Predictors and Outcomes of Cardiac Surgery-Associated Delirium. A Single Centre Retrospective Cohort Study.

BACKGROUND: The predictors and independent outcome association of delirium after cardiac surgery are important and yet poorly characterised. METHODS: We performed a retrospective observational study of cardiac surgery patients between January 2009 and March 2016. We defined delirium using ICD-10 diagnostic codes. Multivariable analysis was conducted to find independent associations between baseline variables, delirium, and key clinical outcomes. RESULTS: We studied 2,447 study patients (28.7% female, median age was 66 [IQR 57-74] years). Delirium was coded for in 12.9% of patients overall, and in 22.9% of those aged >75years. Increasing age, Charlson co-morbidity index, admission not from home, peripheral vascular disease, respiratory disease, preoperative atrial fibrillation, duration of cardiopulmonary bypass and nature of surgery were all independent predictors of delirium. Delirium was independently and strongly associated with increased risk of reintubation (OR 8.18 [95% CI 5.24-12.78]), tracheostomy (OR 10.44 [95% CI 5.91-18.45]), and increased length of stay by 113.7 [95% CI 99.7-127.7] ICU hours and 6.95 [95% CI 5.94-7.95] hospital days, but not 30-day mortality (OR 0.78 [95% CI 0.38-1.59]; p=0.5). CONCLUSIONS: Delirium is common in cardiac surgery patients and increases with age. Delirium was the strongest predictor of reintubation, need for tracheostomy, and prolongation of intensive care unit (ICU) and hospital length of stay. Delirium prevention and attenuation are a priority in cardiac surgery patients.

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed.

Exploring the retinal connectome.

PURPOSE: A connectome is a comprehensive description of synaptic connectivity for a neural domain. Our goal was to produce a connectome data set for the inner plexiform layer of the mammalian retina. This paper describes our first retinal connectome, validates the method, and provides key initial findings. METHODS: We acquired and assembled a 16.5 terabyte connectome data set RC1 for the rabbit retina at ≈ 2 nm resolution using automated transmission electron microscope imaging, automated mosaicking, and automated volume registration. RC1 represents a column of tissue 0.25 mm in diameter, spanning the inner nuclear, inner plexiform, and ganglion cell layers. To enhance ultrastructural tracing, we included molecular markers for 4-aminobutyrate (GABA), glutamate, glycine, taurine, glutamine, and the in vivo activity marker, 1-amino-4-guanidobutane. This enabled us to distinguish GABAergic and glycinergic amacrine cells; to identify ON bipolar cells coupled to glycinergic cells; and to discriminate different kinds of bipolar, amacrine, and ganglion cells based on their molecular signatures and activity. The data set was explored and annotated with Viking, our multiuser navigation tool. Annotations were exported to additional applications to render cells, visualize network graphs, and query the database. RESULTS: Exploration of RC1 showed that the 2 nm resolution readily recapitulated well known connections and revealed several new features of retinal organization: (1) The well known AII amacrine cell pathway displayed more complexity than previously reported, with no less than 17 distinct signaling modes, including ribbon synapse inputs from OFF bipolar cells, wide-field ON cone bipolar cells and rod bipolar cells, and extensive input from cone-pathway amacrine cells. (2) The axons of most cone bipolar cells formed a distinct signal integration compartment, with ON cone bipolar cell axonal synapses targeting diverse cell types. Both ON and OFF bipolar cells receive axonal veto synapses. (3) Chains of conventional synapses were very common, with intercalated glycinergic-GABAergic chains and very long chains associated with starburst amacrine cells. Glycinergic amacrine cells clearly play a major role in ON-OFF crossover inhibition. (4) Molecular and excitation mapping clearly segregates ultrastructurally defined bipolar cell groups into different response clusters. (5) Finally, low-resolution electron or optical imaging cannot reliably map synaptic connections by process geometry, as adjacency without synaptic contact is abundant in the retina. Only direct visualization of synapses and gap junctions suffices. CONCLUSIONS: Connectome assembly and analysis using conventional transmission electron microscopy is now practical for network discovery. Our surveys of volume RC1 demonstrate that previously studied systems such as the AII amacrine cell network involve more network motifs than previously known. The AII network, primarily considered a scotopic pathway, clearly derives ribbon synapse input from photopic ON and OFF cone bipolar cell networks and extensive photopic GABAergic amacrine cell inputs. Further, bipolar cells show extensive inputs and outputs along their axons, similar to multistratified nonmammalian bipolar cells. Physiologic evidence of significant ON-OFF channel crossover is strongly supported by our anatomic data, showing alternating glycine-to-GABA paths. Long chains of amacrine cell networks likely arise from homocellular GABAergic synapses between starburst amacrine cells. Deeper analysis of RC1 offers the opportunity for more complete descriptions of specific networks.

