Autoantibodies to leukocyte alphaMbeta2 integrin glycoproteins in HIV infection.

HIV infection is often associated with polyclonal B-cell activation, autoantibodies, and clinically evident autoimmune disease. Because neutropenia and anti-neutrophil autoantibodies are common clinical features of HIV disease, we studied a series of HIV+ patients to determine whether anti-alphaMbeta2 integrin (MAC-1) specific anti-neutrophil autoantibodies occur in HIV disease, as we have shown to occur in patients with immune neutropenia not associated with HIV. Two new assays specific for anti-alphaMbeta2 IgG were developed to carry out these studies: an ELISA method using affinity-purified alphaMbeta2 integrin protein, and a flow cytometry method using subclones of the 293 human fetal kidney cell line, stably transfected with cDNAs for the alphaM and/or beta2 integrin subunits. In studies of the sera of 20 untreated HIV+ individuals, anti-alphaMbeta2 activity was detected in 9 (45%) by one or the other of these assays and in 5 (25%) by both assays. Seven of the 20 HIV+ study subjects had unexplained neutropenia, and of these, 6 (86%) were positive for anti-alphaMbeta2 autoantibodies. Our findings indicate that anti-alphaMbeta2 integrin autoantibodies are frequent in HIV+ individuals, particularly when unexplained neutropenia is also present, and raise the possibility that these autoantibodies may have a role in the acquired neutrophil dysfunction and increased risk of nonopportunistic bacterial infections observed in HIV disease.

Complement activation in vitro by the red cell substitute, liposome-encapsulated hemoglobin: mechanism of activation and inhibition by soluble complement receptor type 1.

BACKGROUND: Liposome-encapsulated hemoglobin (LEH) has been developed as an emergency blood substitute, yet its effect on human complement has never been explored. Considering that complement activation is a major pathogenic factor in the respiratory distress syndrome that often develops in trauma and shock, LEH-induced complement activation may be a critical safety issue. STUDY DESIGN AND METHODS: Various LEH and corresponding empty liposomes were incubated with normal human sera, and various markers of complement activation (serum levels of C4d, Bb, SC5b-9, and CH50; C5a-induced granulocyte aggregation; membrane deposition of C3b) were measured. Incubations were also performed in the presence of (ethylene-bis[oxyethylenenitrilo]tetraacetic acid) (EGTA) and Mg++ (EGTA/Mg++) and soluble complement receptor type 1. RESULTS: LEH and liposomes activated human complement, as indicated by significant changes in one or more markers. The effect was primarily due to the presence of the phospholipid vehicle; small, unilamellar, highly homodispersed vesicles induced the greatest degree of complement activation. Complement activation was partially inhibited by EGTA/Mg++. The latter finding, together with the parallel increases in serum C4d and Bb, suggests activation of both the classical and alternative pathways. Soluble complement receptor type 1 (0.05-20 micrograms/mL) efficiently inhibited all vesicle-induced complement activation. CONCLUSION: Because of complement activation, the use of LEH for transfusion may require careful evaluation of safety. Soluble complement receptor type 1 may be useful as a prophylactic agent for complement activation-related complications of liposome infusions.

Microcytic anemia with iron malabsorption: an inherited disorder of iron metabolism.

Two siblings were identified with severe hypoproliferative microcytic anemia and iron malabsorption, in the absence of any gastrointestinal disorder or blood loss. These children had severe microcytosis (MCV 48 fl, hemoglobin 7.5 g/dl) with decreased serum iron, elevated serum TIBC, and decreased serum ferritin, despite prolonged treatment with oral iron. An iron challenge study with an oral dose of 2 mg/kg elemental iron as ferrous sulfate documented iron malabsorption. After treatment with intravenous iron dextran, there was an absence of the expected reticulocytosis and only a partial correction of the hemoglobin, hematocrit, and microcytosis. The bone marrow was hypocellular with abnormal iron incorporation into erythroid precursor cells. This appears to be a rare form of inherited anemia characterized by iron malabsorption and disordered iron metabolism that only partially corrects after the administration of parenteral iron. These features resemble those found in the microcytic mouse (mk/mk), which also has severe microcytic anemia and iron malabsorption that partially responds to parenteral iron.

Anti-neutrophil antibodies of the immunoglobulin M class in autoimmune neutropenia.

Anti-neutrophil antibodies of the immunoglobulin G (IgG) class have been implicated in the pathogenesis of autoimmune neutropenia, but few reports have described immunoglobulin M (IgM) anti-neutrophil antibodies. To investigate the prevalence of IgM anti-neutrophil antibodies, sera from 130 patients with possible autoimmune neutropenia were studied for IgG and IgM anti-neutrophil antibodies using an immunofluorescence flow cytometric assay. Twenty-five patients (19%) had IgG anti-neutrophil antibodies exclusively, 21 patients (16%) had both IgG and IgM anti-neutrophil antibodies, and 11 patients (8%) had IgM anti-neutrophil antibodies exclusively. Immunoglobulin M anti-neutrophil antibodies were found in adults and children with isolated chronic neutropenia and in patients with Felty's syndrome, systemic lupus erythematosus, immune thrombocytopenic purpura, and human immunodeficiency virus. Patients with neutropenia with only IgM anti-neutrophil antibodies comprised almost 20% of antibody-positive patients in this study.

