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J W Smit

Publications and source records attributed to J W Smit.

At least 55 records · Page 3Linked to original sources
PubMed1998

Hepatobiliary and intestinal clearance of amphiphilic cationic drugs in mice in which both mdr1a and mdr1b genes have been disrupted.

1. We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (-/-) mice) to study the role of the mdr1-type P-glycoprotein (P-gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug-transporting P-gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow. 2. 3H-labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine. 3. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1-type P-gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri-butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdrla/lb (-/-) mice compared to wild-type. 4. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdrla/lb (-/-) mice compared to wild-type mice. The absence of the mdrl-type P-gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild-type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild-type mice. 5. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdrla/lb (-/-) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expression of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P-glycoprotein in kidney physiology and renal secretory function. 6. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdrl-type P-glycoproteins.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Regulation of p100 (NFKB2) expression in human monocytes in response to inflammatory mediators and lymphokines.

The transcription factor NF-kappaB plays an important role in the regulated expression of cytokines in human monocytes. A p100 subunit of NF-kappaB has IkappaB-like properties by sequestering the p65 transactivating subunit in the cytosol of cells. In transient transfection assays we demonstrated that p100 has an inhibitory effect on the NF-kappaB-dependent IL-6 promoter activity. In view of this finding, we studied the regulation of the p100 subunit in human monocytes in response to LPS, the inflammatory cytokines IL-1beta and TNF-alpha and lymphokines. The results demonstrate that LPS, IL-1beta, and TNF-alpha induce p100 expression at mRNA and protein level while IFN-gamma, IL-3 and IL-4/IL-10 have no effect. The induction of p100 expression was shown to be mediated by a two-fold increase in the p100 transcription rate and a two-fold increase in p100 mRNA stability. Furthermore the p100 mediated upregulation was dependent on a tyrosine kinase dependent pathway rather than the protein kinase C pathway. NF-kappaB is a complex of either p50 homodimers or a p50/p65 heterodimer. The latter is known to strongly autoregulate p100 transcription. We therefore examined the composition of NF-kappaB induced by LPS vs the different lymphokines. LPS-induced NF-kappaB showed a distinct p65 supershift whereas the composition of NF-kappaB induced by different lymphokines did not show a change in p65. We conclude that the p100 subunit of the transcription factor NF-kappaB is induced by different inflammatory mediators while lymphokines fail to induce p100 expression which may be caused by the induction of NF-kappaB predominantly consisting of p50 homodimers.

Cells, Cultured↗
PubMed1998

CD34+/CD36- cells from myelodysplasia patients have a limited capacity to proliferate but can differentiate in response to Epo and MGF stimulation.

Myelodysplasia (MDS) is mostly characterized by a normal or increased number of normoblasts in the bone marrow and an impaired in vitro colony formation. In the present study we analyzed whether this might be due to a disconnection between proliferation and differentiation. CD34+/CD36- sorted bone marrow cells of 18 MDS patients were cultured in a clonogenic and suspension culture assay in the presence of erythropoietin (Epo) and mast cell growth factor (MGF). Burst-forming units erythroid (BFU-E, 75 +/- 88/10(4) CD34+ cells, X +/- s.d.) and colony-forming units E (CFU-E) were observed in eight of the 13 cases (62%) with refractory anemia with or without ring sideroblasts (RA and RARS) and one of the five cases with RA with excess of blasts or in transformation (RAEB and RAEB-T). Suspension cultures with CD34+/CD36- sorted cells with Epo plus MGF demonstrated an 8.9 +/- 6.5-fold expansion after 7 days in cases with >10 BFU-E/10(4) CD34+/CD36- cells while cases with <10 BFU-E/10(4) CD34+/CD36- cells demonstrated 1.0 +/- 0.8-fold expansion especially in cases with RAEB/RAEB-T. FACS and morphology analysis after 7 days of suspension culture demonstrated partial differentiation along the erythroid lineage in cases with RA/RARS (75%) and RAEB/RAEB-T (66%) reflected by the presence of erythroblasts and normoblasts with variable expression of CD34, CD36 and Glycophorin A. In cases with erythroid colony formation 69 +/- 24% of the cells were CD34-/CD36+ and in cases with <10 BFU-E/10(4) CD34+ cells 18 +/- 16% of cells were CD34-/CD36+. Iron staining showed the presence of ring sideroblasts in two cases with RARS indicating that the cells originate from the abnormal erythroid clone. Finally, it was shown that cases with an impaired proliferative response demonstrate an enhanced binding of Annexin-V on CD34+ cells during the first days of the cell suspension culture phase. These results suggest that a defect in the proliferative response is most pronouncedly expressed in MDS whereas a subpopulation of cells retain the capacity to differentiate between transition to a terminated stage.

