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.

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.

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.

Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity.

BACKGROUND: Mucous membrane pemphigoid (MMP), a chronic autoimmune subepithelial blistering disease, is associated with circulating IgG and/or IgA autoantibodies against several basement membrane zone antigens. The heterogeneity of clinical presentation and diversity of target autoantigens have contributed to difficulties in characterizing this condition immunologically. OBJECTIVES: To analyse serum autoantibody profile and HLA class II alleles in MMP patients and to correlate this with the clinical presentation of disease. METHODS: Well-defined subgroups consisting of 124 patients with MMP were examined for IgG and IgA reactivity with immunoblotting using human epidermal, dermal and placental amnion proteins. The results were further analysed on the basis of detailed clinical (sites of involvement and disease severity) and immunopathological criteria (immunofluorescence study and HLA class II alleles). RESULTS: Immunoblot assay revealed that the majority of MMP patients had IgG (93 of 124, 75%) and/or IgA autoantibodies (63 of 124, 51%) to BP180 (including its soluble ectodomains, 120-kDa LAD-1 and 97-kDa LABD97 antigens). Other antigens targeted predominantly by IgG autoantibodies included: BP230 in 34 (27%), beta4 integrin in 26 (21%), and laminin 5 in three (2%). All the BP230+ sera and 23 (88%) beta4 integrin+ sera also reacted with at least one of the BP180 antigens. Over 85% of patients with reactivity to beta4 integrin had ocular involvement. In most cases of MMP, more severe clinical features were associated with antibody reactivity to multiple basement membrane zone antigens, as well as reactivity to multiple BP180 component antigens. Dual BP180/LAD-1 reactivity with IgG and IgA was associated with a more severe phenotype. In addition, the subset-dependent autoantibody reactivity correlated well with specific HLA class II alleles, DQB1*0301, DRB1*04 and DRB1*11. CONCLUSIONS: Our results confirmed that BP180 is a major autoantigen targeted by the sera of patients with MMP. The disease-prevalent HLA class II alleles and humoral autoimmune response against the particular subsets of antigenic epitope(s) within BP180 ectodomain may contribute to the clinicopathological significance and disease severity of MMP.

Evaluation of a BP180-NC16a enzyme-linked immunosorbent assay in the initial diagnosis of bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is the most common subepidermal immunobullous disease, characterized by circulating IgG autoantibodies targeting BP180 and BP230 hemidesmosomal proteins. Several immunological studies have demonstrated that the membrane proximal noncollagenous domain NC16a of BP180 is the immunodominant region targeted by BP autoantibodies. Recently, a commercial BP180 NC16a-specific enzyme-linked immunosorbent assay (ELISA) has become available for detecting pathogenic anti-BP180 autoantibodies in BP sera. However, it remains unclear whether the diagnostic potential of the ELISA is equivalent to that of the 'gold-standard' diagnostic technique of immunofluorescence (IF). OBJECTIVES: To examine the usefulness of a commercially available BP180-NC16a ELISA in the initial serodiagnosis of BP. METHODS: Sera from a large cohort of patients with BP (n = 102) and control subjects (age- and sex-matched normal volunteers, n = 60; pemphigus foliaceus, n = 18; pemphigus vulgaris, n = 16) were assayed by BP180-NC16a ELISA. All BP sera were obtained at presentation before initiation of systemic immunosuppressive therapy. The values of IgG antibody levels measured by ELISA were compared with those measured by indirect IF on salt-split skin. Results Receiver operating characteristic analysis was used to calculate the cut-off value for the ELISA in the diagnosis of BP which maximizes both sensitivity and specificity, and to estimate the diagnostic accuracy of the ELISA as represented by the area under the curve (AUC = 0.965). A cut-off value of 9 was associated with a sensitivity of 89% (91 of 102 BP sera showed a positive result) and a specificity of 98%. Fifty-eight of 60 normal controls and all the pemphigus sera showed a negative result. There was a correlation between the mean ELISA values and indirect IF titres (Spearman rank correlation 0.286; P = 0.004). CONCLUSIONS: Our results suggest that the BP180-NC16a ELISA is a useful tool for the detection of pathogenic anti-BP180 IgG autoantibodies at the initial disease stage of BP. Because it is not only highly sensitive and specific, but is also easy to perform, is objective, and semiquantitative, the ELISA may provide valuable information for the accurate and reliable serodiagnosis of BP.

