Aluminum oxide and zinc oxide induced nanotoxicity in rat brain, heart, and lung.

Nanomaterials or nanoparticles are commonly used in the cosmetics, medicine, and food industries. Many researchers studied the possible side effects of several nanoparticles including aluminum oxide (Al2O3-nps) and zinc oxide nanoparticles (ZnO-nps). Although, there is limited information available on their direct or side effects, especially on the brain, heart, and lung functions. This study aimed to investigate the neurotoxicity, cardiotoxicity, and lung toxicity induced by Al2O3-nps and ZnO-nps or in combination via studying changes in gene expression, alteration in cytokine production, tumor suppressor protein p53, neurotransmitters, oxidative stress, and the histological and morphological changes. Obtained results showed that Al2O3-nps, ZnO-nps and their combination cause an increase in 8-hydroxy-2´-deoxyguanosine (8-OHdG), cytokines, p53, oxidative stress, creatine kinase, norepinephrine, acetylcholine (ACh), and lipid profile. Moreover, significant changes in the gene expression of mitochondrial transcription factor-A (mtTFA) and peroxisome proliferator activator receptor-gamma-coactivator-1alpha (PGC-1alpha) were also noted. On the other hand, a significant decrease in the levels of antioxidant enzymes, total antioxidant capacity (TAC), reduced glutathione (GSH), paraoxonase 1 (PON1), neurotransmitters (dopamine - DA, and serotonin - SER), and the activity of acetylcholine esterase (AChE) in the brain, heart, and lung were found. Additionally, these results were confirmed by histological examinations. The present study revealed that the toxic effects were more when these nanoparticle doses are used in combination. Thus, Al2O3-nps and ZnO-nps may behave as neurotoxic, cardiotoxic, and lung toxic, especially upon exposure to rats in combination.

Immunotherapy for Alzheimer's Disease: Current Scenario and Future Perspectives.

Alzheimer's disease (AD) is a global health concern owing to its complexity, which often poses a great challenge to the development of therapeutic approaches. No single theory has yet accounted for the various risk factors leading to the pathological and clinical manifestations of dementia-type AD. Therefore, treatment options targeting various molecules involved in the pathogenesis of the disease have been unsuccessful. However, the exploration of various immunotherapeutic avenues revitalizes hope after decades of disappointment. The hallmark of a good immunotherapeutic candidate is not only to remove amyloid plaques but also to slow cognitive decline. In line with this, both active and passive immunotherapy have shown success and limitations. Recent approval of aducanumab for the treatment of AD demonstrates how close passive immunotherapy is to being successful. However, several major bottlenecks still need to be resolved. This review outlines recent successes and challenges in the pursuit of an AD vaccine.

Identification of a recombinogenic major histocompatibility complex Q gene with diverse alleles.

Structural diversity enables class Ia molecules to present a diverse repertoire of peptides to the T cell receptor. This diversity is thought to be generated by recombinations between class I genes. We have found that two class Ib Q2 alleles exhibit extremely high sequence diversity, even higher than class Ia alleles. Clustered nucleotide differences between Q2b and Q2k suggest that this sequence diversity was generated by microrecombinations between Q2 genes and other class I genes. The relatively high expression of Q2b in the thymus may be significant and perhaps suggests a novel role for a Q2b product in the education and selection of the T cell repertoire.

Introduction of a disulfide bond in the alpha 1 domain of the H-2Kb molecule confers immunogenicity to the transfected RMA-S tumors.

The use of major histocompatibility complex class I genes is an emerging approach for the immunotherapy of human cancer. The conformational stability of class I molecules is important for their immunologic recognition. We have engineered a disulfide bond in the alpha 1 domain of a murine class I molecule, Kb. The expression of the engineered, but not the wild-type, Kb molecules conferred immunogenicity to a nonimmunogenic and antigen presentation-defective tumor cell line, RMA-S. Mice that rejected the engineered Kb-transfected RMA-S cells developed a long-lived antitumor immune response. These data indicate the possibility of genetically engineering class I molecules to improve their therapeutic potential.

Unique biochemical properties of a mutant MHC class I molecule, H-2Ksm1.

This study describes serological and biochemical properties of a novel MHC class I molecule. The mutant H-2Ksm1 molecule was discovered in a mouse because of loss of reactivity of its peripheral blood lymphocytes to monoclonal antibodies. This mutation in the H-2Ks molecule is the first in vivo mutation described that has altered an amino acid residue (amino acid 107) distant from the regions generally considered to be peptide or TCR contacts. Cell surface expression of the mutant molecules remains high but the Arg107 to Trp substitution appears to alter the native protein conformation, markedly decreasing cell surface association with beta 2-microglobulin light chains and conferring a loss of recognition by Ks specific antibodies.

A single amino acid substitution in the H-2Kb molecule generates a defined allogeneic epitope.

Using Mitomycin C mutagenesis and negative and positive selection with monoclonal antibodies specific for H-2Kb and H-2Kbm10, respectively, a mutant cell line clone, Mitc-182, was isolated. Direct sequencing of uncloned cDNA as well as PCR based cloning and sequencing of the H-2Kb182 transcript from this mutant revealed a single G-->T transversion resulting in the substitution of Trp167 by cysteine. Serologically, the mutant Kb182 and Kbm10 are almost identical as each has lost at least five Kb specific mAb epitopes and gained several new epitopes. Interestingly, the mutant cell line, Mitc-182, is efficiently recognized by alloreactive CTLs raised in reciprocal combinations, e.g. CB6 anti Cbm10 and Cbm10 anti CB6, indicating that Kb182 contains both Kb and Kbm10 specific epitopes. The mutation has not affected the ability of Kb182 to present Kb restricted antigenic peptides of Sendai and vesicular stomatitis viruses. In addition to underscoring the importance of amino acid residue 167 in alloreactivity, these results indicate a positive correlation between the gain of both an mAb epitope and a defined alloreactive CTL epitope.

Rejuvenated expression of H-2Kb in RMA-S cells does not affect alloreactive T cell- and natural killer cell-mediated lysis.

Reduced sensitivity to alloreactive cytotoxic lymphocytes (CTLs) and increased susceptibility to natural killer (NK) cells in vivo and in vitro are two phenotypic characteristics that have been attributed to the reduced major histocompatibility complex (MHC) ligand density on the surface of an antigen-presentation-defective cell line, RMA-S. As RMA-S exhibits both defective processing of antigenic peptides and very low class I expression, it is uncertain which is responsible for the above characteristics. In this report, we show that the phenotype cannot be reversed by increasing the number of Kb + beta 2-M complexes expressed on the cell surface. These findings emphasize the importance of association of MHC class I molecules with endogenously processed peptides for biological interaction with alloreactive CTLs and NK cells.

Effect of vitamin A deprivation on the mitogenic factor activity in the rat testes.

The seminiferous tubules of rat testes contain a protease and heat sensitive factor capable of stimulating (3H)-thymidine incorporation into quiescent cultures of NIH 3T3 mouse fibroblast cells. This mitogenic factor activity was only 4% in the testes of vitamin A deficient-retinoic acid maintained rats as compared to that of normal rats. Supplementation of retinyl acetate to these vitamin A deficient rats for 4 days resulted in a 56% recovery in the mitogenic factor activity while by day 16, the recovery was 80 percent. Incorporation of (3H)-thymidine into DNA of the seminiferous tubules of vitamin A deficient rats was only 46% of that of normal rats which was restored to normal levels by retinyl acetate supplementation for 24 days.

Collagenase induction promotes mouse tumorigenesis by two independent pathways.

Although progress has been made in the understanding of the role of metalloproteinases in tumor progression during metastasis, little is known about their contributions, if any, to tumor formation. Accumulating evidence identified an increased presence of several matrix metalloproteinases in human cancers, but the precise role for interstitial collagenase in tumor formation or progression has not been well defined. Transient induction of collagenase was observed in wild-type mouse skin after treatment with the tumor-promoting agents 12-O-tetradecanoylphorbol-13-acetate (TPA) and chrysarobin, which promote tumorigenesis through protein kinase C-dependent and -independent pathways, respectively. Transgenic mice that constitutively express interstitial collagenase within the epidermis of the skin have an increased susceptibility to tumorigenesis and produced tumors at lower doses of TPA as compared with wild-type mice. Similarly, the transgenic mice showed increased tumorigenesis when promoted with chrysarobin. These results demonstrate that collagenase overexpression can contribute to tumorigenesis via protein kinase C-dependent and -independent pathways. Significantly, compared with wild-type mice, the transgenic mice demonstrated an elevated expression of c-fos in the skin at baseline, before tumor promotion, suggesting a molecular mechanism for the increased tumor susceptibility in collagenase transgenic mice. These findings further support the importance of MMP deregulation in tumorigenesis and suggest that the role of MMP family members is not limited to metastasis but may also contribute to initial tumor development.

