Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules.

Proteinase 3 (PR3), which is also called myeloblastin, the target autoantigen for antineutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis, is a serine proteinase stored in azurophil granules of human neutrophils. We have previously shown that, in contrast to elastase or myeloperoxidase, PR3 is also expressed at the plasma membrane of a subset of unactivated neutrophils and that a high proportion of neutrophils expressing membrane PR3 is a risk factor for vasculitis. The present study demonstrates that the association of PR3 with the plasma membrane is not an ionic interaction and seems to be covalent. Fractionation of neutrophils shows that, besides the azurophil granules, PR3 could be detected both in specific granules and in the plasma membrane-enriched fraction containing secretory vesicles, whereas elastase and myeloperoxidase were exclusively located in azurophil granules. Electron microscopy confirms that PR3 is present along with CR1 in secretory vesicles as well as in some specific granules. In neutrophils stimulated with an increasing dose of FMLP, membrane PR3 expression increased with the degranulation of secretory vesicles, followed by specific granules, and culminated after azurophil granules mobilization. The presence of a readily plasma membrane-mobilizable pool of PR3 contained in the secretory vesicles might play a relevant role in the pathophysiological mechanisms of ANCA-associated vasculitis.

A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis.

It has been shown previously that proteinase 3 (PR3), a neutrophil intracellular protease that is the main antigen of antineutrophil cytoplasm (ANCA) autoantibodies, is present on the plasma membrane of a subset of freshly isolated neutrophils. This study shows that the size of this subset of membrane PR3-positive (mPR3+) neutrophils is a stable feature of a given individual, most likely genetically controlled. It ranges from 0 to 100% of neutrophils and allows us to define a new polymorphism in the healthy population, with three discrete phenotypes corresponding respectively to less than 20% mPR3 + neutrophils (mPR3low) or to a mean percentage of 47% (mPR3intermediate) and 71.5% (mPR3high) mPR3+ neutrophils. The frequency of the mPR3high phenotype was significantly increased in patients with ANCA-associated vasculitis (85% versus 55% in healthy subjects). The percentage of mPR3+ neutrophils was not affected by disease activity, relapses, or therapy, and did not reflect in vivo cell activation. In addition, mPR3+ phenotypes were normally distributed in cystic fibrosis patients, indicating that infection and/or inflammation per se do not lead to a high percentage of mPR3+ neutrophils. The frequency of the mPR3high phenotype was not related to anti-PR3 autoimmunization, since it was increased in vasculitic patients regardless of the ANCA specificity (anti-PR3, anti-myeloperoxidase, or unknown). Interestingly, the frequency of the mPR3high phenotype was also increased in patients with rheumatoid arthritis. It was normal in type I-diabetes, a T cell-dependent autoimmune disease. It is proposed here that a high proportion of membrane PR3-positive neutrophils could favor the occurrence or the progression of chronic inflammatory diseases.

Requirement of caprine arthritis encephalitis virus vif gene for in vivo replication.

Replication of vif-caprine arthritis encephalitis virus (CAEV) is highly attenuated in primary goat synovial membrane cells and blood-derived macrophages compared to the wild-type (wt) virus. We investigated the requirement for CAEV Vif for in vivo replication and pathogenicity in goats by intra-articular injection of either infectious proviral DNA or viral supernatants. Wild-type CAEV DNA or virus inoculation induced persistent infection resulting in severe inflammatory arthritic lesions in the joints. We were unable to detect any sign of virus replication in vif- CAEV DNA inoculated goats, while vif- CAEV virus inoculation resulted in the seroconversion of the goats. However, virus isolation and RT-PCR analyses on blood-derived macrophage cultures remained negative throughout the experiment as well as in joint or lymphoid tissues taken at necropsy. No pathologic lesions could be observed in joint tissue sections examined at necropsy. Goats inoculated with the vif- virus demonstrated no protection against a pathogenic virus challenge. These results demonstrate that CAEV Vif is absolutely required for efficient in vivo virus replication and pathogenicity and provide additional evidence that live attenuated lentiviruses have to establish a persistent infection to induce efficient protective immunity.

