Destruction of articular cartilage by alpha 2 macroglobulin elastase complexes: role in rheumatoid arthritis.

OBJECTIVE: Neutrophil elastase accounts for the ability of some fresh rheumatoid synovial fluids to degrade cartilage matrix in vitro. The aim of this study was to determine if enzyme activity could result from depletion of synovial fluid inhibitors or protection of the enzyme from inhibition. METHODS: The ability of synovial fluids to inhibit porcine pancreatic elastase was investigated together with chemical pretreatments capable of inactivating alpha 1 protease inhibitor (alpha 1PI) or preventing formation of alpha 2 macroglobulin (alpha 2M) elastase complexes. Subsequently, complexes of human neutrophil elastase with alpha 2M were prepared and applied to frozen sections of cartilage. Proteoglycan loss was quantified by alcian blue staining and scanning and integrating microdensitometry. Parallel studies were carried out using a low molecular weight chromogenic elastase substrate. The effects of alpha 1PI and SF on these systems were investigated. Finally, synovial fluids were subjected to gel filtration and the fractions assayed for elastase activity. High molecular weight fractions were pooled, concentrated, and tested for their ability to degrade cartilage sections. RESULTS: All synovial fluids reduced the activity of porcine pancreatic elastase, the inhibition mainly being attributable to alpha 1PI, whereas remaining activity resulted from complexes of elastase with alpha 2M. Complexes of human neutrophil elastase with alpha 2M were shown to cause proteoglycan degradation in frozen sections of human articular cartilage. Alpha 1PI prevented alpha 2M elastase complexes from degrading cartilage but not the chromogenic substrate. The data suggested that alpha 1PI does not inhibit elastase bound to alpha 2M but sterically hinders the complex. However, only one of five synovial fluids was able to completely block the actions of alpha 2M elastase complexes against cartilage. Gel filtration of rheumatoid synovial fluids showed elastase and cartilage degrading activity to be associated with fractions that contained alpha 2M, and not with fractions expected to contain free enzyme. CONCLUSIONS: The data suggest that synovial fluid alpha 2M elastase complexes can degrade cartilage matrix in rheumatoid arthritis.

Oral dosing of rats with OM-89 results in the appearance of specific OM-89 antibodies of the IgG2a isotype: possible significance in the treatment of rheumatoid arthritis.

OM-89 is a glycoprotein-rich extract of Escherichia coli shown to be effective in the treatment of rheumatoid arthritis (RA). It has been reported that oral dosing of animals results in the appearance of specific OM-89 antibodies. In the current study we have investigated some of the immunoglobulin isotypes that may be involved. OM-89 antibodies of IgG1, IgG2a and IgM isotypes were measured by ELISA in serum from rats dosed three times a week for 3 weeks at 4 or 40 mg kg(-1). The results showed a small but significant rise in IgM and a greater rise in IgG2a. The possibility that antigens within OM-89 (e.g. hsp65) may have homology with antigens involved in RA raises the possibility that OM-89 antibodies, particularly of the IgG2 class, may block pathogenic antigens from being recognized by T cells.

Polyarthritis in the rat: effects of tolerance and sensitization to the bacterial extract OM-89 with a possible mode of action.

The Bacterial Extract OM-89 has been shown to significantly reduce paw swelling in an avridine-induced polyarthritis (AvPA) in female Wistar rats whether given orally for 3 weeks or as a series of i.p. injections. Paw swelling was also markedly reduced in groups of adult rats which had received i.p. injections of OM-89 as neonates. Three separate tolerance-inducing regimes were used and all were equally effective. It is suggested that OM-89 can modulate the undesirable immunological reactions to endogenous antigens and since oral administration has been shown to be clinically effective in the human, that the induction of oral tolerance could be a mechanism of action of OM-89.

OM-89 modulation of chronic inflammation: relevance to clinical use.

The modulatory effects of a glycoprotein-rich endotoxin-free extract of Escherichia coli (OM-89) have been studied using the cotton pellet model of chronic inflammation in the male Wistar rat. OM-89 had a suppressive effect on the size of granuloma surrounding implanted cotton pellets at both 4 and 40 mg/kg given three times weekly. The lower dosage of 4 mg was effective throughout and there was little to be gained by increasing the dose as further reduction of granuloma size was not obtained. Whether given prior to, at the same time as, or after an inflammatory stimulus, OM-89 had suppressive effects. However, if given before, animals at first went through a phase of 'sensitization' before suppressive effects were seen on further exposure to OM-89 antigens, a phenomenon which might have bearing on clinical findings in rheumatoid arthritis. In animals presensitized to a cotton pellet, OM-89 was statistically as effective as indomethacin in suppressing a second granuloma. OM-89 combined with indomethacin showed additive effects and was highly effective. The results indicate that OM-89 could be efficacious in the treatment of chronic inflammatory conditions and there is the possibility that in appropriate circumstances OM-89 might replace some drugs currently used and in others reduce their dosage.

Is tolerance induction a possible mode of action of OM-89?

Neonatal Wistar rats were injected intraperitoneally with either 20 mu g OM-89 at birth and on day 3 after birth or 2 mg three times weekly until 200 g body weight. At this weight, groups were either implanted subcutaneously with a cotton pellet to induce granuloma, or had avridine polyarthritis or pleurisy induced. Both OM-89 treatment regimes significantly reduced granuloma weight (p < 0.05 and < 0.01), paw volume (p < 0.001) and pleural exudates (p < 0.01). The results suggest that OM-89 could work via tolerance induction.

In vitro immunogenicity of allogeneic cardiac valves.

The immunogenicity of allogeneic cardiac valves (ACV) has not been previously demonstrated in vitro, though valve failure due to tissue degeneration has been attributed to adverse immunological reactions. A novel in vitro assay has been developed in a Brown Norway (BN; RT1n)-Lewis (RT1; donor-recipient) rat model system that demonstrates the immunogenicity of ACVs. A single cell suspension of viable cardiac valve conduit (CVC) cells was obtained by collagenase treatment of BN rat aortic valve conduits. Brown Norway rat CVC cells (5 x 10(4)) and Lewis responder lymphocytes (10(5)) were co-cultured in 96 well plates in RPMI 1640 plus 2.5% (v/v) non heat-inactivated Lewis rat serum and supplements with appropriate controls. Stimulation of responder lymphocytes by CVC cells was measured by 3H-thymidine incorporation into DNA. The counts obtained between 96-192 h of co-culture in the CVC cell/responder lymphocyte reaction were significantly higher (P < 0.05) than those of responder cell controls as assessed by analysis of variance. These results indicate the presence of potent immunostimulatory cells in viable ACVs and the possibility of using a sensitive and reproducible in vitro assay to evaluate ACV immunogenicity.

The effects of variation of cryopreservation protocols on the immunogenicity of allogeneic skin grafts.

The use of allografts to effect wound closure on full thickness skin injuries is limited by unpredictable graft rejection times. If the period of graft take could be extended, the use of allografts would reduce the morbidity and mortality associated with these wounds. This study evaluated the effects of variation in the cryopreservation protocol on the viability and immunogenicity of skin using a murine model system. Immunogenicity was assessed by the stimulatory activity of C3H (H-2K) skin-derived epidermal cells (EC) in primary one-way EC/lymphocyte reactions with BALB/c (H-2d) and CBA (H-2K) responder lymphocytes. Viability was determined by measuring tetrazolium reductase activity. The following cryopreservation protocols were assessed: freezing at 1, 30, 64, and > 100 degrees C/min in 10 and 15% (v/v) Me2SO and freezing at 30 degrees C/min in 5 to 20% (v/v) Me2SO or glycerol. A cryopreservation protocol of 30 degrees C/min in 15% (v/v) Me2SO proved optimal for murine skin allograft storage and immunomodulation. The viability of skin treated by this protocol was maintained (78% of fresh skin viability, no significant difference analysis of variance). The stimulatory capacity of treated EC for H-2K and H-2d lymphocytes was 5 +/- 4 and 5 +/- 9% (+/- 95% confidence limits) of fresh EC (100%) activity. Langerhans cell numbers in epidermal sheets and EC suspensions did not correlate with the stimulatory capacity of fresh and treated EC for allogeneic lymphocytes. A functional impairment of Langerhans cell immunostimulatory capacity was implied.(ABSTRACT TRUNCATED AT 250 WORDS)

Humoral responses to Malassezia furfur serovars A, B and C in normal individuals of various ages.

A transferable solid-phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class-specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2-64 years; 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2-3 years. There was no difference in either IgM or IgG levels into adulthood. The only age-related differences were lower IgM titres to the three serovars in the 60-64 year age-group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6.9) was significantly higher (P < 0.05) than that to serovar B (mean reciprocal log2 titre of 5.8), but not to serovar C (6.1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6.5) was significantly lower (P < 0.05) than those to serovars B and C (mean reciprocal log2 titre of 8.9 in both cases).

The influence of short-term pre-operative intravenous nutrition upon anthropometric variables, protein synthesis and immunological indices in patients with gastrointestinal cancer.

The influence of short-term pre-operative intravenous nutrition (IVN) upon protein synthesis in human liver and skeletal muscle was investigated in 30 patients with weight loss (>5 kg in preceding 3 months) who underwent resection of a neoplasm of the gastrointestinal tract. Patients were randomised to receive a normal hospital diet (HD group) or a hospital diet supplemented by intravenous nutrition (IVN groups; 0.18 gN/kg/24h as FreAmine III; 126kJ/24h as glucose) for either 3 (IVN(3)) or 7 days (IVN(7)) Nutritional assessment was undertaken by measurement of anthropometric, biochemical and immunological indices. In addition, the rates of protein synthesis in biopsies of liver and skeletal muscle, which were obtained at operation, were determined in vitro from the incorporation into protein of (14)C-leucine. Patients who received a hospital diet only (HD group) were in negative energy balance and continued to lose weight during the pre-operative period. The imbalance between caloric intake and expenditure was met from endogenous fuel stores with a reduction in fat-free mass and arm muscle area. Patients who received IVN maintained body weight and fat-free mass and had higher rates of protein synthesis in liver and skeletal muscle. In addition there was an increase in the plasma concentrations of prealbumin, haptoglobin and complement C3 together with a rise in the concentrations of the circulating immunoglobulins IgM and IgA. The majority of these changes occurred after only 3 days of IVN.

The cutaneous microbiology of haired and hairless mice.

The cutaneous microflora of the mid-dorsal area of hairless and haired mice was studied by processing skin biopsies. In both C3H and CBA hairless genotype animals the prevalence of colonization and the bacterial density were significantly greater than in the haired animals. The dominant bacteria were staphylococci and aerobic coryneforms. No propionibacteria were isolated. Temporal studies with C3H mice showed that from 0 to 9 days after birth the cutaneous microflora reduced and from then on the haired genotype animals maintained a low cutaneous microflora, whilst hairless genotype animals gradually lost hair from head to tail and the microflora density increased. Reciprocal skin grafting between haired and hairless animals showed that the donor skin acquired the microflora characteristics of the recipient animal after 15 d post-grafting even though the donor skin remained morphologically true to genotype.

Differentiation of three serovars of Malassezia furfur.

Malassezia furfur strains were isolated from the clinically normal skin of 10 volunteers by swabbing four different sites (forehead, ear, back and chest). The strains could be divided into three basic groups on the basis of cultural characteristics. Both unabsorbed and absorbed specific rabbit antisera were prepared against nine of the strains, and both species and group specific antigens could be demonstrated. Serologically, three group specific surface antigens could be identified which corresponded to the three groups identifiable on cultural characteristics. The relevance of these findings to previous in vitro results is discussed.

Effects of cryobiological variables on the survival of skin using a defined murine model.

Skin from an inbred strain of hairless mouse was used as a homogeneous model tissue for studies of skin cryopreservation. Tetrazolium reductase enzyme activity was used to assess tissue viability. Hepes-buffered 199 tissue culture medium was confirmed to be a suitable basal medium, to which cryoprotectants were added. Addition of serum to the cryoprotective cocktail had no beneficial effect. Three cryoprotectants, dimethyl sulfoxide, ethanediol, and glycerol were evaluated. There was no evidence of specific toxicity attributable to the cryoprotective agents during the permeation period; however, short permeation times at low temperature were associated with maximum skin viability. Following freezing and thawing, higher viabilities were obtained when using a slow (-1 degree C min-1) or medium (-60 degree C min-1) rather than a fast (immersion in liquid nitrogen) cooling rate. Dimethyl sulfoxide was a marginally better cryoprotectant overall, although this difference was not statistically significant.

Cryopreservation of skin using a murine model: validation of a prognostic viability assay.

In order to evaluate the many variables that can affect cryopreservation success, a simple, highly reproducible model system is required. We have evaluated the use of tetrazolium reductase activity as a prognostic indicator of skin viability in an inbred murine model. Two inbred hairless mouse strains were characterized in studies on autografting and allografting following different skin-storage protocols. Skin tetrazolium reductase (TR) activity correlated well with oxygen consumption, and with graft success--the ultimate performance criterion--following varying degrees of cryogenic injury. The assay was shown to be highly reproducible. In a series of factorial experiments the only factors affecting TR activity were those concerning the mouse donors, i.e., mouse strain, age, sex, and body area. The effects of these factors on TR activity were fully characterized.

Effects of antifungals on the viability of heart valve cusp derived fibroblasts.

Fibroblasts were isolated from fresh porcine aortic cusps and their identity confirmed morphologically and biochemically. The fibroblasts were used to assess the cytotoxicity of nystatin and amphotericin B at room temperature and 4 degrees C, following exposure periods of 4, 24, and 48 h. Except at the highest concentration and the longest exposure period at room temperature, nystatin had little detrimental effect compared with the nystatin free control. In contrast, amphotericin B proved highly cytotoxic. Published protocols for the use of amphotericin B in the disinfection of viable heart valve allografts would result in little residual fibroblast viability. We recommend that amphotericin B is no longer used in this situation.

Autoantibodies to soluble cellular antigens in unexplained recurrent abortion and infertility.

In 36 women with unexplained primary recurrent abortion, 13 with secondary unexpained recurrent abortion, 25 with primary unexplained infertility, 7 with secondary unexplained infertility and two groups of control women, autoantibodies to soluble cellular antigens were measured by Western blotting to a disaggregated HeLa cell antigen preparation, by counter immunoelectrophoresis and by indirect immunofluorescence. Using Western blotting the women with primary infertility and those with secondary recurrent abortion had a significantly higher prevalence of autoantibodies (P less than 0.01 in each case). This was not shown using the other methods. It is possible that these antibodies could be causally related to the pathology of the conditions studied.

Spontaneous glomerular immunoglobulin deposition in young Sprague-Dawley rats.

The frequency, age-onset and distribution of spontaneously deposited immunoglobulins (Igs) in glomeruli of Sprague-Dawley rats has been investigated. Groups of rats (n = 10) were examined at 4-7 day intervals from birth (presuckling) until 30 days of age. Findings were compared with circulating immunoglobulin concentrations in each age group. Immunoglobulins were undetectable in immature kidneys of newborn rats. However, as early as 5 days, scanty IgA and IgM deposits were observed predominantly in mesangial areas of mature glomeruli, corresponding to low circulating concentrations of these immunoglobulins. By contrast, glomerular IgG deposits were not observed until 21 days, despite relatively high concentrations of circulating maternal IgG from birth. Mesangial deposition of immunoglobulins increased with age. Absence of complement C3c or electron dense deposits associated with this mesangial localization suggests that immunoglobulins were not deposited as immune complexes. Accumulation of non-phlogogenic immunoglobulins in the mesangium of normal rats supports the concept that the mesangium is constantly perfused by circulating macromolecules and filtration residues. The results indicate problems of interpretation of the significance of endogenous immunoglobulin deposition in models of experimental glomerulonephritis, even in studies involving weanling rats.

