Immunologic compensation in a patient with a large IgH constant region deletion.

BACKGROUND: Deficiencies of serum Ig of the IgG isotype typically predispose individuals to recurrent infections in some but not all cases. Patients with large deletions of the Ig heavy chain genes are free of recurrent and severe infections. OBJECTIVE: We sought to determine a mechanism of immunologic compensation that would possibly explain the reason for this patient's paucity of infection despite lacking several classes of serum Ig. METHODS: The patient is a 50-year-old white man. Serum Ig levels and specific antibody titers were measured by using various methods, including nephelometry, enzyme immunoassay, and radial immunodiffusion. The status of the Ig heavy chain genes was examined by means of Southern blotting of genomic DNA isolated from EBV-transformed B cells. RESULTS: The patient's serum lacked detectable IgG1, IgG2, IgG4, and IgA1 levels. Southern blot analysis demonstrated a large heavy chain constant (C) region gene deletion that included Cgamma1, Calpha1, psiCgamma, Cgamma2, and Cgamma4. Antibody responses to capsular pneumococcal and hemophilus polysaccharide antigens were essentially absent. However, IgG3 antibodies against the protein antigen tetanus toxoid were present. Relatively high antibody titers were found against pneumococcal surface proteins as well. CONCLUSION: We conclude that our patient's relative freedom from serious infection may be as a result of production of IgG3 antibodies to pneumococcal capsular proteins.

Direct access testing in the clinical laboratory: should laboratories offer testing services directly to the consumer?

This article outlines the author's views on the many issues to consider if a laboratory plans to initiate a program to offer clinical laboratory testing directly to the public. Direct access testing and why is it important are discussed in detail. The regulatory issues are outlined with some alternatives for laboratories in states with restrictive regulations. To illustrate many of the operational issues to consider, the author describes the Personal Diagnostic Center (PDC) in Kansas City. Finally, some of the key business issues are mentioned, including several approaches to promote this type of testing program.

Building-associated pulmonary disease from exposure to Stachybotrys chartarum and Aspergillus versicolor.

The authors present an outbreak of disease associated with exposure to Stachybotrys chartarum and Aspergillus species. A courthouse and two associated office buildings had generated discomfort among employees for two years since initial occupancy. Multiple interventions had been unsuccessful An initial evaluation of 14 individuals identified three with potential asthma and three with symptoms consistent with interstitial lung disease. A clinical screening protocol to identify individuals who should be removed from work identified three likely and seven possible cases of building-related asthma. Detailed environmental and engineering assessments of the building identified major problems in mechanical system design, building construction, and operational strategies leading to excess moisture and elevated relative humidities. Moisture-damaged interior surfaces in both buildings were contaminated with S. chartarum, A. versicolor, and Penicillium species. Aspergillus species, especially A. versicolor, at concentrations of 10(1) to 10(4)/m3 dominated the indoor air under normal operating conditions. Bulk samples also revealed large quantities of Stachybotrys. A questionnaire survey of the three case and two control buildings documented between three- and 15-fold increases in symptoms. A nested case-control study suggested emphysematous-like disease in individuals meeting questionnaire definitions for cases. Replication of analysis strategies used in similar previous investigations suggested an association between worsening symptoms and decreased diffusing capacity of the lung. Performance on neuropsychological measures was similar for both cases and controls, although workers with symptoms reported increased levels of current but not past psychiatric symptomatology. Chemical analyses demonstrated the presence of satratoxins G and H. Cytotoxic laboratory analyses demonstrated the presence of agents with biological effectiveness in bulk materials. No association was seen between IgE or IgG antibodies and the presence of disease. This outbreak represents a likely human response to inhaled fungal toxins in indoor environments. Moisture indoors represents a public health issue currently inadequately addressed by building, health, or housing codes.

Endotoxin as a factor in adverse reactions to latex gloves.

