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Biomedical subjects

J Durham

Publications and source records attributed to J Durham.

At least 19 recordsLinked to original sources
PubMed2000

Hepatocyte transplantation in acute liver failure.

The majority of patients with acute liver failure (ALF) die waiting for orthotopic liver transplantation (OLT). No other treatment modality is shown to improve survival. This study was conducted to assess the safety and feasibility of hepatocyte transplantation (HT) and subsequent engraftment and function of donor cells. Functional and structural integrity of cryopreserved and thawed human hepatocytes were assessed by their morphological characteristics, induction of P-4501A1 transcription, and survival in vivo by xenotransplantation into rats. Five patients with severe ALF underwent intrasplenic (4 patients) and/or intrahepatic (2 patients) HT through angiography under cyclosporine immunosuppression. All patients had grade III to IV encephalopathy and factor V levels less than 0.5 U/mL, were ventilator and dialysis dependent, and were not OLT candidates. Three of the 5 patients who survived 48 hours after HT had substantial improvement in encephalopathy scores, arterial ammonia levels, and prothrombin times. Clinical improvement was paralleled by an increase in aminopyrine and caffeine clearances. All 3 patients lived substantially longer than expected based on clinical experience after HT (12, 28, and 52 days) but eventually died. Postmortem examination showed the presence of transplanted hepatocytes in liver and spleen by light microscopy and fluorescent in situ hybridization (FISH). Cryopreserved and thawed human hepatocytes can be transplanted into recipients with ALF with some acceptable but definite complications. Engraftment of donor hepatocytes was proven by histological examination and FISH by both transjugular biopsy and at autopsy. Improvement in brain edema, encephalopathy grade, and clearance of antipyrine and caffeine suggested function, albeit with a 24- to 72-hour delay posttransplantation.

Adult↗
PubMed2000

Improved method for detecting differentially expressed genes using cDNA indexing.

In cDNA indexing, differentially expressed genes are identified by the display of specific, corresponding subsets of cDNA. Subdivision of the cDNA population is achieved by the sequence-specific ligation of adapters to the overhangs created by class IIS restriction enzymes. However, inadequate specificity of ligation leads to redundancy between different adapter subsets. We evaluate the incidence of mismatches between adapters and class IIS restriction fragments during ligation and describe a modified set of conditions that improves ligation specificity. The improved protocol reduces redundancy between amplified cDNA subsets, which leads to a lower number of bands per lane of the differential display gel, and therefore simplifies analysis. We confirm the validity of this revised protocol by identifying five differentially expressed genes in mouse duodenum and ileum.

Animals↗
PubMed1999

Monocytically differentiating HL60 cells proliferate rapidly before they mature.

1alpha,25-Dihydroxyvitamin D(3) (D(3)) provokes growth arrest and monocytic differentiation in myeloid cells. Although it is usually assumed that the cellular events leading to growth arrest start within one cell cycle of D(3) addition, there is also evidence that D(3) provokes the expression of proliferation-related genes and accelerates cell division. Herein we clarify the relationship between proliferation and maturation in differentiating HL60 cells. Cells were cultured singly, D(3) was added at various stages of the cell cycle, the progeny were counted, and the proportions of mature monocytes were determined. Initially, the D(3)-treated cells proliferated at an accelerated rate, and they matured only later. If cells encountered D(3) early in G1 they divided two to four times before maturing, and if they encountered D(3) later in the cell cycle they underwent an extra division. Indomethacin slows HL60 cell multiplication by prolonging G1, and when these slower-growing cells were exposed to D(3), they matured after the usual period but underwent one division less than indomethacin-free cells. Contrary to common assumptions, we conclude that promyeloid cells do not initiate growth arrest or monocytic maturation immediately after exposure to D(3). Instead, an encounter with D(3) early in G1 sets in train a complex differentiation program. This consists of 2-3 days of rapid proliferation-probably employing cell cycles with a shortened G1 phase-that is followed by growth arrest and maturation. As a result, a single D(3)-treated promyeloid cell gives rise to 10 or more mature monocytes. These observations not only explain why "differentiating" cells express proliferation-related characteristics soon after D(3) addition, but they also show that the process of D(3)-induced monocytic differentiation is much more complex than has previously been realized.

Anti-Inflammatory Agents↗
PubMed1999

Metabolism of 1alpha,25(OH)2D3 and its 20-epi analog integrates clonal expansion, maturation and apoptosis during HL-60 cell differentiation.

