IgG4 as the predominant IgG subclass in pemphigoides gestationis.

BACKGROUND: Pemphigoides gestationis (PG) is a blistering disorder of pregnancy caused by antibodies against basement membrane proteins. They are directed against the 180 kD bullous pemphigoid antigen (BPAg2), towards the epitopes within the NC 16A domain. There are many similarities between pemphigoid gestationis and bullous pemphigoid (BP), but the literature so far indicated different immunofluorescence results in regards with C3 and IgG, and IgG subclasses (IgG4 vs. IgG1). METHODS: We evaluated staining patterns and IgG subclasses, as well as C5b-9 membrane attack complex (MAC) in 10 pregnant patients with PG, using sandwich double antibody immunofluorescence (SDAI) and direct immunofluorescence (DIF). RESULTS: All ten specimens stained with C3 by DIF, but only five had trace amount of IgG reactants by this method. By SDAI, 100% were positive for the IgG4 and C5b-9 MAC, 70% for IgG2, 50% for IgG1, and 40% for IgG3. CONCLUSION: IgG4 was the predominant IgG subtype identified. This finding has not been reported for PG, but it mimics results reported for BP. One explanation is prolonged disease course, as well as blocking of antigenic domains by IgG4. Understanding this completely will help develop therapies and prevention strategies for immunobullous and other autoimmune diseases, and perhaps aid in an exact classification.

Anti-adenosine deaminase antibodies in lupus erythematosus.

Adenosine deaminase (ADA) is an enzyme involved in purine metabolism and has a major role in the development and function of lymphoid cells. Congenital deficiency of ADA results in severe immunodeficiency. Patients with congenital ADA deficiency treated with polyethylene glycol-conjugated bovine ADA develop antibodies to ADA. This leads us to investigate the role of anti-ADA antibodies in patients with systemic rheumatic diseases. Commercially available ADA was used in ELISA and immunoblots for detection of anti-ADA antibodies. Four out of 100 patients examined were positive for anti-ADA antibodies. Two of them had peripheral blood lymphopenia but the antibody levels did not appear to correlate with the lymphocyte counts. Immunoblotting revealed that the antibodies recognized a 40 kDa peptide of ADA, corresponding to ADA1, the major component of ADA. Affinity-purified antibodies were used to locate the distribution of ADA on Hep-2 cells and lymphocytes by indirect immunofluorescence. Anti-ADA antibodies gave a distinct nuclear speckled pattern on acetone-fixed cells. With viable cell immunofluorescence, anti-ADA antibodies also stained the cell surface of HEp-2 cells and lymphocytes, indicating surface expression of ADA. The anti-ADA antibodies failed to gain access into the cytoplasm or nuclei when added to the cultures of HEp-2 cells. In summary, this is the first report of detection of anti-ADA1 autoantibody which is a new type of ANA with discrete, speckled nuclear staining, but which may not be associated with lymphopenia.

The relationship between structures and in vitro properties of a polyanhydride implant containing gentamicin sulfate.

Laboratory scale injection-molding equipment was utilized to fabricate an implant consisting of poly(FAD:SA 1:1) and 20% (w/w) gentamicin sulfate. Characterizations were performed to determine the molecular weight and glass transition temperature of poly(FAD:SA 1:1). A study was carried out to investigate the relationships between the in vitro performance, morphology, and micro-structures of the molded implants. It was found that implants produced with different structures exhibited different physical integrities in water, i.e., cracking or non-cracking. For the non-cracking implants, a skin-core structure formed by an oriented skin layer was observed under a polarized light microscope. The same morphology was not seen in the cracking implants. The crystal orientation in the skin layer of the non-cracking implants was further identified using a wide-angle x-ray diffraction method (WAXD). No crystal orientation could be found in the cracking implants by WAXD. Furthermore, studies were carried out to evaluate the in vitro drug release for implants showing different degrees of integrity in water. The in vitro drug release of the cracking implants was markedly faster than that of the non-cracking implants due to the pronounced initial drug-burst effect as a result of crack formation in the implants.

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton.

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. The cell-surface-expressed nucleolin becomes clustered at the external side of the plasma membrane when cross-linked by the nucleolin-specific monoclonal antibody mAb D3. This clustering, occurring at 20 degrees C and in a well-organized pattern, is dependent on the existence of an intact actin cytoskeleton. At 37 degrees C, mAb D3 becomes internalized, thus illustrating that surface nucleolin can mediate intracellular import of specific ligands. Our results point out that nucleolin should also be considered a component of the cell surface where it could be functional as a cell surface receptor for various ligands reported before.

Cyanine fluorochrome-labeled antibodies in vivo: assessment of tumor imaging using Cy3, Cy5, Cy5.5, and Cy7.

Monoclonal antibodies to two different targetable antigens were conjugated to each of four commercially available cyanine fluorochromes. Equal amounts of all four antibodies were coinjected into tumor-bearing animals and imaged. Small, superficial tumors were adequately labeled using all four fluorochromes. Large tumors were labeled well only by Cy7, probably due to self-masking and dilution effects. Cy7 was superior to other cyanine fluorochromes for visualizing structures located deep within the animal.

Bullous pemphigoid in infancy: case report and literature review.

We report a 2-month-old girl who developed bullous pemphigoid on her hands and feet shortly after receiving her routine immunizations. Infantile bullous pemphigoid has a clinical presentation distinct from bullous pemphigoid seen in older children and should be included in the differential diagnosis of blisters involving the hands and feet. Our patient responded well to topical corticosteroid therapy.

Cytotoxic T cells in basal cell carcinomas of skin.

Previous studies have suggested the importance of CD8+ cytotoxic T-cells of hosts against neoplasms. Earlier studies and our previous investigation showed that a majority of tumor infiltrating T-cells in human basal cell carcinomas (BCCs) belonged to CD4+ T-cells. CD8+ cells were also present in the peritumor areas of human BCCs, but in smaller numbers. Published evidence indicates the importance of cytotoxic T-cells in antitumor immunity. Cytotoxic T-cells have been identified by using monoclonal antibodies against various cytotoxic T-cell components. In this study, we used monoclonal antibodies to perforin to evaluate the role of cytotoxic T-cells in the host response against basal cell carcinomas. Perforin-expressing T-cells could be identified in the infiltrate of BCCs in frozen tissue sections, and also in antigen-retrieved paraffin-embedded sections of BCCs, and the presence of perforin-expressing T-cells correlated with the infiltration of CD8+ T-cells. These results suggest that cytotoxic T-cells play a role in host defense against human BCCs.

Atenolol-induced lupus erythematosus.

Atenolol is a beta-blocker commonly used for treating hypertension. It can induce various kinds of adverse side effects, including psoriasiform skin eruptions, skin necrosis, vasculitis, and (rarely) drug-induced connective tissue disease. We encountered a patient receiving atenolol for his hypertension for 3 years who subsequently acquired connective tissue disease and antihistone antibodies. The initial serologic antinuclear antibody test was negative at a dilution of 1/20 but was positive after further serial dilutions, indicating the prozone phenomenon as the cause of the false-negative result. Six months after discontinuation of atenolol, the skin rash disappeared and antihistone antibody subsided. His skin rash reappeared on rechallenge with atenolol for 3 days, confirming that atenolol was responsible for his lupus erythematosus.

Significance of anti-nuclear matrix antibodies in patients with in vivo speckled antinuclear antibody staining.

OBJECTIVE: Evidence suggests that patients with in vivo speckled antinuclear antibody (ANA) patterns have high titers of circulating ANA, specifically anti-U1 RNP antibody. A small percentage of patients with high titers of anti-U1 RNP antibody have anti-nuclear matrix antibodies, and some also demonstrate in vivo ANA. This study was designed to screen for the presence of anti-nuclear matrix antibodies in patients with in vivo ANA. METHODS: Anti-nuclear matrix antibodies were detected by indirect immunofluorescence on HCI-extracted HEp-2 cell substrate, by enzyme-linked immunosorbent assay, and by immunoblot analysis. RESULTS: All 10 patients with in vivo ANA were found to have anti-nuclear matrix antibody demonstrated using HCI-extracted HEp-2 cell substrate, and all exhibited antibody activity to a 36-kd protein from nuclear matrix antigen. CONCLUSION: These results suggest that anti-nuclear matrix antibodies are a major factor in the development of in vivo ANA.