Oral administration of unmodified colonic but not small intestinal antigens protects rats from hapten-induced colitis.

Colonic administration of a hapten, 2,4,6-trinitrobenzene sulphonic acid (TNBS) has been shown to induce colitis in rats. We are using this model to investigate the role of colonic antigens in the immunopathology. In this study, we show that colitis can be suppressed by oral administration of haptenized colonic antigens prior to the TNBS enema. Moreover, our data suggest that haptenization of the colonic antigens is not essential because oral feeding of non haptenized colonic antigens too protects rats from TNBS-induced colitis. Thus, unmodified colonic antigens may be involved in the induction of oral tolerance, and possibly in the pathogenesis in this model of colitis. Further, we show that the protective immunity or oral tolerance induced by non haptenized colonic antigens can be passively transferred to naïve rats by mesenteric T lymphocytes. Interestingly, oral feeding of small intestinal antigens, haptenized and non haptenized, does not protect rats from colitis, suggesting a specific role for colonic antigens. These data underscore the usefulness of this rat model in the identification of pathogenic antigens in colitis and in the development of therapeutic strategies based on oral tolerance.

Use of mutants to analyze regions on the H-2Kb molecule for interaction with immune receptors.

MHC variants isolated both in vivo (by tissue graft rejection) and in vitro (by antibody selection) were utilized to study sites on the H-2Kb molecule involved in interaction with antibodies and with the TCR. Kb mutants selected by antibodies were found to have single point mutations, which when analyzed in the context of the three-dimensional structure of a Kb molecule modeled from HLA-A2 coordinates showed that the altered residues were localized mostly to the alpha 1 and alpha 2 helices. The side chains of the variant amino acid residues pointed upward and away from the antigenic site. Analysis of the altered amino acids in the previously described tissue-graft-selected Kb mutants showed that the side chains of the variant residues occur either in the alpha-helical regions or in the beta-pleated-sheet floor of the antigen groove, but every mutant contained at least one and sometimes several acid side chains projecting into the antigen-binding groove. Monoclonal antibody studies showed that the available monoclonal antibodies mapped to discrete domain-specific sites. Analysis of CTL recognition sites using cloned mutant anti-parent or allogeneic combinations showed that all CTL clones examined interacted with amino acid side-chain residues on both the alpha 1 and alpha 2 helices. Thus, we concluded that the CTLs must simultaneously interact with the amino acid residues in both the alpha-helical stretches of the alpha 1 and alpha 2 domains. Our analyses with the point mutants imply that the TCR must interact with the MHC molecule over a relatively large surface area in such an orientation that it interfaces with the two alpha helices, as well as with the foreign or self-peptide in the antigen-binding site between the helices. These findings, together with the observation that several of the in vivo Kb mutants induce strong alloreactions yet have changes only in the bottom of the antigen-binding groove and no alterations in the alpha-helical residues, are consistent with the hypothesis that in some cases alloreaction can be the result of T-cell recognition of an altered pattern on the MHC molecule due to a changed peptide in the antigen groove.

Externalization of tropomyosin isoform 5 in colon epithelial cells.

