Differences In Cytokine Profiles Research Articles

Cytokine mRNA profiling of peripheral blood mononuclear cells from trypanotolerant and trypanosusceptible cattle infected withTrypanosoma congolense

To examine differences in cytokine profiles that may confer tolerance/susceptibility to bovine African trypanosomiasis, N'Dama (trypanotolerant, n = 8) and Boran (trypanosusceptible, n = 8) cattle were experimentally challenged with Trypanosoma congolense. Blood samples were collected over a 34-day period, and RNA was extracted from peripheral blood mononuclear cells. The expression levels of a panel of 14 cytokines were profiled over the time course of infection and between breeds. Messenger RNA (mRNA) transcript levels for the IL2, IL8, and IL1RN genes were significantly downregulated across the time course of infection in both breeds. There was an early increase in transcripts for genes encoding proinflammatory mediators (IFNG, IL1A, TNF, and IL12) in N'Dama by 14 days postinfection (dpi) compared with preinfection levels that was not detected in the susceptible Boran breed. By the time of peak parasitemia, a type 2 helper T cells (T(H)2)-like cytokine environment was prevalent that was particularly evident in the Boran. Increases in transcripts for the IL6 (29 and 34 dpi) and IL10 (21, 25, and 29 dpi) genes were detected that were higher in the Boran compared with N'Dama. These findings highlight the implications for using murine models to study the bovine immune response to trypanosomiasis, where in some cases cytokine expression patterns differ. Overall, these data suggest that the trypanotolerant N'Dama are more capable of responding very early in infection with proinflammatory and T(H)1 type cytokines than the trypanosusceptible Boran and may explain why N'Dama control parasitemia more efficiently than Boran during the early stages of infection.

Inconsistencies between Cytokine Profiles, Antibody Responses, and Respiratory Hyperresponsiveness following Dermal Exposure to Isocyanates

Cytokine profiling of local lymph node responses has been proposed as a simple test to identify chemicals, such as low molecular weight diisocyanates, that pose a significant risk of occupational asthma. Previously, we reported cytokine messenger RNA (mRNA) profiles for dinitrochlorobenzene (DNCB) and six isocyanates: toluene diisocyanate, diphenylmethane-4,4'-diisocyanate, dicyclohexylmethane-4,4'-diisocyanate, isophorone diisocyanate, p-tolyl(mono)isocyanate, and meta-tetramethylene xylene diisocyanate. The present study was conducted to test the hypothesis that relative differences in the cytokine profile are predictive of relative differences in total serum immunoglobulin (Ig) E and respiratory responses to methacholine (Mch) following dermal exposure to the chemicals. After a preliminary experiment to determine an exposure regimen sufficient to achieve responses to Mch following dermal diisocyanate exposure, BALB/c mice received nine dermal exposures over a period of 28 days to one of six isocyanates, DNCB, or vehicle. Mice were then challenged with increasing doses of Mch and responsiveness was assessed using whole-body plethysmography. Serum antibody responses and cytokine mRNA profiles in the draining lymph node were also assessed. In separate experiments, cytokine protein assays were performed after five dermal exposures over a 14-day period. The response pattern for interleukin (IL)-4, IL-10, and IL-13 for the different isocyanates was highly reproducible as determined by RNAse protection assay, reverse transcription-PCR, or cytokine protein levels. However, the relative differences in T-helper cytokine profiles were not predictive of relative differences in either total serum IgE or respiratory responses to Mch following dermal exposure. The data suggest that the cytokine profiling approach needs to be further developed and refined before adoption and that other approaches to hazard identification should be pursued as well. Based on the weight of evidence from all the assays performed, it appears that all six isocyanates tested have some potential to cause respiratory hypersensitivity following dermal exposure.

