Referate

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Heterotopic heart transplantation alters high-energy phosphate metabolism irrespective of cardiac allograft rejection

This study was undertaken to validate the potential of ³¹P magnetic resonance spectroscopy (MRS) as a noninvasive alternative for transvenous endomyocardial biopsy in detecting cardiac allograft rejection. Donor hearts from either Lewis rats (L) or Brown-Norway rats (BN) were transplanted into the neck of L rats resulting in a non-rejecting group L-L and a rejecting group L-BN. L-L and L-BN rats were serially studied by means of ³¹P MRS from postoperatine day 1–8. In addition, rejection was confirmed by histology. A similar, marked decrease in phosphocreative/-adenosinetriphosphate (PCr/ATP) ratio from day 1–3 was observed in both L-L and L-BN hearts. This ratio levelled off on postoperative day 3 and remained depressed on subsequent postoperative days in both groups, although histology showed an increase in the severity of rejection in L-BN. However, the PCr signal/noise ratio in L-BN started to decrease after day 4, coinciding with the histologic evidence of severe rejection (score IV), whereas in L-L hearts (score 0) this ratio remained unaltered until day 8. Since high-energy phosphate metabolism is affected by the unloaded status of the heterotopically transplanted heart, irrespective of rejection, the PCr/ATP ratio appears not to be a specific marker for the detection of acute rejection in this model. In contrast, the PCr S/N ratio appears to be a specific and sensitive marker of acute rejection, but only in a late, severe stage.     

High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.     

Environmental Smoking and Smoking Onset in Adolescence: The Role of Dopamine-Related Genes. Findings from Two Longitudinal Studies

Although environmental smoking (i.e., paternal and maternal smoking, sibling smoking, and peer smoking) is one of the most important factors for explaining adolescent smoking behavior, not all adolescents are similarly affected. The extent to which individuals are vulnerable to smoking in their environment might depend on genetic factors. The aim of this study was to examine the interplay between environmental smoking and genes encoding components of the dopaminergic system (i.e., dopamine receptor D2, D4, and dopamine transporter DAT1) in adolescent smoking onset. Data from two longitudinal studies were used. Study 1 consisted of 991 non-smoking early adolescents (mean age = 12.52, SD = .57) whereas study 2 consisted of 365 non-smoking middle to late adolescents (mean age = 14.16, SD = 1.07) who were followed for 16 and 48 months, respectively. Logistic regression analyses were conducted using Mplus. In study 1, we found positive associations between parents'' and friends'' smoking at the first measurement and smoking status 16 months later. In study 2 we found a positive association between friends'' smoking and smoking onset 48 months later. Neither study demonstrated any interaction effects of the DRD2, DRD4, or DAT1 genotypes. In conclusion, the effects of environmental smoking on smoking onset are similar for adolescent carriers and non-carriers of these specific genes related to the dopaminergic system.     

Muscle activity during the active straight leg raise (ASLR), and the effects of a pelvic belt on the ASLR and on treadmill walking

Women with pregnancy-related pelvic girdle pain (PPP), or athletes with groin pain, may have trouble with the active straight leg raise (ASLR), for which a pelvic belt can be beneficial. How the problems emerge, or how the belt works, remains insufficiently understood. We assessed muscle activity during ASLR, and how it changes with a pelvic belt. Healthy nulligravidae (N=17) performed the ASLR, and walked on a treadmill at increasing speeds, without and with a belt. Fine-wire electromyography (EMG) was used to record activity of the mm. psoas, iliacus and transversus abdominis, while other hip and trunk muscles were recorded with surface EMG. In ASLR, all muscles were active. In both tasks, transverse and oblique abdominal muscles were less active with the belt. In ASLR, there was more activity of the contralateral m. biceps femoris, and in treadmill walking of the m. gluteus maximus in conditions with a belt. For our interpretation, we take our starting point in the fact that hip flexors exert a forward rotating torque on the ilium. Apparently, the abdominal wall was active to prevent such forward rotation. If transverse and oblique abdominal muscles press the ilia against the sacrum (Snijders’ “force closure”), the pelvis may move as one unit in the sagittal plane, and also contralateral hip extensor activity will stabilize the ipsilateral ilium. The fact that transverse and oblique abdominal muscles were less active in conditions with a pelvic belt suggests that the belt provides such “force closure”, thus confirming Snijders’ theory.     

The C Terminal Tail of the Histamine H2 Receptor Contains Positive and Negative Signals Important for Signal Transduction and Receptor Down-Regulation

Abstract: To examine the role of the C terminal tail in H₂ receptor regulation, three cDNAs, encoding truncated histamine H₂ receptor mutants (H₂T295, H₂T307, and H₂T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H₂ receptor binding of the H₂T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H₂T307) did not affect the binding properties of H₂ antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H₂T341), which was able to form a ternary complex but was unable to fully activate the G_s protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H₂ receptor down-regulation was more profound for the H₂T307 receptor, indicating that different structural elements of the H₂ receptor protein are involved in the cyclic AMP-dependent and independent pathways of H₂ receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H₂ receptor that act as positive and/or negative signals in H₂ receptor signaling and down-regulation.