The caprine arthritis encephalitis virus tat gene is dispensable for efficient viral replication in vitro and in vivo.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus closely related to visna virus and more distantly to other lentiviruses, such as human immunodeficiency virus. The genomes of visna virus and CAEV contain a tat gene encoding a protein able to weakly transactivate its own long terminal repeat, suggesting that transactivation may be a dispensable function for viral replication. Three different tat gene mutants of an infectious molecular clone of CAEV were used to study their replication after transfection or infection of primary goat synovial membrane cells and of blood-derived mononuclear cells or macrophages. Our results showed no difference between replication of the wild type and either the complete tat deletion mutant or the tat stop point mutant, whereas slower growth kinetics and lower levels of expression of the partial tat deletion mutant that of the wild type were obtained in these cells. Quantitative PCR and reverse transcription-PCR analyses of the different steps of a single replicative cycle revealed an identical pattern of retrotranscription, transcription, and viral production, whereas time course analysis demonstrated that the intracellular level of viral genomic RNA was affected by the partial tat deletion at later time points. We then compared the infectious properties of the wild-type and tat mutant viruses in vivo by direct inoculation of proviral DNAs into the joints of goats. All the animals seroconverted between 27 and 70 days postinoculation. Moreover, we were able to isolate tat mutant CAEV from blood-derived macrophages that was still able to infect synovial membrane cells in vitro. This study clearly demonstrates that the tat gene of CAEV is dispensable for viral replication in vitro and in vivo.

The vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle.

Complex retrovirus genomes contain a variable number of accessory genes, among which is the vif gene. We investigated in vitro the role of the vif gene of caprine arthritis encephalitis virus (CAEV) by studying the phenotype of five vif mutants after infection of primary goat synovial membrane (GSM) cells and blood-derived monocytes/macrophages. Any deletion introduced into the vif gene resulted in slow and low viral replication and production of virions with an infectious titer lower than that of wild-type viral particles. The wild-type phenotype could be restored by the trans expression of the vif gene in a complementation assay. Quantitative PCR and reverse transcription-PCR analyses were performed in order to determine which stage of the replicative cycle was impaired by the vif deletion. Our results demonstrated that CAEV Vif did not act at the level of reverse transcription or transcription but rather at the late stage of virus formation and/or release, as lower amounts of virus were produced after a single replicative cycle. The vif-deleted CAEV produced after 24 h of infection was still able to infect GSM cells, indicating that the vif gene is not essential for virus infectivity but is required for efficient virus production.

Tumor necrosis factor induces a selective shedding of its p75 receptor from human neutrophils.

The effect of tumor necrosis factor alpha (TNF) on the expression of its specific receptors (p55 TNF-R and p75 TNF-R) on the surface of human neutrophils (PMN) and mononuclear cells (MNC) was investigated and compared to the effect of various agonists. PMN and MNC express both p55 and p75 TNF-R on their membranes. Within minutes of incubation with chemotactic factors or calcium ionophore A23187, both types of TNF-R were down-regulated from the surface on both cell populations. At the same time, soluble forms of these TNF-R appeared in supernatants, in amounts proportional to the extent of down-regulation induced by each stimulus, suggesting that shedding is the major mechanism leading to loss of p55 and p75 TNF-R upon activation with these agonists. Likewise, TNF induced 60-80% and 73-90% decreases in PMN surface p55 TNF-R and p75 TNF-R, respectively. However, modulation of the two types of TNF-R by TNF proceeded through different mechanisms. TNF induced a selective shedding of the p75 TNF-R since, by both enzyme-linked immunosorbent assay and Western blot analysis, only the p75 TNF-R was detected in supernatants of cells stimulated with TNF. Down-modulation of surface p55 TNF-R most probably resulted from TNF-induced receptor internalization, since 125I-TNF bound to PMN p55 TNF-R was rapidly internalized with a t1/2 = 5 min and preincubation of PMN with TNF inhibited by 68 +/- 6% the release of p55 TNF-R triggered upon subsequent treatment with A23187. The apparently unique property of TNF to induce a differential modulation of the two types of TNF-R at the surface of PMN and MNC might play an important role in the control of peripheral blood cell responses to TNF.

