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The inhibitory effect of carrageenin, a sulfated algal polygalactose, on humoral factors of natural resistance is discussed. In dependence on dose and time, the influence of non-specific and specific bacteriolysis, on the in vitro and in vivo opsonic activity and on the course of infection was studied.
O6-Methylguanine-DNA methyltransferase (MGMT) is an important DNA repair protein that plays a key role in cancer chemotherapy by alkylating agents such as carmustine (BCNU) and Dacarbazine (DTIC). Therapy by BCNU and DTIC is reduced by dose-limiting hematological toxicity as a result of low MGMT repair activity in bone marrow cells. In this study, we have constructed a Moloney murine leukemia virus retroviral vector containing the human mgmt gene. High-titer retrovirus producer cells lines have been generated. Retroviral-mediated transfer of the human mgmt gene into murine multi-potent hematopoietic stem cells, FDCP-1, resulted in the expression of a high level of MGMT activity. In comparison with the control cells that were transduced with the parent vector, the MGMT-expressing clones were considerably more resistant to the cytotoxicity of the methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroso-N-methyl-urea, and temozolomide, as well as the chloroethylating agents 1-(2-chloroethyl)-1-nitrosourea and BCNU. The protection provided by MGMT could be eliminated by the MGMT inactivator O6-benzylguanine. Thus, the principal lethal lesions produced by these alkylating agents in the murine hematopoietic stem cells and the MGMT deficiency in these cells can be complemented by retroviral-mediated gene transduction.
Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-beta (A beta) plaques. Clusterin also coprecipitates with A beta from CSF, suggesting a physiological interaction with A beta. Given this interaction with A beta, the goal of this study was to determine whether clusterin could modulate A beta neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose-dependent neuroprotection against A beta (1-40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of A beta (1-40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-beta plaque pathology.
The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.
Monoclonal antibody 14G2a (anti-GD2) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD2. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagent N-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor alpha and interferon alpha and chemotherapeutic agents such as cisplatinum and N,N'-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.
Fresh Frozen Plasma is the most important therapeutic agent in acquired coagulation disorders. We investigated the quality of a new virus-inactivated lyophilisated human pool-plasma (VFL) and conventional fresh frozen plasma (FFP). In VFL we found a significantly reduced coagulation activity, a significantly higher pH-value, a highly significant rise of heparin values and a significantly lower number of thrombocytes. Electrolytes, protein levels and complement activity showed no significant difference. In conclusion, this virus-inactivated lyophilisated human pool plasma does not seem to be susceptible to therapy coagulation disorders like the conventional fresh frozen plasma.
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OBJECTIVE: To obtain a synthetic anti-complement inhibitor which has stronger activity than FUT-175 (nafamostat mesilate), as a synthetic ester derivative containing amidino and guanidino groups. METHODS: We synthesized several modified compounds of FUT-175. The anti-complement activities were measured using synthetic substrates and complement-mediated hemolysis in vitro. The anti-complement activity in vivo was evaluated via Forssman systemic shock in guinea pigs. RESULTS: FUT-175 inhibited C1r and C1s with IC50s of 1.7x10(-6) and 3.2x10(-7) M, respectively. Inhibitory activities were decreased by substitution of the amidino group with a hydrogen atom (compound 2), but not the guanidino group with a hydrogen atom (compound 3). Compound 6, in which the benzene ring of compound 3 was substituted with a furan ring, inhibited C1r and the complement-mediated hemolysis in high-diluted serum with higher potency than FUT-175. The inhibitory activity of compound 6 in hemolysis was weakened in low diluted serum. Compound 7 had a guanidino group inserted into compound 6; however, Compound 7 strongly inhibited hemolysis even in low-diluted serum, and suppressed Forssman systemic shock more potently than both FUT-175 and compound 6. CONCLUSIONS: These data suggest that the 2-furylcarboxylic acid derivatives have a strong potential for inhibiting the activities of the complement, and the guanidino group was required to retain high inhibitory activities in vivo, and compound 7 is a hopeful anti-complement agent.
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The informative value of biological characteristics of Staphylococcus aureus reflecting the ability of microbes to inactivate certain factors of natural resistance (lysozyme, complement, immunoglobulins, bactericidal component of interferon) was established in determining the duration of the caused by them pyo-inflammatory diseases such as postinfection abscesses, lactation mastitis. On this basis the heterogeneous successive procedure of pattern recognition was used to develop algorithms allowing prognosis of the duration of pyo-inflammatory diseases of staphylococcal etiology in 84.6-94.4% of the patients after the quantitative and qualitative assessment of the biological properties of the pathogen with not less than 90% reliability.
Complement protein C3 is a central molecule in the complement system whose activation is essential for all the important functions performed by this system. After four decades of research it is now well established that C3 functions like a double-edged sword: on the one hand it promotes phagocytosis, supports local inflammatory responses against pathogens, and instructs the adaptive immune response to select the appropriate antigens for a humoral response; on the other hand its unregulated activation leads to host cell damage. In addition, its interactions with the proteins of foreign pathogens may provide a mechanism by which these microorganisms evade complement attack. Therefore, a clear knowledge of the molecule and its interactions at the molecular level not only may allow the rational design of molecular adjuvants but may also lead to the development of complement inhibitors and new therapeutic agents against infectious diseases.
