Immunofluorescence findings in haematoporphyrin-induced keloid

A case of haematoporphyrin-induced keloid is described in which immunofluorescence (IF) revealed antibodies eluted from circulating lymphoid cells directed against fibroblasts. The antibodies eluted belonged to the main Ig classes IgD and IgM and they did not bind complement in vitro. Their possible activity in stimulating fibroblasts is discussed.     

Distribution patterns of cytoplasmic microtubules in epidermal keratinocytes

The distribution of cytoplasmic microtubules in cultured guinea-pig keratinocytes was investigated using immunofluorescence (IF) microscopy with monospecific anti-tubulin antibodies and electron microscopy (EM). In culture, adherent cells displayed networks of thin fluorescent fibres, while a homogeneous and/or granular cytoplasmic IF was shown in the cells of upper layers as well as in trypsinized cells. By EM many microtubules were shown in adherent cells but there were fewer or none in the upper layers. An increase in calcium ion (Ca²⁺) concentration and the addition of an ionophore (X537A) to the culture medium caused disassembly of microtubules. This effect was cancelled by a calmodulin inhibitor. Cryostat sections of normal human and guinea-pig epidermis stained with anti-tubulin antibodies showed a homogeneous and/or granular cytoplasmic IF from basal to granular layers but no detectable IF was seen in the horny layer. These results suggest that keratinocytes contain a cellular pool of tubulin in various states of polymerization and that microtubule disassembly may occur during differentiation, probably being regulated by Ca²⁺-calmodulin complexes.     

不同底物检测抗核抗体的研究

以大鼠器官印片、人癌印片和人鳞状上皮癌细胞(HEp-2)作底物,采用间接免疫荧光法在59例各种结缔组织病患者中研究抗核抗体.人癌印片检测ANA的敏感性和特异性均高于动物器官底物,人癌印片所见的着丝点抗体经HEp-2细胞底物证实.     

Immunoglobulin-bearing lymphoid cells in primary syphilis. Quantitative and elution studies.

The delay in antibody production in response to infection with Treponema pallidum may be caused by a block in the differentiation of antigen-stimulated B (Bursa-dependent) lymphoid cells towards plasma cells. This hypothesis was tested by a study to detect clonal expansion of immunoglobulin-bearing B lymphoid cells by in-vitro immunofluorescence tests in patients with primary syphilis. In addition, antibodies eluted from circulating lymphoid cells were investigated for treponemal binding by the enzyme-linked immunosorbent assay, the T pallidum immobilisation test, and the immunoglobulin class-specific FTA-ABS test. Results indicated that the number of IgG-bearing lymphoid cells were increased in patients with primary syphilis. However, in only a few cases could antitreponemal antibodies be eluted from isolated lymphoid cells. For this reason, the original hypothesis was rejected.     

Immunoglobulin-bearing lymphoid cells in primary syphilis. Quantitative and elution studies

The delay in antibody production in response to infection with Treponema pallidum may be caused by a block in the differentiation of antigen-stimulated B (Bursa-dependent) lymphoid cells towards plasma cells. This hypothesis was tested by a study to detect clonal expansion of immunoglobulin-bearing B lymphoid cells by in-vitro immunofluorescence tests in patients with primary syphilis. In addition, antibodies eluted from circulating lymphoid cells were investigated for treponemal binding by the enzyme-linked immunosorbent assay, the T pallidum immobilisation test, and the immunoglobulin class-specific FTA-ABS test. Results indicated that the number of IgG-bearing lymphoid cells were increased in patients with primary syphilis. However, in only a few cases could antitreponemal antibodies be eluted from isolated lymphoid cells. For this reason, the original hypothesis was rejected.     

Lymphotoxin, tumor necrosis factor, and gamma interferon are cytostatic for normal human keratinocytes

The effects of crude lymphokine-enriched supernatants, purified recombinant lymphotoxin (LT), tumor necrosis factor-alpha (TNF), and gamma interferon (gamma IF) on proliferating human keratinocytes were assessed using two in vitro culture systems. Activated splenocyte supernatants inhibited keratinocyte colony growth on fibroblast feeder layers and arrested basal keratinocyte DNA synthesis within 24 h. Purified recombinant LT, TNF, and gamma IF inhibited cell proliferation in serum-free medium without noticeably affecting viability. Cytostasis was dose-dependent (up to 90% with LT or TNF and 99% with gamma IF) and was maximal within 24-36 h. Specific antibodies neutralized TNF- and gamma IF-mediated cytostasis. Combined treatment with LT (or TNF) and gamma IF increased the degree of cytostasis, particularly at low lymphokine concentrations. Maximum inhibition of DNA synthesis and the duration of exposure required for this inhibition were comparable for LT and TNF and differed for gamma IF. Each of these lymphokines induced cell enlargement, flattening, and vesiculation, with gamma IF apparently more potent in this respect than LT or TNF. Fusiform keratinocytes with diffusely distributed cytokeratin were observed after prolonged treatment with gamma IF alone or gamma IF plus either LT or TNF. Flow cytometric studies of lymphokine-treated keratinocytes indicated that LT, TNF, and gamma IF could enhance beta-2 microglobulin expression 1.5-fold to threefold, whereas only gamma IF induced class II antigens. Staining for class II and beta-2 microglobulin was reduced on cells treated with high concentrations of gamma IF compared with either optimally treated or untreated cells. The potential relevance of these findings to cutaneous immune defense and disease is discussed.     

Anti-endomysial antibodies. A serologic marker of dermatitis herpetiformis

Direct immunofluorescence (IF) studies of skin biopsies are of value in the diagnosis of most, but not all, cases of dermatitis herpetiformis (DH). Similarly, histologic studies are of help but may be questionable or completely nonspecific. Serologic studies for the presence of IgA-class anti-endomysial antibodies are very specific and are found in 70% of patients with DH and in all untreated patients with celiac disease. The titers of these antibodies are directly associated with the degree of gut disease in these patients. Thus, the presence of these antibodies even in the absence of classic direct IF and histologic findings are diagnostically important. We encountered three cases in which both direct IF and histologic studies were equivocal toward confirming the clinical diagnosis of DH. Serologic studies for the presence of IgA-class anti-endomysial antibodies provided evidence for the diagnosis of DH, and, in each case, results were confirmed by further direct IF studies. Since these antibodies are disease specific for DH and celiac disease and are found in most active cases of DH, they may be considered an adjunct to the direct IF and histologic studies of the skin.     

Intercellular and circulating antibodies in patients with dyskeratosis follicularis, Darier's disease.

Skin biopsies from 6 patients, and biopsies of the palatal mucosa of 4 of these patients with dyskeratosis follicularis (DF) (Dariers disease) were examined for in vivo bound antibodies by means of a direct immunofluorescence (IF) technique. Antibodies located in the intercellular substance of the epidermis were found in the skin lesions of all patients. Immunoglobulins of the classes IgG, IgA and IgM as well as C3 were found in all lesions. No antibodies reacting with the palatal mucosa were found. Sera from 6 patients with DF and 10 control persons were tested by an indirect IF technique for circulating antibodies. Guinea pig lip and normal oral mucosa and skin were used as antigens. All patients sera and one control serum reacted with the basal cell of the guinea pig lip. Three DF sera--but no control sera--reacted with the basal cells of human oral mucosa. None of the sera reacted with human skin.     

Immunologic sequelae of thermal injury autoantibodies in acute adult burn patients

Summary Indirect immunofluorescent (IF) studies have demonstrated the presence of antinuclear antibodies (ANA) and antibodies reactive with the intercellular area (IC), basement membrane zone, and basal cells of autologous and allogeneic unburned skin in the sera of acute adult burn patients. Further demonstration in burn patients of the occurrence of peripheral ANA masquerading as IC antibodies is presented. In keeping with previous IF identifications of autoantibodies in burn patients, there was no significant relationship between the frequency and titer of these antibodies according to the extent of total body surface burn area and third degree burn area. The possible role of epithelial antibodies in the maintenance of host resistance, as previously suggested, is considered in brief.Presented in part at the workshop on the Immune Consequences of Thermal Injury, Lake Arrowhead, California, 2–5 December, 1979     