Regulation of human basophil activation. III. Impairment of the inhibitory effect of Na+ on IgE-mediated histamine release in patients with allergic rhinitis.

We recently observed that external Na+ inhibited the IgE-dependent human basophil histamine release (HR) in normal subjects. In this article we report differences in the Na+ effect on basophil HR between normal subjects (n = 16) and age matched patients with allergic rhinitis (AR) (n = 18). As expected, in vitro anti-IgE-stimulated basophils from the group with AR released greater amounts of histamine than basophils from the normal group. However, removal of external Na+ (and replacement by N-methyl-D-glucamine) abolished this difference between the two groups. HR in the normal group increased to the same high level as that of the group with AR. By contrast, the release of histamine in the group with AR was not further increased by Na+ removal. Although high releasers were more frequent in the group with AR, the absence of effect after Na+ removal was not due to the high basal release level (in the presence of Na+) because no effect after Na+ removal was also observed with medium releasers. These results strongly suggest that increased basophil HR in populations with AR, and possibly in other allergic populations, is linked to a defect in the inhibitory effect of Na+.

Regulation of human basophil activation. II. Histamine release is potentiated by K+ efflux and inhibited by Na+ influx.

Na+ and K+ are the major extra- and intracellular cations, respectively. We have thus studied the role of these ions on human basophil histamine release by modifying their transmembrane gradients or by increasing membrane ion fluxes using ionophores. 1) When external Na+ (reduced to 4 mM) was replaced by the nonpermeating Na+ substitute N-methyl-D-glucamine, the release of histamine was enhanced in 2 mM Ca2+ (from 37.5 +/- 8.0% in 140 mM Na+ to 68.5 +/- 9.1% in low Na+) and became possible in the presence of low Ca2+ (at 1 microM Ca2+: from 0.6 +/- 0.7% in 140 mM Na+ to 36.2 +/- 8.0% in low Na+); moreover, in low Na+, the release of histamine became partly independent on Ca2+ influx. 2) Increasing the Na+ influx with the cation channel-forming gramicidin D inhibited the release of histamine by 33.2 +/- 13.6% (n = 6) in an external Na(+)-dependent manner. 3) Decreasing K+ efflux using K+ channel blockers (4-aminopyridine, quinine, sparteine) inhibited histamine release in a dose-response manner. 4) The K+ ionophore valinomycin, which increases K+ efflux, slightly enhanced IgE-mediated histamine release when used alone, whereas it potentiated the release of histamine from leukocytes previously treated with 4-aminopyridine by 57.0 +/- 18.6% (n = 7). 5) Decreasing K+ efflux by increasing external K+ inhibited IgE-mediated release in a similar manner as Na+ did. The inhibitory effects of Na+ and high K+ were not additive, thus suggesting that both cations inhibited the release by a common mechanism. In conclusion 1) our data evidence that histamine release from human basophils is inhibited by Na+ influx and potentiated by K+ efflux; 2) they suggest that K+ channels are present on the basophil membrane and that Na+ and K+ fluxes act on histamine release most probably via modulation of membrane potential.

Regulation of human basophil activation. IV. Dissociation between cationic dye binding and histamine release: role of Ca2+ ions.

