Biomedical subjects
Publications and source records attributed to R Silverstein.
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The relation between combat-related trauma experience and male ego development and the impact of such trauma on subsequent employment, marital, and legal behavior were investigated empirically. Vietnam combat veterans (N = 52) diagnosed with post-traumatic stress disorder (PTSD) completed the Defense Style Questionnaire (DSQ), the Ego Identity Scale (EIS), and the Impact of Event Scale (IES); 45 noncombat Vietnam-era veterans (not diagnosed with PTSD) completed the DSQ and the EIS. Combat veterans were found to use more maladaptive defenses and have lower levels of identity achievement. Failed marriages, employment problems, and the tendency to experience legal difficulties were more prevalent among the combat veterans. Data analyses showed that identity and defenses do indeed correlate, suggesting that these constitute a unified construct that can be subsumed under the rubric of ego development.
The uniqueness of the ultra-Orthodox Chassidic Jew presents a challenge to the therapist attempting to counsel this population within the group setting. Idiosyncracies of this religious culture highlight the difficulties in prescribing to conventional methodology of group composition and treatment. Proper group composition with an appropriate leader can contribute to the success of group treatment for this population. Suggestions for structuring the group, in consideration of the sensitivities and needs of these members, are presented.
Using tumor necrosis factor (TNF) inhibition in dog blood as a measure of efficacy, and canine emesis as a measure of toxicity, we were able to assign a therapeutic index to rolipram, a prototypic anti-inflammatory compound. Because both assays were performed in the same species, the ambiguities associated with comparing the physiologic effects of drugs on various species was avoided. Rolipram, a standard phosphodiesterase type IV inhibitor, was a prototypic test compound characterized by a number of cardiovascular and central nervous system side effects, as well as its in vitro and in vivo inhibition of TNF. Initial experiments with canine whole blood incubated with lipopolysaccharide resulted in nanogram-per-milliliter concentrations of TNF that could be significantly reduced by in vitro addition of a 0.03 microM concentration of rolipram. Because rolipram inhibited canine TNF production in vitro, a protocol was devised in which TNF inhibitory activity was measured in a series of blood samples from dogs infused with increasingly high doses of rolipram. This yielded the efficacy half of the therapeutic index, whereas the emetogenic dose represented the side effect portion of the index. Rolipram was infused stepwise into conscious dogs at gradually increasing doses. The infusion was stopped when vomiting occurred, and the cumulative dose was reported as the emetic dose. Rolipram caused emesis in dogs at a cumulative dose of 0.1 mg/kg. At each dose of rolipram, blood was collected. The whole blood was incubated in vitro with lipopolysaccharide to induce TNF production, which in turn was quantified by the L929 bio-assay. Theoretically, if the rolipram infusion raised blood values high enough, the rolipram in whole blood would inhibit TNF production and be reflected by a lack of TNF activity in the L929 assay. In this assay system, rolipram's 50% effective dose in the TNF assay was always at least 33-fold lower than its emetic dose of 0.1 mg/kg. This gave rolipram a therapeutic index of at least 33:1 (0.003 versus 0.1 mg/kg) on the basis of its activity in a canine efficacy model (TNF inhibition) and a toxicity model (emesis induction). Experimental compounds were tested for their emetic dose as well as TNF 50% effective dose, with the goal of obtaining a therapeutic index better than that of rolipram. Thus the coupling of cytokine activity with overt toxicity was used to arrive at the therapeutic index of a compound. The therapeutic index was used to rank compounds as to their efficacy/toxicity profile. This ranking was used to eliminate several anti-inflammatory compounds that had a therapeutic index less than that of rolipram.
