Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Identification of a Protective Leishmania Antigen Dihydrolipoyl Dehydrogenase and Its Responding CD4+ T Cells at Clonal Level.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

A Lipoylated Metabolic Protein Released by Staphylococcus aureus Suppresses Macrophage Activation.

The virulence factors of pathogenic microbes often have single functions that permit immune suppression. However, a proportion possess multiple activities and are considered moonlighting proteins. By examining secreted virulence factors of Staphylococcus aureus, we determine that the bacterial lipoic acid synthetase LipA suppresses macrophage activation. LipA is known to modify the E2 subunit of the metabolic enzyme complex pyruvate dehydrogenase (E2-PDH) with a fatty acid derivative, lipoic acid, yielding the metabolic protein lipoyl-E2-PDH. We demonstrate that lipoyl-E2-PDH is also released by S. aureus and moonlights as a macrophage immunosuppressant by reducing Toll-like receptor 1/2 (TLR1/2) activation by bacterial lipopeptides. A LipA-deficient strain induces heightened pro-inflammatory cytokine production, which is diminished in the absence of TLR2. During murine systemic infection, LipA suppresses pro-inflammatory macrophage activation, rendering these cells inefficient at controlling infection. These observations suggest that bacterial metabolism and immune evasion are linked by virtue of this moonlighting protein.

Dihydrolipoamide dehydrogenase-Lpd (Rv0462)-specific T cell recall responses are higher in healthy household contacts of TB: a novel immunodominant antigen from M. tuberculosis.

The partial effectiveness against pulmonary tuberculosis (PTB), displayed by the existing tuberculosis (TB) vaccine, bacillus Calmette-Guérin (BCG), highlights the need for novel vaccines to replace or improve BCG. In TB immunology, antigen-specific cellular immune response is frequently considered indispensable. Latency-associated antigens are intriguing as targets for TB vaccine development. The mycobacterial protein, dihydrolipoamide dehydrogenase (Lpd; Rv0462), the third enzyme of the pyruvate dehydrogenase (PDH) complex, facilitates Mycobacterium tuberculosis to resist host reactive nitrogen intermediates. Multicolor flow cytometry analysis of whole-blood cultures showed higher Lpd-specific Th1 recall response (IFN-γ, TNF-α, and IL-2; P = 0.0006) and memory CD4+ and CD8+ T cells (CCR7+ CD45RA- and CCR7- CD45RA-) in healthy household contacts (HHC) of TB (P < 0.0001), which is comparable with or higher than the standard antigens, ESAT-6 and CFP-10. The frequency of Lpd-specific multifunctional T cells was higher in HHC compared with PTB patients. However, there is no significant statistical correlation. Regulatory T cell (Treg) analysis of HHCs and active TB patients demonstrated very low Lpd-specific CD4+ Tregs relative to ESAT-6 and CFP-10. Our study demonstrates that the Lpd antigen induces a strong cellular immune response in healthy mycobacteria-infected individuals. In consideration of this population having demonstrated immunologic protection against active TB disease development, our data are encouraging about the possible use of Lpd as a target for further TB subunit vaccine development.

Fungal peptides from pneumonitis hypersensitivity etiologic agents are able to induce specific cellular immune response.

PURPOSE: Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH. EXPERIMENTAL DESIGN: A selection of 34 peptides, from the MiDLDH and LcDLDH, able to interact with Major Histocompatibility Complex (MHC) 1 and MHC 2, was obtained using three different epitope prediction websites. By means of ELISPOT assays, we compared the frequency of Interferon gamma (IFNγ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools. Tests were performed using cells from 35 healthy blood donors. RESULTS: One peptide pool containing five peptides from MiDLDH and able to interact with MHC 2 induced a marked IFNγ specific immune response (Pool F, p<0.001, Wilcoxon signed-rank test). CONCLUSION: This study demonstrated that peptides from microorganisms involved in HP were able to induce a high IFNγ specific immune response after stimulation of PBMCs from healthy blood donors which could be useful to develop an effective prevention strategy.

Identification of DLD, by immunoproteomic analysis and evaluation as a potential vaccine antigen against three Vibrio species in Epinephelus coioides.

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.

Proteomic identification of dihydrolipoamide dehydrogenase as a target of autoantibodies in patients with endometrial cancer.