Palliative care and COVID-19 in the Australian context: a review of patients with COVID-19 referred to palliative care.

Objective Palliative care has played a key role in the response to the coronavirus disease 2019 (COVID-19) pandemic in Australia. This review of consecutive patients with COVID-19 referred to the palliative care consultancy service of a tertiary health service in Melbourne describes the palliative care experience with COVID-19 in Australia. Methods The experiences of 55 patients (median age 86 years; interquartile range (IQR) 81-90 years; 55% male; median Charlson comorbidity score 6 (IQR 5-8); 85% with Australia-modified Karnofsky Performance Status ≤50; 67% from residential aged care facilities) were reviewed to collect relevant data points. Results Most patients were referred for end-of-life care with symptoms including dyspnoea (80%) and agitation/delirium (60%). Continuous subcutaneous infusions were commenced in 71% of patients, with the most frequent medications being opioids and benzodiazepines in relatively small doses; 81% required ≤20 mg subcutaneous morphine equivalent and 64% required ≤10 mg subcutaneous midazolam over 24 h. Fifty patients (91%) died in hospital and the median time from palliative care referral to death was 3 days (IQR 1-5 days). Five patients were discharged back to residential aged care facilities. Overall, 80% of referrals were from the aged care team. Conclusion Our patients had similar demographics, symptoms, medication needs and outcomes to patients in similar settings overseas. We found the symptom management of patients with COVID-19 to be generally straightforward. However, the psychosocial needs of patients were predominant and contributed to complexity. This study highlights the need for well-integrated relationships between the palliative care consultancy service and the diverse range of key treating teams involved in the delivery of pandemic health care. What is known about the topic? Palliative care has played a key role in the response to the COVID-19 pandemic in Australia. There is limited research describing the Australian palliative care experience with the COVID-19 pandemic. What does this paper add? Patients with COVID-19 referred to a hospital-based palliative care consultancy service in Australia had similar demographic characteristics, symptoms, medication needs and outcomes to patients with COVID-19 referred to other palliative care services in the UK and the US. There were significant psychosocial issues affecting patients, families and staff in the context of the pandemic. What are the implications for practitioners? This study highlights the need for well-functioning working relationships between the palliative care consultancy service and other hospital teams that can be leveraged at a time of crisis, such as a pandemic, to provide optimal palliative care to patients.

Impact of chronic kidney disease on patient outcome following cardiac surgery.

BACKGROUND: Renal impairment is a major risk factor for cardiovascular disease. This study addressed clinical predictors of outcome following cardiac surgery, focusing on pre-operative renal dysfunction. METHODS: All patients undergoing cardiac surgery at Austin Health from June 1, 2001 to June 30, 2006, were included in the analysis. Logistic regression models were used to evaluate clinical factors predicting "operative mortality" and common post-operative complications. RESULTS: The operative mortality was 1.36% for coronary artery bypass grafting (CABG) alone (n=1027), 5.07% for valve surgery alone (n=217), 4.43% for combined CABG and valve surgery (n=158) and 11.11% for other cardiac surgical procedures (n=270). Amongst CABG alone patients, pre-operative renal impairment was a strong predictor of operative mortality, with a 35-43% increased risk of death (p=0.005) for every 10 ml/min/1.73 m(2) that the glomerular filtration rate was lower. Peripheral vascular disease, recent myocardial infarction and congestive cardiac failure also predicted operative mortality. Pre-operative renal impairment also increased the rate of various post-operative complications, as well as duration of admission. CONCLUSION: Renal dysfunction is significantly associated with increased mortality and morbidity following cardiac surgery and necessitates careful consideration in risk benefit analysis when considering cardiac surgery.

