Determinants of immunogenic response to protein therapeutics

Protein therapeutics occupy a very significant position in the biopharmaceutical market. In addition to the preclinical, clinical and post marketing challenges common to other drugs, unwanted immunogenicity is known to affect efficacy and/or safety of most biotherapeutics. A standard set of immunogenicity risk factors are routinely used to inform monitoring strategies in clinical studies. A number of in-silico, in vivo and in vitro approaches have also been employed to predict immunogenicity of biotherapeutics, but with limited success. Emerging data also indicates the role of immune tolerance mechanisms and impact of several product-related factors on modulating host immune responses. Thus, a comprehensive discussion of the impact of innate and adaptive mechanisms and molecules involved in induction of host immune responses on immunogenicity of protein therapeutics is needed. A detailed understanding of these issues is essential in order to fully exploit the therapeutic potential of this class of drugs. This Roundtable Session was designed to provide a common platform for discussing basic immunobiological and pharmacological issues related to the role of biotherapeutic-associated risk factors, as well as host immune system in immunogenicity against protein therapeutics. The session included overview presentations from three speakers, followed by a panel discussion with audience participation.     

Cr(VI) Imposed Toxicity in Maize Seedlings Assessed in Terms of Disruption in Carbohydrate Metabolism

The present study examined the toxic effects of Cr(VI; 100, 250 and 500 μM) in maize seedlings by investigating the changes in carbohydrate metabolism after 48, 96, and 144 h of exposure. Cr-stress results in severe alterations in the contents of carbohydrates and reducing sugars and the activities of carbohydrate metabolizing enzymes, amylases, phosphatases and phosphorylases, and invertases in maize seedlings. Under Cr stress, the contents of carbohydrates and reducing sugars declined in roots, whereas an increase was noticed in leaves. The catalytic activity of carbohydrate metabolizing enzymes, except invertases, in roots declined in the presence of Cr(VI) in a concentration- and exposure time-dependent manner. In contrast, the activities of these enzymes were enhanced in leaves under Cr(VI) stress. The activity of invertases increased with increasing amount of Cr(VI) but declined with an increase in the time interval. In conclusion, our results show that carbohydrate metabolism is severely affected under Cr(VI) toxicity. The study suggests that Cr-induced perturbations in the carbohydrate metabolism are one of the factors resulting in growth inhibition under Cr(VI) stress.     

BH3-Only Protein BIM Mediates Heat Shock-Induced Apoptosis

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax^−/−Bak^−/− cells and better than either Bid^−/− or dominant-negative caspase-9-expressing cells. Only Bim^−/− and Bax^−/−Bak^−/− cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid^−/− cells, it readily did so in Bim^−/− cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1^−/− cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X_L with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.     

SDS-capped 1-pyrenecarboxaldehyde nanoprobe for selective detection of Cu2+ ion from water samples: Spectroscopic approach

Sodium dodecyl sulfate (SDS)-capped 1-pyrenecarboxaldehyde nanoparticles (PyalNPs) were prepared using a reprecipitation method in an aqueous medium and exhibited red-shifted aggregation-induced enhanced emission (AIEE). The dynamic light scattering (DLS) examination showed narrower particle size distribution with an average particle size of 41 nm, whereas −34.5 mV zeta potential value indicate the negative surface charge and good stability of nanoparticles (NPs) in an aqueous medium. The AIEE was seen at λ_max = 473 nm in a fluorescence spectrum of a PyalNP suspension. In the presence of Cu²⁺ ions, the fluorescence of PyalNPs quenches very significantly, even in the presence of other metal ions like Ba²⁺, Ca²⁺, Cd²⁺, Co²⁺, Al³⁺, Fe²⁺, Hg²⁺, Ni²⁺ and Mg²⁺. The changes in the fluorescence lifetime of PyalNPs in the presence of Cu²⁺ ions suggested that the type of quenching was dynamic. The fluorescence quenching data for the NPs suspension fitted well into a typical Stern–Volmer relationship in the concentration range 1.0–25 μg/ml of Cu²⁺ ions. The estimated value of the correlation coefficient R² = 0.9877 was close to 1 and showed the linear relationship between quenching data and Cu²⁺ ion concentration. The limit of detection (LOD) was found to be 0.94 ng/ml and is far below the tolerable intake limit value of 1.3 μg/ml accepted by the World Health Organization for Cu²⁺ ions in drinking water. The fluorescence quenching approach for a SDS-capped Pyal nanosuspension for copper ion quantification is of high specificity and coexisting ions were found to interfere very negligibly. The developed method was successfully applied for the estimation of copper ions in river water samples.     

An efficient and versatile approach for the construction of oligonucleotide microarrays

A new immobilization chemistry for covalent attachment of phosphorylated oligonucleotides on epoxy-activated glass surface via opening of oxirane ring is described. The proposed strategy results in excellent immobilization efficiency, spot homogeneity, and morphology. The constructed microarray was successfully demonstrated for discrimination of nucleotide mismatches.     

Identification of metabolites of the tryptase inhibitor CRA-9249: observation of a metabolite derived from an unexpected hydroxylation pathway

The metabolites of the tryptase inhibitor CRA-9249 were identified after exposure to liver microsomes. CRA-9249 was found to be degraded rapidly in liver microsomes from rabbit, dog, cynomolgus monkey, and human, and less rapidly in microsomes from rat. The key metabolites included cleavage of an aryl ether, in addition to an unexpected hydroxylation of the amide side chain adjacent to the amide nitrogen. The chemical structures of both metabolites were confirmed by synthesis and comparison to material isolated from the liver microsomes. Several suspected hydroxylated metabolites were also synthesized and analyzed as part of the structure identification process.     

Ack1 Mediated AKT/PKB Tyrosine 176 Phosphorylation Regulates Its Activation

The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery.