Is the variability of nickel patch test reactivity over time associated with fluctuations in the systemic T-cell reactivity to nickel?

BACKGROUND: Patch test reactivity to nickel varies over time. To what extent this variation is associated with fluctuations in the T-cell reactivity to nickel is not known. OBJECTIVES: Our aim was to investigate the relationship between variation over time in the patch test and the systemic T-cell reactivity to nickel. METHODS: Patients (n = 15) with a history of contact allergy to nickel were subjected to three consecutive patch tests at 3-month intervals, utilizing NiSO4 at 10 concentrations ranging from 0.0032% to 12.5%. Prior to each patch test, blood mononuclear cells were analysed for T-cell reactivity to nickel by interleukin (IL)-4 and IL-13 enzyme-linked immunospot assay. RESULTS: Eleven patients reacted positively in all three patch tests, two patients reacted in one or two tests and two remained negative. All 13 positive patients displayed variability over time, in terms of the lowest dose of nickel to which they responded. Also the cytokine response to nickel varied over time but the patients' mean cytokine response was positively correlated with their mean patch test reactivity (r(s) = 0.70, P < 0.01 for IL-4; r(s) = 0.78, P < 0.001 for IL-13). However, although the changes over time in patch test reactivity and the cytokine responses to nickel displayed a similar pattern in many patients, there was no significant correlation between the individuals' variation over time in vivo and in vitro. CONCLUSIONS: The overall magnitude of the T-cell reactivity to nickel and the patch test reactivity are closely associated but fluctuations in the systemic T-cell reactivity cannot be singled out as the major cause of longitudinal variability in nickel patch test reactivity.

Characterization of immune responses to experimental polyvalent subunit vaccines assembled in iscoms.

Immune responses to experimental polyvalent subunit vaccines assembled in a particulate adjuvant/delivery system, iscoms, are described. The fusion protein ZZ-M5 comprises structures of staphylococcal protein A (ZZ) and the Plasmodium falciparum malaria antigen Pf155/RESA (M5). MHC congenic mice were immunized with ZZ-M5 conjugated to iscoms containing human influenza virus antigen (flu ag, M5-flu-isc) or to iscom matrix (iscom particles without flu ag, M5-isc). Comparison of antibody and T-cell responses to M5-isc and M5-flu-isc demonstrated that the flu ag in M5-flu-isc exhibits carrier-related helper functions and that the assembly of immunogens in M5-flu-isc did not result in any apparent antigenic competition. In addition, assembly of ZZ-M5 and flu ag in iscoms induced an alteration of the IgG subclass profile of the antibody response to M5. The results suggest that assembly of immunogens in iscoms may be a useful approach to the design of subunit vaccines but that both quantitative and qualitative aspects of the immunogenic properties of such constructs should be scrutinized.

Predominance of H-2d- and H-2k-restricted T-cell epitopes in the highly repetitive Plasmodium falciparum antigen Pf332.

Genetic restriction of immune responses to malaria antigens is an important issue for a better comprehension of malaria immunity as well as for development of subunit vaccines. To experimentally define the major histocompatibility complex restriction of immune responses to the highly repetitive Plasmodium falciparum high-molecular-weight antigen Pf332, H-2-congenic mice were immunized with EB200, a recombinant fragment of Pf332 consisting of degenerate repeat motifs. Strong B- and T-cell responses were elicited in H-2d and H-2k mice whereas responses in H-2b, H-2q and H-2s mice were of lower magnitude. The T-cell specificity elicited by EB200 was defined by in vitro proliferative responses to a panel of overlapping peptides spanning EB200. Dominant epitopes were identified for H-2d and H-2k mice, respectively, and an additional epitope was recognized by all five mouse strains. Selected EB200-derived peptides were further investigated for their ability to elicit T-cell help when injected as multiple antigen peptides. Defined H-2d- and H-2k-restricted T-cell epitopes generated high antibody levels in the respective mouse strains, as did several peptides lacking defined epitopes indicating the presence of additional H-2d- and H-2k-restricted, cryptic or subdominant T-cell epitopes in EB200. The biased H-2 restriction pattern of T-cell epitopes in Pf332 and, as previously reported, in structurally related repeats in the malaria antigens Pf11.1 and Pf155/RESA may be explained by a shared motif for H-2d and H-2k class II-restricted T-cell epitopes, as revealed by alignment of these sequences.

Differential distribution of isoforms of Leucophaea tachykinin-related peptides (LemTRPs) in endocrine cells and neuronal processes of the cockroach midgut.

