High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats.

UNLABELLED: Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT: Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.

Fatal Disseminated Toxoplasma gondii Infection in a Captive Harbour Porpoise (Phocoena phocoena).

A 7-year-old female harbour porpoise (Phocoena phocoena), born and held in captivity, suffered from reduced consciousness, imprecise and circling swimming movements and long phases of immobility over a period of 3 weeks. The animal died during treatment in a Danish open sea facility. Pathological examination revealed multifocal pyogranulomatous to necrotizing meningoencephalomyelitis, ganglioneuritis, plexus chorioiditis, myocarditis, hepatitis and adrenalitis with few intralesional protozoal tachyzoites and bradyzoites within cysts. Immunohistochemistry was positive for Toxoplasma gondii antigen within the lesions. Using polymerase chain reaction (PCR), the presence of T. gondii-specific genome fragments was confirmed. A multilocus PCR-restriction fragment length polymorphism analysis using nine unlinked marker regions (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) resulted in the identification of T. gondii type II (variant Apico Type I), which is the T. gondii genotype dominating in Germany. This is the first description of disseminated fatal toxoplasmosis in a captive harbour porpoise that lived in an open sea basin. Surface water contaminated with toxoplasma oocysts is regarded as the most likely source of infection.

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

OBJECTIVES: Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. DESIGN AND METHODS: Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. RESULTS: The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, p<0.0001) and lung cancer (96%, p<0.0001) compared to matched controls. CONCLUSIONS: MMP-7-generated elastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases.

IL-6 regulates exercise and training-induced adaptations in subcutaneous adipose tissue in mice.

AIM: The aim of this study was to test the hypothesis that IL-6 regulates exercise-induced gene responses in subcutaneous adipose tissue in mice. METHODS: Four-month-old male IL-6 whole body knockout (KO) mice and C57B wild-type (WT) mice performed 1 h of treadmill exercise, where subcutaneous adipose tissue (AT) was removed either immediately after, 4 h or 10 h after exercise as well as from mice not running acutely. Moreover, AT was sampled at resting conditions after 5 weeks of exercise training. RESULTS: AT leptin mRNA decreased immediately after a single running exercise bout in both genotypes and returned to baseline within 10 h of recovery in IL-6 KO mice, but not WT mice. Leptin mRNA content decreased in WT and increased in IL-6 KO mice with training, but without significant alterations in leptin protein. Acute exercise induced a decrease in the AT TNFα mRNA content in WT, but not in IL-6-KO mice, while training lowered resting levels of TNFα mRNA in both genotypes. In addition, an exercise-induced decline in AT PPARγ mRNA content was absent in IL-6 KO mice and in line training increased PPARγ mRNA only in IL-6 KO mice. CONCLUSION: The present findings indicate a role of IL-6 in regulating exercise- and training-induced leptin and PPARγ expression in adipose tissue. In addition, while IL-6 is required for TNF-α mRNA reduction in response to acute exercise, IL-6 does not appear to be mandatory for anti-inflammatory effects of exercise training in adipose tissue.

Can new pulmonary gas exchange parameters contribute to evaluation of pulmonary congestion in left-sided heart failure?

BACKGROUND: Assessment of pulmonary congestion in left-sided heart failure is necessary for guiding anticongestive therapy. Clinical examination and chest x-ray are semiquantitative methods with poor diagnostic accuracy and reproducibility. OBJECTIVES: To establish reference values, describe reproducibility, and investigate the diagnostic and monitoring properties in relation to pulmonary congestion of new pulmonary gas exchange parameters describing ventilation/perfusion mismatch (variable fraction of ventilation [fA2] or the drop in oxygen pressure from the mixed alveolar air of the two ventilated compartments to the nonshunted end-capillary blood [DeltaPO(2)]) and pulmonary shunt. METHODS: Sixty healthy volunteers and 69 patients requiring an acute chest x-ray in a cardiac care unit were included. The gas exchange parameters were estimated by analyzing standard bedside respiratory and circulatory measurements obtained during short-term exposure to different levels of inspired oxygen. Nine patients were classified as having pulmonary congestion using a reference diagnosis and were followed during 30 days of anticongestive therapy. Diagnostic and monitoring properties were compared with chest x-ray, N-terminal probrain natriuretic peptide (NT-proBNP), spirometry values, arterial oxygen tension, alveolar-arterial oxygen difference and venous admixture. RESULTS: The 95% reference intervals for healthy subjects were narrow (ie, fA2 [0.75 to 0.90], DeltaPO(2) [0.0 kPa to 0.5 kPa] and pulmonary shunt [0.0% to 8.2%]). Reproducibility was relatively good with small within subject coefficients of variation (ie, fA2 [0.05], DeltaPO(2) [0.4 kPa] and pulmonary shunt [2.0%]). fA2, DeltaPO(2) and NT-proBNP had significantly better diagnostic properties, with high sensitivities (100%) but low specificities (30% to 40%). During successful anticongestive therapy, fA2, DeltaPO(2), NT-proBNP and spirometry values showed significant improvements. CONCLUSIONS: The gas exchange parameter for ventilation/perfusion mismatch but not pulmonary shunt can have a possible role in rejecting the diagnosis of pulmonary congestion and in monitoring anticongestive therapy.

