Pathogenesis of DIC in Sepsis

Sepsis can be associated with profound alterations in the hemostatic mechanism. In this article we discuss recent insights into which mediators are involved in the activation of the coagulation system. We focus on studies performed in healthy humans intravenously injected with a low dose of endotoxin, and investigations in nonhuman primates infused with either endotoxin or live gram-negative bacteria. Special emphasis is given to the role of cytokines, in particular tumor necrosis factor-, interleukin (IL)-1, IL-6, IL-10 and IL-12. Moreover, the roles of tissue factor, activated protein C, and the fibrinolytic system are briefly addressed. Disseminated intravascular coagulation likely is the result of complex interactions between several host mediator systems.     

Cyclic AMP Receptor Protein of E. coli: Its Role in the Synthesis of Inducible Enzymes

A cyclic AMP binding protein has been purified over 100-fold from E. coli extracts. Protein purified from wild-type strains binds cyclic AMP with an apparent dissociation constant of 1-2 x 10(-6)M. Two mutant strains that are unresponsive to exogenous cyclic AMP have altered binding activity; the protein purified from one of these mutants has a decreased affinity for cyclic AMP (apparent dissociation constant = 2 x 10(-5)M). Extracts of this mutant are deficient in their ability to support beta-galactosidase synthesis in vitro. The addition of purified, wild-type binding protein to these extracts restores enzyme synthesis toward normal. Because this binding protein appears to be required for cyclic AMP action, we suggest it be called the cyclic AMP receptor protein (CR protein).     

Microparticles in Hematological Malignancies: Role in Coagulopathy and Tumor Pathogenesis

Microparticles (MP) are submicron vesicles released from various cells in response to activation, injury or apoptosis. They contain different structural and functional proteins and RNAs, which contribute to physiological intercellular “crosstalk” and to the pathogenesis of various diseases including cancer. In hematological malignancies, these MPs participate in the initiation and propagation of thrombosis through different pathways. They have a role in the angiogenesis, malignant cell survival and metastasis. MPs act as a mediator of resistance of leukemic cells to chemotherapy. The number of MPs is one of the prognostic factors following stem cell transplant, and studies have also found they contribute to the pathogenesis of graft versus host disease. MPs are being tested as therapeutic options in leukemias and graft versus host disease. Future studies should help us understand the interactions between MPs and cancer cells better, thereby opening new approaches for treatment of hematological malignancies.     

Role of Different Replacement Fluids During Extracorporeal Treatment in a Pig Model of Sepsis

In an extracorporeal combination therapy, the impact of different replacement fluids on survival was tested in a bacterial sepsis model in pigs. In an animal study 19 pigs, weighing 7.5–11.1 kg, were included. All groups received an intravenous lethal dose of live Staphylococcus aureus over 1 h. The animals were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration for 4 h. The extracorporeal circuit was pre‐filled with 400 mL replacement fluid. In the P0 group 100% hydroxyethyl starch 130/0.4 was used as replacement fluid; in the P30 group 30% pig plasma and 70% hydroxyethyl starch; and in the P100 group 100% pig plasma. The observation time was 7 days. All animals of the group P100 survived, while all animals of group P0 and five out of seven animals of the P30 group died during the observation time. Extracorporeal therapy consisting of online centrifugation and plasma filtration with 100% pig plasma as replacement fluid significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged.     

The Role of Protein C in Sepsis

During the past 15 years, several anti-inflammatory treatments have failed to reduce mortality in patients with severe sepsis. However, recent evidence indicates that coagulation abnormalities in sepsis may play a major role in the pathogenesis of multiple organ failure and the high mortality rate in patients with severe sepsis. Interestingly, blockade of the coagulant pathway can inhibit both procoagulant and proinflammatory pathways in sepsis. Protein C, a natural anticoagulant, interrupts several of the pathophysiologic pathways in sepsis. Acquired protein C deficiency is present in the majority of septic patients and is associated with unfavorable outcomes. Protein C replacement therapy was effective in preclinical animal models of sepsis in reducing end-organ damage and mortality. Recent clinical trials of protein C replacement in human meningococcemia resulted in a markedly decreased morbidity and mortality. And, most importantly, in a recently completed large, randomized trial of activated protein C treatment in severe sepsis, mortality was reduced from 30.8% in the placebo group to 24.7% in the treatment group at 28 days. Thus, there is new evidence that mortality can be reduced among patients with severe sepsis through the use of a new therapy that inhibits the procoagulant and the inflammatory cascades.     