BACKGROUND/AIM: Accumulating evidence shows that various types of cancers induce a specific immune response, resulting in the production of antibodies against self-components (autoantibodies). The aim of the present study was to identify antigens for autoantibodies in sera from endometrial cancer patients as novel diagnostic markers for the disease. MATERIALS AND METHODS: The reactivity of individual sera from patients was examined by 2-dimensional (2-D) immunoblotting using HeLa cell lysates as antigens to identify autoantigens. ELISA was established to quantitatively measure autoantibody titer of patients' sera. RESULTS: A mitochondrial protein, dihydrolipoamide dehydrogenase (DLD), was identified as an autoantigen specific to endometrial cancer patients. The levels of immunoglobulin (Ig)A but not IgG autoantibody to DLD were significantly increased in the sera of endometrial cancer patients. CONCLUSION: IgA autoantibody against DLD could be a novel diagnostic marker for endometrial cancer.

External validation of recombinant antigens for serodiagnosis of machine operator's lung.

BACKGROUND: Machine operator's lung (MOL) is a hypersensitivity pneumonitis the diagnosis of which is difficult. Our laboratory previously developed an ELISA test using recombinant antigens from Mycobacterium immunogenum isolated in French plant. The objective was to validate the previous ELISA results with ten new suspected cases from Germany. METHODS: Two serological analyses were performed: ELISA with the six recombinant antigens, and electrosyneresis with crude antigens of M. immunogenum and three other main species isolated from contaminated metalworking fluids. RESULTS: The two recombinant antigens acyl-CoA dehydrogenase and dihydrolipoyl dehydrogenase, combined together, and electrosyneresis are useful in making the diagnosis regardless of the clinical and radiological data. Finally 9 out of the 10 suspected cases were declared as MOL. CONCLUSIONS: Despite the geographical distance, the crude and recombinant antigens produced to investigate the clustered French cases also proved to be useful in diagnosing the suspected cases in Germany.

Dihydrolipoamide dehydrogenase of Pseudomonas aeruginosa is a surface-exposed immune evasion protein that binds three members of the factor H family and plasminogen.

The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18-20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

Anaplasma phagocytophilum dihydrolipoamide dehydrogenase 1 affects host-derived immunopathology during microbial colonization.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization.

Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naïve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.

The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell-surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase.

Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non-electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N- and C-termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti-TiBP polyclonal antibodies by whole cell and protein enzyme-linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were not recognized by these antibodies. Similar results were obtained by fluorescence and scanning electron microscopy. Our postulation that DLDH is located on the surface of Rh. ruber GIN1, serving as a TiO2 binding protein, is in accordance with literary evidence on DLDH in other organisms, Bacteria, Archea, and Eukaryots that suggests it is associated with the outer membranes or cell surfaces. As an exocellular protein DLDH assumes various tasks which are not related to its classical role as a 2-oxoacid dehydrogenase, including serving as an adhesion/binding protein in certain bacteria.

Novelty of the pyruvate metabolic enzyme dihydrolipoamide dehydrogenase in spermatozoa: correlation of its localization, tyrosine phosphorylation, and activity during sperm capacitation.

Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that dihydrolipoamide dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, dihydrolipoamide dehydrogenase along with its host complex, the pyruvate dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of dihydrolipoamide dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, dihydrolipoamide dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.

Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti-neutrophil cytoplamic antibody (ANCA), anti-dihydrolipoamide dehydrogenase (anti-E3), rheumatoid factor (RF), anti-dihydrolipoamide acetyltransferase (anti-E2), anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-cardiolipin (aCL), anti-dsDNA, anti-ssDNA, anti-nuclear antibodies (ANA), anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti-E3 antibody and RF were positive in 278/516 (55.6%), 276/516 (53.3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15.8%, anti-ssDNA in 15.6%, anti-dsDNA in 8.5%, aCL in 5%, anti-SS-B/La in 4.1%, anti-SS-A/Ro (60 kD) in 3.9%, anti-E2 in 3.3% and anti-SSA/Ro (52 kD) in 1.2%, anti-MPO in 4.8%, anti-Topo II and anti-actinin in 0%. All sera with ANCA showed c-ANCA patterns and contained anti-PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and alpha-Fetoprotein (alpha-FP). HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated alpha-FP levels. The prevalence of autoantibodies was not affected by treatment with interferon-alpha (IFN-alpha). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti-E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA-related vasculitis.

Identification of the YMN-1 antigen protein and biochemical analyses of protein components in the mitochondrial nucleoid fraction of the yeast Saccharomyces cerevisiae.