Nematodes include both major parasites of humans, livestock and plants in addition to free-living species such as Caenorhabditis elegans. The nematode nervous system (especially in C. elegans) is exceptionally well defined in terms of the number, location and projections of the small number of neurons in the nervous system and their integration into circuits involved in regulatory behaviours vital to their survival. This review will summarize what is known about the biological activity of neurotransmitters in nematodes: the biosynthetic pathways and genes involved, their receptors, inactivation mechanisms and secondary messenger signalling systems. It will cover the 'classical' transmitters, such as acetylcholine (ACh), GABA, glutamate, serotonin, dopamine, octopamine, noradrenaline and nitric oxide. The localization of peptides throughout the nematode nervous system is summarized, in addition to the isolation of nematode neuropeptides by both traditional biochemical techniques and more modern genetic means. The major contribution of the completion of the C. elegans genome-sequencing program is highlighted throughout. Efforts to unravel neurotransmitter action in various physiological actions such as locomotion, feeding and reproduction are detailed as well as the various inactivation mechanisms for the current complement of nematode transmitters.
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Damaged or old erythrocytes are cleared rapidly from circulation. Because several common biochemical lesions can induce the clustering of integral membrane proteins, we have proposed that formation of microscopic protein aggregates in the membrane might constitute a cell surface marker that promotes removal of the defective/senescent cells. We demonstrate here that treatments that cluster integral membrane proteins in erythrocytes (1 mM ZnCl2, 1 mM acridine orange, and 0.35 microM melittin) induce autologous IgG binding, complement fixation, and phagocytosis by human monocytes in vitro. Removal of the clustering agents prior to incubation in autologous serum or cross-linking of cell surface proteins before addition of clustering agents prohibited the above response, while cross-linking after treatment with the clustering agents preserved the response even if the clustering agents were later removed. Furthermore, subsequent reversal of the chemical cross-link maintaining the clustered distribution also reversed the induction of IgG binding, complement deposition, and phagocytosis. Finally, by deleting or inactivating different steps in the phagocytosis pathway, the chronology of steps was shown to be: (i) integral protein clustering, (ii) IgG binding, (iii) complement deposition, and (iv) phagocytosis.
Natural xenoantibodies are believed to be IgM in nature and are known to play a critical role in the hyperacute rejection of distantly related xenografts. The purpose of this study was to determine whether the reducing agent DL-penicillamine could inactivate baboon natural xenoantibodies to pig splenocytes. Pooled baboon serum was treated with varying concentrations of DL-penicillamine over different lengths of time and a complement-mediated cytotoxicity assay was used to determine the reactivity of baboon natural xenoantibodies to pig splenocytes. A whole-cell ELISA assay was used to assess the binding of both IgG and IgM xenoantibodies to pig splenocytes. In addition, DL-penicillamine-treated serum was dialyzed to assess its potential clinical application. These in vitro experiments indicate that both IgM and IgG baboon natural xenoantibodies bind to pig splenocytes, but only IgM xenoantibody is cytotoxic. The binding of baboon natural IgM xenoantibody can be eliminated, and the cytotoxicity of IgM xenoantibody markedly reduced by DL-penicillamine treatment despite continued binding of IgG xenoantibody to pig splenocytes. In addition, DL-penicillamine can be dialyzed, suggesting that it may be an efficacious clinical treatment, the toxicity of which can be regulated with hemodialysis.
The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.
The antibody-mediated cytotoxicity of complement can be increased by the ribosome-inactivating proteins gelonin and PAP-S. Treatment of human lymphoid cells that express CALLA with an anti-CALLA monoclonal antibody, J5, and then with rabbit complement, leaves about 6% of the cells alive. The same treatment in the presence of a sublethal concentration of gelonin or PAP-S leaves only about 0.02 and 0.3% of the cells alive respectively. This synergistic effect has potential implications for the in vitro elimination of malignant cells or of immunocompetent cells from bone marrow before its transplantation.
Epstein Barr virus (EBV) infection of human B lymphocytes is initiated by selective binding of the virus to the C3d receptor (EBV/C3d receptor) on the cell surface and results in polyclonal proliferation of infected cells. In these studies we examined the fate of the EBV/C3d receptor during viral infection by using an immunotoxin made from a monoclonal antibody (HB5) reactive with the receptor and the potent toxin, gelonin. Binding of the HB5-gelonin conjugate to the EBV/C3d receptor before EBV infection (at concentrations as low as 10(-11) M) significantly inhibited the subsequent polyclonal proliferation of virus-infected B lymphocytes. HB5 antibody and gelonin alone did not inhibit proliferation. Because internalization of gelonin-antibody conjugates is required to cause cytotoxicity, these results indicate that infection of B lymphocytes with EBV selectively induced endocytosis of the EBV/C3d receptor with concomitant internalization of the immunotoxin. Proliferation of B lymphocytes that were activated by prior infection with EBV, or activated by cross-linking of their surface immunoglobulin molecules, was not inhibited by the antibody-toxin conjugate even at concentrations as high as 10(-7) M. Also, the growth of B lymphoblastoid cell lines cultured in the presence or absence of infectious EBV was not inhibited by HB5-gelonin. Thus, our results suggest that the EBV/C3d receptor is internalized only during the infection of normal B lymphocytes by EBV, with co-internalization of immunotoxin, and indicate that internalization of the EBV/C3d receptor-immunotoxin complex does not occur simply as a consequence of activation and proliferation of B lymphocytes. The use of a ligand to induce endocytosis of its receptor offers a new strategy for the selective delivery of immunotoxins to cells and may be more generally applicable.