Protein Protease Inhibitors Research Articles

Purification of a Novel, Heat-Stable Serine Protease Inhibitor Protein from Ovaries of the Desert Locust,Schistocerca gregaria

A protease-inhibitor was isolated from mature ovaries ofSchistocerca gregariaby a combination of trypsin-affinity chromatography and reverse-phase high performance liquid chromatography. It was characterized by aminoterminal amino acid sequencing using Edman degradation based automated microsequencing and by MALDI-TOF mass spectrometry. The N-terminal sequence (Y)XAEXDELA(A)EEY(Y)Q(Q)X(I)(L)M (X being a Cys, an irregular or modified amino acid) revealed no similarities with any other protease inhibitors isolated from invertebrate or vertebrate source. The 14 kDa inhibitor was found to be heat-stable. It shows potent inhibitory activity toward bovine trypsin and chymotrypsin, but not toward pancreatic elastase. It is likely that the characterized inhibitor will serve as an important tool for understanding its role in insect development.

Quail cystatin: Isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B

Quail cystatin, a new cysteine proteinase inhibitor protein of the cystatin superfamily, was purified from egg albumen of Japanese quail Coturnix coturnix japonica. Amino acid sequencing and mass spectrometry revealed the complete 116 amino acid residue primary structure of a phosphorylated form (13 173 Da). The inhibitor has a 90% sequence identity with chicken cystatin. Its interaction with papain is rapid and tight ( K i=4.4 pM; k on=1.8×10 7 M −1 s −1; k off=0.8×10 −4 s −1) and very similar to that of chicken cystatin. Surprisingly, however, cathepsin B was inhibited 15-fold more strongly by quail cystatin ( K i=47 pM; k on=19×10 7 M −1 s −1; k off=9×10 −4 s −1) than by chicken cystatin ( K i=784 pM; k on=2.9×10 7 M −1 s −1; k off=24×10 −4 s −1). Intuitive comparative conformational inspection of related inhibitors and of cognate enzymes suggest that: (i) the 3D structure of quail cystatin is nearly identical to that of chicken cystatin, (ii) quail cystatin can interact with cathepsin B analogous to the stefin B–papain interaction, if the `occluding loop' of cathepsin B possesses an `open' conformation, (iii) the greater inhibition of cathepsin B by quail cystatin compared to chicken cystatins probably arises from two additional ionic interactions between residues Arg 15 and Lys 112 of the inhibitor and Glu 194 and Asp 124 of the enzyme, respectively. The two potential salt bridges are located outside of the known contact regions between cystatins and peptidases of the papain family.

Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families

Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.

Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases.

Bowman-Birk proteinase inhibitor proteins contain two inhibitory regions, each of which is encapsulated within nine-residue disulfide-linked loops. It is known that short cyclic peptides that retain the nine-residue disulfide-bridged motif have inhibitory activity, and can be used as models of the natural inhibitor protein. Two factors are important in determining the effectiveness of such inhibitor peptides: the value of the inhibition constant, and rate at which the inhibitor peptide is hydrolyzed by the proteinase. In this paper we report a study of the inhibitory properties and stability towards proteolytic hydrolysis of a family of synthetic peptides derived from the trypsin reactive site loop of the Bowman-Birk inhibitors. The addition of a single amino acid residue to each end of the nine-residue disulfide-linked loop is found to reduce the rate at which the peptide is hydrolyzed. In addition, changing the P2' residue from Asn-->Ile gives inhibitors with considerably enhanced stability to proteolysis, as well as reduced values of Ki. The implications of these factors for the design of inhibitors based on this loop motif is discussed.

Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3, 4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B. , Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859-14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and beta-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, alpha1-proteinase inhibitor (alpha1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the alpha1PI reactive center P1-P1' bond. Quantitation of kinetic constants for DCI and alpha1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by alpha1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

A target of the HoxB5 gene from the mouse nervous system

An epitope-specific antibody against the protein product of the murine HoxB5 gene was used to select an enriched library of Hox target sequences. Genomic DNA was purified by immunoaffinity chromatography, using glutaraldehyde-cross-linked chromatin from CNS of mouse embryos at gestational day 15. Screening was done by colony hybridization with TAAT-containing oligonucleotides, filter DNA–protein binding, and gel mobility shift assay. Nucleotide sequencing identified a 910 bp DNA fragment, containing a consensus Antennapedia-like binding site, and identical in 640 bps at 3′ end of the clone to the promoter of the SPI3 gene, which encodes a serine protease inhibitor protein [Sun, J., Rose, J.B. and Bird, P., J. Biol. Chem., 270 (1995) 16089–16096]. In situ hybridization experiments were performed to see if a correlation could be found between the expression patterns the SPI3 and the HoxB5 genes. Using a 120 bp cDNA fragment as probe, SPI3 expression was detected mainly in the CNS of 15 day mouse embryos, a pattern which is similar to that of the HoxB5 gene at this stage [Hogan, B.L., Holland, P.W. and Lumsden, A., Cell Diff. Dev., 25 Suppl. (1988) 39–44; Sakach, M. and Safaei, R., Int. J. Dev. Neurosci., 14 (1996) 567–573]. In conclusion, data presented here suggest that the SPI3 gene is a candidate target of the HoxB5 gene in vertebrate embryos.

Protein proteinase inhibitors from avian egg whites.

Avian egg whites are a rich source of protein inhibitors of proteinases belonging to all four mechanistic classes. Ovomucoid and ovoinhibitor are multidomain Kazal-type inhibitors with each domain containing an actual or putative reactive site for a serine proteinase. Cystatin is a cysteine proteinase inhibitor, while ovostatin inhibits proteinases of all four mechanistic classes. In this review we have summarized the general features, isolation, inhibitory mechanism and evolutionary aspects of these inhibitors.

The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non-chondrodystrophoid (greyhound) canine intervertebral disc.

Trypsin inhibitory proteins of low buoyant density (p < or = 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter-alpha-trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti-human alpha1-proteinase inhibitor (alpha-1-PI), alpha2-macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti-BPTI detected an inhibitor species of approximately 85-90 kDa; anti-ITI detected species of 120-250 kDa; biotinylated trypsin detected species of 60-110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti-BPTI.

Probing intermolecular main chain hydrogen bonding in serine proteinase-protein inhibitor complexes: chemical synthesis of backbone-engineered turkey ovomucoid third domain.

Intermolecular main chain H-bonding networks are frequently encountered at the interface of complexes of protein proteinase inhibitors and their cognate enzymes. Studies of X-ray crystal structures of many protein inhibitors complexed with serine proteinases have revealed that the amide NH group of the P1 residue in the inhibitor donates an H-bond to the carbonyl C = O group of Ser214 and Ser195 Oy in the enzyme (Ser125 and Ser221 in subtilisins, respectively). To probe the energetic contribution of this backbone H-bond in the complexes of OMTKY3 with several serine proteinases, native chemical ligation was used for the total synthesis of a backbone-engineered analog of OMTKY3, in which the amide peptide bond between Thr17 (P2) and Leu18 (P1) was replaced by an ester bond, i.e., -CONH-to-COO-. This chemical "mutation" effectively eliminated the backbone H-bond donated by the NH group of Leu18. By measuring association equilibrium constants for synthetic wild-type OMTKY3 and the backbone-engineered ester analog interacting with a panel of six serine proteinases, we have determined that the P1 NH-->O substitution weakens the binding of OMTKY3 to its cognate enzymes by an average of 15-fold, i.e., 1.5 +/- 0.3 kcal/mol. These results place a quantitative value on the contribution of the intermolecular backbone H-bond in enzyme-inhibitor recognition.

Ethylene as a Signal Mediating the Wound Response of Tomato Plants

Plants respond to physical injury, such as that caused by foraging insects, by synthesizing proteins that function in general defense and tissue repair. In tomato plants, one class of wound-responsive genes encodes proteinase inhibitor (pin) proteins shown to block insect feeding. Application of many different factors will induce or inhibit pin gene expression. Ethylene is required in the transduction pathway leading from injury, and ethylene and jasmonates act together to regulate pin gene expression during the wound response.

