Serum and salivary IgG and IgA antibodies to desmoglein 3 in mucosal pemphigus vulgaris.

BACKGROUND: The use of saliva for the diagnosis of pemphigus vulgaris (PV) by enzyme-linked immunosorbent assay (ELISA) using desmoglein (Dsg)3 antigen has not been extensively documented, nor has the detection of serum IgA antibodies to Dsg3. OBJECTIVES: (i) To establish whether whole saliva might provide a suitable alternative to serum for diagnosing and monitoring PV; (ii) to investigate whether anti-Dsg3 IgA antibodies can be detected in serum and saliva and (iii) to establish whether there is an association between serum or saliva anti-Dsg3 antibodies and disease severity. METHODS: Precoated Dsg3 ELISA plates were used to test serum and/or saliva for IgG and IgA antibodies. Matched serum and whole saliva samples were collected from 23 patients with PV, 17 healthy subjects and 19 disease controls. All patients with PV, disease controls and six healthy controls provided matched parotid saliva. RESULTS: Whole saliva IgG antibodies to Dsg3 were detected in 14 of 23 patients (61%) and serum IgG antibodies were detected in 17 of 23 (74%) with a strong positive correlation. Serum IgA antibodies were detected in 14 of 23 patients with PV (61%) with a combined positivity (IgG and/or IgA antibodies to Dsg3) of 78% (18 of 23). We were unable to show IgA anti-Dsg3 antibodies in either whole or parotid saliva of patients with PV. Sequential samples showed that changes in IgG antibody titres in whole saliva were associated with a change in disease severity scores. CONCLUSIONS: Assay of salivary IgG antibodies to Dsg3 offers a diagnostic alternative to serum in the diagnosis and monitoring of PV. The role of anti-Dsg3 IgA antibodies requires further elucidation in the pathogenesis of PV.

Immunobullous dermatosis associated with Waldenström macroglobulinaemia treated with rituximab.

Waldenström macroglobulinaemia (WM) is a chronic lymphoproliferative disorder characterized by the presence of a monoclonal IgM paraprotein. Specific cutaneous features of WM include neoplastic cell infiltrates, IgM storage papules and IgM bullous dermatosis. We report a patient with subepidermal bullous disease associated with WM. Immunofluorescence identified IgM deposition along the basement membrane zone (BMZ) with circulating anti-BMZ IgM antibodies reacting with the dermal side of salt-split skin. The autoantibodies did not react with type VII collagen or laminin 332. Following failed treatment with doxycycline, prednisolone, intravenous immunoglobulin and dapsone, the patient was successfully treated with a modified RCVP regimen (rituximab, cyclophosphamide and prednisolone). To our knowledge, this is the first reported case of IgM bullous disease of WM treated with rituximab.

Bullous pemphigoid in an infant using complementary medicine.

Bullous pemphigoid (BP) is an acquired immunobullous disorder rarely seen in childhood. We report the case of an infant with BP successfully treated with oral corticosteroids. The onset of BP was associated with use of complementary medications and we speculate that these may have been triggering factors.

Inflammatory epidermolysis bullosa acquisita mimicking toxic epidermal necrolysis and dermatitis herpetiformis.

We report a patient with a spectrum of clinical features simulating toxic epidermal necrolysis, bullous erythema multiforme and later, dermatitis herpetiformis (DH). The histological features were suggestive of DH, bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). Direct immunofluorescence results suggested BP or EBA. Indirect immunofluorescence on salt-split skin and immunoblotting analysis on normal human dermal extracts gave results that were diagnostic for EBA.

The distribution of IgG subclasses in pemphigoid gestationis: PG factor is an IgG1 autoantibody.

Using monoclonal antibodies in immunofluorescence techniques, the subclass distribution of anti-basement membrane zone IgG antibodies was studied in the skin, placenta, and serum of patients with pemphigoid (herpes) gestationis. IgG1 was found to be the major IgG subclass in both serum and tissue, being detected in the sera of all pemphigoid gestationis patients studied. In pemphigoid and pemphigus, however, the distribution of IgG subclasses was heterogeneous, with IgG4 being the dominant autoantibody. Pemphigoid (herpes) gestationis factor, the circulating anti-basement membrane zone autoantibody thought to be pathogenic in pemphigoid gestationis, is therefore, an IgG1 antibody, with inferred complement binding capacity. Tissue damage in pemphigoid gestationis is apparently mediated by complement fixation which is detected via the classical complement cascade.

Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis.

We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.

Comparative study of bullous pemphigoid antigens among Japanese, British, and U.S. patients indicates similar antigen profiles with the 170-kD antigen present both in the basement membrane and on the keratinocyte cell membrane.

There are a number of controversies relating to studies of bullous pemphigoid (BP) antigens from different institutions, mainly regarding the detection of the 230-kD and 170-kD BP antigens. In this study, in an attempt to resolve the discrepancies, we have examined and compared the reactivity by immunofluorescence, immunoblotting, and immunoprecipitation among the sera from Japanese, British and U.S. BP patients. Both the 230-kD and 170-kD BP antigens were detected by various sera from all populations with immunoblotting, whereas immunoprecipitation detected only the 230-kD BP antigen but not the 170-kD BP antigen. Immunoprecipitation was more sensitive than immunoblotting to detect the 230-kD antigen. These results indicate that both the 230-kD and 170-kD proteins are BP antigens found in all three populations. By immunofluorescence cell surface staining in the lower epidermis in addition to basement membrane zone staining was shown by a considerable number of patients' sera in all populations. Comparison between the results of immunofluorescence and immunoblotting revealed a clear correlation of this cell surface staining with the presence of antibodies against the 170-kD BP antigen. That the affinity-purified antibodies specific to the 170-kD BP antigen showed this cell surface staining further confirmed this correlation. These results may indicate a different nature of the 170-kD BP antigen from that of the 230-kD BP antigen.

97-kDa linear IgA bullous dermatosis (LAD) antigen localizes to the lamina lucida of the epidermal basement membrane.

Linear IgA bullous dermatosis (LAD) is an autoimmune blistering disease in which IgA autoantibodies develop against the epidermal basement membrane zone. Target antigens of the circulating autoantibodies are thought to be heterogeneous, and their ultrastructural localization has not been fully elucidated. Previous studies with immunoblotting have demonstrated that the 97-kDa autoantigen is detected most frequently in patients' sera and is thought to be a major LAD antigen. Although a recent report suggests that the 97-kDa antigen localized to the hemidesmosomal plaques and the adjacent lamina lucida, discrepancies still exist among previous immunoelectron microscopic findings. To identify the precise localization of the 97-kDa LAD antigen, we used two different low-temperature immunoelectron microscopic techniques. For immunolabeling, we selected five LAD sera that had a high titer of autoantibodies against the 97-kDa LAD antigen. A post-embedding method with cryofixation and freeze substitution failed to immunolabel the 97-kDa LAD antigen. Cryoultramicrotomy with immunoelectron microscopy succeeded in preserving the antigenicity of the 97-kDa LAD antigen. In all cases, the majority of labeling occurred in the lamina lucida beneath the hemidesmosomes. No specific labeling was observed in the hemidesmosomal attachment plaques or the lamina densa or sublamina densa region, including anchoring fibrils. These results indicate that the 97-kDa LAD antigen is a component of the lamina lucida.

Production of mycoplasma-specific antisera in rabbits immunologically tolerized at birth to mycoplasma medium constituents.

Neonatal rabbits were injected intraperitoneally (i.p.) within 12 h of birth followed by similar injections every day for 10 consecutive days and then every second day for a further 8 weeks, with mycoplasma broth medium (tolerogen), to induce immune tolerance. The rabbits were then immunized with the porcine mycoplasmas, M. hyopneumoniae or M. hyorhinis at 9 weeks of age. Immune sera obtained from these rabbits and from normal control rabbits were tested for antibodies against both mycoplasma antigens and for antibodies to medium components by double immunodiffusion in agarose and by enzyme-linked immunosorbent assay (ELISA). Antisera obtained from the tolerized rabbits contained no antibodies to medium components as evidenced by lack of reactivity in both assays. In immunofluorescence tests the antisera obtained from tolerized rabbits permitted specific staining of colonies of the homologous mycoplasma grown on mycoplasma agarose medium. In contrast the antisera obtained from normal rabbits produced strong reactions in all of the tests and non-specific background fluorescence due to reactions with components of the culture medium.

Effect of temperature and lymphokines on mixed lymphocyte and mitogen responses of chicken lymphoid cells in vitro.

