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Biomedical subjects

J D Geiger

Publications and source records attributed to J D Geiger.

At least 37 records · Page 2Linked to original sources
PubMed2000

Selective laparoscopic probing for a contralateral patent processus vaginalis reduces the need for contralateral exploration in inconclusive cases.

BACKGROUND/PURPOSE: Repair of an indirect inguinal hernia is the most common procedure performed by pediatric surgeons, yet the need for contralateral exploration remains controversial. This prospective study evaluated the use of laparoscopy combined with the selective use of probing to determine the presence or absence of a contralateral patent processus vaginalis (CPPV). METHODS: A consecutive series of 75 of the author's patients presenting with a unilateral inguinal hernia were studied. Laparoscopy was performed through the known hernia sac with CO2 insufflation utilizing a 4-mm 70 degrees telescope to inspect the contralateral inguinal region. A patent processus was diagnosed when an obvious opening through the internal ring was identified. In some patients this evaluation was found to be inconclusive because of the inability to determine accurately the length of the patent processus or to rule out a patent processus obscured by a veil of peritoneum. This group of patients was then evaluated selectively with a silver probe placed under direct vision through a 14-gauge intravenous catheter placed through the abdominal wall on the side in question to manipulate the peritoneum and directly measure the length of the potentially patent processus. A patent processus greater then 1.5 cm was considered positive. RESULTS: A total of 54 patients (72%) had a conclusive laparoscopic evaluation of the contralateral inguinal region with a CPPV clearly absent in 38 and an obvious CPPV present in 16. A total of 21 patients (28%) had inconclusive laparoscopy finding and were evaluated further with probing. The probing technique changed the determination of a CPPV in 15 patients (12 positive to negative and 3 negative to positive; P = .029, Fisher's Exact test). Probing resulted in a decrease in the overall rate of identification of a CPPV from 45% to 32%. CONCLUSION: In inconclusive cases, the addition of selective probing to the laparoscopic evaluation for a CPPV reduces the number of patent processus vaginalis found and the need for contralateral inguinal exploration.

Female↗
PubMed2000

A longitudinal analysis of the pediatric surgeon workforce.

OBJECTIVE: To describe the trends in the pediatric surgeon workforce during the last 25 years and to provide objective data useful for planning graduate medical education requirements. SUMMARY BACKGROUND DATA: In 1975, the Study on U.S. Surgical Services (SOSSUS) was published, including a model to survey staffing. A pediatric surgeon workforce study was initiated in conjunction with SOSSUS as a population, supply, and need-based study. The study has been updated every 5 years using the same study model, with the goals of determining the number and distribution of pediatric surgeons in the United States, the number needed and where, and the number of training programs and trainee output required to fill estimated staffing needs. This is the only such longitudinal workforce analysis of a surgical specialty. METHODS: Questionnaires were sent to 100 pediatric surgeons representing the 62 standard metropolitan statistical areas (SMSAs) in the United States with a population of 200,000 or more to verify the names and locations of all active pediatric surgeons and to gain information about the 5-year need for new pediatric surgeons by region. A program was developed to predict the number of pediatric surgeons relative to the total population and the 0-to-17-year-old population in the subsequent 30 years using updated data on the present number and ages of pediatric surgeons, age-specific death and retirement rates, projections of U.S. population by age group, and varying numbers of trainees graduated per year. As each 5-year update was done, previous projections were compared with actual numbers of pediatric surgeons found. The trends during the last 25 years were analyzed and compared and additional information regarding the demographics of practice, trends in reimbursement, and volume and scope of surgery was obtained. RESULTS: The birth rate has been stable since 1994. The 0-to-17-year-old population has been increasing at 0.65% per year; a 0.64% annual rate is projected to 2040. At present, 661 pediatric surgeons are distributed in every SMSA of 200,000 or more population, with an average age of 45 and an average age of retirement 65. The actual number of pediatric surgeons in each 5-year survey has consistently validated previous projections. Trainee output has increased markedly in the past 10 years. The rate of growth of the pediatric surgeon workforce at present is 50% greater than the forecasted rate of increase in the pediatric age group, and during the past 25 years the rate of growth of the pediatric surgeon workforce has been double that of the pediatric population growth. Nationally, significant changes in reimbursement, volume of surgery, and demographics of practice have occurred.

