Smac mimetics as novel promising modulators of apoptosis in the treatment of breast cancer

Breast cancer is the most prevalent cancer in women. Despite improvements in treatment, the rate of breast cancer-related deaths is still high, and this issue needs further, accurate investigations. Although several treatment options are available, none of them are efficient for complete remission, particularly in advanced stages of the disease. It is known that cancerous cells have dysregulated apoptosis-related pathways, by which they can remain alive for a long time, expand freely, and escape from apoptosis-inducing drugs or antitumor immune responses. Therefore, modulation of apoptosis resistance in cancer cells may be an efficient strategy to overcome current problems faced in the development of immunotherapeutic approaches for the treatment of breast cancer. The inhibitors of apoptosis protein (IAPs) are important targets for cancer therapy because it has been shown that these molecules are overexpressed and highly active in various cancer cells and suppress apoptosis process in malignant cells by blockage of caspase proteins. There is evidence of Smac mimetics efficacy as a single agent; however, recent studies have indicated the efficacy of current anticancer immunotherapeutic approaches when combined with Smac mimetics, which are potent inhibitors of IAPs and synthesized mimicking Smac/Diablo molecules. In this review, we are going to discuss the efficacy of treatment of breast cancer by Smac mimetics alone or in combination with other therapeutics.     

Vertebral function during tadpole locomotion

Most anuran larvae show large lateral oscillations at both the tip of the tail and the snout while swimming in a straight line. Although the lateral deflections at the snout have long been considered an inefficient aspect of tadpole locomotion, a recent hydrodynamic model suggests that they may in fact help generate thrust. It is not clear though exactly where this bending takes place. The vertebral column is extremely short and seemingly inflexible in anurans, and any axial flexion that might occur there is hidden within the globose body of the tadpole. Here we test the hypothesis that lateral deflections of the snout correlate with bending of the vertebral column within the torso of tadpoles. To quantify vertebral curvature, three sonomicrometry crystals were surgically implanted along the dorsal midline in locations corresponding to the anterior, middle, and posterior region of the presacral vertebral column. Swimming trials were conducted in a flume where synchronized video recordings were collected in dorsal view. Our results confirm that cyclic lateral bending occurs along the vertebral column during swimming and indicate that vertebral curvature is temporally in phase with lateral oscillation of the snout. Lateral oscillation of the snout increased significantly with increasing vertebral curvature. Similarly, tail beat amplitude also increases significantly with increasing vertebral curvature. Our results suggest that cyclic lateral flexion of the vertebral column, activated by the axial muscle within the torso of tadpoles contributes to snout oscillations and the generation of thrust during undulatory swimming in anuran larvae.     

An Inverse Finite Element Method for Determining the Tissue Compressibility of Human Left Ventricular Wall during the Cardiac Cycle

The determination of the myocardium’s tissue properties is important in constructing functional finite element (FE) models of the human heart. To obtain accurate properties especially for functional modeling of a heart, tissue properties have to be determined in vivo. At present, there are only few in vivo methods that can be applied to characterize the internal myocardium tissue mechanics. This work introduced and evaluated an FE inverse method to determine the myocardial tissue compressibility. Specifically, it combined an inverse FE method with the experimentally-measured left ventricular (LV) internal cavity pressure and volume versus time curves. Results indicated that the FE inverse method showed good correlation between LV repolarization and the variations in the myocardium tissue bulk modulus K (K = 1/compressibility), as well as provided an ability to describe in vivo human myocardium material behavior. The myocardium bulk modulus can be effectively used as a diagnostic tool of the heart ejection fraction. The model developed is proved to be robust and efficient. It offers a new perspective and means to the study of living-myocardium tissue properties, as it shows the variation of the bulk modulus throughout the cardiac cycle.     

Evaluating the efficiency of phiC31 integrase‐mediated monoclonal antibody expression in CHO cells

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site‐specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO‐S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570–1576, 2016     

Prenatal Testosterone Exposure Worsen the Reproductive Performance of Male Rat at Adulthood

The reproductive system is extremely susceptible to environmental insults, for example exogenous steroids during gestational development and differentiation. Experimental induction of androgen excess during prenatal life in female animal models reprograms their reproductive physiology, however the fetal programming of the male reproductive system by androgen excess has not been well studied. We aimed to determine the effect of prenatal exposure of two different doses of testosterone on different gestational days, on the male reproductive system using a rat model. Sixteen pregnant rats were randomly divided into two experimental groups and two control groups. Experimental group І were subcutaneously injected with 3 mg free testosterone on gestational days 16-19 and its controls received solvent for that time; experimental group П were subcutaneously injected with 20 mg free testosterone on day 20 of gestational period and its controls received solvent at the same time. The reproductive system morphology and function of 32 male offspring of these study groups were compared at days 6-30-60 of age and after puberty. The anogenital distance of the male offspring of both experimental groups had no significant differences on the different days of measurement, compared with controls. In the offspring of experimental group І, the testes weight, number of Sertoli, Spermatocyte and Spermatid cells, sperm count and motility and the serum concentration of testosterone after puberty were significantly decreased; except for reduction of sperm motility (p< 0.01), the other effects were not observed in the offspring of experimental group ІІ. In summary, our data show that prenatal exposure of male rat fetuses to excess testosterone disrupted reproductive function, an effect highly dependent on the time, duration and level of exposure. It seems that the reproductive system in individuals exposed to high levels of androgens during fetal life should be evaluated at puberty and likely to be treated.     

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.     

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: Towards tumor-directed oligoclonal T cell therapy

Background

Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells.

Methods

We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated.

Results

The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells.

Conclusions

The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells.

General significance

Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.