Anemia, Refractory↗
PubMed1998

Improved outcome of adult acute lymphoblastic leukaemia by moderately intensified chemotherapy which includes a 'pre-induction' course for rapid tumour reduction: preliminary results on 66 patients.

Sixty-six consecutive adult patients with acute lymphoblastic leukaemia (ALL) were treated with intensified chemotherapy which included a 'pre-induction' course of cytarabine (AraC) and etoposide (VP16) when the white blood cell count (WBC) was > or = 30 x 10(9)/l (18 patients), and maintenance chemotherapy with regular intensifications for a total treatment duration of 3 years. Patients with a mediastinal mass (17) received consolidation courses with intermediate-dose AraC and VP16 followed by mediastinal irradiation. 11 patients underwent allogeneic bone marrow transplantation in first complete remission (CR). 58 patients (87.9%, CI 77.5-94.6) attained CR; with a median follow-up of 7 years, 35 of them (60.3%, CI 46.6-73.0) remain in CR. Toxicity was mild, although three patients died during remission induction, including two who were over 70 years of age. 23 patients (39.7%, CI 27.1-53.4) relapsed, seven of them primarily in the central nervous system (CNS), necessitating intensification of CNS-directed therapy. Only one of 13 patients with WBC 30-100 x 10(9)/l, but eight of nine with WBC > 100 x 10(9)/l, relapsed. The survival of older patients in CR did not differ from younger patients. The outcome of ALL in adult patients could thus be improved by slight intensification of treatment whilst keeping the toxicity within acceptable limits. 'Pre-induction' with AraC and VP16 might improve the prognosis, especially in patients with WBC < 100 x 10(9)/l. Patients with WBC > 100 x 10(9)/l, however, almost always relapse, and the intensified chemotherapy might not be tolerated well by patients over 70 years of age.

Adolescent↗
PubMed1998

Role of integrins in the attachment of metastatic follicular thyroid carcinoma cell lines to bone.

One of the principle targets for metastasis of follicular thyroid carcinoma (FTC) is the skeleton. Because no data are available on the role of the integrin adhesion molecule family in the attachment of FTC to bone, we studied the attachment characteristics of three FTC cell lines to bone and the role of integrins. Three cell lines were used from the same patient, one (FTC-133) from the primary tumor and two (FTC-236 and FTC-238) from metastases. Attachment of FTC cell lines to bone was assessed on conditioned medium of an osteoblastic cell line, coated onto plastic, as an in vitro model of bone matrix. The synthetic RGD (Arg-Gly-Asp) peptide GRGDS impaired attachment of the FTC cell lines to bone matrix, demonstrating the role of integrins in the attachment of FTC to bone. Attachment of FTC-133 to bone matrix was blocked completely by GRGDS, whereas attachment of FTC-236 and FTC-238 could not be impaired completely. Semiquantitative polymerase chain reaction (PCR) of cDNA from the cell lines indicated stronger expression of alpha5 integrin mRNA in FTC-133 than in the other cell lines. In line with this, attachment of FTC-133 to bone matrix could be inhibited almost completely by anti alpha5 and beta1 integrin antibodies, indicating the importance of the fibronectin receptor in the attachment of FTC-133 to bone. Binding of FTC-236 and FTC-238 to bone matrix could not be inhibited completely by anti-integrin antibodies, suggesting an additional role of nonintegrin adhesion molecules in the attachment of FTC-236 and FTC-238 to bone. The synthetic bone sialoprotein cyclic peptide, CNB, revealed antiadhesive effects in the binding of FTC to bone. In conclusion, integrins play an important role in the attachment of metastatic FTC to bone. Differences in the functional involvement of integrins in the attachment to bone are observed between the three cell lines studied. From the present results, antiadhesive interventions with synthetic RGD peptides in FTC may be designed.

Adenocarcinoma, Follicular↗
PubMed1998

Differences in immune response between HCV positive, HIV negative haemophilia A and B patients.

We measured numbers of lymphocytes and subsets in seven HIV negative, HCV positive severe haemophilia B patients, before and after substitution was changed from prothrombin complex concentrate to monoclonally purified concentrate. Data were compared with controls and our previous findings in haemophilia A. At baseline, haemophilia B patients did not differ from controls. After two years, T helper cells showed an increase (p = 0.028), while a rise in B cells approached statistical significance (p = 0.063). Haemophilia A patients showed increased numbers of activated non-B lymphocytes (p = 0.003) and lowered numbers of B cells (p = 0.001) at baseline. After two years activated non-B lymphocytes decreased (p = 0.004), as did the CD4/CD8 ratio (p = 0.002), due to increasing numbers of CD8 positive cells (p = 0.087). Our data suggest minor inhibition of the immune system in haemophilia B patients, which recovers after changing therapy to a monoclonally purified product. These findings contrast with the excessive immune stimulation in haemophilia A. The observed differences might be due to the administered concentrates.

Adolescent↗
PubMed1998

Heterologous expression of various P-glycoproteins in polarized epithelial cells induces directional transport of small (type 1) and bulky (type 2) cationic drugs.

We recently showed that absence of mdr1-type P-glycoprotein (P-gp) in mice resulted in profoundly reduced hepatic and intestinal clearance of type 1 and type 2 cationic drugs compared with that in wild-type mice. These data strongly support the concept that mdr1-type P-gps are involved in the disposition of cationic amphiphilic drugs from the body. We tested the hypothesis that mdr1-type P-gps are involved in the transmembrane transport of organic cations in epithelial cells expressing various drug-transporting P-gps. Therefore, transepithelial transport of the P-gp substrate vinblastine, the steroidal (type 2) cation vecuronium, the relatively small (type 1) cationic compound azidoprocainamide methoiodide and the aliphatic cation tri-n-butylmethylammonium were measured. Apical expression of the mdr1a, mdr1b or MDR1 gene in confluently grown polarized transformed LLC-PK1 cells resulted in highly enhanced apical directed secretion of all the drugs tested compared with controls. The vectorial transport of tri-n-butylmethylammonium in the apical direction in the P-gp (over)expressing cells could be inhibited by vinblastine. The present observations show that apical secretion of type 1 as well as of type 2 organic cations is enhanced significantly in the presence of apical expressed mdr1-type P-gp. These findings provide evidence for the involvement of drug-transporting P-gp in transmembrane transport of various organic cations, including relatively small molecular weight aromatic and aliphatic compounds.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

[Diabetic gastroparesis: is tube feeding an alternative?].

A woman aged 26 with long-standing insulin-dependent diabetes mellitus displayed recurrent periods of nausea, vomiting, abnormal blood sugar levels, weight loss and poor physical condition in spite of a diet and use of propulsive agents. Scintigraphy revealed decelerated gastric evacuation for solid and liquid nutrients. The patient recovered after insertion of a percutaneous endoscopic gastrostomy (PEG) catheter. Diabetic gastroparesis is associated with a vicious circle in which delayed gastric emptying leads to poor glucose regulation with frequent hyperglycaemia, which in its turn adversely affects gastric emptying. Treatment should be aimed at improvement of the gastric motility (for instance by propulsive agents), more accurate glucose regulation and nutritional counselling. If this fails to produce improvement, tube feeding via a permanent nasoduodenal tube or via a PEG catheter constitutes an acceptable alternative for oral nutrition.

Adult↗
PubMed1997

Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine.

In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(-/-) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(-/-) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(-/-) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(-/-) mice. The cumulative fecal excretion (0-96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(-/-) mice. Biliary excretion was not significantly different in wt and mdr1a(-/-) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(-/-) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

A simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy.

We present a simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy. Endothelial and smooth muscle cells were seeded in tissue culture chambers/slides precoated with 30% (w/v) gelatin drops fixed with 0.5% glutaraldehyde. Live endothelial cell layers were labeled with an antibody against the surface membrane protein, anti-CD13. After labeling, the cell layers were fixed and separated from the chambers/slides by lifting all of the samples with a spatula. Sections (1-2 mm) were cut, embedded and processed further for light or electron microscopy. Because of the delicate cell layers and the importance of preserving maximum integrity, labeling was performed under standard culture conditions and treated in situ during the entire procedure. Moreover, the small chamber size of the tissue culture dishes generated the additional advantages of requiring only a limited number of cells, small volumes of media, and little antibody.

Endothelium, Vascular↗
PubMed1996

Association of smooth muscle cell tissue factor with caveolae.

There is still no satisfactory explanation for the low catalytic activity of tissue factor (TF)/factor VII(a) complexes towards coagulation factor X, as found on the apical surface side of cell layers. It has been hypothesized that TF exists in a latent form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in situ and processed for electron microscopy. We showed that, besides internalization and accumulation in lysosomal-like structures, TF remained associated with noncoated, flask-shaped microinvaginations of the plasma membrane. These invaginations were identified as caveolae. In regions in which intercellular contacts were interrupted, more TF-positive caveolae were observed. Enzymatically detached smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase of catalytic activity of apically formed TF/VIIa complexes towards factor X was found on the suspended cells. We speculate that caveolae-associated TF may function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost.

Blood Coagulation↗
PubMed1996

Effects of HMG-CoA reductase inhibitors on growth and differentiation of cultured rat skeletal muscle cells.

HMG-CoA reductase inhibitors have been associated with skeletal muscle myopathy, ranging from asymptomatic elevations of serum creatine kinase (CK) activity to rhabdomyolysis. In this study, we assessed the effects of addition of different concentrations of simvastatin and pravastatin on growth and differentiation of cultured primary rat skeletal muscle cells. Protein concentrations, CK activity and percentage CK-MM, which is a parameter for maturation, were determined. Effects were generally stronger if inhibitors were added to both growth and differentiation medium rather than only to differentiation medium. Addition of 25 microM pravastatin caused only a decrease of CK activity. Addition of 1-5 microM simvastatin resulted in a decrease of protein concentration, CK activity and percentage CK-MM, whereas 25 microM simvastatin resulted in cell death. Addition of mevalonic acid or cholesterol could not prevent the effects of 1 microM simvastatin. In addition, 1 microM simvastatin did not influence the cholesterol and phospholipid content of the cells. Superfusion of cultured cells with simvastatin concentrations of 10 microM and higher caused a transient increase of the cytoplasmic calcium concentration followed by an apparent second rise and cell puncture. The results indicate that HMG-CoA reductase inhibitors may affect skeletal muscle cell regeneration in vivo by a direct toxic effect on growth and differentiation.

Animals↗
PubMed1996

Does the HMG-CoA reductase inhibitor pravastatin influence nucleation of cholesterol crystals in supersaturated model bile?

