The effect of porcine luteinizing hormone in the synchronization of ovulation and corpus luteum development in nonlactating cows.

The objective of this study was to determine the effects of different doses of porcine luteinizing hormone (pLH) versus 100 microg gonadotropin-releasing hormone (GnRH) on ovulatory response (during diestrus and proestrus) and corpus luteum (CL) development in nonlactating cows. In Experiment 1, 75 cows received an intravaginal insert containing 1.9 g progesterone (P4) for 10 d to synchronize estrus (Day 0), with prostaglandin F(2 alpha) (PGF) at insert removal. On Day 5, all follicles >or=8mm were ablated, and on Day 12, cows received 8, 12.5, or 25mg pLH or 100 microg GnRH. Mean (+/-SEM) plasma P4 concentrations on Day 12 did not differ among treatments (5.6+/-0.2 ng/mL). Mean plasma LH concentration was greatest (P<0.01) in cows given 25mg pLH (4.3+/-0.4 ng/mL). The ovulatory response to 25mg pLH (84%) or 100 microg GnRH (72%) was greater (P<0.05) than that to 8 mg pLH (32%), but not different from that of 12.5mg pLH (58%). In Experiment 2, 68 cows were given two injections of PGF 10d apart to synchronize estrus (Day 0). On Day 7, cows received PGF, and, 36 h later, pLH or GnRH (as in Experiment 1). The interval from treatment to ovulation was most variable in cows given 8 mg pLH; only 65% of these cows ovulated during the initial 27 h versus 88% of cows given 25mg pLH (P<0.05). Cows given 25mg pLH or 100 microg GnRH had larger CL area and greater plasma P4 concentrations (P<0.05) than that of those given 8 mg pLH. In summary, diestrous cows given 25mg pLH had the greatest plasma luteinizing hormone concentrations, but ovulatory response did not differ from that of those given 100 microg GnRH. Proestrous cows given 25mg pLH or 100 microg GnRH had greater CL area and P4 concentrations than that of those given 8 mg pLH.

Interaction of cigarette smoke and house dust mite allergens on inflammatory mediator release from primary cultures of human bronchial epithelial cells.

Several studies have shown that exposure to cigarette smoke and/or house dust mite (HDM) can lead to increased airway inflammation in susceptible individuals. The underlying mechanisms, however, are not defined. To investigate the interaction between cigarette smoke and HDM allergen on mediator release from primary cultures of human bronchial epithelial cells. Confluent human bronchial epithelial cell cultures were exposed to cigarette smoke in the absence or presence of HDM allergen and investigated for the release of IL-8, IL-1beta, and sICAM-1. Damage to the epithelial cells themselves was assessed by release of 51Cr. On separate occasions, we investigated the effect of PTL11028, a highly potent and selective Der p1 inhibitor, on HDM allergen-induced release of IL-8, following activation of HDM allergen by incubation with cysteine. The effect of cigarette smoke exposure on the stability of these released mediators in prepared solutions in the absence/presence of reduced glutathione was also studied. Both HDM allergens and short-term (20 min) cigarette smoke exposure led to a significantly increased release of IL-8, IL-1beta and sICAM-1 from the epithelial cell cultures. Longer exposure (1-6 h) to cigarette smoke led to a dramatic decrease in the amount of these mediators detected in the culture medium. Whilst incubation of epithelial cultures with HDM allergen did not cause any significant change in the release of 51Cr from pre-loaded cells, cigarette smoke on its own led to a marked, exposure and incubation-time dependent increase in the release of 51Cr. Incubation with HDM allergen led to a significant, dose and time-dependent increase in the release of IL-8, which was further enhanced when the allergen extract was pre-activated with cysteine. This effect was completely abrogated by PTL11028, a novel Der p1 inhibitor. Prepared solutions of various concentrations of IL-8, IL-1beta and sICAM-1 exposed to cigarette smoke demonstrated a dramatic exposure time-dependent decrease in the detectable amount of these mediators, an effect which was abrogated by GSH. HDM-induced airway inflammation may include Der p-mediated release of inflammatory mediators from epithelial cells. Additionally, short-term cigarette smoke exposure may induce airway inflammation by release of inflammatory mediators from these cells, an effect which may be potentiated by Der p allergens. Longer term cigarette smoke exposure may cause damage to epithelial cells and changes in the structure of inflammatory mediators.

Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. OBJECTIVE: To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. METHODS: The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. RESULTS: PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. CONCLUSION: PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.