Induction of growth arrest and monocyte differentiation of HL-60 leukemia cells by 1alpha,25 dihydroxyvitamin D3 (1alpha,25(OH)2D3) is well established. By contrast, we have observed, that 1alpha,25(OH)2D3 and its metabolites play separate roles in clonal expansion and survival of differentiating HL-60 cells. Cells that had differentiated by 48 h (CD14 positive) grew slower than control cells, whereas CD14 negative cells were growing faster at this time point. Inhibiting 1alpha,25(OH)2D3 or 1alpha,25(OH)2-20-epi-D3 metabolism, by the 25(OH)D3-24-hydroxylase inhibitor ketoconazole, abolished hyperproliferation of CD14 negative cells. Instead, both the onset of differentiation and subsequent apoptosis were enhanced. These events were associated with immediate up-regulation of the cyclin-dependent kinase inhibitor p21(waf1) and a lack of sustained expression, respectively. Stimulation and inhibition of growth by vitamin D3-related compounds was observed to be concentration and metabolite specific. Low amounts of 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated HL-60 cell growth. At higher concentrations, 1alpha,25(OH)2-20-epi-D3 was a more potent inducer than 1alpha,24,25(OH)3-20-epi-D3 of HL-60 differentiation; 1alpha,25(OH)2-20-epi-24-oxo-D3 was exclusively pro-differentiative at all concentrations. 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated proliferation of KG-1a leukemia cells, but neither of these compounds nor 1alpha,25(OH)3-20-epi-24-oxo-D3 exerted pro-differentiative effects on these cells. These findings shed new light on the pro- and anti-proliferative effects of 1alpha,25(OH)2D3 and lead to the postulate that metabolism of 1alpha,25(OH)2D3 and its 20-epi analog regulates different subsets of genes so as to co-ordinate population expansion and the differentiation process. Furthermore, 1alpha,25(OH)2D3 metabolism and/or sensitivity to the effects of metabolites may be altered in transformed cells to derive a clonal advantage.

Apoptosis↗
PubMed1999

Identification and characterization of the human homologue (RAI2) of a mouse retinoic acid-induced gene in Xp22.

We have identified a novel human gene during studies of a 1.3-Mb region of Xp22 between DXS418 and DXS999. A PAC contig spanning the region was constructed, sequenced, and analyzed by gene and exon prediction programs and by homology searches. Further investigation of predicted exons from PAC clone 389A20 led to the identification of a single-exon gene, designated RAI2 (retinoic acid-induced 2). RAI2 mapped 28 kb centromeric to marker DXS7996, between DXS7996 and DXS7997, and was transcribed from centromere to telomere. Northern blot analysis and reverse transcription-polymerase chain reaction analysis revealed expression of a 2.5-kb transcript in four fetal tissues (brain, lung, kidney, and heart) and eight adult tissues (heart, brain, placenta, lung, skeletal muscle, kidney, pancreas, and retina) but not in fetal or adult liver. The 530-amino-acid protein (57 kDa predicted mass) displays 94% homology with a mouse retinoic acid-induced gene product and contains a novel proline-rich (39%) domain of 68 amino acids. Retinoic acid is involved in vertebrate anteroposterior axis formation and cellular differentiation and has been shown to modulate gene expression controlling early embryonal development, suggesting a developmental role for RAI2. RAI2 remains a candidate gene for diseases mapping to the Xp22 region.

Amino Acid Sequence↗
PubMed1998

Characterization of SCML1, a new gene in Xp22, with homology to developmental polycomb genes.

Using exon trapping, we have identified a new human gene in Xp22 encoding a 3-kb mRNA. Expression of this RNA is detectable in a range of tissues but is most pronounced in skeletal muscle and heart. The gene, designated "sex comb on midleg-like-1" (SCML1), maps 14 kb centromeric of marker DXS418, between DXS418 and DXS7994, and is transcribed from telomere to centromere. SCML1 spans 18 kb of genomic DNA, consists of six exons, and has a 624-bp open reading frame. The predicted 27-kDa SCML1 protein contains two domains that each have a high homology to two Drosophila transcriptional repressors of the polycomb group (PcG) genes and their homologues in mouse and human. PcG genes are known to be involved in the regulation of homeotic genes, and the mammalian homologues of the PcG genes repress the expression of Hox genes. SCML1 appears to be a new human member of this gene group and may play an important role in the control of embryonal development.

Amino Acid Sequence↗
PubMed1998

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Antigens, CD↗
PubMed1997

Triiodothyronine blocks potentiation of HL60 monocyte differentiation by anti-inflammatory agents and by steroids and induces apoptosis of all-trans retinoic acid "primed" cells.

