Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.     

Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera

The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis. Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum. Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes. The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels. However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit.     

Demonstration of organ specific antibodies against heart mitochondria (anti-M7) in sera from patients with some forms of heart diseases

Using submitochondrial particles (SMP) from beef heart, pig kidney and rat liver in the ELISA, we detected partial organ specific anti-mitochondrial antibodies (AMA) against heart and kidney SMP in sera from patients with different forms of cardiomyopathies. Serum samples from 50 of 159 patients with congestive or hypertrophic cardiomyopathy (31%) and from two of 15 patients with acute myocarditis (13%) were AMA positive. These AMA could be clearly differentiated from other known AMA (anti-M1-M6) and were therefore named anti-M7. Thirteen of the 52 sera (25%) reacted only with heart SMP (type a) and 39 showed a cross-reaction with kidney, lung and pancreas mitochondria (type b). However, using liver SMP, no positive reaction was found. The anti-M7 type a and b activity was abolished completely by absorption with heart SMP. The anti-M7 antibodies were directed against an antigen which co-purified with the inner mitochondrial membrane and had a molecular weight of 67,000-72,000. They seem to be confined to some forms of cardiomyopathies and myocarditis of unknown aetiology and were not detected in sera from patients with other diseases.     

Anti-mitochondrial antibodies (anti-M7) in heart diseases recognize epitopes on bacterial and mammalian sarcosine dehydrogenase.

The anti-mitochondrial antibody (AMA) anti-M7 has been shown to occur exclusively in sera from patients with acute and chronic myocarditis. Applying different enzymes of the inner mitochondrial membrane to ELISA, anti-M7-positive sera reacted only with sarcosine dehydrogenase (SD) from Pseudomonas aeruginosa. Testing these sera in the Western blot against a commercially available SD as well as against SD prepared from rat liver mitochondria, a determinant at 42 kD and 90 kD, respectively, was visualized. Using submitochondrial particles (SMP) from bovine heart and rat liver another major determinant at 64 kD could be observed with both antigen fractions. Liver SMP also expressed the SD-related, 90-kD epitope. Sera from patients with other AMA-positive and AMA-negative autoimmune diseases were negative with these different determinants. The identity of the 64-kD epitope on heart and liver SMP as well as the 42-kD polypeptide of bacterial SD and the 90-kD epitope on mammalian SD was proven by absorption studies and by elution of antibodies from the antigen bound to the immobilon sheets after immunoblotting. The SD enzyme activity was not affected by anti-64-kD and anti-42-kD antibodies in vitro. It is concluded that anti-M7 antibodies may be stimulated by an antigen expressed on cardiocytes during an infection which shares epitopes with SD, an evolutionary highly conserved protein. SD-sensitized B cell clones could therefore be triggered by the M7-antigen which shows homology to SD.     

Anti-M4 antibodies in primary biliary cirrhosis react with sulphite oxidase, an enzyme of the mitochondrial inter-membrane space.

Testing three anti-M2/anti-M4-positive and three anti-M2 positive/anti-M4-negative primary biliary cirrhosis (PBC) marker sera against different mitochondrial enzymes by ELISA it could be shown that only the anti-M4-positive sera reacted with pyruvate dehydrogenase (M2) and sulphite oxidase (SO), an enzyme of the mitochondrial inter-membrane space in parallel. Absorption of these sera with SO abolished completely the anti-M4 antibodies but had no effect on the anti-M2 activity. The specificity of this reaction was also documented by examining 30 anti-M2/anti-M4-positive sera showing that 28 of them were positive with SO. Among ten anti-M2/anti-M8-positive but anti-M4-negative PBC sera, four became positive when tested against SO, indicating a higher sensitivity of SO for the demonstration of anti-M4. Retesting sera from 76 PBC patients with defined anti-mitochondrial antibody (AMA) profiles who had been followed for up to 18 years against SO by ELISA and complement fixation test (CFT), none of 32 patients with profile A B (positive for anti-M2 and/or anti-M9 by ELISA; benign course) but 33 of 44 patients with profile C/D (anti-M2/anti-M4 and or anti-M8 positive by CFT and or ELISA; progressive course) were positive. These data indicate that sulphite oxidase can be used in the ELISA for the detection of anti-M4 antibodies which may be of prognostic relevance.     

Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.     

Sera from patients with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis.

Anti-M2 antibodies in primary biliary cirrhosis (PBC) have been shown to react with the alpha-ketoacid dehydrogenase complex of the inner mitochondrial membrane consisting of six epitopes (E2 subunit of the pyruvate dehydrogenase complex (PDC), 70 kD; protein X of the PDC, 56 kD; alpha-ketoglutarate dehydrogenase complex, 52 kD; branched-chain alpha-ketoacid dehydrogenase, 52 kD; E1 alpha subunit of PDC, 45 kD; and E1 beta-subunit of PDC, 36 kD). These epitopes are also present in the M2 fraction which is a chloroform extract from beef heart mitochondria. The E2 subunit of the PDC at 70 kD (M2a), especially, is a major target epitope which is recognized by about 85% of all PBC sera. However, analysing sera from 28 patients with active pulmonary tuberculosis it became evident that 12 (43%) also recognized the PDC-E2 subunit (M2a), as shown by Western blotting using the M2 fraction, the purified PDC, and the recombinant PDC-E2. In contrast, only two of 82 patients with other bacterial and viral infections including 25 patients with Escherichia coli infections reacted with the PBC-specific epitope at 70 kD. Naturally occurring mitochondrial antibodies (NOMA) were present in 54% of the patients with tuberculosis and in 50% of patients with other infectious disorders. They recognized either a determinant at 65 kD (epsilon) or at 60/55 kD (zeta/eta). None of the sera from 100 blood donors had anti-M2 but 14 had NOMA. Testing anti-M2 and NOMA-positive marker sera by Western blotting against membrane fractions derived from mycobacteria and E. coli it could be shown that--like mammalian mitochondria--they contain both the PBC-specific M2 antigen as well as the non-PBC-specific naturally occurring mitochondrial antigen system (NOMAg). The observation that PBC-specific antibodies were preferentially induced in patients suffering from a mycobacterial infection may provide some new clues to the still unknown etiology of PBC.     

Characterization of a new mitochondrial antigen-antibody system (M9/anti-M9) in patients with anti-M2 positive and anti-M2 negative primary biliary cirrhosis.

A new antimitochondrial antibody (AMA) against an outer membrane associated antigen on liver mitochondria was detected by ELISA in sera from patients with primary biliary cirrhosis (PBC). This antibody was named anti-M9. There is evidence that it is a partial organ-specific antibody as shown by absorption studies using submitochondrial particles prepared from heart, liver and kidney. A purified M9-fraction was prepared by subjecting a 100,000 g supernatant from rat liver mitochondria to ion exchange chromatography. This fraction was devoid of the previously described M1-M8 antigens except for M4. Trypsin treatment of the fraction enabled a distinction to be made between M4 which was protease resistant, and M9 which was trypsin sensitive. Applying this M9-fraction in Western blotting anti-M9 positive sera recognized two proteins at a molecular weight of 98 kD and 59 kD. Anti-M9 antibodies were detected in 37% of 156 anti-M2 positive as well as in 82% of 22 anti-M2 negative patients with histologically proven PBC. It is concluded that anti-M9 is a new AMA type in PBC which may be helpful especially for the early diagnosis of PBC in patients who are still anti-M2 negative. As one of the earliest immunological signs in PBC further characterization of M9 could provide new insights into the etiopathogenesis of the disease.     

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.     

Distribution of the PBC-specific- (M2) and the naturally-occurring mitochondrial antigen- (NOMAg) systems in plants.