Pre-operative plasma levels of C-reactive protein, albumin and various plasma protease inhibitors for the pre-operative assessment of operability and recurrence in cancer surgery

Pre-operative levels of the acute phase protein C-reactive protein (CRP), albumin (assessing nutritional status), the tumour marker CEA and three plasma protease inhibitors, i.e. C1-esterase inhibitor, alpha-2-macroglobulin and antithrombin III, were prospectively studied in 183 patients with various solid cancers. First, the predictive value of abnormal levels for operability at the primary operation was studied. Secondly, the predictive value of abnormal levels for cancer recurrence and metastases was evaluated during 2 years of follow-up. The results show that malignancy induces increased CRP and C1-esterase inhibitor levels and decreased albumin levels in serum. These changes, as well as raised alkaline phosphatase and lowered haemoglobin levels, also correlate to the 'overall' tumour burden. The most important conclusion is, that increased pre-operative CRP levels (CRP > or = 10 mg/l; sensitivity, 79%; specificity, 71%) and/or low albumin levels (albumin <37 g/l; sensitivity, 94%; specificity, 54%) are seen in inoperable cancer patients compared with patients having operable cancers. The second main important conclusion is, that high pre-operative C1-esterase inhibitor levels (C1-esterase inhibitor >152%; sensitivity, 45%; specificity, 90%), and in some patients a high alkaline phosphatase level, are seen in patients exhibiting early cancer recurrence (within 2 years post-operatively).

Inheritance of Protease Inhibitors in Glycine tomentella Hayata (2n = 38), a Perennial Relative of Soybean

Inheritance of three seed protein protease inhibitors (designated Pi)—Rf 0.4, Rf 0.7, and BBI—in Glycine tomentella (2n = 38), a wild perennial relative of soybean [G. max (L) Merr.], was investigated. Two accessions of G. tomentella, PI 373987 with genotype Rf 0.4+ (PH/PH), Rf 0.7– (pi2l pi2), and BBI' + (Pi3/Pi3) and PI 440998 with genotype Rf 0.4– (pi1/pH), Rf 0.7+ (Pi2/Pi2), and BBI— (pi/pi3), were crossed reciprocally and the allelic segregation in F2 and F3 generations was studied. The recessive alleles pi1 and pi2 encode the lack of trypsin inhibitor activity bands Rf 0.4 and Rf 0.7, respectively, while pi3 encodes the lack of antisoybean Bowman–Birk inhibitor immunocrossreac-tive band BBI'. Alleles at each locus segregated in 3: 1 (presence: absence) ratios for the inhibitor bands, suggesting that each of them was monogenic. Analysis of the F3 progeny from the heterozygous F2 plants confirmed their monogenic nature. A linkage between Pi2 and Pi3 with a recombination frequency of 0.212 ± 0.068 in repulsion phase was observed. This information adds to our understanding of the nature and genetics of protease inhibitors in the genus Glycine.

An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

The binding of BPTI and SBTI with trypsin has been investigated by ESI MS, using the mutant K15V-BPTI and the chemically modified RcamBPTI as controls. Although high cone voltages (+80 V) produce sharp spectra of BPTI, RcamBPTI, SBTI and trypsin alone, the complexes of BPTI, RcamBPTI and SBTI with trypsin undergo partial dissociation due to collisional activation. At lower cone voltages (+40 V) these non-covalent complexes are stable. The charge distribution on the trypsin and the inhibitors produced by gas phase dissociation of the complexes are markedly different from those of the components alone, indicating that ESI MS provides a novel probe for exploring the ionic interactions at the contact surface of proteins. Moreover, by determining the cone voltage at which the gas phase dissociation of complexes occurs it may be possible to use ESI MS to compare the binding energies of closely related complexes.

Regulating aspects of biosoluble and insoluble film release systems containing protein proteinase inhibitor

New types of potent aprotinin-containing medicinal polymer films are elaborated for topical applications. The higher the temperature of drying, the lower the steam absorption, the film swelling, and the velocity of aprotinin in vitro release. The presence of antimicrobials having the basic functional groups contributed to compacting the structure of the films and retention of aprotinin in them. The velocity of aprotinin release from the films is regulated by adding different polymers. Inclusion of polyvinylpirrolidone in the film resulted in acceleration and increase of aprotinin release. This was probably because of increasing the film swelling by a factor of 1.7. Additional retention of the inhibitor in films was achieved by inclusion of sodium alginate and cellulose powder capable of binding aprotinin. Soluble bioadhesive films derived from a copolymer of acrylamide, N-vinyl-pirrolidone, ethyl acrylate (M r 30,000-600,000) and aprotinin were obtained and analyzed. Kinetics of aprotinin release from biosoluble films was studied under various conditions. The duration of aprotinin release was comparable with duration of gradual dissolution of the matrix. Bioavailability of aprotinin from soluble films was linear with time.

Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field

Wounding increases the levels and activities of several defense-related proteins in the foliage of the tomato plant,Lycopersicon esculentum Mill. Evidence indicates that two of these responses, the systemic increases in polyphenol oxidase and proteinase inhibitors, are regulated by an octadecanoid-based signalling pathway which includes the wound hormone, jasmonic acid. It is not known whether other responses to wounding are also regulated by this same signalling pathway. In this paper, we show that application of jasmonates (jasmonic acid or its volatile derivative, methyl jasmonate) in low concentrations to foliage of young tomato plants induced, in a dose-dependent manner, the same protein responses-polyphenol oxidase, proteinase inhibitors, lipoxygenase, and peroxidase-as doesHelicoverpa zea Boddie feeding. Application of jasmonic acid to a single leaflet of four-leaf tomato plants induced these four proteins in a spatial pattern nearly identical to that produced by localized feeding ofH. zea. Exogenous jasmonic acid also decreased suitability of foliage for the beet armyworm,Spodoptera exigua Hubner in the laboratory. Based on these results, we conducted an experiment to measure the effects of jasmonic acid spray under field conditions. We provide the first evidence that jasmonic acid spray on field plants induces production of chemical defenses above the levels found in unsprayed controls. Exogenous jasmonic acid sprayed on plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors. Because application of jasmonic acid induces these defensive compounds at low concentrations in a manner similar to natural wounding, it may prove to be a useful tool for stimulating plant resistance to insects in the field.

Linkage between the loci for serum albumin and vitamin D binding protein (GC) in the Japanese quail

SummaryVitamin D binding protein (GC) and serum protease inhibitor (PI) have been added to genetic markers in the Japanese quail. Both loci were controlled by autosomal codominant alleles named GCA, GCB and PIA, PIB, PIC, respectively. Close linkage between the loci for serum albumin (ALB) and GC protein is reported. Two recombinants were observed in 145 informative offspring of 14 families. The recombination frequency between the loci was estimated as 0.014±0.006. Thus, GC was assigned to linkage group II in the Japanese quail. No signs of linkage were observed among the loci for the ALB‐GC complex, PI. serum prealbumin 2 (PA2), phosphoglucose isomerase (PG1), 6‐phosphogluconate dehydrogenase (PGD) and esterase‐D (ESD).

Identification and Isolation of the Bactericidal Domains in the Proteinase Inhibitor Aprotinin

Aprotinin is a protein proteinase inhibitor of 58 amino acids, located in bovine mast cells which also possesses antibacterial activity. Digestion of aprotinin by clostripain yielded three antimicrobial oligopeptide fragments with the sequences RPDFCLEPPYTGPCK (residues 1–15), IIRYFYNAKAGLCQTFVYGGCR (residues 18–39) and AKRNNFKSAEDCMRTCGGA (residues 40–58). With the exception of residues 16 (alanyl) and 17 (arginyl) they cover the whole aprotinin structure. The three oligopeptides were synthesized and found to exert a broad spectrum of antimicrobial activities. Most potent was oligopeptide IIRYFYNAKAGLCQTFVYGGCR which at a concentration of 7×10−9M exhibited a high bactericidal activity against the eleven gram-positive and gram-negative bacterial strains tested. None of the purified oligopeptides showed any antiproteolytic activity. Our results suggest that aprotinin has multiple antimicrobial domains which are independent of the antiproteolytic function of the original molecule.

Sprouting and abnormal contacts of nonmedullated axons, and deposition of extracellular material induced by the amyloid precursor protein (APP) and other protease inhibitors

We have reported that the local administration of serine protease inhibitors (amyloid precursor protein with the Kunitz insert (APP K + ), aprotinin, and Lupeptin) to the rat sciatic nerve determines a sprouting response of myelinated axons, proliferation of Schwann cells, and demyelination, 5 to 7 days later. Further study of these nerves with the electron microscope revealed (i) a sprouting response of nonmedullated axons, (ii) the appearance of fine axons with a few turns of compact myelin, (iii) abnormal contacts of axons with basal laminae, with fibroblast-like cells, and between them, (iv) the occurrence of hemidesmosome- and desmosome-like junctions between Schwann cell processes, and between Schwann cells and axons, and (v) the appearance of amorphous and fibrillary extracellular deposits alongside the axolemma. The adjacent proximal and distal segments were normal, i.e., axons remained continuous, and the alterations were confined to the segment exposed to the protease inhibitors. Heated APP K + , APP without the Kunitz insert (APP K − ), bovine serum albumin, and saline, did not elicit cytological alterations. Our results suggest that these inhibitors of serine proteases (i) set free a sprouting drive of axons by disrupting an ongoing repressive mechanism; (ii) modify the adhesive properties of axons and Schwann cells, and (iii) alter the natural history of an extracellular material. The imbalance of an extracellular protease system may participate in the pathogenesis of Alzheimer's disease.

Significance of secondary structure predictions on the reactive center loop region of seprins: a model for the folding of serpins into a metastable state

To address how serpins might fold so as to adopt the mechanistically required metastable conformation we have compared the predicted secondary structures of the reactive center loops (RCLs) of a large number of serpins with those of the equivalent regions of other non-serpin protein proteinase inhibitors. Whereas the RCLs of non-serpin inhibitors are predicted to be loop or β-strand, those of inhibitory serpins are strongly predicted to be α-helical. However, non-inhibitory serpins, which also adopt the metastable conformation, show no consistent preference for α-helix. We propose that the RCL primary structure plays little role in promoting the metastable serpin conformation. Instead we hypothesize that preference for the metastable state results from the incorporation of part of the RCL into β-sheet C, which as a consequence precludes incorporation of the RCL into β-sheet A to give the most stable conformation. Consequently the RCL must be exposed and by default will adopt the most stable conformation in this particular context, which is likely to be an α-helix irrespective of the primary structure. Thus the observed correlation between inhibitory properties in serpins and prediction of α-helix in the RCL may instead reflect a need for alanine residues between positions P12 and P9 for functioning as an inhibitor rather than a structural or mechanistic requirement for α-helix.

Accessory cells in Crohn's disease of the terminal ileum

We present a study which describes the immunophenotype and distribution of accessory cells in 13 resections of terminal ileum from patients with Crohn's disease. A panel of antibodies working in paraffin-embedded tissue was employed and these included PGM1 (CD68), S-100 protein, WR18 (HLA class II), factor XIIIa and acid cysteine proteinase inhibitor. This study revealed a heterogeneity of accessory cell populations which was profoundly influenced by local inflammatory and repair mechanisms. Both acid cysteine proteinase activity and S-100 protein positive cells are identified in more actively inflamed areas. The acid cysteine proteinase activity positive dendritic cell population was particularly numerous in ulcer bases. S-100 protein positive dendritic cells had a more limited distribution in close proximity to the epithelium in inflamed but otherwise intact mucosa adjacent to the areas of ulceration. PGM1 revealed normal distribution of macrophages within histologically uninvolved areas and, in addition, also stained granulomas and large numbers of dendritic cells in the inflamed, ulcerated and scarred areas. Factor XIIIa positive dendritic cells were especially numerous in areas of active scarring where they co-localized with PGM1 positive cells. They were largely absent from the more superficial ulcerated areas. HLA class II was strongly expressed on mononuclear inflammatory and dendritic cells. The strength of epithelial staining for HLA class II reflected the intensity of adjacent inflammation, except on ulcer-associated epithelium which consistently showed up-regulation independent of the severity of the inflammatory process. This study shows that localized alterations in the accessory cell distribution in Crohn's disease correlate with different states in the evolution of the inflammatory and repair process of the disease. The more acute lesions are associated with recruitment of acid cysteine proteinase activity and S-100 protein positive dendritic cells while factor XIIIa stained dentritic cells are especially numerous in areas of scarring.