Antibody-based immunotoxins for colorectal cancer therapy

Monoclonal antibodies (mAbs) are included among the treatment options for advanced colorectal cancer (CRC). However, while these mAbs effectively target cancer cells, they may have limited clinical activity. A strategy to improve their therapeutic potential is arming them with a toxic payload. Immunotoxins (ITX) combining the cell-killing ability of a toxin with the specificity of a mAb constitute a promising strategy for CRC therapy. However, several important challenges in optimizing ITX remain, including suboptimal pharmacokinetics and especially the immunogenicity of the toxin moiety. Nonetheless, ongoing research is working to solve these limitations and expand CRC patients’ therapeutic armory. In this review, we provide a comprehensive overview of targets and toxins employed in the design of ITX for CRC and highlight a wide selection of ITX tested in CRC patients as well as preclinical candidates. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

In vivo potential of recombinant granulysin against human melanoma

9-kDa granulysin is a protein expressed into the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It has been shown to exert cytolysis on microbes and tumors. We showed previously that 9-kDa granulysin exerted cell death by apoptosis in vitro on hematological tumor cell lines and also on cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. In addition, we have shown the anti-tumor efficiency of granulysin as a single agent in two in vivo models of human tumor development in athymic mice, the MDA-MB-231 mammary adenocarcinoma and the NCI-H929 multiple myeloma, without signs of overt secondary effects by itself. In this work, we have tested recombinant 9-kDa granulysin in an in vivo and especially aggressive model of melanoma development, xenografted UACC62 cells in athymic mice. Recombinant granulysin was administered once UACC62-derived tumors were detectable and it substantially retarded the in vivo development of this aggressive tumor. We could also detect apoptosis induction and increased NK cell infiltration inside granulysin-treated tumor tissues. These observations are especially interesting given the possibility of treating melanoma by intra-tumor injection

Conjugation of the 9-kDa isoform of Granulysin with liposomes potentiates its cytotoxicity

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni2+ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-xL. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway

Full thermoelectric characterization of a single molecule

Molecules are predicted to be chemically tunable towards high thermoelectric efficiencies and they could outperform existing materials in the field of energy conversion. However, their capabilities at the more technologically relevant temperature of 300 K are yet to be demonstrated. A possible reason could be the lack of a comprehensive technique able to measure the thermal and (thermo)electrical properties, including the role of phonon conduction. Here, by combining the break junction technique with a suspended heat-flux sensor, we measured the total thermal and electrical conductance of a single molecule, at room temperature, together with its Seebeck coefficient. We used this method to extract the figure of merit zT of a tailor-made oligo(phenyleneethynylene)-9,10-anthracenyl molecule with dihydrobenzo[b]thiophene anchoring groups (DHBT-OPE3-An), bridged between gold electrodes. The result is in excellent agreement with predictions from density functional theory and molecular dynamics. This work represents the first measurement, within the same setup, of experimental zT of a single molecule at room temperature and opens new opportunities for the screening of several possible molecules in the light of future thermoelectric applications. The protocol is verified using SAc-OPE3, for which individual measurements for its transport properties exist in the literature

The Acidic Probe LysoSensor™ is not Useful for Acrosome Evaluation of Cryopreserved Ram Spermatozoa

P. 363-367To try new acrosomal probes for assessing ram spermatozoa, we compared the LysoSensor™ probe, which labels acidic organelles, with the frequently used peanut agglutinin acrosomal probe (PNA‐PE; phycoerythrin as fluorescent moiety). The previous microscopic observations showed a lack of relationship of LysoSensor™ with acrosomal status. Semen was obtained from five rams and frozen in four pools. Each pool was analysed carrying out a triple staining propidium ioide/PNA‐PE/LysoSensor™ Green DND‐189 to test acrosome labelling, and a double staining SYBR‐14/PI, to assess sperm viability. Stained samples were analysed by flow cytometry. All measurements were replicated. Data were processed using agreement and repeatability tests. LysoSensor™ labelling did not agree with PNA (mean of differences: 30.8%; coefficient of agreement: 22.6%), confirming microscopic observations. Nevertheless, when LysoSensor™ was compared with SYBR‐14/PI, the agreement was high (mean of differences: −0.05%; coefficient of agreement: 5.07%). Repeatability of both methods was high and similar. LysoSensor™ did not seem to specifically stain the acrosome, but it may accumulate in the cytoplasm and label viable spermatozoa. Therefore, LysoSensor™ might not be used as an acrosomal probe in ram spermatozoa, but it could be used in other kind of studies, taking advantage of its pH sensitivity.S