Evaluation of Internal Medicine Physician or Multidisciplinary Team Comanagement of Surgical Patients and Clinical Outcomes: A Systematic Review and Meta-analysis.

Importance: Older patients who undergo surgery may benefit from geriatrician comanagement. It is unclear whether other internal medicine (IM) physician involvement improves outcomes for adults who undergo surgery. Objective: To evaluate the association of IM physician involvement with clinical and health system outcomes compared with usual surgical care among adults who undergo surgery. Data Sources: MEDLINE, Embase, CINAHL, and CENTRAL databases were searched for studies published in English from database inception to April 2, 2019. Study Selection: Prospective randomized or nonrandomized clinical studies comparing IM physician consultation or comanagement with usual surgical care were selected by consensus of 2 reviewers. Data Extraction and Synthesis: Data were extracted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline by 2 authors independently. Intervention characteristics were described using existing indicators. Risk of bias was assessed using Risk of Bias 2.0 and Risk of Bias in Nonrandomized Studies of Interventions tools. Studies were pooled when appropriate in meta-analysis using random-effects models. Prespecified subgroups included IM physician-only vs multidisciplinary team interventions and patients undergoing elective vs emergency procedures. Main Outcomes and Measures: The prespecified primary outcome was length of stay; other outcomes included 30-day readmissions, inpatient mortality, medical complications, functional outcomes, and costs. Results: Of 6027 records screened, 14 studies (with 1 randomized clinical trial) involving 35 800 patients (13 142 [36.7%] in intervention groups) were eligible for inclusion. Interventions varied substantially among studies and settings; most interventions described comanagement by a hospitalist or internist; 7 (50%) included a multidisciplinary team, and 9 (64%) studied predominantly patients who had elective procedures. Risk of bias in 10 studies (71%) was serious. Meta-analysis showed no significant association with length of stay (mean difference, -1.02 days; 95% CI, -2.09 to 0.04 days; P = .06) or mortality (odds ratio, 0.79; 95% CI, 0.56 to 1.11; P = .18), but multidisciplinary team involvement was associated with significant reduction in length of stay (mean difference, -2.03 days; 95% CI, -4.05 to -0.01 days; P = .05) and mortality (odds ratio, 0.67; 95% CI, 0.51 to 0.88; P = .004). There was no difference in 30-day readmissions (odds ratio, 0.89; 95% CI, 0.68 to 1.16; P = .39). Data could not be pooled for complications or costs. Only 1 study (7%) reported functional outcomes. Conclusions and Relevance: The findings of this study suggest that IM physician comanagement that includes multidisciplinary team involvement may be associated with reduced length of stay and mortality in adults undergoing surgery. Evidence was low quality, and well-designed prospective studies are still needed.

Characterisation of histone variant distribution in human embryonic stem cells by transfection of in vitro transcribed mRNA.

Recent studies, primarily in mouse embryonic stem cells, have highlighted the unique chromatin state of pluripotent stem cells, including the incorporation of histone variants into specific genomic locations, and its role in facilitating faithful expression of genes during development. However, there is little information available on the expression and subcellular localisation of histone variants in human embryonic stem cells (hESCs). In this study, we confirmed the expression of a panel of histone variant genes in several hESC lines and demonstrated the utility of transfection of in vitro transcribed, epitope-tagged mRNAs to characterise the subcellular localisation of these proteins. The subcellular localisations of variant histone H3 (CENP-A, H3.3), H2A (MACROH2A, H2AX, H2AZ, H2ABBD) and H1 (H1A, HB, H1C, H1D) were examined, revealing distinct nuclear localisation profiles for each protein. These data highlight the differences between murine (m) ESCs and hESCs, including the presence of a MACROH2A-enriched inactive X chromosome in undifferentiated XX hESC lines. We also provide the first evidence for MACROH2A accumulation on the Y-chromosome in XY hESCs.

