Effects of bacterial endotoxin on rabbit platelets. IV. The divalent ion requirements of endotoxin-induced and immunologically induced platelet injury.

The divalent ion requirements of rabbit platelet injury by endotoxin have been defined by the use of various anticoagulant solutions and have been compared to the divalent ion requirements of platelet injury produced by addition of antigen to immune platelet-rich plasma. The endotoxin-platelet interaction takes place in citrated blood. Platelet damage by antigen is inhibited by citrate, but preincubation of antigen and immune platelet-poor plasma in the absence of citrate results in a substance, presumably antigen-antibody complement complex, which then does injure platelets in the presence of citrate. Neither endotoxin nor preincubated antigen injures platelets in the presence of sodium EDTA in concentrations sufficient to interact with all divalent cations present in plasma. These observations have been interpreted by viewing the platelet-endotoxin interaction as a consequence of platelet phagocytosis of endotoxin, a reaction not requiring complement but requiring definite small concentrations of divalent cations. The interaction of antigen and platelets is regarded as a two phase reaction, the first requiring the participation of complement and concentrations of divalent cation larger than those provided in citrated plasma, the second requiring smaller concentrations of divalent cation, no further participation of complement, and active in citrated plasma. This second phase is regarded as representing platelet phagocytosis of immune complexes.

Complement-dependent platelet injury by staphylococcal protein A.

A new example of complement-mediated platelet injury has been described. Staphylococcal protein A (SPA) causes rabbit platelet injury as manifested by release of platelet 5-hydroxytryptamine (5HT). This reaction is complement-dependent and occurs over a very small range of SPA concentration, larger amounts being inhibitory. Complement fixation by SPA demonstrates the same narrow SPA concentration requirement whereas precipitation of IgG by SPA is roughly proportional to SPA concentration over a wide concentration range. The reaction can be separated into a sensitization step which requires SPA and plasma but not complement, and a release step which does require complement. Complement-mediated platelet damage induced by SPA is a new biologic property of this common component of the cell wall of pathogenic staphylococci which may contribute to the development of inflammatory and thromboembolic reactions complicating intravascular staphylococcal infection.

Staphylococci-induced human platelet injury mediated by protein A and immunoglobulin G Fc fragment receptor.

Bloodstream infections with staphylococci are accompanied by thromboembolic complications. We have studied the mechanism of the interaction of staphylococci with human blood platelets. Staphylococci that possess protein A, a bacterial receptor for the Fc fragment of immunoglobulin G (IgG), caused aggregation of human platelets in whole plasma accompanied by release of [(3)H]serotonin. These reactions were time and concentration dependent, requiring two or more staphylococci per platelet to give maximal response within 5 min. The interaction between staphylococci and platelets required the presence of cell wall-bound protein A and of IgG with an intact Fc fragment. It did not require an intact complement system. Cell wall-bound protein A (solid phase) was capable of aggregating human platelets in whole plasma. In contrast, free, solubilized protein A (fluid phase) did not cause measurable aggregation, and release of [(3)H]serotonin was reduced. An excess of free, solubilized protein A blocked aggregation of human platelets induced by staphylococci in whole plasma. The role of the Fc fragment of IgG in the staphylococci-human platelet interaction was demonstrated by an experiment in which free, isolated Fc fragment blocked aggregation of platelets in whole plasma induced by staphylococci. Furthermore, binding of (125)I-protein A to human platelets was demonstrated in the presence of complete IgG with intact Fc fragment but not in the presence of the F(ab)(2) fragment. Binding of the protein A-IgG complex to the human platelet Fc receptor was paralleled by the release of [(3)H]serotonin. These results represent a novel example of the interaction of two phylogenetically different Fc receptors, one on prokaryotic staphylococci and the other on human platelets. Their common ligand, IgG, is amplified by one Fc receptor (protein A) to react with another Fc receptor present on human platelets, which results in membrane-mediated aggregation and release reaction occurring in whole plasma. This mechanism can be of significance in the pathomechanism of thromboembolic complications at the site(s) of intravascular staphylococcal infection.

Mediastinal fibrosis complicating histoplasmosis.

Mediastinal fibrosis, the most serious late complication of remote infection by Histoplasma capsulatum, is a thick, dense fibrotic capsule which surrounds a small mediastinal focus of old caseous adenitis. The fibrotic process may accrue over prolonged periods and extend within the lumina of critical mediastinal structures to produce complete occlusion. We summarized clinical and radiographic data for 71 patients with mediastinal fibrosis; the criteria for inclusion were the clinical demonstration of occlusion of major central airways (trachea or mainstem bronchus) or major vessels (pulmonary arteries or veins) and the absence of other disease processes which might cause such obstruction. We selected 65 patients who met these criteria from the medical literature of the last 40 years and report 6 new cases from our experience. The majority of patients were diagnosed between ages 20 and 40. The most common symptoms included hemoptysis, dyspnea, and cough. An accentuated pulmonic component of the second heart sound, wheezing, and localized murmur were among the physical findings reported. Radiographic abnormalities consisted of mass lesions and atelectasis or infiltrates, but were often nonspecific. Chest radiography was deceptively normal in some patients, even in the presence of major central airway or vascular occlusion, especially when the focus was subcarinal. Computed tomography has particular promise to depict the mediastinal abnormalities in this process. Surgery had minimal therapeutic benefit. Because of incomplete followup, the mortality of 30% in this series surely does not represent the true overall mortality of mediastinal fibrosis.

Chronic pulmonary histoplasmosis.

Chronic pulmonary histoplasmosis is best regarded as an opportunist or saprophytic infection of abnormal pulmonary spaces by a fungus of very low human pathogenicity. Tissue disease results from host immune response to dispersions of soluble antigen from these focal sources. There are two distinct types of clinical and radiological response. One is an acute or subacute illness manifested by often large segmental pneumonic lesions which tend to heal and are designated as early lesions. The other, usually developing as a complication of the first, is a chronic disease marked by persistent cavitation, low gard chronic illness, and a tendency to promote pulmonary fibrosis and often progressive pulmonary insufficiency. The early lesion is a segmental interstitial pneumonitis with central areas of infarct-like necrosis often adjacent to bullous disease and often outlining prominent emphysematous spaces which appear as radiolucencies. These radiological findings are further characterized by early clearing of the interstitial components, infarct-like contraction of the necrotic zones, obliteration of much of the contained emphysematous and bullous spaces, and healing attended by considerable loss of lung volume. Symptoms are variable but tend to be mild. Malaise, fatigability, low-grade fever, aching chest pain and mild cough lasting a few days to a few weeks are usual. Symptoms are ameliorated by rest. Rest and diminished activity are recommended as treatment. Under these circumstances, 80% of early lesions heal completely and probably most of these would heal spontaneously. Any subsequent course of the disease depends on whether or nor large air spaces, adjacent to or contained within the area of pneumonitis, become infected and persist as cavities. This occurs in 20% of early lesions. Once established, an infected cavity tends to persist and to be attended by symptoms of chronic bronchitis with chronic cough and sputum, fatigability, anorexia, and weight loss. Persisting thickwalled cavities often induce gradual development of pulmonary fibrosis, particulary in the lung bases, apparently from aspiration of antigenic material. This and the accelerated obstructive bronchopulmonary disease often lead to progressive pulmonary insufficiency. The use of amphotericin B is recommended for all persistent thick-walled cavities and in some circumstances surgical resection may be indicated.

Tuberculosis and acquired immunodeficiency syndrome: a historical perspective on recent developments.

