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The tissue inhibitors of metalloproteinases (TIMPs) represent a family of naturally occurring protein inhibitors of stromelysin and other members of the family of matrix metalloproteinases. A series of peptides based on the N-terminal sequence of natural TIMP-1 was synthesized and assessed for inhibitory activity against purified human stromelysin. Inhibitor peptides were identified in the loop (bounded by the disulfide bonds [C3-C99] and [C13-C124]), e.g., [C3(Acm)-C13], (IC50, 42 microM). It was established that inhibition was due to the free sulfhydryl group of either C13 or C124. However, peptides within [C70(Acm)-C98(Acm)] inhibited stromelysin independently of zinc co-ordination by cysteine. The binding epitope in TIMP-1 may be discontinuous and comprised of sequences from at least 2 loops.

Stromelysin is secreted as an inactive zymogen that is activated in the extracellular space by cleavage of the His81-Phe82 bond with the release of the 81-amino acid propeptide domain. This segment contains a 12-amino acid sequence (MRKPRC75GVPDVG) that is highly conserved in all matrix metalloproteinases. Previous studies have shown that the hexapeptide, Ac-RCGVPD-NH2, and the pentapeptide, Ac-RCGVP-NH2, based on this region retain significant inhibitory activity. This new structure-activity relationship study of both peptides has shown that only Cys75 and Val77 are essential for inhibitory activity. Peptides based on this series inhibited stromelysin and collagenase with equal potency. Additional peptides spanning this region were synthesized in order to focus on these two sites. Significantly, isocysteine was substituted for Cys75 without significant loss of inhibitory activity. Tyr-(2,6-dichlorobenzyl) was substituted for Val77. The introduction of these 2 new residues into Ac-CGVP-NH2 produced a very potent inhibitor, Ac-isoCGY-(2,6 dichlorobenzyl)-P-NH2 with an IC50 of 3 microM. The following factors, acting in combination, determine the inhibitory activity of peptides in this series: distance between the sulfur atom and the peptide backbone, coordination geometry of the thiol side chain with the active-site zinc, and conformational flexibility of the side-chain.

The Asp-Pro sequence of the cyclic peptide Ac-HN-Tyr-Cys*-Asp-Pro-Cys*-OH (1) could be replaced with the achiral dipeptide mimetic 1-(2-aminoethyl)cyclpentylcarboxylic acid with retention of potent inhibition of the VCAM-VLA-4 interaction.

Proliferative responses to the T-cell mitogen, Con A, were markedly suppressed in spleen cells isolated from rats 12-16 days following induction of adjuvant-induced arthritis (AA). These responses were only partially restored following removal of plastic-adherent cells (AC-depleted). Prophylactic treatment of AA rats with a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid, Ro 23-6457, increased mitogen-induced proliferative responses in spleen cells, particularly in AC-depleted cultures. Treatment of AA rats with Ro 23-6457 significantly increased Con A-induced IL-2 production by both unseparated and AC-depleted spleen cells. Although exogenous IL-2 did not restore proliferative responses to Con A-stimulated spleen cells from vehicle-treated AA rats, responses in AC-depleted cells from Ro 23-6457-treated AA rats were further enhanced by the addition of IL-2. Following stimulation with LPS, supernatants from cultures of adherent spleen cells isolated from AA rats contained more IL-1 (expressed as units/ml) than cultures from normal rats. Treatment of AA rats with a high dose of Ro 23-6457 normalised IL-1 levels in these cultures. Treatment of normal rats with Ro 23-6457 had no significant effects on any parameter tested. These data suggest that Ro 23-6457's modulation of certain disease-associated alterations in immune function in AA rats may contribute to its anti-inflammatory activity.

