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PubMed2003

Molecular cloning of the beta subunit of complement component eight of rainbow trout.

Complement-mediated killing of pathogens through the lytic pathway is an important effector mechanism of the innate immune response. C8 is one of the components of the lytic pathway and is composed of an alpha, beta, and gamma subunit. In the present study we report the cloning and characterization of the primary structure of the C8beta subunit in the rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout C8beta shows 72 and 47% identity with that of Japanese flounder and human, respectively. It also contains many of the same structural motifs as those found in mammalian lytic components. The C8beta gene appears to exists as a single copy in the trout genome and is expressed primarily in the liver. The protein encoded by the gene was identified by Western blotting using an anti-peptide antibody and was approximately 65kDa.

Amino Acid Sequence↗
PubMed2001

Contrast in the efficacy of hDAF mouse hearts between ex vivo perfusion and transplantation into primates.

In recent experiments, in which we compared hDAF transgenic rat hearts perfused with 15% human serum in the Langendorff device and hDAF rat hearts transplanted into cynomolgus monkeys, we demonstrated that in the ex vivo heart perfusion model both homozygous and heterozygous hDAF hearts survived longer as nontransgenic controls. Surprisingly, we found that only homozygous hDAF hearts were protected against hyperacute rejection in vivo. The first aim of this study was to determine whether perfusion of mouse hearts with higher human serum concentrations or human blood might explain some of the differences found in survival time of the recently performed experiments with rat heart xenografts. Secondly, we investigated whether the observed differences in survival times of rat xenografts between in vivo and ex vivo transplantation would also hold for mouse hearts transgenic for hDAF. An ex vivo model was used to perfuse hDAF mouse hearts and controls with human serum or blood, and hDAF transgenic hearts and controls were transplanted into cynomolgus monkeys. hDAF transgenic mouse hearts survived significantly longer than their controls when perfused with 15% human serum, but no difference was found when 30% human serum was used, or when these hearts were transplanted into cynomolgus monkeys. However, in both the in vivo and ex vivo models the amount of PMNs adhering to the vascular endothelium was significantly lower in hDAF transgenes as compared with their controls. In conclusion, in the ex vivo situation, the efficacy of hDAF transgenesis in preventing HAR is limited by serum complement concentration.

Animals↗
PubMedDate not supplied

Assembly of two types of tubules with putative cytolytic function by cloned natural killer cells.

The formation of ultrastructural membrane lesions of varying size during cell mediated cytolysis effected by human peripheral blood leukocytes was recently reported by Dourmashkin et al. and Henkart et al. Using cloned mouse natural killer (NK) cells as effectors and YAC-1 cells or rabbit erythrocytes as targets, we now report two types of membrane lesions with inner diameters of 16 +/- 2 nm and approximately 5 nm, respectively. These lesions arise by membrane insertion of tubular complexes that may be assembled from subunits during the cytolytic reaction. The tubules are detected on target membranes by immune electron microscopy and appear to form transmembrane channels as seen in ultrathin sections. Both tubules are partially purified by membrane extraction with SDS and gel filtration in deoxycholate containing buffer. Based on the correlation of tubule assembly and cytolysis and on their detection on target membranes, we suggest that both types of tubules may be related to cytolysis.

Cell Membrane↗
PubMed1983

A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer.

A chemiluminescent derivative of cyclic AMP, aminobutylethylisoluminol succinyl cyclic AMP (ABEI-scAMP), was synthesized in order to develop a homogeneous immunoassay based on non-radiative energy transfer. ABEI-scAMP was chemiluminescent (5.1 X 10(18) luminescent counts X mol-1 at pH 13), pure (greater than 95%) stable and immunologically active. A conventional immunoassay was established using ABEI-scAMP and a solid-phase anti-(cyclic AMP) immunoglobulin G which could detect cyclic AMP at least down to 25fmol. A homogeneous immunoassay for cyclic AMP was established by measuring the shift in wavelength from 460 to 525nm which occurred when ABEI-scAMP was bound to fluorescein-labelled anti-(cyclic AMP) immunoglobulin G. The assay was at least as sensitive as the conventional radioimmunoassay using cyclic [3H]AMP and could measure cyclic AMP over the range 1-1000nM. The homogeneous chemiluminescent energy transfer assay was also able to quantify the association and dissociation of antibody-antigen complexes. Chemiluminescence energy transfer occurred between fluorescein-labelled antibodies and several other ABEI-labelled antigens (Mr values 314-150000) including progesterone, cyclic GMP, complement component C9 and immunoglobulin G. The results provide a homogeneous immunoassay capable of measuring free cyclic AMP under conditions likely to exist inside cells.