OBJECTIVE: To assess whether the presence in bile of HMG-CoA reductase inhibitors, which are secreted predominantly into the bile, influences biliary lithogenicity. DESIGN: Physiologic biliary concentrations of the hydrophilic HMG-CoA reductase inhibitor pravastatin were added to supersaturated model bile (cholesterol saturation index 1.4) and vesicles, the latter with and without the concomitant addition of the nucleation-promoting bile salt taurodeoxycholate. OUTCOME MEASURES: Nucleation time, defined as the number of days after which cholesterol monohydrate crystals are visible by phase contrast microscopy in filtered samples of model bile and vesicles, was assessed. RESULTS: The addition of pravastatin 0.01-1 mg/ml did not influence the nucleation time of supersaturated model bile (mean nucleation time without pravastatin: 8.3 +/- 2.2 days (SD), with pravastatin 1 mg/ml 9.3 +/- 0.4 days and pravastatin 0.01 mg/ml 7.6 +/- 2.3 days). The addition of similar concentrations of pravastatin to vesicle fractions alone did not influence nucleation time (> 20 days), nor could it prevent the nucleation-promoting effect of taurodeoxycholate (nucleation time with or without pravastatin 1 day). CONCLUSION: The results from this in-vitro study indicate that the presence of pravastatin in bile may not influence gallbladder bile lithogenicity. It can be hypothesized that this also applies to other HMG-CoA reductase inhibitors.

Anticholesteremic Agents↗
PubMed1996

Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein.

1. We have used mice with a disrupted mdr 1a P-glycoprotein gene (mdr 1a (-/-) mice) to study the role of P-glycoprotein in the pharmacokinetics of digoxin, a model P-glycoprotein substrate. 2. [3H]-digoxin at a dose of 0.2 mg kg-1 was administered as a single i.v. or oral bolus injection. We focussed on intestinal mucosa and brain endothelial cells, two major pharmacological barriers, as the mdr 1a P-glycoprotein is the only P-glycoprotein normally present in these tissues. 3. Predominant faecal excretion of [3H]-digoxin in wild-type mice shifted towards predominantly urinary excretion in mdr 1a (-/-) mice. 4. After interruption of the biliary excretion into the intestine, we found a substantial excretion of [3H]-digoxin via the gut mucosa in wild-type mice (16% of administered dose over 90 min). This was only 2% in mdr 1a (-/-) mice. Biliary excretion of [3H]-digoxin was not dramatically decreased (24% in wild-type mice versus 16% in mdr 1a (-/-) mice). 5. After a single bolus injection, brain levels of [3H]-digoxin in wild-type mice remained very low, whereas in mdr 1a (-/-) mice these levels continuously increased over a period of 3 days, resulting in a approximately 200 fold higher concentration than in wild-type mice. 6. These data demonstrate the in vivo contribution of intestinal P-glycoprotein to direct elimination of [3H]-digoxin from the systemic circulation and to the pattern of [3H]-digoxin disposition, and they underline the importance of P-glycoprotein for the blood-brain barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Cholesterol synthesis inhibitors in cholesterol gallstone disease.

Cholesterol synthesis inhibitors (HMG-CoA Reductase Inhibitors) are reported to decrease cholesterol saturation index of duodenal bile in hypercholesterolaemic subjects. The dissolution of gallstones in animals on treatment with these drugs created expectations of a therapeutical role for these drugs in cholesterol gallstone disease. However, in prospective studies with these drugs in humans, no effect on number and size of cholesterol gallstones was observed. This is likely the result of the fact that not just biliary secretion of cholesterol is decreased during treatment with these drugs in cholesterol gallstone disease, but phospholipids and bile salts as well. As a consequence, nucleation time of cholesterol crystals in gallbladder bile is not influenced by these drugs. Another important determinant in cholesterol gallstone disease, e.g. gallbladder motility, is not influenced by HMG-CoA reductase inhibitors. Although these drugs and their metabolites are secreted into the bile, they do not influence biliary lithogenicity. In conclusion, there seems to be no therapeutic role for HMG-CoA reductase inhibitors in the treatment of cholesterol gallstone disease, although no negative effects on determinants of cholesterol gallstone formation during treatment with these drugs are observed either.

Animals↗
PubMed1995

Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase.

A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo. We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells. These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells. The cell cultures had been obtained from collagenase-treated human umbilical cord vessels. Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein. We selected chymotrypsin as an alternative for the isolation of endothelial cells. Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed. Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity. Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells. This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.

Base Sequence↗