Single cell multi-omics characterise discrete human tendon cells populations that persist in vitro and on fibrous scaffolds.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

A chemogenomic approach to identify personalized therapy for patients with relapse or refractory acute myeloid leukemia: results of a prospective feasibility study.

Targeted next-generation sequencing (tNGS) and ex vivo drug sensitivity/resistance profiling (DSRP) have laid foundations defining the functional genomic landscape of acute myeloid leukemia (AML) and premises of personalized medicine to guide treatment options for patients with aggressive and/or chemorefractory hematological malignancies. Here, we have assessed the feasibility of a tailored treatment strategy (TTS) guided by systematic parallel ex vivo DSRP and tNGS for patients with relapsed/refractory AML (number NCT02619071). A TTS issued by an institutional personalized committee could be achieved for 47/55 included patients (85%), 5 based on tNGS only, 6 on DSRP only, while 36 could be proposed on the basis of both, yielding more options and a better rationale. The TSS was available in <21 days for 28 patients (58.3%). On average, 3 to 4 potentially active drugs were selected per patient with only five patient samples being resistant to the entire drug panel. Seventeen patients received a TTS-guided treatment, resulting in four complete remissions, one partial remission, and five decreased peripheral blast counts. Our results show that chemogenomic combining tNGS with DSRP to determine a TTS is a promising approach to propose patient-specific treatment options within 21 days.

Post-remission therapy of adults aged 60 and older with acute myeloid leukemia in first complete remission: role of treatment intensity on the outcome.

Although complete remission (CR) is achieved in 50 to 70% of older fit patients with acute myeloid leukemia (AML), consolidation therapy in this age group remains challenging. In this retrospective study, we aimed to compare outcome in elderly patients treated with different post-remission modalities, including allogenic and autologous hematopoietic stem cell transplantation (HSCT), intensive chemotherapy, and standard-dose chemotherapy (repeated 1 + 5 regimen). We collected data of 441 patients ≥ 60 years in first CR from a single institution. Median age was 67 years. Sixty-one (14%) patients received allo-HSCT, 51 (12%) auto-HSCT, 70 (16%) intensive chemotherapy with intermediate- or high-dose cytarabine (I/HDAC), and 190 (43%) 1 + 5 regimen. Median follow-up was 6.5 years. In multivariate analysis, allo-HSCT, cytogenetics, and PS had a significant impact on OS and LFS. In spite of a more favorable-risk profile, the patients who received I/HDAC had no significantly better LFS as compared with patients treated with 1 + 5 (median LFS 8.8 months vs 10.6 months, p = 0.96). In transplanted patients, median LFS was 13.3 months for auto-HSCT and 25.8 months for allo-HSCT. Pre-transplant chemotherapy with I/HDAC had no effect on the outcome. Toxicity was significantly increased for both transplanted and non-transplanted patients treated with I/HDAC, with more units of blood and platelet transfusion and more time spent in hospitalization, but no higher non-relapse mortality. This study shows that post-remission chemotherapy intensification is not associated with significantly better outcome as compared with standard-dose chemotherapy in elderly patients for whom, overall results remain disappointing.

(R)-GEMOX chemotherapy for unfit patients with refractory or recurrent primary central nervous system lymphoma: a LOC study.

Recurrent primary central nervous system lymphomas (PCNSL) have a very poor prognosis. For young and fit patients, intensive chemotherapy followed by autologous stem cell transplantation could be proposed at relapse. In the other cases (unfit or elderly patients), therapeutic options are limited with no consensual regimen. The poly-chemotherapy by (R)-GEMOX is associated with anti-tumor activity in systemic lymphomas and a favorable toxicity profile. Our objective was to evaluate the activity and tolerance of (R)-GEMOX in PCNSL patients enrolled in the French nation-wide LOC cohort. We retrospectively analyzed all refractory or recurrent patients included in the LOC network who benefited from (R)-GEMOX (rituximab 375 mg/m2, gemcitabine 1000 mg/m2, and oxaliplatine 100 mg/m2). Administration, tolerance, and efficacy data were analyzed. Thirteen patients, treated in five different institutions, benefited from the (R)-GEMOX regimen from February 2013 to August 2017. At the initiation of (R)-GEMOX, median age was 71.4 years old (range, 49.5-82.5) and median Karnofsky performance status (KPS) was 60 (range, 40-80). Seven patients were in second line of treatment whereas the six others were in third line or over. All patients had received methotrexate-based polychemotherapy as first-line treatment except one. Overall response rate was 38% with two complete responses and three partial responses. Median progression-free survival was 3.2 months (95%CI: 0.2-6.2), and median overall survival was 8.2 months (95%CI: 0.6-15.8). Toxicity was mainly hematological including grade ¾ neutropenia (38%), lymphopenia (23%), and thrombopenia (23%). Older age (p = 0.046) and low KPS (p = 0.054) tended to be associated with a worse prognosis. (R)-GEMOX is associated with substantial response rate and favorable toxicity profile in unfit patients with recurrent PCNSL. (R)-GEMOX could be considered to be an additional option in patients with recurrent/refractory PCNSL.