The route of administration of an immunodominant peptide derived from heat-shock protein 65 dramatically affects disease outcome in pristane-induced arthritis.

Previous studies have shown that immunization of mice with an immunodominant epitope from heat-shock protein 65 (hsp 65) (amino acids 261-271) can protect from the development of pristane-induced arthritis (PIA) and this protection is mediated by an antigen-specific T helper type 2 (Th2) cytokine response. Here we confirm these findings and show that frequent intranasal administration of this peptide exacerbates disease. In naive mice given peptide intranasally an antigen-specific T-cell population is systemically activated similar to that induced by peptide immunization in incomplete Freund's adjuvant. Thus, a paradox exists whereby apparently similar peptide-specific populations are either associated with protection from, or exacerbation of, PIA. However, comparison of cytokine profiles reveals differences between these two cell populations. Peptide inhalation induces the production of Th1-type cytokines (interferon-gamma) whereas intraperitoneal immunization leads to the production of Th2-type cytokines (interleukin-4, interleukin-5 and interleukin-10) by splenic T cells upon stimulation with peptide. Thus, for the application of nasal 'tolerance' in clinical medicine, it is important to identify antigens and dosing regimes that counteract but do not activate adverse immune responses.

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60.

The hypothesis that T-cell responses to the 60 000 MW family of heat-shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon-gamma and those who did not, although patients whose hsp 60-stimulated T cells produced interleukin-4 (IL-4) and/or IL-10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL-10 production by hsp 60-stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60-stimulated cells produce T-helper 2 type cytokines.

Therapeutic potential of TCR antagonists is determined by their ability to modulate a diverse repertoire of autoreactive T cells.

The use of altered peptide ligands (APL) with TCR antagonist properties holds promise as an antigen-specific therapy for autoimmune disorders. We are investigating the therapeutic potential of APL in experimental autoimmune encephalomyelitis (EAE) using the Ac1-9 peptide of myelin basic protein in H-2u mice. Encephalitogenic T cells recognize Ac1-9 using residues 3Gln and 6Pro as the major TCR contact sites. Use of position 6 APL is compromised by the heterogeneous nature of the Ac1-9-specific repertoire. Here we identify two position 3 APL that act as TCR antagonists on transgenic T cells expressing Ac1-9-specific TCR and that inhibit EAE in H-2u mice. However, the therapeutic capacity of these two APL correlated directly with the ability to maximally inhibit activation of a heterogeneous T cell pool. The implications of these findings for the requirements for EAE induction, the relative contribution of a given T cell subpopulation to pathology and the mechanism underlying EAE inhibition in this model are discussed.

Cigarette smoke potentiates house dust mite allergen-induced increase in the permeability of human bronchial epithelial cells in vitro.

Although studies have suggested that exposure to cigarette smoke (CS) may be associated with the development of atopy, the mechanisms underlying this are not clearly understood. It has been suggested that CS impairs the barrier function of the airway epithelium, leading to increased access of allergens such as those of the house dust mite (HDM) Dermatophagoides pteronyssinus (Der p) to antigen-presenting cells, with subsequent allergic sensitization. In order to test this hypothesis, we established primary explant cultures of human bronchial epithelial cells (HBEC) in cell culture inserts, and exposed these for 20 min, 1 h, 3 h, and 6 h to CS or air in the absence or presence of 300 ng/ml Der p, and then further incubated the cultures over a period of 24 h. The HBEC cultures were assessed for changes in permeability as measured by changes in: (1) electrical resistance (ER); and (2) passage of 14C-labeled bovine serum albumin (14C-BSA) and Der p allergens across the HBEC cultures. We also assessed the effects of protease inhibitors and the antioxidant glutathione (GSH) in this experimental system. Damage to HBEC cultures was assessed by the release of [51Cr]sodium chromate from prelabeled cells, and by release of lactate dehydrogenase (LDH). Twenty minutes of exposure to CS as compared with exposure to air did not significantly alter either the ER or passage of 14C-BSA across the HBEC cultures. In contrast, incubation with Der p led to a significant increase in the permeability of HBEC cultures, an effect that was enhanced by exposure to CS but was abrogated by the specific protease inhibitors and GSH. Passage of Der p was also increased by exposure to CS. Exposure of HBEC cultures to CS led to a significant release of 51Cr and LDH from these cells as compared with cells exposed to air. This effect was augmented further when HBEC cultures were incubated with Der p. Exposure of HBEC cultures for 1 h, 3 h, and 6 h to CS led to a markedly significant dose- and time-dependent increase in the permeability of these cells. These results suggest that exposure to CS significantly enhances Der p-induced decreases in electrical resistance and the increased passage across HBEC cultures of 14C-BSA and of the Der p allergen itself.

Human MAFA has alternatively spliced variants.

Human mast cell function-associated antigen (MAFA) cDNA has been cloned. This molecule is similar to the rat form having an intracellular domain containing a putative immunoreceptor tyrosine inhibition motif and an extracellular C type lectin-like domain. However, in contrast to rat MAFA, the amino acid sequence suggests the presence of two additional extracellular N-linked glycosylation sites. In addition, alternative mRNA transcripts are observed that differ substantially from those found in the rat.

Fine specificity of the myelin-reactive T cell repertoire: implications for TCR antagonism in autoimmunity.

The use of altered peptide ligands (APL) to modulate T cell responses has been suggested as a means of treating T cell-mediated autoimmune disorders. We have assessed the therapeutic potential of TCR antagonist peptides in autoimmunity using murine experimental autoimmune encephalomyelitis (EAE) as a model. The Tg4 transgenic mouse expresses an MHC class II-restricted TCR specific for the immunodominant encephalitogenic epitope of myelin basic protein, Ac1-9 (acetylated N-terminal nonamer). We have used T cell lines derived from Tg4 mice to define the TCR contact residues within Ac1-9. APL with appropriate substitutions at the primary TCR contact residue were effective antagonists of Tg4 T cells. These antagonist APL, however, were found to induce EAE in susceptible, nontransgenic strains of mice. Underlying this, the Ac1-9-specific T cell repertoire of normal mice, rather than reflecting the Tg4 phenotype, showed considerable diversity in fine specificity and was able to respond to the Tg4 antagonist APL. Defining antagonist APL in vitro using T cell clones, therefore, was not a reliable approach for the identification of APL with EAE-suppressing potential in vivo. Our findings highlight the complexities of the autoreactive T cell repertoire and have major implications for the use of APL in autoimmune diseases.

IL-12: a key cytokine in immune regulation.

It is now apparent that interleukin 12 (IL-12) is of central significance in the regulation of immune responses. At a recent meeting, immunologists from preclinical and clinical backgrounds gathered to discuss basic concepts in IL-12 biology, and the potential of IL-12 as a target for immunointervention.

High affinity for class II molecules as a necessary but not sufficient characteristic of encephalitogenic determinants.

A direct binding assay specific for IAs molecules has been developed and its immunological relevance validated by examining, for a panel of nine different synthetic peptides, the correlation between their capacity to bind purified IAs and to inhibit IAs-restricted antigen presentation. The IAs assay thus developed has then been used to study the IAs binding affinity of a set of overlapping peptides spanning the entire myelin basic protein (MBP). It was found that the encephalitogenic MBP region corresponds to peptides with high MHC binding affinities. Other regions of the MBP that have not been described as being pathogenic in the context of IAs molecules have also been found to be high IAs binders, suggesting that variables other than MHC affinity are also involved in determining the pathogenic potential of self-derived determinants.

MHC interaction and T cell recognition of carbohydrates and glycopeptides.

The T cell independence of complex polysaccharide Ag has suggested the possibility that carbohydrates may be incapable of T cell recognition because of a failure to interact with MHC restriction elements and/or a failure of MHC/carbohydrate complexes to interact with and be recognized by Ag-specific TCR. We have used two approaches to obtain information about T cell recognition of carbohydrate. First, we have determined the capacity of a series of oligosaccharides and glycolipids to bind a murine class II MHC molecule, IAd. No significant binding was observed with the 26 compounds tested, but the limitation to these studies was that there was a relatively limited collection of synthetic carbohydrate and glycolipid structures of limited complexity available for analysis. The second approach involved the study of the effect of glycosylation of a known peptide T cell epitope (OVA 323-339) on MHC binding of the peptide and on T cell recognition. Three patterns of effects were observed: 1) no effect on either binding or T cell recognition. This pattern was observed when the carbohydrate was located at residues removed from the core MHC-binding region. When the carbohydrate was located within the core MHC-binding regions, either 2) glycosylation destroyed both MHC binding and T cell recognition; or 3) glycosylation did not ablate MHC binding or T cell recognition. In this latter instance, there was evidence to indicate that the carbohydrate moiety was an important part of the antigenic determinant recognized by T cells.

MHC-antigen-T cell interactions: an overview.

We describe the establishment and validation, both at the biochemical and biological levels, of direct assays to measure the interactions between synthetic peptides and purified HLA class II molecules. The results of several independent approaches to define the structural requirements for the interaction of peptide-class II molecules are also described. Such approaches include random screening, sequence analysis, and site-directed mutagenesis. Finally, in vivo data illustrating the possibility that the competition at the level of the interactions between autoantigenic peptides and class II molecules could be a useful tool in the treatment of T cell-mediated autoimmune diseases are presented.

Inhibition of experimental autoimmune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for IAs molecules.

Blocking of the Ag presenting function of MHC by peptides capable of high affinity binding to this molecule has been proposed as a potential immunotherapeutic intervention in MHC-associated diseases. Recent studies have used this strategy to prevent the induction of experimental allergic encephalomyelitis (EAE) in mice. However, because of the close structural relationship between the inhibitor and encephalitogenic peptides, the results of these previous studies have been difficult to interpret with regard to whether MHC blockade was the mechanism by which the inhibitory peptides functioned. In our study, we have determined the capacity of unrelated peptides capable of binding with high affinity to IAs in inhibiting the induction of EAE in SJL/J mice after immunization with the autoantigenic peptide PLP 139-151. Prevention of the disease was accomplished by two methods: 1) when inhibitor was administered together with the encephalitogenic peptide at the time of immunization, as in previous studies, and 2) when inhibitor was administered at a separate site from the autoantigen 1 day before the immunization with that Ag. Inhibition was due to binding of the inhibitor to IAs, as evidenced by the fact that a control peptide incapable of binding to this MHC had no effect on the course of the disease. The finding that inhibitor could also be efficacious when administered at a separate site has implications for potential use of such a strategy to reverse ongoing autoimmune diseases. The inhibitor had to be present during the time of Ag stimulation, and had no long term inhibitory effects, in that a secondary immune response to the encephalitogenic peptide was not inhibited in animals given the inhibitory peptide before the induction of a primary response. This is compatible with the conclusion that MHC blockade was, in fact, the mechanism of the inhibition, rather than as a result of any long term suppressive effects on immunoreactive T cells. Finally, not only did administration of the inhibitory peptide lead to a prevention of the induction of EAE, but it could also be shown to decrease the T cell proliferative response in vitro to the autoantigen.

A novel approach to the generation of high affinity class II-binding peptides.

We have found that if core regions crucial for class II binding are incorporated in multiple copies in the same peptide molecule ("reiterative motifs"), marked enhancement of the binding capacity occurs. Isotype specificity (IAd vs IEd binding capacities) is retained in all three antigenic determinants so far analyzed (lambda rep 12-26, OVA 323-339, and hen egg lysozyme 105-120). The mechanism involved in such an effect is not clear, but experiments involving introduction of a peptide spacer between two repeated core regions do not support the notion that the effect is mediated by cross-linking of more than one MHC molecule, favoring the possibility that conformational effects or distinct subsites of interaction on the MHC molecule may be involved. Based on reiterative structures, a peptide molecule composed of only two different amino acids (Ala and His) has been produced that still retains a very high binding affinity. An 125I-radiolabeled form of this peptide has been used to demonstrate that the high binding detected is mediated by the same binding site involved in the interaction of IAd and OVA 323-339. Inhibition of Ag presentation studies further supports the immunologic relevance of the phenomena observed. Finally, we observed naturally occurring clustered binding sites in proximity of immunodominant protein regions, raising the possibility that the phenomenon might have a physiologic counterpart.

The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo.

The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.

Inhibition of antigen presentation in vitro and in vivo by MHC antagonist peptides.

A series of analogue peptides have been generated, using as a template the core region of the OVA 323-339 peptide identified as critical in determining binding to I-Ad. Several of these "core extended" peptides had increased affinities for the I-Ad molecule compared to the native sequence, and were able to inhibit activation of an I-Ad-restricted T cell hybridoma in vitro. The induction of a T cell proliferative response to a peptide antigen could be inhibited by co-administration of core-extended peptide with antigen in the same adjuvant emulsion. Furthermore, inhibition also occurred when the inhibitor molecule was delivered separately one day before immunization. Finally, the induction of the autoimmune disease, experimental allergic encephalomyelitis (EAE), in susceptible mice could be reduced by the administration of a core-extended peptide with high affinity for the appropriate class II molecule. These findings have implications for the use of MHC antagonists in the control and treatment of MHC-associated autoimmune conditions in humans.