Conditional Gene Targeting in Mouse Pancreatic ��-Cells: Analysis of Ectopic Cre Transgene Expression in the Brain

OBJECTIVE

Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain.

RESEARCH DESIGN AND METHODS

Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1^tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.

RESULTS

All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)^89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)^1Lphi mice were the only line that lacked Cre activity in the brain.

CONCLUSIONS

Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.In vivo analysis of gene function in the pancreas and β-cells has benefited from the development of mouse lines expressing Cre in all pancreatic compartments or restricted to the islet β-cells. The choice of promoter to drive recombinase expression is critical for controlling the location and timing of gene activity. In addition, inducible versions of Cre recombinase, e.g., CreER, allow temporal control to the manipulation of gene activity, which becomes important when analyzing gene function at specific embryonic and adult stages (1,2). Promoters of the pancreas duodenal homeobox 1 (Pdx1) (3,4) and insulin (Ins1 and Ins2) (5–8) genes have been well characterized to allow the use of regulatory sequences for directing Cre expression to specific pancreatic cell populations. Commonly used transgenic mouse lines that employ rat Ins2 gene promoter sequences to drive Cre expression within the β-cell population include Ins2-Cre/RIP-Cre [Mouse Genome Informatics (MGI): Tg(Ins2-cre)^25Mgn and Tg(Ins2-cre)^1Herr] (9–11) and RIP-CreER [MGI: Tg(Ins2-cre/Esr1)^1Dam] (12). Pdx1 gene promoter sequences have proven useful for directing Cre expression throughout the early pancreatic epithelium (4,10,13,14) and to the endocrine cells of the pancreas (15). The Pdx1 gene is expressed early in pancreas development throughout the endoderm of the dorsal and ventral buds, but expression becomes restricted during development such that high levels of Pdx1 are maintained in the insulin-producing β-cells with lower levels in subpopulations of acinar cells (8,16). Examples of Pdx1-Cre transgenic lines include Pdx1-Cre^early [MGI: Tg(Pdx1-cre)^89.1Dam] (13), Pdx1-Cre^late [MGI: Tg(Ipf1-cre/Esr1)^1Dam/Mmcd] (10), Pdx1-Cre [MGI: Tg(Ipf1-cre)^1Tuv] (14), and Pdx1-CreER [MGI: Tg(Pdx1-cre/ERT)^1Mga] (15).To assess the specificity of recombination and perform lineage tracing analysis, reporter lines such as the ROSA26-stop-lacZ [MGI: Gt(ROSA)26Sor^tm1Sho], also known as R26R (17), or the ROSA26-stop-YFP [MGI: Gt(ROSA)26Sor^tm1(EYFP)Cos] (18) mice have been developed. Upon Cre-mediated recombination, these reporter lines activate expression of a β-galactosidase (β-gal) or a yellow fluorescent protein (YFP) reporter under the control of the ubiquitously active ROSA26 promoter, resulting in expression that is stably inherited by all cell progeny regardless of their differentiation fate.Here we show that most Cre lines currently being used to mediate pancreas or β-cell recombination also direct Cre expression to areas of the brain, and this may lead to altered gene expression in nutrient-sensing neurons that affects nutrient homeostasis.     

After facing traumatic stress: Brain activation, cognition and stress coping in policemen

Introduction

Resilience can be defined as the capacity to recover following stress or trauma exposure by adopting healthy strategies for dealing with trauma and stress. Although the importance of stress resilience has been recognized, the underlying neurocognitive mediators have not yet been identified. Thus, the primary goal of this study was to investigate memory-related brain activity in traumatized policemen who attended a pre-traumatic general stress coping program.

Method

Ten traumatized male police officers were compared to demographically matched non-traumatized officers (n = 15) on associative memory by using a block design paradigm. Participants with either another psychiatric comorbidity or neurological disorder were excluded.During functional brain imaging (1.5-Tesla), face-profession pairs had to be encoded twice. For subsequent retrieval the faces were presented as cue stimuli for associating the category of the prior learned profession. Additionally, clinical pattern, stress coping style, and cognitive parameters were assessed.

Results

Less BOLD activation was found in the hippocampus, parahippocampal gyrus and fusiform gyrus in the trauma group when compared with the non-trauma group during encoding. This was accompanied by slower reaction times in the trauma group during retrieval. Further impairments were found in context memory and in the use of positive cognitive coping strategies.