Adolescent↗
PubMed2000

Synergistic neurotoxicity by human immunodeficiency virus proteins Tat and gp120: protection by memantine.

Human immunodeficiency virus type 1 (HIV-1) proteins Tat and gp120 have been implicated in the pathogenesis of dementia associated with HIV infection. Recently, we showed the presence of Tat protein in brains of patients with HIV-1 encephalitis as well as macaques with encephalitis caused by a chimeric strain of HIV and simian immunodeficiency virus, and that even transient exposure of cells to Tat leads to release of cytopathic cytokines. Now, we report the first demonstration of gp120 protein in brain of patients with HIV encephalitis. We tested the hypothesis that Tat and gp120 would act synergistically to potentiate each protein's neurotoxic effects and determined the extent to which pharmacological antagonists against processes implicated in HIV-1 neuropathogenesis could block HIV-1 protein-induced neurotoxicity. Subtoxic concentrations of Tat and gp120, when incubated together, caused neuronal cell death and prolonged increases in levels of intracellular calcium. A transient exposure of neurons to Tat and gp120 for seconds initiated neuronal cell death, but maximal levels of neuronal cell death were observed with exposures lasting 30 minutes. The neurotoxicity caused by Tat and gp120 applied in combination was blocked completely by memantine, partially by amiloride, and not at all by dipyridamole or vigabatrin.

AIDS Dementia Complex↗
PubMed2000

Caffeine use in sports. A pharmacological review.

Caffeine is the most widely ingested psychoactive drug in the world. As many know, chronic use of caffeine leads to dependence, tolerance, drug craving, and upon abrupt cessation unpleasant withdrawal symptoms. Thus, caffeine fulfills pharmacological criteria by which agents are classified as drugs of abuse. Nevertheless, its use is legal and only at high, but readily attainable, levels is it banned from sport. Its use is widespread by athletes as young as 11 years of age who are seeking athletic advantage over fellow competitors. It is likely that its use will not decline any time soon because it is inexpensive, readily available, medically quite safe, socially acceptable, and by most measures legal. However, at levels allowed in sport, caffeine through its wide-ranging physiological and psychological effects increases endurance in well-trained athletes. If the goal of drug-testing and education programs in sport is to protect the health of athletes, prevent unfair advantage (cheating) and encourage ethical behavior then it seems obvious that the allowable levels of caffeine ingestion should be decreased. The alternative is to continue with policies designed largely to punish only those that get caught.

Animals↗
PubMed2000

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.

Adolescent↗
PubMed2000

Dietary supplement creatine protects against traumatic brain injury.

Creatine, one of the most common food supplements used by individuals at almost every level of athleticism, promote gains in performance, strength, and fat-free mass. Recent experimental findings have demonstrated that creatine affords significant neuroprotection against ischemic and oxidative insults. The present experiments investigated the possible effect of creatine dietary supplementation on brain tissue damage after experimental traumatic brain injury. Results demonstrate that chronic administration of creatine ameliorated the extent of cortical damage by as much as 36% in mice and 50% in rats. Protection seems to be related to creatine-induced maintenance of mitochondrial bioenergetics. Mitochondrial membrane potential was significantly increased, intramitochondrial levels of reactive oxygen species and calcium were significantly decreased, and adenosine triphosphate levels were maintained. Induction of mitochondrial permeability transition was significantly inhibited in animals fed creatine. This food supplement may provide clues to the mechanisms responsible for neuronal loss after traumatic brain injury and may find use as a neuroprotective agent against acute and delayed neurodegenerative processes.

Animals↗
PubMed1999

Dysregulation of adenosine A1 receptor-mediated cytokine expression in peripheral blood mononuclear cells from multiple sclerosis patients.

Cytokines, including tumor necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6), have been implicated in the pathogenesis of multiple sclerosis (MS). The production and release of these cytokines are regulated in part by specific purinergic (adenosine) cell surface receptors. To determine the extent to which the adenosine A1 receptor influenced cytokine expression in peripheral blood mononuclear cells (PBMCs) from MS and control patients, we measured plasma adenosine and TNF alpha levels, A1 receptor messenger RNA (mRNA) and protein amounts, and the effects of activation of A1 receptors on TNF alpha and IL-6 production by PBMCs. Plasma levels of TNF alpha were significantly higher and adenosine levels were significantly lower in MS patients compared with control subjects. Levels of TNF alpha and IL-6 in mitogen-stimulated PBMC culture supernatants from MS patients or control patients were similar. Conversely, treatment of PBMCs with the adenosine A1 receptor agonist R-phenylisopropyladenosine (R-PIA) (1 microM) significantly inhibited mitogen-stimulated production of TNF alpha but not IL-6 in control subjects and significantly inhibited production of IL-6 but not TNF alpha in MS patients. The effects of R-PIA were selectively blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). A1 receptor protein levels were decreased significantly in PBMCs from MS patients. Taken together, these results suggest that decreased levels of adenosine and its A1 receptor modulate TNF alpha and IL-6 levels and may contribute to the pathogenesis of MS.

Adenosine↗
PubMed1999

The market for pediatric surgeons: a survey of recent graduates.

PURPOSE: The aim of this study was to identify the demand for pediatric surgeons as perceived and experienced by recent graduates of North American training programs. METHODS: A survey questionnaire was mailed to every pediatric surgeon who had completed a certified training program in the United States or Canada between 1992 and 1997; 84% of the 165 responded. The data were then analyzed using univariate and bivariate statistics and content analysis. RESULTS: The number trained has risen since 1992 from 21 to 35 per year, exceeding previous definitions of need. However, recently trained pediatric surgeons found positions, and their first-year incomes had risen oven the 6-year period. In contrast, just 54% found first positions in the type of hospital desired, and the percent working in a children's hospital dropped from 65% in 1992 to 32% in 1997; 34% cover between four and ten hospitals. The majority of those in practice for more than 2 years expressed the perception of a decline in market demand with just 30% of those 1996 to 1997 graduates perceiving a strong market. The clinical scope of practice was less than that for which they were trained. Three specified complex cases were managed by fewer than 30% of recent graduates during practice despite more than 60% having had fellowship experience. The scope of practice, as measured by variables of index procedures, was strongly associated with hospital type (children's or general) and by practice region. Although satisfaction with practice is lower for each successive class, 96% of the graduates were satisfied with their training programs, and 98% believed they had been well prepared, although 46% indicated they desired some additional training. Sixty-one percent believed the role of pediatric surgeons will change over the next 5 years. CONCLUSIONS: The market demand was strong as measured by employment and income. This was in contrast to the striking recent changes in the market for new pediatric surgeons, including a migration of practice from children's to general hospitals, a reduced scope of practice, a more negative perception of the pediatric surgery market, and concerns for narrowing of the specialty.

Canada↗
PubMed1999

Role of Na+/H+ exchangers, excitatory amino acid receptors and voltage-operated Ca2+ channels in human immunodeficiency virus type 1 gp120-mediated increases in intracellular Ca2+ in human neurons and astrocytes.

Human immunodeficiency virus type 1 (HIV-1) dementia is the commonest form of dementia in North American people less than 60 years of age. HIV-1 envelope glycoprotein gp120 has been implicated in the neurotoxicity observed in, and the pathogenesis of, HIV-1 dementia. Recombinant gp120 (gp120) was pressure-applied on to cultured human fetal neurons and astrocytes and, by using single-cell calcium imaging, we determined the mechanisms responsible for gp120-induced increases in the levels of intracellular calcium ([Ca2+]i). Significant dose-related increases in [Ca2+]i were observed in neurons and astrocytes. In neurons, 5 pM gp120 increased [Ca2+]i by 290+/-13 nM and increases of 2210+/-211 nM were found at 209 nM, the highest concentration of gp120 tested. The apparent EC50 value for gp120 of 223+/-40 pM in neurons was not significantly different from that in astrocytes. Immunoelution of gp120 with polyclonal anti-gp120 and Ca2+-free conditions blocked increases in [Ca2+]i by gp120. Increases in [Ca2+]i were significantly (P < 0.005) attenuated by the Na+/H+ exchange blocker 5-(N-methyl-N-isobutyl)-amiloride in neurons and astrocytes. The L-type calcium channel blockers nimodipine, diltiazem and CdCl2 + NiCl2 significantly (P < 0.005) reduced increases in [Ca2+]i in neurons, but not astrocytes. Increases in [Ca2+]i by gp120 were not significantly affected by blockers of N-, P- and Q-type calcium channels. The N-methyl-D-aspartate receptor antagonists (+/-)-2-amino-5-phosphonopentanoic acid (AP5), memantine and dizocilpine significantly (P < 0.01) lowered gp120-induced increases in [Ca2+]i in neurons. AP5 and memantine, but not dizocilpine, significantly (P < 0.01) reduced increases in [Ca2+]i by gp120 in astrocytes. Gp120 appears to activate astrocyte Na+/H+ exchangers to release glutamate and potassium and, subsequent to this, increases in [Ca2+]i in neurons and astrocytes result from activation of excitatory amino acid receptors on astrocytes and neurons, and voltage-operated calcium channels on neurons. Drugs that block gp120-induced changes in [Ca2+]i in neurons and astrocytes may help in the treatment of HIV-1 dementia.

