The use of digital dermoscopy in the surveillance of melanocytic skin lesions: A real-life retrospective study comparing university hospital and private practices.

BACKGROUND: Few studies have evaluated the role of digital dermoscopy (DD) in the surveillance of pigmented lesions in real-life practice. PATIENTS AND METHODS: Patients followed with DD by 4 hospital dermatologists (group 1) and 4 private dermatologists (group 2) were retrospectively included if they had had at least 2 DD examinations for a minimum of 4 pigmented lesions. Their characteristics, risk factors, history of excision of benign nevi and melanomas prior to and during the DD follow-up, and characteristics of detected melanomas, were recorded. RESULTS: One hundred and ninety-six patients were included in group 1 and 205 in groups 2. A family history of melanoma (25% vs. 12%, p<0.01), a personal history of melanoma before DD follow-up (47% vs. 15%, p<0.01), and a family (3% vs. 0%, p=0.01) and personal (8% vs. 1%, p<0.01) germline CDKN2a mutation were more frequent in group 1 than in group 2. In both groups, the number of excisions of benign lesions was higher before DD follow-up (380 and 347, respectively) than during DD follow-up (194 and 132). During follow-up, 29 melanomas were detected in group 1, with a median Breslow thickness of 0.4mm, versus 1.3mm for melanomas diagnosed before DD follow-up (p<0.02). In group 2, 4 melanoma and 5 superficial atypical melanocytic proliferations of unknown significance were detected. The median Breslow thickness of newly diagnosed melanomas was 0.35mm vs. 0.6mm before DD follow-up (p=0.1). CONCLUSION: In both populations in real-life practice, DD seemed to allow the detection of thin melanomas and to decrease the rate of "futile" resections.

Real-time mobile teledermoscopy for skin cancer screening targeting an agricultural population: an experiment on 289 patients in France.

BACKGROUND: The incidence of skin cancer has reached epidemic proportions in the white population and is significantly elevated in agricultural populations, who are exposed to ultraviolet radiation during their professional activities. In 2014, the Agricultural Social Insurance Mutual Benefit Fund (MSA) offered its customers who work in agriculture and live in rural areas with reduced access to dermatologists the ability to participate in a 1-day teledermoscopic (TDS) screening event. OBJECTIVE: This study's aim was to assess the feasibility of real-time mobile TDS triage of a large number of agricultural workers by trained medical officers and occupational physicians. METHODS: Fifteen TDS screening centres were located in different areas of France. Individuals older than 18 years who worked in agriculture and lived in rural area near a TDS screening centre were invited to participate in a 1-day screening event and were examined by an MSA physician. In cases of suspicious skin lesions, clinical and dermoscopic images were obtained and transferred immediately to four dermatologists who were simultaneously present at the tele-platform for diagnosis and decision-making. Low-quality images were retaken. RESULTS: Two-hundred eighty-nine patients underwent skin cancer screening. Among 199 patients (69%), 390 suspicious lesions were identified and generated 412 pictures. All lesions were analysed by dermatologists. For 105 patients (53%), no follow-up was required. Seventeen patients were referred to local dermatologists for rapid examination, including 12 cases of suspected malignant melanocytic lesions. Among the 12 patients with suspected melanoma, face-to-face visits were conducted within 10 days for 11 of them, and 1 case of melanoma was confirmed by histopathology. CONCLUSIONS: Our study suggests that teledermoscopy performed in the context of occupational medicine and targeted to agricultural populations is feasible and could be useful for improving skin cancer screening in at-risk populations while avoiding face-to-face examinations by a dermatologist in 53% of cases.

[Clinical patterns and treatment of pyoderma gangrenosum in a French department]. / Caractéristiques cliniques et traitement du pyoderma gangrenosum dans la Marne.

BACKGROUND: Pyoderma gangrenosum (PG) is a rare inflammatory neutrophilic dermatosis for which accurate epidemiological data are limited and therapy remains a challenge. The primary study aim was to examine all cases of PG observed in our regional department over a 15-year period in order to describe the relevant characteristics and outcome under therapy. PATIENTS AND METHODS: The medical records of all patients with PG from 1997 to 2012 in the Marne department of France were studied retrospectively. Clinical and histological characteristics, comorbidities, therapeutic modalities and outcome were analysed. RESULTS: Forty-two patients were included (30 women, 12 men). A classical, ulcerative form was found in 39 cases and PG was multifocal in 28 cases. The number of lesions did not differ according to age or the presence of comorbidities. The most frequent first-line treatments were doxycycline (23 cases) and oral corticosteroids (15 cases), regardless of age, number of lesions or existence of comorbidities. Complete remission of PG was obtained in 38 cases (median time to remission: 3 months), with relapse occurring in 17 patients (median time to relapse: 12 months after treatment withdrawal). After a median follow-up of 46 months, 8 patients had died (median time to death: 26 months after treatment initiation). CONCLUSION: This is the first large French series of patients presenting PG and enabling determination of the annual incidence within the Marne department at around 4.6 cases/1000,000 inhabitants. Our study illustrates the value of first-line treatment with tetracycline, which merits confirmation by further prospective, controlled studies.

Efficacy of a general practitioner training campaign for early detection of melanoma in France.

BACKGROUND: To date, no strategy for improving early diagnosis of melanoma has been evaluated on a population basis in France. OBJECTIVE: To evaluate the efficacy of a general practitioner (GP) awareness and training campaign in a pilot French geographical region (Champagne-Ardenne), including 1.34 million inhabitants, 1241 GPs, 56 dermatologists and a population-based melanoma registry. METHODS: All GPs received repeated awareness postal mailings in 2008 and 398 (32.1%) attended training sessions organized by 27 dermatologists. The pre- (2005-7) and post-campaign (2009-11) periods were compared for the following: primary endpoint - the world-standardized incidence of very thick melanomas (VTM) (Breslow thickness ≥ 3 mm); secondary endpoints--the mean Breslow thickness; the proportions of VTM and of thin (< 1 mm) melanomas among invasive cases; and the ratio of in situ/all melanoma cases. Similar measures were performed in the control area of Doubs/Belfort territory (655,000 ha), where no similar campaign was carried out. RESULTS: The incidence of VTM decreased from 1.07 to 0.71 per 100 000 habitants per year (P = 0.01), the mean Breslow thickness from 1.95 to 1.68 mm (P = 0.06) and the proportion of VTM from 19.2% to 12.8% (P = 0.01). The proportion of thin and in situ melanomas increased from 50.9% to 57.4% (P = 0.05) and from 20.1% to 28.2% (P = 0.001), respectively. No significant variation was observed in Doubs/Belfort territory. CONCLUSION: These results strongly support the efficacy of such a campaign targeting GPs and provide a rationale for a larger public health campaign in France, including training of GPs by dermatologists and encouraging patients to ask their GP for a systematic skin examination.

[Diagnostic modalities of visceral metastases during follow-up of patients with stage I-II melanoma]. / Malades suivis pour mélanome de stade I-II: modalités diagnostiques des métastases viscérales.

INTRODUCTION: Although not recommended in France at the consensus conference of 1994, routine monitoring of patients with stage I melanoma using imaging techniques is commonly carried out. The aim of this retrospective regional study was to define methods for diagnosing transition to the metastatic stage of melanoma. PATIENTS AND METHODS: This was a retrospective study based on questionnaires among dermatologists in the Champagne-Ardenne and southern Aisne regions of France. For each patient with stage IV melanoma between 1987 and 2002, data were collected concerning the primary melanoma (date of diagnosis, clinical picture, histopathologic features), stage of melanoma prior to diagnosis of metastatic melanoma and characteristics of the metastases (date, number, type, site and modern discovery: clinical signs or routine imaging). RESULTS: One hundred and eight patients (63 men and 45 women; mean age: 59 years) were included in the study. The predominant site of the primary melanoma was the trunk for men (n=31) and the lower limbs for women (n=16) and the mean Breslow index was 4.31 mm (SD=4.22), with histologic ulceration being present in 40% of cases. The mean time to transition to stage IV after discovery of the primary tumour was 2.8 years (SD=2.95). The modes of discovery of metastases comprised clinical examination (functional signs or physical examination) in 58 cases and routine imaging in 50 cases, with no significant differences based on whether patients were initially in stage I-II or in stage III. DISCUSSION: This study shows that over half of patients progressing to stage IV melanoma had a suspicious sign or clinical symptom, once again highlighting the importance of clinical monitoring. In contrast, many organ metastases, particularly pulmonary, were discovered by routine imaging examinations carried out as part of patient follow-up, although this is not currently recommended practice in France. CONCLUSION: The role of powerful imaging examinations such as scans, with constantly improving resolution, still remains to be defined in the follow-up of patients with stage I-II melanoma, and further prospective studies are thus required.

Proenzymic C1s associated with catalytic amounts of C1r. Study of the activation process.