The effect of temperature on T cell mitogen and mixed lymphocyte responses (MLR) of chicken lymphoid cells in vitro was examined. Responses at 40 degrees C were much higher than at 37 degrees C. This difference did not appear to be due only to faster kinetics of the responses at 40 degrees C. At the lower temperature the MLR could be enhanced by polyethylene glycol (PEG) for spleen cells and by Con A induced lymphokines for peripheral blood cells (PBL). The positive effect of PEG on the chicken spleen cell MLR appeared to be determined at the stimulator cell level. Responses to mitogens at 37 degrees C of both spleen cells and PBL were enhanced by lymphokines.

Changes in dendritic cell subsets in the lymph nodes of rhesus macaques after application of glucocorticoids.

Corticosteroids are used therapeutically as potent immunosuppressive and antiinflammatory drugs for a broad spectrum of diseases. Although corticosteroids are known to inhibit the production of many cytokines in activated T cells, there is also evidence for increases in IL-4 and in some cases IFNgamma production. These conflicting results may be caused by contrary effects of corticosteroids on different cell types involved in immune regulation, for instance antigen presenting cells (APC) versus T cells. In the present study we simultaneously investigated the effect of local as well as systemic application of glucocorticoids (GCC) on the phenotype of APC in the skin as well as the lymph nodes in a model primate species, the rhesus macaque. Using a range of APC markers, including CD68, HAM56, HLA-DR, CD1a, p55, RFD-1, and costimulatory molecules CD40, CD80, and CD86 we document the close phenotypic resemblance of rhesus and human APC. We noted that topical GCC treatment specifically lead to a marked decrease in the number of CD1a expressing cells in the draining lymph nodes. However, the number of CD1a positive cells in peripheral lymph nodes was not affected by systemic GCC treatment. Importantly, by performing double staining of CD1a with RFD-1 we observed a shift in the expression pattern of these dendritic cell markers in the lymph nodes, with an increase in the number of RFD-1 single positive cells relative to CD1a single positive and CD1a/RFD-1 double positive cells. These findings suggest that GCC treatment results in the presence of phenotypically more mature APC.

Immunoblotting studies of linear IgA disease.

Patients with linear IgA deposits at the basement membrane zone (BMZ) detected by direct immunofluorescence (IF) may show diverse clinical and laboratory findings. The aim of this study was to investigate the issue of target antigens for linear IgA disease (LAD) antibodies. We examined sera from 46 adults and children with exclusive IgA deposits at the BMZ, by both indirect IF on 1 M NaCl split human skin and immunoblotting. IgA anti-BMZ antibodies binding to the epidermal side of the split were found in 31 LAD sera. IgA anti-BMZ antibodies binding to the dermal side of the split were detected only in 4 LAD sera. No sera contained IgA anti-BMZ antibodies binding to both sides of the split. Immunoblotting revealed that 12 epidermal side-positive LAD sera reacted with the 97 kDa protein in the human epidermal extracts. Moreover, we found that 2 dermal side-positive LAD sera reacted with a protein of approximately 255 kDa on immunoblotting of the dermal extract. We conclude that there are at least two types of LAD. However, the nature of target antigens for LAD antibodies remains to be determined.

Analysis of antigens recognized by autoantibodies in herpes gestationis. Usefulness of immunoblotting using a fusion protein representing an extracellular domain of the 180 kD bullous pemphigoid antigen.

Herpes gestationis (HG) is a rare pregnancy-associated disease. The aim of this study was to compare various immunohistochemical and immunobiochemical techniques with respect to their diagnostic sensitivity for HG. We studied 43 HG sera; only half of these reacted with the basement membrane zone (BMZ) with both indirect immunofluorescence (IF) and complement IF of normal human skin. 81% of the sera reacted with the epidermal side of 1 M NaCl-split skin. In general, titers of anti-BMZ antibodies in HG sera were lower than those in bullous pemphigoid (BP) sera. Immunoblot analysis of human epidermal extracts showed that 51% of HG sera recognized the 180 kD BP antigen (BP180) and 26% recognized the 230 kD BP antigen (BP230). We also studied the reactivity of HG sera with fusion proteins representing either the NC16a domain of human BP180 or the C-terminal region of mouse BP230. Whereas 79% of HG sera reacted with the BP180 fusion protein, only 5% recognized the BP230 fusion protein. Our results suggest that indirect IF of 1 M NaCl-split skin and immunoblotting of a fusion protein representing the BP180 NC16a domain are more sensitive techniques for the diagnosis of HG than conventional and complement IF or immunoblotting of crude epidermal extracts.