Astrocytes↗
PubMed1999

Recovery of high-integrity mRNA from brains of rats killed by high-energy focused microwave irradiation.

Following decapitation, during the brief time period of postmortem brain tissue collection, significant changes in neuro-metabolite levels can occur. To circumvent such changes, we routinely kill rats using 1 to 2 sec pulses of focused high-energy microwave irradiation (10 kW). The effects of high-energy microwave irradiation on total RNA and mRNA integrity in brain however, are unknown. Total RNA recovery, per gram wet weight, in brain regions of microwaved rats was less than 50% of that in rats killed by decapitation. Formaldehyde agarose gel electrophoresis showed that ribosomal RNA components were highly degraded in all brain regions of microwaved rats. In contrast, poly A+mRNA, as measured by poly A+driven cDNA synthesis and Northern analysis, in brain samples of microwaved or decapitated rats was of equal integrity and quantity when expressed per mg tissue weight. Furthermore, positive RT-PCR products for GAPDH and TNF-alpha were observed in brain regions of both microwaved and decapitated rats. These observations indicated that high-energy focused microwave irradiation does not reduce mRNA abundance and integrity. Thus, this method of animal sacrifice can be used to simultaneously study, accurately and precisely, levels of brain metabolites as well as molecular biological events in discrete brain regions of experimental animals without postmortem interference.

Adenosine↗
PubMed1999

Secreted form of amyloid precursor protein enhances basal glucose and glutamate transport and protects against oxidative impairment of glucose and glutamate transport in synaptosomes by a cyclic GMP-mediated mechanism.

Synaptic dysfunction and degeneration are believed to underlie the cognitive deficits that characterize Alzheimer's disease, and overactivation of glutamate receptors under conditions of increased oxidative stress and metabolic compromise may contribute to the neurodegenerative process in many different disorders. The secreted form of amyloid precursor protein (sAPPalpha), which is released from neurons in an activity-dependent manner, can modulate neurite outgrowth, synaptic plasticity, and neuron survival. We now report that sAPPalpha can enhance glucose and glutamate transport in synaptic compartments. Treatment of cortical synaptosomes with nanomolar concentrations of sAPPalpha resulted in an attenuation of impairment of glutamate and glucose transport induced by exposure to amyloid beta-peptide and Fe2+. The protective effect of sAPPalpha was mimicked by treatment with 8-bromo-cyclic GMP and blocked by a cyclic GMP-dependent protein kinase inhibitor, suggesting that protective action of sAPPalpha is mediated by cyclic GMP. Our data suggest that glucose and glutamate transport can be regulated locally at the level of the synapse and further suggest important roles for sAPPalpha and cyclic GMP in modulating synaptic physiology under normal and pathophysiological conditions.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Distribution of equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporters (ENT1) in brain.