RegII is a beta-cell protein and autoantigen in diabetes of NOD mice.

The Reg family of proteins has been studied in the context of growth and regeneration in several organs including pancreatic islets. We previously suggested that Reg proteins act as autoantigens in type 1 diabetes, based on evidence that a member of the Reg family (hepatocellular carcinoma intestine pancreas [HIP]/pancreatitis-associated protein [PAP]) was overexpressed in the islets of a patient who died after sudden onset of type 1 diabetes, and that, in NOD mice, Reg-specific T-cells adoptively transferred diabetes. In the current study, we developed antisera to detect individual Reg members in mouse islets and found that RegIIIalpha was present in the non-beta-cell portion of the islets, while RegII was predominantly expressed in beta-cells. Vaccination of NOD mice with the separately expressed N-terminal (NtfrII) or C-terminal (CtfrII) portion of RegII revealed a dichotomy: NtfrII vaccination accelerated and CtfrII vaccination delayed type 1 diabetes. Vaccination with CtfrII was more effective when given at later stages in the pathogenesis of type 1 diabetes, a time dependency different from that seen with other antigen-dependent vaccine strategies in NOD mice, which might have therapeutic implications. In conclusion, RegII is a novel beta-cell-derived autoantigen in NOD mice. The autoimmune response against this protein may convert a regenerative into an islet-destructive process accelerating development of type 1 diabetes.

Effective and efficient characterization of Chinese hamster ovary production cell lines using automated intracellular staining and statistical modeling.

Mammalian cell line development is critical to bioproduct manufacturing. Success requires selecting a line with desirable performance characteristics, including consistent expression throughout the proposed manufacturing window. Given the genetic and phenotypic flux inherent to immortalized lines such as Chinese hamster ovary cells, clonally-derived cell line characterization is vital. We describe here the development and implementation of a novel addition to our characterization approach to ensure production cell line suitability: automated intracellular staining with statistical modeling. Case studies are presented which highlight this method's sensitivity to epigenetic expression effects, closing a gap left by our historically-leveraged genetic suitability characterization. Additionally, we demonstrate how an orthogonal, complimentary assay can help identify opportunities for improvement in even a well-established methodology such as our genetic suitability assessment. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:570-583, 2018.

Activation of AMP-activated protein kinase within the ventromedial hypothalamus amplifies counterregulatory hormone responses in rats with defective counterregulation.

Defective counterregulatory responses (CRRs) to hypoglycemia are associated with a marked increase in the risk of severe hypoglycemia. The mechanisms leading to the development of defective CRRs remain largely unknown, although they are associated with antecedent hypoglycemia. Activation of AMP-activated protein kinase (AMPK) in the ventromedial hypothalamus (VMH) amplifies the counterregulatory increase in glucose production during acute hypoglycemia. To examine whether activation of AMPK in the VMH restores defective CRR, controlled hypoglycemia ( approximately 2.8 mmol/l) was induced in a group of 24 Sprague-Dawley rats, all of which had undergone a 3-day model of recurrent hypoglycemia before the clamp study. Before the acute study, rats were microinjected to the VMH with either 5-aminoimidazole-4-carboxamide (AICAR; n=12), to activate AMPK, or saline (n=12). In a subset of rats, an infusion of H(3)-glucose was additionally started to calculate glucose turnover. Stimulation of AMPK within the VMH was found to amplify hormonal CRR and increase endogenous glucose production. In addition, analysis of tissue from both whole hypothalamus and VMH showed that recurrent hypoglycemia induces an increase in the gene expression of AMPK alpha(1) and alpha(2). These findings suggest that the development of novel drugs designed to selectively activate AMPK in the VMH offer a future therapeutic potential for individuals with type 1 diabetes who have defective CRRs to hypoglycemia.

Engineering chromosomes for delivery of therapeutic genes.

The ability to create fully functional human chromosome vectors represents a potentially exciting gene-delivery system for the correction of human genetic disorders with several advantages over viral delivery systems. However, for the full potential of chromosome-based gene-delivery vectors to be realized, several key obstacles must be overcome. Methods must be developed to insert therapeutic genes reliably and efficiently and to enable the stable transfer of the resulting chromosomal vectors to different therapeutic cell types. Research to achieve these outcomes continues to encounter major challenges; however recent developments have reiterated the potential of chromosome-based vectors for therapeutic gene delivery. Here we review the different strategies under development and discuss the advantages and problems associated with each.

A Reg family protein is overexpressed in islets from a patient with new-onset type 1 diabetes and acts as T-cell autoantigen in NOD mice.

Genes overexpressed in pancreatic islets of patients with new-onset type 1 diabetes are potential candidates for novel disease-related autoantigens. RT-PCR-based subtractive hybridization was used on islets from a patient who died at the onset of type 1 diabetes, and it identified a type 1 diabetes-related cDNA encoding hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP). This protein belongs to the family of Reg proteins implicated in islet regeneration; its gene contains a putative interleukin-6 (IL-6) response element. Islets from healthy cadaveric human donors released HIP/PAP protein into the culture medium, and this release was enhanced by the addition of IL-6. The expression pattern of mouse homologues of HIP/PAP was determined in pancreata of prediabetic and diabetic NOD mice. Both groups showed positive immunostaining for HIP/PAP in islets and ductal epithelium. To test whether HIP/PAP is a target of islet-directed autoimmunity, we measured splenic T-cell responses against HIP/PAP in NOD mice. Spontaneous proliferation was detected after 4 weeks. Lymphocytes from islet infiltrates and pancreatic lymph nodes from 7- to 10-week-old NOD mice were used to establish an HIP/PAP-specific I-A(g7)-restricted T-cell line, termed WY1, that also responded to mouse islets. WY1 cells homed to islets of NOD-SCID mice and adoptively transferred disease when coinjected with purified CD8(+) cells from diabetic NOD mice. Our conclusion was that differential cloning of Reg from islets of a type 1 diabetic patient and the response of Reg to the cytokine IL-6 suggests that HIP/PAP becomes overexpressed in human diabetic islets because of the local inflammatory response. HIP/PAP acts as a T-cell autoantigen in NOD mice. Therefore, autoimmunity to HIP/PAP might create a vicious cycle, accelerating the immune process leading to diabetes.

Factors influencing the distribution of larval blacklegged ticks on rodent hosts.

Because of differences among hosts in reservoir competence for tick-borne diseases, the distribution of larval blacklegged ticks on hosts might determine tick infection prevalence and disease risk to humans. We conducted a three-part study to determine the factors responsible for greater burdens of larval blacklegged ticks on white-footed mice than on eastern chipmunks. A microhabitat study indicated that questing ticks have higher encounter rates with mice than with chipmunks. Laboratory experiments demonstrated that ticks oriented more strongly toward mice. However, larval ticks fed more successfully from chipmunks. Our results strongly suggest that mice are both more likely to use larval tick-infested microhabitats and to attract questing larvae than are chipmunks, leading to a dramatically higher initial infestation rate, which is then reduced by greater grooming activity by mice. The high mortality rate of larvae that were experimentally introduced onto mice suggests that grooming is a significant cause of mortality to larval blacklegged ticks.

Temporal pattern of herpes simplex virus type 1 infection and cell death in the mouse brain stem: influence of guanosine nucleoside analogues.