Effect of rapamycin on cytokine profile in kidney transplant recipients treated with triple drug therapy

The aim of this study was to explore differences in the cytokine profile among de novo kidney transplant recipients treated with either Rapamycin (Rapa) + cyclosporine (CsA) + prednisone (P) or CsA + azathioprine (Aza) + P. Patients and methods Among the 13 adult kidney transplant recipients studied, seven received Rapa + CsA + P while the remaining six received CsA + Aza + P with their living donors serving as controls ( n = 13). Spontaneous production of IL-2, IFNγ, IL-10, and TGF-β were measured by ELISA in supernatants from 24-hour cultured unstimulated peripheral blood mononuclear cell (PBMC) at time zero (the day before the transplant), and at 3 and 6 months posttransplant. Cytokines were also measured 1 month after CsA withdrawal in the Rapa + CsA + P group. Results From time zero to the end of the study, IL-2, IFNγ, and IL-10 were present at low or undetectable levels in all three groups. TGF-β tended to increase in supernatants from patients under Rapa + CsA + P at 6 months posttransplant and at 1 month after CsA withdrawal without correlation to Rapa blood levels. TGF-β remained stable throughout the study period for patients included in the CsA + Aza + P group. There was no difference in this cytokine level between these study groups at any given time. Conclusions This study showed no differences in the spontaneous cytokine profiles evaluated in patients treated with both therapeutic schemes.

Characterization of proliferative responses and cytokine mRNA profiles induced by Vespula venom in patients with severe reactions to wasp stings.

The reasons why severe allergic reactions to bee and wasp stings develop in only a small portion of exposed individuals are incompletely understood, but differences in T cell responses to venom antigens comparing allergic and non-allergic individuals are likely to be important. To identify such differences, venom-induced proliferative responses and cytokine mRNA production by blood mononuclear cells from Vespula venom-allergic patients and non-allergic individuals were compared. Mononuclear cells from most venom-allergic patients proliferated in response to alkylated Vespula venom (7275 +/- 8387 ct/min, n = 19), and the extent of proliferation was greater for patients with a history of multiple prior stings and those with high levels of venom-specific IgE. Although mononuclear cells from non-allergic subjects showed little or no proliferation in response to venom (926 +/- 711 ct/min, n = 8), production of mRNAs coding for IL-2, IL-4, IL-5, IL-10 and interferon-gamma (IFN-gamma) in response to Vespula venom by cells from non-allergic subjects was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), indicating that these individuals had been previously sensitized to venom antigens. In contrast to the Th0 cytokine mRNA profile observed for non-allergic individuals, venom-allergic patients released a more restricted profile of cytokines following stimulation with venom. Only IFN-gamma mRNA expression was detected in all individuals evaluated, whereas IL-2 mRNA was not detected during the first 48 h of stimulation, and T cells from only one of three venom-allergic individuals produced detectable IL-4 or IL-5 mRNA. The difference in cytokine profiles observed comparing venom-allergic patients and non-allergic controls could not be attributed to intrinsic differences in T cells from these individuals, because polyclonal stimulation with phorbol myristate acetate (PMA) + ionophore induced similar cytokine mRNA profiles in the two groups. These studies demonstrate clear differences in the T cell responses of venom-allergic subjects, that may contribute to the development of severe allergic reactions in these individuals.

The proportion of Th1 cells, which prevail in gut mucosa, is decreased in inflammatory bowel syndrome.

T lymphocytes and their cytokines have an important role in the regulation of immune responses in the gut and in the pathogenesis of intestinal inflammation such as in Crohn's disease. The aim of this study was to analyse the Th1/Th2 cytokine profile (IFN-gamma, IL-2, IL-4 and IL-10) in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) in Crohn's disease (CD) and ulcerative colitis (UC) in relation to healthy controls (C). Colonic and ileal biopsy specimens were obtained from controls (n = 13) and patients with CD (n = 32). Colonic biopsies were obtained from patients with UC (n = 11). Intracytoplasmic IFN-gamma, IL-2, IL-4 and IL-10 were determined by flow cytometry after PMA-ionomycin stimulation in IEL and LPL. In colonic LPL, a significant proportional decrease of IFN-gamma and IL-2 producing CD3+ cells was observed in patients with CD and UC compared to controls. In ileal LPL, a similar tendency was found although differences were not significant. In IEL no differences in cytokine profiles could be observed. Flow cytometric analysis of intracytoplasmic cytokines at single cell level showed a proportional decrease of IFN-gamma and IL-2 producing T cells in colonic lamina propria in patients with inflammatory bowel disease.

Cytokine Profile of Cervical Cancer Cells

Objective. In patients with cervical carcinoma, the presence of cytokines produced by TH2 cells, and the presence of an eosinophilic inflammatory infiltrate, has been associated with a less effective immune response and tumor progression. In the present study, we have investigated the cytokine profile of cervical carcinoma cells. In addition, we have measured whether differences in cytokine profiles between normal and malignant cervical epithelial cells are present.Methods. For this purpose we have determined the mRNA expression patterns of 20 relevant cytokines by RT-PCR and Southern blotting in 3 normal primary cervical epithelial cell cultures (NPE) and 10 cervical cancer cell lines (CCCL).Results. TGF-β1, IL-4, IL-12p35, and IL-15 were produced by all CCCL and NPE. TNF-α, IL-10, IL-5, and RANTES were present in most NPE, but not in any of the CCCL. MCP-1 was expressed in all CCCL but in only one NPE. The presence of the anti-inflammatory cytokine TGF-β1 in cervical carcinomas was confirmed by RNA in situ hybridization on tissue sections of carcinomas from which the CCCL originated.Conclusions. Our results suggest that cervical carcinoma cells produce immunomodulatory cytokines and that cytokine expression patterns change after malignant transformation. The implications of locally produced cytokines by cervical cancer cells are further discussed.

Cytokine responses to mitogen and Schistosoma haematobium antigens are different in children with distinct infection histories.

Prevalence of Schistosoma haematobium infection in children from two neighbouring villages in Zimbabwe was 77.1% and 40.3%, respectively. The age-intensity data indicated peak intensities of infection at a lower age in the high prevalence village. This study investigated whether the difference in infection histories was reflected in a difference in cytokine profiles between children resident in these two villages. Blood samples were taken to assay for cytokine secretion 1 year after treatment for schistosomiasis. They were cultured with phytohaemagglutinin (PHA), schistosome egg antigens (SEA) or cultured without stimulant and tested for the presence of interleukin (IL)-4, IL-5, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma. Blood samples from children from the low prevalence village were more likely to produce IL-4 (P < 0.0001) and produced higher levels of IFN-gamma (P < 0.02) and GM-CSF (P < 0.03) when cultured with PHA for 24 h. Residence in the high prevalence village was associated with production of IL-10 (P < 0.006) and GM-CSF (P < 0.04) in response to culture with SEA and IL-5 (P < 0.02) with PHA for 48 h. The interaction between age and village was not significant for these results; however, there was a significant interaction between age and village for IL-5 detected in blood samples cultured with PHA for 24 h (P < 0.01). These results concur with previous observations that major patterns of cytokine production can be related to immunosuppression, but also indicate an underlying pattern which reflects the importance of history of infection to the immune response.

Cytokine responses to treponema pectinovorum and treponema denticola in human gingival fibroblasts.

Human gingival fibroblasts were challenged with Treponema pectinovorum and Treponema denticola to test three specific hypotheses: (i) these treponemes induce different cytokine profiles from the fibroblasts, (ii) differences in cytokine profiles are observed after challenge with live versus killed treponemes, and (iii) differences in cytokine profiles are noted from different gingival fibroblast cell lines when challenged with these treponemes. Three normal gingival fibroblast cell cultures were challenged with T. pectinovorum and T. denticola strains, and the supernatants were analyzed for cytokine production (i.e., interleukin-1alpha [IL-1alpha], IL-1beta, IL-6, IL-8, IL-10, gamma interferon, macrophage chemotactic protein 1 [MCP-1], platelet-derived growth factor, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor). Unstimulated fibroblast cell lines produced IL-6, IL-8, and MCP-1. T. pectinovorum routinely elicited the greatest production of these cytokines from the fibroblast cell lines, increasing 10- to 50-fold over basal production. While T. denticola also induced IL-6 and IL-8 production, these levels were generally lower than those elicited by challenge with T. pectinovorum. MCP-1 levels were significantly lower after T. denticola challenge, and the kinetics suggested that this microorganism actually inhibited basal production by the fibroblasts. No basal or stimulated production of the other cytokines was observed. Significant differences were noted in the responsiveness of the various cell lines with respect to the two species of treponemes and the individual cytokines produced. Finally, dead T. pectinovorum generally induced a twofold-greater level of IL-6 and IL-8 than the live bacteria. These results supported the idea that different species of oral treponemes can elicit proinflammatory cytokine production by gingival cells and that this stimulation did not require live microorganisms. Importantly, a unique difference was noted in the ability of T. pectinovorum to induce a robust MCP-1 production, while T. denticola appeared to inhibit this activity of the fibroblasts. While the general cytokine profiles of the fibroblast cell cultures were similar, significant differences were noted in the quantity of individual cytokines produced, which could relate to individual patient variation in local inflammatory responses in the periodontium.

Modulation of cytokine profiles by the Mycoplasma superantigen Mycoplasma arthritidis mitogen parallels susceptibility to arthritis induced by M. arthritidis.

Mycoplasma arthritidis mitogen (MAM) is a potent superantigen secreted by M. arthritidis, an agent of murine arthritis. Here we compare the abilities of MAM to induce a panel of cytokines in vitro and in vivo in BALB/c and C3H/HeJ mouse strains that differ in susceptibility to mycoplasmal arthritis. Splenocytes from both mouse strains produced high levels of all cytokines by 24 h following in vitro exposure to MAM. No differences in cytokine profiles were seen irrespective of the MAM dose. However, there were striking differences in cytokine profiles present in supernatants of splenocytes that had been collected from mice after intravenous (i.v. ) injection of MAM and subsequently rechallenged with MAM in vitro. Splenocytes collected 24 and 72 h after i.v. injection of MAM and challenged in vitro with MAM showed the most marked divergence in the secreted cytokines. Type 1 cytokines were markedly elevated in C3H/HeJ cell supernatants, whereas they were depressed or remained low in BALB/c cell supernatants. In contrast, the levels of type 2 cytokines were all greatly increased in BALB/c cell cultures but were decreased or remained low in C3H/HeJ supernatants. Interleukin-12 mRNA and protein was also markedly elevated in C3H/HeJ mice, as were the levels of immunoglobulin G2a. The data indicate a major skewing in cytokine profiles to a type 1 inflammatory response in C3H/HeJ mice but to a protective type 2 response in BALB/c mice. These cytokine changes appear to be associated with the severe arthritis in C3H/HeJ mice following injection of M. arthritidis in comparison to the mild disease seen in injected BALB/c mice.

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low-dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL-4 and IL-5, and low levels of IFN-gamma and IL-2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high-dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL-4 defective (IL-4-/-) C57BL/6 mice infected with 5 metacercariae produced less IFN-gamma and more IL-5 compared to their wild-type C57BL/6 counterparts, suggesting that IL-4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN-gamma and IL-2 was completely suppressed in the high-dose infection and this was also observed in IL-4-/- mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL-4 and IL-5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.

Pathology, immunohistology, and cytokine responses in early phases of human granulocytic ehrlichiosis in a murine model.

Human granulocytic ehrlichiosis (HGE) results in fever, pancytopenia, and mild liver injury. We used a mouse model to examine immunity in the pathogenesis of HGE. HGE agent-infected C3H/HeJ mice were necropsied over 21 days. Histologic, immunohistologic, and serologic analyses, blood culture, tissue and blood polymerase chain reaction (PCR), cell counts, serum chemistries, and plasma cytokine ELISAs were performed. No clinical signs were detected. Ehrlichiae were identified in neutrophils in hematopoietic tissues maximally on day 7. Interleukin (IL)-10 levels were high throughout, whereas interferon (IFN)-gamma levels peaked on days 7 and 10 and dropped thereafter. Hepatic lymphohistiocytic aggregates with apoptoses were maximal at day 14. HGE-agent infection of mice induces pathologic changes similar to those in infected humans, despite differences in cytokine profile. The IFN-gamma peak prior to maximal pathologic change, when ehrlichiae are absent in tissues, suggests a role for host immunity in the pathogenesis of HGE.

Cytokine gene polymorphism in human disease: on-line databases.

The pathologies of many infectious, autoimmune and malignant diseases are influenced by the profiles of cytokine production in pro-inflammatory (TH1) and anti-inflammatory (TH2) T cells. Interindividual differences in cytokine profiles appear to be due, at least in part, to allelic polymorphism within regulatory regions of cytokine gene. Many studies have examined the relationship between cytokine gene polymorphism, cytokine gene expression in vitro, and the susceptibility to and clinical severity of diseases. A review of the findings of these studies is presented. An on-line version featuring appropriate updates is accessible from the World Wide Web site, http://www.pam.bris.ac.uk/services/GAI/cytokine4.htm.

Antigenic stimulation in T-cell cultures in cardiomyopathies: differences in cytokine profiles.

Immune-mediated mechanisms are involved in the pathogenesis of cardiomyopathies. In this study, we investigate which pattern of immune response (Th1 or Th2) lies behind these diseases by analysing the basic cytokines secreted from PHA-cultured T lymphocytes and determining what differences, if any, exist between dilated cardiomyopathy (DMC) and hypertrophic cardiomyopathy (HCM). Two groups of patients were studied: 10 patients with DCM and 10 patients with HCM. Age- and sex-matched healthy individuals were used as controls. PHA-cultured T lymphocytes in the presence or absence of different myocardial antigen (MA) concentrations were measured. Interleukine-2 (IL-2), Interleukine-6 (IL-6) and Interferon-gamma (IFN-gamma) levels were measured in culture supernatants by an ELISA method. At the same time, delayed-type hyperactivity (DTH) against the same antigenic preparation was measured by the leukocyte migration inhibitory index technique. Patients were subdivided into DTH-positive and DTH-negative and re-examined for IL-2 cytokine expression. IL-6 levels were found to increase both in the presence and in the absence of MA in the patient groups compared to the controls. IL-2 levels were decreased in both groups, in an antigen dose-related manner. Anergic patients showed a further reduction in IL-2 levels for both groups of patients. IFN-gamma remained unaffected in the patient groups. Almost half of the patients exhibited anergy to the DTH reaction against MA. We conclude that, upon antigenic stimulation, the initially mounted immune response (increased IL-6) is somehow blocked/switched off in patients, resulting in an immunologic tolerance/unresponsiveness to MA (IL-2 decreased, IFN-gamma unchanged). Finally, increased IL-6 could lead to a perpetuation of immunologic injury through the release of oxygen-free radicals with a cytotoxic effect on the myocardium. We hypothesize an antigen-related, defective macrophage-Th1 cell reaction, which accounts for the differences in the IL-2 profile between the DCM and HCM groups, that might cause local immune responses to lead to immunosuppression (immune tolerance effect), thus contributing to the pathogenesis of cardiomyopathies.

Blister fluid cytokines in cutaneous inflammatory bullous disorders.

Cytokines are important regulators of immune and inflammatory reactions in the skin, and may contribute to inflammatory blister induction. We examined the profiles of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) in fluid of spontaneous blisters in the immune-based inflammatory disorders bullous pemphigoid (8 patients), allergic contact dermatitis (5 patients) and toxic epidermal necrolysis (5 patients). These were compared with levels in 9 patients with burns, i.e. inflammatory blisters of non-immune aetiology, and 4 patients with blisters of physical origin. Very high levels of IL-6 were found in bullous pemphigoid and toxic epidermal necrolysis (p<0.001) compared with non-inflammatory and burn blisters. TNF-alpha levels were high in bullous pemphigoid and burns, but undetectable in non-inflammatory blisters. The pattern in bullous pemphigoid (very high IL-6, high TNF-alpha) differed substantially from toxic epidermal necrolysis (very high IL-6, low TNF-alpha), while burns and allergic contact dermatitis showed lesser elevation of both cytokines. Hence, differences in cytokine profiles were identified, although the relevance to underlying pathomechanisms is uncertain.