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties

Stress Hormone Exposure Reduces mGluR5 Expression in the Nucleus Accumbens: Functional Implications for Interoceptive Sensitivity to Alcohol

Escalations in alcohol drinking associated with experiencing stressful life events and chronic life stressors may be related to altered sensitivity to the interoceptive/subjective effects of alcohol. Indeed, through the use of drug discrimination methods, rats show decreased sensitivity to the discriminative stimulus (interoceptive) effects of alcohol following exposure to the stress hormone corticosterone (CORT). This exposure produces heightened elevations in plasma CORT levels (eg, as may be experienced by an individual during stressful episodes). We hypothesized that decreased sensitivity to alcohol may be related, in part, to changes in metabotropic glutamate receptors-subtype 5 (mGluR5) in the nucleus accumbens, as these receptors in this brain region are known to regulate the discriminative stimulus effects of alcohol. In the accumbens, we found reduced mGluR5 expression (immunohistochemistry and Western blot) and decreased neural activation (as measured by c-Fos immunohistochemistry) in response to a moderate alcohol dose (1 g/kg) following CORT exposure (7 days). The functional role of these CORT-induced adaptations in relation to the discriminative stimulus effects of alcohol was confirmed, as both the systemic administration of 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) an mGluR5 positive allosteric modulator and the intra-accumbens administration of (R,S)-2-Amino-2-(2-chloro-5-hydroxyphenyl)acetic acid sodium salt (CHPG) an mGluR5 agonist restored sensitivity to alcohol in discrimination-trained rats. These results suggest that activation of mGluR5 may alleviate the functional impact of the CORT-induced downregulation of mGluR5 in relation to sensitivity to alcohol. Understanding the contribution of such neuroadaptations to the interoceptive effects of alcohol may enrich our understanding of potential changes in subjective sensitivity to alcohol during stressful episodes

Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis ‘knock-in’ strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis

Activation of mGluR2/3 following stress hormone exposure restores sensitivity to alcohol in rats

Sensitivity to the interoceptive effects of alcohol is blunted following a period of exposure to the stress hormone corticosterone (CORT), an effect that is suggested to be related, in part, to glutamatergic neuroadaptations. Group II metabotropic glutamate receptors (subtypes 2 and 3; mGluR2/3) modulate several drug- and alcohol-related behaviors, including the interoceptive (discriminative stimulus) effects of alcohol. Therefore, we sought to determine if manipulation of mGluR2/3 would restore sensitivity to the interoceptive effects of alcohol following CORT exposure. Using a two-lever drug discrimination task, male Long-Evans rats were trained to discriminate alcohol (1 g/kg, intragastric [IG]) vs. water. First, the effect of mGluR2/3 antagonism on the discriminative stimulus effects of alcohol was determined using LY341495 (0.3–3.0 mg/kg; intraperitoneal [IP]). Next, the effects of mGluR2/3 antagonism and activation were assessed in discrimination-trained animals exposed to CORT (300 μg/mL) in the home cage drinking water or water only, for 7 days. Following CORT exposure, decreased sensitivity to alcohol (1 g/kg) was observed. Pretreatment with the mGluR2/3 agonist LY379268 (1.0–3.0 mg/kg; IP), but not the mGluR2/3 antagonist (0.3–1.0 mg/kg; IP), restored sensitivity to alcohol. Additionally, in Water controls, mGluR2/3 antagonism and mGluR2/3 activation disrupted expression of the discriminative stimulus effects of alcohol. Together, these findings suggest that blunted sensitivity to the interoceptive effects of alcohol following an episode of heightened stress hormone levels may be due to adaptations in mGluR2/3-related systems. The ability of mGluR2/3 activation to restore sensitivity to alcohol under these conditions lends further support for the importance of these receptors under stress-related conditions