Detection of active kallikrein in induced blister fluids of hereditary angioedema patients.

Six suction-induced blister fluids obtained from five patients with hereditary angioedema (HAE) contained active kallikrein, whereas only two blister fluids obtained from eight normal volunteers contained small amounts of this activity. Kallikrein was present in large amounts of HAE blister fluids as assessed by its ability to liberate smooth-muscle-contracting activity from purified high molecular weight kininogen. It was inhibited by purified antibodies specific for plasma prekallikrein and also by purified C1 inhibitor, but not by antibodies specific for C1s. These observations suggest that activation of the Hageman-factor-dependent pathways occurs in the tissues of HAE patients, and once generated, active kallikrein persists in these tissues.

Activation of complement by serum-resistant Neisseria gonorrhoeae. Assembly of the membrane attack complex without subsequent cell death.

Interaction of the human complement system in normal human serum (NHS) with serum-resistant and -sensitive Neisseria gonorrhoeae was evaluated to better understand the mechanism of serum-resistance. Complement activity (CH50) was depleted from NHS in a dose-dependent fashion by both serum-resistant and -sensitive N. gonorrhoeae. No detectable CH50 remained in NHS incubated with 10(9) colony-forming units (CFU)/ml serum of either resistant or sensitive strains. When smaller numbers of bacteria were incubated with NHS, lesser, yet comparable, amounts of CH50 were depleted by both resistant and sensitive strains. Hemolytic C2 activity was diminished by 33% in the case of resistant N. gonorrhoeae (10(8) CFU/ml serum) and by 48% in the case of a sensitive strain. No detectable decreases in hemolytic C4 or C7 activities were found with either sensitive or resistant strains at this concentration. Both resistant and sensitive strains activated C1s in NHS. Resistant strains specifically activated 19-21% of radiolabeled C1s in NHS, whereas sensitive strains activated 18-32%. Both resistant and sensitive strains also activated C5 in NHS. In binding assays using radiolabeled C5 and C9 in NHS, resistant and sensitive strains bound comparable amounts of C5 and C9. The number of bound C5 and C9 molecules varied according to the number of bacteria or amount of serum used in the assay. The ratio of C9/C5 bound to a sensitive strain was 6.8, and to a resistant strain was 8.2, suggesting that C5 and C9 were incorporated into membrane attack complexes (MAC). Electron microscopic examination of resistant and sensitive strains incubated with NHS revealed that MAC is bound to the surfaces of the resistant strain as well as the sensitive strain.

Demonstration of modified inactive first component of complement (C1) inhibitor in the plasmas of C1 inhibitor-deficient patients.

The first component of complement (C1) inhibitor plays a critical role in the regulation of the classical complement pathway and the contact system, and the deficiency of C1 inhibitor protein or function is associated with recurrent angioedema. In this study we evaluated the size of the C1 inhibitor antigens present in the plasmas of C1 inhibitor-deficient patients. We found that the C1 inhibitor in the plasmas existed in three forms: high molecular weight forms in complex with proteases, native 110-kD C1 inhibitor, and a modified inactive 94-kD form. The proportion of the total C1 inhibitor in the 94-kD form was 28% in nine hereditary angioedema patients, 92% in five acquired C1 inhibitor-deficiency patients, and 1.2% in five normal controls. In vitro activation of normal plasma with kaolin, but not heat-aggregated gamma-globulin generated 94-kD C1 inhibitor from 110-kD C1 inhibitor. Neither kaolin activation nor heat-aggregated gamma-globulin activation generated 94-kD C1 inhibitor in Hageman factor-deficient plasma. These results suggest that 94-kD C1 inhibitor is generated in vitro by activation of the contact system. The in vivo mechanism of 94-kD C1 inhibitor generation in C1 inhibitor-deficient patients is not known.

Immunoglobulin E-rheumatoid factor in the serum of patients with rheumatoid arthritis, asthma, and other diseases.

A solid-phase radioimmunoassay was developed to detect immunoglobulin (Ig)E antibodies that bound to human IgG. IgE-rheumatoid factor activity was found in the serum of 18 of 20 patients with seropositive rheumatoid arthritis, 1 of 4 patients with seronegative rheumatoid arthritis, 3 of 32 patients with seronegative rheumatoid arthritis, 3 of 32 patients with asthma, and in 1 patient with hypocomplementemic vasculitis and iodide sensitivity. Immunopathologic implications of IgE-rheumatoid factor are discussed.

Potent toxicity of 2-chlorodeoxyadenosine toward human monocytes in vitro and in vivo. A novel approach to immunosuppressive therapy.