IgG anti-50 kd lymphocyte membrane peptide antibody in patients with Sézary syndrome.

BACKGROUND: Recent evidence suggests that as cutaneous T-cell lymphoma progresses, cell-mediated immunity is reduced and humoral responses are augmented. OBJECTIVE: The present study was designed to compare the IgG response in Sézary syndrome with that in mycosis fungoides. METHODS: The IgG antilymphocyte response was studied in six patients with Sézary syndrome and in 11 patients with mycosis fungoides by means of immunoblot analysis, enzyme-linked immunosorbent assay, immunohistochemistry, and flow cytometry. RESULTS: An IgG antilymphocyte response to a 50 kd peptide was seen in five of the six patients with Sézary syndrome; however, none of the 11 patients with mycosis fungoides expressed this response. CONCLUSIONS: An enhanced IgG immune response to 50 kd lymphocyte peptide may be helpful in identifying disease progression in patients with cutaneous T-cell lymphoma.

Immune-associated cells in basal cell carcinomas of skin.

Increased numbers of mast cells (MCs) and lymphocytes infiltrating in basal cell carcinomas (BCCs) have been observed. The presence of these infiltrating cells has been considered a sign of an immunologic anti-tumor response in the host, but the relationship of these two cell populations has not been examined. To elucidate this possible relationship, 30 non-ulcerated BCCs were analyzed. Frozen sections of the tumors were stained with monoclonal antibodies for Langerhans' cells, lymphocyte subsets and natural killer cells. Fluorescein isothiocynate (FITC)-avidin as well as anti-tryptase and anti-CD45RO monoclonal antibodies were used on formalin-fixed, paraffin-embedded sections for mast cell and T cell identification, respectively. B cells and natural killer cells were rarely observed in these tumors. MCs and T cells were quantified by direct enumeration and expressed as number of cells per high power field (hpf). FITC-avidin and anti-tryptase antibodies were equivalent in their ability to identify MCs. MC content in BCCs ranged from 1.0 to 31 cells/hpf. The number of T cells ranged from 0 to 50 cells/hpf with helper/suppressor cell ratios of 0.2 to 10. There was no correlation between helper/suppressor ratios and mast cell numbers; however, an inverse relationship was observed between the numbers of T cells and the number of mast cells in these tumors. These studies indicate that T cells and MCs are the primary immune cell populations responding to BCCs, and that decreased numbers of T cells are associated with more aggressive tumors.

Internalization of anti-nucleolin antibody into viable HEp-2 cells.

Anti-nucleolin antibodies have been detected in patients with systemic connective tissue diseases (SCTD) including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In vivo bound autoantibodies to nucleoli of epidermal keratinocytes have been demonstrated in skin from patients with SCTD. In this study, monoclonal antibody to nucleolin (D-3) was used to determine the distribution of nucleolin in different culture cells including HEp-2, HepG2, HRCC, Molt-4 and Wil2 cells. Nucleolin was found to be present on the surface of HEp-2 and HepG2 cells, but not on the surface of HRCC and lymphoblastoid (Molt-4 and Wil2) cells; in contrast, nucleolin was detected in the nucleoli of all permeabilized cells examined. In immunoprecipitation, using extracts from 32P-labeled HEp-2 cells as antigenic source, cell membrane as well as nuclear nucleolins were found to be phosphorylated with a molecular weight of 105 kDa. Viable HEp-2 and HepG2 cells were cocultured with IgG fraction of D-3 in a CO2 incubator for 1 to 24 h, and then permeabilized with acetone followed by immunofluorescence staining with FITC-labeled goat anti-mouse IgG antibodies. Nucleolar staining was observed in cells after 10 h or longer of coculture. These data indicated that D-3 antibody reacted with cell membrane nucleolin and subsequently gain access into cells in a process related to pinocytosis.

Antigen retrieval in immunofluorescent testing of bullous pemphigoid.

Direct immunofluorescence is an immunopathological technique frequently utilized for diagnosis of vesiculobullous disease such as bullous pemphigoid. Fresh-frozen tissue is required for immunofluorescent testing, making retrospective analysis difficult. In this study, we compared two methods of antigen retrieval in formalin-fixed, paraffin-embedded skin tissue from patients with bullous pemphigoid to determine if archival tissue, after use of an unmasking antigen, can be substituted for fresh-frozen tissue in the immunopathological study of skin. Paraffin-embedded tissue blocks from patients with bullous pemphigoid and patients with eosinophilic spongiotic dermatitis as the prodromal stage of bullous pemphigoid were obtained. Sections were mounted on poly-L-lysine-coated slides and the slides were deparaffinized. The methods of antigen retrieval included incubation with trypsin (0.1%) and microwave irradiation in urea (6 M). Antigen retrieval was followed by indirect immunofluorescence. Microwave irradiation was more effective in antigen retrieval than was incubation with trypsin (0.1%). Microwave irradiation in urea (6 M) produced more intense immunofluorescent staining than did trypsinization. Overall, positive basement membrane zone immunofluorescent staining was found in 60% of patients with a diagnosis of classical bullous pemphigoid and in 50% of patients with eosinophilic spongiotic dermatitis as the prodromal stage of bullous pemphigoid. Although the frozen-tissue method appeared more effective than the antigen-retrieval method in immunofluorescent testing of skin, the antigen-retrieval method can certainly be considered an option in retrospective studies. Antigen retrieval may be particularly advantageous in patients with eosinophilic spongiotic dermatitis in whom the diagnosis of bullous pemphigoid may not be suspected initially.

Thiazide diuretics induce cutaneous lupus-like adverse reaction.

One of the side effects reported in patients taking thiazide diuretics is photosensitivity. We report two patients who developed lupus-like skin lesions while taking thiazide diuretics. One patient developed erythematous scaling papules, patches and plaques on the upper extremities and trunk resembling subacute cutaneous lupus erythematosus. Histopathology of a skin biopsy from the trunk showed basal cell layer liquefaction and lichenoid interface changes suggestive of lupus erythematosus. The skin lesions resolved completely within two months of discontinuing thiazide therapy. The second patient developed multiple flesh colored urticarial plaques on the trunk one year after beginning thiazide therapy. Slight lichenoid interface changes were noted on a skin biopsy, along with dense mucin deposition in the papillary and deep dermis, suggestive of tumid lupus erythematosus. The skin lesions persisted despite discontinuing thiazide therapy, necessitating systemic corticosteroid treatment. Both patients had circulating anti-SSA/Ro autoantibodies and antinuclear antibodies. These two patients illustrate that thiazide diuretics may induce a cutaneous lupus erythematosus-like adverse reaction and production of anti-SSA/Ro autoantibodies as demonstrated by immunodiffusion, immunoblot and immunoprecipitation testing.

Proliferation cell nuclear antigen and soluble interleukin 2 receptor levels in cutaneous T cell lymphoma: correlation with advanced clinical diseases.

Cutaneous T-cell lymphoma and leukemias (CTCL) are malignant clonal proliferation of T lymphocytes which have a predilection to home to and proliferate in skin. There are no clinical and laboratory parameters which consistently correlate with stage of disease, which varies from patch, plaque, tumor, or erythroderma. Soluble IL-2 receptor (sIL2-R) levels are elevated both in benign and malignant diseases involving immune activation. Proliferation cell nuclear antigen (PCNA) is a marker of the G1 and G/S phases of cell cycle and can be used to quantitate proliferation. We studied 43 skin biopsies of CTCL in various clinical stages for the presence of PCNA via immunoperoxidase techniques to establish a relationship between PCNA and the stage of disease. In addition, sIL2-R levels were determined in 14 patients. PCNA reactivity was detected in the nuclei of infiltrating cells in a total of 25 patients (58%). According to clinical stage there were 2/12 patch (12%), 9/17 plaque (53%), 4/4 tumor (100%) and 9/10 erythrodermic (90%) stage patients with PCNA positive cells. Thus PCNA positivity correlated with advanced clinical stage. sIL2-R levels were elevated in 14 of 14 patients and the degree of elevation correlated with advanced clinical stage of disease and with increased numbers of PCNA positive cells. Immunohistochemical studies for PCNA and serum sIL2-R levels can be used as laboratory parameters to correlate with clinical stage of disease and enhance prognostication in CTCL.

Eosinophilic spongiosis: a clinical, histologic, and immunopathologic study.

BACKGROUND: Epidermal spongiosis with exocytosis of eosinophils (ES) has been reported in biopsy specimens from patients with various dermatoses. Its diagnostic value and the patient's outcome remain poorly understood in those cases in which ES is not associated directly with diagnostic features of a bullous dermatosis. OBJECTIVE: We evaluated the clinical, histopathologic, and immunopathologic findings and their clinical correlation in patients who had ES in a biopsy specimen but who had no evidence of a bullous dermatosis. METHODS: A retrospective study of 150 cases with ES was performed. Clinical, histopathologic, direct immunofluorescence, and subsequent follow-up data were collected to assess final diagnosis and outcome. RESULTS: A total of 144 patients had generalized eruptions; of these, 34 (24%) had autoimmune bullous disease. Fourteen (41%) of those patients had neither a bullous nor a vesicular eruption. Other diagnoses included eczematous dermatitis, arthropod bites, scabies, and drug eruption. CONCLUSION: The majority of patients whose biopsy specimen revealed only ES had either dermatitis or autoimmune bullous disease, often in the prodromal phase. Direct immunofluorescence is often necessary to distinguish these diseases, and repeated testing may be needed for final diagnosis.

Elevated serum soluble interleukin-2 receptor levels in subacute cutaneous lupus erythematosus.

Subacute cutaneous lupus erythematosus (SCLE) is a subset of lupus erythematosus which is characterized by unique cutaneous manifestations and immunological abnormalities. Soluble IL-2 receptor (sIL-2 R) is the shed product of membrane Il-2 R, a product of T cell activation. It is measured by enzyme-linked immunosorbent assay (ELISA) and has been found to correlate well with disease activity in systemic lupus erythematosus (SLE) patients. The objective of the present work was to determine whether there is a correlation between sIL-2 R levels and disease activity in SCLE. Serum samples were obtained from 25 SCLE patients and then measured for sIL-2 R levels of ELISA. Fifteen of 25 SCLE patients tested had normal levels of sIL-2 R, while 10 of 25 SCLE patients had elevated sIL-2 R levels. The serum sIL-2 R level in SCLE patients correlated well with disease activity and the number of American College of Rheumatology criteria for SLE. These findings indicate that sIL-2 R levels can be used as a valuable laboratory parameter in managing SCLE patients.

Affinity of SSB/La nuclear antigen to small nuclear ribonucleic acids.

Anti-SSB/La antibody is one kind of antinuclear antibody and is found predominantly in patients with Sjögren's syndrome and systemic lupus erythematosus. SSB/La antigen is present in the nucleus as ribonucleoprotein in particle form and is composed of peptide and RNAs. SSB/La antigen can be partially purified through several sequential procedures including salt fractionation and ion exchange chromatography. SSB/La antigen is capable of binding to 4 RNAs from uninfected KB cells and 6 RNAs from Epstein-Barr virus transformed WiL2 cells. These RNAs can bind to SSB/La peptide in vitro and form ribonucleoprotein complexes which could be reprecipitated by anti-SSB/La antibody. SSB/La associated RNAs are labile and subjected to degradation easily. Anti-SSB/La antibody may be a useful probe to investigate the potential role of Epstein-Barr virus involvement in cutaneous pathology.