Lichen planus pemphigoides evolving into pemphigoid nodularis.

Lichen planus pemphigoides (LPP) and pemphigoid nodularis are rare clinical variants of bullous pemphigoid (BP), which are characterized by histological findings of lichen planus (LP) and nodular prurigo, respectively, and the finding of linear deposits of IgG and/or C3 at the basement membrane zone in perilesional skin. In both cases bullae may arise at the site of pre-existing LP-like or nodular prurigo-like eruptions, and clinically uninvolved skin. The disease spectrum of LPP and pemphigoid nodularis differs from that of classical BP phenotype, and their presentations are often indolent. LPP may predominantly affect a younger age group and is responsive to standard treatments used in acquired autoimmune bullous diseases, while pemphigoid nodularis is more common in elderly women and is relatively resistant to therapy. We describe a patient who had LPP for nearly two decades and subsequently developed a nodular eruption with a concurrently detected antibullous pemphigoid antigen 2 (BP180) autoantibody. His overall clinicopathological features were indicative of LPP evolving into another BP variant, pemphigoid nodularis.

Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting.

BACKGROUND: Identification of antigens by immunoblotting techniques, using epidermal and dermal extracts, is regarded as essential for making a definitive diagnosis in autoimmune bullous diseases (AIBDs). These procedures involve epidermal-dermal separation for subsequent protein extraction, which may result in partial loss of some antigenic polypeptides and changes in the conformational epitopes targeted by autoantibodies in AIBDs. It may therefore be necessary to use different substrates for consistent results. Objectives To evaluate the usefulness of human placental amnion extract as a substrate for immunoblotting in the diagnosis of AIBDs. METHODS: We checked the structural components of the desmosomes and basement membrane zone (BMZ) of amnion by electron microscopy. Using immunofluorescence and immunoblotting techniques, we tested the amnion immunoreactivity with antibodies to desmosomal and BMZ proteins, and with sera from 76 patients with AIBDs including pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (BP), pemphigoid gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, paraneoplastic pemphigus and mucous membrane pemphigoid. RESULTS: The desmosomes and BMZ of the amnion tissue were ultrastructurally similar to those in skin. Antigen mapping confirmed that amnion contains all the proteins that were recognized by a panel of monoclonal and polyclonal antibodies. Immunoblotting showed that the antibodies clearly detected bands corresponding to desmogleins 1 and 3, desmocollins 1 and 2, desmoplakins 1 and 2, three subunits (alpha3, beta3 and gamma2) of laminin 5, BP antigens 1 and 2, the 97-kDa LAD antigen and type VII collagen. In addition, most of the patient sera (82%) reacted exclusively with their respective antigens. CONCLUSIONS: Harvesting proteins from amnion does not require epidermal-dermal separation, and a sufficient yield of desmosomal and hemidesmosomal proteins can be obtained. Therefore, amnion may be a more reliable source of substrate than skin samples for immunoblot analysis of AIBDs.

Psoriasis bullosa acquisita.