Lymphoid cells were thought to be uniquely susceptible to excess 2'-deoxyadenosine (dAdo), when exposed to inhibitors of adenosine deaminase (ADA). However, we now find that human monocytes are as sensitive as lymphocytes to dAdo or to the ADA-resistant congener 2-chloro-2'-deoxyadenosine (CldAdo). Monocytes exposed in vitro to CldAdo, or to dAdo plus deoxycoformycin rapidly developed DNA strand breaks. Both the DNA damage and the toxicity of CldAdo or dAdo toward monocytes were blocked by deoxycytidine, but not by inhibitors of poly(ADP-ribose) polymerase. A partial decrease in RNA synthesis and a gradual decline of cellular NAD were early biochemical events associated with monocyte DNA damage. Low CldAdo concentrations (5-20 nM) inhibited monocyte phagocytosis and reduced the release of interleukin 6. Higher CldAdo concentrations led to a dose- and time-dependent loss of monocyte viability. Circulating monocytes disappeared within 1 wk in patients with cutaneous T cell lymphoma or with rheumatoid arthritis during continuous CldAdo infusion. The marked sensitivity of human monocyte function and survival to CldAdo in vitro, together with the monocyte depletion in patients receiving CldAdo chemotherapy, suggests that CldAdo or other dAdo analogues offer a novel therapeutic strategy for chronic inflammatory and autoimmune diseases characterized by inappropriate monocyte deployment or function.

Meningococcemia and acquired complement deficiency. Association in patients with hepatic failure.

We treated two patients with severe hepatic failure complicated by meningococcemia. Serum complement profiles performed on these patients found low total hemolytic complement assays, normal concentrations of C1q, and low or undetectable concentrations of C3 through C6, C8, C9, and factors B and I. These studies suggest that these patients developed meningococcemia in the setting of acquired complement deficiency from impaired synthesis of multiple complement system proteins.

Direct quantitation of activated C3 in human plasma with monoclonal anti-iC3b-C3d-neoantigen.

We developed a microtiter solid-phase radioimmunoassay for quantitating C3 breakdown products (iC3b, C3dg, C3d) in human plasma with a unique monoclonal antibody specific for a neoantigen present on iC3b and C3d (MoAb 130). This monoclonal antibody reacts with a neoantigen which appears when C3b is converted to iC3b. The neoantigen is also present on the C3dg and C3d fragments derived from iC3b. The concentration of the neoantigen is elevated in the plasma of most patients with rheumatoid arthritis and systemic lupus erythematosus as compared to normal volunteers. Some patients with glomerulonephritis also had elevated concentration of the neoantigen in their plasma.

Serologic changes during induction of lupus-like disease by procainamide.

Procainamide-induced lupus is a well-recognized syndrome, but the events leading up to clinical symptoms are obscure. In the present study, serologic changes in a 69-year-old man were monitored during his treatment with procainamide and after discontinuation of procainamide because of symptoms of drug-induced lupus. Antihistone antibodies of unique specificity and in vivo complement activation were detected after one year of procainamide therapy during a period prior to development of significant clinical symptoms. Antihistone antibodies and complement activation substantially increased during a full-blown episode of lupus-like symptoms. Progressive return to normal laboratory findings occurred after procainamide was discontinued. The antihistone/complement profile may be useful in the diagnosis of drug-induced lupus and warn of impending clinical deterioration in patients with minimal symptoms.

Detection and quantitation of cleaved and uncleaved high molecular weight kininogen in plasma by ligand blotting with radiolabeled plasma prekallikrein or factor XI.

A method for the quantitative assay of native single chain and kallikrein cleaved two-chain high molecular weight (HMW)-kininogen in plasma is described. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma is followed by electrotransfer of the electropherogram to nitrocellulose membranes and detection of the blotted HMW-kininogen with its physiologic ligands, radiolabeled plasma prekallikrein or radiolabeled factor XI. Using unreduced SDS-PAGE cleaved two-chain HMW-kininogen (Mr approximately 107,000 and 95,000), is electrophoretically separated from uncleaved single chain HMW-kininogen (Mr approximately 150,000). Counting the radioactivity of the nitrocellulose pieces corresponding to cleaved HMW-kininogen permits its quantitative measurement by comparison with standards consisting of decreasing amounts of fully dextran sulfate activated normal human plasma. Single chain HMW-kininogen is similarly assayed using reduced SDS-PAGE and unactivated normal human plasma standards. This technique is highly specific and sensitive to about 50 ng of either cleaved or uncleaved HMW-kininogen. Varying amounts of cleaved HMW-kininogen were found in a small series of plasmas from patients suffering from various inflammatory conditions. Higher levels of in vivo cleaved HMW-kininogen were observed during acute attacks of hereditary angioedema due to Cl-inhibitor deficiency. This technique may be useful for the assessment of the degree of in vitro or in vivo activation of the contact system.

Prolonged, complete remission after 2-chlorodeoxyadenosine therapy in a patient with refractory essential mixed cryoglobulinemia.

Essential mixed cryoglobulinemia is a systemic disorder in which cutaneous vasculitis and frequently glomerulonephritis are associated with cryoprecipitable serum immune complexes. Typically, the treatment regimen consists of plasmapheresis, high-dose corticosteroids, and cytotoxic chemotherapy, as well as interferon alfa for hepatitis C virus-related cryoglobulinemia. Herein we describe a man with progressive, symptomatic cryoglobulinemia and multisystem organ dysfunction in whom corticosteroid and alkylating therapy failed; however, he had a complete and long-lasting remission after administration of 2-chlorodeoxyadenosine (cladribine).

Immunodiffusion assay of C1 inhibitor function in serum: prospective analysis in angioedema-urticaria.

An immunodiffusion assay for detecting C1 inhibitor function in human serum was described recently by Ziccardi and Cooper. In our present study, the applicability of this assay for C1 inhibitor deficiency or C1 inhibitor dysfunction was evaluated. Of the 39 patients evaluated, all eight patients with the common (C1 inhibitor deficiency) form of hereditary angioedema and all three patients with the variant (dysfunctional C1 inhibitor) form of hereditary angioedema were identified correctly. Treatment of patients with hereditary angioedema with stanozolol or danocrine increased their serum C1 inhibitor concentrations and normalized the immunodiffusion assay for C1 inhibitor function. In addition, the assay allowed the correct identification of three patients with the acquired form of C1 inhibitor deficiency, because the sera of these patients exhibited a distinctive pattern. The 25 samples from patients (chronic angioedema, chronic urticaria, or hypocomplementemic vasculitis) without C1 inhibitor deficiency had normal assays.

Rocket immunoelectrophoresis of C4 and C4d. A simple sensitive method for detecting complement activation in plasma.

Activation of complement component C4 has recently been measured by the quantitation of C4 and C4d (a cleavage fragment of C4) by electroimmunodiffusion in gels containing specific precipitating antibodies for C4 and C4d (Rocket immunoelectrophoresis, RIE). Quantitative measurements of the complement component C4 and its fragment C4d were determined in rocket immunoelectrophoresis (RIE) and compared with measurements of total hemolytic complement activity (CH50) or concentrations of C4 as determined by single radial immunodiffusion (RID). This newly developed RIE assay shows activation of the classical complement pathway and involves electroimmunodiffusion in gels containing specific precipitating antibodies for C4 and its cleavage fragment, C4d. In 37 plasma samples, excellent correlation was demonstrated between the C4 in RIE and CH50 (r = .70) and C4 by RID (r = .87). In vivo activation of C4 was determined by measuring the ratio of C4d to C4; 21 of the plasma samples assayed had ratios greater than 1.1 indicating activation of C4. In 13 of the plasma samples there were correspondingly low CH50 values, whereas 8 had normal CH50 levels. Therefore, activation can be detected in those instances when other measurements (CH50 and C4 quantitation) are normal. Thus the RIE assay for plasma C4 activation appears to be the most sensitive method available for assessing in vivo activation of the classical pathway of complement.

Effects of protamine administration after cardiopulmonary bypass on complement, blood elements, and the hemodynamic state.

Nineteen patients were prospectively selected and studied before and after the administration of protamine sulfate following cardiopulmonary bypass (CPB). After protamine administration, C3a, C4a, and C4d were elevated; the peak levels of C3a and C4a were in samples taken 10 minutes after protamine administration while those of C4d were in those obtained at 5 hours. Only C3a was elevated after CPB and before protamine administration. In vitro, only the combination of protamine sulfate and heparin, and neither alone, resulted in increased C3a and C4a. Administration of protamine was associated with small and transient decreases in total white blood cells, granulocytes, and platelets, and with small and transient reductions in systemic and pulmonary arterial and left and right atrial pressures. Systemic vascular resistance fell (p = 0.07), and pulmonary vascular resistance rose but the change could be due to chance (p = 0.29). These data and those reported by others support the inference that complement activation occurs during CPB by the alternative pathway and again during protamine administration by the classic pathway; and that this accompanies a whole-body inflammatory reaction with blood cell and hemodynamic changes which, when extreme, could result in a severe hemodynamic derangement.

The hereditary and acquired deficiencies of complement.

The identification of hereditary and acquired complement deficiencies in humans has led to a better understanding of the biologic importance of the complement system in immunity and autoimmune disease. Although the understanding of the relevance of complement in the pathogenesis of disease is incomplete, several characteristic clinical syndromes associated with complement deficiencies have been recognized and should be known to the practicing clinician. In allergic diseases, one need recognize the C1 inhibitor deficiency syndromes which can present as severe, recurrent angioedema in childhood or in the adult as recurrent angioedema in association with a lymphoid malignancy or autoimmune disease. Complement analyses allow one to readily diagnose C1 inhibitor deficiency in angioedema. Correct diagnosis is critical because safe effective therapy is available. Chronic urticaria is also uncommonly associated with complement deficiencies, particularly acquired C1q deficiency. Again, effective therapy for hypocomplementemic urticarial vasculitis and C1q deficiency is available and differs significantly from the usual management of chronic urticaria. Homozygous and acquired deficiencies of C3 are associated with severe immune deficiency and recurrent infections with gram-positive and gram-negative bacteria. Recurrent meningococcemia and gonococcemia are being identified frequently in patients with a deficient membrane attack mechanism relating to deficiency of C5, C6, C7, or C8. Nearly one third of the patients developing meningococcemia may have an associated complement deficiency indicating the importance of complement determinations in understanding the treatment and prognosis for these patients. Deficiency of almost every complement component has been reported in association with one or more rheumatic diseases, particularly systemic lupus erythematosus. Extensive studies of C2 deficiency and limited studies of C4 deficiency indicate that these components of the classical pathway of complement are important in preventing the development of SLE or are linked to other genes predisposing to SLE. The clinical presentations of SLE in association with C2 or C4 deficiency are relatively uniform. The patients exhibit typical skin manifestations suggestive of SLE and DLE and often exhibit antibodies to SSA (Ro). The association of complement deficiencies with clinical syndromes is important for today's physician. The syndromes and deficiencies described here are the beginning of an expanding knowledge relating to the pathobiology of complement in human disorders.

Extraspinal causes of lumbosacral radiculopathy.

Twelve of 12,125 patients who had been referred during a seven-year period to a specialist in spinal disorders were found to have an extraspinal cause of radiculopathy or neuropathy of the lower extremity. The records of these twelve patients were reviewed retrospectively. The average age of the twelve patients was sixty-five years (range, forty-two to seventy-seven years). The cause of the symptoms was an occult malignant tumor in nine patients and a hematoma, an aneurysm of the obturator artery, or a neurilemoma of the sciatic nerve in the others. The average time from the onset of symptoms to the final diagnosis was eight months (range, one month to two years). The most useful test for determination of the correct diagnosis was computed tomography or magnetic resonance imaging of the abdomen and pelvis. Computed tomography or magnetic resonance imaging of the spine and bone-scanning of the whole body were of little help in localizing the disease. In four of the twelve patients, an operation was performed on the basis of an incorrect diagnosis. In dealing with elderly patients who have radiculopathy, one should be suspicious that the cause is outside the spine.

Atlantoaxial lateral mass osteoarthritis. A frequently overlooked cause of severe occipitocervical pain.

Localized C1-C2 lateral mass osteoarthritis is a degenerative disorder of the upper cervical spine that has a natural history markedly different from that of degenerative afflictions of the lower cervical spine. Atlantoaxial lateral mass arthritis is a distinct cause of occasionally severe occipitocervical pain in elderly persons. In this series, the diagnosis was suggested by the medical history of nine elderly patients who presented with severe occipitocervical pain (frequently diagnosed as occipital neuralgia). Physical examination demonstrated marked restriction of rotation of the cervical spine to the affected side, and localized tenderness unilaterally at the occipitocervical junction. The diagnosis was confirmed by plain radiographs of the C1-C2 articulation (open-mouth view), demonstrating marked, usually unilateral joint-space narrowing, osteophyte formation, and subchondral sclerosis. Bone scanning demonstrated focal uptake unilaterally at the occipitocervical junction. Additional imaging studies, including computed tomography, magnetic resonance imaging, or cervical myelogram, were performed to rule out coexisting intraspinal pathology. Conservative treatment was usually successful; however, C1-C2 arthrodesis was successful for severe occipitocervical pain due to atlantoaxial lateral mass arthritis not responsive to conservative treatment.

A systematic approach to disorders of the cervical spine.

Pain in the neck may arise from a clear-cut cause (e.g., an auto accident) or without warning or apparent reason. It may limit exercise, preclude driving, or interfere with sleep, and at its worst make any movement excruciating. Common denominators are rare, although age is often a fundamental factor. It is usually best to begin management conservatively.