BACKGROUND: Endotoxin is an inflammatory made by gram negative bacteria that can irritate the skin, induce respiratory problems, fever, and shock. It is an adjuvant for both delayed hypersensitivity and IgE production and has been shown to magnify antigen specific mediator release. Since many of the clinical problems associated with natural latex products involve similar clinical sequelae, we investigated the possibility that latex gloves might be contaminated with endotoxin. OBJECTIVE: To measure the endotoxin content of a variety of natural latex gloves, investigate the its distribution and origin, associated with latex proteins, and determine the particle sizes associated with its release. METHODS: Endotoxin, protein, and allergen were measured using a quantitative kinetic Limulus assay, modified Lowry, and RAST inhibition, respectively. Particle size and density were determined using an Anderson multistage air sampler and CsCl2 gradient. RESULTS: Endotoxin was found to be a highly significant contaminant of some latex gloves. Levels ranged from 0.09 ng to 2.8 micrograms/g of glove. Protein levels ranged from < 25 to 1150 micrograms/g of glove while allergen levels ranged from < 1 to 837 micrograms/g of glove. Endotoxin and protein eluted rapidly from the interior of the gloves tested. Greater than 70% of the endotoxin was found to be associated with particles in the < 7 microns aerodynamic diameter range. The highest levels of endotoxin were found in nonsterile examination gloves with a tendency towards powdered gloves containing more endotoxin and protein. A slurry containing cross-linked dextran through which gloves were dipped revealed very high endotoxin contamination (64 micrograms/mL) while unused cross-linked dextran has very little associated endotoxin. CONCLUSIONS: These data demonstrate that some natural rubber latex gloves, particularly nonsterile examination gloves, are contaminated with high amounts of endotoxin and proteins. These were found mostly on the inside of gloves and were released as very small respirable particles that were not physically associated with the powder. These findings support the hypothesis that endotoxin may be responsible for some of the tissue irritation associated with latex glove use. In addition, this material may be responsible for the enhancement of delayed and immediate hypersensitivity reactions to chemicals and proteins found in these products and offers a possible explanation for the disproportionate severity of these reactions.

A nurse with anaphylaxis to stone fruits and latex sensitivity: potential diagnostic difficulties to consider.

BACKGROUND: There have been several recent reports describing a cross-reactivity between latex and antigens derived from a number of fruits, including those of the stone fruit family. The relationships between the allergic reactions to the antigens from these plant products are still being defined. OBJECTIVE: To characterize the IgE reactivity of a patient who had anaphylactic reactions following the ingestion of several members of the stone fruit family (ie, plum, peach, and nectarine) and had a positive clinical history for latex allergy. METHODS: The patient's serum was tested for the presence of specific IgE for latex, stone fruits, and a panel of other foods reported to be cross-reactive with latex. Prick testing was also performed with freshly prepared extracts from the implicated fruits. Finally, the immunochemical relationship between this patient's fruit and latex sensitivity was investigated by RAST inhibition. RESULTS: The patient had strongly positive skin tests to the freshly prepared fruit extracts but the in vitro food-specific IgE tests were equivocal or very low positive. In vitro latex-specific IgE tests were strongly positive. The stone fruit extracts were shown to be inhibitors of the patient's latex specific IgE by RAST inhibition. CONCLUSION: Skin testing with freshly prepared fruit extracts was more sensitive than the in vitro tests with this patient. The inhibition data support an immunochemical relationship between the patient's latex allergy and sensitivity to stone fruits.

Elevated postmortem tryptase in the absence of anaphylaxis.

Elevations in tryptase, a recently discovered mast cell enzyme, have been proposed as a postmortem indicator of fatal anaphylaxis. The previous studies had limited numbers of controls and thus the specificity of the test with postmortem samples was not known. Therefore, tryptase was evaluated in postmortem blood samples from 49 autopsy cases where there was no evidence of fatal anaphylaxis. The tryptase was above the normal serum threshold of 1 nanogram/mL (ng/mL) in 31 of these cases. Twenty-four cases had values in the 1 to 5 ng/mL range, two cases were between 5 and 10 ng/mL, and five were greater than 10 ng/mL. One autopsy specimen had a tryptase value of 106 ng/mL. The postmortem interval and the specimen storage condition did not appear to correlate with these elevations in tryptase. Although elevations in the postmortem tryptase remain an important supporting finding in the diagnosis of fatal anaphylaxis, it should not be used alone as the sole criterion for the postmortem diagnosis of anaphylaxis.

Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response.