A new analysis, the Projective Invariant Analysis, was developed for the evaluation of magnetic resonance images of the temporomandibular joint. This method was used because it takes into account factors such as linear and angular measurements that simple Euclidean geometric methods fail to address. Forty "normal" joints (20 subjects) and 65 "abnormal" joints were analyzed. Joints were defined as normal if they had a Class I skeletal pattern and a Class I occlusion. In addition, a history was obtained and a clinical examination was performed on each of these subjects to rule out any temporomandibular joint dysfunction, masticatory muscle myalgia, or cervicalgia. The images of the 40 normal joints were compared with 65 previously taken images of 53 patients with conditions that had been diagnosed as anteriorly displaced disks with reduction. These two groups were compared with the use of the analysis method described by Drace and Enzmann that uses angular measurements. The groups were also analyzed with the use of a new method in which the structural relationships of the articular eminence, gleniod fossa, mandibular condyle, and articular disk were related to each other using projective geometry. For the projective invariant analysis, a 95% confidence ellipse was constructed on the basis of the normal data. All but one of the 40 normal points fell within this ellipse, and only three of the 65 abnormal points were inside the ellipse. Both analyses showed a significant difference between the normal and abnormal groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Hydrazine sulfate pretreatment has previously been shown in our laboratory to protect normal mice against endotoxin and D-galactosamine-sensitized mice against both exogenous tumor necrosis factor (TNF) and endotoxin. An intact pituitary is required for manifestation of the protective effects. Further, we have demonstrated that hydrazine sulfate pretreatment specifically modulates the TNF response to lipopolysaccharide (LPS) in mouse macrophages in vitro. This in vivo study was performed to test whether a reduced TNF response and/or increased glucocorticoid response may contribute to hydrazine sulfate protection against LPS-induced lethality in vivo. The results presented here establish that hydrazine sulfate pretreatment selectively attenuates circulating TNF levels following LPS challenge. Moreover, adrenalectomy abrogates hydrazine sulfate protection but does not prevent hydrazine sulfate attenuation of circulating TNF levels in response to LPS. Hydrazine sulfate-mediated protection is, however, restored permissively by corticosterone. Thus, the mechanism by which hydrazine sulfate protects against LPS lethality in adrenalectomized mice includes TNF modulation in response to endotoxin, as well as a pivotal requirement for glucocorticoid.
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PURPOSE: To describe the major pathophysiologic mechanisms underlying inherited and secondary hypercoagulable states and to evaluate the frequency, natural history, diagnosis, and management of the various clinical disorders. DATA SOURCES AND STUDY SELECTION: Relevant clinical literature obtained from bibliographies in hematology textbooks and from computerized indexes was reviewed. A hypothesis was formed based on this literature review and on recent developments from a number of experimental studies. DATA SYNTHESIS: Hypercoagulable states include various inherited as well as acquired clinical disorders characterized by an increased risk for thromboembolism. Primary hypercoagulable states include relatively rare inherited conditions that lead to disordered endothelial cell thromboregulation. These conditions include decreased thrombomodulin-dependent activation of activated protein C, impaired heparin binding of antithrombin III, or down-regulation of membrane-associated plasmin generation. The major, inherited, inhibitor disease states include antithrombin III deficiency, protein C deficiency, and protein S deficiency and should be considered in patients who have recurrent, familial, or juvenile deep-vein thrombosis or occlusion in an unusual location such as a mesenteric, brachial, or cerebral vessel. Secondary hypercoagulable states may be seen in many heterogeneous disorders. In many of these conditions, endothelial activation by cytokines leads to loss of normal vessel-wall anticoagulant surface functions with conversion to a proinflammatory thrombogenic phenotype. Important clinical syndromes associated with substantial thromboembolic events include the antiphospholipid syndrome, heparin-induced thrombopathy, the myeloproliferative syndromes, and cancer. CONCLUSIONS: Physiologic thromboregulation occurs at the vessel-wall surface. Quantitative and qualitative deficiencies of normal, steady-state endothelial anticoagulant activities are associated with primary hypercoagulable states. Activated endothelial cell surfaces express a thrombogenic phenotype and contribute to secondary or acquired hypercoagulability.
Adenosine (ADO) and its structurally related analogues are known to regulate the activities of immune and inflammatory cells, including a number of key functions of mononuclear phagocytes. In this study ADO and the synthetic ADO analogue MDL201112 inhibited TNF-alpha, but not IL-1, production by activated mouse peritoneal macrophages and the macrophage-like cell lines J774 and RAW-264. Northern blot analysis indicated that MDL201112 selectively inhibited the expression of steady-state TNF-alpha RNA in LPS+IFN-gamma-activated J774 and RAW-264 cells. This effect could not be attributed to changes in TNF-alpha RNA stability. In contrast, ADO had no effect on RNA levels for TNF-alpha and IL-1, suggesting that ADO acts at a post-transcriptional biosynthetic step. To determine whether either compound inhibited TNF-alpha-mediated inflammatory responses, mice were treated with ADO or MDL201112 and challenged with a lethal dose of endotoxic LPS and D-galactosamine, an hepatotoxin that sensitizes mice to lethal LPS challenge. A single i.p. injection of MDL201112 (100 mg/kg) protected over 90% of the mice, whether injected 1 h before or at the time of LPS challenge. MDL201112 also inhibited the appearance of TNF-alpha in the serum of LPS-challenged animals. The compound did not block D-galactosamine sensitization nor did it prevent lethality caused by the injection of rTNF-alpha. ADO failed to protect animals against endotoxin lethality, most likely due to the rapid metabolism of the nucleoside in vivo. These results establish ADO and MDL201112 as potent inhibitors of TNF-alpha biosynthesis and suggest that MDL201112 or similar analogues warrant further study as potential agents for the treatment of endotoxin shock and other diseases in which TNF-alpha plays an important pathogenic role.
Leukocytes form zones of close apposition when they adhere to ligand-coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein-coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti-fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.
Endotoxin sensitivity and dexamethasone protection have been assessed in mice that were adrenalectomized and also treated with D-galactosamine at the time of endotoxin challenge. Our data establish that adrenalectomy did not detectably alter the magnitude of the increased sensitivity induced by D-galactosamine alone. Furthermore, protection provided by acute exogenous glucocorticoid treatment was still demonstrable in these mice and was not influenced by chronic experimentally induced glucocorticoid deficiency. Our data confirm that the adrenalectomized mouse model of endotoxin lethality is characterized by increased sensitivity to endotoxin and establish that the magnitude of this sensitizing effect is more than 100-fold. We also show for the first time that adrenalectomy causes an appreciable kinetic shift in the endotoxic crisis and that dexamethasone, given at the time of endotoxin challenge, will significantly reverse the increased sensitivity to lethality. Our results indicate that the protective effects of corticosteroids may involve important chronic as well as acute responses. In particular, we conclude that endogenous glucocorticoid need not always increase host resistance to endotoxin, nor does such a circumstance eliminate the possibility for exogenous glucocorticoid-mediated protective effects.
Lipopolysaccharide (LPS), a component of gram-negative bacterial outer cell walls, can stimulate lymphoreticular cells to produce cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. One of these proinflammatory cytokines, IL-6, induces hepatic synthesis of a class of proteins termed acute-phase proteins. D-Galactosamine inhibits acute-phase protein synthesis and concurrently sensitizes mice to a lethal dose of LPS approximately 10,000-fold. From these observations, we hypothesized that the acute-phase response may serve as a defense mechanism for protection of the host against the deleterious effects of LPS. To test this hypothesis, murine recombinant IL-6 (mrIL-6) was used to induce an acute-phase response prior to a lethal LPS challenge in both D-galactosamine-treated and normal mice. Induction of the acute-phase response by mrIL-6 was quantitated by measuring the concentrations of fibrinogen and complement component C3, two well-characterized acute-phase proteins, in the circulation. The effect of acute-phase and normal serum on TNF-alpha release by peritoneal macrophages stimulated with LPS in vitro was also examined. The results of these studies confirmed the induction of the acute-phase response by mrIL-6, as reflected in an approximate doubling in circulating levels of fibrinogen and C3. However, when either D-galactosamine-sensitized or normal mice were challenged with a lethal dose of LPS at various times after mrIL-6 administration, the acute-phase response induced by mrIL-6 did not alter either cumulative lethality or the kinetics of lethality. Additionally, compared with normal serum, acute-phase serum did not affect TNF-alpha release by peritoneal macrophages following LPS-mediated stimulation in vitro. Collectively, these studies would not support a dominant role for an IL-6-mediated acute-phase response as contributing to the resistance of normal mice compared with D-galactosamine-sensitized mice in LPS-induced lethal toxicity.