1. Proenzymic C1s isolated from human plasma by euglobulin precipitation and DEAE-cellulose chromatography is associated with trace amounts of C1r (0.5--1% on a molar basis). Incubation for 2 h at 37 degrees C leads to the proteolytic activation of C1s. The proteolysis is characterized by the sigmoidal appearance of C1s esterase activity and of the typical heavy (57 000-dalton) and light (28 000-dalton) fragments of C1s on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. 2. The C1s activation process observed is markedly temperature and concentration dependent, and the rate of activation is decreased by calcium and high ionic strength (I = 0.9). Diisopropyl phosphorofluoridate, benzamidine, polyanethol sulfonate and pentosane polysulphate inhibit the activation, which is also sensitive to C1-inactivator and anti-C1r IgC. From the kinetic experiments and from the inhibition characteristics, the activation of C1s can be attributed to the presence of C1r, which appears to undergo activation and then to activate secondarily C1s.

Purified proenzyme C1r. Some characteristics of its activation and subsequent proteolytic cleavage.

1. Upon incubation for 1 h at 37 degrees C, proenzymic C1r was activated by a proteolytic cleavage comparable to that observed in vivo; after reduction and alkylation, two fragments of apparent molecular weights 57 000 and 35 000 were evident on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The activation kinetics were slightly sigmoidal and nearly independent of C1r concentration. They were characterized by a marked thermal dependence (activation energy = 45 kcal/mol). The reaction was inhibited by calcium and p-nitrophenyl-p'-guanidinobenzoate, but poorly sensitive to di-isopropyl phosphorofluoridate. The dependence of the activation rate on pH was unusual; it decreased progressively in the acid range (pH 4.5-6.5) which coincides with the dissociation of the C1r-C1r dimer. Above pH 6.5, the rate increased slightly and showed no clear maximum. These results are consistent with an intramolecular autocatalytic activation mechanism involving the pro-site of each subunit of the C1r-C1r dimer. 2. During a 5 h incubation period at 37 degrees C, C1r underwent two proteolytic cleavages which led to the successive removal of two fragments, alpha (35 000) and beta (7000-11 000) from each subunit, leaving a dimeric molecule of reduced size (Mr = 110 000; s20,w = 6.1 S). The proteolytic process was nearly independent of C1r concentration and characterized by a pH optimum at 8.5-9.0, and a high activation energy (36.8 kcal/mol). Calcium and p-nitrophenyl-p'-guanidinobenzoate, and also di-isopropyl phosphorofluoridate and benzamidine were inhibitors of this reaction. The product, C1r II, retained the original antigenic properties of C1r and a functional active site, but lost the capacity to bind C1s. These results are consistent with an autocatalytic intramolecular proteolysis mediated by the active site of each subunit of the C1r-C1r dimer.

A study on the structure and interactions of the C1 sub-components C1r and C1s in the fluid phase.

1. Both proenzyme and activated C1r, which are dimers at pH 7.4, dissociated into monomers at pH 5.0 (C1r) and 4.0 (C1r), as shown by the decrease of apparent molecular weight and of sedimentation coefficient, which was shifted from 7.1 S (dimer) to 5.0 S (monomer). 125I-labelling of C1r in the presence of lactoperoxidase occurred, for the dimer, 16-20% in the A chain and 80-84% in the B chain, whereas the distribution was 67.5% and 32.5%, respectively, for the monomer. It appears likely that the two monomers of C1r interact through their A chain and that the A and B chains are relatively independent from each other. 2. 125I-labelling of C1s in the presence of lactoperoxidase confirmed the calcium-dependent dimerization of this subcomponent. In the monomer, the B chain appears to be embedded in the A chain, as shown by the 125I- distribution in these chains, which was 5% and 95%, respectively. This changed after dimerization to 25% and 75%, respectively, which suggests that interactions occur through the A chain of each monomer and lead to an unfolding of the B chain. 3. C1r dimer and C1s monomer were found to interact in the absence of calcium to form a C1r2-C1s complex (7.7 S), whereas in the presence of calcium the two sub-components were associated into a C1r2-C1s2 complex (8.7S). It appears likely that the formation of this tetrameric complex involves both calcium-dependent, and calcium-independent binding forces, and that C1r and C1s interact through their respective A chain which, in the case of C1s, is hidden upon association.

Purification of proenzymic and activated human C1s free ofC1r. Effect of calcium and ionic strength on activated C1s.

