Cognitive disorders in migrants: retrospective analysis in a Center for Cognitive Disorders and Dementia in Milan.

BACKGROUND: The number of migrants with dementia in Italy might increase considerably over the coming years due to the increasing flow of immigration and the aging of the population. AIMS: We retrospectively registered rate and characteristics of demented migrant outpatients referred to one hospital in Milan from 2001 to 2017. METHODS: Information about country of origin of migrants attending general neurology and memory clinics was obtained from their Italian tax code. Socio-demographic, cultural, and clinical characteristics were derived from their medical records. RESULTS: Migrants with cognitive decline represented a minimal fraction (3.1%) of demented outpatients, but a grow rate of 400% was registered within the period of observation. A linguistic barrier resulted as the main obstacle for the application of available diagnostic tools for dementia. DISCUSSION/CONCLUSION: Given the above-reported data, the implementation of strategies (such as transcultural diagnostic instruments) and policies dedicated to this growing health problem appears a priority for our health systems.

Robot-assisted partial nephrectomy using the novel Hugo™ RAS system: Feasibility, setting and perioperative outcomes of the first off-clamp series.

INTRODUCTION: Hugo Robot-Assisted Surgery (RAS) System has been conceived with enhanced modularity but its role for nephron-sparing surgery setting still remains poorly explored. We aimed to describe our experience in robot-assisted partial nephrectomy (RAPN) with a three-arms setting for the first off-clamp series using the new Hugo RAS System. METHODS: Patients were placed on an extended flank position at the margin of the surgical bed with a slightly flexion (45°). The first 11 mm robotic trocar (camera port) was placed along the pararectal line 14 ± 2 cm far from the umbilicus. The pneumoperitoneum was then induced through the AirSeal system (SurgiQuest, Milford, Connecticut, USA©). Two more 8 mm operative robotic ports were placed under direct vision, either 8 ± 1 cm far from optic's port. Two 12 mm laparoscopic ports for bed-assistant were placed between robotic ports. Monopolar curved shears, fenestrated grasper, and large needle driver were used in a three-instruments configuration. RESULTS: Off-clamp RAPN was successfully performed in seven patients with cT1 renal masses using a trans-peritoneal route. Median port placement and docking time was 6 min (IQR, 4-8 min). Hemostasis was achieved through renorraphy using a single transfix stitch with sliding clips technique. There was no need for additional ports placement. No intraoperative complications occurred, no clashing of robotic instruments or between the robotic arms was observed. No technical failures of the system occurred. Median console time was 83 min (IQR, 68-115 min). Median estimated blood loss were 200 ml (IQR, 50-400 ml). All patients were discharged between post-operative day 2 and 3, without the need of hospital readmission. No complications were recorded within the first 30 post-operative days. CONCLUSIONS: We performed the first series of off-clamp RAPN using the novel HUGO RAS System. This novel robotic platform showed an easy-friendly docking system, providing excellent perioperative outcomes with a simple three-arms configuration.

Mast cells are critically involved in serum-mediated vascular leakage in chronic urticaria beyond high-affinity IgE receptor stimulation.

BACKGROUND: Chronic urticaria (CU) is one of the most common skin disorders whose pathogenic mechanisms are not fully clarified. Autoimmune aetiology can be ascribed to 45% of patients with CU, and basophil histamine release is positive in 40% of cases. Our aim was to use a novel approach to evaluate the serum permeabilizing effect to identify the mediators of endothelial cell (EC) leakage and to define the role of mast cells (MCs) in the process. METHODS: Permeabilizing activity of sera from 19 patients with CU and 11 healthy blood donors was evaluated by measuring serum-induced degranulation of two MC lines, expressing (LAD2) or lacking (HMC-1) the IgE receptor. Mast cell supernatant (SN) was then incubated with an EC monolayer, and endothelial permeability was evaluated by Fluorescein isothiocyanate-bovine serum albumin leakage in a transwell system. RESULTS: All 19 patient sera failed to induce direct EC leakage, but 15/19 and 17/19 promoted degranulation of HMC-1 and LAD2, respectively. Interestingly, 85% of autologous serum skin test-negative sera were able to cause MC degranulation. Also, 17/19 SNs from HMC-1 and all SNs from LAD2 incubated with CU sera increased endothelial permeability. Endothelial cell leakage remained unchanged after Ig depletion and was prevented by antihistamine, platelet-activating factor or leukotriene antagonist. CONCLUSIONS: Our study shows that CU sera are able to degranulate MCs through an IgE- and IgG-independent mechanism. The nature of histamine-releasing factors involved is still unclear, but our finding opens new ways to the understanding of the pathogenesis of CU, particularly in patients not showing circulating autoantibodies to FcεRI or IgE.

Hepatocyte nuclear factor 1alpha controls the expression of terminal complement genes.

The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1alpha (HNF1alpha) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8beta, C8gamma, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1alpha-deficient mice showed negligible hemolytic activity of both C5 and C8alpha-gamma subunits. The activity of C8beta was severely affected despite only a 50% reduction in C8beta mRNA levels in the liver. This is reminiscent of C8alpha-gamma-deficient patients who accumulate extremely low levels of the C8beta subunit. Our results demonstrate that HNF1alpha plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.

The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

Anti-phospholipid antibody mediated fetal loss: still an open question from a pathogenic point of view.

Antiphospholipid antibodies (aPL) are associated with recurrent miscarriages and pregnancy complications, however their pathogenic mechanisms are still matter of research. Thrombotic events at the placental level cannot explain all of the clinical manifestations. It has been suggested that aPL may be responsible for a local acute inflammatory response mediated by complement activation and neutrophil infiltration eventually leading to fetal loss. However histological and immunohistological studies on human placental samples do support such a mechanism only in part and with no any clear relationship with the pregnancy outcome. A direct effect of aPL on both maternal and fetal placental tissues has been reported through the reactivity of the antibodies with beta2 glycoprotein I (beta2GPI) expressed on the cell membranes. These events do not require an inflammatory response and can be in part related to the inhibition of growth factors favouring a physiological placentation. Understanding the different pathogenic mechanisms of aPL-associated miscarriages may help in improving our therapeutic approach particularly in recurrent cases not responsive to the usual treatment.

Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors.

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca2+]i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T. M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371-1380). To determine whether stimulation of [Ca2+]i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca2+]i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca2+]i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti-P- and anti-E-selectin mAb, as well as anti-VCAM-1 mAb, induced transient increases in EC [Ca2+]i that were comparable to those induced by 200 microM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca2+]i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca2+]i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca2+]i changes in the 5 h-treated EC, whereas the anti-VLA-4 mAb alone was sufficient to inhibit [Ca2+]i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti-PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca2+]i, i.e. , mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.

Higher interleukin-18 and mannose-binding lectin are present in uterine lumen of patients with unexplained infertility.

The uterine luminal environment was explored with regard to interleukin-18 (IL-18) and mannose-binding lectin (MBL) and the possibility that the procedure of flushing the uterine cavity would optimize the physiological initial pseudo-inflammatory uterine reaction. Uterine flushings were performed among 175 IVF/intracytoplasmic sperm injection (ICSI) patients at the time of oocyte retrieval and the cycles were compared with a control group matched for age, number of previous attempts and type of assisted reproductive procedure (IVF or ICSI) in which no flushing were performed (n = 175). Samples collected were divided into two groups according to the presence/absence of endometrial cells in samples. IL-18 and MBL expressions were explored by enzyme-linked immunosorbent assay. Implantation rates were significantly higher in those patients who underwent the uterine flushing compared with controls (P = 0.04). Luminal concentrations of IL-18 and MBL were higher if endometrial cells were present in flushings, suggesting endometrial origin of the secretion. Both concentrations of MBL and IL-18 were higher in patients with unexplained infertility compared with patients involved in IVF/ICSI for male or tubal infertility (P = 0.005 and 0.02, respectively). The exploration of the endoluminal environment before oocyte retrieval may enhance pregnancy rates and show distinct features in patients with unexplained infertility.

Selection and characterization of a novel agonistic human recombinant anti-TRAIL-R2 minibody with anti-leukemic activity.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its death receptors, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricidal activity. The anti-TRAIL-R2 MB2.23 efficiently and specifically bound to membrane-associated TRAIL-R2 on different leukemic cell lines and could act as a direct agonist in vitro, initiating apoptotic signaling as well as complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, providing a rationale for further investigations of MB2.23 in anticancer therapy.

Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed.

Two types of dysfunctional eighth component of complement (C8) molecules in C8 deficiency in man. Reconstitution of normal C8 from the mixture of two abnormal C8 molecules.

Restoration of hemolytic activity was examined in sera from seven unrelated eighth component of complement (C8)-deficient subjects. The sera fell into two groups, depending on whether hemolytic activity was restored by the addition of the beta-chain (group 1) or the alpha-gamma-subunit (group 2) purified from normal human C8. Antigenic analysis of these sera by double-immunodiffusion using anti-human C8 confirmed previous findings of a dysfunctional C8 in the four sera of group 1 and established the presence of a different dysfunctional C8 in one of the sera of group 2 when tested at a high concentration. Further characterization of the dysfunctional C8 molecules in the two sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that group 1 sera were missing the beta-subunit and group 2 sera were missing the alpha-gamma-subunit of the C8 molecule. Sera from either of these two groups alone did not produce hemolysis in hemolytic plates containing sheep erythrocytes coated with antibody and complement components up to C7 (EAC1-7) and C9. When sera from the two groups were added to adjacent wells in the hemolytic plates, a zone of hemolysis developed between the wells. The contribution of C8 alpha-gamma from the sera of group 1 and of C8 beta from those of group 2 to the lysis of EAC1-7 in the presence of C9 was confirmed by the inhibitory effect of specific antibodies against the two C8 subunits. In experiments in which hemolytic activity was reconstituted by mixing sera from group 1 with sera from group 2, the serum source of C8 beta (group 2) was the limiting reagent. The dysfunctional C8 molecule in this serum was able to bind to EAC1-7. Chromatographic analysis demonstrated that the generation of hemolytic activity in the mixture of the two sera resulted from the reconstitution of the C8 molecule rather than the sequential action of the two C8 subunits.

Fibular nerve palsy after hip replacement: Not only surgeon responsibility. Hereditary neuropathy with liability to pressure palsies (HNPP) a rare cause of nerve liability.

Mononeuropathy after surgery may occur and hereditary neuropathy with liability to pressure palsies is a possible pathological condition related to paresis after hip surgery. We present a case of 66-year-old man presenting severe weakness at inferior limb muscles after hip prosthesis revision. Clinic and electrophysiology showed severe right fibular nerve damage and ultrasound found a marked enlargement of the same nerve, associated with focal enlargements in other nerves. A diagnosis of hereditary neuropathy with liability to pressure palsies was suspected and confirmed by genetic test. The patient gradually recovered returning to a normal daily active life. Ultrasound was crucial for diagnosis. The suspicion and diagnosis of latent neuropathy, which can occur after surgical intervention, may lead to a better understand of the risks of the surgery, specific for the patient, and avoid the wrong attribution to surgical malpractice.

Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

: Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. SIGNIFICANCE: This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from patients with Duchenne muscular dystrophy (DMD), using a microengineered model. An ad hoc experimental strategy was designed to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. This microscaled in vitro model, which validated previous in vivo preclinical work, revealed that mesoangioblasts showed unexpected efficiency as compared with myoblasts in dystrophin production. Consequently, this model may be used to predict efficacy of new drugs or therapies aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo.

The possible role of pancreatic proteases in the turnover of intestinal brush border proteins.

1. Intestinal brush border enzymes have heterogeneous rates of turnover, the largest proteins having the fastest turnover. Since the membrane faces the intestinal lumen, the effects of pancreatic factors were examined in mediating this turnover. Surgical subtotal pancreatectomy was used as an experimental model to study the turnover of brush border proteins in the absence of most pancreatic secretions. 2. Subtotal (95%) pancreatectomy of rats was found to cause elevations by about 50% of total activity and specific activities of certain brush border enzymes (maltase, sucrase, lactase), but not of others (alkaline phosphatase, trehalase). Rats were judged to be functionally deficient in pancreatic proteolytic enzymes (a) by demonstration of vitamin B-12 malabsorption, which was corrected by trypsin, and (b) by the finding of only about 20% of proteolytic activity appearing in the lumen after a test meal when compared to control. 3. To measure protein turnover in vivo the method of double labelling was used, where [3H]- and [14C]valine were administered intraduodenally in sequence 10 h apart. With this technique, a high 3H/14C ratio is correlated with rapid turnover. Proteins with apparent molecular weights of about 200 000-270 000 were found to turn over more rapidly than smaller proteins. 3H/14C ranged from 4.7 to 6.2 in animals without pancreatic insufficiency. In the face of decreased pancreatic proteolysis, the 3H/14C ratio was 2.3-3.1, similar to that of proteins with a slow half life. 4. Estimates of relative synthetic rates of large brush border proteins were lower than normal in pancreatectomized animals, but were constant over the period of the labelling experiment. The high enzyme levels in the face of lower synthetic rates confirms that, at the new steady rate, degradation rates must be slower for large brush border proteins in pancreatic insufficiency. 5. In vitro, using purified brush borders, unfractionated pancreatic enzymes were found to remove sucrase, maltase and lactase, but not alkaline phosphatase and trehalase. The enzyme most potent in this respect was the pancreatic protease, elastase. Non-proteolytic enzymes (amylase, lipase, phospholipase A) were inactive in removing enzyme from the brush border. The addition of elastase to pancreatectomized animals in vivo restored the rapid turnover rate of large brush border proteins. 6. A model is thus proposed for the normal catabolism of some large intestinal brush border proteins. It is suggested that the surface of intestinal absorptive cells is being constantly remodelled, and that certain surface enzymes are in part removed from the membrane by the action of pancreatic proteases. A possible special role for elastase is suggested.

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. II. Presence of the NADPH2 oxidizing activity in a myeloperoxidase-deficient subject.

1. The biochemical properties of leucocytes from a myeloperoxidase-deficient subject were compared with those of leucocytes from healthy subjects. 2. Myleoperoxidase-deficient leucocytes responded to phagocytosis of heat-killed bacteria with increased respiration, increased oxidation of glucose through the hexose monophosphate shunt and increased production of H2O2 as normal leucocytes do. 3. The ability of granules isolated from myeloperoxidase-deficient leucocytes to oxidize nicotinamide coenzymes was comparable to that of granules isolated from normal leucocytes. 4. The results argue against the hypothesis that oxidation of NADPH2 in leucocytes is performed by myeloperoxidase.

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. I. Evidence for superoxide anion involvement in the oxidation of NADPH2.

1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Beta chain deficiency in three patients with dysfunctional C8 molecules.

Structural and functional studies were performed on a dysfunctional C8 molecule present in the serum of two siblings and an unrelated individual. The C8 in these three sera exhibited a pattern of partial immunologic identity with C8 in normal serum but was devoid of functional activity. The C8 was immunoprecipitated from the three sera and from a control serum with an antihuman C8 antiserum and analyzed by SDS-PAGE using highly purified human C8 as a reference. A selective absence of a band of 62,000 mol. wt was observed in the immunoprecipitates from the sera containing dysfunctional C8. Experiments performed with the purified alpha-gamma and beta subunits showed that the hemolytic activity of the C8 deficient sera could be reconstituted by the addition of the beta chain but not the alpha-gamma dimer. Binding of the dysfunctional C8 to C567 was excluded by the following observations: (1) EAC1-7 treated with the C8 deficient sera and then washed could not be lysed after the addition of the beta subunit and C9; and (2) the abnormal molecules did not interfere with the consumption of normal C8 by the soluble complex SC5b-7.

Complement-endothelial cell interactions: pathophysiological implications.

The function of the endothelial cells can be modulated by humoral factors present in the circulation and in the extravascular fluid, including proteins of the complement system. This review examines the multiple interactions between the complement system and the endothelial cells and their functional consequences on inflammation, coagulation and regulation of vascular tone. The implications of these interactions in the induction and progression of the vascular lesions occurring in atherosclerosis, ischemia/reperfusion and xenotransplantation and the possible therapeutic approaches in terms of complement regulation are also discussed.

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.