In previous studies we observed that in vitro histamine release from human basophils could be dissociated from the loss of affinity of basophil granules for a cationic dye, toluidine blue. In the present study we further explored the intracellular signals leading to the decrease in toluidine blue positive basophil (TB+) numbers, with or without histamine release. Since Ca2+ mobilization is a crucial event in secretion and particularly in histamine release, we studied the role of Ca2+ in histamine release as compared to TB+ decrease. In the presence of external Ca2+ (2 mM): i) Ca2+ channel antagonists verapamil and nifedipine up to 10 microM were without effect on IgE-mediated histamine release and TB+ decrease; ii) loading of the leucocytes with Quin2 or preincubation with TMP-8, an internal Ca2+ antagonist, significantly inhibited the release of histamine and the decrease of TB+ basophils. In the absence of added external Ca2+:i) histamine release was abolished whereas the decrease of TB+ was not modified, even in the presence of EGTA;ii) the decrease of TB+ could be inhibited by prolonged EGTA preincubation, by Quin2 loading and incubation with TMB-8. We conclude that histamine release requires both external Ca2+ influx and mobilization of internal Ca2+. In contrast, no influx of external Ca2+ is required for TB+ decrease in which, however, internal Ca2+ mobilization appears to play an important role.

Regulation of human basophil activation. I. Dissociation of cationic dye binding from histamine release in activated human basophils.

Human basophil activation was demonstrated by histamine release (HR) and by the decrease of the toluidine blue-positive basophils (TB+). In four experimental systems, TB+ number decreased in the absence of HR (1) in basophils from atopic subjects stimulated by allergen concentrations below the threshold for HR, (2) in basophils sensitized by anti-2,4-dinitrophenyl IgE stimulated by noncovalently linked 2,4-dinitrobenzene sulfonic acid-human serum albumin (also, the threshold for decrease of TB+ required lower concentrations of sensitizing anti-2,4-dinitrophenyl IgE than for HR), (3) in low Ca++ medium, and (4) in the presence of the Na+/H+ exchanger, monensin. These results suggest that (1) there is a lower threshold for TB+ decrease than for HR in allergen concentration, number of membrane IgE molecules, and number of IgE cross-linkings; moreover, external Ca++ requirement is lower for decrease of TB+ than for HR and (2) TB+ decrease reflects either granule exocytosis or, in the absence of HR, biochemical changes (most probably cation exchanges) altering the interaction of the basic dye with the granules. Thus, monitoring decrease in TB+ allows detection of basophil activation in the absence of HR.

Bimodal IgG4-mediated human basophil activation. Role of eosinophils.

The role of IgG4 antibodies in allergic disorders is suspected. Yet, their presence on human basophil membrane has not been demonstrated and the mechanism of the degranulation induced by anti-IgG4 antibodies remains unclear. As previously reported, we observed that monoclonal anti-IgG4 (10 to 100 micrograms/ml) induced histamine release in the presence of D2O from leukocytes of normal and atopic subjects. The release was accompanied by a decrease of the number of toluidine blue-positive basophils (TB+). Histamine release and TB+ decrease were also observed with lower concentrations of anti-IgG4 (1 to 100 pg/ml). Since basophil activation assessed by TB+ decrease was more sensitive than histamine release, we thus used the former method to further study the mechanisms of the anti-IgG4- vs anti-IgE-induced basophil activation. Basophil activation by anti-IgG4 at 1 to 100 pg/ml, but not by anti-IgG4 at 10 to 100 micrograms/ml or anti-IgE, required the presence of polymorphonuclear cells. Furthermore, anti-IgG4-stimulated purified eosinophils, but not neutrophils, released basophil-activating factors identified as cationic proteins from eosinophils. Thus, the human basophil can be activated by anti-IgG4 via two different mechanisms according to the antibody concentration. At high concentrations (10 to 100 micrograms/ml) basophil activation does not require the presence of polymorphonuclear cells whereas at lower concentrations (1 to 100 pg/ml) the presence of eosinophils is necessary. We propose that in the latter concentration range, basophil activation is a two-step process: 1) release by anti-IgG4 of eosinophil cationic proteins that 2) will, in turn, activate human basophils. This study lends support to the role of IgG4 and eosinophils in anaphylactic reactions.

Effect of sodium cromoglycate and nedocromil sodium on anti-IgE-induced and anti-IgG4-induced basophil degranulation.