Stromelysin is secreted as an inactive zymogen that is activated in the extracellular space by cleavage of the His81-Phe82 bond with the release of the 81-amino acid propeptide domain. This segment contains a 12-amino acid sequence (MRKPRC75GVPDVG) that is highly conserved in all matrix metalloproteinases. Previous studies have shown that the hexapeptide, Ac-RCGVPD-NH2, and the pentapeptide, Ac-RCGVP-NH2, based on this region retain significant inhibitory activity. This new structure-activity relationship study of both peptides has shown that only Cys75 and Val77 are essential for inhibitory activity. Peptides based on this series inhibited stromelysin and collagenase with equal potency. Additional peptides spanning this region were synthesized in order to focus on these two sites. Significantly, isocysteine was substituted for Cys75 without significant loss of inhibitory activity. Tyr-(2,6-dichlorobenzyl) was substituted for Val77. The introduction of these 2 new residues into Ac-CGVP-NH2 produced a very potent inhibitor, Ac-isoCGY-(2,6 dichlorobenzyl)-P-NH2 with an IC50 of 3 microM. The following factors, acting in combination, determine the inhibitory activity of peptides in this series: distance between the sulfur atom and the peptide backbone, coordination geometry of the thiol side chain with the active-site zinc, and conformational flexibility of the side-chain.

Accurate kinetic characterization of stromelysin (MMP-3) inhibitors is critical in the design of potent inhibitors of this enzyme. We have successfully modified a previously described assay [1] which used an internally quenched peptide substrate (Dnp-PYAYWMR) that, upon cleavage by MMP-3, produces the products, Dnp-PYA (quiet) and YWMR (a fluorophore at 360 nm). This improved assay uses purified human MMP-3 in the presence of either 5% methanol or 5% DMSO. Fluorescence intensities associated with total hydrolysis of substrate by enzyme have been successfully mimicked using a combination of the product peptides as a standard. We have determined a Km of 39.2 microM and Kcat/Km of 4.6 microM/h for MMP-3 (in 5% MeOH) using this peptide substrate. This assay was used successfully to characterize Ro 31-4724 ((N-[(N-[2-[(N-hydroxycarbamoyl)methyl]-4-methyl-valeryl]-L-leucyl ] - L-alanine ethyl ester) as a reversible, tightly binding, inhibitor with a Ki of 26 nm.

Prostromelysin, a member of the family of matrix metalloproteinases, is secreted as a zymogen which is activated after cleavage of the His81-Phe82 bond. The 82 amino acid propeptide that is removed during activation contains 12 amino acids, MRKPRC75GVPDVG, that are highly conserved in all MMPs. We evaluated a series of peptides that span this region for their ability to inhibit stromelysin. The hexapeptide, Ac-RCGVPD, and the pentapeptide, Ac-RCGVP had IC50 values of approx. 10 microM. The tetrapeptide, Ac-RCGV, was somewhat less potent with an IC50 of 60 microM. Smaller peptides, e.g. Ac-RCG, were significantly less potent as inhibitors. Substitutions of Cys75 with Ser resulted in a complete loss of inhibitory activity. The peptides in this series also inhibited human fibroblast collagenase.

Thirty-six percent of Old English rabbits fed pasteurized cow’s milk developed early rheumatoid-like synovial lesions. All rabbits drinking milk developed high titres of serum and synovial fluid C1q-binding activity. This has been shown to be due to specific antibody to C1q, produced in response to Clq in cow’s milk. In addition, these rabbits responded to other bovine proteins present in cow’s milk, including conglutinin, β-lactoglobulin and IgG. There was no correlation between serum or synovial fluid titres of antibody to bovine milk proteins and synovial lesions.

The arthritogenic effect of dietary cow’s milk, egg albumin and soya milk were compared in Old English rabbits. The 12-week cow’s milk feeding regimen produced the highest incidence of significant joint lesions. Lesions were evident but mild at 5 weeks and the synovitis had resolved by 32 weeks. It is suggested that the transient nature of the synovitis may be due to the development of specific secretory IgA antibodies which were detectable in faecal pellet extracts. Sandy Lop rabbits were less susceptible to the arthritogenic effect than were Old English rabbits. Dietary ovalbumin was less arthritogenic than cow’s milk despite high titres of serum and synovial fluid antibodies and immune complexes. The rabbits were ‘tolerant’ to dietary soya due probably to pre-existing levels of soya protein in their diet. Lewis and Wistar strain rats, CBA, Balb/c and C57/BL6 mice fed on cow’s milk for 3 months did not develop serum antibodies or synovial lesions. It is suggested that this allergic synovitis is not a model for early rheumatoid joint disease because of the transience of the lesions and lack of stimulation of rheumatoid factor. It may well, however, be a model for the arthralgia seen in patients with certain food allergies.

We have employed a cultured crypt-like intestinal epithelial rat cell line (IEC-18) to assess whether killing by nonspecific cytotoxic cells contributes to epithelial cell destruction in Nippostrongylus brasiliensis (N.b.) infection. Spleen cells from N.b. -infected Sprague-Dawley rats, 11–13 days postinfection when villus atrophy and crypt hyperplasia were evident, killed IEC-18 in a 4-hr 51 Cr-release assay. In contrast, intraepithelial leukocytes (IEL) from N.b. -infected rats showed decreased killing of IEC-18 compared to controls in a 16-hr assay. Spleen NK activity was increased in N.b. -infected rats whereas IEL NK activity was decreased. We conclude that direct cytotoxicity of crypt-like intestinal epithelial cells by nonspecific cytotoxic cells appears to play no significant role in intestinal injury in N.b. infection.

Rabbits receiving repeated intravenous injections of killed bacteria (Escherichia coli or Bacillus subtilis) developed IgM rheumatoid factor which reacted with autologous heat-aggregated IgG. In addition, 5/7 'Old English' and 7/8 'Sandy Lop' rabbits receiving killed E. coli developed rheumatoid-like synovial lesions. 'Old English' rabbits developed lesions of a more severe nature. Three of eight 'Sandy Lop' rabbits injected with killed B. subtilis had high levels of rheumatoid factor but only mild joint lesions.

Low concentrations (40 microM) of the purine nucleoside metabolite 2'deoxyguanosine (2'dGuo), were shown to enhance both phytohaemagglutinin- and concanavalin A-induced T cell transformation. Evidence is presented which indicates that this enhancement may be due to the inhibition of suppressor T cell function resulting in the promotion of T cell growth factor production. T cell responses were only inhibited at concentrations of 2'dGuo which were toxic to the lymphocytes.

In isolated normal rat jejunum, platelet-activating factor (PAF) induced a dose-dependent increase in short-circuit current (Isc) that was reduced in chloride-free buffer and inhibited by the Cl- channel blocker, diphenylamine-2-carboxylate. An immediate rise in Isc (early phase) occurred that fell to a new elevated base line by 15 min (late phase). These responses to PAF occurred only when experiments were conducted at or before approximately 9 A.M. Early phase responses were blocked by the specific PAF antagonists, BN52021 and WEB2086, and were inhibited by the neurotoxin, tetrodotoxin. Early and late phases were also reduced by cyclooxygenase inhibitors and by doxantrazole, a mast cell stabilizing drug. However, histamine and serotonin antagonists were ineffective. We conclude that PAF causes changes in ion transport that include Cl- secretion and acts on the epithelium possibly via an intermediate cell and enteric nerves. In addition, known PAF receptors are involved in one component of the response that appears to follow a circadian rhythm.

This paper reports the spontaneous development of synovitis in 'Old English' rabbits concurrent with serum IgM rheumatoid factor. Female rabbits developed lesions at a higher incidence than males (42% compared with 17%) and the pathology was of a more severe nature. In the female rabbits synovitis was associated with concurrent high serum IgM rheumatoid factor and high serum antibody levels to Pasteurella multocida. There was no histological evidence that the joints were directly infected.

Rheumatoid-like lesions developed in 9 of 25 (36%) Old English rabbits drinking cow's milk for 12 weeks. The incidence of lesions in male and female animals was similar. The majority of rabbits drinking cow's milk developed increased numbers of nucleated cells and raised percentages of T lymphocytes in their synovial fluids, compared to control rabbits. The cell counts and T cell percentages correlated with the severity of the histological lesions. No evidence of glomerulonephritis was observed in any of the experimental animals.

Addition of 40 microM 2'deoxyguanosine (2'd Guo) results in enhancement of human lymphocyte cultures stimulated with pokeweed mitogen, assessed by staining intracytoplasmic immunoglobulins. Furthermore, B cell responses were enhanced under experimental conditions favouring T cell suppression rather than help. Experiments with enriched B cell fractions indicate that 2'dGuo does not directly stimulate B cell responses. The enhancement by 2'dGuo was still observed in the presence of irradiated (non-proliferating) T cells. The mechanism of enhancement by 2'dGuo is discussed in terms of its effect on proliferation-independent suppressor T cells.