The incidence of tuberculosis has increased in recent years, at least in part as a result of the ongoing worldwide epidemic of acquired immunodeficiency syndrome (AIDS). In addition, the occurrence of outbreaks caused by multidrug-resistant Mycobacterium tuberculosis organisms has greatly heightened concern. In retrospect, a number of seminal studies that have appeared during the past decade have helped to define changing concepts concerning the epidemiology, pathogenesis, approaches to preventive care, diagnosis, and treatment of tuberculosis in HIV-infected persons. Such reports have shown that the variable clinical manifestations of tuberculosis in patients with AIDS are greatly influenced by the degree of HIV-induced immunosuppression. Explosive outbreaks of tuberculosis occurring in closed environments have emphasized that patients with AIDS and pulmonary tuberculosis may be highly contagious, especially when diagnosis and implementation of appropriate infection control measures are delayed. The extent to which homelessness and illicit drug use complicate management of tuberculosis have been examined, and the high risk of persons who are both tuberculin-positive and HIV-positive ultimately developing active tuberculosis, unless chemoprophylaxis is completed, has been clearly shown. The utility of sputum smears, bronchoscopy, and newer technologies such as polymerase chain reaction for diagnosis has been examined. The risk of relapse appears to be low when patients with AIDS with drug-sensitive tuberculosis complete appropriate multiple-drug therapy. Recent reports have addressed important hospital infection control, tuberculin testing, and chemoprophylaxis issues. This paper describes this evolution of understanding, focusing on reports that we believe have been conceptually important.

Apical localization of pulmonary tuberculosis, chronic pulmonary histoplasmosis, and progressive massive fibrosis of the lung.

We have reviewed the accumulated evidence for the explanation of the apical localization of pulmonary tuberculosis, chronic pulmonary histoplasmosis, and massive fibrosis of the lung due to silicosis and coal-workers pneumoconiosis. The effect of gravity on the erect human lung results in greatly diminished pulmonary artery blood flow in the apical and subapical areas. This in turn results in higher oxygen tensions but also impairment of tissue clearance mechanisms in these areas. Analysis of the accumulated evidence better supports the theory of lymph stasis and impaired clearance of antigenic substances as the major determinant of the apical localization of pulmonary tuberculosis rather than the presently favored oxygen tension theory. The impaired clearance theory also best explains the apical localization of chronic pulmonary histoplasmosis and progressive massive fibrosis of the lung.

The role of pyrazinamide in tuberculosis chemotherapy.

Pyrazinamide is an antituberculosis drug synthesized in the 1950s and formerly used only as salvage therapy. Recent developments have elevated it to a central role in tuberculosis chemotherapy as the essential addition to isoniazid and rifampin which makes it possible to successfully complete treatment in six months. This is accomplished with no increase in hepatotoxicity. The only substantial side effect of this drug given at the dosage and for the duration used in these six-month regimens is a polyarthralgia which is only bothersome and not sufficient to warrant interruption of therapy. More rarely, acute gout is produced. The early history and pharmacology of this now first line antituberculosis drug are reviewed herein.

Tuberculous meningitis.

Tuberculous meningitis is an uncommon but potentially devastating form of tuberculosis. Current antituberculous drugs are highly effective when treatment is initiated early, before the onset of altered mentation or focal neurologic deficits. Because the clinical outcome depends greatly on the stage at which therapy is initiated, early recognition is of paramount importance. Patients with the meningoencephalitis syndrome and CSF findings of low glucose levels, elevated protein levels, and pleocytosis should be treated immediately if there is evidence of TB elsewhere in the body, or if prompt evaluation fails to establish an alternative diagnosis. Examination of CSF is the best diagnostic approach; with sufficient diligence, serial AFB smears and cultures will usually yield positive results, even days after therapy has been started. The CT scan is an important and highly effective tool for the diagnosis and management of patients with TBM. In a patient with compatible clinical features, the combination of basilar meningeal enhancement and any degree of hydrocephalus is strongly suggestive of the diagnosis of TBM. Serial evaluation by CT scanning is useful for following the course of hydrocephalus and tuberculoma, particularly in reference to the need for, or response to, adjunctive therapy with corticosteroids and surgery. The decision to administer corticosteroids should be based on careful correlation of the clinical and radiographic features of the case. Surgical shunting should be considered early in the patient with hydrocephalus and symptoms of raised intracranial pressure. Tuberculomas are best treated medically, often in conjunction with corticosteroids where cerebral edema is believed to contribute to neurologic decline. The recommended chemotherapy regimen is isoniazid and rifampin in all patients, together with pyrazinamide for the first 2 months.

Plasma components are required for platelet activation by the toxin of Loxosceles reclusa.

We have used a partially purified toxin from the venom of the brown recluse spider, Loxosceles reclusa, to study its effects on human platelets isolated from plasma proteins. This toxin, which produced skin necrosis in rabbits, contained sphingomyelinase D activity. The toxin induced platelet aggregation and secretion of [3H]serotonin in human plasma but not in buffer or in human neonate plasma. Ca2+ was required for the interaction of toxin, platelets, and plasma factor(s). The addition of C-reactive protein restored aggregation and serotonin release of platelets incubated in human neonate plasma. The ADP-degrading enzyme, apyrase, and the non-steroidal, anti-inflammatory drug, indomethacin, inhibited platelet aggregation, suggesting that ADP secreted from platelet storage granules and indomethacin-sensitive pathway(s) are involve in the toxin-induced human platelet activation (aggregation and serotonin release). Generation of platelet activating factor (PAF) from the platelet by brown recluse toxin is not likely since the PAF receptor antagonist, BN 52021, did not inhibit platelet aggregation induced by the brown recluse toxin.

CNS tuberculosis.

Tuberculous meningitis is a rare, treatable neurologic disorder, in which early recognition is paramount because outcome depends greatly on the speed with which therapy is initiated. Patients with meningitis and CSF findings of low glucose, elevated protein and pleocytosis with evidence of tuberculosis elsewhere in the body (chest radiographs, positive tuberculin skin test), or a history of exposure to tuberculosis should be treated immediately with antituberculous medication. When the diagnosis remains uncertain, serial examination of the CSF for tuberculous organisms will often yield positive results. The CT scan may show hydrocephalus, a basilar arachnoiditis, or intraparenchymal lesions: tuberculomas. Hydrocephalus may respond to early shunting. Tuberculomas are best treated medically. Therapy should include INH and rifampin; ethambutol and pyrazinamide are suggested for the first 2 months of therapy. Steroids may be useful in diminishing the inflammatory response when altered consciousness or focal neurologic signs are present.

Interaction of Histoplasma capsulatum with human platelets.

Thrombocytopenia is a common accompaniment of disseminated histoplasmosis. The yeast form of Histoplasma capsulatum does not directly injure human platelets freed of plasma. Preincubation of H. capsulatum with plasma enabled it to induce prompt platelet aggregation and selective release of [3H]serotonin without release of lysosomal beta-glucuronidase and the cytoplasmic marker, lactate dehydrogenase. Platelet aggregation was mediated by adenosine diphosphate, as shown by the blocking of the reaction by apyrase. Indomethacin inhibited both aggregation and serotonin release, indicating their dependence on prostaglandin synthesis by platelets. Plasma IgG conferred [3H]serotonin-releasing activity after complexing with yeasts, and plasma fibrinogen was necessary for platelet aggregation; classical and alternative complement pathways were not involved. The interaction of H. capsulatum with human platelets, mediated by IgG and fibrinogen without complement, represents a new attribute of this fungal pathogen and may contribute to thrombocytopenia complicating disseminated histoplasmosis.

Alternate complement pathway induction of aggregation and release of 5-hydroxytryptamine and adenosine diphosphate by rabbit platelets.

The present studies investigated patterns of rabbit platelet aggregation and release of 5-hydroxytryptamine (5HT) utilizing nine variables: three different types of challenge, soluble antigen and antibody (AG-AB), zymosan (Z), an agent known to activate the alternate complement pathway (ACP), and Z preincubated in lightly heparinized plasma so as to become coated with complement (ZC); three different types of platelet-rich plasma (PRP), lightly heparinized PRP in which both complement pathways are active, ethylene glycol tetraacetic acid-PRP (EGTA-PRP) in which only the ACP is active, and ethylene diamine tetraacetic acid PRP (EDTA-PRP), which inhibits both complement pathways; three different types of inhibitors, cobra venom factor (CoF), which causes activation of C3 proactivator (C3PA) to C3 activator (C3A) and fluid phase decomplementation of C3 and C5 through C9, adenosine monophosphate (AMP), a specific antagonist of ADP, and tosyl arginine methyl ester (TAME), an inhibitor thought to act not only on the first component of complement, but also on a platelet membrane site of mediating complement-induced platelet injury as well as on C3PAse. In heparinized PRP, both AG-AB and Z produced biphasic aggregation and prompt and extensive 5HT release. A brief lag period noted with both AG-AB and Z challenge was not observed with ZC challenge, indicating that this lag period represented time required for generation of the necessary complement-dependent membrane-injuring activity. Prior decomplementation by CoF entirely prevented both aggregation and release by either AG-AB or Z but by ZC, indicating first that fluid-phase ACP activation did not produce platelet injury, and second that ZC had on its surface an activity capable of producing immediate biphasic aggregation and prompt 5HT release without the further participation of later acting complement components. Both AMP and TAME eliminated the second phase of aggregation and diminished or eliminated 5HT release with all three challenges, suggesting that both inhibitors might be operative on similar or identical platelet membrane receptors mediating complement-dependent platelet injury. In EGTA-PRP, AG-AB and Z produced delayed monophasic aggregation and delayed and diminished 5HT release, whereas ZC produced immediate although monophasic aggregation but delayed and diminished 5HT release. This suggested that all three challenges were capable of producing ACP-mediated platelet injury.

Function of the classical and alternate pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgCl2-ethylene glycol tetraacetic acid.

An immunochemical and functional analysis of the classical and alternate complement pathways in human serum was performed in the presence of 10 mM ethylene glycol tetraacetic acid (EGTA) and MgCl(2)-EGTA (MgEGTA), chelating agents which have been recently utilized as a means of distinguishing between these two complement pathways. Total hemolytic activity, integrity of the C1 complex, hemolytic activity of C2, conversion of factor B (C3 proactivator), and complement-dependent bactericidal activity were studied. The effect of these chelators on activation of complement pathways by Escherichia coli, by sensitized erythrocytes as a prototype of activators of the classical pathway, and by zymosan as a prototype of alternate (properdin) pathway activators was studied. Human serum containing 10 mM EGTA, which provides almost no ionized calcium and considerably less ionized magnesium than unchelated serum, allowed consumption of complement via the alternate (properdin) pathway, but blocked the classical pathway as judged by disintegration of the C1 complex and lack of utilization of C2. However, activity of the alternate complement pathway in EGTA serum, as judged by conversion of factor B and bactericidal activity against gram-negative bacteria, was distinctly suboptimal. Addition of magnesium ion in a concentration equimolar to EGTA (MgEGTA serum), while still providing conditions in which the C1 complex dissociated, significantly enhanced alternate complement pathway-mediated bactericidal activity. However, in MgEGTA serum considerable fluid-phase activation of the alternate pathway, as indicated by decrease in 50% hemolytic complement (CH(5 0)) titers and conversion of factor B to its active form in the absence of any activating challenge, was observed. Moreover, some fluid-phase consumption of C2 was observed in MgEGTA serum, even though, as mentioned, the C1 complex was shown to be dissociated under these conditions. MgEGTA-related activation of C2 and of the alternate (properdin) pathway of complement was significantly enhanced by the presence of zymosan and E. coli. These results indicate that use of the chelating agents EGTA and MgEGTA to differentiate between classical and alternate pathway activation of human complement is more complex than has hitherto been suggested. In EGTA serum, spontaneous activation of either pathway does not occur but bactericidal activity, as a measure of biologic function of complement, is suboptimal. In MgEGTA serum, bactericidal activity is fully expressed, but there is considerable instability, in terms of fluid-phase activation, in Mg(2+)-dependent components of both pathways. Thus, caution is indicated in the use and interpretation of the effects of these chelating agents on biologic functions mediated by either pathway of human complement.