Hemidesmosomes, collagen VII, and intermediate filaments in basal cell carcinoma

We have undertaken an analysis of hemidesmosomes (HD) and their associated structures, intermediate filaments (IF) and anchoring fibrils (AF), in various types of basal cell carcinoma (BCC). Using a combination of electron microscopy and immunofluorescence microscopy we show that there is a correlation between the loss of HD and tumor type (i.e., in solid and infiltrative BCC hemidesmosomes are present, sometimes in reduced numbers), while there appears to be a lack of hemidesmosomes in cells of sclerosing specimens. Moreover, even though there is a loss of cytoplasmic constituents of the HD in sclerosing forms of BCC, this is not the case with regard to collagen VII, a component of AF, which are normally associated with the extracellular side of the HD. Collagen VII is localized to the basement membrane zone of tumor cells in the absence of the cytoplasmic constituents of HD. Furthermore, deposits of collagen VII occur in the connective tissue close to tumor cell populations in all but one of the BCC specimens we analyzed. In addition to modifications in HD and AF in BCC tissue, there are changes in the cytoskeletal elements of both tumor cells and the normal appearing epidermis that overlies tumor areas. In sclerosing BCC microfilaments are commonly observed along the basal portions of tumor cells where they abut the connective tissue. IF are often found interacting with these microfilaments. Indirect immunofluorescence analysis of tumor tissue using a monoclonal keratin antibody preparation, AE1, which in normal epidermis stains basal cells, reveals that AE1 antibodies only weakly stain tumor cells. Moreover, in the epidermis that overlies tumor cell regions AE1 antibodies stain suprabasal cells and not basal cells. This change in staining pattern generated by AE1 antibodies appears to depend upon the proximity of tumor cells. These results are discussed in relation to the organization of the HD and its associated AF and IF. The possibility that HD, IF, and AF antibody preparations may be of diagnostic use is raised.     

Possible drug-induced pemphigus-like antibodies with the clinical manifestation of erythema multiforme

A patient with bullae and target lesions on the extremities and mucous membranes was seen with the clinical picture of erythema multiforme following an episode of pneumonia and a course of penicillin G potassium and tobramycin sulfate therapy. An unusually high titer of intercellular circulating (IC) antibodies was identified in the serum by indirect immunofluorescence (IF) microscopy, but direct lesional IF microscopy study results were negative. These IC antibodies were not true pemphigus antibodies and can best be termed pemphigus-like antibodies. These antibodies were characterized by their ability to fix complement, in contrast to pemphigus antibodies, which apparently fail to do so.     

A Case of Pemphigus Herpetiformis with Only Immunoglobulin G Anti-Desmocollin 3 Antibodies

Pemphigus represents a group of autoimmune blistering diseases caused by autoantibodies against desmogleins (Dsgs), a class of desmosomal cadherins. Recently, several pemphigus patients only with desmocollin (Dsc) 3-specific antibodies have been reported. Here, we report a case of pemphigus herpetiformis (PH), where only anti-Dsc3-specific antibodies but not anti-Dsg antibodies were detected. A 76-year-old woman presented with a 3-year history of blister formation. Physical examination revealed pruritic erythemas with vesicles on the trunk and legs, but no lesions of the oral mucosa. A skin biopsy specimen revealed intraepidermal blister containing neutrophils, eosinophils, and lymphocytes. Direct immunofluorescence (IF) showed immunoglobulin G (IgG) and complement 3 (C3) depositions on the keratinocyte cell surfaces. Indirect IF showed IgG anti-keratinocyte cell surface antibodies. These findings hinted at a diagnosis of pemphigus. However, repeated enzyme-linked immunosorbent assays (ELISAs) for both anti-Dsg1 and 3 antibodies proved to be negative. Immunoblotting of normal human epidermal extracts revealed Dsc antibodies, and recently established ELISAs using human Dsc1-Dsc3 recombinantly expressed in mammalian cells detected anti-Dsc3 antibodies. Based on these clinical, histopathological, and immunological findings, the patient was diagnosed as PH with only anti-Dsc3 antibodies. Treatment with corticosteroid prednisolone and steroid-sparing agent dapsone accomplished complete clinical remission of the patient.     

