Monoclonal Antibodies against Aβ42 Fibrils Distinguish Multiple Aggregation State Polymorphisms in Vitro and in Alzheimer Disease Brain

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.     

What Can Be Learnt about Selection from Gene Frequency Distribution?

Polymorphism has been studied at the Esterase 6 locus in the Yellow Fever mosquito Aedes aegypti (L.) in laboratory stocks. At least 12 alleles are present, with up to four coexisting in a stock. The allele frequency distribution is quite sharply peaked at a mode of about 0.25. The experimental data are compared with the results of simulation based on two models, one in which the initial global distribution is taken to be the stationary distribution obtained from the neutral model assuming M = 4 mu Ne = 1 and the other in which the initial global distribution is generated from the experimental populations studied. The results suggest that the patterns observed are not likely to arise through random fluctuation of frequencies in neutral alleles, but that some kind of selection maintains polymorphism, either in the wild or in the laboratory, or both.     

Inhibitors of arachidonic acid metabolism act synergistically to signal apoptosis in neoplastic cells

Arachidonic acid (AA) and its metabolites are intimately linked to carcinogenesis. Inhibitors of AA metabolic enzymes have demonstrated anti-carcinogenic effects in vivo and induce apoptosis of many cancer cell lines in vitro. The mechanism by which AA influences carcinogenesis, however, remains unresolved. The current study explores the growth inhibitory potential of Triacsin C, PLT-98625, and NS-398 which inhibit three distinct metabolic enzymes that control intracellular AA levels: fatty acid coenzyme-A ligase 4 (FACL-4), coenzyme-A independent transacylase (CoA-IT), and cyclooxygenase (COX), respectively. Results reveal the anti-proliferative effects of these inhibitors in a number of human cancer cell lines. Further studies in the SK-MES-1 cell line demonstrate that all three inhibitors induce accumulation of unesterified AA which correlates with induction of apoptosis. Addition of exogenous AA also induces apoptosis. Furthermore, in combination, these inhibitors act cooperatively to induce AA accumulation which correlates to a synergistic reduction in cell viability. Taken together, these results suggest that accumulation of unesterified AA is a common mechanism in the induction of cancer cell apoptosis by various inhibitors of AA metabolism, confirm that previously described AA remodeling pathways are valid in cancer cells, and indicate that combination treatment strategies utilizing these inhibitors may represent a novel approach to blocking cancer cell growth. Further study is required to determine the downstream pathway(s) whereby high cellular burdens of unesterified AA promote apoptosis.     

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.     

Mechanical Disruption of Tumors by Iron Particles and Magnetic Field Application Results in Increased Anti-Tumor Immune Responses

The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor in situ. This was accomplished by intratumor injection of magneto-rheological fluid (MRF) consisting of iron microparticles, in Balb/c mice bearing orthotopic 4T1 breast cancer, followed by local application of a magnetic field resulting in immediate coalescence of the particles, tumor cell death, slower growth of primary tumors as well as decreased tumor progression in distant sites and metastatic spread. This treatment was associated with increased activation of DCs in the draining lymph nodes and recruitment of both DCs and CD8(+)T cells to the tumor. The particles remained within the tumor and no toxicities were observed. The immune induction observed was significantly greater compared to cryoablation. Further anti-tumor effects were observed when MRF/magnet therapy was combined with systemic low dose immunotherapy. Thus, mechanical disruption of the primary tumor with MRF/magnetic field application represents a novel means to induce systemic immune activation in cancer.     

Characterization,Release Profile and Antimicrobial Properties of Bioactive Polyvinyl Alcohol-Alyssum homolocarpum Seed Gum- Nisin Composite Film

A common goal of active packaging is to improve the shelf life, safety, or quality of packaged foods. The integrity of an active package must be remained in order to prevent the growth of microorganisms on the surface of food. Therefore, active polyvinyl alcohol-Alyssum homolocarpum seed gum (PVA-AHSG) composite films with different nisin concentrations (3000, 5000 and 10,000 IU) were prepared and their physico-chemical and antimicrobial properties were determined. Addition of nisin to the composite films increased their water vapor permeability (WVP), elongation at break (EB) and opacity, whereas their total color difference (ΔE), glass transition temperature (Tg), melting temperature (Tm), tensile strength (TS) and young modulus (YM) were decreased. Increasing the nisin concentration remarkably increased the chain mobility, interactions between polymers and water molecules and also the free volume of polymer matrix. The antimicrobial potential of film against L. monocytogene, S. aureus and E. coli as well as the release of nisin into phosphate buffer solution (pH 7.2) were investigated. Films containing nisin had inhibition effect against gram positive pathogens among which L. monocytogenes was the most sensitive bacterium. In liquid media, all films containing nisin prevented the growth of L. monocytogenes and S. aureus, but it was only the film with 10,000 IU nisin content which was able to control 100% of the microbial population during incubation time. Nisin release and diffusion coefficient (D) increased as its concentration increased in the film matrix due to the interaction of nisin with film polymer chains. Therefore, the resultant film had appropriate controlled release property and suitable microbial inhibitory against gram positive bacteria.

Composite bioactive films based on polyvinyl alcohol-Alyssum homolocarpum seed gum blend and Nisin: Physiochemical characterization and antimicrobial properties. Monjazeb et al. (2017).