune response (9-20 days after immunization) abrogated disease, but blockade during antigen priming (1-10 days after immunization) exacerbated disease. Upon culture with PLP and compared with immunized controls, splenocytes produced either decreased interferon-gamma (IFN-gamma, in efferent blockade) or excessive IFN-gamma (in priming blockade). PLP-specific immunoglobulin G1 was decreased in animals treated with anti-ICOS during antigen priming, but not in other groups.

Primary T cell activation requires B7-CD28 and CD40-CD154 costimulation, but effector T cell functions are considered to be largely independent of these costimulatory pathways. Although blockade of costimulation with cytolytic T lymphocyte-associated antigen 4-immunoglobulin (CTLA-4-Ig) or monoclonal antibody (mAb) to CD154 prolongs allograft survival, chronic rejection follows, which suggests that additional key costimulatory pathways are active in vivo. We found that both antibody to inducible costimulator (anti-ICOS) and an ICOS-Ig fusion protein suppressed intragraft T cell activation and cytokine expression and prolonged allograft survival in a manner similar to that in ICOS-/- allograft recipients. The combination of anti-ICOS therapy and cyclosporin A led to permanent engraftment. In addition, ICOS-B7RP-1 costimulation was required for the development of chronic rejection after CD40-CD154 blockade. These data demonstrate a key role for the ICOS-B7RP-1 pathway in acute and chronic rejection and highlight the benefits of targeting this pathway in combination with the use of conventional immunosuppressive agent.

We examined the requirement for and cooperation between CD28 and inducible costimulator (ICOS) in effective T helper (TH) cell responses in vivo. We found that both CD28 and ICOS were critical in determining the outcome of an immune response; cytolytic T lymphocyte-associated antigen 4-immunoglobulin (CTLA-4-Ig), ICOS-Ig and/or a neutralizing ICOS monoclonal antibody attenuated T cell expansion, TH2 cytokine production and eosinophilic inflammation. CD28-dependent signaling was essential during priming, whereas ICOS-B7RP-1 regulated TH effector responses, and the up-regulation of chemokine receptors that determine T cell migration. Our data suggests a scenario whereby both molecules regulate the outcome of the immune response but play separate key roles: CD28 primes T cells and ICOS regulates effector responses.

VCAM-1 and ICAM-1 are endothelial adhesion molecules of the Ig gene superfamily that may participate in atherogenesis by promoting monocyte accumulation in the arterial intima. Both are expressed in regions predisposed to atherosclerosis and at the periphery of established lesions, while ICAM-1 is also expressed more broadly. To evaluate functions of VCAM-1 in chronic disease, we disrupted its fourth Ig domain, producing the murine Vcam1(D4D) allele. VCAM-1(D4D) mRNA and protein were reduced to 2-8% of wild-type allele (Vcam1(+)) levels but were sufficient to partially rescue the lethal phenotype of VCAM-1-null embryos. After crossing into the LDL receptor-null background, Vcam1(+/+) and Vcam1(D4D/D4D) paired littermates were generated from heterozygous intercrosses and fed a cholesterol-enriched diet for 8 weeks. The area of early atherosclerotic lesions in the aorta, quantified by en face oil red O staining, was reduced significantly in Vcam1(D4D/D4D) mice, although cholesterol levels, lipoprotein profiles, and numbers of circulating leukocytes were comparable to wild-type. In contrast, deficiency of ICAM-1 either alone or in combination with VCAM-1 deficiency did not alter nascent lesion formation. Therefore, although expression of both VCAM-1 and ICAM-1 is upregulated in atherosclerotic lesions, our data indicate that VCAM-1 plays a dominant role in the initiation of atherosclerosis.