Both anti-IgE and anti-IgG4 induce human basophil degranulation as assessed by toluidine blue staining. Anti-IgG4 has been recently shown to act on the human basophil by a 2-step process: anti-IgG4 induces the release from eosinophils of eosinophil cationic proteins which in turn induce human basophil degranulation. In the present study, we show that sodium cromoglycate and nedocromil sodium have no direct effect on human basophil degranulation but inhibit the anti-IgG4-induced degranulation. This effect was dose-dependent and significant inhibitions were obtained at 2.5 to 25 mumol/L for sodium cromoglycate and 25 mumol/L for nedocromil sodium. No drug effect was observed on the basophil degranulation induced by supernatants from anti-IgG4-stimulated eosinophils. However, the release of basophil degranulating factors (eosinophil cationic proteins) by anti-IgG4 from purified eosinophils was significantly inhibited after preincubation with 25 mumol/L of sodium cromoglycate or nedocromil sodium. Taken together, these results indicate that both sodium cromoglycate and nedocromil sodium have an inhibitory effect on the first step of anti-IgG4-induced human basophil degranulation, thus strongly suggesting that these drugs inhibit the release of eosinophil cationic proteins from human eosinophils.

Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells.

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.

Decrease in IgE Fc receptor expression on mouse bone marrow-derived mast cells and inhibition of paf-acether formation and of beta-hexosaminidase release by dexamethasone.

The effect of dexamethasone (DM) on the immunologic and nonimmunologic release of paf-acether and of the granule marker beta-hexosaminidase (BHEX) from mouse bone marrow-derived mast cells (BMMC) was studied. BMMC (1 X 10(6] in a modified Tyrode's solution containing 0.25% bovine serum albumin (BSA) were sensitized with an optimal dose of dinitrophenyl (DNP)-specific monoclonal IgE, and were washed before challenge with 40 ng/ml of DNP coupled to BSA. Preincubation of BMMC for 24 hr with 1 nM to 1 microM DM inhibited in a dose-dependent fashion the immunologic release of paf-acether and of BHEX as compared with control cells, with a half-maximal effect at 20 nM and 4 nM respectively. By contrast, the ionophore A23187 (1 microM)-induced release of paf-acether and of BHEX was unaffected by DM pretreatment. Finally, the antigen-induced increase in acetyltransferase activity, used as an index of cellular activation, was inhibited by 37 +/- 16% in 1 microM DM-treated BMMC as compared with untreated cells. Preincubation of BMMC with DM for 24 hr caused a dose-dependent inhibition of 125I-IgE binding to the cells, with a half-maximal effect at 14 nM. As determined by Scatchard analysis, the number of IgE Fc receptors was decreased by 55% in 1 microM DM-treated BMMC as compared with untreated cells, although the dissociation constants were comparable (control: 12.6 +/- 4.1 nM; DM-treated cells: 14.1 +/- 6.7 nM; mean +/- 1 SD; n = 3). Cytofluorometer analysis of BMMC sensitized with a saturating amount of purified monoclonal IgE, followed by addition of a fluoresceinated anti-mouse IgG (heavy and light chains), revealed a single cellular population for both DM-treated and untreated BMMC. This demonstrates that the DM-induced decrease in IgE Fc receptor expression was exhibited by every BMMC. The possible link between the decreased sensitization of the cells consequent to the reduction in IgE Fc receptor expression and the alteration of the secretory response and acetyltransferase activity was investigated. BMMC were incubated with IgE under experimental conditions giving half-sensitization of the cells. Upon antigen challenge, a 10.5 +/- 3.7% decrease in acetyltransferase activity and a 29.2 +/- 3.5% decrease in paf-acether release were observed with half-sensitized cells as compared with cells sensitized with a saturating amount of IgE.(ABSTRACT TRUNCATED AT 400 WORDS)

[Human basophil degranulation test in the diagnosis and prevention of peranesthetic anaphylactoid complications]. / Le test de dégranulation des basophiles humains dans le diagnostic et la prévention des accidents anaphylactoïdes peranesthésiques.