We report a 51-year-old man with a 20-year history of chronic plaque psoriasis who developed an autoimmune subepidermal blistering eruption that had clinical features of bullous pemphigoid, erythema multiforme and epidermolysis bullosa acquisita. Investigations revealed a 1 : 400 titre circulating and in vivo bound IgG autoantibody that mapped to the dermal side of 1 m NaCl-split skin and localized to the lower lamina lucida/upper lamina densa on immunogold electron microscopy. Immunoblotting, using dermal extracts, showed serum binding to antigens of approximately 200- and approximately 260 kDa. Indirect immunofluorescence microscopy, using the patient's serum on archival skin sections taken from selected individuals with different forms of inherited epidermolysis bullosa as substrate, showed normal basement membrane labelling on all samples apart from recessive dystrophic epidermolysis bullosa skin (with inherent mutations in the type VII collagen gene): in these cases there was a complete absence of immunostaining. Clinically, the patient responded rapidly to combination treatment with intravenous immunoglobulin and oral corticosteroids, dapsone and mycophenolate mofetil. Autoimmune subepidermal blistering has been reported in other patients with psoriasis, although no specific target antigen has ever been determined. Our study provides preliminary evidence that, for this patient at least, the autoantibody may be targeted against a skin component closely associated with type VII collagen (the epidermolysis bullosa acquisita antigen). Therefore, we propose the term 'psoriasis bullosa acquisita' for this and possibly other patients with similar skin eruptions.

Pemphigoid nodularis (non-bullous): a clinicopathological study of five cases.

Pemphigoid nodularis is a rare clinical variant of pemphigoid characterized by overlapping clinical features of both prurigo nodularis lesions and pemphigoid-like blisters. In pemphigoid nodularis, the immunopathological findings are identical to those of bullous pemphigoid (BP). We describe five patients who presented with the typical clinical phenotype of prurigo nodularis, who were found to have circulating and tissue-bound antibasement membrane zone autoantibodies. By immunoelectron microscopy and Western immunoblotting studies, the circulating antibodies were shown to target the hemidesmosome and specifically the BP antigens 1 and 2 (BP180 and BP230). In contrast to the majority of reported cases, none of these patients has ever developed blisters. The role of antibasement membrane zone antibodies in the development of the eruption, or the role of the eruption in the development and persistence of autoantibodies, is not clear. These cases demonstrate that the presence of these antibodies is not sufficient for the development of blisters.

The transition of pemphigus vulgaris into pemphigus foliaceus: a reflection of changing desmoglein 1 and 3 autoantibody levels in pemphigus vulgaris.

The transition of pemphigus vulgaris (PV) into pemphigus foliaceus (PF) is rare and the immunological changes underlying this event are not well understood. We report a 44-year-old woman who presented with oral and cutaneous erosions typical of PV. Over a 9-year period, the clinical features evolved into those of PF. To examine whether quantitative changes in desmoglein (Dsg) antibodies were associated with this transition, Dsg1 and Dsg3 antibody levels were measured by enzyme-linked immunosorbent assay in 82 sequential serum samples collected over this period. At presentation, when the phenotype was PV with oral and cutaneous erosions, antibodies to both Dsg1 and Dsg3 were detected. The disappearance of oral involvement was associated with a decline in Dsg3 antibodies, which are now undetectable, while the development of more severe skin involvement was associated with rising Dsg1 antibody levels. These data strongly suggest that the change in clinical features is a reflection of qualitative and quantitative changes in antibody profile. It is not known whether the transition to PF is permanent or whether disease relapses in the future may be associated with the re-emergence of Dsg3 antibodies, oral ulceration and a PV phenotype.

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes.

Delivery of genes to the lung has enormous potential in a wide variety of illnesses, from lung cancer to genetic deficiency diseases. Many delivery systems have been utilized, each with its own advantages and limitations. Polyethylenimine is a polycation capable of binding and compacting DNA, enabling intravascular plasmid delivery to normal tissues in such a way that the plasmid can be expressed in a proportion of the exposed cells. We have developed a novel intravenous method to deliver small amounts of plasmid to lung tissue, using nontoxic quantities of polyethylenimine in combination with albumin (or other soluble proteins). Injection of 1 microg or less of plasmid resulted in highly efficient gene expression in lung interstitial and endothelial tissues (0.5 to 1 ng luciferase per microg plasmid DNA), while larger quantities of plasmid reduced relative gene expression. Using luciferase as a reporter gene, single injections had maximal gene expression between 24 and 48 h, with a rapid decline thereafter. In contrast to some other delivery systems, however, no inhibition of gene expression occurred during multiple rounds of plasmid administration through 20 days. As a result, this method may have useful applications in diseases that could benefit from recurrent therapeutic gene delivery.