Horseradish peroxidase (HRP) was covalently coupled to the binding subunit of cholera toxin (CTB) via a two-step glutaraldehyde procedure. The HRP-CTB conjugate was characterized by physiochemical as well as immunochemical methods. Mice were immunized intraduodenally with the HRP-CTB conjugate, with HRP alone, or with a mixture of uncoupled CTB and HRP. The functionally active dose of CTB was 50 micrograms and the HRP dose was in the 30- to 90-micrograms range. Both IgA and IgG antibody responses were measured in serum, intestinal washes, and bile by using a solid phase immunoradiometric assay. Mice immunized with the HRP-CTB conjugate showed a significantly higher level of IgA anti-HRP in intestinal washes and bile, as well as increased levels of serum IgG anti-HRP. Animals that received only HRP or the mixture of CTB and HRP had reduced levels of HRP-specific antibody of either class in both gut washes and bile. The IgA anti-HRP responses in the gut washes were 33- to 120-fold higher when the conjugate was used as the immunogen in comparison with immunization with the CTB + HRP or the HRP alone. Vaccines to stimulate mucosal immunity to any antigenic determinant might thus be prepared by covalent conjugation to effective mucosal immunogens such as CTB.

An assay to measure antigen-specific immune complexes in food-allergy patients.

An allergen- and isotype-specific assay to quantify the presence of serum immune complexes is described. This assay is based on a two-site recognition system so that only molecular complexes containing both antigen and antibody are counted. In this study affinity-purified antibodies to egg and milk whey were used on paper discs in conjunction with a soluble, labeled anti-immunoglobulin (IgE or IgG specific). To evaluate the assay, immune complexes were prepared in vitro by mixing antigen at different concentrations with serum from a patient with high levels of antibody. Complexes were detectable over a 5 log range of antigen concentration. The lower limit of sensitivity of the assay was estimated to be 5.8 ng of complexed IgE per milliliter of patient serum. To determine if the assay could detect natural in vivo-formed complexes, sera from patients with positive RAST scores to egg white or milk were selected for a preliminary study. Three of the nine egg white-positive sera and two of the nine milk-positive sera were found to contain significant levels of IgE immune complexes. The versatility and sensitivity of the assay should now make it feasible to design experiments to elucidate the role, if any, of circulating immune complexes in the etiology of food allergy.

Expression of Fc gamma binding sites on human extraplacental membranes.

Binding sites for the Fc portion of immunoglobulin G (IgG) molecules (Fc gamma BS) were identified on amnionic epithelial cells and chorionic trophoblast cells in human extraplacental membranes using direct immunofluorescence with labeled human IgG. The binding sites are similar to FC gamma BS on placental trophoblast in their specificity and high degree of relative binding affinity for IgG monomers. Neither immunoglobulin A (IgA) nor IgM bound to a significant degree to the membranes. Binding was completely inhibited with unlabeled IgG but not F(ab')2 fragments of IgG. Studies on isolated amnion cells demonstrated that the binding sites are expressed on the cell membrane. At the level of sensitivity of the immunohistologic methods used, aggregated IgG or antigen-antibody complexes failed to bind amnionic epithelial cells or chorionic trophoblasts. This is in contrast to Fc gamma BS on macrophages which, being present in the same tissue, failed to exhibit significant binding of deaggregated IgG but bound complexes avidly.

Metabolism and clearance of antibody-excess immune complexes in lactating mice.

The metabolism of antibody-excess immune complexes was compared in lactating and normal female BALB/c mice. The initial rate of clearance of the antigen, dinitrophenyl- (DNP) ovalbumin, when complexed with mouse anti-DNP antibody was considerably faster in lactating mice. The half-life was 4 min in lactating mice compared with 8 min in normal female mice. Tissue distribution studies revealed that by 2 hr the mammary gland had sequestered immune complexes in threefold to fourfold excess over the levels in the liver. These lactating mice were shown to transfer immune complexes to their milk, and small quantities of the complexes could be demonstrated in the circulation of the nursing neonates.

Metabolism of immunoglobulin A in lactating mice: origins of immunoglobulin A in milk.

The metabolism of albumin and IgA was studied in normal and lactationg mice. Lactation resulted in significant changes in the metabolism of these proteins. The serum albumin concentration was lowered from 47 mg/ml in normals to 24 mg/ml in lactating mice. However, only a slight decrease in the serum concentration of IgA was observed during lactation. The proportion of polymeric and monomeric IgA in serum and milk was evaluated by gel exclusion chromatography. The onset of lactatin led to a rise in the proportion of polymeric IgA (P-IgA) in serum from 37% to 51%. The proportion of P-IgA in milk was 65% and remained constant throughout lactation. P-IgA and albumin were shown to be efficiently transferred from the serum of lactating mice into their milk. Serum decay studies were performed to evaluate the turnover of the serum pools during lactation. The rates of disappearance from the serum of isotopically labeled albumin and P-IgA were observed to increase dramatically during lactation, suggesting that both of these two mild proteins might be derived at least in part from the serum. The sites of synthesis of mild IgA (local vs. extra-mammary gland) were evaluated by determining the extent of dilution of isotopically labeled serum IgA during transport through the mammary gland into the milk. Early in lactation, the majority of the IgA in mouse milk appeared to be derived from distant sites and transferred via the blood to the mammary gland. However, by day 8 of lactation, the isotopically labeled P-IgA in milk was significantly diluted by the IgA synthesized in the mammary gland. Albumin and IgG were not diluted by local synthesis indicating that these proteins were exclusively serum-derived.

Network theory in autoimmunity. In vitro suppression of serum anti-DNA antibody binding to DNA by anti-idiotypic antibody in systemic lupus erythematosus.

Regulation of serum anti-DNA antibody in systemic lupus erythematosus (SLE) by an antiidiotypic antibody was evaluated. Various sera from SLE patients in active and inactive states of their disease, as well as sera from normal individuals, were first completely depleted of anti-DNA and of DNA by affinity chromatography. The suppressive capacity of equimolar concentrations of the various depleted sera (blocking sera) on target lupus sera were determined. The target sera were from lupus patients with known DNA-binding capacity. Blocking sera from inactive SLE suppressed the binding of autologous anti-DNA antibody to [(3)H]DNA (n = 19,P < 0.01). Blocking sera from active SLE (n = 19), as well as human serum albumin, did not suppress. Sera from normal donors who had no contact with lupus patients or with lupus sera did not suppress (n = 14, P > 0.5), whereas those from normal donors who had contact with lupus patients or sera did suppress the binding (n = 5,P < 0.02). The anti-anti-DNA antibody suppressive activity in the inactive lupus serum was shown to be localized within the F(ab')(2) portion of immunoglobulin (Ig)G and could not be removed upon adsorption by normal human gammaglobulin. Furthermore, immune complexes could be detected by a Clq binding assay when the inactive lupus blocking sera were incubated with the anti-DNA antibody containing target sera. The specificity of the suppressive serum factor was shown by its inability to block the binding of tetanus toxoid to antitetanus antibody and its ability to block the binding of DNA to F(ab')(2) fragments of active lupus IgG. Regulation of serum anti-DNA antibody levels by anti-antibodies could induce and maintain disease remission in lupus patients and prevent disease expression in normals.

Immune complexes facilitate uptake of antigen from the gut.

Radiolabeled DNP-ovalbumin was injected with and without DNP-specific antibody into ligated gut segments of neonatal rats. The transfer of this labeled protein to mesenteric lymph nodes and binding to gut tissue was examined. When the antigen, DNP-ovalbumin, was present in the gut in an immune complex from a selective localization of antigen in the mesenteric lymph node was observed. These data suggest that immune complexes in milk could potentially modulate neonatal immunological development.

Transport of immunoglobulins from serum into colostrum.

Oligomeric, J-chain-containing immunoglobulins were observed to be transferred selectively from serum into colostrum. These studies suggest that, in the case of the mammary gland secretion, a significant role for extraglandular synthesis of IgA merits consideration. Thus, for example, colostrum may contain antibodies synthesized locally as well as antibodies synthesized in the much larger lymphoid tissues such as the gut lamina propria.

DNA binding activity of serum from patients with systemic lupus erythematosus.

The reaction conditions essential for reproducible use of the cellulose ester membrane filter assay for anti-DNA antibody levels in patients with systemic lupus erythematosus are described. A dependence of DNA-binding capacity on serum concentration was observed in the assay, requiring that serum concentrations be comparable in determinations of DNA-binding activity of different sera and when comparing different published studies. The DNA-binding capacity of serum from lupus patients was found to be significantly different from that of healthy controls. However, the binding capacities were not significantly different between lupus patients with and without nephritis. The relative avidity of the anti-DNA antibodies were estimated from plots of 1/DNA bound vs 1/DNA free and these data indicate that the avidities of the antibodies from the two groups of lupus patients are not significantly different. This observation suggests that the tightness of binding between the DNA and the serum anti-DNA antibodies cannot be used to predict immune complex-induced nephritis in lupus patients.