Essential structural requirements for triggering of mast cells by a synthetic peptide comprising a sequence in the C epsilon 4 domain of human IgE.

A range of synthetic analogues of the peptides Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-PheNH2 (human IgE epsilon-chain 497-506 decapeptide) and Lys-Thr-Lys-Gly-Ser-Gly-Phe-PheNH2 (epsilon-chain 497-504 octapeptide) were tested for activity as releasers of 5-hydroxytryptamine from rat peritoneal mast cells. The following structural modifications were found to abrogate activity: N-acetylation of the alpha-amino group of the N-terminal lysine residue; substitution of the two lysine residues by either serine or glutamine; depletion of the two C-terminal hydrophobic residues (Val-Phe) of the decapeptide; and substitution of phenylalanine by alanine in the C-terminal position of the octapeptide. These observations point to a requirement for positively charged amino acids and hydrophobic amino acids at the N- and C-terminus respectively for triggering of mast cells by these short-chain peptides. Releasing activity was also found to depend on the stereospecific conformation of the positively charged region, since substitution of L-isomeric amino acids by D-isomeric forms in the three N-terminal positions of the decapeptide led to loss of potency. Inactive analogues of the decapeptide and octapeptide, at concns up to 10(-4) M, failed to antagonise the mediator-releasing effects of the active decapeptide at concns of 3 X 10(-6) - 10(-4) M.

Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to protein-reactive drugs and metabolites: criteria for identification of antibody activity. Detection and hapten specificity of anti-DNP, anti-captopril and anti-sulphanilamidobenzoic acid.

Certain hypersensitivity reactions to drugs are thought to depend on coupling of reactive species (the drug itself or a metabolite) to macromolecules, leading to the formation of hapten-carrier conjugates. In assays for the detection of antibodies directed against such reactive species the drug or metabolite must be used in conjugated rather than free form. We describe ELISAs for the detection of anti-dinitrophenyl (DNP), anti-captopril (CP) and anti-sulphanilamidobenzoic acid (SABA) antibodies, in which the wells of microtitre plates are coated with hapten conjugated to protein. We define coating conditions and the following 3 criteria for identification of anti-hapten activity: Immunoglobulin in the test sample binds to the immobilised hapten-protein conjugate, but not to the immobilised protein alone. Binding is inhibited by preincubation of the test sample with protein conjugates incorporating the test hapten, but not by preincubation with the same unconjugated proteins, nor protein conjugates incorporating haptenic groups unrelated to the test hapten. The inhibitory hapten-protein conjugates are shown to be inactive in unrelated antigen-antibody interactions. Binding is blocked by preincubation of the test sample with low molecular weight chemical derivatives of the reactive hapten. The inhibitory derivatives must be shown to be inactive in unrelated antigen-antibody interactions. On the basis of these criteria, IgG anti-DNP and IgG anti-CP were detected in the sera of immunized rabbits. The IgG anti-DNP antibody recognised protein-conjugated DNP, DNP-lysine, N-acetyl-DNP-lysine and DNP-S-glutathione, whereas the IgG anti-CP antibody recognised CP-S-S-protein and CP-S-S-CP. By the same criteria IgG anti-SABA was detected in the sera of immunized mice. The antibody recognised free and protein-conjugated SABA, but not free sulphanilamide.

Neuraminidase- and benzalkonium chloride-dependent inhibition of basic peptide-induced rat mast cell secretion.

Two types of inhibition of basic peptide-induced rat mast cell secretion are reported. Pretreatment of rat peritoneal mast cells with Vibrio comma neuraminidase, an enzyme which cleaves sialic acid from oligosaccharides, led to inhibition of 5-hydroxytryptamine release induced by the basic peptides polylysine, corticotropin 1-24 and a decapeptide sequence of human IgE. Inhibition was similarly observed when mast cells were challenged in the presence of the cationic cell membrane-active substance benzalkonium chloride. It is postulated that both of these experimental procedures inhibit basic peptide-induced secretion by depletion of cell surface negative charge. Sialic acid itself does not act as a specific receptor for basic peptides, since a molar excess of sialic acid in free solution failed to inhibit secretion by binding to basic peptides in the fluid phase.