Deletion of GARP on mouse regulatory T cells is not sufficient to inhibit the growth of transplanted tumors.

GARP is a transmembrane protein that presents latent TGF-ß1 on the surface of regulatory T cells (Tregs). Neutralizing anti-GARP monoclonal antibodies that prevent the release of active TGF-ß1, inhibit the immunosuppressive activity of human Tregs in vivo. In this study, we investigated the contribution of GARP on mouse Tregs to immunosuppression in experimental tumors. Unexpectedly, Foxp3 conditional garp knockout (KO) mice challenged orthotopically with GL261 tumor cells or subcutaneously with MC38 colon carcinoma cells did not show prolonged survival or delayed tumor growth. Also, the suppressive function of KO Tregs was similar to that of wild type Tregs in the T cell transfer model in allogeneic, immunodeficient mice. In conclusion, garp deletion in mouse Tregs is not sufficient to impair their immunosuppressive activity in vivo.

[Haematological malignancies (HM) in Basse-Normandie (BN): incidence and clinical and biological characteristics over the period 1997-2005 in patients less than 25 years old]. / Épidémiologie des hémopathies malignes en Basse-Normandie : incidence et caractéristiques cliniques et biologiques chez l'enfant et l'adulte jeune de moins de 25ans (1997-2005).

BACKGROUND: The study was designed to present the incidence of all the haematological malignancies (HM) in Basse-Normandie (BN) over the period 1997 to 2005 in patients less than 25 years old. BN is an administrative region in the North-West of France, composed of three departments: Calvados, Manche and Orne. We extracted data from the Registre régional des hémopathies malignes de Basse-Normandie, a French registry which belongs to the Association of the French Cancer Registries (Francim). METHODS: All the HM were coded using the third edition of the International classification for oncologic diseases (ICD-O-3). We compared the clinical and biological descriptive data in patients less than 15 years old to those of young adults (15-24 years old). RESULTS: A total of 305 new cases of HM were recorded over the period 1997 to 2005. HM were more frequent in men (168 cases) than in women (137 cases). Patients less than 25 years old accounted for 4.1% of all HM cases, whereas patients less than 15 years old and young adults (15-24 years old) represented 2.2% and 1.9% of all cases, respectively. In patients less than 25 years old, the overall world-standardized incidence rates (WSR) were 7.67/100,000 (95% CI: 6.31-9.04) in BN (8.08 [6.15-10.02] for men and 7.24 [5.31-9.17] for women). In patients less than 15years, the overall WSR was 7.38/100,000 (6.23-8.52), with no difference between boys (7.57) and girls (7.17). Acute lymphoblastic leukaemia (ALL) was the most frequent HM, WSR=4.02/100,000 (3.16-4.88) (4.30 [3.08-5.53] in men, 3.73 [2.52-4.93] in women), with similar clinical and biological criteria between patients less than 15 years and young adults. In young adults, the overall WSR was 8.21/100,000 (7.47-8.96), similar between men and women (9.02 [7.93-10.12] and 7.37 [6.35-8.38], respectively). Their highest WSR was obtained for Hodgkin lymphomas (HL): 3.37/100,000 (2.89-3.85), similar between men (3.49 [2.8-4.17]) and women (3.25 [2.58-3.93]). The study did not show any significant difference between the Calvados, Manche and Orne departments (except for HL, which seems more frequent in Manche department for 15-24 years old cases). There was no evidence of an increased risk for ALL in the subdistricts Beaumont-Hague and Les Pieux, which respectively have a nuclear waste reprocessing plant and a nuclear power plant on their territory. The subtype of HM was dependent on age whereas clinical and biological data were the same, whatever the age. CONCLUSION: These results contribute to HM monitoring in an area where the nuclear industry is present and to improve the organization and follow-up of medical care.

[Screening contacts for latent tuberculosis infection (LTBI) using an interferon test in Paris in 2009]. / Dépistage de l'infection tuberculeuse latente (ITL) à Paris à l'aide d'un test interféron chez des sujets contact en 2009.

There are two reasons for screening contacts: one is to identify cases of secondary tuberculosis disease (TB) and the other is to identify new cases of latent tuberculosis infection (LTBI). The tuberculin skin test (TST) and the interferon-gamma-release assay (IGRA) have their limitations when used for the detection of LTBI. They neither allow a definite diagnosis of LTBI nor provide information as to the date of onset. The present study was observational, multi-centre (four centers) and retrospective. Six hundred and one contacts were included. The results of the QFT test showed 88 positive (15 %). Among the 144 index cases, all presented with pulmonary disease and 89 cases were sputum positive. In our series, 101 contacts belonged to the family circle. The four factors that had a significant positive impact on the result of the QFT test were: increasing age, the region of birth of the contact (high incidence areas), both of which may indicate old infection, while contact within the family and sputum positivity of the index case probably indicate recent infection. Only sputum positivity influenced the decision to treat the LTBI. We propose a tool aimed at facilitating the decision making process in QFT positive cases. Estimation of the duration of LTBI should help the physician to decide on the need for preventative treatment as well as a search for factors that increase the risk of progression to TB disease.

[Factors associated with the lack of proposition for HIV-AIDS and hepatitis B and C screening to underprivileged immigrants]. / Facteurs liés à l'absence de proposition de dépistage du VIH-sida et des hépatites B et C aux immigrés en situation de précarité

BACKGROUND: Underprivileged immigrants from endemic areas cumulate risk factors for infections by HIV-AIDS and hepatitis B and C. Free primary care consultations are available to them in the four health centers of the city of Paris. The objective of our study was to identify socio-demographic and medical factors related to the lack of screening proposition for HIV-AIDS and hepatitis B and C to new immigrant patients in these centers in 2003. METHODS: For each disease, the absence of screening proposition was analyzed according to geographical origin, length of stay in France, type of accommodation, type of health insurance and symptom motivating the encounter in logistic mixed models adjusted on sex and age. RESULTS: About 500 patients were included in the analysis. Three-quarters of them were male and from Sub-Saharan Africa. They were 36years old on average. Half of them lived in shelters for homeless or immigrants. Their median stay lasted two years. They rarely came for screening (1%), sometimes for asthenia (6%) and two-thirds of them for uro-genito-digestive signs. The results were similar for the three screenings. The lack of screening proposition was about 45% and varied significantly between physicians. Factors significantly associated with the lack of screening proposition were: coming from non-Sub-Saharan Africa (especially from North Africa and Middle East; OR=1.7 to 3.6) and having a health insurance (OR=2.4 to 2.6) regardless of the disease; being a female (OR=2.0 to 2.3) in the case of hepatitis; and having a length of stay in France greater than or equal to five years (OR=1.9) for hepatitis B. CONCLUSIONS: Our results should encourage practitioners to provide more screening to underprivileged immigrants and draws attention to immigrants from non-Sub-Saharan origin and those with health insurance. Factors that might explain doctor and gender-related variability observed in hepatitis are highlighted.

[Haematological malignancies: incidence in Basse-Normandie, France, for 1997-2004]. / Epidémiologie des hémopathies malignes en Basse-Normandie.

BACKGROUND: The study was designed to present the incidence of all the haematological malignancies in Basse-Normandie over the period 1997-2004. We extracted the data from the "Registre régional des hémopathies malignes de Basse-Normandie (RRHMBN)", a French registry which belongs to the Association of the French Cancer Registries (FRANCIM). All the malignant haematological diseases were coded using the third edition of the International Classification for Oncologic Diseases (ICD-O-3) and the ADICAP classification, a special version adapted in 2001 for haematology. Five thousand five hundred and ten new cases of malignant haematological disorders were registered over the period 1997-2004. Whatever the department constituting the Basse-Normandie (Calvados, Manche and Orne), no significant difference of incidence was detected. In men, the more frequent malignant disorders were non-Hodgkin's malignant lymphomas (NHML) followed by chronic lymphocytic leukemia and other mature neoplasms, myelodysplastic syndromes (MDS), multiple myeloma (MM), myeloproliferative disorders (MPD), acute myeloid leukemias (AML), Hodgkin's lymphomas (HL), Waldenström's macroglobulinemia (WM) and acute lymphoblastic leukemia (ALL). In women, MM is the third more frequent haematological disorders after NHML and lymphocytic leukaemia and other mature neoplasms, MPD, MDS, AML, Hodgkin's lymphomas, WM and ALL. The other haematological disorders are very rare. We provide the incidence for the main haematological disorders and for the first time we also present the incidence of the different subtypes of the Hodgkin's and non-Hodgkin's malignant lymphomas, mature lymphoid neoplasms, MPD and also MDS. These results are useful for the organization and follow-up of medical care. The development of specialized haematology and active protocols can optimize the management of the older patients. A high quality of the collected data remains necessary for a continuous watching and research on patients with malignant haematological diseases.

Cancer incidence and mortality in France over the period 1980-2005.

BACKGROUND: The objective of this study was to provide updated estimates of national trends in cancer incidence and mortality for France for 1980-2005. METHODS: Twenty-five cancer sites were analysed. Incidence data over the 1975-2003 period were collected from 17 registries working at the department level, covering 16% of the French population. Mortality data for 1975-2004 were provided by the Inserm. National incidence estimates were based on the use of mortality as a correlate of incidence, mortality being available at both department and national levels. Observed incidence and mortality data were modelled using an age-cohort approach, including an interaction term. Short-term predictions from that model gave estimates of new cancer cases and cancer deaths in 2005 for France. RESULTS: The number of new cancer cases in 2005 was approximately 320,000. This corresponds to an 89% increase since 1980. Demographic changes were responsible for almost half of that increase. The remainder was largely explained by increases in prostate cancer incidence in men and breast cancer incidence in women. The relative increase in the world age-standardised incidence rate was 39%. The number of deaths from cancer increased from 130,000 to 146,000. This 13% increase was much lower than anticipated on the basis of demographic changes (37%). The relative decrease in the age-standardised mortality rate was 22%. This decrease was steeper over the 2000-2005 period in both men and women. Alcohol-related cancer incidence and mortality continued to decrease in men. The increasing trend of lung cancer incidence and mortality among women continued; this cancer was the second cause of cancer death among women. Breast cancer incidence increased regularly, whereas mortality has decreased slowly since the end of the 1990s. CONCLUSION: This study confirmed the divergence of cancer incidence and mortality trends in France over the 1980-2005 period. This divergence can be explained by the combined effects of a decrease in the incidence of the most aggressive cancers and an increase in the incidence of less aggressive cancers, partly due to changes in medical practices leading to earlier diagnoses.

[Evaluation of four immunoenzymatic tests for detecting Clostridium difficile toxins A and B]. / Evaluation de quatre réactifs immunoenzymatiques pour la recherche des toxines A et B de Clostridium difficile.

Four immunoenzymatic tests for detecting Clostridium difficile toxins A and B were studied: two rapid tests (Tox A/B QUIK CHEK-Techlab and NoviView Toxine-A-Hiss diagnostics) and two Elisa tests (C. difficile TOX A/B II -Techlab and Toxin A+B Elisa Test, Novitec-Hiss diagnostics). The results were compared to those obtained with ImmunoCard Tox A+B -ICTAB (Meridian), C. difficile Toxine A (Oxoid) for rapid test and Elisa Premier A+B Meridian for Elisa. A total of 41 stools and 16 isolates were studied with rapid tests. On stools, the sensitivity and specificity of QUIK CHEK test was 94.1% and 100% respectively compared to the test ICTAB. On the isolates, sensitivity and specificity was 100%. With the Noviview test, the sensitivity on stools and isolates was respectively 88.2 and 85.7% and the specificity was 100% compared to Oxoid. A total of 38 stools were studied with Elisa tests. With Techlab test compared to the test Premier, sensitivity and specificity was 100%. The Novitec test gave five false negative reactions with consequently a sensitivity of 70.6%.

Biodiagnóstico de las diarreas agudas bacterianas y virales / Biodiagnóstico of acute bacterial and viral diarrhea

El diagnóstico de las diarreas agudas bacterianas se realiza mediante la prescripción de coprocultivos típicos u orientados, realizados después del interrogatorio. Después del examen macroscópico y microscópico de las heces (diagnóstico de orientación), el cultivo puede poner en evidencia Salmonella, Shigella, Campylobacter o Yersinia, que serán siempre considerados como patógenos, mientras que, salvo excepción notable, la presencia de E. coli forma parte de la flora cólica comensal. En el niño, los agentes responsables de las gastroenteritis virales son, por orden devreciente de frecuencia, los rotavirus (sobre todo los del grupo A), calicivirus, astrovirus y adenovirus. En el diagnóstico virológico de rutina sólo se detectan rotavirus y adenovirus 40-41. Algunos coprocultivos se realizan en el curso de un cotexto epdemiológico-clínico particular en búsqueda de bacterias enteropatógenas como Vibrio cholerae, Clostridium difficile o E. Coli enterohemorrágico. Este patotipo de E. Coli puede ser responsable del síndrome hemolítico y urémico ligado a la producción de toxinas. Su responsabilidad a menudo se establece por cultivo después de serotipado, por detección de las toxinas producidas o más raramente por métodos serológicos. La diarrea aguda es un fenómeno patológico definido según la OMS por la emisión de al menos tres heces líquidas o blandas al fía con menos de 14 días de antelación. Puede tener diferentes orígene, infecciosos o no. Cuando el origen de la diarrea aguda es infeccioso puede ser bacteriana, viral o parasitaria. Es frecuente en el niño,en el que representa una causa mayor de morbilidad y mortalidad en el mundo. En Frencia, cerca de tres millones de pacientes consultan por una diarrea aguda y sólo del 3 al 4% de estas consultas originan la prescripción de un coprocultivo (1). Es importante para el microbiólogo conseguir informació de los signos clínicos (dolores abdominales, fiebre, aspecto de las heces) y del contexto epidemiológico...

[Schistosoma haematobium among immigrants consulting in municipal free clinics in Paris in 2003]. / La schistosomose urinaire chez des migrants ayant consulté dans des centres médico-sociaux de Paris en 2003.

The "Consultations de Diagnostic et d'Orientation" (CDO) are free medical consultations for precarious populations, proposed by the Health Department of Paris. More than two-thirds of the CDO patients come from Sub-Saharan Africa. Schistosoma haematobium (SH) is one of the most frequent infectious diseases detected within CDO. More than a thousand people have consulted for the first time in CDO in 2003 in one of the municipal free Clinics which proposes this service. Parasitologic test of urine has been performed among 220 patients and found 24 positive results: viable eggs of SH (10.8%). All 24 patients are male, most of them are under 35 years-old and come from the region of the Senegal River that lies in the junction of Mali, Senegal and Mauritania. We want to remind physicians in non-tropical setting to think of SH when they see a patient originating from Africa. To ask him if he presents haematuria and if not, to prescribe a parasitologic test of urine. If all patients from endemic regions had undergone this screening in 2003, we would have detected about 20 more cases of SH. If treated early enough, it could avoid severe uronephrological complications, which are rare but represent a high health care cost (bladder tumor, renal failure).

Identification and characterization of a novel retroviral-like aspartic protease specifically expressed in human epidermis.

Proteases play a pivotal role in epidermal differentiation and desquamation. Separation of a total protein extract from human reconstructed epidermis by two-dimensional gel electrophoresis and subsequent peptide analysis of a specific protein spot identified a new protein exhibiting similarities with the retroviral aspartic protease family. Cloning of the corresponding full-length cDNA revealed an open reading frame encoding for a new protease of 343 amino acids, containing a putative aspartic protease catalytic domain. We named this protein Skin ASpartic Protease (SASPase). RT-PCR and northern blot analysis of various human tissues revealed that SASPase was specifically expressed within the epidermis. Immunohistochemical analysis showed a particularly intense expression restricted to the granular layers, whereas in diseased skin, its expression was changed. Western blot analysis, using a monoclonal antibody, revealed the expression of two forms of the enzyme: a 28 kDa putative proform and the active 14 kDa form. Recombinant truncated SASPase (SASP28) was generated from a prokaryotic expression system in Escherichia coli as a fusion protein with GST. SASP28 degraded insulin and to a lesser extent casein with a pH optimum of 5. As seen for retroviral proteases, an auto-activation processing was evidenced, generating a 14 kDa protein (SASP14). Site-directed mutagenesis inhibited auto-activation of the enzyme. Indinavir, a potent HIV protease inhibitor used in AIDS therapy, had a significant inhibitory effect on rSASPase auto-activation, which could explain its side effects on skin.

[Characterization of an extracellular protease from Clostridium difficile]. / Caractérisation d'une protéase extracellulaire de Clostridium difficile.

Clostridium difficile is an intestinal pathogen, which produces two main virulence factors, the exotoxins A and B. Other bacterial structures have been implicated in the colonization of the gastrointestinal tract, which is the first step of the pathogenic process. C. difficile expresses adherence factors and also, displays some surface-associated proteolytic activity, which could play a role in the physiopathology of this bacterium. The aim of this work was to study the protein named Cwp84 which displays significant homologies with many cysteine proteases. The coding catalytic domain of this protein has been cloned in the expression system pGEX-6P-1, as an in-frame fusion with the gluthatione S-transferase, and subsequently purified. The purified fraction showed proteolytic activity on gelatine and BAPNA, but not on azocoll, suggesting a highly selective substrate specificity. The results obtained from inhibition experiments confirmed that Cwp84 belongs to the cysteine protease family. Cwp84 could play a role in degrading some specific host proteins or in the maturation of surface-associated bacterial proteins.

Analysis of proteins with caseinolytic activity in a human stratum corneum extract revealed a yet unidentified cysteine protease and identified the so-called "stratum corneum thiol protease" as cathepsin l2.

Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was to investigate if human stratum corneum contains proteases different from the above, exhibiting similar properties. After gel filtration, we identified four distinct proteolytic activities in a human stratum corneum extract, a cathepsin-E-like activity (80 kDa), a cathepsin-D activity (40 kDa), a yet unknown cathepsin-L-like form (28 kDa) exhibiting the highest caseinolytic activity, and a chymotrypsin-like protein (24 kDa) containing the acidic activity of the well described stratum corneum chymotryptic enzyme. We named the new 28 kDa protease stratum corneum cathepsin-L-like enzyme. Characterization of stratum corneum cathepsin-L-like enzyme provided clear evidence that this new protease, despite its membership to the cathepsin-L-like family, is distinct from cathepsin L and from the recently described stratum corneum thiol protease. Its ability to hydrolyze corneodesmosin, a marker of corneocyte cohesion, was in favor of a role of stratum corneum cathepsin-L-like enzyme in the desquamation process. A more detailed analysis did not allow us to identify stratum corneum cathepsin-L-like enzyme at the molecular level but revealed that stratum corneum thiol protease is identical with the recently described cathepsin L2 protease. Reverse transcription polymerase chain reaction studies and the use of a specific antibody revealed that, in contrast to earlier reports, expression of stratum corneum thiol protease in human epidermis is not related to keratinocyte differentiation. Our results indicate that the stratum corneum thiol protease is probably expressed as a pro-enzyme in the lower layers of the epidermis and in part activated by a yet unidentified mechanism in the upper layers during keratinocyte differentiation.

Purification and characterization of the endoglycosidase heparanase 1 from human plantar stratum corneum: a key enzyme in epidermal physiology?

A protein exhibiting endoglycosidase activity was purified from plantar stratum corneum to apparent homogeneity in two sequential column chromatographic steps. Protein sequencing revealed its identity with the recently cloned human heparanase 1, an enzyme, the expression of which is reported to be related to the metastasic potential of tumor cells. By using a heparanase 1 specific antibody we were able to demonstrate that, in the plantar stratum corneum, heparanase 1 exists in two forms, the active 50 kDa protein and the inactive 63 kDa form, probably a proform of the enzyme. The antibody also decorated numerous degradation fragments. Reverse transcription polymerase chain reaction studies as well as immunohistochemical analysis using reconstructed and normal human epidermis demonstrated clearly a keratinocyte differentiation related expression of heparanase 1. Interestingly, the antibody also strongly decorated dendritic cells, which after double labeling could be identified to be a subpopulation of the epidermal Langerhans cells. Based on our findings and the known history of this enzyme, we advanced the hypothesis that heparanase 1 has multiple physiologic functions in the epidermis: (i) it plays an important role in epidermal differentiation, possibly by modulating the liberation of heparan sulfate bound (growth) factors; (ii) in the stratum corneum, the endoglycosidase activity of heparanase 1 might be indispensable and represent the first step in the desquamation process; and (iii) in Langerhans cells, its catalytic activity is required for the trans-tissue migration of these cells.

Alterations of epithelial permeability by Helicobacter and IL-1beta in vitro: protective effect of rebamipide.

Infection with Helicobacter increases the transcellular passage of macromolecules across the epithelium, and this effect can be prevented by a gastroprotective agent rebamipide. The aim was to gain insight into the mechanisms involved. The HT29-19A intestinal epithelial cells grown on microporous filters as monolayers were incubated in the presence or absence of rebamipide (1 or 2 mM) with: (1) suspension of a wild H. pylori strain, (2) IL-1beta (0.5 ng/ml) + IFN-gamma (2 units/ml). After incubation, the monolayers were submitted to evaluation of apoptosis by using the apoptotic cell death detection ELISA kit and to assessment of epithelial permeability in Ussing chamber where the ionic conductance (G), fluxes of mannitol (J(Man)) and of horseradish peroxidase in both intact (J(HRPi))- and degraded (J(D)) form, were measured. H. pylori increased the intact HRP fluxes across the barrier (J(HRPi) = 17 +/- 20 vs 97 +/- 70 ng/hr/cm2, P < 0.007), an effect prevented by rebamipide (J(HRPi) = 33 +/- 34 ng/hr/cm2, P < 0.006). IL-1beta increased the ionic conductance (G = 5.5 +/- 1.0 and 21.0 +/- 7.0 mS/cm2, P < 0.006), the intact HRP fluxes (J(HRPi) = 18 +/- 15 and 476 +/- 344 ng/hr/cm2, P < 0.006), and the apoptotic index of the cells (AI = 1 +/- 0 vs 3.7 +/- 0.8), all effects prevented by rebamipide (G = 12 +/- 4.9 mS/cm2, J(HRPi) = 79 +/- 38, AI = 1.6 +/- 0.6, P < 0.03 as compared to IL-beta-treated cells). In basal conditions, rebamipide increased the integrity of the barrier (G = 7.5 +/- 2.3 vs 6.0 +/- 1.8 mS/cm2 for controls, P < 0.007). In conclusion, H. pylori as well as IL-1beta, may alter epithelial permeability and rebamipide may exert its protective effect on gastric mucosa by reinforcing the epithelial barrier in normal conditions and by counteracting the deleterious effect of Helicobacter pylori and IL-1beta on macromolecular permeability.

[Prevalence of Clostridium difficile and toxin A in feces of HIV infected patients]. / Prévalence de Clostridium difficile et de la toxine A dans les selles de patients VIH positifs.

Patients with AIDS are immunodeficient, receive multiple antibiotic treatments, occasionally anti-cancer chemotherapy and are often hospitalised; thus they are susceptible to develop a Clostridium difficile infection. The aim of this study was to evaluate the role of C. difficile in diarrhoea in this patient population. Therefore, C. difficile and toxin A which plays a major role in pathogenicity were examined in faecal samples of HIV infected patients. Between January 1991 and June 1992, 102 stool samples from 67 patients were studied. Ninety p. cent of these patients were hospitalised (length > 3 days), 80% had a diagnosis of AIDS stage IV, and 66% had diarrhoea. Nineteen point four p. cent of the patients were carriers of C. difficile. Different associations were found: 1) presence of non toxigenic strains and absence of toxin A in stool samples (6 patients), 2) presence of toxigenic strains and absence of toxin A in stool samples (6 patients), 3) presence of toxigenic strains and toxin A in stool samples (2 patients). None of the patients developed a colitis or pseudomembranous colitis. The carrier rate was identical to those found in other hospitalised populations without AIDS. The prevalence of C. difficile diarrhoea or colitis is low. In this study, AIDS patients do not seem to constitute a risk group for C. difficile intestinal pathology. However, carriers of C. difficile were subjected to strict hygiene rules to prevent nosocomial spread.