In previous studies it was demonstrated that antibodies in sera from patients with primary biliary cirrhosis (PBC) and their relatives can recognize two different antigen systems in the ATPase fraction prepared from beef heart mitochondria, namely the PBC-related M2- and the naturally occurring mitochondrial antigen (NOMAg)-related epitopes. Since separation of these two antigen systems could not be achieved using mammalian mitochondria, mitochondria from a wide spectrum of plants were analysed with respect to the presence of mitochondrial antigens. Mitochondria from 29 species of plants were prepared and tested by ELISA and Western blot using marker sera from patients with PBC reacting in the Western blot with M2a,b,c,d (alpha-ketoacid-dehydrogenase complex) and NOMAg-specific sera recognizing the three major epitopes epsilon, zeta, and eta at 65, 61 and 58 kD. Naturally occurring mitochondrial antibody (NOMA)-positive marker sera reacted in the ELISA with mitochondria from all plants, and the zeta/eta positive sera gave also a positive reaction at 61/58 kD in the Western blot while the epsilon epitope could not be visualized by this method. In contrast, the M2 antigen was detected preferentially in lower plants such as algae, fungi, and ferns. Analysing these data with respect to the evolution of proteins one would have to assume that the M2 antigen was lost in most higher plants or underwent some structural alterations. Furthermore, considering the fact that the M2- and the NOMAg-related epitopes could be only partially separated, i.e. there were no plant mitochondria showing only M2 but no NOMAg, one could speculate that anti-M2 antibodies are derived from the pool of naturally occurring antibodies.     

Mitochondrial antigens and autoantibodies: from anti-M1 to anti-M9

Summary The specificity and clinical relevance of nine antimitochondrial antibodies (AMA) — anti-M1 to anti-M9 — are described. All nine AMA types react with antigens which are associated either with inner (M1, M2, M7) our outer mitochondrial membranes (M3, M4, M5, M6, M8, M9) derived from rat liver or beef heart mitochondria. These antigens can be clearly distinguished by their different physical and chemical properties. Anti-M1 to anti-M9 can be related to distinct clinical entities: anti-M1, anti-M5, and anti-M7 are found in nonhepatic disorders, such as syphilis (anti-M1), undefined collagen diseases (anti-M5), and some forms of cardiac diseases (anti-M7). Anti-M3 and anti-M6 are detected in drug-induced disorders, such as phenopyrazon-induced pseudolupus syndrome (PLE; anti-M3) and iproniazid-induced hepatitis (anti-M6). Anti-M2, anti-M4, anti-M8, and anti-M9 are confined to primary biliary cirrhosis (PBC).Anti-M2 is a specific marker for the diagnosis of PBC; 96% of PBC patients (n=752) were anti-M2 positive. Anti-M4 and anti-M8 seem to reflect disease activity. Anti-M9 antibodies occur preferentially in early PBC. The clinical course of PBC was analyzed with respect to four different AMA profiles: profile A: only anti-M9 positive in the ELISA; profile B: anti-M9 and anti-M2 positive in the ELISA; profile C: anti-M2 positive in ELISA and complement fixation test (CFT), but anti-M4 and anti-M8 positive only in the ELISA; and profile D: anti-M2, anti-M4, anti-M8 positive in ELISA and CFT. Patients with profile A und B were found to have a rather benign course while those patients with profile C and D showed a rather progressive course when followed over a period of 6–15 years.Considering the similarities between bacterial and mitochondrial membranes, it is suggested that the formation of AMA of different specificities in PBC, especially of the anti-M2 type, may be induced by cross-reacting antigens.Abbreviations ALAT Alanine aminotransferase - AMA Antimitochondrial antibodies - ANT Adenine nucleotide translocator - AP Alkaline phosphatase - ASAT Aspartate aminotransferase - ATPase Adenosinetriphosphatase - CAH Chronic active hepatitis - CFT Complement fixation test - DNA Deoxyribonucleic acid - ELISA Enzyme linked immunosorbent assay - ID Immunodiffusion - IFL Immunofluorescence test - PBC Primary biliary cirrhosis - PLE Pseudolupus syndrome - RNA Ribonucleic acid - SLE Systemic lupus erythematosus - SMP Submitochondrial particles - VDRL Veneral Disease Research Laboratory Dedicated to Prof. H.D. Waller on the occasion of his 60th birthday     

Autoantibodies in the sera of patients with rheumatic heart disease: characterization of myocardial antigens by two-dimensional immunoblotting and N-terminal sequence analysis

The concept of antigenic mimicry in autoimmune diseases such as rheumatic fever has been under investigation for decades and the range of cross-reactive tissue antigens for streptococcal-induced antibodies identified in rheumatic heart disease is still expanding. To identify heart tissue-reactive antigens which may be implicated in the secondary immunopathogenesis of rheumatic fever, sera from 56 patients with acute rheumatic heart disease were probed in two-dimensional Western blots for reactivity against heart tissue antigens. After two-dimensional immunoblot analysis, proteins were submitted to N-terminal amino acid sequence analysis. This analysis identified creatine kinase, two mitochondrial proteins and, at a low level, various stress proteins as cross-reactive myocardial antigens. Therefore, in addition to myosin, creatine kinase may represent another major antigen for autoreactive antibodies in rheumatic heart disease. Mitochondrial proteins have been implicated in the pathogenesis of inflammatory heart disease for some years, and in this study we have identified two mitochondrial proteins as relevant antigens in rheumatic heart disease.     

Structure and analysis of the human dimethylglycine dehydrogenase gene

Dimethylglycine dehydrogenase (DMGDH; E.C. 1.5.99.2) is an enzyme involved in the catabolism of choline, catalyzing the oxidative demethylation of dimethylglycine (DMG) to form sarcosine. Subsequently, sarcosine dehydrogenase (SDH; E.C. 1.5.99.1) converts sarcosine to glycine via a similar reaction. Both enzymes are found as monomers in the mitochondrial matrix, and both contain 1 mol of covalently bound flavin adenine dinucleotide. DMGDH and SDH also utilize a noncovalently bound folate coenzyme that receives the "1-carbon" groups that are removed by DMGDH and SDH, forming "active formaldehyde." We have recently described a new inborn error of metabolism of DMGDH characterized by an unusual fish-like body odor. To augment our study of this new disorder, we have isolated two human genomic clones that together contain 16 exons of coding sequence for the hDMGDH gene. Fluorescent in situ hybridization analysis of the hDMGDH gene indicates that it is found on chromosome 5q12.2-q12.3. In addition, several polymorphisms have been identified in the hDMGDH cDNA sequence. Population analysis of two Ser/Pro polymorphisms found 367 amino acids apart reveals a skew of alleles, with the haplotypes Ser/Pro or Pro/Ser (79%) overrepresented compared to the number of Ser/Ser or Pro/Pro alleles observed. Possible functional consequences of these findings are discussed. Characterization of the gene structure for hDMGDH will aid in the study of patients with inherited defects of this enzyme.     

Inhibition of enzyme function by human autoantibodies to an autoantigen pyruvate dehydrogenase E2: different epitope for spontaneous human and induced rabbit autoantibodies.

Antibodies to the mitochondrial autoantigen M2, characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), react with the E2 subunit of the pyruvate dehydrogenase enzyme (PDH-E2). We examined the effect of disease sera on the enzyme activation catalysed by the PDH complex. Inhibition of enzyme activity was observed in 19 of 24 sera of patients with PBC with a level of greater than 90% inhibition in 14 at a serum dilution of 1/50. The onset of inhibition by serum was rapid, within the time of mixing, and the inhibitory activity was shown to reside in the immunoglobulin fraction of the serum. The immunoglobulin fraction of control sera from patients with other liver diseases (n = 26) and healthy persons (n = 8) failed to produce inhibitory activity. In addition sera from four rabbits, intensively immunized with a recombinant human M2 autoantigen, gave anti-M2 reactions by fluorescence, ELISA and immunoblotting, but did not inhibit the activity of PDH. The failure of experimentally induced M2 antibodies in rabbits to inhibit is interesting in view of the reactivity of the natural M2 autoantibodies of PBC with the highly conserved site on the enzyme which carries the essential lipoic acid cofactor.     

Use of western blot to analyze the reactivity of sera from patients with mediterranean spotted fever

The reactivity of antigenic components ofRickettsia conorii with sequentially obtained sera from 20 adult Spanish patients with Mediterranean spotted fever was analyzed by Western blot. The major rickettsial antigens reacting with the serum samples corresponded to molecular weights of 135 and 115 kDa. These antigens constantly exhibited higher staining intensity than the other antigens, and reacted with 100 % and 86.7 %, respectively, of acute sera and with 100 % of convalescent phase samples. Rickettsial lipopolysaccharide antigens reacted with 94.7 % of sera collected in the fourth and fifth week after onset of symptoms. Other major antigens reactive in the blots had molecular sizes of 160, 100, 90 and 60 kDa, and a relatively frequent humoral immune response was also seen to antigens of 80, 73 and 55 kDa.     

Epitope mapping of anti-proteinase 3 and anti-myeloperoxidase antibodies.

Anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies are present in many patients with Wegener's granulomatosis (WG) and microscopic polyarteritis. The aim of this study was to determine whether these antibodies bound to linear peptide sequences on their target antigens. If common linear epitopes were demonstrated, then these could be manufactured and used in diagnostic ELISAs for anti-PR3 and anti-MPO antibodies. In addition, any homology between these epitopes and bacterial or viral sequences might implicate those microorganisms in the development of these antibodies and the pathogenesis of the associated diseases. The presence of linear epitopes on PR3 and MPO was suggested by the binding of the corresponding autoantibodies to these proteins after they had been reduced with beta-mercaptoethanol (beta-ME) and denatured with SDS or boiling, and digested with proteases. Four of the 22 sera with anti-PR3 antibodies bound to PR3 in Western blots after treatment with SDS, beta-ME and boiling for 5 min. Thermal denaturation reduced the amount of binding more than other forms of denaturation. One serum with anti-PR3 antibodies bound to Lys-C and Glu-C-digested PR3 in dot blots. Linear epitopes could not be further defined by their binding in an ELISA using overlapping peptides corresponding to the PR3 molecule because of non-specific binding. Three of the five sera with anti-MPO antibodies bound to MPO in Western blots after treatment with SDS, beta-ME and boiling for 5 min. One serum with anti-MPO antibodies bound to Lys-C and Glu-C-digested MPO in dot blots. Again, linear epitopes could not be further defined using an ELISA with overlapping peptides because of non-specific binding. Some anti-PR3 and anti-MPO antibodies are likely to recognize linear epitopes, but these cannot be defined by use of a PIN ELISA system.     

Human B lymphocytes produce leukocyte interferon after interaction with foreign cells.

Culture filtrate antigens of Micropolyspora faeni grown in a synthetic medium in a stirred fermentor were characterized. The culture filtrate antigens were fractionated by preparative isoelectric focusing with a pH gradient of 3.5 to 5.5. The fractions were pooled according to their reaction with rabbit anti-M. faeni sera. A pool containing two major antigens which were resolved by analytical isoelectric focusing and polyacrylamide disc gel electrophoresis was obtained. One antigen was stainable with Coomassie blue and periodic acid-Schiff stain and was determined to have a mass of 51,000 daltons. The other antigen was stainable only with Coomassie blue and was determined to have a mass of 29,000 daltons. When used at 1 mg/ml, this pool reacted with the sera from all patients with farmer's lung disease by immunodiffusion but failed to react with control sera.     

