Characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the Clostridium botulinum neurotoxin type A.

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10(3) and 10(4) times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D(5)) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K(5)). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

Induction of human B2 bradykinin receptor mRNA and membrane receptors by IFNgamma.

A potential mechanism for the increased sensitivity of inflamed tissues to bradykinin is the upregulation of bradykinin receptor expression. We report that recombinant human IFNgamma stimulated a concentration-dependent increase in cell surface bradykinin receptor expression in intact T24 human epithelial-like cells, determined by radioligand binding analysis. Analysis of specific [3H]-bradykinin binding revealed that IFNgamma-treated cells had a two- to threefold increase in bradykinin receptor number compared to the controls with no effect on receptor affinity. The ability of IFNgamma to stimulate increased bradykinin receptor expression was abrogated by treatment with either the transcription inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. IFNgamma enhanced steady-state human B2 bradykinin receptor mRNA expression in the T24 cells in a dose-dependent manner. B2 bradykinin receptor mRNA expression was increased as early as 1 h following IFNgamma stimulation, and continued to accumulate for 24 h. Bradykinin-stimulated intracellular calcium mobilization was also increased in IFNgamma-treated T24 cells compared to controls. The ability of IFNgamma to upregulate B2 bradykinin receptors in primary epithelial cells was demonstrated using cultured human airway epithelial cells. These observations suggest that increasing IFNgamma levels during inflammation may upregulate the expression of B2 bradykinin receptors, leading to increased sensitivity to bradykinin.

Molecular cloning of a novel 97-kd Golgi complex autoantigen associated with Sjögren's syndrome.

OBJECTIVE: To identify a Golgi complex autoantigen bound by Sjögren's syndrome (SS) autoantibodies. METHODS: Serum from a patient with secondary SS and anti-Golgi antibodies was used as a probe to isolate a complementary DNA (cDNA) insert from a HeLa cDNA library. RESULTS: A 3.7-kb cDNA encoding a 56-kd recombinant protein was immunoprecipitated by the human anti-Golgi serum and immune rabbit serum. Western blot analysis showed that the immune rabbit sera recognized a protein of 97 kd (golgin-97), suggesting that the isolated clone contained a partial cDNA. The 5' upstream sequence was obtained by rapid amplification of the cDNA ends. The complete cDNA contained 4,860 basepairs, encoding a protein with a calculated Mr of 88 kd. Antibodies to golgin-97 were found in 12 (20%) of 60 sera known to have anti-Golgi autoantibodies, and the majority of these sera (8 of 12, or 75%) were from patients who had secondary SS. CONCLUSION: Golgin-97 is a unique Golgi complex antigen that appears to be a target of SS autoantibodies.

Analysis of an exon 1 polymorphism of the B2 bradykinin receptor gene and its transcript in normal subjects and patients with C1 inhibitor deficiency.

The B2 bradykinin receptor (B2BKR) mediates most of the inflammatory actions of bradykinin. To evaluate its potential role in allergic diseases, we assessed the structure of the human B2BKR gene. Screening a human placenta genomic DNA library identified only clones containing exons 2 and 3. Human placenta and colon tissues were used for 5' rapid amplification of complementary DNA ends to identify nine exon 1 clones, each containing one 9 bp and two 1 bp deletions compared with published sequences. Exon 1 genomic polymerase chain reaction of human leukocyte DNA revealed two distinct products, which were shown to differ by the presence or absence of the 9 bp deletion. Alleles with the 9 bp deletion were designated as (-)21-29, whereas alleles without the deletion were designated as (+)21-29. Genomic polymerase chain reaction in 39 Caucasian, 31 African-American, and 32 Asian normal subjects revealed a highly significant difference in the allelic frequency of the two genotypes, primarily because of an absence of the (+)21-29 allele in Asian subjects. Analysis of steady-state B2BKR messenger RNA levels by reverse-transcription polymerase chain reaction in heterozygous normal subjects revealed consistently higher expression of (-)21-29 transcripts. To investigate the potential clinical significance of the exon 1 polymorphism, 21 patients with angioedema and C1 inhibitor deficiency were genotyped. None were homozygous for the (+)21-29 allele (p = 0.0088 compared with normal subjects). In contrast, two patients with immunochemical evidence of hereditary angioedema without history of clinical angioedema were (+)21-29 homozygous. These results suggest that the B2BKR genotype may influence clinical status in diseases characterized by involvement of bradykinin.

Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human fibroblasts.

Bradykinin (BK), a pluripotent nonameric peptide, is known for its proinflammatory functions in both tissue injury and allergic inflammation of the airway mucosa and submucosa. To understand the mechanisms by which BK serves as an inflammatory mediator, the human lung fibroblast cell line WI-38 was stimulated with BK and the expression of IL-1beta gene was examined. BK at nanomolar concentrations induced a marked increase in immunoreactive IL-1beta, detectable within 2 h in both secreted and cell-associated forms. BK-induced IL-1beta synthesis was inhibited by a B2-type BK receptor antagonist and by treatment of the cells with pertussis toxin, indicating the involvement of a BK receptor that couples to the G(i)/G(o) class of heterotrimeric G proteins. Whereas cycloheximide and actinomycin D both inhibited BK-induced IL-1beta synthesis, results from Northern blot and nuclear run-on assays suggested that BK acted primarily at the transcription level which led to the accumulation of IL-1beta message in stimulated cells. Gel mobility shift assays were used with nuclear extracts from stimulated WI-38 cells to examine the transcription mechanism for BK-induced IL-1beta expression. A DNA binding activity specific for the decameric kappaB enhancer was detected and was found to contain the p50 and p65 subunits of the NF-kappaB/rel protein family. BK-induced NF-kappaB activation correlated with IL-1beta message upregulation with respect to agonist concentration, time course, sensitivity to bacterial toxins, and blockade by the B2 receptor antagonist. After BK stimulation, a significant increase in the activity of chloramphenicol acetyltransferase was observed in WI-38 cells transfected with a reporter plasmid bearing the kappaB enhancers from the IL-1beta gene. Deletion of the kappaB enhancer sequence significantly reduced BK-induced chloramphenicol acetyltransferase activity. These findings suggests a novel function of BK in the activation of NF-kappaB and the induction of cytokine gene expression.

Antibodies against malondialdehyde (MDA) in MRL/lpr/lpr mice: evidence for an autoimmune mechanism involving lipid peroxidation.

Previous studies have shown that lipid peroxidative processes may play a role in disease pathogenesis in lupus-prone MRL/lpr mice. Studies were thus performed to determine if an immune response against malondialdehyde (MDA), a highly reactive byproduct of lipid peroxidation, was present in these mice. By using MDA-modified mouse serum albumin (MSA) as antigens in ELISA, we found that these mice produce high levels of MDA-specific antibodies in the complement-fixing IgG2a and IgG2b subclasses. Anti-MDA antibodies were also found in MRL/+ mice but in significantly lower levels. The specificity of these antibodies was verified by inhibition ELISA. MDA may contribute to disease pathogenesis in these mice by altering the immunogenicity of self molecules, eliciting an immune response and forming immune complexes that may deposit in tissues.

Glycosylation enhances malondialdehyde binding to proteins.

To determine whether glycosylation of proteins increases their susceptibility to modification with malondialdehyde (MDA), bovine serum albumin, which was pretreated with 500 mg/dl dextrose at 37 degrees C for 0, 1, 2, and 4 weeks, were incubated with 100 mM MDA at 37 degrees C for 24 h. The MDA content of the protein samples were determined after dialysis using thiobarbituric acid (TBA) assay. In addition, a specific anti-MDA protein antiserum was used to demonstrate MDA proteins with immunoblotting technique. The MDA content of BSA preincubated with dextrose for 4 weeks and reincubated with MDA (0.0649 +/- 0.0019 microgram MDA/mg protein) was significantly higher (p < .001) than the MDA content of BSA preincubated with dextrose for only one (0.0227 +/- 0.0031 microgram/mg) or two (0.0347 +/- 0.0034 microgram/mg) weeks or the MDA content of nonglysolated BSA incubated with MDA at the same experimental conditions (0.0201 +/- 0.0029 microgram/mg). These differences could also be found in the immunoblots. However, the correlation of TBA assay with the estimates on immunoblots was poor. It is likely that the immunoblotting assay is more of an estimate of the number of BSA molecules modified with MDA, rather than MDA content of each BSA molecule. It is concluded that in vitro glycosylation of proteins increases their susceptibility to MDA-modification. This may well be an additional pathway of diabetes-related modification of proteins.

Molecular characterization of Golgin-245, a novel Golgi complex protein containing a granin signature.

The serum from a Sjögren's syndrome patient with anti-Golgi antibodies was used as a probe to isolate a 4.6-kilobase pair cDNA insert from a HeLa cDNA library. Expression of the cDNA in Escherichia coli and the in vitro translation products of the cDNA yielded a recombinant protein that migrated in SDS-polyacrylamide gel electrophoresis at 180 kDa. This protein was immuno-precipitated by the human anti-Golgi serum and by immune rabbit serum but not by normal human serum or preimmune rabbit serum. Western blot analysis showed that the prototype human and immune rabbit sera recognized a 245-kDa protein, suggesting that the isolated clone contained a partial cDNA. The 5'-upstream sequence obtained by the rapid amplification of cDNA ends methodology using human placental cDNA and the combined HeLa cDNA contained 6965 base pairs and combined HeLa cDNA contained 6965 base pairs and encoded a protein of 245 kDa and, like other Golgi autoantigens described earlier, is highly rich in coiled-coils. The deduced amino acid sequence included the decapeptide ESLALEELEL, which was identified as one of two signature sequences previously reported in a family of peptide hormones and neuropeptides known as "granins". This is the first report of a Golgi complex autoantigen that bears structural similarities to the granin family of proteins.

Cholesterol enriched diet enhances malondialdehyde modification of proteins in cerebral microvessels of rabbits.

To determine the role of dietary cholesterol on malondialdehyde (MDA) modification of proteins, the cerebral microvessels of rabbits fed a cholesterol enriched diet for 8 weeks were compared to control rabbits. The MDA proteins were estimated with immunoblotting using a specific polyclonal antiserum against MDA proteins. Cholesterol-fed rabbits had a significantly increased MDA protein band at 175 kDa compared to control rabbits (65.6 +/- 6.8 OD versus 16.5 +/- 2.5 OD, P < 0.01). An increase in MDA protein was also found in rabbits intravenously injected with 2 mg of MDA-modified low density lipoproteins or MDA-modified rabbit serum albumin (RSA) at 0, 2, and 4 weeks of observation. The MDA proteins were not increased in cerebral tissue of cholesterol-fed rabbits. It is concluded that a high cholesterol diet is associated with increased MDA modification of proteins in cerebral microvessels. The mechanism could be related to increased circulatory MDA proteins since exogenously administered MDA-LDL or MDA-RSA resulted in similar increases in MDA proteins in cerebral microvessels.

Diabetes-related changes in the protein composition of rat cerebral microvessels.

To determine the effect of diabetes mellitus on cerebral microvessel protein composition, post translational modification of proteins with glucose and malondialdehyde (MDA) was determined and the abundant protein species found in cerebral microvessels isolated from control and streptozotocin-induced diabetic rats were studied. Two dimensional gel electrophoresis and computer assisted densitometry revealed that only one out of 25 quantitated proteins was significantly altered in diabetic rats after 5 weeks of uncontrolled hyperglycemia. The level of glycosylation of cerebral microvessel protein mixture was significantly increased in diabetic rats compared to control rats (168.8 +/- 25 vs 109.5 +/- 4.8 nmol/mg) (p < 0.05). Western blot analysis of cerebral microvessel proteins from diabetic rats using a specific antibody against MDA-modified proteins revealed three protein spots with molecular weights of approximately 60,000 Kd. These were shown not to be contaminants from cerebral tissue or plasma proteins modified with MDA. It is concluded that short duration of streptozotocin-induced diabetes mellitus in rats is associated with some qualitative changes in protein composition of cerebral microvessels. These changes may contribute to the diabetes-related alterations in the blood-brain barrier.

Tissue-specific distribution of malondialdehyde modified proteins in diabetes mellitus.

A potential mechanism of diabetes-related tissue damage is modification of various proteins by lipid peroxidation by-products such as malondialdehyde (MDA). To determine the extent of MDA derivatization of various proteins in diabetes mellitus, Western blots were carried out using a specific anti-MDA antiserum to study proteins in plasma and various tissues of control and streptozotocin (STZ)-induced diabetic rats. The concentration of MDA-proteins was highest in plasma compared to other tissues tested. Diabetes was associated with a reduction in MDA-protein content of plasma, lung and liver while in the heart, testicle, cerebrum and kidney the MDA-protein concentration was not altered. Insulin treatment of diabetic rats normalized MDA-protein content of plasma but not in the lung or liver. A large interindividual variability in various protein species was observed within a group. This was partly attributed to polymerization of MDA-proteins. It is concluded that although diabetes is associated with increased lipid peroxidation the content of MDA-proteins in plasma and in some tissues is decreased.