Antibodies to myeloid precursor cells in autoimmune neutropenia.

Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further studies, antibodies from 12 sera bound to mononuclear CD34+ cells that had been purified from normal human bone marrow by an immunomagnetic separation procedure. Moreover, two of these sera suppressed the growth of granulocyte-macrophage colony-forming units (CFU-GM) in methylcellulose cultures. The presence of antibodies to primitive hematopoietic cells in the sera of some patients with suspected immune neutropenia suggests that these antibodies may have a role in the pathogenesis of the neutropenia observed.

Detection of antineutrophil autoantibodies by flow cytometry: use of unfixed neutrophils as antigenic targets.

Antineutrophil antibodies may be found in the sera of patients with chronic neutropenia as well as in the sera of a variety of patients with neutropenia and associated autoimmune or infectious disorders. We evaluated an immunofluorescent flow cytometric technique for the measurement of antineutrophil antibodies in serum. Sera from patients with suspected immune neutropenia were studied and compared with a group of sera from normal healthy individuals, as well as with sera from patients with rheumatoid arthritis and systemic lupus erythematosus. Of 159 patients with suspected immune neutropenia and a variety of associated clinical disorders, 59 (37%) were found to have evidence for enhanced binding of IgG to normal target neutrophils, interpreted as positive for antineutrophil antibodies. Whereas 0/37 non-neutropenic patients with typical RA had positive results, 51/244 (21%) of sera from nonneutropenic patients with SLE or other collagen vascular disorders showed enhanced IgG binding to neutrophils. Living neutrophils were used to study the effects of cellular activation, and increased antibody binding was observed with certain sera that contained IgG directed against activation-dependent antigens. We found that, under controlled conditions, flow cytometry can be reliably used to detect antineutrophil autoantibodies, with unfixed, living neutrophils as antigenic targets.

Identification of autoantibodies specific for the neutrophil adhesion glycoproteins CD11b/CD18 in patients with autoimmune neutropenia.

Anti-neutrophil antibodies have been described in a variety of clinical conditions associated with neutropenia. However, relatively little is known about the antigenic specificities of naturally occurring anti-neutrophil autoantibodies. We investigated the possibility that anti-neutrophil antibodies specific for the neutrophil adhesion glycoprotein (GP) complex CD11b/CD18 might be present in the sera of some patients with autoimmune neutropenia. These membrane GPs have been shown to be highly immunogenic in the production of murine monoclonal antibodies against neutrophil antigens. Moreover, autoantibodies to the platelet membrane GP complex IIb/IIIa, another member of the integrin family of cell adhesion proteins, have been demonstrated in immune thrombocytopenic purpura. Sera from 50 patients known to have anti-neutrophil IgG antibodies were evaluated using an immunobead "antigen capture" assay, modeled after a method used to identify anti-platelet GPIIb/IIIa autoantibodies. This assay detected anti-CD11b/CD18 autoantibodies in seven of the 50 sera. Each of these seven sera demonstrated decreased IgG binding to the neutrophils of a patient with congenital deficiency of CD11b/CD18. The patient with the highest levels of anti-CD11b/CD18 suffered recurrent skin infections and cellulitis, and died of respiratory failure during one of multiple episodes of pneumonia. Purified IgGs from five of these patients demonstrated effects on adhesion and/or opsonin receptor-mediated functions when tested with intact neutrophils in vitro. Our findings indicate that some patients with autoimmune neutropenia have autoantibodies specific for the functionally important neutrophil adhesion proteins CD11b/CD18. Our findings also raise the possibility that these autoantibodies may, in some cases, interfere with neutrophil function, thereby amplifying the risk of infection associated with neutropenia.

Prognostic value of histopathology in Ewing's sarcoma. Long-term follow-up of distal extremity primary tumors.

The pathologic material from 56 patients diagnosed initially as Ewing's sarcoma of the distal extremity and treated on National Cancer Institute protocols between 1968 and 1984 was reviewed and correlated with clinical outcome. Histologically, the tumors were categorized, based on recent pathologic criteria, into three diagnostic groups: (1) typical Ewing's sarcoma, (2) atypical Ewing's sarcoma, and (3) other (predominantly peripheral neuroepithelioma [PN]). Thirty-two patients (57%) had typical Ewing's, 13 (23%) were atypical, and 11 (20%) were in the "other" diagnostic category (seven [13%] PN, two primitive rhabdomyosarcoma, one primitive sarcoma of bone, and one synovial cell sarcoma). No cases of metastatic neuroblastoma, osteosarcoma, or lymphoma were found. Forty-five patients had localized disease at diagnosis; 11 had metastases. Patients with typical Ewing's sarcoma were less likely to have metastatic disease at the time of diagnosis. Only two of 32 patients with typical Ewing's sarcoma had metastases compared with nine of 24 patients in the two other groups. The pattern of relapse was also different in these other groups compared with typical Ewing's patients; five patients developed lymph node metastases and two patients developed brain metastases. Although the presence of metastatic disease at diagnosis was a strong negative prognostic factor, our histologic grouping was independently prognostic of clinical outcome in patients with localized disease. Patients with typical osseous Ewing's sarcoma had an improved overall survival (P2 = 0.03) and patients with other tumors (neither typical nor atypical Ewing's sarcoma) had a poorer disease-free survival (P2 = 0.02). Since no generally accepted histopathologic prognostic criteria exist for Ewing's sarcoma, the potential value of our proposed classification should be evaluated in a larger retrospective and a prospective study.