Animals↗
PubMed2001

Rhabdomyolysis, acute renal failure, and death after monensin ingestion.

We report a case of human monensin intoxication; to our knowledge, this is the first reported case in the medical literature. The patient took a dose of monensin three times higher than a dose considered lethal for cattle and developed a clinical picture similar to that reported in veterinary medicine. There was an early and extremely severe rhabdomyolysis followed by acute renal failure, heart failure, and death. The main changes observed at autopsy were extensive skeletal muscle necrosis, complement deposition at the myocardial level, pulmonary edema, and acute tubular damage.

Acute Kidney Injury↗
PubMed1977

Low-titer cold-hemagglutinin disease. Mechanism of hemolysis and response to corticosteroids.

We studied two patients with a low-titer cold-hemagglutinin disease syndrome to investigate the mechanism of hemolysis and the therapeutic response to corticosteroids. The antierythrocyte antibody was of the IgM class, had a high thermal amplitude and had enough activity of 37 degrees C to account for the hemolysis. The capacity of peripheral blood monocytes to increase the osmotic fragility of C3-coated erythrocytes suggests that macrophage interaction with C3-coated erythrocytes explains the observed in vivo spherocytosis. Both patients responded to high-dose corticosteroids. The data suggest that the steroid effect is probably due to alteration of macrophage complement-receptor function. These studies demonstrate the importance of antibody activity at body temperature in producing hemolysis, particularly in this variant of cold-hemagglutinin disease. The response to steroids suggests the efficacy of corticosteroid therapy in alleviating hemolysis due to macrophage recognition of erythrocytes coated with IgM and C3.

Adrenal Cortex Hormones↗
PubMed1986

Extracellular release of lymphocyte cytolytic pore-forming protein (perforin) after ionophore stimulation.

The cytolytic pore-forming protein (PFP, perforin) of lymphocyte granules has recently been isolated and characterized. The lytic activities expressed by both the isolated granules and the purified PFP require the presence of Ca2+. Here, we report on the extracellular release of PFP after stimulation of lymphocytes with the Ca2+ ionophore A23187, which degranulates the cells. The secreted protein associates with lipid to form structural and functional channels and supramolecular complexes that partially resist dissociation by sodium dodecyl sulfate and reducing agents. Immunoblots of the released material reveal positive identification with antibodies specific for mouse PFP and human complement component C9, indicating cross-reactivity between these two molecules. By using these specific antibodies as immunoadsorbents, the lymphocyte PFP has been affinity purified from the supernatant of stimulated cells. The extracellular release of PFP is associated with simultaneous formation of functional ion-nonselective channels with conductances of 550-600 pS in 0.15 M NaCl, as measured in planar model bilayers. In the absence of extracellular Ca2+, 15% of the maximal release activity is observed. Ca2+ appears to be required to elicit both secretion by lymphocytes and the assembly of the released PFP into tubular polymers. Similar secretion of PFP may occur during cell killing by lymphocytes, resulting in its assembly on target membranes to form tubular transmembrane lesions.

Animals↗
PubMed1988

Identification and sequencing of cDNA clones encoding the granule-associated serine proteases granzymes D, E, and F of cytolytic T lymphocytes.

Cytoplasmic granules of cytolytic T lymphocytes contain at least six related serine esterases (granzymes) that are released together with perforin, a pore-forming protein related to complement component C9, during target-cell lysis. Polyclonal antibodies were used to isolate a large number of cDNA clones from an expression library derived from cytolytic-T-cell mRNA. Three distinct full-length cDNA clones coding for granzymes D, E, and F were identified by restriction site mapping and nucleotide sequencing. The three deduced amino acid sequences are highly similar to one another (between 72% and 90% amino acid identities) and to the sequences of granzymes B and C, cathepsin G, and rat mast-cell proteases I and II (between 43% and 57% amino acid identities). Cysteine residues capable of forming intramolecular disulfide bonds are conserved, as are the catalytic-site residues characteristic of serine proteases. Comparison of the cDNA-derived protein sequences with the amino termini of the isolated granzymes provides evidence that they are stored in a fully processed, activated form after removal of the signal peptide and two additional residues (propeptide) at the amino terminus. Immunoelectron microscopic studies demonstrated that granzymes D, E, and F are present in the same morphologically distinct cytoplasmic granules in which perforin has been found previously.

Amino Acid Sequence↗
PubMed1989

Lipoprotein binding and endosomal itinerary of the low density lipoprotein receptor-related protein in rat liver.