Age-related changes in tissue content of malondialdehyde-modified proteins.

One of the possible mechanisms of the age-related modifications of proteins is the result of reaction with malondialdehyde (MDA), a lipid peroxidation byproduct. To determine the effect of age on the extent of MDA derivatization of proteins in plasma and various tissues, male Fisher 344 rats at 4, 12 and 26 months of age were studied. Protein electrophoresis and immunoblotting was carried out using a specific antiserum against MDA-protein complexes. The concentration of MDA-proteins in plasma (mean +/- SD of optical density in 50 micrograms protein) was 201.6 +/- 47.7 in 4 month old rats, 197.4 +/- 67 in 12 month old rats and 101.4 +/- 22.7 in 26 month old rats (P < 0.01). The MDA protein content of testicle, liver and heart was increased in 12 month old rats compared to 4 and 26 months old rats. There were no age-related differences in MDA-protein content of lung, brain, and kidney. Because of the interindividual variability of MDA-protein profiles within an age group distinct age-related changes in the distribution of various MDA protein bands could not be documented.

Malondialdehyde modified proteins and their antibodies in the plasma of control and streptozotocin induced diabetic rats.

One of the possible mechanisms of diabetes-related tissue damage is modification of various proteins via lipid peroxidation byproducts such as malondialdehyde (MDA). To determine the extent of MDA derivatization of plasma proteins, Western blots were carried out using anti-MDA antisera to study plasma proteins in control and streptozotocin (STZ)-induced diabetic rats. Since MDA can modify proteins and may alter or enhance their antigenicity, we screened plasma samples for anti-MDA antibodies using enzyme-linked immunosorbent assay (ELISA), and confirmed antibody specificity by inhibition ELISA. Circulating immune complexes containing MDA were also assayed. This study is the first demonstration of the existence in plasma of MDA-modified proteins with a molecular weight of approximately 100 Kd. Both control and diabetic rats have similar concentrations of plasma anti-MDA antibodies and circulating immune complexes. These results do not support the notion that diabetes alters the immune response to MDA modified proteins. Whether MDA modification of proteins participate in immunological processes that lead to tissue injury remains to be demonstrated.

Immunochemical properties of malondialdehyde-protein adducts.

Malondialdehyde (MDA), a product of lipid peroxidation, can bind to and modify proteins by adduct formation. To determine whether MDA adducts were immunogenic, MDA was added to rabbit serum albumin (RSA) in order to characterize MDA-proteins and to immunize rabbits. Bound MDA was proportional to the concentration of MDA added in the range of 2.5-20 mM as measured by thiobarbituric acid reactivity. MDA adducts of RSA migrated further toward the anode than native serum protein in zone and immunoelectrophoresis indicating increased negative charge. Rabbits immunized with MDA-RSA produced high titers of IgG antibodies to MDA-RSA as measured by enzyme-linked immunosorbent assay (ELISA). Hapten specificity of the antibody was demonstrated by antisera reactivity with MDA-RSA but not with unaltered RSA. Our findings support the possibility that MDA may serve as a hapten to form neoantigens which may represent a pathway by which lipid peroxidation could produce tissue damage via an immunologic mechanism.

Acetaldehyde-albumin adduct formation: possible relevance to an immunologic mechanism in alcoholism.

Acetaldehyde (A), an ethanol metabolite, was incubated with rabbit serum albumin (RSA) and human serum albumin (HSA) to form the corresponding soluble haptenized proteins, A-RSA and A-HSA respectively. Both protein adducts showed conjugation with acetaldehyde evidenced by more rapid anodal migration compared to RSA and HSA. A-RSA immunized rabbits produced titers greater than 1:100,000 by enzyme-linked immunosorbent assay (ELISA). Rabbit serum antibodies to A-RSA diluted 1:10,000 showed high specificity for the hapten when reacted with acetaldehyde conjugates of RSA, HSA, bovine serum albumin, bovine gamma globulin and human gamma globulin. Our findings support the possibility that acetaldehyde may serve as a hapten to form neoantigens relevant to an immunologic mechanism which might be associated with alcoholic liver disease.