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Biomedical subjects

John Kelly

Publications and source records attributed to John Kelly.

At least 37 records · Page 2Linked to original sources
PubMed2005

Specialized staff and equipment for weight loss surgery patients: best practice guidelines.

OBJECTIVE: To provide evidence-based guidelines on the specialized personnel, equipment, and physical plant required for safe and effective care of severely obese weight loss surgery (WLS) patients. RESEARCH METHODS AND PROCEDURES: We examined MEDLINE (Ovid and PubMed) and the Cumulative Index of Nursing and Allied Health Literature for articles on facilities resources for care of WLS patients published in English between January 1980 and March 2004. We queried several web sites for appropriate references; these included the Agency for Healthcare Research and Quality and the American College of Surgeons. The majority of reference material was descriptive and not specific to facilities resources for WLS patients. We identified a substantial body of literature on the general subject of patient safety; three of these articles were used to develop recommendations on the use of technology for medical error reduction. All other recommendations are based on 11 expert opinion reports. RESULTS: We recommended adequate training and credentialing for all medical staff; dedicated support and administrative personnel; and specialized interventional, diagnostic, operating room, and transport equipment. We specified needed adaptations to the physical plant and developed evidence-based guidelines for medical error reduction and systems improvements. DISCUSSION: Specialized resources and dedicated staff are needed to protect the health of WLS surgery patients and staff. Adaptations include preoperative preparation for safe means of patient transport; techniques of anesthesia and intraoperative exposure; provisions for postoperative recovery; and measures to assure postoperative patient safety, hygiene, and comfort.

Credentialing↗
PubMed2005

Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT-nanoLC-MS/MS.

Cerebral ischemia rapidly initiates structural and functional changes in brain vessels, including blood-brain barrier disruption, inflammation, and angiogenesis. Molecular events that accompany these changes were investigated in brain microvessels extracted using laser-capture microdissection (LCM) from Sprague-Dawley rats subjected to a 20 min transient global cerebral ischemia followed by 1, 6, or 24 h reperfusion. Proteins extracted from approximately 300 LCM captured microvessels (20-100 microm) were ICAT-labeled and analyzed by nanoLC-MS. In-house software was used to identify paired ICAT peaks, which were then sequenced by nanoLC-MS/MS. Pattern analyses using k-means clustering method classified 57 differentially expressed proteins in 7 distinct dynamic patterns. Protein function was assigned using Panther Classification system. Early reperfusion (1 h) was characterized by down-regulation of ion pumps, nutrient transporters, and cell structure/motility proteins, and up-regulation of transcription factors, signal transduction molecules and proteins involved in carbohydrate metabolism. The up-regulation of inflammatory cytokines and proteins involved in the extracellular matrix remodeling and anti-oxidative defense was observed in late reperfusion (6-24 h). The up-regulation of IL-1beta and TGF-1beta in ischemic brain vessels was confirmed by ELISA, quantitative PCR, and/or immunohistochemistry. A biphasic postischemic (1 and 24 h) BBB opening for (3)H-sucrose was evident in the same model. Differentially expressed proteins identified in brain vessels during reperfusion are likely involved in orchestrating functional vascular responses to ischemia, including the observed BBB disruption.

Animals↗
PubMed2005

A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine.

Francisella tularensis subsp. tularensis (type A) strain SCHU S4 is a prototypic strain of the pathogen that is highly virulent for humans and other mammals. Its intradermal (i.d.) 50% lethal dose (LD50) for mice is <10 CFU. We discovered a spontaneous mutant, designated FSC043, of SCHU S4 with an i.d. LD50 of >10(8) CFU. FSC043 effectively vaccinated mice against challenge with a highly virulent type A strain, and the protective efficacy was at least as good as that of F. tularensis LVS, an empirically attenuated strain which has been used as an efficacious human vaccine. Comparative proteomics was used to identify two proteins of unknown function that were identified as defective in LVS and FSC043, and deletion mutants of SCHU S4 were created for each of the two encoding genes. One mutant, the DeltaFTT0918 strain, failed to express a 58-kDa protein, had an i.d. LD50 of approximately 10(5) CFU, and was found to be less capable than SCHU S4 of growing in peritoneal mouse macrophages. Mice that recovered from sublethal infection with the DeltaFTT0918 mutant survived when challenged 2 months later with >100 LD50s of the highly virulent type A strain FSC033. This is the first report of the generation of defined mutants of F. tularensis subsp. tularensis and their use as live vaccines.

