Patricia C. Giclas

Patients with Nontuberculous Mycobacterial Lung Disease Exhibit Unique Body and Immune Phenotypes

RATIONALE Among patients with nontuberculous mycobacterial lung disease is a subset of previously healthy women with a slender body morphotype, often with scoliosis and/or pectus excavatum. We hypothesize that unidentified factors predispose these individuals to pulmonary nontuberculous mycobacterial disease. OBJECTIVES To compare body morphotype, serum adipokine levels, and whole-blood cytokine responses of patients with pulmonary nontuberculous mycobacteria (pNTM) with contemporary control subjects who are well matched demographically. METHODS We enrolled 103 patients with pNTM and 101 uninfected control subjects of similar demographics. Body mass index and body fat were quantified. All patients with pNTM and a subset of control subjects were evaluated for scoliosis and pectus excavatum. Serum leptin and adiponectin were measured. Specific cytokines important to host-defense against mycobacteria were measured in whole blood before and after stimulation. MEASUREMENTS AND MAIN RESULTS Patients with pNTM and control subjects were well matched for age, gender, and race. Patients with pNTM had significantly lower body mass index and body fat and were significantly taller than control subjects. Scoliosis and pectus excavatum were significantly more prevalent in patients with pNTM. The normal relationships between the adipokines and body fat were lost in the patients with pNTM, a novel finding. IFN-γ and IL-10 levels were significantly suppressed in stimulated whole blood of patients with pNTM. CONCLUSIONS This is the first study to comprehensively compare body morphotype, adipokines, and cytokine responses between patients with NTM lung disease and demographically matched controls. Our findings suggest a novel, predisposing immunophenotype that should be mechanistically defined.

Differential effects of the complement peptides, C5a and C5a des Arg on human basophil and lung mast cell histamine release.

The ability of purified anaphylatoxins to induce human lung mast cell mediator release was investigated. In eight anti-IgE responsive (histamine release = 22 +/- 5%, mean +/- SEM) mast cell preparations of 1-96% purity, C5a and C5a des Arg (0.55 pg/ml to 55 micrograms/ml), failed to elicit or potentiate histamine release; lung fragments were similarly unresponsive. The related peptide C3a was also inactive. All anaphylatoxins failed to induce mast cell leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) release. LTC4 release was also negligible from basophils where C5a was a potent histamine release stimulus. Supernatants from C5a-challenged mast cells remained fully active on basophils, excluding carboxypeptidase inactivation of C5a as an explanation for the lung mast cell results. In contrast to lung, skin mast cells were C5a-responsive (histamine release = 8 +/- 1%, at 55 micrograms/ml, n = 2). We conclude that C5a, though devoid of activity on the human lung mast cell, is a human basophil and skin mast cell secretagogue. These findings demonstrate significant organ-specific heterogeneity in mast cell responsiveness.

Rush immunotherapy results in allergen-specific alterations in lymphocyte function and interferon-γ production in CD4+ T cells

BACKGROUND Allergen immunotherapy results in a number of changes in clinical, inflammatory, and immunologic parameters. However, the basis for the specificity of this form of therapy is unknown, especially in the context of changes in T- and B-lymphocyte function after desensitization to specific allergens. OBJECTIVE This study was designed to determine the immunologic consequences of rush immunotherapy. METHODS We studied 10 patients who had positive skin test responses to the house dust mite Dermatophagoides pteronyssinus (Dpt) and cat dander extract. Each received rush immunotherapy to mite, but not cat dander, over a 2- to 4-week period until maintenance was achieved. Patients were evaluated before and when maintenance was achieved for skin test and nasal reactivity to mite and cat dander; antibody levels to the allergen were monitored, as were lymphocyte proliferative responses and cytokine production. RESULTS Rush immunotherapy to house dust mite resulted in a significant reduction in skin and nasal reactivity to mite allergen, but not to cat allergen, in 10 of 10 patients. This was accompanied by a rise in serum anti-Dpt IgE, whereas anti-cat IgE was not altered (7 of 7 patients). In seven of seven patients there was an increase in anti-Dpt IgG4 levels. T-cell proliferative responses to mite antigen were suppressed, and numbers of CD8+ T cells increased in frequency. There was a marked increase in interferon-gamma production, particularly by CD4+ T cells in 10 of 10 patients. The correlation between the increases in interferon-gamma production and the changes in cutaneous reactivity was highly significant. CONCLUSION We show that rush immunotherapy is immunologically specific in eliciting changes in T- and B-cell responses to the desensitization antigen. The specificity and potential benefit of immunotherapy may be linked to the increase in interferon-gamma production by allergen-activated CD4+ T lymphocytes.

An international serum standard for application in assays to detect human complement activation products

The importance of the complement system in clinical medicine has become evident during the last decades and complement therapeutics has now reached the clinic. Thus, there is an increased interest in and need for assays to evaluate complement activity and dysfunction. Pathologically increased complement activation can indirectly be evaluated by quantification of complement components, but in order to exactly measure such activation, assays for quantification of products formed during activation are required. Progress in this field is hampered by lack of standardization. Therefore, members of the International Complement Standardization Committee, a joint initiative of the International Complement Society and the International Union of Immunological Societies (IUIS), prepared a defined standard for application in assays for complement activation products. We here report on the production and properties of this International Complement Standard #2 (ICS#2). ICS#2 was made from a pool of sera from healthy blood donors (ICS#1) that was activated with a combination of heat-aggregated IgG and zymosan, and subsequently stabilized by adding EDTA and nafamostat mesylate. The protocol was optimized to make the standard applicable in the following activation product assays: C1rs-C1-inhibitor complexes, C4a, C4bc, C4d, Bb, C3bBbP, C3a, C3bc, C3dg, C5a and the soluble terminal C5b-9 complement complex (SC5b-9, TCC). ICS#2 was defined as containing 1000 complement activation units (CAU)/mL for all activation products measured. All activation products were stable after 10 times thawing and freezing and most of the activation products were stable during storage at 4°C for up to 21 days. ICS#2 was produced large-scale and is considered a valuable tool for standardization, calibration and reference control for complement activation assays, providing the necessary prerequisite for quality assessments between complement laboratories.

Early elevations of the complement activation fragment C3a and adverse pregnancy outcomes.

OBJECTIVE: To estimate whether elevations of complement C3a early in pregnancy are predictive of the subsequent development of adverse pregnancy outcomes. METHODS: A plasma sample was obtained from each enrolled pregnant woman before 20 weeks of gestation. The cohort (n=1,002) was evaluated for the development of adverse pregnancy outcomes defined as hypertensive diseases of pregnancy (gestational hypertension or preeclampsia), preterm birth (before 37 weeks of gestation), premature rupture of the membranes, pregnancy loss (during the embryonic and fetal period), intrauterine growth restriction, and the composite outcome of any adverse outcome. RESULTS: One or more adverse pregnancy outcomes occurred in 211 (21%) of the cohort. The mean levels (ng/mL) of C3a in early pregnancy were significantly (P=<.001) higher among women with one or more adverse outcomes (858±435) compared with women with an uncomplicated pregnancy (741±407). Adjusted for parity and prepregnancy body mass index, women with levels of C3a in the upper quartile in early pregnancy were three times more likely to have an adverse outcome later in pregnancy compared with women in the lowest quartile (95% confidence interval, 1.8–4.8; P<.001). The link between early elevated C3a levels and adverse pregnancy outcomes was driven primarily by individual significant (P<.05) associations of C3a with hypertensive diseases of pregnancy, preterm birth, and premature rupture of the membranes. CONCLUSION: Elevated C3a as early as the first trimester of pregnancy is an independent predictive factor for adverse pregnancy outcomes, suggesting that complement-related inflammatory events in pregnancy contribute to the subsequent development of poor outcomes at later stages of pregnancy. LEVEL OF EVIDENCE: II

IgG antibodies produced during subcutaneous allergen immunotherapy mediate inhibition of basophil activation via a mechanism involving both FcγRIIA and FcγRIIB

The majority of human subjects who receive subcutaneous allergen immunotherapy (IT) develop decreased sensitivity to their allergens. Multiple factors may explain the efficacy of IT, some evidence support a role for allergen specific IgG antibodies. There is controversy whether such antibodies act by blocking allergen binding to IgE or initiation of active inhibitory signaling through low affinity IgG receptors (FcgammaRIIB) on mast cells and basophils. In this study, we addressed this question using peripheral blood from cat non-allergic, cat allergic, and immunotherapy-treated cat allergic subjects. Blood from subjects who received IT contain IgG antibodies that mediate inhibition of basophil activation by a mechanism that is blocked by antibodies specific for the inhibitory IgG receptor FcgammaRIIB. Surprisingly, inhibition was also blocked by aglycosylated, putatively non-FcR binding, antibodies that are specific for the FcgammaRIIA, suggesting a contribution of this receptor to the observed effect. Consistent with a cooperative effect, ex vivo basophils were found to express both IgG receptors. In other studies we found that basophils from subjects who were both chronically exposed to allergen and were producing both cat allergen specific IgE and IgG, are hyporesponsive to allergen. These studies confirm that IgG antibodies produced during IT act primarily by stimulation of inhibitory signaling, and suggest that FcgammaRIIA and FcgammaRIIB function cooperatively in activation of inhibitory signaling circuit. We suggest that under normal physiologic conditions in which only a small proportion of FcepsilonRI are occupied by IgE of a single allergen specificity, FcgammaRIIA co-aggregation may, by providing activated Lyn, be required to fuel activation of inhibitory FcgammaRIIB function.

Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients.

Activation of complement by plicatic acid, the chemical compound responsible for asthma due to western red cedar (Thuja plicata).

Plicatic acid, a low-molecular-weight compound responsible for western red cedar (Thuja plicata) asthma was tested for its ability to activate complement and to generate chemotactic activity from pooled normal human serum (NHS). Dose-dependent complement consumption was found as determined by hemolytic assay (CH50). Activation of complement by plicatic acid was also confirmed by the demonstration of conversion of C3 to C3b on immunoelectrophoresis. This activation was completely prevented by pretreating the serum with either edetate (EDTA) or ethylene glycol tetraacetic acid (EGTA), suggesting that complement was activated via the classical pathway. No conversion of factor B was seen in any of the samples. Leukocyte chemotactic activity was also generated when serum was incubated with plicatic acid. The consumption of C3 and CH50 was unimpaired in two samples of serum from patients with severe, untreated hypogammaglobulinemia and thus appears to be immunoglobulin independent. These observations suggest that plicatic acid could activate complement in vivo, thereby inducing an inflammatory response in the airways and contributing to the higher prevalence of industrial chronic bronchitis in exposed subjects. The pathogenetic role of complement activation in red cedar asthma has yet to elucidated.

Immunoglobulin G, total and subclass, in children with or without recurrent otitis media

Total IgG and subclasses IgG1, IgG2, IgG3, and IgG4 were measured in 89 subjects with recurrent otitis media. There was no significant difference between the groups with respect to the arithmetic or geometric mean levels for total IgG or subclasses IgG1, IgG2, IgG3, or IgG4.

Immunomodulatory effects of intravenous immunoglobulin in severe steroid-dependent asthma

Intravenous immunoglobulin (IVIG) has become an accepted mode of therapy in immune diseases with the potential to act as an immune enhancer or immunomodulator. A 14-year-old girl with severe steroid-dependent asthma was enrolled in a trial of high dose intravenous immunoglobulin because of the unremitting nature of her illness and severe steroid side effects. She received 2 g/kg of IVIG every month for six months. In the 4 months preceding IVIG therapy she had repeated exacerbations of her asthma. In contrast, during the 6 months of IVIG therapy, she only had one exacerbation of her asthma. Her steroid dose was tapered by 75% and her FEV1 improved by 100%. To detect alterations in immune reactivity, we monitored serial skin tests to an individualized panel of antigens, RAST tests to the same panel, and specific IgG levels. Serial titrations of her prick skin tests showed a 2-3 log increase in threshold for skin reactivity to all five antigens on her panel. In parallel, RAST results to the same antigen panel showed a decrease in specific IgE production. The significant clinical improvement and steroid reduction in this patient appear due, at least in part, to the immunomodulatory effects of high dose IVIG therapy.