Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies.

The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis

Background/aims: Chronic infections such as those caused by Chlamydia pneumoniae and periodontopathic bacteria such as Porphyromonas gingivalis have been associated with atherosclerosis, possibly due to cross-reactivity of the immune response to bacterial GroEL with human heat shock protein (hHSP) 60. Methods: We examined the cross-reactivity of anti-GroEL and anti-P. gingivalis antibodies with hHSP60 in atherosclerosis patients and quantified a panel of six pathogens in atheromas. Results: After absorption of plasma samples with hHSP60, there were variable reductions in the levels of anti-GroEL and anti-P. gingivalis antibodies, suggesting that these antibodies cross-reacted with hHSP60. All of the artery specimens were positive for P. gingivalis. Fusobacterium nucleatum, Tannerella forsythia, C. pneumoniae, Helicobacter pylori, and Haemophilus influenzae were found in 84%, 48%, 28%, 4%, and 4% of arteries, respectively. The prevalence of the three periodontopathic microorganisms, P. gingivalis, F. nucleatum and T. forsythia, was significantly higher than that of the remaining three microorganisms. Conclusions: These results support the hypothesis that in some patients, cross-reactivity of the immune response to bacterial HSPs including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may be a possible mechanism for the association between atherosclerosis and periodontal infection.

Measurement of HNE-protein adducts in human plasma and serum by ELISA-Comparison of two primary antibodies

There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions.The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples.After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group. © 2013 The Authors.

Anti-N-methyl-D-Aspartate Receptor Encephalitis with Positive Serum Antithyroid Antibodies, IgM Antibodies Against Mycoplasma Pneumoniae and Human Herpesvirus 7 PCR in the CSF

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune.

Interrogation of human monoclonal antibodies induced by 4C-MenB to identify protective antigens contained in the OMV component

Neisseria meningitidis is a gram negative human obligated pathogen, mostly found as a commensal in the oropharyngeal mucosa of healthy individuals. It can invade this epithelium determining rare but devastating and fast progressing outcomes, such as meningococcal meningitidis and septicemia, leading to death (about 135000 per year worldwide). Conjugated vaccines for serogroups A, C, W135, X and Y were developed, while for N. meningitidis serogroup B (MenB) the vaccines were based on Outern Membrane Vesicles (OMV). One of them is the 4C-MenB (Bexsero). The antigens included in this vaccine’s formulation are, in addition to the OMV from New Zeland epidemic strain 98/254, three recombinant proteins: NadA, NHBA and fHbp. While the role of these recombinant components was deeply characterized, the vesicular contribution in 4C-MenB elicited protection is mediated mainly by porin A and other unidentified antigens. To unravel the relative contribution of these different antigens in eliciting protective antibody responses, we isolated human monoclonal antibodies (mAbs) from single-cell sorted plasmablasts of 3 adult vaccinees peripheral blood. mAbs have been screened for binding to 4C-MenB components by Luminex bead-based assay. OMV-specific mAbs were purified and tested for functionality by serum bactericidal assay (SBA) on 18 different MenB strains and characterized in a protein microarray containing a panel of prioritized meningococcal proteins. The bactericidal mAbs identified to recognize the outer membrane proteins PorA and PorB, stating the importance of PorB in cross-strain protection. In addition, RmpM, BamE, Hyp1065 and ComL were found as immunogenic components of the 4C-MenB vaccine.

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA). A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027) in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera Hydroelectric Plant, São Paulo, Brazil

Yellow fever (YF) is an acute viral infectious disease transmitted by mosquitoes which occurs in two distinct epidemiological cycles: sylvatic and urban. In the sylvatic cycle, the virus is maintained by monkey's infection and transovarian transmission in vectors. Surveillance of non-human primates is required for the detection of viral circulation during epizootics, and for the identification of unaffected or transition areas. An ELISA (enzyme-linked immunosorbent assay) was standardized for estimation of the prevalence of IgG antibodies against yellow fever virus in monkey sera (Alouatta caraya) from the reservoir area of Porto Primavera Hydroelectric Plant, in the state of São Paulo, Brazil. A total of 570 monkey sera samples were tested and none was reactive to antibodies against yellow fever virus. The results corroborate the epidemiology of yellow fever in the area. Even though it is considered a transition area, there were no reports to date of epizootics or yellow fever outbreaks in humans. Also, entomological investigations did not detect the presence of vectors of this arbovirus infection. ELISA proved to be fast, sensitive, an adequate assay, and an instrument for active search in the epidemiological surveillance of yellow fever allowing the implementation of prevention actions, even before the occurrence of epizootics.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera hydroeletric plant, São Paulo, Brazil

A febre amarela (FA) é doença infecciosa aguda de origem viral transmitida por mosquitos. No ciclo silvestre, o vírus é mantido por meio da infecção de macacos e da transmissão transovariana nos vetores. A vigilância sobre populações de primatas não humanos torna-se necessária para detectar a circulação viral, quando ainda está restrito a epizootias, e para determinar sua presença em regiões indenes ou de transição para a doença. Padronizou-se a técnica ELISA (Enzyme Linked Immunosorbent Assay) para determinar a prevalência de anticorpos da classe IgG contra o vírus da FA em soros de bugios (Alouatta caraya) da região do reservatório da Usina Hidrelétrica de Porto Primavera, SP. Foram testados soros de 570 macacos sendo que nenhuma amostra mostrou-se reativa para a presença de anticorpos contra o vírus da FA. Os resultados são coerentes com a epidemiologia da FA na região. Mesmo sendo área de transição, não se conhece, até o momento, ocorrência de epizootia ou surto de FA em humanos e investigações entomológicas não apontaram a presença de vetores para esta arbovirose. A técnica mostrou-se sensível, rápida e útil à vigilância epidemiológica como instrumento de busca ativa permitindo desencadear ações preventivas, como vacinação, antes mesmo do surgimento de epizootias