The enhanced inflammatory response in non-small cell lung carcinoma is not reflected in the alveolar compartment

An inflammatory response has been observed in lung cancer both locally and systemically. The aim of the present study was to investigate whether the alveolar compartment was involved in the inflammatory response in non- small cell lung carcinoma (NSCLC). Both inflammatory mediators in bronchoalveolar lavage fluid (BALF) and cytokines produced by alveolar macrophages (AM) were investigated. Twenty patients with newly detected NSCLC and nine control subjects were studied. The patients had not been treated with chemotherapy, radiotherapy or with systemic or inhaled corticosteroids. All patients and control subjects were current smokers or stopped smoking recently. BAL was performed in the affected lung as well as in the contralateral lung of NSCLC patients, and only unilaterally in control subjects. Comparable results were demonstrated for the levels of the inflammatory mediators TNF-α, Interleukin (IL)-6, IL-8, both soluble TNF receptors and the soluble adhesion molecules E-selectin and intercellular adhesion molecule (ICAM)-1 between the affected lung and the contralateral lung in the NSCLC population as well as between the NSCLC population and the control subjects. Moreover, no significant differences in cytokine profiles of AM were found between AM obtained from the affected lung and from the contralateral lung. Although BAL is a useful tool in the diagnostic procedure for NSCLC, the present findings suggest that BAL does not reflect the enhanced inflammatory state, as reported in plasma and in the interstitial compartment around the tumour cells in NSCLC.

T-helper-cell cytokines in the early evolution of murine Lyme arthritis.

Genetic susceptibility to murine Lyme arthritis has been correlated with the dominance of T-helper (Th1)- or Th2-cell-associated cytokines. To determine when commitment of the Th cell phenotype occurs, we examined the kinetics of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production by lymph node T cells of disease-susceptible C3H/HeN and disease-resistant BALB/c mice from days 2 through 30 of infection, a period encompassing the evolution of disease and early regression. BALB/c mice produced more IFN-gamma on day 2 of infection than did C3H/HeN mice, whereas IL-4 was first detected on day 14. In contrast, only IFN-gamma could be detected in C3H/HeN mice, and the levels steadily increased from day 2 to surpass those seen in BALB/c mice by day 14 of infection. Despite the difference in cytokine profiles, both BALB/c and C3H/HeN mice developed comparable arthritis assessed at 14 days of infection. Arthritis regressed by day 30 in BALB/c mice but persisted in C3H/HeN mice. These studies are the first to demonstrate that the Th2 response to Borrelia burgdorferi infection of BALB/c mice is preceded by a Th1 cytokine response. Moreover, the timing of the appearance of IL-4 suggests that its primary effect is not in preventing disease, as suggested by others, but, rather, in hastening the resolution of inflammation. The implications of these findings for the orchestration of host defense against B. burgdorferi infection are discussed.

Cytokine Profiles of In Vivo Activated Thyroid-Infiltrating T Cells Cloned in the Presence or Absence of Interleukin 4

We compared Th1 and Th2 cytokines secreted by randomly selected, intrathyroidal CD4+ T cell clones isolated from a patient with Graves' disease using IL-4 + IL-2 versus IL-2 alone. Prior to T cell isolation, PCR of cDNA from the intact thyroid tissue generated IL-4 and IL-10, but not IFN-γ, products. As controls, IL-4, IFN-γ and IL-10 cDNA was amplified from stimulated, but not unstimulated, PBMC. All 21 of the nine IL-2 clones and twelve IL-2 + IL-4 clones isolated from the thyroid tissue were CD4+. With the exception of one clone in the IL-2 group, all clones produced IL-10 on stimulation with anti-CD3 and phorbol-12-myristate 13-acetate (PMA) with similar mean values for both groups. The majority of clones in both groups also produced IFN-y and IL-4 after stimulation. However, the IL-4: IFN-γ ratios were significantly higher in clones isolated using IL-2 + IL-4 than in those isolated with IL-2 alone. Furthermore, the distribution of Thl, ThO and Th2 type clones, defined by their IL-4: IFN-γ ratios, was also significantly different between those isolated using IL-2 alone and those isolated using IL-2 + IL-4. Of note, although Th0 clones predominated in both groups, Th1 clones were only obtained using IL-2 alone and Th2 clones were only obtained with IL-2 + IL-4.In conclusion, the presence of IL-4 together with IL-2 induces a shift away from a Th1-towards a Th2-response in T cells cloned from in vivo-activated thyroid-infiltrating lymphocytes. This difference in cytokine profile emphasizes that IL-4 is required for cloning T cells representative of a response involving both Th1 and Th2 cells, as occurs in autoimmune thyroid disease.