The high affinity of 45Ca binding to the low density lipoprotein receptor (LDL-R) and the LDL-R-related protein (LRP) was utilized to study the subcellular distribution of these two proteins in rat liver. Like the LDL-R, LRP was manyfold enriched in rat liver endosomal membranes with a relative distribution in early and late endosomal compartments consistent with recycling between endosomes and the cell surface. The high concentration of LRP in hepatic endosomal membranes greatly facilitated demonstration of Ca-dependent binding of apolipoprotein E- and B-containing lipoproteins in ligand blots. LRP was severalfold more abundant than the LDL-R in hepatic parenchymal cells, showed extensive degradation in hepatic endosomes, and was found in high concentrations in the Golgi apparatus and endoplasmic reticulum. These data suggest a high rate of synthesis of LRP that appeared to be unaffected by treatment of rats with estradiol. The repeating cysteine-rich A-motif found in the ligand-binding domain of LRP appeared to be responsible for Ca binding by LRP, LDL-R, and complement factor C9 and accounted for immunological cross-reactivity among these proteins. Weaker ligand-blotting properties and an extraordinary susceptibility to proteolysis most likely contribute to the difficulty of detecting LRP in conventional assays for lipoprotein receptors. Our data suggest an extensive proteolytic processing of this protein and are consistent with a functional role of LRP in lipoprotein metabolism.

Animals↗
PubMed1979

C5b-9 dimer: isolation from complement lysed cells and ultrastructural identification with complement-dependent membrane lesions.

The membrane attack complex (MAC) of complement was extracted from the membranes of cells lysed by human complement and its properties were compared with those of the fluid phase complex SC5b-9. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical analysis, the two isolated complexes had identical subunit compositions, except that the MAC lacked the S-protein. The sedimentation coefficient and molecular weight of the extracted and isolated MAC were, respectively, 33.5 S and 1.7 x 10(6) daltons, compared to 23 S and 1.0 x 10(6) dalton for SC5b-9. Because the molecular weight of the MAC is approximately two times greater than that of C5b-0 (800,000 daltons), the MAC is considered the dimer of C5b-9. Under specified conditions, the 33.5 S dimer could be converted to the 23 S monomer without dissociation of subunits. The MAC had the electron microscopic appearance and dimensions that are characteristic for the complement produced ultrastructural membrane lesions. SC5b-9 had a different ultrastructure that is dissimilar to the morphology of the lesions. The isolated MAC could be reincorporated into phospholipid bilayers and assumed on the surface of the resultant lipid vesicles the orientation and appearance of typical complement lesions.

Animals↗
PubMed2004

Paradoxical alteration of acute-phase protein levels in patients with chronic hepatitis C treated with IFN-alpha2b.

Previously we observed elevation of the serum concentration of two acute-phase protein (AFP) complement components (C9 and C1-inhibitor) in patients with chronic hepatitis C who responded (R) to IFN-alpha therapy, but not in non-responders (NR). In the present study we investigated the effect of high-dose IFN-alpha therapy on serum concentrations of two positive [orosomucoid (OROSO) and C-reactive protein (CRP)] and two negative [transferrin (TF) and fetuin/alpha2HS-glycoprotein (AHSG)] AFP in an outpatient setting. We investigated blood samples of 40 patients with chronic hepatitis C at the onset and at the end of a 3-month treatment with high-dose IFN-alpha2b (5 MIU/day for 6 weeks, followed by 5 MIU t.i.w.) and of 52 healthy individuals. Serum concentrations of OROSO, TF and AHSG were measured by radial immunodiffusion; CRP levels were determined by immunotubridimetry. Compared to controls, patients with chronic hepatitis C had significantly lower OROSO and CRP, and higher AHSG levels. By the end of treatment, OROSO concentration increased in R (P = 0.0054), but not in NR patients. In contrast, TF levels decreased in R (P = 0.0040), but did not change in NR patients. Similarly, in R patients, AHSG levels tended to decrease (P = 0.0942) following IFN-alpha treatment. We conclude that the acute-phase reaction is suppressed in patients with chronic hepatitis C that may be potentially related to the responsiveness to IFN-alpha therapy.

Acute-Phase Proteins↗
PubMed1998

High-level endothelial expression of human CD59 prolongs heart function in an ex vivo model of xenograft rejection.