Analysis of antigens targeted by circulating IgG and IgA autoantibodies in 50 patients with cicatricial pemphigoid.

In this study we investigated sera from 50 typical cicatricial pemphigoid (CP) patients. By indirect immunofluorescence on 1 M NaCl-split human skin sections, IgG of 17 sera and IgA of 22 sera reacted with the epidermal side of the split, while IgG of two sera reacted with the dermal side. These latter two sera were later confirmed to be anti-epiligrin CP. By immunoblotting of epidermal extracts, IgG of 14 sera reacted with the 230 kD bullous pemphigoid (BP) antigen (BP230). IgG of 15 sera and IgA of 11 sera reacted with the 180 kD BP antigen (BP180). Interestingly, a bacterial fusion protein containing the BP180 NC16a domain was recognized by IgG of 18 sera but not by IgA of any sera. Fusion proteins containing the C-terminal region of BP180 were recognized by IgG of 20 sera, but it was detected by IgA of only two sera. Our results suggest that, although CP sera show very low titers of autoantibodies, a considerable number of sera contain IgG antibodies to BP180 (either NC16a or C-terminal domain), confirming previous studies. In addition, we showed that greater numbers of IgA antibodies react with BP180, seemingly with different types of epitopes from those for IgG antibodies. Because the specificity of IgG antibodies is not very different from those in BP, IgA antibodies may play a specific role for the development of characteristic clinical features in CP. Future studies should elucidate the pathogenic role of the IgA antibodies in CP.

Neutrophil mediated and IgA dependent antibacterial immunity against enteropathogenic Escherichia coli in the porcine intestinal mucosa.

The role of polymorphonuclear neutrophils (PMN) in the antibacterial immunity against enteropathogenic Escherichia coli (EEC) 0:149 in the porcine intestine was studied using intestinal Thiry-Vella loop (T-V loop) as a model. Intraluminal immunizations of T-V loops resulted in elevated levels of immunoglobulin A (IgA) anti-EEC 0:149 antibody in the loop secretions, an infiltration of PMN in the lumen of the loops and an increase in the concentrations of lactoferrin (LF), lysozyme (LY), cationic proteins (CP), and a specific bactericidal response in the immunized loops. PMN were observed by electron microscopy (EM) to be actively phagocytic in the lumen of the immune loops. EM observations of loop fluids as well as the abrogating effect of iron on the in vivo bactericidal response strongly suggest that the pMN played an important role in the bactericidal response in the loops against EEC. In addition to phagocytosis by PMN and subsequent intracellular killing, disintegration of PMN in the lumen of the loops and extracellular killing of EEC by the antibacterial products of PMN such as LF, LY and CP, with and/or without synergistic effect of IgA antibodies, also contribute to the bactericidal response of the immunized loops.

Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections.

A genetically engineered Eimeria tenella antigen (GX3262), produced as a fusion protein with beta-galactosidase and identified with a monoclonal antibody, induced partial but significant protection in young broiler chickens against experimental E. tenella and Eimeria acervulina infections. The antigen appears to share a T-helper cell epitope with the parasite as evidenced by (a) booster inoculation with either the recombinant antigen or with a small number of live oocysts enhanced the protective immunity in GX3262 primed chickens, and (b) ability of the antigen to induce in vitro stimulation of T-cells from chickens immunized with antigen or parasite. These observations suggest the feasibility of a single vaccination of 1 or 2-day-old broilers with GX3262 to induce an acceptable degree of protective immunity. The implications of the observations reported here are far reaching in terms of a practical coccidiosis vaccine for poultry, and show for the first time that 1-day-old broiler chickens can be efficiently vaccinated with a recombinant antigen against one or more species of Eimeria.

Immunopathological techniques in the diagnosis of bullous disorders.