Nucleoside transport processes may play a role in regulating endogenous levels of the inhibitory neuromodulator adenosine in brain. The cDNAs encoding species homologues of one member of the equilibrative nucleoside transporter (ENT) gene family have recently been isolated from rat (rENT1) and human (hENT1) tissues. The current study used RT-PCR, northern blot, in situ hybridization, and [3H]nitrobenzylthioinosine autoradiography to determine the distribution of mRNA and protein for ENT1 in rat and human brain. Northern blot analysis indicated that hENT1 mRNA is widely distributed in adult human brain. 35S-labeled sense and antisense riboprobes, transcribed from a 153-bp segment of rENT1, were hybridized to fresh frozen coronal sections from adult rat brain and revealed widespread rENT1 mRNA in pyramidal neurons of the hippocampus, granule neurons of the dentate gyrus, Purkinje and granule neurons of the cerebellum, and cortical and striatal neurons. Regional localization in rat brain was confirmed by RT-PCR. Thus, ENT1 mRNA has a wide cellular and regional distribution in brain, indicating that this nucleoside transporter subtype may be important in regulating intra- and extracellular levels of adenosine in brain.

Adenosine↗
PubMed1999

Involvement of inositol 1,4,5-trisphosphate-regulated stores of intracellular calcium in calcium dysregulation and neuron cell death caused by HIV-1 protein tat.

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.

2-Amino-5-phosphonovalerate↗
PubMed1999

Specialty networking in pediatric surgery: a paradigm for the future of academic surgery.

OBJECTIVE: To review retrospectively a 4-year experience with pediatric surgical networking at a major academic medical center in the Midwest. BACKGROUND: The growth of managed care in the United States during the past decade has had a major impact on the practice of medicine in general, but especially on academic medicine. In some academic medical centers, the loss of market share has not only affected clinical activity but has also compromised the educational and research missions of these institutions. METHODS: At the authors' institution, a networking strategy in pediatric surgery was established in 1993 and implemented on July 1, 1994. In 1994, one new satellite practice was established; over the next 4 years, four additional practices were added, including one in another state. To assess the impact on financial status, clinical activity, education, and academic productivity, the following parameters were analyzed: gross and net revenue, surgical cases, clinic visits, ranking of the pediatric surgery residency, publications, grant support, and development and endowment funds. RESULTS: Gross and net revenue increased from $3,273,000 and $302,000 in 1993 to $10,087,000 and $2,826,000, respectively, in 1998. Surgical cases and clinic visits increased from 1240 and 3751 in 1993 to 5872 and 11,604, respectively, in 1998. At the medical center's children's hospital, surgical cases and clinic visits increased from 1240 and 3751 to 2592 and 4729 during the same time period. During this 4-year period, the faculty increased from 4 to 11. Since 1997, the National Resident Matching Program has provided data on how pediatric surgery residency candidates ranked a training program. In 1997, this program received the second-most one to five rankings; in 1998, it tied for first. This exceeds the faculty's perception of previous years' rankings. Publications increased from 26 in 1993 to a peak number of 62 in 1996; in 1997 and 1998 the publications were 48 and 37, respectively. External grant support increased from $139,882 in 1993 to a total of $6,109,971 in 1998. Development and endowment funds increased from $103,559 in 1993 to $2,702,2777 in 1998. CONCLUSIONS: Pediatric surgical networking at the authors' institution has had a markedly positive impact on finances, clinical activity, education, and academic productivity during a 4-year period. The residency training program appears to have improved in popularity among candidates, probably because of the increased referral of complex cases to the medical center from the various networking satellites. External grant support and basic laboratory research significantly increased, most likely because of the greater number of faculty with protected time for research recruited. Development and endowment funds dramatically grew because of the excellent fiscal health of the pediatric surgical program. This experience may serve as a model for other academic surgical specialties.

Academic Medical Centers↗
PubMed1999

Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells.

Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A(2). In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C(16:0)) or stearoyl (C(18:0)) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A(2) inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A(2) from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca(2+)-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were pre-incubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A(2) which results in the enhancement of arachidonate release in H9c2 cells.

Animals↗
PubMed1998

Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.

Caffeine↗
PubMed1998

The role of tumor necrosis factor alpha in modulating the quantity of peripheral blood-derived, cytokine-driven human dendritic cells and its role in enhancing the quality of dendritic cell function in presenting soluble antigens to CD4+ T cells in vitro.

Because dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor alpha (TNFalpha) at day 7 resulted in a twofold higher yield of DC compared with non-TNFalpha-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFalpha (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non-TNFalpha-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFalpha from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFalpha over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFalpha added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.

Antigen Presentation↗