Upon encounter with specific antigen, naïveT helper precursor (THP) cells become activated. This event is regulated not only by engagement of the T cell receptor (TCR) with peptide presented in the context of major histocompatibility complex (MHC) class II molecules but by a number of costimulatory signals. CD28 engagement by B7-1 and B7-2 on resting THP cells provides a critical signal for initial cell cycle progression, interleukin 2 production and clonal expansion. However, largely as a consequence of the unraveling of the human genome, it is becoming clear that B7-1 and B7-2 are part of a larger family T of related counter-receptors that play an essential role in regulating the fate of primed, rather then resting,THP cells. These molecules play an important sequential role and act, together with B7-1- and B7-2-primed T cells, in the acquisition of effector function and/or tolerance induction.

The orphan receptor T1/ST2, a member of the IL-1R family, is preferentially expressed on the surface of murine Th2 cells. In this study, we analyzed the kinetics and function of T1/ST2 expression on Th2 cells in vitro. Whereas naive CD4+ cells did not express T1/ST2, most CD4+ cells became T1/ST2+ upon repeated antigenic stimulation under Th2-polarizing conditions. Flow cytometric analyses revealed that the kinetics of T1/ST2 expression on Th2 cells was delayed compared with the kinetics of type 2 cytokine production. Exogenous IL-6, IL-5, IL-1, and TNF-α enhanced the expression of T1/ST2 on Th2 cells, and IL-6 was by far most effective in this regard. However, the expression of T1/ST2 did not depend on the presence of IL-6 and was also detected in IL-6-deficient mice. Most important, cross-linking of T1/ST2 provided a costimulatory signal for Th2 but not Th1 cells and directly induced proliferation and type 2 cytokine production. Thus, T1/ST2 is not only a Th2 cell marker but also plays an important role in the activation of Th2 cells.

The understanding of the relative contribution of particular chemokines to the selective accumulation of leukocyte subsets to an organ site during an inflammatory response is made difficult by the simultaneous presence of multiple chemokines with partially overlapping functions at the inflammatory site. The study of several chemokine pathways (expression and function) during the development of a mouse model of allergic airway disease (AAD) has revealed differential expression regulation with distinct cellular sources for individual chemokines with functional bias for the recruitment/localization of regulatory and/or effector leukocyte subsets. In the present review, we propose that distinct functional groups of chemokines co-operate to generate the complete inflammatory response in the lung during AAD. We will also extend these concepts to the specific recruitment of a key cellular subset such as T helper type 2 (Th2) lymphocytes. We propose that the long term recruitment of antigen-specific Th2 cells to target organs, such as airways during chronic lung inflammation, is the result the sequential involvement of several chemotactic axes. Specifically, the CCR3/eotaxin and the CCR4/MDC pathway act in a coordinated co-operative manner, with the CCR3/eotaxin pathway being critical in the acute/early stages of a response, followed by the CCR4/MDC pathway, which ultimately dominates in the recruitment of antigen-specific Th2 cells. Other chemokines/receptors participate in this process possibly by amplifying/priming the Th2 recruitment response.

T cells are critical mediators of inflammation and as such, their migration to inflammatory sites is a tightly controlled process involving a complex series of molecules expressed by a variety of cell types. As our appreciation of the mechanisms governing T cell surveillance, activation, differentiation, and subsequent homing to sites of inflammation has advanced, the opportunity to develop novel therapeutic agents that modulate the immune system has increased. Importantly, the possibility of specifically targetting subpopulations of effector cells raises the exciting potential for the development of novel agents that selectively modify the immune response to allergens, without resulting in generalized immune suppression.

It has been shown that certain pathogens can trigger efficient T cell responses in the absence of CD28, a key costimulatory receptor expressed on resting T cells. Inducible costimulator protein (ICOS) is an inducible costimulator structurally and functionally related to CD28. Here, we show that in the absence of CD28 both T helper cell type 1 (Th1) and Th2 responses were impaired but not abrogated after infection with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and the nematode Nippostrongylus brasiliensis. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization. Blocking of ICOS alone had a limited but significant capacity to downregulate Th subset development. In contrast, cytotoxic T lymphocyte (CTL) responses, which are regulated to a minor and major extent by CD28 after LCMV and VSV infection, respectively, remained unaffected by blocking ICOS. Together, our results demonstrate that ICOS regulates both CD28-dependent and CD28-independent CD4+ subset (Th1 and Th2) responses but not CTL responses in vivo.