We analyzed the protein components contained in the mitochondrial nucleoid (mt-nucleoid) fraction of the yeast Saccharomyces cerevisiae. Immunoblotting with anti-Abf2p antibody demonstrated the association of Abf2p, a major mitochondrial DNA-binding protein, with the mt-nucleoids. In contrast, porin and cytochrome c oxidase subunit III (CoxIIIp) were not detected by immunoblotting in the mt-nucleoid fraction. The YMN-1 monoclonal antibody recognized a 48 kDa protein of the mt-nucleoid fraction. The N-terminal amino acid sequence of the protein and immunological evidence showed that the YMN-1 monoclonal antibody recognizes dihydrolipoyl transsuccinylase (KE2), which is one of the constituents of the alpha-ketoglutarate dehydrogenase complex (KGDC). alpha-Ketoglutarate dehydrogenase (KE1) and dihydrolipoyl dehydrogenase (E3), which are other subunits of KGDC, were also detected in the mt-nucleoid fraction. An enzyme assay of the mt-nucleoid fraction showed that cytochrome c oxidase and fumarase activity were barely detected in the fraction, but the specific activity of KGDC in the mt-nucleoid fraction was relatively high and was approximately 60% of the specific activity in the mitochondrial fraction. Three components of KGDC were detected in the DNA-binding protein fractions after DNA-cellulose column chromatography of mt-nucleoid proteins. These results suggested that a part of KGDC in the mitochondrial matrix is associated with mt-nucleoids in vivo.

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis.

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.

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.

Autoreactive cytotoxic T cells in mice are induced by immunization with a conserved mitochondrial enzyme in Freund's complete adjuvant.

Standard methods to generate autoimmune reactions in mice, by immunization with antigens emulsified with adjuvants, stimulate strong helper (CD4) T-cell and antibody responses but are not reported to induce cytolytic CD8 T cells. The aim of this study was to assess whether specific autoreactive CD8 T cells could be readily generated after immunization with a 'weak' autoantigen in adjuvant. Mice were immunized intraperitoneally three times with the E3 subunit of the mitochondrial 2-oxoacid dehydrogenase enzyme complexes (dihydrolipoamide dehydrogenase) emulsified with Freund's complete adjuvant. Splenic and lymph node lymphocytes were harvested after 14 days for in vitro functional studies. T lymphocytes were tested for proliferative responses and cytotoxicity against antigen-loaded isogeneic target cells. An autoreactive cytolytic T lymphocyte (CTL) response was detectable only after the in vitro restimulation of lymphocytes with E3 antigen-loaded syngeneic splenocytes. These CTL were identified as H-2-restricted CD8+ T cells. A proliferative response to E3 was demonstrable against antigen-pulsed syngeneic splenocytes. Immunized mice also generated strong antibody responses to E3. Liver histology showed portal infiltrates interpreted as a response of the liver to a non-specific immunological stimulus. It is concluded that autoreactive cytolytic T cells can be generated experimentally upon appropriate stimulation of the immune system, and can be identified in vitro upon release from the controlling mechanisms that are likely to regulate them in vivo.

Humoral and cellular immune responses to dihydrolipoamide dehydrogenase (E3): lack of specificity for primary biliary cirrhosis.

Immune responses to dihydrolipoamide dehydrogenase, the E3 subunit which is a common component of 2-oxoacid dehydrogenase complexes, have been suggested to be associated with the etiology of primary biliary cirrhosis (PBC). However, since an antibody to E3 could be detected in Caucasian patients with PBC, but was not specific for the disease, the proposal is not evident at the antibody level. We have identified the antibody also in Japanese patients with PBC by immunoblotting with sera at a 1:100 dilution and have assessed cellular immune responses to E3 by proliferation assay of peripheral blood lymphocytes. Anti-E3 antibody was detected more frequently in 25 of 43 PBC (58.1%) than in normal controls (p < 0.01) and in chronic liver diseases (p < 0.05), but the antibody was not specific for PBC as reported in Caucasian PBC. Anti-E3 antibody-positive sera of PBC patients or normal controls and their IgG fraction did not inhibit the enzyme activity of E3. Lymphocyte blastogenesis to E3 in PBC was significantly greater than normal controls (p < 0.05), but was not significant as compared with chronic liver disease or non-hepatic autoimmune diseases. Thus, these data do not support the hypothesis that the immune response to the E3 subunit is associated with etiology of PBC.

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon.

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.