Assessment of anti-stratum corneum antibody titres in pustulosis palmaris et plantaris

Titres of anti-stratum corneum (SC) antibodies, determined by an indirect immunofluores-cence (IF) technique, were significantly higher on average in pustulosis palmaris et plantaris (PPP) and pompholyx than in normal controls. By a complement IF technique, the antibodies fixed complement (C₃) in high title in PPP, but to a much lesser degree in pompholyx and normal controls. Direct IF microscopy studies showed the deposition of IgG and C₃ in the SC in some of the biopsy specimens of the pustular lesions. These findings suggest that in PPP anti-SC antibodies may be involved at least in part in activation of the complement system and the subsequent accumulation of polymorphonuclear leukocytes.     

Reactivity of lupus erythematosus antibodies with leukemic helper T cells.

Immunoabsorbent columns, containing membrane fragments of either leukemic "helper" T cells or B cell lymphoblasts, were used to isolate and study antilymphocyte antibodies from plasmas of 2 patients with systemic lupus erythematosus (SLE). Both plasmas contained IgG which bound to and could be eluted from the "helper" T cell column. These antibodies significantly inhibited normal lymphocyte proliferative responses to microbial and histocompatibility antigens. The findings indicate that these SLE plasmas contain immunoglobulins of the IgG class which react with leukemic "helper" T cells and inhibit normal effector T cell function.     

Immunoglobulin deposits in lepromatous leprosy skin. Presence of deposits in apparently uninvolved skin and occurrence of serum antiepithelial antibodies.

Immunoglobulin deposits were detected in ten of 13 biopsy specimens from apparently uninvolved skin of patients with lepromatous leprosy. There were deposits of IgM at the dermoepidermal junction in the skin of five patients, and deposits of IgM along the dermal collagen and elastic fibers in the skin of the other five. The deposits were eluted with acid buffers and high molarity salt solution. Circulating IgG antibodies to intercellular substance of epithelial cells, similar to those present in pemphigus vulgaris, were found in 25% of patients with lepromatous leprosy who were studied. These antibodies appeared to be different from the skin-bound immunoglobulin deposits.     

Studies of developing human hair shaft cells in vitro

Methods for studying aspects of hair formation in vitro have been devised on the basis of isolating developing hair shaft cells. These cells were obtained using a sterile microdissection technique. Plucked anagen follicles were dissected free of surrounding tissues (inner and outer root sheaths), and presumptive hair shaft cells (including germinal epithelia) were cultured directly on mammalian fibroblasts or in media preconditioned by fibroblasts. Specimens were cultured either as dispersions or in whole tissue pieces. Trypsinized whole tissue specimens in culture were sometimes observed to form increased bulk, while dispersed cells appeared to elongate and form larger colonies. In sections of these colonies examined by transmission electron microscopy, intracellular hard keratin intermediate filaments (IFs) together with IF-matrix hard keratin complexes were observed. Radiolabelled cysteine [35S] was added to cultures (3-20 days), showing a continuing but reduced synthesis of hard keratin IF proteins (low-sulfur) over the period of study. Matrix protein (high-sulfur) production was drastically reduced after 3 days. Monoclonal antibodies directed against hair keratin IF components were used in Western transfers and immunofluorescent studies to help assess the specificity of proteins synthesized in culture. Our observations indicate that, with some refinement, the presently described methods enable preparation of hair shaft precursor cells suitable for observing certain hair-forming processes in vitro.     

Substrate specificity of anti-epithelial antibodies of pemphigus vulgaris and pemphigus foliaceus sera in immunofluorescence tests on monkey and guinea pig esophagus sections

The indirect immunofluorescent (IF) reactivity of the pemphigus antibodies in sera of 21 cases of pemphigus vulgaris (PV), 15 cases of pemphigus foliaceus (PF), and 14 cases of Brazilian PF (BPF) was compared on 2 substrates, notably monkey esophagus (ME) sections and guinea pig esophagus (GPE) sections. The IF reactions of the pemphigus antibodies of PV could be distinguished from those of PF or BPF by differences in their reactivity on ME and GPE sections in 98% of the cases examined in this study. In most cases, the pemphigus antibodies of PV cases gave higher titers and stronger IF staining reactions on ME sections, while those of PF and BPF cases gave stronger reactions on GPE sections. In addition, most (13 of 21) PV sera react with the lowest 3-4 cell layers of ME sections, while most (13 of 15) PF sera failed to do so but did react with the upper layers of the sections. Importantly, in 8 of the 50 cases examined by IF, the choice of substrate affected the detectability of the pemphigus antibodies, i.e., 4 of 15 PF and 2 of 14 BPF sera reacted only with GPE and 2 of 21 PV sera reacted only on ME. These research findings point to the need for an evaluation of the combined use of ME and GPE in routine diagnostic studies of pemphigus antibodies.     

Coexistence of bullous pemphigoid and systemic lupus erythematosus.

A vesiculobullous eruption with clinical and histological features of bullous pemphigoid developed in a 28-year-old woman with proven systemic lupus erythematosus (SLE). Serum of this patient contained elevated titers of antinuclear antibodies but basement membrane antibodies could not be detected at first, though they did appear in blister fluid. Normal monkey skin explants cultured on this patient's sera gave positive direct immunofluorescence (IF) at the basement membrane zone (BMZ) for IgG deposits. The use of tissue culture methods may be helpful because of the capacity of this test system to reveal the presence of the antibodies to the BMZ despite the presence of the antinuclear antibodies that appear to interfere with their demonstration in standard indirect IF tests.     

Serologic studies of erythema chronicum migrans Afzelius and acrodermatitis chronica atrophicans with indirect immunofluorescence and enzyme-linked immunosorbent assays

To determine whether antibodies to Borrelia spirochetes were present, sera from 88 patients with uncomplicated erythema chronicum migrans Afzelius (ECMA), from 9 patients with ECMA-related extracutaneous complications and from 26 patients with acrodermatitis chronica atrophicans (ACA) were submitted to an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence (IF) assay. The assays were calculated to be 95% specific. There was good correlation between the IF test with a polyvalent conjugate and IgG ELISA. Of patients with uncomplicated ECMA, 18% were seropositive by IgG ELISA and 11% by IgM ELISA, and 15% showed elevated IF titers. Elevated serum antibody levels of IgG as measured by ELISA and elevated IF titers were found in all patients with extracutaneous complications and in the patients with ACA. Declining IgG titers were observed at follow-up 6-12 months after therapy, but the majority of the patients with ACA were still seropositive.     

Autoimmunity in psoriasis

Immunofluorescence (IF) studies using the test providing information on the reactivity of stratum corneum (SC) antibodies and their in vivo binding have been performed in 193 cases of psoriasis and 89 cases with other dermatoses. It has been shown that: 1. Essentially all fully developed, active psoriatic lesions had IgG deposits in the stratum corneum at the sites of the SC antigen, presumably due to in vivo binding of SC antibodies. 2. In various forms of psoriasis SC antigen sites appeared to be completely or almost completely satured with in vivo deposits of IgG as seen in tests with SC antibodies. 3. In most but not all lesions complement components C3 and/or C4 was found in a comparable pattern in the SC, especially when multiple specimens of single cases were studied. Partial or complete saturation of the SC antigen could be observed by the performance of complement indirect IF tests for SC antibodies in such specimens. 4. In lesions with typical histology of psoriasis the above-mentioned immunologic characteristics appeared to be a constant finding. However, in specimens of recent lesions which had not yet developed typical histology a proportion (2 of 17) were negative both for IgG and complement deposits. In receding lesions weak deposits of IgG were present only in a few specimens and complement deposits were as a rule negative. 5. In a group of 89 control specimens of other dermatoses only occasional cases gave the psoriasiform IF pattern, but about 30% of the specimens gave positive reactions in the SC though these were usually of a different pattern.