Immunogenicity of JHM Virus Proteins: Characterization of a CD4+T Cell Epitope on Nucleocapsid Protein Which Induces Different T-helper Cell Subsets

The CD4+T-cell-restricted recognition of the structural proteins in the JHM strain of MHV (JHMV) was compared between two mouse strains. Following immunization with inactivated JHMV, all proteins elicited an immune response in both C57BL/6 and BALB/c mouse strains, except the nucleocapsid (N) protein, which was not immunogenic in C57BL/6 mice. T-cell lines and clones were derived from BALB/c mice immunized with UV-inactivated JHMV, to determine the epitope(s) for CD4+T cells. The N protein appears immunodominant, since all T-cell lines and clones derived therefrom recognized this protein. To locate epitope-containing domains within the N protein, truncated N-protein fragments expressed in vaccinia constructs were used to stimulate the T-cell clones. Five independent T-cell clones recognized three separate N-protein domains: 1–133, 134–249, and 250–306. Since three of five clones recognized the last domain, its sequence was studied in more detail by constructing overlapping synthetic peptides covering this region. A single epitope was localized within the peptide comprising the N-protein residues 266–279. Its restriction element was identified as I-Edusing mAb to I-Edand I-Ad. In addition, peptide N266–279 contains the motif for binding to I-Ed. This peptide elicited proliferative responses following immunization with JHMV, confirming its recognition in the complete virus. In addition, peptide N266–279 was recognized by T-cell clones that express differences in cytokine profile as well as in TCR Vβ usage.

The pathogenesis of tuberculosis.

Tuberculosis patients relapse if treatment is not continued for 6 months, because chemotherapy fails to convert the patients' response from the necrotizing pattern characteristic of disease (Koch phenomenon) to the nonnecrotizing bactericidal function required for optimal immunity. We need to understand the nature of these two immunological states and how to convert one to the other. Studies in mice and humans implicate differences in cytokine profiles and in metabolism of adrenal steroids. Either enhanced susceptibility or protection can be evoked in mice with appropriate doses of a killed environmental saprophyte. This emphasizes the importance of shared epitopes and may explain the geographically variable efficacy of Mycobacterium bovis Bacillus Calmette Guérin vaccination. Unlike soluble antigens of M. tuberculosis itself, which tend to evoke necrosis, the shared mycobacterial epitopes evoke little skin-test reactivity in patients. Preparations of these epitopes show potential as immunotherapeutic agents to convert the response from necrotic to bactericidal mode.

Patterns of disease activity in multiple sclerosis patients: a study with quantitative gadolinium-enhanced brain MRI and cytokine measurement in different clinical subgroups.

In this study we assessed the subclinical disease activity in 45 patients with primary progressive, secondary progressive or relapsing-remitting multiple sclerosis (MS). The patients had gadolinium-enhanced brain MRI scans, which were analysed using a semiquantitative method both for lesion load and for degree of enhancement. At the same time cerebrospinal fluid (CSF) and serum samples were collected and, from these, cytokine levels were measured in most cases by enzyme-linked immunoassay using commercially available kits. Enhancing lesions on MRI were found in 73% of the patients. The sensitivity of this test was greatly increased by our method of analysis as far as the primary progressive patients are concerned (70% vs 40% for conventional evaluation). CSF interleukin-1 beta (IL-1 beta) levels were above the normal range in 22% and IL-6 levels in 13% of patients, while tumour necrosis factor alpha (TNF-alpha) was undetectable or below the upper normal limits in all the samples tested. Serum IL-1 beta was above the normal limits in 40%, IL-6 in 42% and TNF-alpha in 7% of patients. No significant differences in cytokine profiles were found between the clinical subgroups. This study confirms the high sensitivity of gadolinium-enhanced MRI in detecting MS activity, which was further increased by our method of analysis. Longitudinal studies performed with more sensitive immunological techniques are needed to define better the relationship between cytokine, clinical and MRI data in MS patients.