A Possible Role of Phospholipid in Nerve Excitation

A possible role of phospholipid in regulating the ionic permeability of excitable membranes is proposed. It is assumed that each excitable transport channel in the nerve fiber is lined with two chains of phospholipid dipoles in either parallel or antiparallel directions; a molecular mechanism of nerve excitation is developed that is qualitatively consistent with most of the relevant observations reported in the literature.     

Protective effects of murine monoclonal antibodies in experimental septicemia: E. coli antibodies protect against different serotypes of E. coli

Murine monoclonal antibodies that bind outer membrane antigens of the J5 mutant of Escherichia coli O111:B4 were derived from spleen cells of BALB/c mice immunized with killed whole cells and boosted with lipopolysaccharide (LPS) and LPS-associated proteins. Seven hybridomas were selected for their reactivity against the J5 LPS; they cross-reacted with O111, O55, O127, and O128 E. coli LPS. One (B7B3) also reacted with the Serratia marcescens LPS and Klebsiella pneumoniae lipid A. A protective effect was obtained with D6B4 antibody in a lethal endotoxemia model induced by LPS from O111, O127, and O128 E. coli serotypes in D-galactosamine-sensitized mice. D6B4 and D6B3 antibodies protected mice infected with E. coli O111:B4, when administered before infection. The D6B4 antibody was also protective when administered after infection. The antibodies D6B3 and D4B5 were protective in heterologous infection induced by E. coli O2:K1.     

内皮细胞微粒对动脉粥样硬化发生发展的作用

内皮细胞微粒（EMP）是内皮细胞在活化或凋亡时释放的脂质膜小囊泡, 其在内皮细胞损伤、促炎症反应及细胞与细胞之间信号传导等方面发挥重要作用。在一系列疾病中,如动脉粥样硬化、冠心病、糖尿病、脓毒血症和恶性高血压等都有EMP的增高。动脉粥样硬化是很多疾病发生发展的病理基础,具有很高的主要死亡率和发病率。本文就EMP对动脉粥样硬化发生发展的作用作一概述。     

Escherichia coli Osteomyelitis after Sepsis in Regional Enteritis: Report of a Case

A patient receiving immunosuppressive drugs (prednisone and azathioprine) for regional enteritis developed Escherichia coli sepsis followed in several months by E. coli osteomyelitis of a distal femur. This unusual complication may represent another potential hazard of immunosuppressive therapy for inflammatory bowel disease.     

Pathophysiological Role of Pro- and Anti-Inflammatory Cytokines in Sepsis

The appearance of detectable pro- as well as anti-inflammatory cytokines in the blood stream during sepsis is indicative of their exacerbated production. While pro-inflammatory cytokines are a prerequisite for initiating an effective anti-infectious process, they are also associated with harmful effects which can lead to multiple organ dysfunction and death. While anti-inflammatory cytokines are a prerequisite for controling the inflammatory response, they also lead to a depression of the immune system of patients. Furthermore, new evidence suggests that these so-called anti-inflammatory cytokines may not always behave as such. All in all, the analysis of the interplay between cytokines reveals that a very complex network of interactions is occuring and that pieces are still missing from the sepsis puzzle.     