Administration, Cutaneous↗
PubMed2005

An interactive framework for developing simulation models of hospital accident and emergency services.

Discrete-event simulation can be a valuable tool in modelling health care systems. This paper describes an interactive framework to model and simulate a hospital accident and emergency department. An interactive spreadsheet (Excel) facilitated the user-friendly input of data such as patient pathways, arrival times, service times and resources into the discrete event simulation package (SIMUL8). The framework was enhanced further by configuring SIMUL8 to visually show patient flow and activity on a schematic plan of an A&E. The patient flow and activity information included patient icons flowing along A&E corridors and pathways, processes undertaken in A&E work areas and queue activity. One major benefit of visually showing patient flow and activity was that modellers and decision makers could visually gain a dynamic insight into the performance of the overall system and visually see changes over the model run cycle. Another key benefit of the interactive framework was the ability to quickly and easily change model parameters to trial, test and compare different scenarios.

Accidents↗
PubMed2004

Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells.

Regulation of the rolipram-sensitive cAMP-specific phosphodiesterase 4 (PDE4) gene family was studied in rat pulmonary microvascular endothelial cells (RPMVECs). Total PDE4 hydrolysis was increased within 10 min after addition of forskolin (10 microM), reached a maximum at 20-40 min, and then gradually declined in the cells. A similar activation of PDE4 activity was observed using a protein kinase A (PKA) activator, N(6)-monobutyryl cAMP. Both the forskolin and the N(6)-monobutyryl cAMP activated PDE4 activities were blocked by the PKA-specific inhibitor, H89. This forskolin-stimulated and PKA-mediated short-term activation of PDE4 activity was further confirmed by in vitro phosphorylation of 87kDa PDE4A6 and 83kDa PDE4B3 polypeptides using exogenous PKA Calpha. Increased immunoreactivity of phosphorylated PDE4A6 in situ was detected in Western blots by a PDE4A-phospho antibody specific to the putative PKA phosphorylation sites. Following long-term treatment of RPMVECs with rolipram and forskolin medium (RFM) for more than 60 days, PDE4 activity reached ten-fold higher values than control RPMVECS with twenty-fold increases detected in intracellular cAMP content. The RFM cells showed increased immunoreactivities of the constitutive 4A6 and 4B3 isoforms plus two novel splice variants at 101kDa (4B1) and 71kDa (4B2). Treatment with H89 did not inhibit the PDE4 elevation in RFM cells. In addition to the increased levels of PDE4 in RFM cells, immunofluorescence showed a translocation of PDE4A and 4B to a nuclear region, which was normally not observed in RPMVECs. The PDE4 activity in RFM cells decayed rapidly with an even faster decline of intracellular cAMP content when forskolin/rolipram were removed from the medium. These results suggest that both the activation (short-term) and induction (long-term) of PDE4A/4B isoforms in RPMVECs are closely modulated by the intracellular cAMP content via both post-translational and synthetic mechanisms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2004

A pentavalent single-domain antibody approach to tumor antigen discovery and the development of novel proteomics reagents.

Proteomics research has delivered many novel tumor targets. However, due to key limitations, it does not specifically identify targets that are most accessible for drug delivery, such as cell-surface antigens. A novel tumor antigen discovery platform based on screening a single domain antibody (sdAb) library against tumor cells and subsequently identifying the corresponding antigens of the isolated antibodies is described. An sdAb, AFAI, specific for non-small cell lung carcinoma (A549 cell line) was isolated from a phage library derived from the heavy chain antibody repertoire of a llama. The homopentamerization property of a non-toxic verotoxin B-subunit was exploited to make the ES1 pentabody, a pentameric form of AFAI. Pentamerization improved the binding of the AFAI to A549 cells dramatically and greatly facilitated antigen identification by a Western blotting/mass spectrometry approach. The antigen of ES1, which is present only in the hydrophobic, not in the hydrophilic, fraction of A549 cellular proteins, was identified as carcinoembryonic antigen-related cell adhesion molecule 6 (CEA6). CEA6 was observed to be acidic and highly glycosylated, and to exist in multiple glycoforms. The results show that the platform described here should find wide application in antigen discovery, and demonstrated that the pentabodies are very useful immunological reagents for proteomics.

Amino Acid Sequence↗
PubMed2004

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9.

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.

Adenosine Triphosphate↗
PubMed2004

A role for thymic stromal lymphopoietin in CD4(+) T cell development.

Thymic stromal lymphopoietin (TSLP) signals via a receptor comprising the interleukin (IL)-7 receptor alpha chain and a distinctive subunit, TSLP receptor (TSLPR), which is most related to the common cytokine receptor gamma chain, gamma(c). We have generated TSLPR knockout (KO) mice and found that although these mice had normal lymphocyte numbers, gamma(c)/TSLPR double KO mice had a greater lymphoid defect than gamma(c) KO mice. This indicates that TSLP contributes to lymphoid development and accounts for some of the residual lymphoid development in gamma(c) KO mice and presumably in patients with X-linked severe combined immunodeficiency. Injection of TSLP into gamma(c) KO mice induced the expansion of T and B cells. Moreover, sublethally irradiated TSLPR KO mice showed weaker recovery of lymphocyte populations than wild-type (WT) littermates, even when neutralizing anti-IL-7 antibodies were injected. Interestingly, TSLP preferentially stimulated the proliferation and survival of CD4(+) single positive thymocytes and peripheral T cells in vitro. Additionally, CD4(+) T cells from TSLPR KO mice expanded less efficiently than WT CD4(+) T cells in irradiated hosts, and TSLP preferentially expanded CD4(+) T cells both in vitro and in vivo. Thus, as compared with other known cytokines, TSLP is distinctive in exhibiting a lineage preference for the expansion and survival of CD4(+) T cells.

Animals↗
PubMed2004

Viral FLIP impairs survival of activated T cells and generation of CD8+ T cell memory.

Viral FLIPs (vFLIPs) interfere with apoptosis signaling by death-domain-containing receptors in the TNFR superfamily (death receptors). In this study, we show that T cell-specific transgenic expression of MC159-vFLIP from the human Molluscum contagiosum virus blocks CD95-induced apoptosis in thymocytes and peripheral T cells, but also impairs postactivation survival of in vitro activated primary T cells despite normal early activation parameters. MC159 vFLIP impairs T cell development to a lesser extent than does Fas-associated death domain protein deficiency or another viral FLIP, E8. In the periphery, vFLIP expression leads to a specific deficit of functional memory CD8(+) T cells. After immunization with a protein Ag, Ag-specific CD8(+) T cells initially proliferate, but quickly disappear and fail to produce Ag-specific memory CD8(+) T cells. Viral FLIP transgenic mice exhibit impaired CD8(+) T cell responses to lymphocytic choriomeningitis virus and Trypanosoma cruzi infections, and a specific defect in CD8(+) T cell recall responses to influenza virus was seen. These results suggest that vFLIP expression in T cells blocks signals necessary for the sustained survival of CD8(+) T cells and the generation of CD8(+) T cell memory. Through this mechanism, vFLIP proteins expressed by T cell tropic viruses may impair the CD8(+) T cell immune responses directed against them.

Animals↗
PubMed2004

Genome-wide expression analyses of Campylobacter jejuni NCTC11168 reveals coordinate regulation of motility and virulence by flhA.

We examined two variants of the genome-sequenced strain, Campylobacter jejuni NCTC11168, which show marked differences in their virulence properties including colonization of poultry, invasion of Caco-2 cells, and motility. Transcript profiles obtained from whole genome DNA microarrays and proteome analyses demonstrated that these differences are reflected in late flagellar structural components and in virulence factors including those involved in flagellar glycosylation and cytolethal distending toxin production. We identified putative sigma(28) and sigma(54) promoters for many of the affected genes and found that greater differences in expression were observed for sigma(28)-controlled genes. Inactivation of the gene encoding sigma(28), fliA, resulted in an unexpected increase in transcripts with sigma(54) promoters, as well as decreased transcription of sigma(28)-regulated genes. This was unlike the transcription profile observed for the attenuated C. jejuni variant, suggesting that the reduced virulence of this organism was not entirely due to impaired function of sigma(28). However, inactivation of flhA, an important component of the flagellar export apparatus, resulted in expression patterns similar to that of the attenuated variant. These findings indicate that the flagellar regulatory system plays an important role in campylobacter pathogenesis and that flhA is a key element involved in the coordinate regulation of late flagellar genes and of virulence factors in C. jejuni.

Animals↗
PubMed2004

Discovering novel phenotype-selective neurotrophic factors to treat neurodegenerative diseases.

Astrocytes and neurons in the central nervous system (CNS) interact functionally to mediate processes as diverse as neuroprotection, neurogenesis and synaptogenesis. Moreover, the interaction can be homotypic, implying that astrocyte-derived secreted molecules affect their adjacent neurons optimally vs remote neurons. Astrocytes produce neurotrophic and extracellular matrix molecules that affect neuronal growth, development and survival, synaptic development, stabilization and functioning, and neurogenesis. This new knowledge offers the opportunity of developing astrocyte-derived, secreted proteins as a new class of therapeutics specifically to treat diseases of the CNS. However, primary astrocytes proliferate slowly in vitro, and when induced to immortalize by genetic manipulation, tend to lose their phenotype. These problems have limited the development of astrocytes as sources of potential drug candidates. We have successfully developed a method to induce spontaneous immortalization of astrocytes. Gene expression analysis, karyotyping and activity profiling data show that these spontaneously immortalized type-1 astrocyte cell lines retain the properties of their primary parents. The method is generic, such that cell lines can be prepared from any region of the CNS. To date, a library of 70 cell lines from four regions of the CNS: ventral mesencephalon, striatum, cerebral cortex and hippocampus, has been created. A phenotype-selective neurotrophic factor for dopaminergic neurons has been discovered from one of the cell lines (VMCL1). This mesencephalic astrocyte-derived neurotrophic factor (MANF) is a 20 kD, glycosylated, human secreted protein. Homologs of this protein have been identified in 16 other species including C. elegans. These new developments offer the opportunity of creating a library of astrocyte-derived molecules, and developing the ones with the best therapeutic indices for clinical use.

Animals↗
PubMed2004

Iontophoresis with glycopyrrolate for the treatment of palmoplantar hyperhidrosis.

To determine the comparative efficacy of tap water iontophoresis to iontophoresis with the anticholinergic glycopyrrolate, we undertook a single-blinded right-left comparison study in 20 patients with palmoplantar hyperhidrosis. Most patients had their palms treated and one patient had the soles treated. We compared the duration of symptom relief following iontophoresis with glycopyrrolate unilaterally to iontophoresis with glycopyrrolate bilaterally. Patients filled in daily efficacy assessment cards. Each palm was rated as 'dry', 'slightly wet', 'moderately wet' or 'very wet'. Following treatment with unilateral tap water iontophoresis, unilateral glycopyrrolate and bilateral glycopyrrolate, patients reported hand dryness for a median of 3, 5 and 11 days, respectively. As the data was paired, treatment differences were analysed using a sign-rank test. Bilateral glycopyrrolate was superior to both unilateral glycopyrrolate and tap water in most patients. Unilateral glycopyrrolate was superior to tap water in most patients. All differences between groups were found to be statistically significant. We postulate that the increased efficacy of bilateral glycopyrrolate when compared with unilateral glycopyrrolate relates to its systemic absorption. We conclude that glycopyrrolate iontophoresis is more effective than tap water iontophoresis in the treatment of palmoplantar hyperhidrosis and that glycopyrrolate iontophoresis has both local and systemic effects on perspiration.

Adolescent↗
PubMed2004

Laparoscopic Roux-En-Y gastric bypass is a safe and effective operation for the treatment of morbid obesity in patients older than 55 years.

BACKGROUND: Bariatric surgery in patients >50 years has been controversial. We investigated the safety and efficacy of laparoscopic Roux-en-Y gastric bypass (LRYGBP) in patients >55 years of age. METHODS: Prospective data on 71 patients (54 females and 17 males) undergoing LRYGBP were reviewed. The patients were followed for a mean of 17 months (range 2-35 months). RESULTS: The mean age was 59 years (range 55-67 years), and the mean preoperative BMI was 50.2 kg/m2 (range 37-65 kg/m2). There were no conversions to open technique. Mean percent of excess weight loss (%EWL) was 20%, 48%, 64% and 67% at 1, 6, 12 and 24 months respectively. 89% of patients had at least a 50% EWL at 1 year postoperatively. There was a significant decrease in the number of patients requiring medical treatment for co-morbidities associated with morbid obesity: diabetes mellitus 87%, hypertension 70% and sleep apnea 86%. There was no inpatient mortality. 1 patient died suddenly 2 weeks postoperatively of possible myocardial infarction or pulmonary embolism. 16 patients developed 22 complications. The median length of hospital stay was 3 days. CONCLUSION: LRYGBP is a safe and well-tolerated surgical option for the treatment of morbid obesity in patients >55 years old. These patients demonstrate a satisfactory weight loss and resolution of co-morbidities.

Age Factors↗
PubMed2003

A role for Stat5 in CD8+ T cell homeostasis.

Cytokine signals are known to contribute to CD8+ memory T cell homeostasis, but an exact understanding of the mechanism(s) has remained elusive. We have now investigated the role of Stat5 proteins in this process. Whereas Stat5a and Stat5b KO mice have decreased numbers of CD8+ T cells, Stat5-transgenic mice have an increased number of these cells. Stat5b-transgenic mice exhibit increased Ag-induced cell death of CD4+ T cells and augmented proliferation and Bcl-2 expression in CD8+ T cells, providing a basis for this finding. Moreover, CD8+ memory T cells are substantially affected by Stat5 levels. These findings identify Stat5 proteins as critical signaling mediators used by cytokines to regulate CD8+ T cell homeostasis.

Animals↗
PubMed2003

Multicentric Castleman's disease treated with antivirals and immunosuppressants.

A patient negative for human immunodeficiency virus (HIV) developed multicentric Castleman's disease (MCD) and Kaposi's sarcoma (KS) associated with active human herpesvirus 8 (HHV-8) infection. He was treated with sequential antiviral therapy, chemotherapy, and corticosteroids. HHV-8 levels were monitored throughout the course of the patient's illness, and were found to rise on relapse. No consistent change in HHV-8 levels was found with antiviral therapy. We demonstrate that in this patient antiviral therapy was clinically ineffective, and did not alter HHV-8 levels, but that corticosteroid and combination chemotherapy led to clinical improvement. Despite the implication of HHV-8 as a cause of MCD, few studies have correlated HHV-8 levels with clinical response.

Aged↗
PubMed2003

Immune deficiencies due to defects in cytokine signaling.

Severe combined immunodeficiency (SCID) represents a syndrome comprising the most severe forms of inherited immunodeficiencies. Defects in cytokine signaling pathways can result in impaired development of lymphoid cells and/or defective functioning of these cells, and most cases of SCID result from defective signaling through the common cytokine receptor g chain (g(c)) or associated molecules and signaling pathways. Studies of these patients and the analysis of gene-targeted mice provide insight into the underlying signaling defects in inherited immunodeficiencies. The identification of the genetic defects in humans with SCID provides the basis for future therapies for these patients. More subtle deficiencies in cytokine signaling have also been found as causes of other forms of immunodeficiency, and the knowledge learned could lead to novel approaches to antimicrobial therapy.

Child↗