Antibodies to actin in autoimmune neutropenia.

In an effort to characterize the cellular antigens recognized by anti-neutrophil antibodies in autoimmune neutropenia, we studied sera, purified immunoglobulin G (IgG) and isolated F(ab')2 from 70 neutropenic patients suspected of this diagnosis. Anti-neutrophil antibodies were found in the sera of 36 of these patients by either 125I-staph A binding or immunofluorescence cytometric techniques that detected increased binding of patients' IgG to normal neutrophils. Anti-neutrophil antibody positive sera were then evaluated for specific binding to electrophoretically separated neutrophil membrane-associated proteins by immunoblotting. A 43-Kd protein was consistently identified by eight anti-neutrophil antibody positive sera. The specificity of binding to this protein was confirmed with affinity purified IgG and F(ab')2 fragments prepared from these sera. Sera from 20 healthy normal controls and from 22 non-neutropenic, anti-neutrophil antibody negative rheumatoid arthritis patients failed to bind this protein. Separate studies identified the 43-Kd protein as actin. Purified Acanthamoeba actin comigrated with the protein and was specifically bound by anti-neutrophil antibody positive IgG. Moreover, two actin-specific monoclonal antibodies bound to the 43-Kd membrane-associated protein in immunoblots. In addition, a rabbit anti-actin antiserum not only bound to this same 43-Kd protein but also expressed anti-neutrophil antibody activity against normal human neutrophils, as did purified human anti-actin IgG prepared by affinity chromatography from the serum of one of the index patients. These studies indicate that the anti-neutrophil antibodies of certain patients with autoimmune neutropenia include autoantibodies specific for actin. The molecules on the surface of neutrophils, which have actin-like antigenic epitopes and are recognized by these anti-actin antibodies, remain to be characterized.

Homozygous protein C deficiency: early treatment with warfarin.

We present a case of homozygous protein C deficiency with neonatal purpura fulminans and disseminated intravascular coagulopathy (DIC) starting shortly after birth. In addition, the infant had vitreal eye hemorrhages and intraparenchymal brain infarction, apparently as intrauterine events. Within 15 hours of institution of fresh frozen plasma (FFP) infusions the DIC resolved and the progression of purpura fulminans reversed. Warfarin (0.4 mg/kg/day) was started on the fifth day of life, followed by gradual tapering of the FFP infusions. There were no recurrences of purpura, areas of skin necrosis healed without the need for skin grafting, and the areas of brain infarction resolved without apparent sequelae. The eye and brain lesions may be intrauterine events and appear to be a regular feature of this syndrome. Family studies are essential to establish the diagnosis, although there may be no family history of thromboembolic events, as in this case. Homozygous protein C deficiency is a rare disorder, but one in which early recognition and intervention may be lifesaving. Ours is the youngest patient yet reported to be treated with warfarin anticoagulation. We were thus able to avoid the complications of long-term plasma therapy as well as the potential thrombotic complications of central venous catheter placement.

Antimicrobial therapy of Broviac catheter infections in pediatric hematology oncology patients.

Long-term therapy of pediatric oncology patients has been facilitated by permanent indwelling venous catheters. Over a 3-year period, 54 Broviac catheters were placed in 43 oncology patients and two hemophiliacs. There were 20 episodes of sepsis in 14 patients and the most common bacteria were S epidermidis (4), S aureus (4), and K pneumoniae (3). Catheter exit site infections occurred ten times in six patients; S aureus eight of ten. Antibiotic therapy without catheter removal was successful in 18 of 20 children with catheter sepsis and 8 of 10 patients with exit site infections. These data strongly suggest that although catheter-related infections are common, removal of Broviac catheters is not required for successful treatment of the infection.

Use of the radioallergosorbent test for the study of coelenterate toxin-specific immunoglobulin E.

The radioallergosorbent test (RAST) was adapted for use to screen the immune response to sea nettle (Chrysaora quinquecirrha) and Protuguese man-o'-war (Physalia physalis) toxins. The results of a preliminary screening of serum from patients varying in their reaction to envenomations indicate the potential of these toxin proteins to induce an allergic state in humans and illustrate the use of the RAST as a screening device to detect persons sensitive to coelenterate stings.