Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

A trial of using timentin (ticarcillin/clavulanate) in treating abdominal surgical infection]

Efficacy of timentin was studied in the treatment of 19 patients with peritonitis of various etiology and clinical and laboratory signs of systemic inflammatory reaction characteristic of abdominal sepsis. The clinical and bacteriological effects were recorded in 84.2 and 87.5 per cent of the cases respectively. The drug was administered intravenously dropwise for 30 minutes in a dose of 3.1 g every 4 hours. The treatment course was 4-11 days. The treatment failed in 3 patients. One of them had general peritonitis of gynecological etiology. In the other no significant regression of abdominal sepsis was observed, Pseudomonas aeruginosa strains were isolated from the abdominal cavity, the antibiotic was changed, still incurable polyorganic insufficiency developed and the patient died. The third patient had perforation of the large intestine due to tumor. No adverse reactions to the use of timentin in any of the cases was observed.     

Hemolytic,cytotoxic and complement inactivating properties of extracts of different species of Aspergillus

Some of the biological properties of saline extracts of the mycelia of several species of the Aspergillus genus, namely, A. fumigatus, A. flavus, A. niger, A. nidulans, A. parasiticus and A. glaucus, were studied. Only the extract prepared from A. fumigatus was found to be hemolytic for sheep red blood cells. In contrast, all the extracts with the only exception of that of A. glaucus, had cytotoxic effects on Vero cells. Both, the hemolytic and cytotoxic constituents of the extracts were removable by adsorption with activated carbon. Heating of the extracts at 100°C for 30 minutes also resulted in detoxification. In vivo studies, performed only with detoxified extracts of A. fumigatus, showed these were capable of depleting complement levels in guinea pigs. Complement inactivation was also found to occur in vitro and was caused by all the extracts tested. Also triggered by the extracts was the conversion of serum C3 but not of purified C3, indicating that other serum factors are essential in the process. Despite the similarity in this respect with cobra venom factor, differences in activity after heating-negative in cobra venom factor-indicate that the complement inactivating substance/s present in the Aspergillus extracts differ from those of the snake product.     

Emile Brumpt's contribution to the characterization of parasitic diseases in Brazil, 1909-1914

During the first thirty years of the XXth century, parasitologists and epidemiologists who were at the origin of the nosography and etiology of parasitic diseases were faced with several overlapping problems. A person can be infected simultaneoulsy by several different parasites. The delineation of clinical signs is an essential step, in the field and without the help of the laboratory, to identify the various parasitic pathologies and to propose the most likely diagnoses. The use of photography as a nosographic tool enabled the French parasitologist Emile Brumpt (1877-1951) to set up clinical pictures, given the multiplicity of pathologies on a given patient (for instance, goitre and ankylostomiasis, ankylostomiasis and myxoedema, "sylvan" leishmaniasis and palpebral oedema, goitre and Chagas' disease). We will set the paper on a part of Brumpt's photographic archives which he constitued during his missions to South America between 1913 and 1914.     

Isolation and identification of the third component of complement of Japanese quails

The identification of the third component of complement (C3) of Japanese quails was attempted by using rabbit antiserum prepared against quail serum-treated zymosan (ZX) as an initial reagent. This antiserum (anti-ZX) had agglutinating activity on rabbit erythrocytes reacted with quail antibody and quail complement (EACq) but not on EAq, and developed two precipitin lines against quail serum at beta- and gamma-regions in crossed immunoelectrophoresis. Subsequently, monospecific antisera to each of these precipitin lines were prepared in rabbits, and quail serum proteins reactive with these antisera were purified by salt precipitation followed by Sephadex gel filtration and DEAE cellulose column chromatography. One protein with a m.w. of 184,000 (184K) resembled mammalian C3 in that: 1) monospecific antiserum (anti-184K protein serum) agglutinated EACq but not EAq; 2) treatment of fresh quail serum with either inulin or zymosan resulted in the conversion of the precipitin line developed against 184K protein from gamma to beta in crossed immunoelectrophoresis; 3) the 184K protein was shown to consist of two polypeptide chains of 110K and 73K linked by disulfide bonds. Furthermore, the 184K protein in serum was cleaved through the incubation with inulin to 174K and 140K proteins that might correspond to C3b and C3c of human complement; 4) the 184K protein bound to zymosan was eluted with hydrazine or methylamine but not with Nonidet P-40, indicating that 184K protein binds to zymosan by a covalent bond but not by a hydrophobic one; and 5) by treatment of fresh quail serum with methylamine, complement reactivity was reduced, although its activity was restored by the addition of purified 184K protein. These results suggest the 184K protein is the quail's equivalent to mammalian C3. When quail serum was reacted with cells that had complement-activating capacity, quail C3 deposited on their membrane as in mammalians; however, no conversion of quail C3 was noted by the reaction with CVF. Antibody to quail C3 failed to cross-react with that in mammals.     

