Blockade of the erbB2 Receptor Induces Cardiomyocyte Death through Mitochondrial and Reactive Oxygen Species-dependent Pathways

Overexpression of the receptor tyrosine kinase erbB2 (Her2 in humans) is correlated with a poor prognosis in breast and ovarian cancers. Treatment with trastuzumab (a monoclonal antibody against erbB2) improves survival; however, it also causes cardiomyopathy. We hypothesized that blockade of the erbB2 receptor induces cardiomyocyte death through a mitochondrial pathway that is dependent on the production of reactive oxygen species (ROS). We first showed that levels of erbB2 receptor are significantly decreased in an animal model of ischemic heart disease and in human ischemic cardiomyopathy. We treated neonatal rat cardiomyocytes with an inhibitory erbB2 antibody to study the mechanism behind the deleterious effects of erbB2 blockade. These cells displayed a dose-dependent increase in ROS production and cell death compared with control IgG-treated cells; these processes were reversed by the antioxidant, N-acetylcysteine. The effects of erbB2 antibody on both cell death and ROS production were also reversed by cyclosporine A and diazoxide, chemicals that regulate the pro- and anti-apoptotic channels in the mitochondria, respectively. Furthermore, mouse embryonic fibroblasts lacking Bax and Bak (proteins that mediate cell death through a mitochondrial pathway) were resistant to the deleterious effects of erbB2 antibody. These effects of erbB2 blockade appear to occur through a pathway involving AKT and PKC-α. Our results suggest that erbB2 plays a role in cardiomyocyte survival, and that the deleterious effects of trastuzumab on the heart occur through a mitochondrial pathway and is mediated by ROS production. Manipulation of redox signaling may be beneficial in cancer patients receiving trastuzumab.The Her-2/neu oncogene, also known as erbB2 in nonhuman organisms, is a transmembrane receptor tyrosine kinase that belongs to the epidermal growth factor receptor family (1, 2). Overexpression of Her2 is seen in ∼30% of breast cancer patients and is associated with poor survival, increased metastasis, and resistance to chemotherapy (3–5). Transgenic mice overexpressing erbB2 develop focal mammary tumors, thus implicating this protein in tumorigenesis (6). Trastuzumab (Herceptin, Genentech, CA) is a monoclonal antibody (Ab)² that binds to Her2 with high affinity and improves survival of patients with advanced breast cancer (7). Trastuzumab is clinically efficacious both as a single agent or in combination with standard chemotherapy regimens (4–6). However, this agent is cardiotoxic on its own, and especially when administered with anthracyclines, where it can cause cardiomyopathy (CM) in up to 27% of patients (8).The importance of erbB2 in normal cardiac development and physiology was demonstrated in mice by cardiac-specific knock-out of erbB2 (9, 10). The mice were initially normal, but developed CM as adults. One study demonstrated no difference between the wild-type and knock-out mice in the degree of cardiac cell death as assessed by TUNEL staining (10). However, in another study that used a more sensitive PCR-based DNA fragmentation assay increased DNA fragmentation was reported in the hearts of erbB2-knock-out animals (9). Recently, Grazette et al. (11) studied the effects of erbB2 blockade on cardiomyocyte survival, and showed that erbB2 antibody (erbB2-Ab) caused a loss of mitochondrial membrane potential and an increase in cell death.The mechanism for the deleterious effects of erbB2 blockade remains unclear, but a recent report showed that activation of erbB2 reduces doxorubicin-induced oxidative stress in cardiomyocytes (12). Therefore, we hypothesized that erbB2-Ab-induced cell death in cardiomyocytes is a mitochondrial dependent process that involves ROS production. In this report, we show that erbB2 levels are decreased in an animal model of myocardial ischemia and in patients with ischemic CM. We then demonstrate that erbB2 blockade in cardiomyocytes leads to ROS production, and that the antioxidant N-acetylcysteine (NAC) protects against the damage induced by erbB2-Ab. We also find that erbB2 signaling in cardiomyocytes occurs through a mitochondrial, AKT-, and PKCα-dependent pathway. Moreover, the deleterious effects caused by the loss of erbB2 function require the pro-apoptotic proteins Bax and Bak. Finally, by using an erbB2-specific siRNA, we demonstrate that the effects of erbB2 blockade evolve from the specific inhibition of the erbB2 pathway rather than through nonspecific effects of the antibody. Together, our results suggest that erbB2 blockade increases ROS through a mitochondrial pathway.     

Ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves

Most estrous cycles in cows consist of 2 or 3 waves of follicular activity. Waves of ovarian follicular development comprise the growth of dominant follicles some of which become ovulatory and the others are anovulatory. Ovarian follicular activity in cows during estrous cycle was studied with a special reference to follicular waves and the circulating concentrations of estradiol and progesterone. Transrectal ultrasound examination was carried out during 14 interovulatory intervals in 7 cows. Ovarian follicular activity was recorded together with assessment of serum estradiol and progesterone concentrations. Three-wave versus two-wave interovulatory intervals was observed in 71.4% of cows. The 3-wave interovulatory intervals differed from 2-wave intervals in: 1) earlier emergence of the dominant follicles, 2) longer in length, and 3) shorter interval from emergence to ovulation. There was a progressive increase in follicular size and estradiol production during growth phase of each wave. A drop in estradiol concentration was observed during the static phase of dominant anovulatory follicles. The size of the ovulatory follicle was always greater and produced higher estradiol compared with the anovulatory follicle. In conclusion, there was a predominance of 3-wave follicular activity that was associated with an increase in length of interovulatory intervals. A dominant anovulatory follicle during its static phase may initiate the emergence of a subsequent wave. Follicular size and estradiol concentration may have an important role in controlling follicular development and in determining whether an estrous cycle will have 2 or 3-waves.     

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N - acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.      

First record of lily thrips, Liothrips vaneeckei Priesner, in Australia (Thysanoptera: Phlaeothripidae)

The first Australian record of the lily thrips, Liothrips vaneeckei Priesner, is reported from a bulb farm in Warragul South, Victoria. It is an occasional pest of Lilium bulbs, both in the field and in storage, particularly in the USA and several European countries, and is also infrequently found in considerable numbers on the corms of orchids.     

CD40 signaling replaces CD4+ lymphocytes and its blocking prevents chronic rejection of heart transplants

Chronic rejection remains the major obstacle to long term survival in heart transplant recipients. The cellular and molecular mechanisms that underlie chronic rejection are not known, and their discovery can form the basis of clinical intervention. Several investigators have suggested that the development of chronic rejection in solid organ transplants is dependent on help mediated by CD4(+) lymphocytes. Importantly, the mechanism through which help is provided has not been fully delineated in transplant rejection. Using a murine heterotopic heart transplant model without immunosuppression, this study defines the functional role of CD4(+) lymphocytes in chronic rejection. In an MHC class II-mismatched model, we demonstrate that chronic rejection was absolutely contingent on the presence of CD4(+) lymphocytes. Importantly, here we report that signaling through CD40 can replace the requirement of CD4(+) lymphocytes, demonstrated by the development of chronic rejection in CD4 knockout recipients treated with a CD40-activating mAb (FGK45). The return of rejection appears to be a CD8(+) lymphocyte-dependent process, noted by the absence of rejection in FGK45-treated recombinase-activated gene knockout (CD4(+) and CD8(+) lymphocyte-deficient) recipients. The CD40 signaling pathway works independently of B7-CD28 costimulation, as indicated by the development of severe chronic rejection in CD28 knockout recipients. Importantly, this study provides evidence that CD40 ligand-targeted therapies may prevent chronic rejection only in strain combinations where CD4(+) lymphocyte help is absolutely required.