The severity of cutaneous and oral pemphigus is related to desmoglein 1 and 3 antibody levels.

BACKGROUND: Pemphigus vulgaris (PV) and foliaceus (PF) are characterized by antibodies to the desmosomal proteins desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1), respectively. Past studies using indirect immunofluorescence (IIF) as a measure of pemphigus antibody levels have failed to demonstrate consistently a relationship between disease severity and IIF titres. However, IIF is not able to measure separately Dsg1 and 3 antibodies, unlike enzyme-linked immunosorbent assays (ELISA), which utilize recombinant proteins. OBJECTIVES: To compare independently Dsg1 and 3 antibody levels with the severity of both cutaneous and oral involvement in PV and PF. Patients and methods Four hundred and twenty-four serum samples were analysed from 80 subjects with PV and 24 with PF. IgG antibodies to Dsg1 and 3 were measured by ELISA. For every sample analysed, the associated severity of skin and oral disease were graded from 0 to 3; quiescent, mild, moderate and severe. RESULTS: A relationship between Dsg1 antibodies and skin severity was demonstrated such that a 10-unit increase in Dsg1 ELISA value was associated with a 34% chance of having a higher severity score [95% confidence interval (CI), 25-45%, P < 0.0005]. This was observed in both PV and PF. Oral severity was associated with Dsg3 antibody levels and a 10-unit increase in the Dsg3 ELISA value was associated with a 25% chance of a higher oral severity score (CI 17-33%, P < 0.0005). We were unable to demonstrate a relationship between Dsg1 antibodies and oral severity, even after adjusting for the effect of Dsg3 antibodies. Similarly, there was no relationship between Dsg3 antibodies and skin severity. CONCLUSIONS: This study suggests that the clinical phenotype of pemphigus, in particular the balance of skin and oral disease, is determined principally by the quantities of Dsg1 and 3 autoantibodies, respectively.

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.

Dermatitis herpetiformis evolving into bullous pemphigoid: a probable example of 'epitope spreading'.

We report a case of dermatitis herpetiformis which, 11 years after its original diagnosis, evolved into bullous pemphigoid. Only a few similar cases supported by immunofluorescence studies have been reported previously, and we believe that they represent examples of 'epitope spreading', an increasingly recognized phenomenon used to explain the coexistence of autoimmune diseases.

Pericapillary fibrin deposits and skin hypoxia precede the changes of lipodermatosclerosis in limbs at increased risk of developing a venous ulcer.

This study investigated the possibility that pericapillary fibrin deposition, found in the calf skin of patients with venous ulceration and lipodermatosclerosis, might already be present in the dermis of the gaiter area of apparently healthy limbs before any skin changes were visible. The apparently healthy limbs of 19 consecutive patients with a healed venous ulcer on one leg and no history of ulceration or clinical evidence of lipodermatosclerosis in the opposite calf, were studied. Bipedal ascending phlebography and foot volume plethysmography were performed, and systemic fibrinolytic activity and fibrinogen levels were calculated. Transcutaneous oxygen measurements were expressed as a ratio of levels from a fixed position in the gaiter skin over a control site on the arm. Biopsies of a standard site in the gaiter skin and the thigh were assessed for the presence of laminin, fibrinogen and fibronectin using immunofluorescent microscopy. The extent of pericapillary fluorescence was expressed as a ratio of the number of capillaries with deposits divided by the total number of capillaries staining with laminin (fibrin and fibronectin scores). Pericapillary fibrin deposits were observed in the dermis in 16 of the biopsies of the gaiter region (median score 0.20), and in eight of the biopsies from the thigh (median score 0.0). This difference was highly significant (P<0.01, Wilcoxon signed rank test). The transcutaneous oxygen ratio correlated negatively with the fibrin score (Spearman rank correlation coefficient -0.62, P<0.01), and there was a weak negative correlation between the half volume refilling time on foot volume plethysmography (an indicator of venous reflux) and the fibrin score (Speraman rank correlation coefficient -0.47, P<0.05). No such correlation could be shown between the fibrin score and the indicators of calf pump function, the euglobulin clot lysis time or the plasma fibrinogen. The presence of significant numbers of pericapillary fibrin deposits within the dermis of the gaiter skin has been demonstrated in 84% of this cohort of 'at risk' limbs before there is any evidence of clinical lipodermatosclerosis.

A study of desmoglein 1 autoantibodies in pemphigus vulgaris: racial differences in frequency and the association with a more severe phenotype.

BACKGROUND: Pemphigus vulgaris (PV) is characterized by pathogenic autoantibodies to desmoglein (Dsg) 3, but additional antibodies to Dsg1, the pemphigus foliaceus antigen, are detectable in some cases. OBJECTIVES: To investigate the clinical significance of the presence of both Dsg 1 and 3 antibodies. METHODS: In 79 subjects with PV, enzyme-linked immunosorbent assays were used to detect IgG autoantibodies reactive with the ectodomain of Dsg1 and Dsg3. RESULTS: There was a clear association between the clinical phenotype and the Dsg antibody profile. All subjects had Dsg3 autoantibodies and 61% had coexisting Dsg1 antibodies (Dsg3+/Dsg1+). PV limited entirely to the mucosal surfaces was seen only in Dsg3+/Dsg1- patients, while additional Dsg1 antibodies (Dsg3+/Dsg1+) predicted cutaneous in addition to mucosal involvement. Although minor cutaneous involvement was observed in most Dsg3+/Dsg1- patients, severe cutaneous involvement was seen only in Dsg3+/Dsg1+ patients. Dsg1 antibodies were detectable early in the course of disease and their appearance did not relate to the use of systemic therapy. The proportion of Dsg1+ patients was higher in those of Indian origin compared with white northern Europeans (P < 0.05). CONCLUSIONS: These data suggest that the presence of Dsg1 antibodies is predictive of a potentially more severe disease and that genetic factors may determine the Dsg antibody profile.

The use of two substrates to improve the sensitivity of indirect immunofluorescence in the diagnosis of pemphigus.

The aim of this study was to re-evaluate indirect immunofluorescence (IIF) comparing two substrates, normal human skin (HS) and monkey oesophagus (MO) using serum from 29 pemphigus patients classified according to the presence of serum autoantibodies to either desmoglein (Dsg) 1 or Dsg3 detected by enzyme-linked immunosorbent assay (ELISA). Overall, the sensitivity of IIF was 83% on HS and 90% on MO. When data from both substrates were combined, the sensitivity increased to 100%. When sera from pemphigus foliaceus (PF) patients were studied, which contained Dsg1 antibodies only, the sensitivity of IIF was greatest on HS and titres were on average 4.8 doubling dilutions higher than on MO. In contrast, when sera containing autoantibodies only to Dsg3 from pemphigus vulgaris (PV) patients was studied, the sensitivity was greatest on MO and titres were on average 4.4 doubling dilutions higher than on HS. There was a significant correlation between Dsg1 antibody levels and IIF titres on HS and between Dsg3 antibody levels and IIF titres on MO. The investigation of immunobullous disorders in the future is likely to move towards antigen-specific techniques such as the Dsg ELISAs used in this study. However, in laboratories which currently rely on IIF for detecting pemphigus autoantibodies, the data presented in this study strongly suggest that two substrates should be used for IIF screening: one rich in Dsg1, such as HS, and the other rich in Dsg3, such as MO. This combination of substrates should not only increase the sensitivity of detecting pemphigus antibodies, but will aid in the differentiation of PV from PF. It is also possible that the data might be more useful for disease monitoring.

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses.

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.