DNA polymorphism related to the idiopathic hemochromatosis gene: evidence in a recombinant family

Summary The metabolic error involved in idiopathic hemochromatosis, as well as the underlying genetic defect remain unknown. It has, however, been recently shown that this genetic lesion occurs at a locus linked to the major histocompatibility complex, probably close to the HLA-A locus, and that the disease is recessively transmitted. Therefore, in a family where one subject has idiopathic hemochromatosis his HLA-identical siblings should also be affected. We present here the restriction polymorphism with two MHC class I probes and one DR probe in an exceptional family with three HLA-identical siblings: one (the proband) has a major form of idiopathic hemochromatosis, while the other two are free of any clinical or biochemical signs of the disease. The restriction patterns observed after DNA digestion by enzymes EcoRI, EcoRV, BglII, BamHI, PvuII, TaqI, HincII, and HindIII led to the conclusion that one of the proband's chromosome 6 had undergone two alterations: one, a deletion in the DR region, was revealed by missing fragments all correlated with DR5; the other was an unbalanced cross-over or a genetic conversion in the MHC class I region. This latter alteration was revealed by modifications in the patterns of high molecular weight HindIII bands which hybridize with probe pHLA2 and also by the absence of a HindIII fragment of 7.4 kb hybridized by another class I probe. This latter alteration most likely involved the hemochromatosis gene and could be the first step toward a molecular approach to this gene.     

Association of Dengue Hemorrhagic Fever With Multiple Risk Factors for Pituitary Apoplexy

ObjectiveTo describe pituitary apoplexy that developed during the course of dengue hemorrhagic fever.MethodsWe describe the clinical findings, laboratory test results, imaging findings, and clinical course of the study patients.ResultsPatient 1 was a 40-year-old man who developed clinical signs and symptoms of dengue, which was confirmed by serologic testing. He presented with thrombocytopenia and developed severe headache and vomiting. During hospitalization, acromegaly was suspected because of the characteristic disease phenotype. Magnetic resonance imaging confirmed the diagnosis of pituitary apoplexy. Subsequently, the biochemical diagnosis of acromegaly was confirmed, and the patient underwent transsphenoidal surgery. Histopathologic examination showed signs of recent bleeding. Patient 2 was a 38-year-old man with a macroprolactinoma, who had been treated with cabergoline for 10 weeks and had shown improvement on laboratory testing and imaging. The patient then presented with clinical symptoms of dengue (confirmed serologically) and thrombocytopenia. He developed bilateral hemianopsia, and magnetic resonance imaging showed enlargement of the pituitary adenoma with signs of intratumoral bleeding. The patient underwent transsphenoidal surgery, and histopathologic examination documented a pituitary adenoma diffusely infiltrated by blood cells.ConclusionsWe describe dengue as a probable novel condition for pituitary apoplexy because it may be associated with multiple risk factors for pituitary infarction or bleeding. Physicians should suspect pituitary apoplexy in patients with dengue hemorrhagic fever who develop a rapid onset of severe headache and vision defects, even in those without known pituitary adenomas. (Endocr Pract. 2012;18:e97-e101)     

Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitt's lymphoma and infectious mononucleosis.

Cultured human lymphoblastoid cell lines derived from patients with Burkitt's lymphoma or infectious mononucleosis were shown to activate the alternative pathway of complement fixation. This reaction does not require any conventional antibody directed against the cells. Although the reaction showed an absolute dependence on the presence of factor B it was relatively independent of the presence of factor D or of properdin. To this extent activation of the alternative pathway by lymphoblastoid cells resembles that produced by “C3-nephritic factor.” Rat and mouse complement were activated in a manner similar to human complement, but guinea pig complement was inactive. Chicken complement, unlike any of the mammalian complements tested, was able to bring about lysis of the lymphoblastoid cell lines by the alternative pathway.     

Interpretation of diagnostic data: 5. How to do it with simple maths.