BACKGROUND: Hyperacute rejection of discordant xenografts is dependent on activation of the complement system of the recipient. Transgenic expression of recipient complement regulatory factors in donor tissue has proved to be a promising approach to dealing with hyperacute rejection, although the relationship between the level of complement regulatory factor expression and the degree of protection is not well established. Here, we examine this relationship using CD59 transgenic mouse hearts in an ex vivo model of xenograft rejection. METHODS: The level of expression of CD59 in two lines of transgenic mice, in which CD59 is expressed under the control of either the murine H2Kb (MHC class I) promoter (line CA-17) or the endothelium-specific human intercellular adhesion molecule-2 promoter (line 237-7), was compared by immunohistochemistry and flow cytometry. Hearts from both groups and wild-type controls were perfused ex vivo with human plasma, and mean heart work for each group was compared over a 60-min period. RESULTS: CD59 expression on cardiac endothelial cells isolated from homozygous CA-17 mice was 25- to 30-fold lower than that on cardiac endothelial cells from heterozygous 237-7 mice. CA-17 hearts perfused with 6% human plasma exhibited a reduction in deposition of the membrane attack complex, but not a prolongation of function, compared with nontransgenic mouse hearts. In contrast, 237-7 hearts showed significantly prolonged function during perfusion with 20% plasma. CONCLUSIONS: High-level endothelial-specific expression of CD59 was effective in prolonging the function of mouse hearts perfused with 20% human plasma, whereas low-level, broader expression did not provide protection from 6% plasma.

Animals↗
PubMed2006

Differential macrophage/microglia activation in neocortical EAE lesions in the marmoset monkey.

Recent studies revealed an important involvement of the cerebral cortex in multiple sclerosis (MS) patients. Cortical lesions in MS were reported to be less inflammatory and to show less structural damage than white matter lesions. Animal models reflecting the histopathological hallmarks of cortical demyelinated lesions in MS are sparse. Induction of experimental autoimmune encephalomyelitis (EAE) in the common marmoset has turned out to be an attractive non-human-primate model for MS. In the present study we investigated the presence and detailed cellular composition of cortical inflammatory demyelinating pathology in the common marmoset upon immunization with myelin oligodendrocyte glycoprotein (MOG). Extensive cortical demyelination reflecting the topographically distinct cortical lesion types in MS patients was revealed by immunohistochemistry for myelin basic protein (MBP). We explored the density of T- and B-lymphocytes, MHC-II expressing macrophages/microglia cells and early activated macrophages (MRP14) at perivascular and parenchymal lesions sites in neocortex and subcortical white matter. Despite a similar density of perivascular inflammatory infiltrates in the demyelinated neocortex, a considerable lower fraction of macrophages was found to express MRP14 in the neocortex indicating a different activation pattern in cortical compared with white matter lesions. Furthermore, cortical EAE lesions in marmoset monkeys revealed immunoglobulin leakage and complement component C9 deposition in intracortical but not subpial demyelination. Our findings indicate that the inflammatory response, especially macrophage and microglia activation, may be regulated differently in gray matter areas in primate brain.

Animals↗
PubMed1981

Circulating immune complexes, serum immunoglobulins, and acute phase proteins in psoriasis and psoriatic arthritis.

Raised levels of circulating immune complexes were found in the plasma of 47% of patients with psoriasis and in 58% of those with psoriatic arthritis. The mean levels were significantly raised when compared with normals, but there was no difference between the 2 patient groups. The levels of acute phase proteins (C-reactive protein, fibrinogen, alpha-1-antitrypsin, and the 9th component of complement) were normal in those patients with psoriasis but were significantly raised in patients with psoriatic arthritis. Serum immunoglobulin G and A levels were equally raised in both patient groups, immunoglobulin M being normal. C-reactive protein and fibrinogen gave the best correlation with the clinical index of disease activity.

Antigen-Antibody Complex↗
PubMed1991

Atypical structured glomerular deposits: an immunohistochemical study.

Atypical structured glomerular deposits were identified in sub-epithelial, sub-endothelial and mesangial areas in biopsy tissue from a female, aged 33, who presented with anaemia and was found to have proteinuria and microscopic haematuria. Histological examination showed that the deposits were periodic acid Schiff positive and silver negative whilst stains for amyloid were negative. Immunofluorescent staining for all immunoglobulins was negative, and only C3 showed moderate labelling. Conventional electron microscopy revealed that all deposits contained microtubular structures of variable length but with an average diameter of 25 nm and a periodicity of approximately 16 nm. The glomerular basement membrane was interrupted in many areas by deposits, and also contained 'myelin-like' structures. Free microtubular structures were also seen in the urinary space. Immunoelectron microscopy using protein-A-gold confirmed the immunofluorescent findings with immunoglobulins and fibrinogen, showed marked positive labelling of deposits with C1s and C3d and also intense labelling of coiled microtubular structures with C9. Other complement components C1q, C1r, C3c, C4 and C5 showed weak or negative results. Although these organised glomerular deposits contain complement components, their pathogenesis remains uncertain.

Adult↗