Ulcerative colitis (UC) is associated with autoantibody response to a cytoskeletal protein, human tropomyosin (hTM) isoform-5 (hTM5). Because hTM5 is an intracellular protein, it may remain inaccessible to the autoantibodies. Therefore, we have investigated the possibility of externalization of hTM5 in colon epithelial cells. Freshly isolated colonic and small intestinal epithelial cells and LS-180 colon cancer cell line were examined for surface expression of hTM5 by flow cytometric analysis using hTM isoform-specific MoAbs. The extracellular release of hTM5 was determined by Western blot and radioimmunoprecipitation analyses. Physical association of hTM5 with a membrane-associated colon epithelial protein (CEP) was examined by co-immunoprecipitation of hTM5 with anti-CEP MoAb, and CEP with anti-hTM5 MoAb. Cell surface expression of hTM5 was observed in colonic epithelial and LS-180 cells but not in small intestinal epithelial cells. LS-180 cells spontaneously released hTM5 as well as CEP into the culture medium that was significantly stimulated by a calcium ionophore, A23187, but inhibited by phorbol-12-myristate-13-acetate, monensin and methylamine. Co-immunoprecipitation experiments revealed that hTM5 forms a complex with CEP. We conclude that hTM5 is externalized in colon but not in small intestinal epithelial cells. The physical association of hTM5 with CEP suggests a possible chaperone function of CEP in the transport of hTM5, a putative target autoantigen in UC.

Molecular basis for the lack of bilirubin-specific and 3-methylcholanthrene-inducible UDP-glucuronosyltransferase activities in Gunn rats. The two isoforms are encoded by distinct mRNA species that share an identical single base deletion.

Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. In addition, a UDPGT isoform which is active toward 4-nitrophenol and is induced by 3-methylcholanthrene (3-MC) in normal rats, is produced in a nonfunctional truncated form in Gunn rats due to the deletion of a single guanosine residue in the coding region of its mRNA. The hepatic concentration of bilirubin-UDPGT mRNA was lower in Gunn rats than in congeneic normal rats. However, bilirubin-UDPGT mRNA was of apparently normal length and was induced by clofibrate, a known inducer of bilirubin-UDPGT activity. 3' regions of bilirubin- and 3-MC-inducible UDPGT mRNAs have identical nucleotide sequences; the single base deletion in the 3-MC-inducible UDPGT in Gunn rats occurs within this region. Using oligonucleotide primers corresponding to the identical and unique regions of the two mRNAs, and polymerase chain reaction, we amplified segments of mRNAs for the bilirubin- and 3-MC-inducible UDPGTs from normal and Gunn rat livers. Both amplified DNAs in Gunn rats lacked the restriction site for BstNI. Nucleotide sequence determination revealed that bilirubin- and 3-MC-inducible UDPGT mRNAs in Gunn rats contain an identical frame-shift deletion of a single guanosine residue within the common region of their coding sequences.

Mechanism of the lack of induction of UDP-glucuronosyltransferase activity in Gunn rats by 3-methylcholanthrene. Identification of a truncated enzyme.

Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. 4-Nitrophenol glucuronidation is mediated by several UDPGT isoforms that are distinct from bilirubin-UDPGT, one of which is induced after 3-methylcholanthrene (3-MC) administration in normal, but not in Gunn rats. In normal rats, 3-MC-inducible UDPGT mRNA concentration increased 15-fold in the liver and 3-fold in kidney after 3-MC (140 mg/kg) administration. Concentration of this mRNA is much lower in Gunn rat liver and kidney compared to normal. However, this mRNA was normally induced after 3-MC administration. By RNA blot hybridization, the mRNA in Gunn rat liver and kidney appeared to be of normal size. Nuclear run-on studies showed that the transcription rate for 3-MC-inducible UDPGT was 3-fold higher in Gunn rat liver and kidney than in normal and increased 3- to 5-fold after 3-MC administration. Immunotransblot studies revealed an Mr = 56,000 3-MC-inducible UDPGT in liver and kidney of normal, but not in Gunn rats. However, a new immunoreactive UDPGT band (Mr = 43,000) was present in Gunn, but not in normal rats. Cell-free translation of kidney mRNA from 3-MC-treated Gunn rats showed that the Mr = 43,000 UDPGT is synthesized as an Mr = 45,000 protein. Prior hybridization of the mRNA with an isoform-specific oligonucleotide spanning the initiation codon abolishes synthesis of this protein. These results suggest that a sequence abnormality in the 3-MC-inducible UDPGT mRNA in Gunn rats results in reduced mRNA concentration and synthesis of a truncated, enzymically inactive UDPGT.

Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor.

Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues.