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Explicit guidelines are needed to develop safe and effective patient portals. This paper proposes general principles, policies, and procedures for patient portal functionality based on MyHealthAtVanderbilt (MHAV), a robust portal for Vanderbilt University Medical Center. We describe policies and procedures designed to govern popular portal functions, address common user concerns, and support adoption. We present the results of our approach as overall and function-specific usage data. Five years after implementation, MHAV has over 129,800 users; 45% have used bi-directional messaging; 52% have viewed test results and 45% have viewed other medical record data; 30% have accessed health education materials; 39% have scheduled appointments; and 29% have managed a medical bill. Our policies and procedures have supported widespread adoption and use of MHAV. We believe other healthcare organizations could employ our general guidelines and lessons learned to facilitate portal implementation and usage.

Background: The blood product administration process has been subject to various quality improvement initiatives aimed at reducing errors, including blood product labels that are missing, inaccessible, unreadable, or mismatched to orders and/or patients. This article reports the results of a formal simulation-based usability test of two comparable technologies designed to reduce blood product administration errors., Study Design and Methods: Nineteen nurses and three anesthesia providers evaluated one of the two products during simulated use in realistic scenarios during 90-minute test sessions. Both products required additional learning despite 15 minutes of dedicated vendor-provided pretest training., Results: There were significant effectiveness differences between the two products, but use of both devices was less efficient than manual checking. Usability issues included poor access to subtasks, lack of process feedback, inadequate error messaging, and confusing device interactions., Conclusion: While clinicians' subjective ratings of both devices were similarly high, both products had significant usability issues likely to lead to clinician frustration and workarounds during actual use. This study suggests that usability testing is a valuable and more effective method than preference surveys of determining the ability of blood administration products to meet clinicians' needs in the complex world of patient care., (© 2011 American Association of Blood Banks.)

Treatment of Arabidopsis (Arabidopsis thaliana) leaves by extended darkness generates a genetically activated senescence program that culminates in cell death. The transcriptome of leaves subjected to extended darkness was found to contain a variety of reactive oxygen species (ROS)-specific signatures. The levels of transcripts constituting the transcriptome footprints of chloroplasts and cytoplasm ROS stresses decreased in leaves, as early as the second day of darkness. In contrast, an increase was detected in transcripts associated with mitochondrial and peroxisomal ROS stresses. The sequential changes in the redox state of the organelles during darkness were examined by redox-sensitive green fluorescent protein probes (roGFP) that were targeted to specific organelles. In plastids, roGFP showed a decreased level of oxidation as early as the first day of darkness, followed by a gradual increase to starting levels. However, in mitochondria, the level of oxidation of roGFP rapidly increased as early as the first day of darkness, followed by an increase in the peroxisomal level of oxidation of roGFP on the second day. No changes in the probe oxidation were observed in the cytoplasm until the third day. The increase in mitochondrial roGFP degree of oxidation was abolished by sucrose treatment, implying that oxidation is caused by energy deprivation. The dynamic redox state visualized by roGFP probes and the analysis of microarray results are consistent with a scenario in which ROS stresses emanating from the mitochondria and peroxisomes occur early during darkness at a presymptomatic stage and jointly contribute to the senescence program.

Purpose: In this paper, the psychosocial oncology-themed Cross Cancer Institute Multidisciplinary Summer Studentship in Palliative and Supportive Care in Oncology is described from the perspective of the first participants and supervising faculty., Methods: This 6-week inter-professional elective exposed pre-licensure students to issues facing patients and their families following a diagnosis of cancer, through treatment, recovery, recurrence, palliation, and end-of-life., Results: Participants gained experience in team-based skills and compassionate care, were introduced to other disciplines, and formed collaborative partnerships. The Studentship encompasses the features of best practice cooperative learning through clinical experience, facilitated weekly discussion, an exploratory investigation and presentation.The authors' backgrounds and interest in this area are discussed, as well as pre-existing expectations and goals, reflections on their interactions, challenges faced, and lasting impressions from their experiences seeing through patients' eyes, framed by the essential tenets of psycho-oncology practice., Conclusions: Our multidisciplinary placement is feasible, successful, and potentially transferable to other academic settings.

Background: The standard surgical procedure for polar renal tumors is nephron-sparing surgery, currently performed laparoscopically in most cases. This technically challenging surgery involves clamping of the hilar vessels and warm kidney ischemia. This study tested the feasibility of performing polar nephrectomy using a cable tie without hilar clamping or any other hemostatic procedure., Methods: General anesthesia and pneumoperitoneum were induced in four farm pigs, and three trocars were inserted intraperitoneally. A kidney pole (either a lower or an upper pole of either the left or right kidney) was exposed and encircled with a plastic cable tie. The pole then was transected sharply with scissors, opening the urinary collecting system and major blood vessels. Neither tissue sealants nor any form of energy was used throughout the cases. The animals were killed 3 weeks later., Results: All the pigs tolerated the surgery well. The renal part of the surgery required less than 12 min, and blood loss was minimal (<30 ml) for all the animals. The postoperative course was benign. Laparotomy performed 3 weeks later showed no peritoneal or retroperitoneal abnormalities. The plastic cable tie remained in the same position covered with a thin layer of mesothelial cells. A thin sector of coagulation necrosis was found under the pressure of the cable., Conclusions: In a porcine model, polar nephrectomy using a plastic cable tie is a very easy and rapid procedure. Cheap and bloodless, it eliminates the need for renal reconstruction. Hilar dissection and renal ischemia are avoided. "Cable-tie partial nephrectomy" may become a method for removing a tumor-bearing renal pole.

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3'-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.

Redox-sensitive GFP (roGFP) localized to different compartments has been shown to be suitable for determination of redox potentials in plants via imaging. Long-term measurements bring out the need for analyzing a large number of samples which are averaged over a large population of cells. Because this goal is too tedious to be achieved by confocal imaging, we have examined the possibility of using a fluorometer to monitor changes in roGFP localized to different subcellular compartments during development and dark-induced senescence. The degree of oxidations determined by a fluorometer for different probes was similar to values obtained by confocal image analysis. Comparison of young and old leaves indicated that in younger cells higher levels of H(2)O(2) were required to achieve full roGFP oxidation, a parameter which is necessary for calculation of the degree of oxidation of the probe and the actual redox potential. Therefore, it is necessary to carefully determine the H(2)O(2) concentration required to achieve full oxidation of the probe. In addition, there is an increase in autofluorescence during development and extended dark stress, which might interfere with the ability to detect changes in oxidation-reduction dependent fluorescence of roGFP. Nevertheless, it was possible to determine the full dynamic range between the oxidized and the reduced forms of the different probes in the various organelles until the third day of darkness and during plant development, thereby enabling further analysis of probe oxidation. Hence, fluorometer measurements of roGFP can be used for extended measurements enabling the processing of multiple samples. It is envisaged that this technology may be applicable to the analysis of redox changes in response to other stresses or to various mutants.

Mutational analysis of c-KIT or PDGFRA has become an important laboratory assay for patients with gastrointestinal stromal tumors (GISTs) because the results are useful in predicting the responsiveness to imatinib. To assess the diagnostic usefulness of denaturing high-pressure liquid chromatography (DHPLC) in this setting, we performed DHPLC and DNA sequencing to study exons 9, 11, 13, and 17 of c-KIT and exons 12 and 18 of PDGFRA in 54 consecutive cases of GIST collected from a single population. Most (40/54 [74%]) carried c-KIT mutations, and 7 (13%) carried PDGFRA mutations. These results were similar to those described in the literature. It is important to note that DHPLC was found to be highly sensitive, detecting all of the mutations in these 6 exons that were identified by DNA sequencing. Our data suggest that DHPLC is a cost-effective, rapid, and sensitive test for screening for mutations of c-KIT and PDGFRA in GISTs.

We report the case of a patient with metastatic hormone refractory prostate cancer in whom "indirect" cauda equina syndrome developed concurrent with multilevel spinal cord compression (SCC). Three months after his first positive bone scan, a 65-year-old otherwise healthy man presented with severe back pain, bilateral lower extremity paresthesias, leg weakness and urinary retention. Magnetic resonance imaging (MRI) showed a dural-based mass causing SCC at the T9, T10 and T11 vertebrae, with a normal cauda equina. He received corticosteroids and palliative external beam radiotherapy, resulting in good pain control and gradual improvement in his neurological symptoms. He did well for 8 months, at which time his residual bilateral leg weakness abruptly worsened and he experienced numbness, paresthesias, urinary incontinence and constipation. Repeat MRI showed progression of epidural metastatic disease compressing the spinal cord or thecal sac at 7 thoracic vertebral levels. The cauda equina was also distorted and flattened without evidence of direct solid tumour impingement. We hypothesized that the etiology was increased intrathecal pressure due to disrupted cerebrospinal fluid flow resulting from multiple levels of upstream thecal sac compression. It is essential to image the entire spinal cord and cauda equina when patients with metastatic bone disease present with neurological symptoms to institute correct treatment and preserve function and mobility.

Aims: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Encoded in the genome of most eukaryotes, miRNAs have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. For the first time, we sought to test how myocardial ischaemia-reperfusion (IR) changes miR expression., Methods and Results: Following 2 and 7 h of IR or sham operation, myocardial tissue was collected and subjected to miRNA expression profiling and quantification using a Bioarray system that screens for human-, mice-, rat-, and Ambi-miR. Data mining and differential analyses resulted in 13 miRs that were up-regulated on day 2, 9 miRs that were up-regulated on day 7, and 6 miRs that were down-regulated on day 7 post-IR. Results randomly selected from expression profiling were validated using real-time PCR. Tissue elements laser-captured from the infarct site showed marked induction of miR-21. In situ hybridization studies using locked nucleic acid miR-21-specific probe identified that IR-inducible miR-21 was specifically localized in the infarct region of the IR heart. Immunohistochemistry data show that cardiac fibroblasts (CFs) are the major cell type in the infarct zone. Studies with isolated CFs demonstrated that phosphatase and tensin homologue (PTEN) is a direct target of miR-21. Modulation of miR-21 regulated expression of matrix metalloprotease-2 (MMP-2) via a PTEN pathway. Finally, we noted a marked decrease in PTEN expression in the infarct zone. This decrease was associated with increased MMP-2 expression in the infarct area., Conclusion: This work constitutes the first report describing changes in miR expression in response to IR in the mouse heart, showing that miR-21 regulates MMP-2 expression in CFs of the infarct zone via a PTEN pathway.

Purpose: Dissection of the seminal vesicles during radical prostatectomy has the potential to damage the pelvic plexus, thus compromising trigonal, bladder neck and cavernous innervation, and contributing to delayed gain of continence and erectile function. The rate of prostate cancer invasion into the seminal vesicles in currently operated patients is low and in most it may be predicted preoperatively. This situation calls for seminal vesicle sparing radical prostatectomy in select patients, leaving a distal remnant of the seminal vesicles in place. We investigated the fate of the seminal vesicle remnant after proximal transection or ligation in an animal model., Materials and Methods: The right seminal vesicle in 36 anesthetized male rats was divided by suture ligation or by transection. The left seminal vesicle served as a control. Six rats per group were sacrificed 1, 2 and 4 weeks after division, respectively. Seminal vesicle morphology was evaluated macroscopically and microscopically., Results: All rats tolerated surgery well and gained weight postoperatively. Transected seminal vesicles were similar in weight and morphology to control contralateral glands. One week after seminal vesicle ligation the remnants became significantly heavier and showed balloon dilatation of the hollow spaces, while the lining epithelium became significantly flattened. Two and 4 weeks after ligation half of the animals showed gland shrinkage and half demonstrated persistent dilatation., Conclusions: Seminal vesicle transection preserves the gland remnant in a relatively normal morphology, while ligation leads to severe and inconsistent morphological changes. When considering seminal vesicle sparing radical prostatectomy, seminal vesicle transection may be preferred to ligation.

Objective: A Dicer knockdown approach was used to test the significance of miRNA in regulating the redox state and angiogenic response of human microvascular endothelial cells (HMECs)., Methods and Results: Lowering of miRNA content by Dicer knockdown induced vascular endothelial growth factor expression but diminished the angiogenic response of HMECs as determined by cell migration and Matrigel tube formation. Such impairment of angiogenic response in the Matrigel was rescued by exogenous low micromolar H2O2. Dicer knockdown HMECs demonstrated lower inducible production of reactive oxygen species (ROS) when activated with either phorbol ester, tumor necrosis factor-alpha, or vascular endothelial growth factor. Limiting the production of ROS by antioxidant treatment or NADPH oxidase knockdown approaches impaired angiogenic responses. Experiments to identify how ROS production is limited by Dicer knockdown identified lower expression of p47phox protein in these cells. This lowering of cellular miRNA content induced expression of the transcription factor HBP1, a suppressor transcription factor that negatively regulates p47phox expression. Knockdown of HBP1 restored the angiogenic response of miRNA-deficient HMECs., Conclusions: This study provides the first evidence that redox signaling in cells is subject to regulation by miRNA. Specifically, p47phox of the NADPH oxidase complex has been identified as one target that regulates the angiogenic properties of endothelial cells.

Selenium is recognized as essential in animal and human nutrition. Several hypotheses have been advanced for its biological activity. The aim of this study was to investigate the in vivo effect of selenium on rat liver manganese superoxide dismutase (MnSOD), a key antioxidant enzyme, under naïve and inflammatory conditions. Rats received sodium selenite supplementation and LPS injection. Whole-liver samples, isolated hepatocytes, Kupffer cells and blood samples were subjected to protein, RNA and biochemical analysis. Liver enrichment with selenium increased whole-liver MnSOD levels due to an increase in MnSOD transcription in hepatocytes. This was due to an increase in the ratio of specificity protein 1 to activating enhancer binding protein 2 DNA-binding activity. The inflammatory stimulus further elevated MnSOD levels in the whole-liver that was abrogated in sodium selenite supplementation due to reduced transcription of MnSOD in Kupffer cells. Moreover, selenium enrichment decreased Kupffer cells IL-6 transcription in LPS-injected animals. Anti-inflammatory activity of selenium was demonstrated by normalized blood levels of ALT and IL-6 in LPS-injected animals. In conclusion, selenium up-regulates hepatocytes MnSOD expression, probably improving their anti-oxidant defense, while decreasing MnSOD and IL-6 transcription in Kupffer cells in the presence of inflammatory stimuli, attenuating their inflammatory response. This selective mechanism may explain the anti-inflammatory and hepato-protective effect of selenium.

Objective: Computerized clinical reminders (CRs) were designed to reduce clinicians' reliance on their memory and to present evidence-based guidelines at point of care. However, the literature indicates that CR adoption and effectiveness has been variable. We examined the impact of four design modifications to CR software on learnability, efficiency, usability, and workload for intake nursing personnel in an outpatient clinic setting. These modifications were included in a redesign primarily to address barriers to effective CR use identified during a previous field study., Design: In a simulation experiment, 16 nurses used prototypes of the current and redesigned system in a within-subject comparison for five simulated patient encounters. Prior to the experimental session, participants completed an exploration session, where "learnability" of the current and redesigned systems was assessed., Measurements: Time, performance, and survey data were analyzed in conjunction with semi-structured debrief interview data., Results: The redesign was found to significantly increase learnability for first-time users as measured by time to complete the first CR, efficiency as measured by task completion time for two of five patient scenarios, usability as determined by all three groupings of questions taken from a commonly used survey instrument, and two of six workload subscales of the NASA Task Load Index (TLX) survey: mental workload and frustration., Conclusion: Modest design modifications to existing CR software positively impacted variables that likely would increase the willingness for first-time nursing personnel to adopt and consistently use CRs.

Repair of a defect in the human skin is a highly orchestrated physiological process involving numerous factors that act in a temporally resolved synergistic manner to re-establish barrier function by regenerating new skin. The inducible expression and repression of genes represents a key component of this regenerative process. MicroRNAs (miRNAs) are approximately 22-nucleotide-long endogenously expressed non-coding RNAs that regulate the expression of gene products by inhibition of translation and/or transcription in animals. miRNAs play a key role in skin morphogenesis and in regulating angiogenesis. The vascular endothelial growth factor signaling path seems to be under repressor control by miRNAs. Mature miRNA-dependent mechanisms impair angiogenesis in vivo. It is critically important to recognize that the understanding of cutaneous wound healing is incomplete without appreciating the functional significance of wound-induced miRNA. Ongoing work in our laboratory has led to the observation that the cutaneous wound healing process involves changes in the expression of specific miRNA at specific phases of wound healing. We hypothesize that dysregulation of specific miRNA is critical in derailing the healing sequence in chronic problem wounds. If tested positive, this hypothesis is likely to lead to completely novel diagnostic and therapeutic strategies for the treatment of problem wounds.