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A.

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

Cycloheximide treatment of mouse mast cells inhibits serotonin release. Evidence of a requirement for newly synthesized protein in the exocytotic response.

Treatment of mouse peritoneal mast cells and mouse bone marrow-derived cloned mast cells with the protein synthesis inhibitor cycloheximide led to a marked abrogation of serotonin (5-hydroxytryptamine; 5-HT) release induced by a range of activators including antigen, anti-immunoglobulin E (anti-IgE) antibody, calcium ionophore A23187, and the polycation polylysine. Significant inhibition (28%) of IgE-mediated secretion was attained after incubation of peritoneal cells for only 2 hr, and inhibition progressed over a 24 hr period, reaching greater than 80% after 15 hr. When peritoneal mast cells were exposed to cycloheximide and then washed and returned to culture conditions, a substantial recovery of responsiveness to anti-IgE was seen after 5 hr. Under the same conditions to those used in functional studies, cycloheximide inhibited protein synthesis, measured as incorporation of [35S]-methionine, by purified peritoneal mast cells and cloned mast cells to 18.5% and 7.9% of control levels, respectively. These results show that synthesis of new protein over a period of a few hours is required to render mast cells fully responsive to stimuli that act via the IgE receptor, and to certain other stimuli that are receptor-independent.

Drug-protein conjugates--XVII. The effect of storage on the antigenicity and immunogenicity of benzylpenicillin in the rat.

The disposition and immunogenicity of freshly prepared and stored solutions of benzylpenicillin (BP) and benzylpenicillenic acid (BPE), a degradation product of BP, were studied. No IgG anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA) following daily i.p. or i.m. administration to male Wistar rats of BP (2.7 mmol/kg) freshly dissolved in 0.5% glucose, for 4 consecutive days at 4-week intervals. In contrast, IgG anti-BPO antibodies were detected following both chronic i.p. and i.m. administration of BP (2.7 mmol/kg) stored for 24 hr at room temperature in 0.5% glucose. An IgG anti-BPO response was obtained only after the high dose, following daily i.m. administration of BPE (27 mumol/kg, 2.7 mumol/kg, 0.24 mumol/kg). The specificity of the IgG antibody for the BPO-determinant was confirmed by ELISA inhibition with BPO-amino-caproate. Circulating BPO plasma-protein antigens were detected by a modified ELISA following i.p. and i.m. administration of both stored and fresh BP. Significantly lower BPO-antigen levels were detected in serum following BPE administration. Irreversible binding of BP to 75% rat plasma proteins was of the same magnitude when freshly dissolved in phosphate buffer or in 0.5% glucose (2.63 +/- 0.32% and 2.55 +/- 0.25% bound, respectively after 3 hr incubation at 37 degrees). Irreversible binding was significantly greater (P less than 0.05) when the BP was stored prior to incubation with the protein (3.81 +/- 0.27%). The major degradation product of stored BP was benzylpenicilloic acid; a small amount of BPE (0.2% of incubated BP) was detected in stored but not fresh BP. Thus, the increased immunogenicity of BP stored for 24 hr at room temperature may be due to the formation of reactive degradation products such as BPE in vitro, which can then form immunogenic drug-protein conjugates in vivo. These experiments also show that although BP and BPE form drug-protein conjugates in vivo, circulating levels of antigen do not relate to the immunogenicity of either of the compounds.

Drug-protein conjugates--IX. Immunogenicity of captopril-protein conjugates.