Immunopathological techniques have become essential to the investigation and management of autoimmune vesiculobullous diseases. In this article the role and practical application of direct immunofluorescence, indirect immunofluorescence and complement C3 binding indirect immunofluorescence techniques, and their relevance to the diagnosis and management of the individual blistering diseases will be reviewed. The diagnostic value and limitations of the new split skin indirect method will also be described. Furthermore, the contribution of immunoelectron microscopy and immunoblotting studies to our knowledge of the pathogenesis of these diseases will be discussed.

Concomitant occurrence of circulating IgA anti-intercellular and anti-basement membrane zone antibodies in autoimmune blistering diseases: immunofluorescence and immunoblot studies.

Recently, cases with circulating IgA anti-intercellular antibodies have been described. The objective of this study was to present immunofluorescence and immunoblot findings in three cases of bullous diseases with concomitant circulating IgA anti-intercellular and anti-basement membrane zone antibodies. Direct immunofluorescence, indirect immunofluorescence on intact and 1M NaCl-split skin, immunoblotting of epidermal extracts from dispase- and EDTA-separated (two different procedures) human skin, and immunoblotting of the bovine desmosome preparation were performed. All three cases had IgA anti-intercellular and anti-basement membrane zone antibodies. However, immunoblot results were divergent. Case 1 had antibodies against the 150 kD pemphigus foliaceus antigen (IgG), the 170 kD protein (IgG and IgA), and the 97 kD antigen (IgG and IgA). Case 2 had IgG antibodies reactive with the 230 kD and the 170 kD bullous pemphigoid antigens, while case 3 had IgA antibodies against the 97 kD antigen only. The results of immunofluorescence and immunoblot studies in our patients widen the spectrum of laboratory features in blistering skin diseases mediated, at least in part, by antibodies of the IgA class.

Duration of anti-adhesive and bactericidal activities of milk from vaccinated sows on Escherichia coli O149 in the digestive tract of piglets during the nursing period.

Piglets were exposed orogastrically to Escherichia coli to enable study of the duration of anti-adhesive and bactericidal activities of milk of sows vaccinated with a K88 enriched E coli vaccine. There was a marked increase in the number of the challenge strain in the digestive tract of weaned piglets of all ages (between 888 and 2144 per cent). In contrast, there was a decrease in their number (75 per cent) in the day-old colostrum-fed piglets. When the piglets were two weeks old milk was still capable of reducing the rate of proliferation of the pathogen but at five weeks it proliferated at equal rates in the digestive tract of both suckling and weaned litter-mates. The rate of adhesion of the K88 positive E coli to the small intestine of colostrum deprived piglets was high (5 x 108/g). Rate of adhesion fell gradually in weaned piglets from 5.4 x 107/g at two weeks to 2.0 x 106/g at four to five weeks of age. In contrast, resistance of the small intestine of suckling pigs to adhesion by K88-positive E coli remained relatively stable through the five week period of nursing bacterial counts ranging from 5 x 104/g to 3 x 104/g of tissue.

The effect of colostrum or past colibacillosis on the adhesion of Escherichia coli to the small intestine of the pig.

Nursing litters of vaccinated (K88 antigen) and non-vaccinated gilts as well as weaned piglets, which had recovered from natural colibacillosis, were exposed to Escherichia coli O149: K91(B), K88ac(L) orogastrically and 3-4 h after exposure a proportion of each group was killed for adhesion studies; others were kept for clinical observations. From killed piglets duodenal contents were collected and after washing of the duodenum by standardised techniques it was homogenised. Viable counts of E coli O149 in these specimens were carried out and compared. Colicounts in the duodenal contents and in the homogenate of washed duodenum of naturally infected piglets were also compared. There was less than tenfold difference in counts of K88-positive E coli between duodenal contents and washed duodenum of piglets which were (A) naturally infected, (B) colostrum deprived and experimentally infected, or (C) were from colostrum fed groups where those litter mates which were kept for clinical observation died of colibacillosis. There was 10(3)-10(5)-fold difference in E coli O149 counts between comparable specimens from piglets which (a) recently recovered from K88-positive E coli infection at the time of oral challenge or (b) were from colostrum fed groups where those litter mates which were kept for clinical observation survived oral challenge. It is concluded that adhesion of K88-positive E coli to the epithelium of the anterior small intestine of the pig is a feature of natural as well as experimental colibacillosis. Such adhesion may be prevented by (i) earlier natural infection of K88-positive E coli or (ii) by the ingestion of colostrum of K88 antigen vaccinated dams.