Isolated peripheral blood CD4 cells from allergic individuals express CC chemokine receptor (CCR)3 and CCR4 after expansion in vitro. In addition, human T helper type 2 (Th2) cells polarized in vitro selectively express CCR3 and CCR4 at certain stages of activation/differentiation and respond preferentially to the ligands eotaxin and monocyte-derived chemokine (MDC). However, controversy arises when the in vivo significance of this distinct expression is discussed. To address the functional role of CCR3/eotaxin and CCR4/MDC during the in vivo recruitment of Th2 cells, we have transferred effector Th cells into naive mice to induce allergic airway disease. Tracking of these cells after repeated antigen challenge has established that both CCR3/eotaxin and CCR4/MDC axes contribute to the recruitment of Th2 cells to the lung, demonstrating the in vivo relevance of the expression of these receptors on Th2 cells. We have shown that involvement of the CCR3/eotaxin pathway is confined to early stages of the response in vivo, whereas repeated antigen stimulation results in the predominant use of the CCR4/MDC pathway. We propose that effector Th2 cells respond to both CCR3/eotaxin and CCR4/MDC pathways initially, but that a progressive increase in CCR4-positive cells results in the predominance of the CCR4/MDC axis in the long-term recruitment of Th2 cells in vivo.

Mesangial cells are specialized cells of the renal glomerulus that share some properties of vascular smooth muscle cells and macrophages. They are implicated in the pathogenesis of many forms of nephritis. The murine CXC-chemokines macrophage inflammatory protein-2 (MIP-2) and KC induce migration of mouse mesangial cells. Mesangial cells also exhibit a unique chemokine feedback mechanism. Treatment with nanomolar concentrations of MIP-2 or KC markedly up-regulates monocyte chemoattractant protein-1 and RANTES expression in mesangial cells. Autoinduction of MIP-2 and KC mRNA was also noted. Low levels of MIP-1α, MIP-1β, and IFN-γ-inducible protein-10 were induced following treatment with higher doses of MIP-2 or KC. These effects are specific to mesangial cells, as MIP-2 or KC treatment of renal cortical epithelial cells or peritoneal macrophages failed to induce chemokine production. This cascade of chemokine interactions may contribute to renal infiltration and leukocyte activation. The abilities of MIP-2 or KC to stimulate their own synthesis may also contribute to the maintenance and chronic course of glomerular inflammation. The mesangial cell receptor for MIP-2 and/or KC is unknown but is not CXC-chemokine receptor-2.

The cloning, expression, and function of the murine (m) homologue of human (h) monocyte-derived chemokine (MDC) is reported here. Like hMDC, mMDC is able to elicit the chemotactic migration in vitro of activated lymphocytes and monocytes. Among activated lymphocytes, Th2 cells were induced to migrate most efficiently. mMDC mRNA and protein expression is modulated during the course of an allergic reaction in the lung. Neutralization of mMDC with specific Abs in a model of lung inflammation resulted in prevention of airway hyperreactivity and significant reduction of eosinophils in the lung interstitium but not in the airway lumen. These data suggest that mMDC is essential in the transit/retention of leukocytes in the lung tissue rather than in their extravasation from the blood vessel or during their transepithelial migration into the airways. These results also highlight the relevance of factors, such as mMDC, that regulate the migration and accumulation of leukocytes within the tissue during the development of the key physiological endpoint of asthma, airway hyperreactivity.

