John P. Leddy

Long-term prospective study in children after respiratory syncytial virus infection

We have prospectively evaluated for the past 8 years 29 children who were hospitalized during infancy with acute lower respiratory tract illness caused by respiratory syncytial virus (RSV). No differences in the prevalence of a family history of atopy or breast-feeding in these infants compared with controls were noted. However, a history of parental smoking was significantly associated with hospital admission for RSV lower respiratory tract disease. Evidence of atopy, as defined by serum IgE levels and radioallergosorbent testing, have developed in only three (10%) of 29 children. Six children (21%) continue to have recurrent lower respiratory tract disease. Fifty-five percent of these children had abnormally low oxyhemoglobin levels (SaO2) measured by ear oximetry for the first 3 to 4 years after the acute illness. Twenty-one percent have persistently low SaO2 levels during the eighth year of follow-up. Spirometric values indicate evidence of peripheral airway obstruction. These studies suggest that an association between RSV lower respiratory tract infections and chronic abnormalities of pulmonary function may be detected sequentially through the first 8 years of life. These abnormalities are not limited to those children developing an atopic state during that same time period.

Hereditary deficiency of the fifth component of complement in man. I. Clinical, immunochemical, and family studies.

The first recognized human kindred with hereditary deficiency of the fifth component of complement (C5) is described. The proband, a 20-year-old black female with systemic lupus erythematosus since age 11, lacked serum hemolytic complement activity, even during remission. C5 was undetectable in her serum by both immunodiffusion and hemolytic assays. Other complement components were normal during remission of lupus, but C1, C4, C2, and C3 levels fell during exacerbations. A younger half-sister, who had no underlying disease, was also found to lack immunochemically detectable C5. By hemolytic assay, she exhibited 1-2% of the normal serum C5 level and normal concentrations of other complement components. C5 levels of other family members were either normal or approximately half-normal, consistent with autosomal codominant inheritance of the gene determining C5 deficiency. Normal hemolytic titers were restored to both homozygous C5-deficient (C5D) sera by addition of highly purified human C5. In specific C5 titrations, however, it was noted that when limited amounts of C5 were assayed in the presence of low dilutions of either C5D serum, curving rather than linear dose-response plots were consistently obtained, suggesting some inhibitory effect. Further studies suggested that low dilutions of C5D serum contain a factor (or factors) interfering at some step in the hemolytic assay of C5, rather than a true C5 inhibitor or inactivator. Of clinical interest are (a) the documentation of membranous glomerulonephritis, vasculitis, and arthritis in an individual lacking C5 (and its biologic functions), and (b) a remarkable propensity to bacterial infections in the proband, even during periods of low-dose or alternate-day corticosteroid therapy. Other observations indicate that the C5D state is compatible with normal coagulation function and the capacity to mount a neutrophilic leukocytosis during pyogenic infection.

Inhibition of the Lytic Action of Cell-bound Terminal Complement Components by Human High Density Lipoproteins and Apoproteins

Human serum lipoproteins are known to participate in or modify several immunologically relevant responses, including the inhibition of target cell lysis initiated by fluid-phase C5b-7 (reactive lysis). We now report that human high density lipoproteins (HDL) can inhibit the complement (C) lytic mechanism after C5b-7, C5b-8, and even C5b-9 have been bound to the target membrane. This inhibitory activity of serum or plasma copurifies in hydrophobic chromatography with antigenically detected apolipoprotein A-I (apoA-I), the major HDL apoprotein, and with HDL in CsCl density gradient ultracentrifugation. Although HDL is more active than its apoproteins in fluid-phase inhibition of C5b-7-initiated reactive lysis, the HDL apoproteins are more effective after C5b-7, C5b-8, or C5b-9 have become bound to human or sheep erythrocytes (E). Highly purified HDL apoproteins, apoA-I and apoA-II, both have greater inhibitory activity than whole HDL on a protein weight basis, and some evidence has been obtained that apoA-I dissociating spontaneously from HDL may be the principal inhibitory moiety in physiological situations. HDL lipids themselves are inactive. The HDL-related inhibitors are ineffective when incubated with EC5b-7 and removed before C8 and C9 are added, and only minimally effective on cell-bound C5b-8 sites before C9 is added. They exert their most prominent inhibitory activity after C9 has been bound to EC5b-8 at low temperature, but before the final temperature-dependent, Zn(++)-inhibitable membrane damage steps have occurred. Therefore, HDL or its apoproteins do not act to repair already established transmembrane channels, but might interfere either with insertion of C9 into the lipid bilayer or with polymerization of C9 at C5b-8 sites. This heat-stable inhibitory activity can be demonstrated to modify lysis of erythrocytes in whole serum, i.e., it does not depend upon artificial interruption of the complement membrane attack sequence at any of the above-mentioned stages. Contributions of the target membrane itself to the mechanism of inhibition are suggested by the observations that, in contrast to sheep or normal human E, lysis of guinea pig E or human E from patients with paroxysmal nocturnal hemoglobinuria is inhibited poorly. This is the first description of a naturally occurring plasma inhibitor acting on the terminal, membrane-associated events in complement lysis. Although further study is required to assess the physiologic or immunopathologic significance of this new function of HDL, the HDL apoproteins or their relevant fragments should be useful experimentally as molecular probes of the lytic mechanism.

Listeriosis complicating lymphoma: Report of four cases and interpretive review of pathogenetic factors

Abstract Described herein are three adult patients with Hodgkins disease and one with lymphosarcoma in whom a serious intercurrent infection with Listeria monocytogenes developed. An analysis of predisposing factors points out the behavior of Listeria as a facultative intracellular parasite, a category of pathogens including the tubercle bacillus. The critical role of host macrophages in defense against this group of pathogens is emphasized and discussed in relation to the underlying disease, corticosteroid therapy and cytotoxic drugs. Finally, hypothetical comments are offered concerning host resistance in Hodgkins disease or lymphosarcoma, and its possible relationship to the expression of delayed hypersensitivity.

Complement Lysis of Human Erythrocytes: DIFFERING SUSCEPTIBILITY OF TWO TYPES OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA CELLS TO C5b-9

Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated. The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding. The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.

Hereditary deficiency of the fifth component of complement in man. II. Biological properties of C5-deficient human serum.

The first known human kindred with hereditary deficiency of the fifth component of complement (C5) was documented in the accompanying report. This study examines several biological properties of C5-deficient (C5D) human serum, particularly sera obtained from two C5D homozygotes. The proband, who has inactive systemic lupus erythematosus is completely lacking C5, while her healthy half-sister has 1-2% of normal levels. Both sera were severely impaired in their ability to generate chemotactic activity for normal human neutrophils upon incubation with aggregated human gamma-globulin or Escherichia coli endotoxin. This function was fully restored in the siblings serum, and substantially improved in the probands serum, by addition of highly purified human C5 to normal serum concentrations. Sera from eight family members who were apparently heterozygous for C5 deficiency gave normal chemotactic scores. The ability of C5D serum to opsonize Saccharomyces cerevisiae (bakers yeast) or Candida albicans for ingestion by normal neutrophils was completely normal. In addition, C5D serum was capable of promoting normal phagocytosis and intracellular killing of Staphylococcus aureus. The probands serum was incapable of mediating lysis of erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria in both the sucrose hemolysia and acid hemolysis tests, and also lacked bactericidal activity against sensitized or unsensitized Salmonella typhi. The siblings serum, containing only 1-2% of normal C5, effectively lysed S. typhi, but only at eightfold lower serum dilutions as compared to normals. These findings underscore the critical role of C5 in the generation of chemotactic activity and in cytolytic reactions, as opposed to a nonobligatory or minimal role in opsonization, at least for the organisms under study.