Discussion

Support was provided for the presence of memory-related disturbances in brain activity associated with trauma even in a resilient population. The contribution of the changes in stress coping ability needs to be further examined in longitudinal studies.     

Effect of protein binding on the pharmacological activity of highly bound antibiotics

During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used protein supplements. Free, unbound ceftriaxone and ertapenem concentrations were determined in bacterial growth medium with and without bovine/human serum albumin, as well as adult bovine serum and human plasma using in vitro microdialysis. The corresponding antimicrobial activity was determined in MIC and time-kill curve experiments using Escherichia coli ATCC 25922 and Streptococcus pneumoniae ATCC 6303 as test strains. A semimechanistic maximum effect model was simultaneously fitted to the data and respective EC₅₀ (concentration at half-maximum effect) values compared. Protein binding differed significantly for ceftriaxone (P < 0.05) between human plasma (76.8 ± 11.0%) and commercially available bovine (20.2 ± 8.3%) or human serum albumin (56.9 ± 16.6%). Similar results were obtained for ertapenem (human plasma, 73.8 ± 11.6%; bovine serum albumin, 12.4 ± 4.8%; human serum albumin, 17.8 ± 11.5%). The MICs and EC₅₀s of both strains were significantly increased (P < 0.05) for ceftriaxone when comparing human and bovine serum albumin, whereas the EC₅₀s were not significantly different for ertapenem. Free, unbound antibiotic concentrations differed substantially between plasma and protein supplements and correlated well with antimicrobial efficacy. Therefore, free, active concentrations should be measured in the test system instead of correcting for literature protein binding values.     

Overexpression of CXCL16 and its receptor CXCR6/Bonzo promotes growth of human schwannomas

Chemokines and their receptors play a decisive role in tumor progression and metastasis. Here, we describe the expression of the CXCL16-CXCR6-system in human schwannomas of different localization and in malignant peripheral nerve sheath tumors. The transmembrane chemokine CXCL16 and its receptor CXCR6/Bonzo were overexpressed on the mRNA and protein levels in all tumor samples investigated as compared with normal peripheral or 8th cranial nerve tissues. Chromogenic immunostaining and confocal laser microscopy revealed that CXCL16 and CXCR6 were localized mainly on S-100 positive schwannoma cells. Cultured schwannoma cells responded to CXCL16-stimulation by phosphorylation of kinases p42/44 (Erk 2/1) that could be inhibited by the MEK1/2-inhibitor U0126 indicating an involvement of the mitogen-activated protein kinase signal transduction pathway. As a biological response, CXCL16 increased proliferation and induced migration of schwannomas. Hence, CXCL16 appears to be a novel growth factor for schwannomas of different localization.     

Intraoperative mapping of language functions: a longitudinal neurolinguistic analysis

OBJECT: This prospective longitudinally designed study was conducted to evaluate language functions pre- and postoperatively in patients who underwent microsurgical treatment of tumors in close proximity to or within language areas and to detect those patients at risk for a postoperative aphasic disturbance. METHODS: Between 1991 and 2005, 153 awake craniotomies with subsequent cortical mapping of language functions were performed in 149 patients. Language functions were assessed using a standardized test battery. Risk factors were obtained from multivariate logistic regression models. RESULTS: Language mapping was able to be performed in all patients, and complete tumor resection was achieved in 48.4%. Within 21 days after surgery a new language deficit (aphasic disturbance) was observed in 41 (32%) of the 128 cases without preoperative deficits. There were a total of 60 cases involving postoperative aphasic disturbances, including cases both with and without preoperative disturbances. Risk factors for postoperative aphasic disturbance were preoperative aphasia (p<0.0002), intraoperative complications (p<0.02), language-positive sites within the tumor (p<0.001), and nonfrontal lesion location (p<0.001). In patients without a preoperative deficit, a normal (yet submaximal) naming performance was a powerful predictor for an early postoperative aphasic disturbance (p<0.0003). Seven months after treatment 10.9% of the 128 cases without preoperative aphasic disturbances continued to demonstrate new postoperative language disturbances. A total of 17.6% of all cases demonstrated new postoperative language disturbances after 7 months. Risk factors for persistent aphasic disturbance were increased age (>40 years, p<0.02) and preoperative aphasia (p<0.001). CONCLUSIONS: Every attempt should be undertaken to preserve language-relevant areas intraoperatively, even when they are located within the tumor. New postoperative deficits resolve in the majority of patients, which may be a result of cortical mapping as well as functional reorganization.