Anaphylactoid reaction to vecuronium followed by systemic reaction to skin testing.

An unusual case is presented of a systemic anaphylactoid reaction to tubocurarine and subsequently to vecuronium. Intradermal testing with vecuronium following the latter response was negative at recommended test dose levels but at a higher concentration it initiated a hazardous systemic response. The laboratory investigations and possible mechanisms involved in this unusual case are discussed in detail since they may relate to other patients who experience anaphylactoid responses to anaesthetic drugs and who then undergo intradermal testing.

Glomerular inflammation: a consequence of sequential challenge of the rat mesangium with particulate macromolecules.

Imposil pretreatment (50 mg/100 g bodyweight) of male Sprague-Dawley rate (100-150 g), 24-48 h before an albumin-coated latex challenge resulted 24 h later in markedly reduced mesangial endocytosis of latex, compared with control animals receiving latex-albumin alone. This reduced capacity to ingest latex was associated with increased glomerular infiltration by Ia+ macrophages and mesangial localisation if immunoglobulins and complement C3c. In sharp contrast, pretreatment with albumin-coated latex had little effect on the subsequent mesangial uptake of Imposil. In this reverse situation macrophage infiltration was minimal, immunoglobulin deposition was similar to that in untreated control animals, and C3c was not detectable. Similarly, extending the interval between Imposil pretreatment and albumin-coated latex challenge up to 144h resulted in only minor infiltration by Ia+ macrophages and immunoglobulin localisation without accompanying C3c deposition, even though mesangial latex uptake was still limited. These observations demonstrate the consequences of secondary challenge following mesangial impairment due to a predisposing insult. Glomerular inflammation was dependent upon the timing and nature of the sequential challenge. The results support the concept that mesangial impairment following an initial insult may be a major factor in the predisposition to some forms of human glomerular inflammation.

Antibodies to P. acnes and P. acnes exocellular enzymes in the normal population at various ages and in patients with acne vulgaris.

Total serum IgM and IgG agglutinins to P. acnes and neutralizing antibodies to P. acnes lipase, hyaluronate lyase and acid phosphatase were measured in normal individuals of different age groups. Agglutinins to P. acnes were detected in infants at 4 months of age and were present at a high level throughout life. A switch from predominantly IgM agglutinins in children, to IgG agglutinins in adults, occurred during adolescence. Anti-P. acnes lipase antibodies were present in 20% of teenagers and 17-42% of adults. Anti-P. acnes hyaluronate lyase antibodies were found in adults only (4-17%). Antibodies to acid phosphatase were not detected. Agglutinins to P. acnes were measured in individuals with mild, moderate and severe acne, and in normal controls. Only patients with severe acne had significantly higher titres than the controls. IgM and IgG agglutinins were determined in 13-14-year-olds with mild, moderate and severe acne, and in normal controls. Thirty-three per cent, 60% and 100% of the acne patients, respectively, but none of the normal controls, had predominantly IgG agglutinins. No difference in the prevalence or titre of antibodies to P. acnes exocellular enzymes was observed when patients with severe acne were compared with normal controls. There was no evidence to suggest a role for antibodies to P. acnes exocellular enzymes in the initiation of inflammatory acne.

Preparation, purification and analysis of BSA-coated latex particles for in vivo studies. Non-ionic adsorbance of unbound protein ligand.

A procedure for the preparation, purification and analysis of small, BSA-coated carboxylate-modified latex (CML) particles, suitable for use in in vivo studies has been developed. Following conjugation, uptake of unbound BSA by Amberlite XAD-8 non-ionic adsorbent beads has been shown to be an effective method by which unbound protein ligands may be removed from coated latex preparations.