Immunological effect of E. coli L-asparaginase

Summary The effect of asparaginase on cellular immunity was investigated on human peripheral blood lymphocytes incubated in the culture system. Results showed that asparaginase inhibits lymphocyte blastogenesis, both when stimulated with PHA or PKW, and in the mixed lymphocyte cultures. This happened when the enzyme was added directly to the culture medium, as well as when the enzyme was administered i.v. in humans, and then the blood lymphocytes were removed and put into the PHA-culture medium added to the lymphocyte donor's plasma. Crossed experiments showed that: a) lymphocytes from asparaginase injected subjects which were set up in a PHA-culture-medium to which plasma (20%) from non-injected subjects had been added, undergo a normal amount of blastogenesis; and b) lymphocytes from non-injected subjects, set up in a PHA-culture-medium with plasma (20%) from asparaginase injected subjects show an inhibition in their blastic transformation.The effect of asparaginase on the cell precursors of humoral immunity was investigated on the spleen lymphatic follicles of adult rabbits i.v. injected with that enzyme preparation. It appeared that asparaginase causes, on the one hand, a decrease in cellular density and size until there is a complete disappearance of the lymphatic collars, and, on the other hand, an increase in the cellular growth and size of the germinal centers, accompanied by an increase of the plasma-cell frequency.These results, taken as a whole, show that asparaginase is a depressing agent of the lymphocyte-depending immunity, but not of the plasma cell-system, i.e., the system which provides the humoral immunity.

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Zusammenfassung Die Wirkung von Asparaginase auf die zelluläre Immunität wurde an menschlichen Blutlymphozyten untersucht, die in dem Kultur-System inkubiert wurden. Die Ergebnisse zeigten, daß Asparaginase die Blastogenese der Lymphozyten inhibiert, sowohl bei PHA- oder PKW-Stimulation, als auch in der gemischten Lymphozytenkultur. Dies geschah sowohl bei Zufügen des Enzyms direkt zum Kulturmedium, als auch bei i.v.-Verabreichung des Enzyms beim Menschen. In letzterem Fall wurden die Lymphozyten isoliert und die Kultur-Medien hinzugefügt. Gekreuzte Experimente zeigten, daß a) Lymphozyten von Asparaginase-injizierten Personen, die in ein PHA-Medium gebracht wurden, welchem das Plasma (20%) von nicht-injizierten Personen zugefügt worden war, eine normale Blastogenese durchmachten; und b) Lymphozyten von nicht-injizierten Personen, die einem PHA-Kulturmedium mit Plasma (20%) von Asparaginase-injizierten Personen zugesetzt wurden, eine Inhibition in ihrer blastogenetischen Transformation zeigen.Die Wirkung der Asparaginase auf die Zell-Vorläufer der humoralen Immunität wurde an den lymphatischen Follikeln der Milz von ausgewachsenen Kaninchen, denen diese Enzym-Präparation i.v. verabreicht worden war, untersucht. Es wurde gezeigt, daß die Asparaginase einerseits eine Abnahme der Zelldichte und-größe bis zum vollkommenen Verschwinden des lymphatischen Ringwalls verursachte, andererseits aber eine Zunahme des Zellwachstums und Vergrößerung der Keimzentren zusammen mit einer Vermehrung der Plasmazellen auslöst.Diese Ergebnisse zusammengenommen zeigen, daß Asparaginase eine depressorische Wirkung auf die lymphozyten-abhängige Immunität ausübt, nicht aber auf die Plasmazelluläre, d. h. humorale Immunität.

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Supported by The Blood Researarch Foundation, Washington D.C./USA.

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Paper delivered at the XIII International Congress of Hematology, Munich/Germany, Aug. 2nd–8th, 1970.

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Part of this study was performed with grants from A.Nattermann, Cologne-Braunsfeld, Germany, and fromCarlo Erba, S.p.A., Milano, Italy.     

人胸苷激酶基因在大肠杆菌中的表达

目的　在大肠杆菌中表达人胸苷激酶 (hTK)。方法　用内切酶将hTKTA克隆中的hTK基因切下 ,并与同样内切酶切的载体PET2 8a 连接 ,转化大肠杆菌 ,筛选阳性克隆 ,酶切和DNA序列分析鉴定 ,用IPTG诱导培养阳性克隆 ;SDS -PAGE鉴定hTK在大肠杆菌中的表达。结论　hTK基因重组入PET2 8a 的EcoRI和XhoI位点 ,IPTG可以明显诱导hTK在大肠杆菌中表达。