The human basophil degranulation test (HBDT) studies the allergic response of effector cells. In this work, it was used in the diagnosis of anaphylactoid reactions during anaesthesia. Thirteen patients having had an anaphylactoid shock during general anaesthesia were investigated. An HBDT was performed with every suspected drug used during the anaesthesia. Ten patients had a positive HBDT and in five cases the drug involved was suxamethonium. In one case, the IgE-dependency of the accident was asserted by the ability of the plasma to sensitize in vitro alien basophils. These sensitized basophils degranulated in the presence of Althesin and suxamethonium. Passive sensitization was abolished when using plasma heated for 2 h at 56 degrees C. Two patients had a negative HBDT to 11 drugs tested. To assess the validity of these results, and to rule out unspecific degranulation, thirteen control patients who were to undergo general anaesthesia were investigated. HBDT was performed immediately before anaesthesia in a double-blind method with every drug to be used during the anaesthesia. The twelve patients who had a negative HBDT to every drug had an uneventful anaesthesia. One patient had a positive test to thiopentone and displayed bronchospasm and generalized urticaria during the anaesthesia. In conclusion, the HBDT appears to be a reliable test in the diagnosis of anaphylactoid reactions during anaesthesia.

[Frequent positivity of the human basophil degranulation test in idiopathic nephrotic syndromes (minimal glomerular changes and segmental and focal glomerulosclerosis)]. / Fréquente positivité du test de dégranulation des basophiles humains dans les syndromes néphrotiques idiopathiques (lésions glomérulaires minimes et glomérulosclérose segmentaire et focale).

Atopic factors play an important role in minimal changes idiopathic nephrotic syndrome ( MCINS ). We therefore studied human basophil degranulation test ( HBDT ) in MCINS and also in patients with segmental and focal glomerulosclerosis ( SFGSNS ) whose relation with MCINS is debated. HBDT was performed with 5 or 6 allergens chosen according to the history of patients and results of cutaneous tests. HBDT was positive in 16 out of 28 MCINS and in 14 among 18 SFGSNS subjects. The difference versus 29 Immune Complex glomerulonephritis and 11 blood donors was significant with the chi 2 test. Because the basophil count was low (less than 10/microliter) in 5 cases, 4 of which had a severe nephrotic syndrome, the HBDT was performed later. The allergen most frequently involved was house dust. This was responsible for a significant degranulation in 10 MCINS (45%) and in 11 SFGSNS (68%) patients; degranulation was obtained more frequently with 2 allergens than with a single one. Obviously such a basophil sensitisation does not prove the responsibility of these allergens.

Basophil sensitisation in idiopathic nephrotic syndrome.

The degranulation of basophils in the presence of a specific allergen (human basophil degranulation test, HBDT) is an index of IgE-dependent cellular response and it was tested in 46 unselected idiopathic nephrotic syndrome (INS) patients with or without atopic manifestations. 28 of the INS patients had "minimal change" nephrotic syndrome (MCNS) and 18 had focal segmental glomerular sclerosis (FSGS). 29 patients with different types of primary glomerulonephritis (GN) and 11 healthy donors acted as controls. The HBDT was positive in 16 (57%) of 28 MCNS and in 14 (77.8%) of 18 FSGS patients. Only 5 (17.2%) of 29 GN and 1 (6%) of 11 blood donors exhibited positive HBDT. House dust was the commonest allergen to which MCNS and FSGS subjects were sensitive, whereas sensitivity to grass pollen, moulds, and animal danders was less common. 8 MCNS and 8 FSGS subjects were sensitive to house dust plus another allergen. 4 MCNS and 1 FSGS patient had fewer than 5/microliters basophils; 4 of these 5 patients were having a relapse of their nephrotic syndrome at the time. The demonstration of enhanced IgE-basophil sensitisation in INS patients suggests than an anaphylactic process plays a part in the pathogenesis of INS.