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Biomedical subjects

J F Nicolas

Publications and source records attributed to J F Nicolas.

At least 55 records · Page 3Linked to original sources
PubMed1997

Diagnostic value of indirect immunofluorescence on sodium chloride-split skin in differential diagnosis of subepidermal autoimmune bullous dermatoses.

OBJECTIVE: To determine the diagnostic value of indirect immunofluorescence on sodium chloride-split skin (SSS) in differentiating the pemphigoid group of subepidermal autoimmune bullous dermatoses, including bullous pemphigoid (BP), cicatricial pemphigoid, and pemphigoid gestationis, from epidermolysis bullosa acquisita (EBA). DESIGN: Serum samples were tested using immunofluorescence on SSS and immunoblot assay on epidermal and dermal extracts, a recombinant protein corresponding to the C-terminal end of the 230-kd BP antigen, and purified laminin-5. SETTING: An immunodermatology laboratory. PATIENTS: One hundred forty-two serum samples from patients with BP (n = 98), cicatricial pemphigoid (n = 23), pemphigoid gestationis (n = 10), EBA (n = 10), and anti-type IV collagen (n = 1). MAIN OUTCOME MEASURES: Binding sites of serum to the epidermal and/or dermal sides of SSS were correlated with their antigenic specificities. RESULTS: Epidermal staining on SSS was highly specific for pemphigoid. Alternatively, a poor correlation was found for the dermal-reacting serum samples and the diagnosis of EBA; of the 19 serum samples with dermal staining on SSS, only 10 reacted with the EBA antigen. The remaining serum samples were from patients with cicatricial pemphigoid having antibodies to the alpha 3 or beta 3 chains of laminin-5 (n = 5) or patients with BP having antibodies to the 180-kd BP antigen (n = 2). One sample recognized exclusively a 185-kd dermal antigen corresponding to type IV collagen. One more BP serum sample with dermal staining did not recognize any dermal or epidermal antigen. CONCLUSION: In case of immunofluorescent dermal staining, the precise diagnosis should be confirmed by identification of the involved antigen, since it may reveal antibodies to laminin-5 or type XVII or IV collagen, in addition to the EBA antigen.

Antibody Specificity↗
PubMed1997

Autoantibody formation against a 190-kDa antigen of the desmosomal plaque in pemphigus foliaceus.

We report changes in the antigen recognition pattern of sera from two pemphigus foliaceus patients with a long-term follow-up. The patients' sera were analysed by immunoblotting using different antigenic sources: cultured human keratinocytes, bovine tongue epithelium and a recombinant protein corresponding to the C-terminal end of the 230-kDa bullous pemphigoid antigen. While initial serum samples reacted exclusively with the 160-kDa desmoglein 1, the later sera reacted both with desmoglein 1 and a 190-kDa antigen immunolocalized to the desmosomal plaque, previously demonstrated to be recognized by sera of some patients with paraneoplastic pemphigus. IgG subclass analysis further showed that antidesmoglein 1 antibodies were of IgG1 and/or IgG4 subclasses, while anti-190-kDa antibodies were IgG3. The patients were free of malignancy.

Aged↗
PubMed1996

IgE autoantibodies directed against the major bullous pemphigoid antigen in patients with a severe form of pemphigoid.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized in part by circulating and tissue-bound IgG autoantibodies directed against the basement membrane zone. In addition, most of the patients with BP have increased serum IgE levels which seem to be correlated with the disease activity, whereas the presence of circulating anti-basement membrane zone IgE Abs has been reported in some patients. To elucidate whether IgE-dependent mechanisms play a role in the physiopathology of BP, we looked for the presence of IgE Abs specifically directed against the major BP Ag (BPAg1) in sera of BP patients at the onset and after remission of the disease. A radioimmunoassay and a 55-kDa recombinant protein (rBP55) obtained from a cDNA sequence, encoding the C-terminal region of the BPAg1 and containing the BPAg1 immunodominant epitopes, were used. Anti-rBP55 IgE Abs were found in 12 of the 19 sera tested. When the patients were divided into two groups according to the disease severity, anti-rBP55 IgE Abs were found only in patients with a severe form of the disease. Cytophilic IgE was detected on approximately 20% of peripheral blood eosinophils purified from BP patients. Immunohistochemistry studies suggested that some of the IgE-bearing cells in the lesional skin of BP patients are eosinophils. Immunostaining experiments revealed the existence of FcepsilonRI on both peripheral blood and tissue eosinophils. Taken together, these results suggest that IgE-dependent mechanisms could participate in the constitution of the lesions in BP.