Rodriguez, J.L., Gutierrez, C., Gonzalez, J. and Brooks, D.L. 1999. Clinicopathological and haematological study of experimentally infected goat kids with Mycoplasma capricolum subsp. capricolum. J. Appl. Anim. Res., 15: 169–174. Goat kids experimentally infected with Mycoplasma capricolum subsp. capricolum showed anorexia, high fever, swollen-painful joints, lameness, dyspnea, depression, cellulitis and septicaemia. They exhibited anemia, leukocytosis with neutrophilia at early stage and a leukopaenia at later stage of infection, increase in prothrombin time, thrombin time, activated partial thromboplastin time and fibrinogen, and decrease of antithrombin III. No pulmonary changes of caprine pleuropneumonia were seen in the kids, but they had diffuse interstitial pneumonia, congestion and oedema. Disseminated intravascular coagulation was not found.

Mortality from asthma increased and is now declining in some countries, but little is known about these trends in South America.We aimed to assess trends in mortality from asthma in southern Brazil in children and young adults.Death certificates of 425 people in the state of Rio Grande do Sul aged between 5 and 39 years in whom asthma was reported to be the underlying cause of death during the period 1970 to 1992 were reviewed. Population data were available in 10-year age groups. Testing for trends in mortality rates was conducted using linear and log-linear regression procedures.Asthma mortality rates in the age groups 5 to 19 and 20 to 39 years ranged between 0.04 and 0.39/100,000 and 0.28 to 0.75/100,000, respectively, and were nonuniformly distributed over the study period. The mean annual increase in rate in 5- to 19-year olds was +0.01 (95% CI 0.003 to 0.016), an average annual percentage increase of +6.8% (95% CI 3% to 11%), with a total increase of 352% between 1970 and 1992. This increase was not due to a shift in labeling from bronchitis to asthma. In the 20 to 39-year age group, asthma and bronchitis mortality rates showed no trend to increase or decrease.Asthma mortality in southern Brazil is low, but rose significantly between 1970 and 1992 in the 5 to 19-year age group. This trend differs from that found in other states of Brazil and several other Latin American countries. Reasons for this difference remain unclear.Levels of mortality due to asthma are declining in some countries. To measure trends in mortality from asthma in southern Brazil among children and young adults, the death certificates of 425 people in the state of Rio Grande do Sul aged 5-39 years in whom asthma was reported to be the underlying cause of death during the period 1970-92 were reviewed. Asthma mortality rates among people aged 5-19 and 20-39 years were 0.04-0.39/100,000 and 0.28-0.75/100,000, respectively, and were nonuniformly distributed over the study period. The mean annual increase in mortality rate among 5-19 year olds was 0.01, an average annual percentage increase of 6.8%, with a total increase of 352% during 1970-92. This increase was not due to a shift in labeling from bronchitis to asthma. Among people aged 20-39 years, asthma and bronchitis mortality rates showed no trend of increase or decrease. Reasons for the dramatic increase in asthma-related mortality among 5-19 year olds are unclear.

T1/ST2 is an orphan receptor of unknown function that is expressed on the surface of murine T helper cell type 2 (Th2), but not Th1 effector cells. In vitro blockade of T1/ST2 signaling with an immunoglobulin (Ig) fusion protein suppresses both differentiation to and activation of Th2, but not Th1 effector populations. In a nascent Th2-dominated response, anti-T1/ST2 monoclonal antibody (mAb) inhibited eosinophil infiltration, interleukin 5 secretion, and IgE production. To determine if these effects were mediated by a direct effect on Th2 cells, we next used a murine adoptive transfer model of Th1- and Th2-mediated lung mucosal immune responses. Administration of either T1/ST2 mAb or T1/ST2-Ig abrogated Th2 cytokine production in vivo and the induction of an eosinophilic inflammatory response, but failed to modify Th1-mediated inflammation. Taken together, our data demonstrate an important role of T1/ST2 in Th2-mediated inflammatory responses and suggest that T1/ST2 may prove to be a novel target for the selective suppression of Th2 immune responses.