Saccharopolyspora erythraea is used for the industrial-scale production of the antibiotic erythromycin A, derivatives of which play a vital role in medicine. The sequenced chromosome of this soil bacterium comprises 8,212,805 base pairs, predicted to encode 7,264 genes. It is circular, like those of the pathogenic actinomycetes Mycobacterium tuberculosis and Corynebacterium diphtheriae, but unlike the linear chromosomes of the model actinomycete Streptomyces coelicolor A3(2) and the closely related Streptomyces avermitilis. The S. erythraea genome contains at least 25 gene clusters for production of known or predicted secondary metabolites, at least 72 genes predicted to confer resistance to a range of common antibiotic classes and many sets of duplicated genes to support its saprophytic lifestyle. The availability of the genome sequence of S. erythraea will improve insight into its biology and facilitate rational development of strains to generate high-titer producers of clinically important antibiotics.

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C(4) plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 +/- 0.8 muM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C(4) plant but no effect on a model C(3) plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.

Diuraphis noxia (Russian wheat aphid, RWA) is a major pest on wheat in South Africa and most other wheat growing countries. Being a probing-sucking insect, RWAs insert their stylets into the phloem sieve elements and feed on the phloem sap. This feeding causes necrotic lesions in resistant varieties, or decoloration of leaves and death in susceptible varieties. In an effort to broaden our understanding on the response of the plant to RWA feeding, we synthesized and analyzed expressed sequence tags (ESTs) from suppression subtractive hybridization (SSH) libraries. These libraries were constructed using near isogenic wheat lines susceptible "Tugela" and resistant "TugelaDN" (Dn1) to RWA, as well as accession lines PI137739 (Dn1) and PI294994 (Dn5). Analysis of 200 ESTs from the libraries revealed the involvement of transcripts encoding genes involved in cell maintenance, growth and regulation, plant defense and signaling, photosynthesis and energy production, and of unknown function. A selection of these ESTs, in combination with clones obtained from other sources, were used on a custom array to study the expression profiles of 256 candidate wheat sequences putatively involved in plant defense against RWA. The selected sequences included wheat genomic clones with putative nucleotide binding site (NBS) motifs, rapid amplification of cDNA ends PCR (RACE-PCR), and cDNA clones from RWA induced libraries. Genomic banana and flax clones that were obtained using representative difference analysis (RDA), and suspected to be involved in abiotic stress responses, were also spotted onto the microarray slides. The spotted custom arrays were then hybridized against cDNA isolated from a resistant cultivar "TugelaDN" on 0, 2, 5, and 8 days after infestation, post-labeled with Cy3- or Cy5-fluorescent dyes. The subsequent expression profiling using DNA microarray, RT-PCR, and Northern Blot analysis identified 29 transcripts associated with the feeding response. These transcripts encoded proteins functioning in direct defense and signaling, oxidative burst, cell wall degradation, cell maintenance, photosynthesis, and energy production. Results indicate that plants co-ordinately regulate gene expression when attacked by RWA. It is hypothesized that the NBS-LRR proteins are important in receptor recognition and signaling, which enable the plant to overcome the stresses inflicted by RWA feeding. It is further suggested that the ability to maintain photosynthetic function with resultant energy production is one of the determining factors ensuring the survival of the resistant varieties when coping with the RWA feeding.

Advances in electronic medical record capabilities enable clinical reminders to inform providers when recommended actions are "due" for a patient. Despite evidence that they improve adherence to guidelines, the Veteran's Health Administration (VHA) has experienced challenges in having providers consistently use clinical reminders as intended. In this paper, we describe how multiple methods were used to opportunistically triangulate, or "bootstrap," an understanding of barriers to the effective use of clinical reminders in the VHA. In an initial study using ethnographic observations and semi-structured interviews of HIV clinical reminders, we identified six barriers to effective use: workload, time to remove inapplicable reminders, false alarms, training, reduced eye contact, and the use of paper forms rather than software. In a second study, we collected open-ended and closed-ended data regarding barriers and facilitators to the use of clinical reminders in general in the VHA through a survey of 261 participants at a national informatics meeting, where 104 of 142 VHA health care facilities were represented. The findings from the second study extended our understanding of the previously identified barriers. In addition, four new barriers were identified: ease of use issues, accessibility of workstations, resident physicians and trainees, and administration benefiting more than providers from clinical reminder use. We discuss potential implications regarding the similarities and differences in study findings for factors to consider in planning interventions to improve clinical reminder use.

Selenium can activate cell death. However, the mechanism of action is not yet fully defined. We hypothesized that selenium may impede mitochondrial superoxide dismutation to H2O2 and O2, leading to cell death in macrophages and that this effect may be relevant to antiinflammatory treatment by selenium. In this study, the mechanism of action of selenium was investigated in nonactivated and activated (immune-stimulated) J774.2 macrophages. Sodium selenite treatment decreased dichlorodihydrofluorescein-reacting intracellular reactive oxygen species (ROS) (mainly peroxides and hydroxyl radicals), with no correlation to glutathione peroxidase activity. However, selenite decreased the transcription and expression of manganese superoxide dismutase (MnSOD) and uncoupling protein 2 (UCP2). This cellular effect was due to inhibition of specificity protein-1 (Sp1) binding to its DNA binding site. Following immune stimulation of macrophages using lipopolysaccharides plus interferon-gamma, MnSOD was up-regulated. Activated macrophages showed higher mitochondrial membrane potential, intracellular ROS levels, and cellular resistance to cell death. Selenite treatment attenuated all of these parameters. Selenite prevented nuclear factor-kappaB (NF-kappaB) activation as a mechanism of its inhibitory activity on MnSOD expression in the immune-stimulated cells. In addition, overexpression of human MnSOD protected against death induced by selenite treatment. It is therefore concluded that selenium at high nanomolar to low micromolar concentrations shifts the balance between inflammatory response and cell death toward the latter, through a direct effect on the transcription factors Sp1 and NF-kappaB, and down-regulation of MnSOD and UCP2.

Sodium selenite, a common dietary form of selenium, is recognized as essential in animal and human nutrition. Mechanisms regulating the inflammatory response of the immune system involve regulation of apoptosis and control of reactive oxygen species (ROS) production. In this study, the effect of sodium selenite on ROS production and cell-death rates in macrophages and T cells was investigated. Exposing Jurkat T cells or J774.2 macrophages to >5 micro M sodium selenite induced cell death. In both Jurkat T cells and J774.2 macrophages, rapid loss of the cell's capacity to generate dichlorofluorescein-sensitive ROS preceded cell death. The main cellular source of ROS was found to be the mitochondria electron-transfer chain. DEVDase activity in the cells remained unchanged and even decreased with time, as well as DNA fragmentation level, which was almost unaffected, indicating cell death with necrotic characteristics. tert-Butyl hydroperoxide at a concentration of 5 micro M was beneficial in attenuating the rate of cell death. The superoxide scavenger Tiron was tested for its ability to protect the cells against selenium. Tiron completely protected the J774.2 macrophage cell line against selenium and attenuated the cell death effect in Jurkat T cells. In the presence of the superoxide dismutase-mimicking compound tempol, selenium's macrophage-killing effect was inhibited. Therefore, our results show that, at least in vitro, selenite induces changes in the balance between mitochondrial superoxide and hydrogen peroxide production, which can facilitate cell death in immune system cells. This may be one mechanism by which selenium down-regulates the immune system's inflammatory response and protects against overproduction of peroxides.

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill-defined. Here, we show that ROS levels play a role in moderating Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or (mitochondrial uncoupler) and Fas-activating antibody (CH11) facilitated rapid cell death that was not associated with decreased ATP production or increased DEVDase activity and cytochrome c release. However, a decrease in cellular ROS production was associated with CH11 treatment, and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS production. Thus, decreased ROS production is correlated with enhanced cell death. A transition from state 3 to state 4 mitochondrial respiration accounted for the attenuated ROS production and membrane potential. Similar observations were demonstrated in isolated rat liver mitochondria. These data show that ROS production is important in receptor-mediated apoptosis, playing a pivotal role in cell survival.

There is a known connection between selenium supplementation and chemo-protective anti-cancer activity. This biological phenomenon may be due to the ability of selenium to instigate cellular apoptosis. However, the mechanism by which selenium promotes cellular apoptosis is still obscure. The present study shows that sodium selenite, a common dietary form of selenium, promotes the mitochondrial permeability transition (MPT) in isolated rat liver mitochondria both in vitro and following in vivo supplementation. A low selenium concentration (0.1-10 microM) strongly induced cyclosporin A-sensitive mitochondrial swelling. Selenium also promoted both calcium release from the matrix of isolated mitochondria and uncoupled respiration. The MPT-inducing effect of selenium provoked the release of cytochrome c, a pro-apoptotic factor, into the incubation medium. Selenium did not increase intra-mitochondrial peroxide production, but did consume endogenous mitochondrial glutathione. Moreover, the effect of MPT induction was greatly potentiated in the presence of thiol-bearing antioxidants, e.g. N -acetylcysteine and lipoamide. During MPT progression, selenium induced NADH oxidation via electron acceptance from complex I. Supplementation for 20 days with 16 p.p.m. selenium in the drinking water of rats increased the propensity of mitochondria to undergo the MPT. More marked mitochondrial swelling in response to calcium and lower calcium-uptake capacity were observed, in the absence of liver damage or the intensive oxidation of reduced glutathione. Therefore selenite facilitates MPT pore opening via its thiol- and NADH/complex I-dependent reduction, and thereby may provide chemo-protection by potentiation of the capacity of the mitochondria to regulate programmed cell death. Data from the present study suggest that selenium can regulate important mitochondrial functions both in vivo and in vitro.

It has been suggested that one key feature of mitochondrial permeability transition (PT) regulation is its control by the proton electrochemical gradient and that depolarization favors pore opening, swelling, and reactive oxygen species (ROS) production. Moreover, ROS have been suggested to facilitate the process of mitochondrial PT pore opening. The aim of this study was to show that collapsing the mitochondrial membrane potential with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), at concentrations of up to 10 microM, does not induce mitochondrial swelling and, in fact, stabilizes mitochondria exposed to oxidant, protecting them from tert-butyl hydroperoxide (TBH)-induced high-amplitude swelling. FCCP decreased polyethylene glycol-induced mitochondrial contraction following exposure to TBH, indicating closing of the PT mega-channel. In the presence of the calcium uniporter inhibitor ruthenium red, FCCP induced PT due to suppression of calcium efflux. Under PT-favorable conditions, ROS production was evaluated in mitochondria following treatments with TBH, inorganic phosphate, or FCCP (with or without ruthenium red). FCCP alone and in combination with ruthenium red attenuated mitochondria-derived ROS production. FCCP also decreased the augmented ROS production induced by inorganic phosphate. It is concluded that mitochondrial depolarization protects and prevents high-amplitude swelling and PT-derived ROS production.

Objectives: To analyze the clinical characteristics, associated risk factors, and outcome of hypoglycemia in nondiabetic hospitalized older patients., Design: A retrospective case control study., Participants: Sixty patients, aged 65 years and older, in the acute medical and geriatric wards who developed hypoglycemia. A control group was composed of 83 older patients, sex and age matched, in orthopedic and surgery wards who were undergoing corrective surgery for hip fracture or hernioplasty., Measurements: For all patients, data for the following variables were abstracted from the charts: age, sex, degree of hypoglycemia, clinical presentation of hypoglycemia, number and duration of hypoglycemia episodes, nutritional state, and blood chemistry analysis. Risk factors were defined as nutritional state, heart failure, renal or liver disease, malignancy, and infection or sepsis., Results: Mean blood glucose in hypoglycemic cases was 38.9 +/- 7 mg/dL. Symptoms and signs of hypoglycemia were noted in only 38.4% (23/60) of patients. All identified risk factors except cachexia were found significantly more frequently in the hypoglycemic patients than in the control group. Mean total number of risk factors was greater in the hypoglycemic group than in the control group, 2.97 +/- 1.1 versus 1.64 +/- .8, respectively (P < .001). In a multivariant logistic model, low plasma albumin level, liver disease, malignancy, and congestive heart failure were significant predictors of hypoglycemia. In-hospital mortality rate was higher among the hypoglycemic patients, 48% versus 18.1% (P < .001), and was independent of the degree of hypoglycemia or the number of hypoglycemic episodes. Mortality was correlated significantly with the number of risk factors (3.4 +/- 1.1 vs 2.5 +/- 1.1; P = .006). Hypoglycemia remained a significant predictor of mortality (OR = 3.67; 95% CI, 1.2-11.2) even after the adjustment for other risk factors., Conclusions: Hypoglycemic episodes occur even among nondiabetic hospitalized older patients. Symptoms and signs of hypoglycemia were noted in only two-fifths of the patients. Albumin less than 3.0 g%, liver disease, renal insufficiency, malignancy, congestive heart failure, and sepsis were statistically significant predictors of developing hypoglycemia. The overall mortality rate was significantly higher among the hypoglycemic patients and was independent of hypoglycemia levels. Mean total number of risk factors was significantly higher among those who died compared with hypoglycemic patients who survived. Based on the present study, the estimated odds of mortality in an older patient with hypoglycemia were 3.67 times higher than in those without hypoglycemia.

We developed a 24 hour intravenous dexamethasone suppression test for the differential diagnosis of Cushing's syndrome. Basal ACTH and cortisol levels were measured at 8 and 9 AM; a bolus of 8 mg dexamethasone phosphate (in children 5 mg/m2) was administered intravenously, and cortisol levels were measured hourly until 3 PM, then every 2 hours until midnight, and the next morning at 8 and 9 AM. We studied 13 patients with an ACTH-secreting pituitary adenoma, four with an autonomous adrenal adenoma, a 10 year-old girl with primary adrenocortical nodular dysplasia, one male with an ACTH-secreting medullary carcinoma of the thyroid, and one male with an ACTH-secreting non-small cell carcinoma of the lung, and compared their results to those obtained in 8 lean and 12 obese normal individuals (controls). The clinical diagnosis was first ascertained by the response to the oral administration of dexamethasone in low and high doses (standard Liddle test), then by the intravenous dexamethasone suppression test, and finally confirmed surgically. Although both controls and patients with an ACTH-secreting pituitary adenoma significantly suppressed their cortisol levels within hours after the injection (50% reduction of basal value at 2 hours, and 75% at 4 hours, p < 0.0001), levels remained suppressed the next morning only in the controls, while in the patients they returned to basal values. No suppression was observed in any of the patients with an adrenal adenoma and the child with primary adrenocortical nodular dysplasia (whose ACTH levels were low), or in the patients with ectopic ACTH secretion tumors (whose ACTH levels were high).(ABSTRACT TRUNCATED AT 250 WORDS)

A 65-year-old man was admitted because of severe cervical pain radiating to both shoulders and bouts of fever, which followed pneumonia. Bone scan showed increased focal uptake in C6-7, while computerized tomography showed destruction of the 7th intervertebral disk, with bone sequesters and soft tissue swelling. Fine needle aspiration drew purulent material with gram-positive bacteria on direct staining. Culture grew Staphylococcus aureus, coagulase positive. Treatment with IV cloxacillin for 3 weeks, followed by oral treatment for another 3, resulted in complete remission.

Compensated hypothyroidism was diagnosed in a 36-year-old female who presented with breast tenderness and mild galactorrhoea. T4 was 5.8 mcg dl-1 and T3RU was 22.5%, while TSH and prolactin were very mildly elevated (6.5 mU ml-1 and 26.1 ng ml-1, respectively). The TRH test showed an exaggerated response. TSH increased to 43 mU ml-1, and prolactin levels reached 161 ng ml-1. Treatment with T4 decreased the TSH and prolactin levels to within the normal range, and prevented the galactorrhoea. The case presented here demonstrates that galactorrhoea can be present even with mild hypothyroidism.

Abnormal regulation of growth hormone (GH) secretion has been reported in some patients with insulin-dependent diabetes (IDD). We compared the GH responses in 32 healthy subjects (age 25 +/- 2 SE years) and in 23 IDD patients (28 +/- 1.9 years old, diabetes duration 10.4 +/- 2 years, and glycohemoglobin levels 9.3 +/- 2.0%). During acute, severe hypoglycemia (glucose less than 40 mg/dl), the mean GH levels were similar. When prolonged mild hypoglycemia was induced (58.0 +/- 2.0 mg/dl in the controls and 54.0 +/- 2.0 mg/dl in the IDD patients), the mean GH levels were similar, although the increase in GH was delayed in the latter group. During brief (30 min) exercise at 40-50% of VO2max, GH rose comparably in both groups (IDD patients maintained euglycemia with basal insulin infusion). However, with more prolonged and intense exercise using a glucose clamp to maintain euglycemia, GH rose to 5.4 +/- 2.2 ng/ml in controls and 26.4 +/- 12.6 ng/ml in the diabetics (P less than 0.05). When the combination of intense exercise and hypoglycemia (approximately 55 mg/dl) was used, GH rose to a peak of 21.7 +/- 2.7 ng/ml in the controls and to 33 +/- 3.0 ng/ml in the diabetics (P = NS). Our data show that in insulin-infused IDD patients made euglycemic for these experiments: a) The GH response to acute, severe hypoglycemia was identical to that in the controls and the response to mild, prolonged hypoglycemia was delayed, but of similar magnitude compared with controls; b) Exercise-induced GH responses were observed in both groups, but exaggerated in the diabetics at a higher exercise intensity; c) Hypoglycemia during exercise produced an additive effect on GH secretion in the controls but not in the IDD patients. We conclude that the wide range of abnormal GH secretory responses in type I diabetes reflects a central, possibly hypothalamic, defect in GH regulation.

A decline in plasma insulin and an increase in glucagon are known to occur during intense and/or prolonged exercise. However, it is not established whether changes in insulin and glucagon secretion are involved in the precise matching of hepatic glucose production to the enhanced glucose uptake by muscle during brief, low intensity exercise. We studied the effects of 30-min cycle exercise at 40% of maximal aerobic capacity in healthy subjects and C-peptide-deficient subjects with type 1 diabetes (IDDM) using [3-3H]glucose to estimate glucose turnover. Diabetic subjects were studied during continuous iv insulin infusion, which normalized glucose kinetics before experimental perturbations. In control (saline-infused) experiments, endogenous glucose appearance (Ra) increased by 80-90% above baseline to match the increase in glucose disappearance in both normal and IDDM subjects, even though the latter exercised at fixed levels of plasma free insulin, averaging 203 +/- 19 pmol/L. In other experiments, somatostatin was infused, and glucagon (1.0 ng/kg.min) and insulin (at two different rates) were maintained at constant levels. Infusion of insulin in normal subjects at doses sufficient to maintain constant peripheral plasma insulin was associated with no apparent effect on glucose turnover (plasma insulin, 80 +/- 21 pmol/L, compared to 52 +/- 5 pmol/L during saline; P = NS). However, insulin infusion at doses that normalized the portal insulin concentration (approximately 208 pmol/L) together with glucagon replacement inhibited the rise in glucose production in both normal and IDDM subjects. There were similar 45-55% reductions (P less than 0.03) of the increase in Ra seen with exercise in control experiments. When peripheral plasma free insulin (and presumably portal levels as well) were increased by about 20% in this experimental setting in IDDM (278 +/- 43 pmol/L), the suppression of Ra was even more profound, and Ra failed to increase at all with exercise. We conclude that the hormonal regulation of Ra in brief duration exercise in man does not necessitate the decrements in portal venous insulin observed under more intense exercise conditions as long as an exercise-induced glucagon secretory response can occur. Glucagon secretion alone cannot prevent hypoglycemia when portal venous insulin concentrations are increased by minimal amounts, such as in insulin-treated diabetics.

The effect of prolonged treatment with chromium on glucose homeostasis was assessed in rabbits. Thirty-one male rabbits were divided into 6 groups. All were fed for 30 days with a standard ad libitum diet. Twenty-seven rabbits were injected daily during a further 120 days with different doses of potassium chromate (3-20 micrograms) while 4 rabbits served as controls. No significant rise in serum glucose levels was observed, insulin levels were the same in the different groups, and glycosylated hemoglobin concentration also did not change after the administration of chromium. Serum chromium levels increased proportionally to the dose injected to an almost a 14 fold increase in the 20 micrograms chromium treated group. Although others have shown that chromium may have a hyperglycemic effect with inhibition of insulin secretion, we have not been able to confirm this finding.

A patient with methimazole-induced aplastic anemia is described. Despite severe pancytopenia and the complete disappearance of hemopoietic elements from the bone marrow, recovery of hemopoiesis has been observed within 14 days of discontinuing methimazole therapy. In vitro studies of CFU-C inhibition of peripheral mononuclear cells harvested in remission, were performed by incubation with autologous sera collected at earlier phases of the disease. These studies provide evidence in favor of a humoral, and most probably autoimmune mechanism as the cause of transient bone marrow aplasia.

The aim of the present study was to measure the PRL response to metoclopramide (MET) and chlorpromazine (CPZ) in seventeen patients with primary testicular failure and eight patients with isolated gonadotrophin deficiency (IGD). The responses were compared with those to TRH. Basal gonadotrophins and peak responses to LHRH were increased in testicular failure and reduced in IGD. Basal PRL levels were normal in both groups of patients. However, when compared with controls, the PRL response to both MET and CPZ as well as to TRH was exaggerated in primary testicular failure, whereas the responses wee decreased in IGD. In both patient groups, as well as in the controls, the PRL response to MET exceeded that to TRH and CPZ. It is suggested that alterations in the steroid milieu are responsible for the exaggerated PRL response to MET, CPZ and TRH in primary testicular failure and the reduced response observed in IGD.

We have assessed the gonadotropin, TSH and PRL responses to the non aromatizable androgens, mesterolone and fluoxymestrone, in 27 patients with primary testicular failure. All patients were given a bolus of LHRH (100 micrograms) and TRH (200 micrograms) at zero time. Nine subjects received a further bolus of TRH at 30 mins. The latter were then given mesterolone 150 mg daily for 6 weeks. The remaining subjects received fluoxymesterone 5 mg daily for 4 weeks and 10 mg daily for 2 weeks. On the last day of the androgen administration, the subjects were re-challenged with LHRH and TRH according to the identical protocol. When compared to controls, the patients had normal circulating levels of testosterone, estradiol, PRL and thyroid hormones. However, basal LH, FSH and TSH levels, as well as gonadotropin responses to LHRH and TSH and PRL responses to TRH, were increased. Mesterolone administration produced no changes in steroids, thyroid hormones, gonadotropins nor PRL. There was, however, a reduction in the integrated and incremental TSH secretion after TRH. Fluoxymesterone administration was accompanied by a reduction in thyroid binding globulin (with associated decreases in T3 and increases in T3 resin uptake). The free T4 index was unaltered, which implies that thyroid function was unchanged. In addition, during fluoxymesterone administration, there was a reduction in testosterone, gonadotropins and LH response to LHRH. Basal TSH did not vary, but there was a reduction in the peak and integrated TSH response to TRH. PRL levels were unaltered during fluoxymesterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

An improved chromatic rotatable refraction test for simultaneous spheric and astigmatic refraction is described. Its new features are interposition of a bright yellow stripe between the conventional red and green fields and a pair of parallel test lines, which traverse vertically all colored fields. Thereby astigmatic errors become easily visible.

An elevation of serum aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) may be produced in patients treated with i.v. full-dose HEPARIN. We studied the influence of low-dose s.c. HEPARIN (5,000 IU X 2) in 34 patients with acute myocardial infarction (AMI) and in 7 with cerebrovascular accidents or calf thrombophlebitis. Twelve patients (all males) with AMI showed a secondary elevation of GOT and GPT at about the sixth or seventh day after the commencement of therapy that persisted throughout the period of treatment. Four patients (two males and two females) with cerebrovascular accidents or thrombophlebitis showed similar increases of GOT and GPT.

To investigate prolactin (PRL) and thyrotropin-stimulating hormone (TSH) secretion in ovarian failure, 14 women with primary ovarian failure were challenged with luteinizing hormone-releasing hormone (LHRH) (100 micrograms) and thyrotropin-releasing hormone (TRH) (200 micrograms) given intravenously at 30-minute intervals. Responses were compared with those of 13 healthy female controls. In the patient group, basal follicle-stimulating hormone (FSH), LH, and peak gonadotropin responses to LHRH were higher and basal estrone and estradiol levels were lower than in the controls (P less than .001). Mean basal PRL levels were similar in the 2 groups. However, the mean peak and integrated PRL responses in the patients were greater than in the controls (P less than .05). Ten patients had a markedly exaggerated PRL response to TRH. The mean basal TSH levels and the peak TSH response to TRH were similar to those of the controls. Estrogens are known to stimulate PRL secretion. These subjects had increased PRL responses with low circulating estrogens. The mechanism underlying the findings is not known, but could be related to increased aromatization of androgens to estrogens in the hypothalamus. Alternatively, other factors could be responsible for the exaggerated PRL responses to TRH noted in these patients with primary ovarian failure.

We studied two sisters who developed large non-toxic goitres in adolescence. Deiodinase deficiency was diagnosed by a rapid thyroid uptake of radioactive iodine (RAI) at 2 hours associated with a marked fall in thyroidal 131I by 24 hours. Serial RAI scans in the second patient documented evolution of the iodine-deficient state. Conservation of intra-thyroidal iodine stores was maintained by avid iodine uptake and failure to release organified 131I. With progressive loss of inorganic iodine, hypothyroidism developed, associated with a rise in serum TSH which further exacerbated the loss of iodine. Treatment with L-thyroxine resulted in an improvement of thyroid function, but normalization was achieved only after small doses of Lugol's iodine were administered. These studies illustrate the variable nature and late onset of an inborn error of thyroid metabolism. This family supports an autosomal recessive mode of inheritance for deiodinase deficiency. We have documented progression from a euthyroid to hypothyroid state resulting from decompensation of iodine conservation mechanisms.

Hypophosphatemia in diabetic ketoacidosis is well recognized, but is believed to be usually of moderate severity. We describe 2 patients in whom acute hemolytic anemia secondary to severe (0.19-0.35 mmol/l) hypophosphatemia has developed 1-2 days following treatment for diabetic ketoacidosis. Our experience indicates that severe hypophosphatemia requiring phosphate supplementation does occur in diabetic patients, and calls for increased awareness for the clinical and laboratory manifestations of this complication of diabetic ketoacidosis.