The use of simple maths with the likelihood ratio strategy fits in nicely with our clinical views. By making the most out of the entire range of diagnostic test results (i.e., several levels, each with its own likelihood ratio, rather than a single cut-off point and a single ratio) and by permitting us to keep track of the likelihood that a patient has the target disorder at each point along the diagnostic sequence, this strategy allows us to place patients at an extremely high or an extremely low likelihood of disease. Thus, the numbers of patients with ultimately false-positive results (who suffer the slings of labelling and the arrows of needless therapy) and of those with ultimately false-negative results (who therefore miss their chance for diagnosis and, possibly, efficacious therapy) will be dramatically reduced. The following guidelines will be useful in interpreting signs, symptoms and laboratory tests with the likelihood ratio strategy: Seek out, and demand from the clinical or laboratory experts who ought to know, the likelihood ratios for key symptoms and signs, and several levels (rather than just the positive and negative results) of diagnostic test results. Identify, when feasible, the logical sequence of diagnostic tests. Estimate the pretest probability of disease for the patient, and, using either the nomogram or the conversion formulas, apply the likelihood ratio that corresponds to the first diagnostic test result. While remembering that the resulting post-test probability or odds from the first test becomes the pretest probability or odds for the next diagnostic test, repeat the process for all the pertinent symptoms, signs and laboratory studies that pertain to the target disorder. However, these combinations may not be independent, and convergent diagnostic tests, if treated as independent, will combine to overestimate the final post-test probability of disease. You are now far more sophisticated in interpreting diagnostic tests than most of your teachers. In the last part of our series we will show you some rather complex strategies that combine diagnosis and therapy, quantify our as yet nonquantified ideas about use, and require the use of at least a hand calculator.     

A complement receptor locus: genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to 1q32

The alternative or classical pathways for complement system component C3 may be triggered by microorganisms and antigen-antibody complexes. In particular, an activated fragment of C3, C3b, covalently attaches to microorganisms or antigen-antibody complexes, which in turn bind to the C3b receptor, also known as complement receptor 1. The genes encoding the proteins that constitute the C3-activating enzymes have been cloned and mapped to a "complement activation" locus in the major histocompatibility complex, and we demonstrate in this study such a locus on the long arm of chromosome 1 at band 1q32.     

Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b

Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef.     

Hematin promotes complement alternative pathway-mediated deposition of C3 activation fragments on human erythrocytes: potential implications for the pathogenesis of anemia in malaria

Childhood malaria caused by Plasmodium falciparum is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported, based on clinical observations and in vitro models, that complement control proteins on erythrocytes such as CR1, the immune adherence receptor specific for C3b, may be reduced in childhood malaria, suggesting a possible role for complement in erythrocyte destruction. Intravascular lysis of iE by P. falciparum leads to release of erythrocyte breakdown products such as hemoglobin and hematin, which have inflammatory properties. In the present article, we demonstrate that in serum and in anticoagulated whole blood, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on erythrocytes. The degree of C3 fragment deposition is directly correlated with erythrocyte CR1 levels, and erythrocytes opsonized with large amounts of C3dg form rosettes with Raji cells, which express CR2, the C3dg receptor which is expressed on several types of B cells in the spleen. Thus, the reaction mediated by hematin promotes opsonization and possible clearance of the youngest (highest CR1) erythrocytes. A mAb specific for C3b, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this mAb in nonhuman primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of targeted therapies based on inhibiting the alternative pathway of complement.     

Allergy and autoimmunity: Molecular diagnostics,therapy, and presumable pathogenesis

Allergic and autoimmune diseases represent immunopathological reactions of an organism to antigens. Despite that the allergy is a result of exaggerated immune response to foreign antigens (allergens) and autoimmune diseases are characterized by the pathological response to internal antigens (autoantigens), the underlying mechanisms of these diseases are probably common. Thus, both types of diseases represent variations in the hypersensitivity reaction. A large percentage of both the adult and pediatric population is in need of early diagnostics of these pathologies of the immune system. Considering the diversity of antibodies produced in allergic and autoimmune disease and the difficulties accompanying clinical diagnosing, molecular diagnostics of these pathological processes should be carried out in several stages, including screening and confirmatory studies. In this review, we summarize the available data on the molecular diagnostics and therapy of allergic and autoimmune diseases and discuss the basic similarities and differences in the mechanisms of their development.     

Atypical systemic leishmaniasis to be considered in the differential of patients presenting with depressed immunity

Background

Systemic leishmaniasis has been known to present with prolonged fever, hepatosplenomegaly and wasting. Beside this classical form, a sub-clinical form has been identified. It is described with either one or two of the above symptoms missing; other findings have been reported instead, such as lymphadenopathy and anemia. In this report, we reveal a third unsuspected form which we are referring to as “atypical”.

Methodology/Principal Findings

Patients suspected to be immune-deficient were referred to our immunology specialized laboratory to study some aspects of their immune functions (not normally covered in the general laboratory). Multiple specialized tests were performed, including microscopic examinations using appropriate stains, and mainly cultures of biopsies on several types of specialized media. 19·4% of 160 patients were found to have close to normal laboratory profiles, but exhibited dysfunctional macrophages laden with Leishmania parasites.