Identification and Purification of Specific Penicillium marneffei Antigens and Their Recognition by Human Immune Sera

Disseminated infection with the dimorphic pathogenic fungus Penicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.     

The non-collagenous domains of the alpha 3 and 4 chains of type IV collagen and their relationship to the Goodpasture antigen

The Goodpasture antigen is the target recognized by anti-glomerular basement membrane (GBM) antibodies in anti-GBM disease or Goodpasture's syndrome. This structure is present in all normal GBM, but when serum containing anti-GBM antibodies is used to examine renal tissue from most males with classical Alport's syndrome, the Goodpasture antigen appears to be missing. The nature of the Goodpasture antigen is uncertain although it has been putatively and controversially localized to the non-collagenous domain of a novel type IV collagen chain (alpha 3) by one group, and a short peptide sequence has been published (M2). We have performed several experiments to determine whether M2 represents the Goodpasture antigen and we have also studied the corresponding sequence of the alpha 4 chain of type IV collagen (M3). Firstly, we demonstrated by polymerase chain reaction (PCR) amplification using specific priming oligonucleotides that mRNAs corresponding to M2 and M3 were found within the kidney and that the published sequences were correct. When heterologous antibodies were raised against M2 and M3 these bound specifically to GBM in an ELISA based on collagenase-digested basement membrane and this binding could be inhibited by incubation with collagenase-digested GBM but not with ovalbumin. On further examination of the target molecules using Western blots, the anti-M2 antibody bound to a single high molecular weight band of collagen-digested GBM in contrast to the anti-M3 antibody that bound to the same bands as Goodpasture serum. We then established ELISAs for anti-M2 and anti-M3 activity using the peptides M2 and M3. While rabbit anti-M2 and M3 antibodies bound specifically to their respective peptides in these ELISAs, there was no binding of three high titre Goodpasture's syndrome sera or two sera from Alport's syndrome patients with inhibitable anti-GBM antibody post-renal transplant. We have shown that the sequences of M2 and M3 correspond to proteins present within the collagenase-resistant part of the GBM, suggesting that these do represent parts of novel type IV collagen chains. However, sera containing anti-GBM antibodies did not bind to either peptide in solid-phase ELISAs, and these antibodies may recognize a different peptide sequence, features of the tertiary structure of these peptides or interactions between collagen chains.     

Antigenic Variation and Cross-Reactivity in Bacteroides forsythus Clinical Isolates Detected by Western Blot

Bacteroides forsythus is one of the etiologic agents of destructive periodontal diseases. Determining which antigenic components of the bacterium are recognized in the immune response of periodontitis patients is an important step in assessing strategies for vaccine development. The aim of this study was to identify the major strain-variable and cross-reactive antigens of B. forsythus clinical isolates recognized by serum IgG from patients with early-onset rapidly progressive periodontitis. Ten patient sera with measurable IgG against antigenic components of the species were identified by Western blot. Positive sera were tested by checkerboard ELISA to identify those most responsive to strain-variable antigens in nine clinical isolates and ATCC strain 43037. Correlation analysis of the ELISA data suggested that different subsets of isolates were preferentially recognized by different sera. Western blots revealed that certain sera also recognized major shared components across all the isolates, but preferential recognition of different isolate subsets by different patients was clearly confirmed. To determine if the variable antigens recognized were nonprotein, proteinase K-digested isolates were compared to undigested controls by Western blot. The main strain-variable antigens were proteinase resistant, while proteins at 200 and 210 kDa were identified as the major shared components. Two-dimensional SDS-PAGE revealed that these proteins are the quantitatively dominant heat-modifiable components of the cell envelope. Even though variable antigens are prominent in the immune response of patients, a cross-protective vaccine based on the shared envelope proteins of B. forsythus seems feasible in light of these observations.