The immunogenicity of captopril (CP), conjugated to heterologous proteins, was investigated in male New Zealand White rabbits by monthly injections of CP-protein conjugates in Freund's Complete Adjuvant. Anti-CP antibody activity was readily detected by immunodiffusion in sera of rabbits immunized with the amide-linked CP-HSA (23:1) conjugate. Hapten inhibition studies revealed that the antigenic determinant contained CP and a lysine residue from the protein carrier. When rabbits were immunized with disulphide-linked CP-S-S-HSA (9:1) and CP-S-S-KLH (160:1) conjugates, anti-CP antibody activity was detected by a sensitive ELISA method, but not by immunodiffusion and radioligand binding assays. The specificity of the serum IgG anti-CP activity after immunization with disulphide-linked CP-S-S-protein conjugates was confirmed since anti-CP activity was inhibited by preincubation of the antisera with CP conjugated to an unrelated protein carrier (CP-S-S-OVA), but not by the corresponding unconjugated protein, nor by penicillamine-S-S-protein conjugates. These results show that disulphide-linked CP-protein conjugates are sufficiently stable to induce humoral (B lymphocyte) anti-hapten responses under the experimental conditions employed. In a separate study, delayed-type skin hypersensitivity reactions to topically applied CP were demonstrated in the guinea pig. The specific and sensitive immunochemical technique (ELISA) described here could be useful in future studies for determining whether or not patients taking CP produce antibodies to the drug.

Drug-protein conjugates--XV. A study of the disposition of D-penicillamine in the rat and its relationship to immunogenicity.

The disposition of [14C]D-penicillamine (PA) was investigated in vitro and in vivo with male Wistar rats. Irreversible binding of [14C]PA to isolated rat plasma proteins in vitro reached a maximum of 20.6% of total radioactivity at 6 hr. Irreversibly bound [14C]PA could be dissociated with dithiothreitol, demonstrating that conjugation was via disulphide linkage. Three hours after i.v. administration of [14C]PA (27 mumol/kg) to rats 100% of plasma radioactivity was irreversibly bound, representing approximately 3.5% of the dose. Further studies on the disposition of PA-plasma protein conjugates showed that dissociation occurred readily in vivo: the plasma half-life of the conjugate was approximately 3 hr. Free [14C]PA was the major urinary metabolite after administration of both free and conjugated drug. These studies show that the disposition of PA is similar to that reported for the structurally related sulphydryl drug captopril (CP). Free PA (340 mumol/kg and 3.4 mmol/kg) administered i.p. and i.m. daily for 4 days at one monthly intervals, and also PA-KLH conjugate (100 micrograms/rat) administered by single i.p. injection at monthly intervals with and without Freund's complete adjuvant, failed to induce PA-specific IgG or IgM antibody responses detectable by ELISA. In contrast, CP (270 mumol/kg), administered by the same protocol as free PA, induced a CP-specific IgG antibody response after the third series of monthly injections. These data suggest that the difference in immunogenicity between PA and CP arises from a difference in the intrinsic immunogenicity of the haptens, rather than from their disposition.

Drug-protein conjugates--XI. Disposition and immunogenicity of dinitrofluorobenzene, a model compound for the investigation of drugs as haptens.

The conjugation of drugs to autologous proteins is thought to be a key step in the hapten mechanism of drug hypersensitivity. We have studied the mild arylating agent dinitrofluorobenzene (DNP-F) as a model compound with which to investigate the relationship between the disposition and immunogenicity of drug haptens in two species, the rat and rabbit. Intramuscular administration of DNP-F (0.027-27 mumol/kg/day) for 4 days to male Wistar rats produced a dose-dependent (ED50 2.7 mumol/kg) IgG anti-DNP antibody response, measured by enzyme-linked immunosorbent assay. Subsequent monthly administrations (for 4 days) increased both the frequency and titre of antibody response. Intravenous administration of [3H]DNP-F (0.27 or 2.7 mumol/kg) for 9 days to male New Zealand White rabbits produced an IgG and IgM anti-DNP response in all animals from day 9 onwards. Formation of circulating (serum) DNP-protein conjugates was determined by radiometric analysis, and found to reach steady state (0.12-0.17% dose/ml) between days 6 and 8 and decline with a half-life of 7.4 days. The immunogenicity of fully characterized haptenated, autologous proteins was investigated in further experiments in which dinitrophenylated serum protein conjugates (DNP-RSP) and albumin conjugates (DNP-RSA) were prepared ex vivo and then administered (50 mg/kg; i.v.) to the rabbit from which the protein had been obtained. The plasma clearance and immunogenicity of DNP-RSA conjugates was dependent on epitope density. Anti-DNP antibodies were detected after administration of an RSA-DNP15 conjugate but not after either RSA-DNP0.5 or RSA-DNP5. Plasma concentrations of RSA-DNP15 conjugate declined slowly initially, but then fell rapidly between days 8 and 10. The plasma clearance of DNP-RSP conjugates showed a dependence on epitope density from day 1 onwards and anti-DNP antibodies were detectable after administration of all conjugates investigated (range of epitope densities 0.5-30 DNP/albumin equivalent). Thus conjugates derived from proteins other than albumin are likely to be the effective immunogens, for the model hapten DNP. These studies show that DNP-F is a useful model compound in studies of the disposition and immunogenicity of drugs acting as haptens, and may therefore be used as a positive control in experiments designed to assess the potential immunogenicity of drugs and other xenobiotics.

Drug-protein conjugates--XII. A study of the disposition, irreversible binding and immunogenicity of penicillin in the rat.

The disposition, irreversible binding and immunogenicity of benzylpenicillin (BP) were studied in male Wistar rats. [3H]BP, administered i.v. to anaesthetized rats at two doses (27 mumol/kg, 2.7 mmol/kg), showed dose-dependent kinetics: plasma and tissue concentrations of total BP were disproportionately increased at the higher dose. BP was rapidly cleared from the plasma at both doses (less than 0.05% of administered dose/ml plasma after 3 hr). In spite of the disproportionately elevated levels of total BP after the higher dose, covalent binding to plasma proteins was quantitatively similar as a percentage of the dose at both doses. Three hours after i.v. injection of 27 mumol/kg and 2.7 mmol/kg of the drug, 5.6% +/- 1.7% and 3.3% +/- 1.1% respectively of circulating BP was covalently bound, representing less than 0.004% of the administered dose bound per ml of plasma in each case. Covalent binding of BP to rat plasma proteins in vitro was of a similar magnitude to that observed in vivo: 1.6% +/- 0.4% of BP was bound to 25% rat plasma after 3 hr incubation at 37 degrees. In a separate series of experiments the immunogenicity of BP was studied by chronic administration of the drug to rats. Following daily i.v. or i.m. administration of BP (27 mumol/kg, 270 mumol/kg, 2.7 mmol/kg) for 4 consecutive days at 4-week intervals (three series of injections) neither IgG nor IgM anti-benzylpenicilloyl (BPO) antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Intravenous administration of the high dose of BP was discontinued after the first series of injections due to local necrosis. In contrast to free BP, BPO-keyhole limpet haemocyanin (BPO-KLH, 42 nmol BP bound/mg KLH) administered by single i.v. injection at 4-week intervals at two doses (20 and 200 micrograms conjugate/kg, corresponding to 0.84 and 8.4 nmol BPO/kg) readily induced IgG and IgM anti-BPO antibody responses (median IgG titres were 872 and 5470 one week after the third injection of the low and high dose of conjugate respectively; corresponding IgM titres were 4513 and 22,866). The specificity of the IgG and IgM antibodies for the BPO determinant was confirmed by ELISA inhibition with BPO-aminocaproate. These experiments show that BP binds irreversibly, but to a limited extent, to plasma proteins in vivo, and that such a degree of conjugation appears to be insufficient to elicit a detectable anti-BPO antibody response.

Regulation of the Secretory Function of Mouse Peritoneal Mast Cells by IL-3, IL-4 and IFN-γ.

We investigated IL-3 IL-4 and IFN-γ for regulatory effects on the IgE-mediated exocytotic secretory function of mast cells in vitro. Mouse peritoneal mast cells were incubated under cell culture conditions with purified mouse recombinant cytokines, either alone or in various combinations, for 48 h, and monoclonal IgE antibody was added for the second 24 h of culture. Exposure of the cells to IL-3 or IL-4 at only a few nanograms per millilitre led to an enhancement in 5-HT release upon subsequent challenge of the cells with antigen. In contrast, IFN-γ at comparable concentrations strongly inhibited 5-HT release, regardless of whether the cells had been maintained in the absence or presence of IL-3 and/or IL-4. We conclude that IL-3/IL-4 reciprocate with IFN-γ to regulate degranulation.

The mast cell response to substance P: effects of neuraminidase, limulin, and some novel synthetic peptide antagonists.

We have investigated certain aspects of the mechanism whereby substance P triggers secretion of 5-hydroxytryptamine (5-HT) from rat peritoneal mast cells in vitro. Substance P-induced release of 5-HT was inhibited following pretreatment of rat peritoneal cells with 0.01-1.0 units/ml neuraminidase; secretion induced by anti-IgE antibody was inhibited by pretreatment with 1.0 units/ml but not by lower concentrations of enzyme. Addition of the sialic acid-rich substances N-acetyl-neuraminlactose (up to 1.0 mM) and mucin (up to 1.0 mg/ml) to substance P in free solution failed to block the activity of the neuropeptide. Limulin, a sialic acid-specific lectin, failed to block substance P-induced secretion of 5-HT, but was found to possess intrinsic non-lytic secretory activity (at 5-20 micrograms/ml). Release of 5-HT induced by limulin was independent of that induced by substance P. A range of octapeptides incorporating the C-terminal sequence Gly-Ser-Phe-Phe, but differing in degree of cationicity and positioning of cationic residues in the four N-terminal positions, were tested for their capacity to antagonise the mast cell-triggering activity of substance P. A peptide incorporating two lysine residues at the N-terminus was found to have partial substance P antagonist activity; no effects on IgE-mediated secretion were observed.

Ureterovesical reimplantation in children. Surgical results in 491 children.

The experience with ureterovesical reimplantation in 491 children is presented. A success rate of 92% using the Paquin technique is reported. The ineffectiveness of ureteral reimplantation in the face of massive hydronephrosis and thick-walled contracted bladder is stressed.

Neomycin-induced neuromuscular blockade.

The unusual complication of neuromuscular blockade secondary to neomycin absorption is described. The syndrome characterized by acute muscle flaccidity, diaphragmatic breathing, and central nervous system depression presents a potentially fatal situation. Appropriate treatment of this complication includes respiratory assistance and calcium gluconate administration (IV). A review of the pediatric literature reveals 12 previous cases, of which 2 were secondary to urologic procedures.

Comparison of Pilcher bag technique with standard suprapubic prostatectomy.

A total of 930 cases of open prostatectomies done at Lenox Hill Hospital from 1965 to 1974 are reviewed. The Pilcher bag technique utilized in 830 cases is compared with 100 cases of suprapublic prostatectomy with suturing of the bladder neck. Operating time, intra- and postoperative blood transfusions, hospital stay, and complications are compared. Operative time and intra- and postoperative blood loss are significantly less with the Pilcher bag technique.

Experimental use of free grafts of bladder mucosa in canine bladders. Successful closure of recurrent vesicovaginal fistula utilizing bladder mucosa.

Three to 4 cm-diameter, circular-shaped full thickness sections of the anterior wall of canine bladders were resected. Free grafts of bladder mucosa were lifted from the muscular wall and sewn back as a patch onto the original defect. Fusion of the graft and normal urothelium occurred, and at eight weeks a dense fibrotic external layer and normal urothelium was observed. No diverticuli occurred. A successful closure of recurrent multiple vesicovaginal fistulas in a woman using mucosa alone is reported.