Conclusions/Significance

Findings such as the ones we obtained allowed us to uncover the presence of patients with an atypical form of systemic leishmaniasis. It presents with symptoms masquarading a condition in which the immune system is non functional. This predisposes patients to recurrent secondary infections resulting in clinical pictures with a great variety of signs and symptoms. These findings alerted us to the fact that systemic leishmaniasis presents with a much wider spectrum of signs and symptoms than so far suspected and is far more common than diagnosed to date. Furthermore, among these 31 patients was a number of adults. This proved that in our area systemic leishmaniasis is surely not limited to the pediatric age group. Our recommendation is to entertain the diagnosis of atypical systemic leishmaniasis in any patient with an unexplained depressed immunity state and in whom no obvious immunologic defect can be identified.     

35例肺隐球菌病临床分析

目的提高肺隐球菌病早期诊断与治疗的水平。方法对浙江大学医院附属第一医院2006年9月至2009年9月收治的35例病理确诊为肺隐球菌病患者的性别、年龄分布、职业情况、基础疾病、诱因、临床表现、实验室检查、影像学特征、治疗方案及预后特点等进行回顾性分析。结果患者以中青年男性为主,多数患者有基础疾病或诱因,临床症状相对轻微,其中40%无症状,一般实验室检查,气管镜检查及病原学检查阳性率极低,影像学表现以单发或多发结节肿块影为主(共占65.7%),氟康唑治疗3～9个月一般均能治愈,但亦有17.6%迁延不愈或无效者。结论中青年患者,无发热性肺部感染性病变,症状轻微,血象正常,血沉正常,痰细菌学检查阴性,考虑肺隐球菌病可能,尽早取得组织病理学依据,治疗首选氟康唑,疗程3～9个月,手术病理明确并病灶切除者,术后仍需常规应用氟康唑治疗。     

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcgamma receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels > 150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.     

Clinical value of the pudendal somatosensory evoked potential

The pudendal evoked potential was recorded in 126 patients who had presented with various uro-genital complaints. The patients were divided into two groups depending on whether or not there were clinical signs of neurological disease. Group I consisted of 83 patients (66%) who on clinical examination were neurologically normal. In group 11 there were 43 patients (34%) who had physical signs suggesting underlying neurological pathology. The pudendal evoked potential was abnormal in 10 patients from the group with neurological signs (group 11) but only 1 patient from group I (a man who had made an excellent recovery from previous transverse myelitis). The conclusion of this study is that the pudendal evoked potential is of no greater value than the clinical examination in the assessment of patients with uro-genital dysfunction. The recommendation that the potential should be recorded as part of the routine assessment of patients suspected of having a neurogenic disorder of the bladder and sexual function should be reconsidered.     

Pavlov, psychoanalysis, and neuroses

Pavlov's discovery of experiment neurosis was serendipitous, yet it was made under the influence of Breuer and Freud's case of Anna O. In 1914, Pavlov's disciple N. R. Shenger-Krestovnikova, exploring the limits of visual discrimination in dogs, noticed that when the discrimination was difficult, the dogs' behavior became disorganized. Pavlov drew an analogy between the condition of Shenger-Krestovnikova's dogs and their disorganized behavior with Anna O.'s situation and her neurotic reaction. Pavlov concluded that he had demonstrated in the laboratory the elements of neurosis in animals and human alike. Schilder's criticism of his position, his later study of human neuroses in clinical settings, and the views of Janet may have induced Pavlov to differentiate between animal and human neuroses.     

C3d binding to the myelin oligodendrocyte glycoprotein results in an exacerbated experimental autoimmune encephalomyelitis

The complement system is known to contribute to demyelination in multiple sclerosis and experimental autoimmune encephalomyelitis. However, there are few data concerning the natural adjuvant effect of C3d on the humoral response when it binds to myelin Ags. This study addresses the effect of C3d binding to the myelin oligodendrocyte glycoprotein (MOG) in the induction of experimental autoimmune encephalomyelitis in C57BL/6J mice. Immunization with human MOG coupled to C3d was found to accelerate the appearance of clinical signs of the disease and to enhance its severity compared with MOG-immunized mice. This finding was correlated with an increased infiltration of leukocytes into the central nervous system accompanied by increased complement activation and associated with areas of demyelination and axonal loss. Furthermore, B cell participation in the pathogenesis of the disease was determined by their increased capacity to act as APCs and to form germinal centers. Consistent with this, the production of MOG-specific Abs was found to be enhanced following MOG/C3d immunization. These results suggest that binding of C3d to self-Ags could increase the severity of an autoimmune disease by enhancing the adaptive autoimmune response.