Allergic reactions to cyclophosphamide: Delayed clinical expression associated with positive immediate skin tests to drug metabolites in five patients

BACKGROUND Cyclophosphamide (CP) is one of the relatively few drugs implicated in systemic allergic reactions for which the metabolites are well known. Formation of CP metabolites is a multistep, time-dependent process (hours) with significant interindividual differences. Although allergic reactions to CP have been recorded in 17 previous reports, skin testing with CP or its metabolites has been included in only five. We now describe five patients receiving monthly cycles of intravenous CP whose allergic reactions included clinical features of type I hypersensitivity but were atypical in their markedly delayed onset (i.e., 8 to 16 hours in patients 1 to 4 and 10 days in patient 5). OBJECTIVE The objective was to investigate these late-developing clinical reactions by skin testing with CP and two of its major metabolites. METHODS The five patients and a control group receiving intravenous CP uneventfully were studied by the same skin test protocol. RESULTS The four individuals in the control group were unreactive to CP or its metabolites. All five patients with late-onset allergic reactions had positive immediate skin test results to CP metabolites but not to CP itself. We propose that the allergic reactions in patients 1 to 4 were mediated, wholly or in major part, by IgE antibodies reactive with allergens derived from time-dependent drug metabolites. The 10-day lag time in patient 5 is unexplained. Immunomodulation by the underlying malignancies or by the immunosuppressive drugs could have contributed. CONCLUSION IgE-mediated allergic drug reactions may have a delayed onset if the allergen is a time-dependent drug metabolite, illustrated in this study by CP.

beta-adrenergic receptors of human leukocytes: studies with intact mononuclear and polymorphonuclear cells and membranes comparing two radioligands in the presence and absence of chloroquine

Owing to the large differences in reported values for beta-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[3H]dihydroalprenolol (DHA) and (+/-)-[125I]iodohydroxybenzylpindolol (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10-24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000-60,000 sites/cell) of low affinity (Kd 12-15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 microM chloroquine not only in PMN cells but also in the lysome-poor MN cells (greater than or equal to 90% lymphocytes), leaving 2000-3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (+/-)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 microM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent Kd. Mean (+/- S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 +/- 100 sites/MN cell and 1135 +/- 129 sites/PMN cell (Kd 143-153 pM) using (-)-DHA; and 1487 +/- 210 sites/MN cell and 1065 +/- 69 sites/PMN cell [avg. Kd(+/-) 224-274 pM] using (+/-)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the beta 2-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (+/-)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (+/-)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[3H]DHA, 1 microM (-)-propranolol and 10 microM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for beta-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature.

Hereditary deficiency of the sixth component of complement in man. II. Studies of hemostasis.

Prompted by previous observations of defective blood clotting in rabbits deficient in the sixth component of complement (C6), an evaluation was made of the hemostatic functions of the homozygous proband of a newly recognized human kindred with hereditary C6 deficiency. This human subject, who had no clinical evidence of a bleeding disorder, exhibited a total lack of C6 by functional and immunoprecipitin assays of serum or plasma. Standard tests of hemostatic function were normal; however, when the whole blood clotting time was measured at 25 degrees C in plastic tubes, it was at the upper range of our normal values. In confirmation of this observation, prothrombin consumption, when performed at 37 degrees C in plastic tubes, was at the lower range of normal. Inulin and endotoxin, in concentrations shown to cause activation of human complement, had little or no effect on clotting times or prothrombin consumption of normal or C6-deficient human blood. These observations indicate that absence of C6 does not have a significant effect on hemostatic function in man. In the light of other investigations, the observed differences in clotting function between C6-deficient human blood and C6-deficient rabbit blood could be due to species differences governing the susceptibility of platelets to complement activation.

Inactivation of the skin-sensitizing antibodies of human allergy by thiols.

Summary In vitro exposure of 16 sera from patients with atopic allergy to several different sulfhydryl compounds resulted in inactivation of their skin-sensitizing (reagin) activity, as measured by passive transfer to normal human skin (Prausnitz-Küstner reaction).