Chronic loss of adrenocorticosteroid activity by adrenalectomy (10 days) or acute inhibition of glucocorticoid receptor function by injection of an anti-progestin (RU-38486) increased sensitivity to lipopolysaccharide about 200-fold, compared to that of control animals. Daily subcutaneous injection of a mixture of the natural glucocorticoid corticosterone and the mineralocorticoid deoxycorticosterone did not restore resistance to the endotoxin. The largest dose of steroid, 400 micrograms corticosterone + 200 micrograms deoxycorticosterone, provided only partial protection against 20 micrograms LPS, which was 10% of the LD50 for animals with adrenals. The mechanisms involved in lack of effectiveness of natural corticosteroids will require further experimentation.
Polymorphonuclear leukocytes express multiple surface receptors that mediate their adhesion to extracellular matrices and to other cells. These receptors also play roles in cell migration and phagocytosis. We have studied the role of one class of polymorphonuclear leukocytes surface receptors, the beta 2 integrins, in the interactions of these cells with fibrinogen. We have found that the beta 2 integrins, CD11b/CD18 (complement receptor three) and CD11c/CD18 mediate attachment of polymorphonuclear leukocytes to fibrinogen-coated surfaces. Polymorphonuclear leukocytes must be stimulated with chemoattractants, such as fMLP, or with cytokines, such as tumor necrosis factor to bind to fibrinogen via these integrins. Moreover, each of these integrins interacts with a different segment of the fibrinogen molecule. PMN adherence to fibrinogen via CD11b/CD18 depends on the carboxy terminus of fibrinogen, whereas adherence via CD11c/CD18 depends on the amino terminus of fibrinogen's alpha-chain. One of the physiological consequences of these interactions is that polymorphonuclear leukocytes stimulated with either chemoattractants or TNF form protected compartments at their interface with fibrinogen-coated surfaces and that elastase released into these compartments is inaccessible to protease inhibitors present in the plasma. These protected compartments exclude plasma proteins of > 40,000 daltons such as alpha 1 anti-proteinase, thereby allowing polymorphonuclear leukocyte proteases to degrade matrix proteins within this compartment without interference by plasma anti-proteinases.
Hydrazine sulfate (HS) pretreatment protects mice against the lethal effects of bacterial endotoxin lipopolysaccharide (LPS) through mechanisms yet to be established. The liver was examined as a model organ to determine HS effects on (a) LPS activation of leukocyte (Kupffer cell) interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) genes and (b) subsequent cytokine-mediated induction of the acute-phase response as measured by hepatic metallothionein (MT) gene expression. The utility of this model was documented by in situ hybridization which showed that acute induction by LPS of the IL-1 beta gene occurred in cells found in liver sinusoids, consistent with Kupffer cells, whereas induction of the MT gene occurred in hepatocytes. The cell specific expression of these genes was further verified by Northern blot hybridization to LPS-treated liver RNA which showed that the LPS-mediated increase in hepatic cytokine mRNA levels, unlike that of MT, was not prevented by D-galactosamine (D-GalN) treatment. Northern blot hybridization established that HS pretreatment did not block the acute induction of hepatic cytokine mRNAs (IL-1 beta and TNF-alpha) by LPS nor did it induce these cytokine mRNAs in the absence of LPS. Northern blot hybridization further established that HS did not prevent LPS-mediated activation of hepatocyte MT gene expression. Thus, HS does not prevent LPS from activating liver leukocytes. These results also suggest that HS pretreatment neither prevents the general release of cytokines from LPS activated leukocytes nor the general induction of acute-phase protein gene expression in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