Eotaxin is a potent chemoattractant for eosinophils during inflammation and allergic reactions in the adult, but its role in the embryonic development of the hematopoietic system has not been examined. We report here that eotaxin and its receptor, CCR-3, are expressed by embryonic tissues responsible for blood development, such as fetal liver (FL), yolk sac (YS), and peripheral blood. We found that eotaxin acts synergistically with stem cell factor to accelerate the differentiation of embryonic mast cell progenitors, and this response can be suppressed by pertussis toxin, an inhibitor of chemokine-induced signaling through Gi protein and chemotaxis. Eotaxin promotes the differentiation of fetal mast cell progenitors into differentiated mast cells as defined by the expression of mast cell specific proteases. Furthermore, in combination with stem cell factor (SCF), it promotes the growth of Mac-1+myeloid cells from embryonic progenitors. These studies suggest that eotaxin may be involved in the growth of granulocytic progenitors and the differentiation and/or function of mast cells during embryogenesis and/or pathological conditions that induce high levels of eotaxin, such as allergic responses.© 1998 by The American Society of Hematology.

Interleukin-5 (IL-5) is a potent eosinophilopoietic factor implicated in the chronic inflammatory cell accumulation accompanying bronchial asthma. However, its role in stimulating eosinophil differentiation within the bone marrow following allergen exposure remains to be elucidated. The aims of our study were to determine the expression of IL-5 within the bone marrow of sensitized and control mice after allergen exposure, and to investigate the cellular phenotype of IL-5-producing cells. Sensitized Balb/c mice were challenged with either ovalbumin (OVA) or sterile saline. After 6 h, the mice were exsanguinated and the bone marrow prepared for cytospins. Bone marrow-derived cells from OVA-sensitized mice exhibited an increase in IL-5 immunoreactivity and mRNA compared with those from nonsensitized control mice (p0. 05). After allergen challenge, there was a further increase in IL-5 expression (p0.05) within the bone marrow. Both sensitization and allergen challenge resulted in an increase in the number of cells expressing major basic protein (MBP) (p0.05). In nonsensitized mice, the IL-5 mRNA was expressed predominantly by CD34-positive (CD34+) progenitor cells. Following sensitization and allergen challenge, CD3-positive (CD3+) T lymphocytes were the major source of this cytokine. These results demonstrate the presence of IL-5 within the bone marrow of normal Balb/c mice. After sensitization and allergen challenge, the increase in IL-5-producing cells within the bone marrow is attributed by T lymphocytes.

During inflammatory processes, inflamed tissues signal the bone marrow (BM) to produce more mature leukocytes in ways that are not yet understood. We report here that, during the development of lung allergic inflammation, the administration of neutralizing antibodies to the chemotactic cytokine, Eotaxin, prevented the increase in the number of myeloid progenitors produced in the BM, therefore reducing the output of mature myeloid cells from BM. Conversely, the in vivo administration of Eotaxin increased the number of myeloid progenitors present in the BM. Furthermore, we found that, in vitro, Eotaxin is a colony-stimulating factor for granulocytes and macrophages. Eotaxin activity synergized with stem cell factor but not with interleukin-3 or granulocyte-macrophage colony-stimulating factor and was inhibited bypertussis toxin. We report also that CCR-3, the receptor for Eotaxin, was expressed by hematopoietic progenitors (HP). Thus, during inflammation, Eotaxin acts in a paracrine way to shift the differentiation of BM HP towards the myeloid lineage.

Multiple adhesion molecules of the selectin, integrin, and immunoglobulin-like families are involved in the migration of leukocytes out of the bloodstream into inflamed tissues. This study addresses the question of which adhesion molecules are specifically involved in endotoxin-induced uveitis.Mice genetically deficient in p-selectin, ICAM-1, beta2-integrin, or controls received intravitreal injections of endotoxin. Eyes were harvested 24 h later and inflammation was evaluated by histologic and immunohistochemical assays of infiltrating cells.Mice lacking either P-selectin or beta2-integrin had less inflammation than controls (median cells/section: 64 for P-selectin knockout vs 130 for controls, p=0.02, n=17 per group: 244 for beta2-integrin knockouts, n=14, vs 355 for controls, n=17, p=0.05). Neither gene deletion significantly changed the ratio of infiltrating neutrophils to macrophages. ICAM-1 knockouts tended to have fewer infiltrating cells (median 22 cells/section) compared to controls (median 132 cells/section), but this difference was not statistically significant (p=0.25, n=9 per group).P-selectin, beta2-integrin, and possibly ICAM-1 are involved in the ocular inflammatory response to endotoxin. The lack of complete inhibition of leukocyte infiltration with the complete loss of each adhesion molecule is in accord with the notion that alternative adhesion molecules also participate in this process.

The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1α, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.

We examined brain sections from ICAM-1 deficient mice (−/−) and their nontransgenic littermates (+/+) after focal cerebral ischemia and reperfusion (I/R) for the presence of apoptosis. Despite the reduction in necrosis, the −/− mice had apoptotic cells in the ischemic hemisphere as shown by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining and DNA laddering. ICAM-1 deficiency minimizes necrosis but not apoptosis after temporary MCAO in mice, thereby leaving the potential for delayed neuronal cell death despite ICAM-1 inactivation.

This article presents the clinical and laboratorial findings in an outbreak of abortions and high neonatal mortality attributable to Trypanosoma evansi infection in camels. A total of 16 females were diagnosed, 2 of which showed moderate signs of chronic form, particularly hyporexia and intolerance to exercise. The main laboratorial findings were regenerative anemia (hemolytic anemia), lymphocytic and monocytic leukocytosis, hyperproteinemia, hyperglobulinemia, hypoglycemia, serum urea increased, and serum iron decreased. The most characteristic finding in the examined females would be the uremia, probably due to the higher protein metabolism.

Mac-1 (alphambeta2), a leukocyte adhesion receptor, has been shown in vitro to functionally interact with Fcgamma receptors to facilitate immune complex (IC)-stimulated polymorphonuclear neutrophil (PMN) functions. To investigate the relevance of Mac-1-FcgammaR interactions in IC-mediated injury in vivo, we induced a model of Fc-dependent anti-glomerular basement membrane (GBM) nephritis in wild-type and Mac-1-deficient mice by the intravenous injection of anti-GBM antibody. The initial glomerular PMN accumulation was equivalent in Mac-1 null and wild-type mice, but thereafter increased in wild-type and decreased in mutant mice. The absence of Mac-1 interactions with obvious ligands, intercellular adhesion molecule 1 (ICAM-1), and C3 complement, is not responsible for the decrease in neutrophil accumulation in Mac-1- deficient mice since glomerular PMN accumulation in mice deficient in these ligands was comparable to those in wild-type mice. In vitro studies showed that spreading of Mac-1-null PMNs to IC-coated dishes was equivalent to that of wild-type PMNs at 5-12 min but was markedly reduced thereafter, and was associated with an inability of mutant neutrophils to redistribute filamentous actin. This suggests that in vivo, Mac-1 is not required for the initiation of Fc-mediated PMN recruitment but that Mac-1-FcgammaR interactions are required for filamentous actin reorganization leading to sustained PMN adhesion, and this represents the first demonstration of the relevance of Mac-1-FcgammaR interactions in vivo. PMN-dependent proteinuria, maximal in wild-type mice at 8 h, was absent in Mac-1 mutant mice at all time points. Complement C3-deficient mice also had significantly decreased proteinuria compared to wild-type mice. Since Mac-1 on PMNs is the principal ligand for ic3b, an absence of Mac-1 interaction with C3 probably contributed to the abrogation of proteinuria in Mac-1-null mice.

Eotaxin is a potent chemoattractant for eosinophils during inflammation and allergic reactions in the adult, but its role during development has not been studied. We report that eotaxin and its receptor, CCR-3, are expressed by embryonic tissues responsible for blood development, including the yolk sac, fetal liver, and fetal blood. We also found that eotaxin acts synergistically with stem cell factor (SCF) to accelerate the differentiation of embryonic mast cell progenitors and to promote the growth of Mac-1+/Gr-1- cells from progenitors isolated at 10-12 days of gestation. This response is diminished by Pertussistoxin, the Giα inhibitor. These studies suggest that eotaxin is involved in the growth of myeloid cell progenitors and the differentiation of mast cells during embryogenic development.

Chemokines are small secreted proteins that stimulate the directional migration of leukocytes and mediate inflammation. During screening of a murine choroid plexus complementary DNA library, we identified a new chemokine, designated neurotactin. Unlike other chemokines, neurotactin has a unique cysteine pattern, Cys-X-X-X-Cys, and is predicted to be a type 1 membrane protein. Full-length recombinant neurotactin is localized on the surface of transfected 293 cells. Recombinant neurotactin containing the chemokine domain is chemotactic for neutrophils both in vitro and in vivo. Neurotactin messenger RNA is predominantly expressed in normal murine brain and its protein expression in activated brain microglia is upregulated in mice with experimental autoimmune encephalomyelitis, as well as in mice treated with lipopolysaccharide. Distinct from all other chemokine genes, the neurotactin gene is localized to human chromosome 16q. Consequently we propose that neurotactin represents a new delta-chemokine family and that it may play a role in brain inflammation processes.

We dissected the functions of the microenvironment of bone marrow (BM) and fetal liver (FL) at the cellular level by cloning individual stromal calls and characterizing their phenotypical and functional features. Stromal cell clones derived from FL are large in size (mean forward light scatter intensity [mFSC] of 450), express the surface antigen Thy-1 but not Sca-1 and 6 out of 6 are able to differentiate into fat accumulating adipocytes. BM derived stromal cell clones are either small (mFSC of 250) or large (mFSC of 450), express Sca-1 but not Thy-1 and only 2 out of 7 differentiate towards adipocytes. Heterogeneity in terms of vascular adhesion molecule-1, intracellular adhesion molecule-1 and heat stable antigen expression was found among the different cell clones. Functional assays using long- and short-term cocultures of stromal and hematopoietic calls revealed: (1) the capacity of 8 out of 12 stromal cell clones to support the expansion of primitive hematopoietic progenitors (colony forming unit spleen day 12) more than 10 weeks. Fat accumulation but not expression of stem cell factor by stromal cells did correlate with this supportive function. (2) Better support of granulocyte maturation and proliferation by BM- compared to FL-derived stromal cell clones. However, stromal cell clones from both organs expressed macrophage-colony stimulating factor. (3) The ability of 4 out of 12 stromal cell clones (derived from both, FL and BM) to support the expansion of Interleukin-7 dependent pre-B cells from the BM. Pre-B cell growth stimulating factor was not restricted to supporters. (4) Mutual exclusiveness of myeloid and lymphoid support in that a given stromal cell clone supported either pre B-cell or granulocyte expansion. Experiments comparing the support of BM- and FL-derived hematopoietic progenitors showed identical responses of late (B220+/c-kit-) but strikingly different responses of early (B220+/c-kit+) pre-B cells, revealing different proliferation requirements for FL- versus BM- derived early pre-B cells in vitro.

Survival after lung transplantation is limited by bronchiolitis obliterans syndrome (BOS). Oxidative stress (OxS) can be associated with BOS due to chronic inflammation. The type of fat and antioxidant intakes may also contribute to OxS. Our aim was to compare OxS and nutritional intakes in non-BOS versus various stages of BOS. Fifty-eight lung recipients with versus without BOS were prospectively classified as: non-BOS; BOS Op-1 (mild), and BOS 2-3 (severe). We measured nutritional intake and plasma vitamins A, C, and E. Among a subgroup of 37 patients, OxS was assessed by measuring lipid peroxidation (LPO micromol/L MDA) and oxidized glutathione (GSSG) in bronchoalveolar lavage BAL fluid (BALF). One-way analysis of variance was used to compare groups. Results are reported as mean values +/- standard errors of the mean. There was no significant difference in demographic features on time posttransplant among groups. Although there were comparable cell counts in BALF, severe BOS patients showed significantly higher BALF LPO concentrations when compared with milder stage of BOS or with non-BOS (P = .001, for both). Severe BOS recipients also displayed higher BALF GSSG concentrations compared to milder stage of BOS (P = .001) or non-BOS (P = .007). In conclusion, patients with severe BOS were more oxidatively stressed compared with mild and non-BOS recipients.

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by ∼70% of CD4+ T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4+ T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4+ effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.

Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IkappaB kinase beta (IKKbeta) in mucus synthesis in vitro and in vivo. Tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-alpha stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein. TNF-alpha stimulation induced IKKbeta-dependent p65 nuclear translocation, mucus synthesis, and production of cytokines from epithelial cells. TNF-alpha, but not transforming growth factor-alpha, induced mucus production dependent on IKKbeta-mediated NF-kappaB activation. In vivo, TNF-alpha induced NF-kappaB as determined by whole mouse body bioluminescence. This activation was localized to the epithelium as revealed by LacZ staining in NF-kappaB-LacZ transgenic mice. TNF-alpha-induced mucus production in vivo could also be inhibited by administration into the epithelium of an IKKbeta dominant negative adenovirus. Taken together, our results demonstrated the important role of IKKbeta in TNF-alpha-mediated mucus production in airway epithelium in vitro and in vivo.

It is now well established that passive exposure to inhaled OVA leads to a state of immunological tolerance. Therefore, to elicit allergic sensitization, researchers have been compelled to devise alternative strategies, such as the systemic delivery of OVA in the context of powerful adjuvants, which are alien to the way humans are exposed and sensitized to allergens. The objectives of these studies were to investigate immune-inflammatory responses to intranasal delivery of a purified house dust mite (HDM) extract and to evaluate the role of GM-CSF in this process. HDM was delivered to BALB/c mice daily for 10 days. After the last exposure, mice were killed, bronchoalveolar lavage was performed, and samples were obtained. Expression/production of Th2-associated molecules in the lymph nodes, lung, and spleen were evaluated by real-time quantitative PCR and ELISA, respectively. Using this exposure protocol, exposure to HDM alone generated Th2 sensitization based on the expression/production of Th2 effector molecules and airway eosinophilic inflammation. Flow cytometric analysis demonstrated expansion and activation of APCs in the lung and an influx of activated Th2 effector cells. Moreover, this inflammation was accompanied by airways hyper-responsiveness and a robust memory-driven immune response. Finally, administration of anti-GM-CSF-neutralizing Abs markedly reduced immune-inflammatory responses in both lung and spleen. Thus, intranasal delivery of HDM results in Th2 sensitization and airway eosinophilic inflammation that appear to be mediated, at least in part, by endogenous GM-CSF production.

Infections with gastrointestinal helminths elicit potent Th2 responses, which ultimately result in their expulsion. However, during expulsion of Trichinella spiralis this Th2 response also induces a severe enteropathy characterized by villus atrophy and crypt hyperplasia. Inducible costimulator (ICOS), a homologue of CD28, interacts with B7-related protein 1, and has been shown to be important in T-B cell interactions and antibody class switching. Significantly, ICOS appears to be involved in the induction of both Th1 and Th2 responses, but may be of heightened importance in Th2 responses. Here we employed a blocking antibody against ICOS to investigate the contribution of ICOS costimulation to the development of the protective and pathological immune responses induced during infection with T. spiralis. We show that, although blocking ICOS resulted in a decrease in TNF-alpha and the Th2 cytokines IL-4 and IL-5 and serum levels of total IgE, it did not affect the expulsion of the adult parasites. Surprisingly, levels of IL-9, IL-13 and IL-10 were elevated and protection against the larval muscle stage of the parasite was enhanced. Importantly, these findings may relate to the fact that ICOS blockade significantly ameliorated the enteropathy that usually accompanies expulsion of the adult parasite.