Antibody Specificity↗
PubMed1996

Lack of oral tolerance but oral priming for contact sensitivity to dinitrofluorobenzene in major histocompatibility complex class II-deficient mice and in CD4+ T cell-depleted mice.

Oral tolerance is defined by immune unresponsiveness after oral administration of soluble antigens and by antigen-specific inhibition of peripheral immune responses induced by prior antigen feeding. The aim of this study was to investigate the implication of the major histocompatibility complex (MHC) class II presentation pathway to CD4+ T cells in oral tolerance of contact sensitivity (CS) to the hapten dinitrofluorobenzene (DNFB). We used MHC class II knockout (AB0/0) and invariant chain knockout (Ii0/0) mice, which have, respectively, a total or partial defect in class II-restricted activation of CD4+ T cells, as well as normal C57BL/6 mice depleted of CD4+ T cells by injection of a specific antibody. Intragastric administration of DNFB prior to skin sensitization induced specific inhibition of contact sensitivity to DNFB in A beta +/0 and Ii+/0 heterozygotes comparable to that observed in C57BL/6 mice. In contrast, no oral tolerance was observed in either MHC class II-deficient A beta 0/0 and Ii0/0 homozygote mutants or in syngeneic anti-CD4-depleted C57Bl/6 mice. Moreover, a single oral administration of DNFB, without skin sensitization, could prime A beta 0/0, Ii0/0 as well as anti-CD4-depleted C57BL/6 mice for DNFB-specific CS. These findings demonstrate that the class II/CD4 pathway is involved in oral tolerance manifested both as the inhibition of CS by hapten feeding prior to skin sensitization, and as immune unresponsiveness of normal mice to oral administration of hapten. Furthermore, our data provide evidence that a single oral feeding with DNFB is able to prime mice for hapten-specific CS, provided that the class II/ CD4 pathway is bypassed.

Administration, Oral↗
PubMed1996

Identification of a 168-kDa mucosal antigen in a subset of patients with cicatricial pemphigoid.

This study describes the presence of antibodies in sera from patients with cicatricial pemphigoid specific for a 168-kDa antigen expressed by buccal mucosa. Six cicatricial pemphigoid sera unreactive, with epidermal or dermal proteins in immunoblot assay were tested on mucosal protein extracts. Four of these sera labeled a mucosal 168-kDa antigen (M168) under reducing conditions. An additional cicatricial pemphigoid serum with circulating antibodies to 180-kDa bullous pemphigoid antigen (BPAg2) also labeled M168. None of these cicatricial pemphigoid sera reacted with the alpha, beta, or gamma subunits of laminin-5. Nitrocellulose elution studies showed that the M168 antigen is a basement membrane antigen and labeled the epidermal side of salt-split skin. Immunoaffinity-purified anti-M168 antibodies did not bind to the 230-kDa bullous pemphigoid antigen (BPAg1) or to the 180-kDa BPAg2. None of the control sera from healthy individuals or from bullous pemphigoid, pemphigus vulgaris, or pemphigus foliaceus patients reacted with Ml68. This study demonstrates the specificity of some cicatricial pemphigoid sera against a 168-kDa antigen that is different from the laminin-5 subunits and shares no epitopes with the antigens of bullous pemphigoid (BPAg1, BPAg2) or the epidermolysis bullosa acquisita.

Autoantibodies↗
PubMed1996

Suboptimal non-inflammatory concentrations of haptens may elicit a contact sensitivity reaction when used as a mix.

Contact dermatitis is a cutaneous inflammatory reaction mediated by hapten-specific T cells. We used a murine model to investigate the contact sensitivity reaction elicited by different concentrations (optimal and suboptimal) of the haptens DNFB and oxazolone applied singly or in combination. The simultaneous application of DNFB and oxazolone at optimal concentrations (0.2% and 0.4% respectively) did not significantly increase the ear swelling response induced by each of the allergens when applied singly. No contact sensitivity response was observed when the haptens were tested individually at subthreshold concentrations (0.05% and 0.1% respectively). However, mixing the 2 molecules at the same concentrations gave rise to a clinical contact sensitivity reaction. The simultaneous application of the haptens at a 2 x higher, but still suboptimal concentrations (0.1% and 0.2% respectively), elicited an inflammatory response that was significantly greater than the responses elicited by either of the haptens when applied separately. These results suggest that a "false-positive" reaction to a mix may reveal a genuine sensitization to the constituents.

Animals↗
PubMed1996

Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site.

Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence. However, these elements are not useful in the integrated provirus and can be the cause of problems. We have developed a vector which eliminates the majority of these viral elements. This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage. The Cre-lox system is neutral for eukaryotic cells. The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers). Following the LTR-mediated loxP duplication, the LTRs can be recombined by the Cre enzyme. The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced. If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute. If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector.

3T3 Cells↗
PubMed1996

Evidence in the mouse for self-renewing stem cells in the formation of a segmented longitudinal structure, the myotome.

A novel method of clonal analysis has been used in the mouse to define the cellular events that lead to the formation of a segmented longitudinal structure, the myotome. Progenitor cells of the myotome were randomly marked during development by intragenic homologous recombination in transgenic mice expressing a reporter laacZ gene. 153 clones corresponding to 7829 cells, that is 20% of the myotomal population of one embryo, were obtained from 3000 E11.5 embryos. Their analysis leads to the hypothesis that, at E11.5, the 41 segments of the myotome have been mainly produced from a unique, spatially organised pool of self-renewing stem cells that accompanies the formation of the anterior-posterior axis.

Animals↗
PubMed1996

Comparative sensitivity of indirect immunofluorescence to immunoblot assay for the detection of circulating antibodies to bullous pemphigoid antigens 1 and 2.

The sera of 263 patients with bullous pemphigoid (BP) were tested by indirect immunofluorescence (IIF) on salt-split skin (SSS) and immunoblot (IB) assay, in order to assess the diagnostic sensitivity of these techniques. Among the 263 sera tested, 198 sera (75%) contained antibasement membrane zone antibodies demonstrable by IIF reacting to the epidermal (98%) or both the dermal and epidermal sides (2%) of SSS. One hundred and eighty-two of the 263 sera (69%) reacted by IB with BP antigens (Ag), most commonly the BPAg1 (93 cases, 51%), and a complex of BPAg1 and the 180 kDa minor BP antigen (BPAg2) (47 cases, 26%). BPAg2 alone was found in 42 cases (23%). A good correlation was found between the detection of autoantibodies by IIF and labelling of BPAg1 and/or BPAg2 by IB assay, in which 152 of 198 sera with an epidermal pattern in IIF identified a BP antigen. IB analysis of the 65 sera negative by IIF yielded positive results in 30 cases (46%). Thirty-one percent (13 of 42) of sera recognizing by IB BPAg2, were negative by IIF, as compared with 12% (11 of 93) of those recognizing BPAg1 (P < 0.01). Comparing the sensitivity of the two tests, IIF (75%) was found to be more sensitive than IB (69%). Thirty-five of the 263 sera (13%) remained negative by both techniques. It can be concluded from this study that IIF on SSS appears to be a sensitive and reliable assay for screening BP; IB should be performed for the sera that are negative by IIF as it may reveal circulating antibodies, particularly to BPAg2.

Adult↗
PubMed1996

[SLTR, a new family of retroviral vectors for gene transduction].

In this article, we describe two novel methods to modify the genome of mammalian cells. The first method utilizes a new family of recombinant retroviruses (RRV) which leads to transduction in the mammalian genome of, not a complete recombinant retroviruses, but a single LTR (sLTR) that carries the foreign gene. Therefore, all cis viral regulatory elements of the virus, including those necessary for the control of transcription, reverse transcription and integration, are absent from the transduced material. In addition to being safer because of their inability to retrotranspose, the sLTR family of recombinant retrovirus (sLTR-RRV) overcome the problems associated with the presence of viral sequences that negatively control the expression of the transduced material in stem cells.

DNA, Recombinant↗
PubMed1995

Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene.

Contact sensitivity (CS) is a form of delayed-type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed-type hypersensitivity responses, by CD4+ T helper-1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8+, CD4+, or both, T cells. MHC class I single-deficient and MHC class I/class II double-deficient mice, both of which have a drastic reduction in the number of CD8+ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II-deficient mice and normal mice treated with an anti-CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti-CD8 mAb depletion of class II-deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4+ T cells in class II-deficient mice by anti-CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I-restricted CD8+ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II-restricted CD4+ T cells down-regulate this inflammatory response.

Animals↗
PubMed1995

Major antigenic epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein. Evidence using a 55 kDa recombinant protein.

In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M(r) (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.

Animals↗
PubMed1995

Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.

3T3 Cells↗
PubMed1995

In vitro T cell response to staphylococcal enterotoxin B superantigen in chronic plaque type psoriasis.

Recent studies have demonstrated the important role of CD4+ T cells in the pathophysiology of psoriasis. One of the current hypotheses is that triggering of the psoriatic inflammatory process could be secondary to CD4+ T cell activation by bacterial superantigens in the skin. In this study, IL-2-derived T cell lines were recovered from the blood and the skin of 4 patients with chronic plaque type psoriasis and of 2 patients with allergic contact dermatitis (ACD). Blood and skin T cell lines were tested for their ability to proliferate in vitro to staphylococcal enterotoxin B (SEB) presented by MHC class II expressing antigen-presenting cells. The results showed a significantly higher SEB-induced T cell proliferation in skin T cell lines as compared to blood T cell lines in 3 out of 4 psoriatic patients and in one of the 2 ACD patients. No difference between the skin and blood T cells for their response to phytohemagglutinin was observed. Furthermore the blood T cell lines from both patients and control individuals responded equally well to SEB. Thus psoriatic skin T cell lines were characterized by an enrichment in SEB-responding T cells. Since similar enhancement of SEB-responsive T cells was occasionally found in ACD patients, we propose that SEB could be an environmental factor associated with rather than responsible for psoriatic inflammation.

Antigen-Presenting Cells↗
PubMed1995

[Study of epitopic sites of the major bullous pemphigoid antigen (BPAg1)].

Bullous pemphigoid is an acquired blistering skin disease associated with the production of IgG autoantibodies to 230 kDa BP Ag (BPAg1) and to 180 kDa BP Ag (BPAg2). The aim of our work was to better characterize the epitopes of BPAg1. For this, we firstly generated a bullous pemphigoid recombinant protein of 55 kDa Mr (rBP55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Ninety-two percent of sera from patients with autoantibodies to the 230 kDa polypeptide recognized the rBP 55 protein. These results confirm that this 555 aminoacid segment corresponding to rBP 55 contains major epitopes. In order to study the epitopes of the amino-terminal portion of the BPAg1 antigen, we generated immortalized B cell lines secreting human monoclonal antibodies to BPAg1 from two patients whose sera reacted with native BPAg1 but not with the recombinant rBP55 carboxy-terminal peptide. Blocking immunofluorescence experiments and phylogenetic studies showed that these human monoclonal antibodies recognize different epitopes of BPAg1. According to these studies, we can conclude on the wide variety of epitopes recognized by BPAg1 autoantibodies.

Antibodies, Monoclonal↗
PubMed1995

[Pemphigoid mimicking epidermolysis bullosa acquisita].

INTRODUCTION: Subepidermal autoimmune bullous dermatoses form a clinical entity for which there is not always an individualized clinical and pathological description. CASE REPORT: A patient presented with bullous skin disease of atypical nature. There was an almost total desepidermization of the legs, vast areas of erosion on the trunk and arms with a Nikolski sign in an area of healthy skin, buccal involvement, multiple milium cysts and ungueal dystrophies with nail loss. DISCUSSION: This clinical presentation in this patient suggested acquired bullous epidermolysis. However, according to the recently defined clinical criteria for pemphigoid, the probability of correct diagnosis of pemphigoid was greater than 95 p. 100 since nearly three fourths of the major criteria were present. This diagnosis was confirmed by reference techniques (electron microscopy, indirect electron immunomicroscopy and immunoblotting). Thus, bullous autoimmune diseases of the dermoepidermal junction can be reliably differentiated on the bases of the clinical features, together with direct and indirect immunofluorescence on salt-split skin.

Aged↗