Five isoforms of tachykinin-related peptides (TRPs), designated LemTRP-1-5, have been identified in the midgut of the cockroach Leucophaea maderae. These peptides have a conserved C-terminus hexapeptide (GFX1GX2Ramide; X1 and X2 are variable residues) and variable N-termini. Here, we address the question of whether these five isoforms are all colocalized in the two types of cells in the cockroach midgut, the endocrine cells and the neuronal processes. We also investigate whether the N-terminally extended isoforms LemTRP-2 and -3, which contain putative endoproteolytic cleavage sites, are expressed in intact form or are cleaved in the midgut cells. To this end, we used two approaches. (1) Extracts from portions of the midgut containing each of the cell types were subjected to reverse-phase high performance liquid chromatography (HPLC) and the fractions monitored in a radioimmunoassay (RIA) with an antiserum to the conserved C-terminus of insect TRPs. (2) Antisera were raised to the variable N-termini of the extended LemTRP-2 and -3 and used for immunocytochemistry. The HPLC-RIA and immunocytochemical findings indicate that LemTRP-1 and 4-5 are present in the neuronal processes and in endocrine cells of the midgut proper and of the gastric cecae. The two extended forms LemTRP-2 and -3 display a differential distribution: LemTRP-2 was found in endocrine cells of midgut and gastric cecae, but not in neuronal processes, whereas LemTRP-3 was seen in neuronal processes and endocrine cells of the midgut proper, but not in the gastric cecae. LemTRP-3 and -4 have not been identified in the brain, suggesting further cell- and tissue-specific expression of LemTRPs. The mechanisms behind the cell-specific expression of the LemTRPs are not yet understood, but the demonstration of differential distribution of the peptide isoforms provide a first indication that the isoforms may have different actions.

Synthesis of a diepitope multiple antigen peptide containing sequences from two malaria antigens using Fmoc chemistry.

Multiple antigen peptides (MAP) consist of lysine residue cores with branching peptide arms and have been demonstrated to be efficient immunogens as well as useful antigens for ELISA. Synthesis of diepitope MAPs with two different branching peptides has previously been described using combined Boc and Fmoc chemistry. Here, the synthesis of a tetrameric diepitope MAP based on Fmoc chemistry is described. A lysine core was synthesized with N alpha- and N epsilon-amino groups othogonally protected by Fmoc and a recently described protection group, Dde, respectively. On the N alpha-amino groups, a sequence from the Plasmodium falciparum antigen Pf332 was synthesized with a capped N-terminus. After removal of Dde, a sequence from the P. falciparum circumsporozoite protein was synthesized on the core. Amino acid analysis of the MAP displayed equimolar amounts of the two peptide sequences, indicating the reliability of the protection group Dde. In ELISA, antibodies specific for either of the two malarial sequences reacted with the MAP. The major advantages of this approach for synthesis of diepitope MAPs are that only a panel of Fmoc-amino acid derivatives is required and that the more complicated cleavage procedure for Boc chemistry can be avoided.

Generation of antibodies to human IL-12 and amphiregulin by immunization of Balb/c mice with diepitope multiple antigen peptides.

Six peptide sequences derived from the human proteins/oligopeptides IL-12, amphiregulin and FALL-39 were synthesized in order to raise specific antibodies in Balb/c mice. Although peptides are valuable tools for generating specific antibodies, they are often poor immunogens due to their small size and lack of relevant T-cell epitopes. To circumvent these limitations, the human peptides were co-synthesized in diepitope multiple antigen peptides (MAP) with a known H-2d-restricted T helper-cell epitope. The importance of including a T-cell epitope in the diepitope MAPs was demonstrated by the fact that only one of the human peptides was immunogenic as a monoepitope MAP, lacking the T-cell epitope. Conversely, all diepitope MAPs generated potent antibody responses to the desired human peptides as well as to the T-cell epitope. A certain degree of variability of the antibody responses to the diepitope MAPs indicated that the alterable component, i.e. the human B-cell epitope, influenced the T-cell help elicited by the T-cell epitope. Still, the relative conformity of the B-cell responses suggests that this strategy is generally applicable for a rational production of specific antibodies. Moreover, antiserum to four diepitope MAPs recognized the corresponding full-length human protein/oligopeptide as did monoclonal antibodies made against IL-12-and amphiregulin-based MAPs.

Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2.

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1-8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine.

Solid-phase gene assembly of constructs derived from the Plasmodium falciparum malaria blood-stage antigen Ag332.

A general method for solid-phase gene assembly on streptavidin-coated magnetic beads has been developed. The introduction of biotin in the 5'-end of the initiation oligonucleotide enables anchoring to the bead by means of the streptavidin-biotin interaction. The immobilization of one oligonucleotide enables controlled, stepwise annealing/ligation of successive 5'-phosphorylated oligonucleotides to rapidly build up predesigned gene constructs. In this report, we have assembled gene constructs of different lengths derived from the Plasmodium falciparum malaria blood-stage antigen Ag332. The encoded gene products were subsequently expressed in Escherichia coli using two parallel expression systems based on staphylococcal protein A and streptococcal protein G, respectively.

Analysis of a human monoclonal antibody reactive with multiple Plasmodium falciparum antigen repeat sequences using a solid phase affinity assay.

A solid-phase affinity assay was set up for the determination of the affinity of the interaction between the human monoclonal antibody (mAb) 33G2 and peptides corresponding to repeated sequences in three blood stage antigens of the malaria parasite Plasmodium falciparum. The epitope of this mAb is of interest due to the parasite blocking capacity of the mAb. Previous studies with PEPSCAN have defined the minimal epitope for the mAb as the pentapeptide VTEEI, a sequence frequently found in antigen Pf332. In the previous study, epitopes responsible for the cross-reactivity of the mAb with antigens Pf155/RESA and Pf11.1 were also identified. In the affinity assay described herein, the mAb was coated on a solid phase and binding of a labelled peptide was displaced by homologous or heterologous peptides. The affinity of peptides corresponding to Pf332 increased with increasing length, and the highest affinity was displayed by a dimer (23 amino acids) of a Pf332 repeat (K = 1.9 x 10(8) M-1). Peptide length did not influence the binding of peptides corresponding to the Pf155/RESA and Pf11.1 repeats, which had lower affinities comparable to that of the shortest Pf332 octapeptide (K = 2.2 x 10(4) M-1). Only peptides containing binding sites as defined by PEPSCAN analysis showed a measurable binding. When using peptides as inhibitors in peptide ELISA, binding correlated with the affinity of the peptides, but only the high affinity peptides were inhibitory. In contrast, a poor correlation was found when peptides were used directly for coating in ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)

B- and T-cell responses in congenic mice to repeat sequences of the malaria antigen Pf332: effects of the number of repeats.

The Plasmodium falciparum antigen Pf332 comprises degenerated 11-amino-acid repeats with regularly spaced pairs of glutamic acid. Epitopes formed by such repeats are recognized by polyclonal and monoclonal antibodies that interfere with the life cycle of the blood stages of the malaria parasite. In order to study the immunogenicity of one such Pf332 repeat sequence (SVTEEIAEEDK), fusion proteins containing ZZ (two IgG binding domains of staphylococcal protein A) and dimers, trimers or tetramers of the malarial sequence were injected into mice. To analyse possible major histocompatibility complex class II restrictions of the immune response, mice of different H-2 haplotypes were used. A significant antibody response was elicited by administration of all the three fusion proteins in mice expressing the I-Ak allele (B10.BR, B10.A(2R) and B10.A(4R)) whereas B10 and C57BL/6 (H-2b) mice were low responders. In comparison, B10.D2 (H-2d) mice were low responders to fusion proteins with 2 or 3 repeats but responded well to the protein containing 4 repeats. Lymph node cells from B10.BR (H-2k) mice, primed in vivo with ZZ-fusion proteins containing either 2 or 4 repeats, proliferated in vitro in response to repeat sequences fused to ZZ or to an unrelated fusion partner, as well as to a synthetic peptide containing less than two repeats. In contrast, a response of lymph node cells from B10.D2 (H-2d) mice was only obtained when a fusion protein containing 4 repeats was used both for in vivo priming and in vitro restimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential induction of immunoglobulin G subclasses by immunization with DNA vectors containing or lacking a signal sequence.