Sensitivity of the human promyeloid cell line HL60 to physiological differentiating agents [e.g. all-trans retinoic acid (all-trans RA) and 1 alpha, 25-dihydroxyvitamin (D3)] is increased by exposure of cells to "anti-inflammatory agents" (e.g. indomethacin) and to steroids [e.g. medroxyprogesterone acetate (MPA)] and post "priming" with a low dose (10(-8) M) of all-trans RA. Co-treatment of serum free-grown HL60 cells (HL60-ITS) with indomethacin and D3 reduces the dose of D3 required for monocyte differentiation from 10(-7) to 6.25 x 10(-9) M. This potentiating effect was observed to be almost absent when experiments where undertaken using serum-grown HL60 cells (HL60-FCS). The agent present in serum that interferes with indomethacin- and MPA-potentiation of the sensitivity of HL60 cells to D3 has been identified as the thyroid hormone 3,5,3'-L-triiodothyronine (T3). "Priming" of HL60-ITS cells with a low dose of all-trans RA reduces the amount of D3 required for the induction of monocyte differentiation to the same degree as co-treatment with either indomethacin or MPA (to 5 x 10(-9) M). However, the combined effect of all-trans RA "priming" and T3 treatment of HL60-ITS cells was induction of apoptosis. Treatment with either agent alone did not result in increased levels of apoptotic cells. These data reveal that T3 has an important influence on the capacity of HL60 cells to undergo differentiation and can promote apoptosis of these cells. Drug combinations, such as a differentiation potentiating agent, for example, indomethacin or MPA, and a differentiation inducer, for example, all-trans RA or D3, may have important therapeutic significance. Serum levels of T3 would be anticipated to influence the outcome.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

m-Calpain activation/depletion is associated with androgen-induced reduction of protein kinase C and proliferation of male accessory sex organ smooth muscle cells.

PURPOSE: In the guinea pig seminal vesicle smooth muscle (SVM), androgen-dependent proliferation and terminal differentiation appear to be coupled to protein kinase C (PKC). This is based on the observations that both the soluble (cytosolic) enzyme and the Triton X-100 solubilizable form of the particulate enzyme were reduced during proliferation but were androgen-resistant in the amitotic state of adults. The purpose of the present investigation was to determine if the reduction in PKC activity was linked to the translocation of the activated enzyme to acceptor sites in the Triton X-100 insoluble fraction of the cell or reflected enzyme depletion due to proteolysis by androgen-dependent activation of the u- and/or m-calpains. MATERIALS AND METHODS: SVM was harvested from treated animals, homogenized and separated into soluble and particulate components. The particulate material was further fractionated into Triton X-100 soluble and insoluble fractions. PKC activity was determined in all fractions using radioactive ATP. Cultures of pure smooth muscle cells from both human prostate and SVM were also employed to assess the role of calpain in smooth muscle growth. RESULTS: During androgen-induced proliferation, instead of a translocation of PKC activity to the Triton X-100 insoluble particulate fraction of the cell, PKC activity in this fraction was significantly reduced. The m-calpain was the only isoform detected in SVM. At the peak of androgen-induced DNA synthesis in pre-pubertal castrate animals, m-calpain decreased 45% whereas in proliferative resistant SVM of adult castrates the protease was not significantly affected by androgen treatment. In pure smooth muscle cultures from the SVM as well as human prostate glands calpeptin the cell permeable inhibitor of calpains produced a concentration-dependent inhibition (IC50 approximately equal to 35 microM) of cellular proliferation. CONCLUSIONS: Given that biochemical assays of calpain quantify the residual proenzyme and that upon activation calpain is rapidly degraded, our findings indicate that m-calpain activation occurs in association with androgen-induced degradation of PKC and SMC proliferation. Thus in vitro inhibition of m-calpain activation is antiproliferative. The relative resistance of m-calpain in adult SVM may be an important component of the terminal differentiation process in normal smooth muscle.

Androgens↗
PubMed1996

Potentiation of myeloid differentiation by anti-inflammatory agents, by steroids and by retinoic acid involves a single intracellular target, probably an enzyme of the aldoketoreductase family.