In previously published studies, we had demonstrated that hydrazine sulfate pretreatment protected mice against the lethal effects of endotoxin and that this protection was accompanied by a sustained increase in hepatic phosphoenolpyruvate carboxykinase activity (Silverstein, R., C.A. Christoffersen, and D.C. Morrison. 1989. Infect. Immun. 57:2072). The same hydrazine sulfate pretreatment has now been found to protect mice against endotoxin in the D-galactosamine model with an increase in the endotoxin LD50 of approximately four orders of magnitude. Elimination of the pretreatment period, or administration of an additional dose of D-galactosamine at the time of hydrazine sulfate pretreatment, renders the mice refractory to the protection. Given the sensitivity of phosphoenolpyruvate carboxykinase regulation to several hormones, we investigated the possibility that protection may have been hormone mediated. In addition to determining the effect of hydrazine sulfate on the plasma levels of phosphoenolpyruvate carboxykinase regulating hormones, we have investigated the effects of hydrazine sulfate on endotoxin lethality in mice whose capacity to respond hormonally to external stimuli has been compromised by hypophysectomy. Our results show a significant enhancement in circulating levels of plasma corticosterone 30 min after hydrazine sulfate injection. Moreover, hypophysectomy results in a marked increase in sensitivity of mice to endotoxin challenge as well as an abrogation of the protection against endotoxin lethality mediated by hydrazine sulfate. Although hydrazine sulfate protection distinguishes between sensitivity brought on, individually, by D-galactosamine and by hypophysectomy, mice sensitized by both hypophysectomy and D-galactosamine are not protected against endotoxin lethality by hydrazine sulfate. We conclude that hydrazine sulfate protection against endotoxin lethality is endocrine dependent, with the available evidence implicating a pituitary/adrenal axis, with glucocorticoid involvement. In as much as D-galactosamine is known to act directly in the liver in disrupting protein synthesis, it is proposed that events in the liver are critical to the hydrazine sulfate-mediated protection against endotoxin and are possibly the target of the endocrine involvement. Hydrazine sulfate pretreatment also protects D-galactosamine-sensitized mice against the lethal effects of injected tumor necrosis factor/cachectin.
Specific endotoxic lipopolysaccharide (LPS) binding sites on the cell membranes of murine lymphocytes and macrophages that may serve as functional receptors for LPS have recently been identified using photoactivatable cross-linking LPS derivatives. A monoclonal antibody (Mab 5D3) with specificity for this 80-kDa protein has also been generated and characterized. The capacity of MAb 5D3 to protect mice against the lethal effects of endotoxin was investigated. Pretreatment of CF1 mice with as little as 15 micrograms of MAb 5D3 provided virtually complete protection against a dose of endotoxin 10-fold greater than that required to kill all mice in an untreated control group using the galactosamine sensitization model. Significant protection was also afforded normal mice given MAb 5D3 relative to saline. Several lines of evidence suggest that MAb 5D3-mediated protection is due to the agonist properties of this antibody rather than a receptor blockade mechanism.
Although the precise mechanism of endotoxin lethality has yet to be defined, it is well recognized that the amount of hepatic phosphoenolpyruvate carboxykinase is reproducibly and significantly reduced after challenge with endotoxin. Hydrazine has been shown to be a specific inhibitor of gluconeogenesis, causing a metabolic crossover at the step catalyzed by phosphoenolpyruvate carboxykinase. More recently, it has also been shown that hydrazine sulfate may be of potential therapeutic value against cancer cachexia. The experiments described in this paper demonstrate that treatment of CF1 mice with hydrazine sulfate 5 h prior to challenge with endotoxin from Salmonella enteritidis significantly improved survival. Furthermore, such treatment counteracted the drop in hepatic phosphoenolpyruvate carboxykinase activity in isolated cytosol otherwise evident at 6 h and 12 h after endotoxin challenge. Despite this, there was no corresponding improvement in the plasma glucose, measured at 6, 12, and 24 h following endotoxin challenge. It is suggested that the endogenous response to the metabolic crossover initiated by hydrazine may contribute to the protection. The response to hydrazine sulfate has yet to be fully elaborated but does include the increase in phosphoenolpyruvate carboxykinase activity. In contrast with the protection seen upon hydrazine sulfate pretreatment, injecting a corresponding dose of hydrazine sulfate after the endotoxin resulted in more fatalities.