The route and method used to immunize mice with antigen-expressing DNA plasmids have an impact on the resulting T-helper cell response and IgG subclass distribution. Previous findings further indicate that the intracellular targeting of expressed antigens influences the differentiation of naive T-cells into either a Th1 or a Th2 type of response. In the present study, we analyzed the levels of IgG1 and IgG2a antibodies, as correlates of Th2 and Th1 responses, respectively, after intramuscular injection of mice with plasmids encoding a chimeric protein containing a Plasmodium falciparum blood stage antigen expressed in two different forms. One plasmid expresses the antigen in a secreted form as it is preceded by a signal sequence while expression from the other plasmid, lacking this sequence, results in cytoplasmic localization of the antigen. Mice immunized with the plasmid encoding secreted antigen responded with predominantly IgG1 antibodies. In contrast, sera from mice immunized with the plasmid expressing cytosolic protein displayed a mixed IgG1/IgG2a profile. In line with previous findings, our results suggest that the intracellular targeting of proteins expressed by DNA plasmids is an important factor for the differentiation of Th cells and the resulting subclass pattern of IgG responses.

Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332.

The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines.

Comparative study of DNA-based immunization vectors: effect of secretion signals on the antibody responses in mice.

The presence of a signal sequence preceding the gene encoding a target antigen in a DNA vaccine should facilitate secretion of the in vivo translated antigen. The immune responses elicited upon injection with such a vector could differ from those induced by the same vector lacking a signal sequence. In the present study, the humoral responses elicited in mice immunized with two plasmids, either containing or lacking the human tissue plasminogen activator signal sequence, were compared. Both plasmids encode the chimeric antigen ZZN4, containing a malaria antigen Pf332-derived sequence (N4) linked to a bacterial fusion partner (ZZ). In vitro transfection of COS cells with each plasmid and treatment of the transfectants with brefeldin A confirmed that secretion of ZZN4 via the endoplasmic reticulum and Golgi pathway only occurred in cells transfected with the signal peptide-encoding plasmid. Repeated intramuscular injections of mice with either of the plasmids elicited comparable antibody responses to ZZN4 with regard to kinetics, specific IgG levels and persistence. These results indicate that in vivo transfection of muscle cells by either of these two plasmids generated comparable levels of antigen available for B-cell recognition and for uptake by antigen-presenting cells, despite the differential intracellular targeting of the encoded antigen. The relevance of these findings for the design of DNA vaccine vectors is discussed.

T- and B-cell responses of malaria immune individuals to synthetic peptides corresponding to non-repeat sequences in the N-terminal region of the Plasmodium falciparum antigen Pf155/RESA.

While the C-terminal repeat region of Pf155/RESA, a Plasmodium falciparum vaccine candidate has been extensively studied for B- and T-cell reactivities, little is so far known about the non-repeat region in this respect. The present study aimed at investigating the non-repeat sequence 171-227 of Pf155/RESA for T- and B-cell epitopes. Eight overlapping peptides were synthesised and assayed for their ability to stimulate peripheral blood mononuclear cells obtained from P. falciparum-immune donors to proliferate and to induce secretion of interferon-gamma (IFN-gamma) and/or interleukin 4 (IL-4) using the ELISPOT assay. The plasmas of the corresponding donors were tested for antibody reactivity with the same peptides in ELISA. The individual cellular responses to the different peptides varied and in general they were not correlated, emphasising the importance of including several parameters for T-cell activation. The most frequent T-cell responses (proliferation, IFN-gamma and/or IL-4) were seen with two partially overlapping peptides corresponding to the sequences 171-185 and 181-195 that induced responses in 71 and 62% of the donors, respectively. Although, the frequency of responders was high, the magnitude of the responses was generally low. Two overlapping peptides corresponding to the sequence 186-206 bound antibodies from a large number of plasma samples. IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide. However, there was no absolute correlation and many donors having anti-peptide antibodies could also be induced to produce IFN-gamma. In conclusion, the non-repeat region of Pf155/RESA contains several epitopes inducing functionally distinct T-cell responses. The sequence 171-206 was found to contain both B- and T-cell epitopes recognised by almost all individuals naturally primed to malaria. Thus, this sequence should be a useful tool in future immuno-epidemiological studies and/or for inclusion into a subunit vaccine against the asexual blood stages of the P. falciparum parasite.

The lipophilic hapten parthenolide induces interferon-gamma and interleukin-13 production by peripheral blood-derived CD8+ T cells from contact allergic subjects in vitro.

BACKGROUND: Peripheral blood mononuclear cells (PBMC) from persons with contact allergy to nickel react in vitro predominantly with nickel-induced CD4+ T cell-mediated production of both T-cell type 1 and 2 cytokines. OBJECTIVES: The aim of the present study was to investigate if the contact allergen parthenolide, a sesquiterpene lactone of lipophilic character, elicits an immune response which differs from that induced by water-soluble nickel ions. PATIENTS AND METHODS: Ten allergic subjects with strong (n = 6), moderate (n = 2), or weak (n = 2) patch-test reactivity to parthenolide and five patch test-negative control subjects participated in the study. PBMC from the subjects were analysed for in vitro reactivity with parthenolide by an enzyme-linked immunospot (ELISpot) assay, measuring cytokine production at the single-cell level. RESULTS: The allergic group, but not the control group, responded to parthenolide with increased numbers of cells producing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5 (P < 0.05 for all) and IL-13 (P < 0.01). The responses manifested by T-cell type 1 (IFN-gamma and IL-2) and type 2 (IL-4, IL-5 and IL-13) cytokines were positively correlated between cytokines. Subjects with a strong or moderate, but not weak or negative, patch-test reaction displayed detectable in vitro responses. In contrast to the CD4+ T cell-mediated peripheral reactivity induced by nickel, cell depletion experiments identified the parthenolide-reactive IFN-gamma- and IL-13-producing cells as CD8+ T cells. CONCLUSIONS: The finding that the PBMC reactivity to parthenolide in humans involves a CD8+ T cell-mediated type 1 and 2 cytokine response warrants further studies on the relationship between the chemical nature of a hapten and the resulting immune response.

Nickel, cobalt, chromium, palladium and gold induce a mixed Th1- and Th2-type cytokine response in vitro in subjects with contact allergy to the respective metals.

Nickel (Ni), the main cause of contact allergy to metals, induces in vitro production of both Th1- and Th2-type cytokines in peripheral blood mononuclear cells (PBMC) from allergic subjects. Because the knowledge of the cellular immune response to other metals involved in contact allergy has been limited, we investigated the cytokine profile induced by Ni, cobalt (Co), chromium (Cr), palladium (Pd) and gold (Au) in PBMC from patients with patch test reactivity to the respective metals. PBMC from patients with patch test reactivity to Ni, Co, Cr, Au and/or Pd (n = 31) and non-allergic controls (n = 5) were stimulated in vitro with corresponding metal salts. Th1- [interleukin (IL)-2 and interferon (IFN)-gamma] and Th2- (IL-4 and IL-13) type cytokine responses were measured by enzyme-linked immunospot (ELISpot) and/or enzyme-linked immunosorbent assay (ELISA). All metals induced a mixed Th1- and Th2-type cytokine production in PBMC from individual patients with patch test reactivity to the corresponding metal, but not in control PBMC. Significantly higher responses in the patient versus controls were found for Cr (IL-2 and IL-13), Pd (IL-2 and IL-4), Au (IL-13 and IFN-gamma) (all P < 0.05) and Ni (all four cytokines; P < 0.01) but not Co. Overall, 71% (37/52) and 89% (81/91) of the positive and negative patch test reactivities to metals, respectively, were matched by the in vitro reactivity. In conclusion, our data suggest that sensitization to Co, Cr, Pd and Au results in a cellular immune response of a character similar to the mixed Th1- and Th2-type cytokine profile shown previously to be induced by Ni.

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel.

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.

Down-regulation of interferon-gamma parallels clinical response to selective leukocyte apheresis in patients with inflammatory bowel disease: a 12-month follow-up study.

BACKGROUND & AIMS: Pilot studies have indicated a therapeutic role for an apheresis device (Adacolumn) that selectively adsorbs leukocytes in patients with inflammatory bowel diseases. It may also exert immunoregulatory effects contributing to its clinical efficacy. This study aimed to correlate the clinical response to leukocyte apheresis with the expression of key cytokines in mucosal tissue, in peripheral leukocytes, and in plasma. METHODS: Ten patients (seven with Crohn's disease and three with ulcerative colitis, median age: 31 years) with mild to moderately chronic activity were recruited to an open study. Patients were refractory to or had a relapse despite conventional treatment including azathioprine. Leukocyte apheresis was performed once a week for five consecutive weeks. Clinical efficacy was assessed on week 7 and after 12 months. Colonoscopy with multiple biopsies was performed at the start of the study and after 7 weeks for semiquantitative immunohistochemical analyses of cytokines. Cytokine levels in blood and the proportion of cytokine producing CD4+ and CD8+ lymphocytes were determined. RESULTS: The apheresis procedures were well tolerated and no major adverse events were encountered. The median clinical activity score decreased from 12 to 7 on week 7 (P=0.031, n=9) and to 4 after 12 months (P=0.004, n=9). Five patients were in clinical remission at the 12th month. Tissue interferon (IFN)-gamma-positive T-cells decreased in clinical responders (P=0.027) after apheresis. In parallel, significantly lower levels of IFN-gamma-producing lymphocytes were detected in peripheral blood. IFN-gamma-positive cells in pretreatment biopsies completely disappeared or decreased in posttreatment biopsies sampled on week 7 in responders (P=0.027) and appeared to predict the maintenance of long-term remission or response after 12 months. CONCLUSIONS: Leukocyte apheresis is a novel and safe nonpharmacological adjunct therapy that may prove useful in steroid refractory or dependent patients when conventional drugs have failed. Down-regulation of IFN-gamma in mucosal biopsies and in peripheral leukocytes may be a predictive marker for sustained, long-term response.

Nickel elicits concomitant and correlated in vitro production of Th1-, Th2-type and regulatory cytokines in subjects with contact allergy to nickel.

Nickel (Ni2+) elicits production of functionally distinct cytokines in vitro, but the relation between the cytokine profile and the degree of the allergic reaction in vivo needs to be better defined in order to improve the understanding of the immunological mechanisms involved in contact allergy and to facilitate development of in vitro diagnostics. The aim of the study was to define Th1-type [interferon-gamma (IFN-gamma)], Th2-type [interleukin-4 (IL-4), IL-5 and IL-13] and regulatory (IL-10) cytokine responses to Ni2+ in peripheral blood mononuclear cells (PBMC) from subjects with varying patch test reactivity to Ni2+. The study included subjects with strong (+3), moderate (+2), weak (+1) or negative (controls) patch test reactivity to Ni2+ (n = 10 per group). All +3 and +2 subjects but only three +1 subjects had a clinical history of contact allergy to Ni(2+). Cytokine production of PBMC stimulated with Ni(2+) was determined by enzyme-linked immunospot and/or enzyme-linked immunosorbent assay. Ni2+ elicited significant production of all cytokines in PBMC from patch-test-positive subjects versus controls with a positive correlation between each cytokine and the patch test reactivity as well as with other cytokines. More subjects responded to Ni2+ above cut-off values with Th2-type cytokines as compared with IFN-gamma or IL-10; 100% of +3, 80% of +2, 50% of +1 and 0% of control subjects displayed reactivity to Ni2+ based on IL-4 and IL-13 assays. Despite the prevailing view of Ni2+ allergy as a type-1-mediated condition, the in vivo reactivity to Ni2+ correlated with a mixed Th1-type, Th2-type and regulatory cytokine response to Ni2+in vitro. The results accentuate the importance of type 2 responses in contact allergy and also demonstrate that IL-4 and IL-13 are reliable markers for Ni2+ allergy.

Mapping of B-cell determinants in the nucleocapsid protein of Puumala virus: definition of epitopes specific for acute immunoglobulin G recognition in humans.

The complete amino acid sequence of the Puumala (PUU) virus nucleocapsid protein (N), deduced from the genome of the prototype strain Sotkamo, was synthesized as decapeptides with 5-amino-acid overlaps. By use of the PEPSCAN method, 86 peptides were examined for reactivity with sera from serologically confirmed nephropathia epidemica (NE) patients and 11 PUU virus N-specific bank vole monoclonal antibodies. The human sera showed reactivity with several different regions, while only one of the monoclonal antibodies reacted with one single peptide. Sequences were selected by this PEPSCAN analysis of human antibody reactivities, and five 15-amino-acid peptides were synthesized and evaluated as antigens by an enzyme-linked immunosorbent assay (ELISA). Peptide-reactive antibodies of the immunoglobulin M (IgM) class were measured in serum samples drawn from patients with acute NE. In comparison with the results of a mu-capture IgM ELISA using native PUU virus antigen, only a few serum samples were found positive (sensitivity, 2 to 10%). Interestingly, when antibodies of the IgG class were measured, the sensitivities of the five peptide ELISAs were found to be 79, 46, 2, 100, and 40%, respectively, as compared with the sensitivity of an IgG ELISA based on native viral antigen. The IgG reactivities of sequentially drawn sera from NE patients with the two peptides giving the highest assay sensitivities were analyzed and compared with their reactivities with native viral antigen. All patients had detectable anti-peptide IgG in the acute-phase sample, which, however, had totally declined in samples drawn after 2 years. The opposite pattern was seen with native viral antigen, in which case all patients showed the highest levels of specific IgG after 2 years. The results suggest the presence of epitopes specific for the acute IgG response.