1. A rapid method for the purification of the proenzymic and activated forms of C1s is presented. In the case of proenzymic C1s, di-isopropyl phosphorofluoridate (0.5--5 mM) is added at all stages of the purification procedure, which includes euglobulin precipation followed by DEAE-cellulose chromatography and affinity chromatography on anti-C1r IgG-Sepharose 6B. The final step completely removes contaminant traces of C1r and/or C1r, ensuring that the final preparation of C1s is stable in the proenzyme form and suitable for activation studies. 2. The apparent molecular weight of C1s and C1s determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis is 85 000 +/- 2000. Reduction followed by alkylation of C1s gives two fragments of apparent molecular weights 57 000 and 28 000. Results of N-terminal amino acid determination and labelling with di-iso[3H]propyl phosphorofluoridate are consistent with previous reports. 3. The influence of calcium and ionic strength on the structure and activity of C1s has been investigated. Calcium leads to a shift of the sedimentation coefficient from 4.3 to 5.6 S, whereas variation in ionic strength has no effect on this parameter. The thermal inactivation curve is profoundly modified both by calcium and ionic strength. In contrast, the esterase activity is only slightly influenced as judged from the absence of gross modification of Km and V.

Interaction of C1-inhibitor with the C1r and C1s subcomponents in human C1.

1. Insoluble IgG-ovalbumin aggregates were used to bind and activate C1 from human serum. The bound C1 provided a useful reagent for studying the interaction of C1 subcomponents with C1-inhibitor. 2. C1-inhibitor bound to both subcomponents (C1r and C1s in C1 and formed stable complexes of respective apparent molecular weights 197,000 and 185,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The binding reaction proceeded more readily with C1s than with C1r and was correlated with the inhibition of C1s esterase activity. 3. At physiological ionic strength, binding of C1-inhibitor to subcomponents C1r and C1s caused release of these subcomponents from the C1-immune aggregates complex, indicating that C1-inhibitor binding decreased the inter-subcomponent binding forces in C1. At low ionic strength, however, this release did not occur.

A study of a covalent-like interaction between soluble nascent C4b and C4-binding protein.

In the classical pathway of complement, the interaction between C4b and C4bp can be considered as a control of the C3 convertase formation. Purified C4-binding protein (C4bp) interacts with soluble nascent C4b to form covalent-like complexes; the interaction is also possible with nascent C4b-like C4, but not with C4, C4b or C4b-like C4. Formation of the complexes upon incubation of C4bp, C4 and C1s appears to involve a single link between a subunit of C4bp and the alpha' chain of C4b, as observed by SDS-polyacrylamide gel electrophoresis in reducing conditions (160 000 dalton band). In non-reducing conditions, a mixture of C4b-C4bp complexes is observed as a function of the C4b:C4bp molar ratio, with apparent molecular weights differing by a value of 210 000 and reflecting different C4b-C4bp associations. A maximum of five molecules of C4b are bound per molecule of C4bp, which appears to consist of 10 subunits of apparent molecular weight 72 000. The link between C4b and C4bp is partially destroyed by 1 M hydroxylamine at pH 9.0; its formation is strongly inhibited by 3.5 mM hydroxylamine or 60 mM methylamine at pH 9.0. These findings suggest an ester or amide bond between the activated carboxyl group of the thioester bridge in the alpha' or alpha chain of nascent C4b or C4b-like C4 and a hydroxyl or amino group of C4bp. Thus, C4bp might compete with other C4b acceptors such as membranes or IgG.

Fluid phase activation of proenzymic C1r purified by affinity chromatography.

1. Proenzymic C1r was purified from human plasma in a two-step technique involving indirect affinity chromatography on Sepharose Ig anti-C1s. The capacity of C1r to monomerize at pH 5.0 and to redimerize at neutral pH was used for selective elution of C1r. The yield in purified C1r was 39% from plasma; no trace of contaminating serine proteases was detected from [3H]diisopropyl phosphorofluoridate labelling of C1r. 2. C14 was able to undergo a two-way autoactivation: an intramolecular catalytic process catalysed by proenzymic C1r itself and an intermolecular reaction catalysed by activated C1r formed in the process of the reaction. DFP (5mM) and C1 Inh at a C1 Inh/C1r ratio of 1:1 were effective on the solely intermolecular activation, leading to partial inhibition of the autoactivation from proenzymic C1r: C1r formed during the activation was titrated by the inhibitors. Calcium, high ionic strength or acid pH decreased C1r activation. The pH effect was characterized by a slowed-down reaction below pH 6.0 and no net influence at values as high as 10.5. The two types of activation developed similarly as a function of pH. 3. Peripheral iodination of C1r revealed differences in label distribution between proenzymic (A chain moiety 48%, B chain moiety 52%) and activated C1r (A chain 20%, B chain 80%). Two different conformational states of C1r were also suggested by 125I-labelling at different temperatures.

Neutron scattering studies of subcomponent C1q of first component C1 of human complement and its association with subunit C1r2C1s2 within C1.

Neutron scattering studies are reported on subcomponent C1q of component C1 of human complement, and on C1, the complex of C1q with subunit C1r2C1s2. For C1q, the molecular weight was determined as 460,000. The radius of gyration at infinite contrast RC is 12.8 nm. The RC values for the proteolytically cleaved forms of C1q, namely the heads and the stalks, are 1.5 to 2 nm and 11 nm, respectively, and thus the axis-to-arm angle of C1q is estimated at 45 degrees. Neutron data for subunit C1r2C1s2 are published elsewhere. The neutron data on C1 lead to an RC value of 12.6 nm for proenzymic C1 and a molecular weight of 820,000. The wide-angle scattering curve of C1q exhibits a minimum at Q = 0.28 nm-1 and a maximum at 0.39 nm-1; on the addition of C1r2C1s2, this minimum disappears. The neutron data on C1 indicate that C1q and C1r2C1s2 have complexed with a large conformational change in one or both parts. No conformational changes can be detected on the activation of C1 by this method.

Neutrophil activation by anti-beta 2 glycoprotein I monoclonal antibodies via Fc gamma receptor II.

Murine monoclonal antibodies (mAbs) to human beta 2-glycoprotein I (beta 2GPI), a plasma protein required for the binding of some antiphospholipid antibodies, have been shown to possess lupus anticoagulant properties and to activate platelets via Fc gamma receptor (Fc gamma R) crosslinking. Here we investigated their ability to induce polymorphonuclear leukocyte (PMN) functional responses. The six mAbs (IgG1 isotype) tested in combination with beta 2GPI led to a concentration-dependent activation of human PMNs as appreciated by granule release, H2O2 production, and cytosolic Ca2+ increase. This activation process was accompanied by the enhancement of PMN-mediated heparan sulfate loss from the endothelial cell line EA.hy 926 without evidence for cell lysis or detachment. F(ab')2 fragments of one of the mAbs bound to PMNs in a beta 2GPI-dependent manner but were devoid of activating effects. Carbamylated beta 2GPI was unable to mediate PMN-antibody binding and subsequent activation. In addition, cationization of beta 2GPI or removal of its sialic acid groups led to higher efficiency in binding to the PMN surface and triggering activation in comparison with the untreated protein. Thus, the process of PMN activation depends on mAb binding to these cells through both Fab (via beta 2GPI) and Fc domains, as confirmed by the suppression of all responses upon treatment with an anti-Fc gamma RII, but not anti-Fc gamma RIII, antibody. Our data suggest a model of cellular activation by beta 2GPI-dependent antiphospholipid antibodies.

Analysis of low molecular weight intracellular associations of a human mannan binding lectin (MBL).

Human mannan binding lectin (MBL) is a member of the collectins, a group of proteins that contain a dual structure with a lectin and a collagenous moieties. The collectins are considered as major actors of innate immunity. We report the presence of low molecular weight intracellular MBL forms in human hepatocytic cell lysates, with binding capacities associated to its lectin and/or its collagen moiety. Competition with D-mannose and with antibodies directed against the lectin binding site of MBL indicate that the 60 kDa form represents an intracellular association of MBL through its lectin moiety. The effects of collagenase or MBL associated serine proteases (MASPs) from a MBL deficient plasma, gave evidence that the 60 KDa form contains also collagen and suggested the binding of a ligand to this collagen part. These results show that this intracellular form of MBL shares binding properties with circulating MBL. The binding potential of the lectin and the collagenous parts of precursor forms of intracellular MBL may suggest that they behave as molecular chaperone. The complexity of MBL structure and functions deserves further investigation on other intracellular forms of MBL.

OH. treatment of tetanus toxin reduces its susceptibility to limited proteolysis with more efficient presentation to specific T cells.

At inflammatory sites, before their processing, antigens are exposed to oxygen free radicals released by activated cells. The effect of hydroxyl radicals (OH.) on the structure of a protein antigen, tetanus toxin (TT) was investigated, as well as the consequences on processing and presentation. A chemical system composed of Fe-EDTA, ascorbate and H2O2 was used to produce physiological amounts of OH. radicals. TT exposed to OH. radicals presented a marked decrease of its intrinsic fluorescence with a concomitant increase of the content of bityrosine, but no fragmentation of the protein was detected by SDS-PAGE. Processing of the modified TT was analysed, by incubating TT at acidic pH with fractions enriched in plasma membranes and lysosomes obtained from a lymphoblastoid cell line (LCL). Proteolysis of OH.-treated TT was less important than proteolysis of native TT, especially upon prolonged incubations. Oxidized TT presented by LCL cells induced a greater proliferation of three different TT specific T cell clones, compared to native TT. When proteolytic digests of TT were presented by fixed LCL cells to a homologous T cell line, the proliferative response obtained in the presence of digests of OH.-treated TT was sustained, even in the case of prolonged proteolysis, whereas the response to digests of native TT fell rapidly. The relative resistance of OH.-treated TT to proteolysis appears thus responsible for its greater presentation to specific T cells, probably by protecting epitopes.

Covalent binding of non-proteolysed C3 to Jurkat T cells.

Purified C3 binds covalently to Jurkat T cells upon incubation at neutral pH. This binding does not appear to involve proteolysis of C3; it leads to high-molecular-weight associations, preferentially through ester linkages, which are disrupted upon incubation with hydroxylamine at alkaline pH. Part of the association also appears to involve disulfide links between C3 and Jurkat cells. Similarly, plasma membranes purified from these cells bind C3 with no evidence for proteolysis of C3. Binding of C3 appears to be "catalysed" by Jurkat cells, and is not due to the well-known spontaneous hydrolysis of C3. Binding of C3 involves hydrolysis of its thioester bond, as titratable--SH groups are available in soluble C3 after incubation of purified C3 with Jurkat plasma membranes; loss of C3 haemolytic activity confirms this finding. These observations give evidence for the binding of C3b-like C3 to Jurkat cells, conferring on these cells the potential to interact with other complement receptor-bearing cells such as B cells.

Major involvement of cathepsin B in the intracellular proteolytic processing of exogenous IgGs in U937 cells.

Monoclonal antibodies used for diagnostic and therapeutic purposes behave as antigens when injected into patients. They are recognized by T cells in a processed form and in a major histocompatibility complex class II restricted fashion. Monoclonal murine IgG2a were used as a model to analyse the early phase of antigen processing in U937 cells. IgG2a prebound to cell surface Fc receptors were rapidly internalized in the cells. During internalization, they were proteolysed with a time-dependent intracellular accumulation of 26, 25, 24, 22 and 14 kDa fragments. Comparison of in vitro IgG2a proteolysis by U937 subcellular fractions or by purified cathepsin B and their intracellular processing indicated that a major cathepsin B like protease is responsible for IgG2a intracellular processing in endo-lysosomal compartments of U937 cells.

Associated complement C3b. Towards an understanding of its intracellular modifications.

Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.

C3b covalently associated to tetanus toxin modulates TT processing and presentation by U937 cells.

Complement protein C3, like C4 and alpha 2-macroglobulin (alpha 2M), is a potentially bivalent ligand: (1) its proteolytic fragment, C3b, is able to interact covalently with antigens, and (2) this bound fragment is able to interact non-covalently with specific complement receptors of antigen presenting cells (APC). The formation of antigen-C3b complexes frequently occurs in vivo at inflammatory sites during the early stages of an immune response. Tetanus toxin (TT)-C3b covalent complexes, prepared from purified proteins, were used to study how C3b association influences the handling of TT by U937 cells used as APC. TT-specific T cell proliferation following TT-C3b processing was observed at a concentration when TT alone was inefficient. Whereas TT pinocytic uptake was low, TT-C3b uptake, through the help of complement receptor CR1, was three times higher. Free TT was rapidly transported to the lysosomes where it was proteolysed, whereas TT-C3b complexes were first retained in the endosomes and underwent only limited proteolysis. While the ester link of the complexes was fairly stable in the endosomes, it was gradually hydrolysed in the lysosomes with ensuing efficient proteolysis of the two proteins. This reflects the fact that associated C3b escorts TT during intracellular trafficking in the APC, and influences antigen processing. A triple role of C3b escorting antigen residues at the level of antigen uptake, routing, and proteolysis inside U937 cells, thus modulating antigen-dependent T cell proliferation.