Renal transplantation between HL-A identical siblings with partial nephrectomy and machine preservation for ossified renal cell carcinoma.

A case of renal transplantation between HL-A identical siblings is reported in which the donor kidney was found to have a calcified mass in the upper pole. Because an immediate pathologic diagnosis could not be made at the time of nephrectomy, the kidney was preserved with pulsatile perfusion for fifty-four hours after excision of the upper pole. At that time the diagnosis was still not available, and transplantation was performed only to have the report of ossified renal cell carcinoma established the following day.

The bladder mucosal graft technique for hypospadias repair.

A technique for the repair of severe hypospadias is described. The essential features include: chordee release and cosmetic repair of the previously tethered penis; the construction of a new urethra using bladder mucosa in a subcutaneous tunnel; and an oblique anastomosis of the graft and true urethra as deep within the shaft of the penis as possible. One stricture has developed utilizing this technique. The remainder of the children appear to have obtained a good result. Meatal stenosis requiring dilatation remains a minor problem. The use of bladder mucosal grafts in cases in which previous repairs have failed or when adequate foreskin for urethral reconstruction is lacking is feasible and has been successfully performed in adults. The combination of a chordee release and bladder mucosal inlay graft as a single-stage procedure in a small child had not yet been attempted.

Nitric oxide in immunity and inflammation.

Nitric oxide (NO) is synthesised by many cell types involved in immunity and inflammation. The principal enzyme involved is the inducible type-2 isoform of nitric oxide synthase (NOS-2), which produces high-level sustained NO synthesis. NO is important as a toxic defense molecule against infectious organisms. It also regulates the functional activity, growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and natural killer cells. However, the role of NO in nonspecific and specific immunity in vivo and in immunologically mediated diseases and inflammation is poorly understood. NO does not act through a receptor-its target cell specificity depends on its concentration, its chemical reactivity, the vicinity of target cells and the way that target cells are programmed to respond. At high concentrations as generated by NOS-2, NO is rapidly oxidised to reactive nitrogen oxide species (RNOS) that mediate most of the immunological effects of NOS-2-derived NO. RNOS can S-nitrosate thiols to modify key signalling molecules such as kinases and transcription factors. Several key enzymes in mitochondrial respiration are also inhibited by RNOS and this leads to a depletion of ATP and cellular energy. A combination of these interactions may explain the multiple actions of NO in the regulation of immune and inflammatory cells.

The effects of mercuric chloride on growth, cytokine and MHC class II gene expression in a human leukemic mast cell line.

We have examined the effects of mercuric chloride (HgCl2) on growth and IL-4, IL-8, TNF-alpha and MHC class II gene expression in the HMC-1 human leukemic mast cell line. Proliferation, measured by [3H]thymidine incorporation or production of a formazan product (MTT assay), was substantially inhibited by HgCl2 at concentrations of 10(-6) M and above. Inspection of the DNA by agarose gel electrophoresis from HgCl2-treated cells revealed that it was intact, indicating inhibition of DNA synthesis, but not denaturation. HgCl2 inhibited expression of mRNA for IL-8, TNF-alpha and MHC class II at 4 x 10(-6) M and inhibited expression of IL-4 mRNA at 8 x 10(-6) M and above. At a concentration of 10(-5)M, HgCl2 almost completely blocked mRNA expression for IL-4, IL-8, TNF-alpha and MHC class II, but produced negligible inhibition of expression of mRNA encoding the housekeeping gene beta-actin, thus demonstrating selective toxicity for the cytokine and MHC class II genes studied. Pre-exposure of the cells to human recombinant IL-4 prior to treatment with HgCl2 had no effect on expression levels of any of the genes examined. The effects seen in this study are consistent with previous reports showing immunotoxic effects of HgCl2 on other cell types, therefore, the HMC-1 mast cell line may prove useful in further studies of mast cell cytokine gene expression and the mechanisms involved in cytokine gene toxicity.