HL60 cells are human promyeloid cells that can be induced to differentiate by physiological stimuli (e.g. all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 (D3), granulocyte colony-stimulating factor (G-CSF)) and by non-physiological agents such as dimethysulphoxide (DMSO) and protein kinase C-activating phorbol esters. The sensitivity of HL60 cells to physiological differentiating agents, but not to DMSO, is enhanced when cells are exposed to 'anti-inflammatory agents' (e.g. indomethacin) or are 'primed' (pretreated) with a small amount of ATRA: alone, neither treatment induces differentiation. We earlier suggested that indomethacin might act by inhibiting the endogenous formation of a differentiation-suppressing prostanoid (Bunce, C.M., et al. (1994) Leukemia 8, 595-604). Studies of the formation of prostanoids by HL60 cells and of the effects of prostanoids on these cells failed to identify any prostanoid that could be implicated in sensitization by indomethacin. 3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) is another target of such 'anti-inflammatory agents'. Steroid inhibitors of 3 alpha-HSD sensitized HL60 cells to inducers of differentiation in a manner similar to indomethacin. 3 alpha-HSD is a member of the aldoketoreductase enzyme family, which comprises many enzymes of similar size and primary sequence. A protein that was recognised by an antiserum to 3 alpha-HSD was found in HL60 cells, but the cells showed no detectable 3 alpha-HSD activity. The 3 alpha-HSD-like protein was strikingly down-regulated by 'priming' doses of ATRA. When treatment with a differentiation-sensitizing 'anti-inflammatory agent' or steroid was combined with ATRA "priming', the effects of the different treatments were not additive: the resulting increase in sensitivity equalled that achievable by either treatment alone. We conclude that interference with a single intracellular regulatory mechanism underlies the increases in sensitivity of cells to differentiating agents that are caused by anti-inflammatory agents, by certain steroids and by 'priming' with ATRA. Decreased activity of a yet-to-be-identified member of the aldoketoreductase family of dehydrogenases is likely to be a central feature of a previously unrecognised mechanism that controls the responsiveness of cells to environmental stimuli such as retinoids and D3.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Exposure for laparoscopic cholecystectomy dissection adversely alters biliary ductal anatomy.

BACKGROUND: Exposure for open cholecystectomy entails lateral, caudal traction on the gallbladder infundibulum, which results in opening the angle between the cystic and hepatic ducts. Laparoscopic cholecystectomy (LC), as initially described, is done with cephalad traction on the gallbladder. We hypothesized LC exposure technique narrows the angle between the cystic and hepatic ducts, placing them at increased risk of injury. METHODS: Twenty-three patients had routine LC. Cystic duct cholangiography (IOC) was done with a flexible 5-Fr catheter via a percutaneous introducer placed anterior to the gallbladder. Exposure of Calot's triangle was maintained with cephalad traction on the gallbladder fundus. IOC was repeated after allowing the organ to assume the anatomic position. The cholangiograms were inspected for significant differences, and the angle of the cystic to the hepatic duct (CDHD) was measured by a blinded radiologist. RESULTS: The mean angle of the cystic to hepatic duct was 30 degrees +/- 19 degrees in the IOCs taken with cephalad traction on the gallbladder fundus vs 59 degrees +/- 22 degrees, P < 0.001, in the cholangiograms taken without traction. A filling defect at the cystic-hepatic duct junction was present in 39% of IOC taken with traction vs none without traction. The intrahepatic ducts were seen in all films without traction, whereas the intrahepatic ducts were not visualized in 13% of IOCs taken with traction. CONCLUSIONS: From these data we conclude (1) extra-hepatic biliary ducts may be at increased risk of injury during LC because of the exposure technique and (2) imaging bile ducts in the anatomic position may convey misleading information about the relative location of important structures. Optimal exposure for dissection of Calot's triangle should utilize a second clamp on the infundibulum with lateral, caudal traction.

Bile Ducts, Intrahepatic↗
PubMed1996

Growth of single HL60 cells in liquid culture: analysis of the influences of differentiative agents.

Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.

Cell Differentiation↗
PubMed1995

Assessment of compliance with home cardiorespiratory monitoring in infants at risk of sudden infant death syndrome. Collaborative Home Infant Monitoring Evaluation (CHIME).

OBJECTIVES: Documented monitoring was used to evaluate prospectively (1) the level of compliance among infants in whom cardiorespiratory monitoring was clinically indicated and (2) factors that might influence compliance: diagnosis, socioeconomic status, maternal age and education, and alarms. STUDY DESIGN: Sixty-seven infants (51% female, 49% term) were sequentially enrolled, and monitoring was prescribed for the following indications: siblings of sudden infant death syndrome victims (16%), apnea of prematurity (45%), and apparent life-threatening events or apnea of infancy (39%). Demographic data, alarm and event data, and a summary report of monitor use from the first monitor download were obtained. RESULTS: Maternal age, education, and insurance status did not differ significantly by indication for monitoring. The median number of monitor alarms per 10 hours of use was 0.7 for apnea or bradycardia and 0.6 for loose lead alarms. Monitors were available for use in the home from 2 to 106 days (median, 11 days). Median hours of monitor use per full day in the home was 15.5 hours. Of 67 infants, 58 used the monitor for at least part of every day in the home. The number of hours of monitor use per day did not differ significantly by diagnostic category, chronologic age, alarms, maternal age, education, or insurance type. This study population of infants at increased risk of sudden infant death syndrome had excellent compliance; 75% of the infants were monitored more than 10.5 hours per day, and 25% were monitored more than 21 hours per day. CONCLUSIONS: Documented monitoring provides an objective measure of compliance. These data provide a potential goal for level of compliance with home cardiorespiratory monitoring.

Analysis of Variance↗