Levels of bystander death occurring in herpes simplex virus type 1 (HSV-1)-infected mouse brain stems were studied, as well as the extent to which bystander death is influenced by guanosine nucleoside analogue treatment. Consecutive sections from brain stems of HSV-1-infected mice were stained alternately for (i) viral infection and (ii) cell death (TUNEL assay). Virus antigen was detectable in brain stems on day 3 of infection, while TUNEL staining was comparatively lower. An increase in the extent of TUNEL staining was observed on day 4 of infection. Despite this increase, however, the ratio of TUNEL-stained to infection marker-stained tissue still indicated that the amount of TUNEL staining remained lower than infection staining at this time point. On days 5 and 6 of infection, TUNEL staining continued to increase and the TUNEL/infection marker ratio switched on day 6 in favour of excess TUNEL staining, which was observed in and around the foci of infection, suggesting bystander death. The excess TUNEL staining on day 6 of infection was further increased on treatment with antivirals. The significance and implications of these results are discussed with respect to the nature and mechanism of action of the TUNEL assay, dynamics of primary HSV-1 infection, immunological influences and potential effects of antiviral treatment. The potential problems of the TUNEL assay are considered in the context of viral infection and the TUNEL assay, in combination with infection marker staining, may potentially provide a model system for quantitative analysis of true bystander death during HSV infection in vivo.

An evaluation of functional outcomes (speech, swallowing) in patients attending speech pathology after head and neck cancer treatment(s): results and analysis at 12 months post-intervention.

We have earlier reported establishing a computerized database to audit functional outcomes in patients who underwent head and neck cancer treatment in Victoria, Australia and attended speech pathology services from April 1997-April 1999. This paper presents the statistical analyses and results from this study. Speech pathologists collected, prospectively, functional outcome data on 293 patients who underwent head and neck cancer treatment, and sent these for analysis to La Trobe University. Clinician and patient assessments of outcomes: speech, swallowing, activity, pain, employment, health, QOL status were made. Initial data on 293 patients were collected and data on mortality and morbidity were compiled at three, six and 12 months post-treatment. Within twelve months, 74 patients had died. Three, six and/or 12-month follow-up data was available on 219 patients, with both clinician and patient assessments of status completed. The status forms are presented as appendices to this paper. Complete status forms on 179 patients at 12 months were obtained. This clinical audit of functional outcomes represents the first study of this kind, collecting data from speech pathologists and patients in a multi-centre study of patients with head and neck cancer. We present data to demonstrate optimal recovery of function at six months, such that this may represent a good reference point for reporting and comparison of functional outcomes.

Vaccination with single chain antigen receptors for islet-derived peptides presented on I-A(g7) delays diabetes in NOD mice by inducing anergy in self-reactiveT-cells.

To develop a vaccination approach for prevention of type 1 diabetes (T1D) that selectively attenuates self-reactive T-cells targeting specific autoantigens, we selected phage-displayed single chain antigen receptor libraries for clones binding to a complex of the NOD classII MHC I-A(g7) and epitopes derived from the islet autoantigen RegII. Libraries were generated from B-cell receptor repertoires of classII-mismatched mice immunized with RegII-pulsed NOD antigen presenting cells or from T-cell receptor repertoires in pancreatic lymph nodes of NOD mice. Both approaches yielded clones recognizing a RegII-derived epitope in the context of I-A(g7), which activated autoreactive CD4(+) T-cells. A receptor with different specificity was obtained by converting the BDC2.5 TCR into single chain form. B- but not T-cells from donors vaccinated with the clones transferred protection from diabetes to NOD-SCID recipients if the specificity of the diabetes inducer cell and the single chain receptor were matched. B-cells and antibodies from donors vaccinated with the BDC2.5 single chain receptor induced a state of profound anergy in T-cells of BDC2.5 TCR transgenic NOD recipients while B-cells from donors vaccinated with a single chain receptor specific for I-A(g7) RegII peptide complexes induced only partial non-responsiveness. Vaccination of normal NOD mice with receptors recognizing I-A(g7) RegII peptide complexes or with the BDC2.5 single chain receptor delayed onset of T1D. Thus anti-idiotypic vaccination can be successfully applied to T1D with vaccines either generated from self-reactive T-cell clones or derived from antigen receptor libraries.

Key role for AMP-activated protein kinase in the ventromedial hypothalamus in regulating counterregulatory hormone responses to acute hypoglycemia.

OBJECTIVE: To examine in vivo in a rodent model the potential role of AMP-activated protein kinase (AMPK) within the ventromedial hypothalamus (VMH) in glucose sensing during hypoglycemia. RESEARCH DESIGN AND METHODS: Using gene silencing technology to selectively downregulate AMPK in the VMH, a key hypothalamic glucose-sensing region, we demonstrate a key role for AMPK in the detection of hypoglycemia. In vivo hyperinsulinemic-hypoglycemic (50 mg dl(-1)) clamp studies were performed in awake, chronically catheterized Sprague-Dawley rats that had been microinjected bilaterally to the VMH with an adeno-associated viral (AAV) vector expressing a short hairpin RNA for AMPKalpha. RESULTS: In comparison with control studies, VMH AMPK downregulation resulted in suppressed glucagon ( approximately 60%) and epinephrine (approximately 40%) responses to acute hypoglycemia. Rats with VMH AMPK downregulation also required more exogenous glucose to maintain the hypoglycemia plateau and showed significant reductions in endogenous glucose production and whole-body glucose uptake. CONCLUSIONS: We conclude that AMPK in the VMH plays a key role in the detection of acute hypoglycemia and initiation of the glucose counterregulatory response.

Fructose-1,6-bisphosphatase overexpression in pancreatic beta-cells results in reduced insulin secretion: a new mechanism for fat-induced impairment of beta-cell function.

OBJECTIVE: Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic beta-cell lines exposed to high fat. However, whether specific beta-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet beta-cell FBPase can result in reduced glucose-mediated insulin secretion. RESEARCH DESIGN AND METHODS: To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet beta-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic beta-cell lines (MIN6) stably overexpressing huFBPase. RESULTS: FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels. CONCLUSIONS: Taken together, these results suggest that upregulation of FBPase in pancreatic islet beta-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.

5'AMP-activated protein kinase alpha deficiency enhances stress-induced apoptosis in BHK and PC12 cells.

5'AMP-activated protein kinase (AMPK) activation occurs under a variety of stress conditions but the role of this enzyme in the promotion or inhibition of stress-induced cell death is unclear. To address this issue, we transformed two different cell lines with shRNA-expressing plasmids, targeting the alpha subunit of AMPK, and verified AMPKalpha downregulation. The cell lines were then stressed by exposure to medium without glucose (PC12 cells) or with the viral thymidine kinase-specific DNA replication inhibitors: acyclovir, penciclovir and ganciclovir (herpes simplex virus thymidine kinase-expressing Baby Hamster Kidney cells). In non-AMPK-downregulated cells, these stress treatments induced AMPK upregulation and phosphorylation, leaving open the question whether the association of AMPK activation with stress-induced cell death reflects a successful death-promoting or an ineffective death-inhibiting activity. In AMPKalpha-deficient cells (expressing AMPKalpha-specific shRNAs or treated with Compound C) exposure to low glucose medium or DNA replication inhibitors led to an enhancement of cell death, indicating that, under the conditions examined, the role of activated AMPK is not to promote, but to protect from or delay stress-induced cell death.

Technical white paper: bone densitometry.

The Conference of Radiation Control Program Directors (CRCPD) Task Force on Bone Densitometry (H-30) was assigned by the Healing Arts Council on Emerging Issues to address issues of bone densitometry. Issues for clarification included the practice of precision testing, in which multiple bone density determinations are performed on one patient; the use of quantitative computed tomographic (CT) densitometry; and radiation dose to patients and operators. This paper is a condensation of the white paper produced by the task force, which addresses the various methods of measuring bone density, the qualifications and responsibilities of personnel, the rationale for precision testing, and the doses patients and operators may receive. The white paper is available in its entirety on the CRCPD's Web site (http://crcpd.org).