Glutamine supplementation further enhances exercise-induced plasma IL-6.

Exercise stimulates the production and release of interleukin-6 (IL-6) from skeletal muscle. Glutamine is also synthesized in skeletal muscle and is involved in protein synthesis within this tissue. During exercise, plasma levels of glutamine decline, and this may affect the concentration of plasma IL-6 via a decrease in IL-6 synthesis and release from muscle. We hypothesized that glutamine supplementation would attenuate the exercise-induced decrease in plasma glutamine concentration and, thus, further enhance levels of plasma IL-6. Eight healthy men participated in a randomized, double-blind, crossover study in which they performed 2 h of cycle ergometry at 75% of peak O2 uptake. They received glutamine, glutamine-rich protein, or placebo supplementation at intervals during and 2 h after exercise. Exercise induced an 11-fold increase in plasma IL-6, which was further enhanced by glutamine (18-fold) and glutamine-rich protein (14-fold) supplementation, administered at doses that attenuated the exercise-induced decrease in plasma glutamine concentration.

Elevated plasma levels of vascular endothelial growth factor and plasminogen activator inhibitor-1 decrease during improvement of psoriasis.

OBJECTIVE AND DESIGN: An evaluation of angiogenesis related molecules during open treatment of psoriasis. MATERIALS AND SUBJECTS: Plasma samples and skin biopsies from 16 patients with psoriasis and plasma samples from 13 healthy controls. TREATMENT: Ranitidine 300 mg orally twice daily for 6 months. METHODS: Vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were determined by ELISA methods in plasma collected from the patients before treatment and after 1, 3 and 6 months. Vessel counts were performed in biopsies from affected skin areas taken before treatment and after 3 and 6 months. The results were compared to simultaneous PASI scores. RESULTS: Pre-treatment plasma levels of VEGF and PAI-1 were significantly elevated in patients compared with levels in healthy persons (p = 0.02 and p = 0.04, respectively). The plasma levels decreased significantly during treatment (p = 0.03 and p = 0.01, respectively), and the decrease in combined levels correlated with the decrease in PASI score. However, the vessel density in affected skin did not change during treatment. CONCLUSIONS: Increased pre-treatment levels of VEGF and PAI-1 and decrease during improvement of the disease suggest that the two molecules may play a role in pathogenesis of psoriasis.

Activity of CHS 828 in primary cultures of human hematological and solid tumors in vitro.

CHS 828 is a pyridyl cyanoguanidine that has shown promising preclinical anticancer activity against various experimental tumor models and is presently being tested in a phase II trial in man. In the present study the fluorometric microculture cytotoxicity assay was used for in vitro evaluation of CHS 828 activity in primary cell cultures from hematological and solid tumors. In total, 156 samples from various diagnoses were tested with 72-h continuous drug exposure. CHS 828 showed high relative in vitro activity against tumor cells from chronic lymphocytic leukemia as well as from acute leukemia and high-grade lymphoma. Activity was also observed in several solid tumor cell samples, although the group as a whole appeared less responsive. CHS 828 was significantly more active against hematological malignancies compared to normal lymphocytes. Correlation analysis with standard drugs revealed low to moderate correlation coefficients. The results show that CHS 828 has potent antitumor activity against primary cultures of human tumor cells from patients and might have a unique mechanism of action.

Strenuous exercise decreases the percentage of type 1 T cells in the circulation.

Prolonged strenuous exercise is followed by a temporary functional immune impairment. Low numbers of CD4+ T helper (Th) and CD8+ T cytotoxic (Tc) cells are found in the circulation. These cells can be divided according to their cytokine profile into type 1 (Th1 and Tc1), which produce interferon-gamma and interleukin (IL)-2, and type 2 (Th2 and Tc2) cells, which produce IL-4. The question addressed in the present study was whether exercise affected the relative balance between the circulating levels of these cytokine-producing T cells. Nine male runners performed treadmill running for 2.5 h at 75% of maximal oxygen consumption. The intracellular expression of cytokines was detected following stimulation with ionomycin and phorbol 12-myristate 13-acetate in blood obtained before, during, and after exercise. The percentage of type 1 T cells in the circulation was suppressed at the end of exercise and 2 h after exercise, whereas no changes were found in the percentage of type 2 T cells. Plasma epinephrine correlated negatively with the percentage of circulating CD8+ T cells producing IL-2, whereas peak IL-6 correlated with the percentage of CD8+ IL-4-producing T cells in the circulation. Peak plasma IL-6 correlated with plasma cortisol postrunning. In conclusion, the postexercise decrease in T lymphocyte number is accompanied by a more pronounced decrease in type 1 T cells, which may be linked to high plasma epinephrine. Furthermore, IL-6 may stimulate type 2 T cells, thereby maintaining a relatively unaltered percentage of these cells in the circulation compared with total circulating lymphocyte number.

Plasma interleukin-6 during strenuous exercise: role of epinephrine.

Exercise induces increased levels of plasma interleukin-6 (IL-6) as well as changes in the concentration of lymphocytes and neutrophils. The aim of this study was to investigate a possible role for epinephrine. Seven healthy men participated in an exercise experiment. One month later they received an epinephrine infusion. The exercise consisted of treadmill running at 75% of maximal O(2) consumption for 2.5 h. The infusion trial consisted of 2.5 h of epinephrine infusion calculated to reach the same plasma epinephrine levels seen during the exercise experiment. The plasma concentration of IL-6 increased 29-fold during exercise, with peak levels at the end of exercise. The increase in plasma IL-6 during epinephrine infusion was only sixfold, with the peak value at 1 h after infusion. The lymphocyte concentration increased to the same levels during exercise and epinephrine infusion. The lymphocyte count decreased more in the postexercise period than after epinephrine infusion. The neutrophil concentration was elevated threefold in response to exercise, whereas no change was found in response to epinephrine infusion. In conclusion, the exercise-induced increase in plasma IL-6 could not be mimicked by epinephrine infusion. However, epinephrine induced a small increase in IL-6 and may, therefore, partly influence the plasma levels of IL-6 during exercise. In addition, the results support the idea that epinephrine plays a role in exercise-induced changes in lymphocyte number, whereas epinephrine does not mediate exercise-induced neutrocytosis.

A mechanism for increased contractile strength of human pennate muscle in response to strength training: changes in muscle architecture.

1. In human pennate muscle, changes in anatomical cross-sectional area (CSA) or volume caused by training or inactivity may not necessarily reflect the change in physiological CSA, and thereby in maximal contractile force, since a simultaneous change in muscle fibre pennation angle could also occur. 2. Eleven male subjects undertook 14 weeks of heavy-resistance strength training of the lower limb muscles. Before and after training anatomical CSA and volume of the human quadriceps femoris muscle were assessed by use of magnetic resonance imaging (MRI), muscle fibre pennation angle (theta(p)) was measured in the vastus lateralis (VL) by use of ultrasonography, and muscle fibre CSA (CSA(fibre)) was obtained by needle biopsy sampling in VL. 3. Anatomical muscle CSA and volume increased with training from 77.5 +/- 3.0 to 85.0 +/- 2.7 cm(2) and 1676 +/- 63 to 1841 +/- 57 cm(3), respectively (+/- S.E.M.). Furthermore, VL pennation angle increased from 8.0 +/- 0.4 to 10.7 +/- 0.6 deg and CSA(fibre) increased from 3754 +/- 271 to 4238 +/- 202 microm (2). Isometric quadriceps strength increased from 282.6 +/- 11.7 to 327.0 +/- 12.4 N m. 4. A positive relationship was observed between theta(p) and quadriceps volume prior to training (r = 0.622). Multifactor regression analysis revealed a stronger relationship when theta(p) and CSA(fibre) were combined (R = 0.728). Post-training increases in CSA(fibre) were related to the increase in quadriceps volume (r = 0.749). 5. Myosin heavy chain (MHC) isoform distribution (type I and II) remained unaltered with training. 6. VL muscle fibre pennation angle was observed to increase in response to resistance training. This allowed single muscle fibre CSA and maximal contractile strength to increase more (+16 %) than anatomical muscle CSA and volume (+10 %). 7. Collectively, the present data suggest that the morphology, architecture and contractile capacity of human pennate muscle are interrelated, in vivo. This interaction seems to include the specific adaptation responses evoked by intensive resistance training.

Stromal-mediated down-regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients.

The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down-regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation-dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.

Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise.

The present double-blinded, placebo-controlled study investigated whether antioxidant vitamin supplementation was able to modulate the cytokine and lymphocyte responses after strenuous eccentric exercise. Furthermore, muscle enzyme release was examined to see whether antioxidant treatment could reduce muscle damage. Twenty male recreational runners randomly received either antioxidants (500 mg of vitamin C and 400 mg of vitamin E) or placebo for 14 days before and 7 days after a 5% downhill 90-min treadmill run at 75% .VO(2 max). Although the supplemented group differed significantly with regard to plasma vitamin concentration before and after exercise when compared with the placebo group, the two groups showed identical exercise-induced changes in cytokine, muscle enzyme, and lymphocyte subpopulations. The plasma level of interleukin (IL)-6 and IL-1 receptor antagonist increased 20- and 3-fold after exercise. The plasma level of creatine kinase was increased sixfold the day after exercise. The concentrations of CD4+ memory T cells, CD8+ memory and naïve T cells, and natural killer cells increased at the end of exercise. The total lymphocyte concentration was below prevalues in the postexercise period. In conclusion, the present study does not support the idea that exercise-induced inflammatory responses are induced by free oxygen radicals.

Randomized comparison of conventional and gasless laparoscopic cholecystectomy: operative technique, postoperative course, and recovery.

The positive CO2 pneumoperitoneum needed to create the working space for laparoscopic surgery induces cardiovascular, neuroendocrine, and renal changes. Concern about these pathophysiologic changes has led to the introduction of a gasless technique. Fifty consecutive patients with symptomatic gallstones were randomized to conventional (CLC) or gasless laparoscopic cholecystectomy (GLC), with special reference to overall patient satisfaction, technical difficulties, duration of surgery, postoperative pain, and recovery. The overall exposure of the operative field was extremely poor in the GLC group, whereas the duration of surgery, steps involved in the cholecystectomy technique, length of hospital stay, and postoperative pain score did not differ significantly. After discharge, the median time to complete relief of pain tended to be shorter in the gasless group (5 days [range 1 to 15]) vs. the conventional group (8 days [range 1 to 15]). The period to return to normal activity was shorter in the GLC group (6 days [range 1 to 15]) compared to the CLC group (8.5 days [range 1 to 15]) (P = 0.031). No differences were found in terms of fatigue, dizziness and nausea, and overall satisfaction with the outcome. This study demonstrates a significantly shorter convalescence after laparoscopic cholecystectomy by means of the gasless technique compared to the conventional CO2 technique. Exposure of the operative field was less than optimal using the gasless technique.

Single site polymorphisms and alternative splicing of the human CD13 gene--different splicing frequencies among patients with acute myeloid leukaemia and healthy individuals.

Within the haematopoietic system, CD13/aminopeptidase N (APN), a transmembrane glycoprotein, is expressed on the surface of early committed progenitors of granulocytes and monocytes and by all cells of these lineages as they mature. CD13 is expressed on the majority of leukaemic myeloblasts in acute myeloid leukaemia (AML), and on leukaemic lymphoblasts in a small percentage of acute lymphoid leukaemia cases. Thus, anti-CD13 monoclonal antibodies are used as diagnostic markers in leukaemia typing. By systematically amplifying overlapping reverse transcription polymerase chain reaction (RT-PCR) amplicons throughout the CD13 mRNA, we identified two splice variants in which exon 3 and exon 14 were lost. Fourteen healthy individuals and 34 patients with AML were screened for these splice variants. All healthy individuals, and the majority of AML patients, had both splice variants but they represented less than 10% of the total RT-PCR-amplified CD13 product. Increased expression of both truncated CD13 mRNA forms were observed in 6% of AML patients, whereas no detectable exon 3 or exon 14 splice variants could be generated in 26% and 9% of AML patients respectively. The different splicing frequencies may reflect altered processing of pre-mRNA or expansion of certain cell types for some AML patients, even though no correlation existed to blast percentage, FAB classification, surface antigens or cytogenetic characteristics. In addition, we identified an intron of 506 bp between exon 1 and exon 2 as well as two sites of single nucleotide polymorphism with a heterozygosity index of about 0.5, making them useful as genetic markers.

Temporal effects of the novel antitumour pyridyl cyanoguanidine (CHS 828) on human lymphoma cells.

CHS 828, a novel pyridyl cyanoguanidine, has shown potent antitumour activity both in vitro and in vivo and is currently undergoing phase I evaluation in humans in collaboration with the European Organization for Research and Treatment of Cancer (EORTC). Here we study the temporal effects of CHS 828 on cytotoxicity, protein and DNA synthesis, cellular morphology and ultra structure using the lymphoma cell line U-937 GTB as the primary tumour model. In vitro analysis of tumour cell survival in response to CHS 828 revealed a cytotoxic effect progressively increased as a function of exposure time with maximum efficacy observed after 72 h. Activity of CHS 828 on U-937 GTB cells grown in vivo was also found. CHS 828 induced-cell death was dependent on intact protein synthesis and most cells appeared to lose their membrane integrity in the presence of a relatively well preserved nuclear structure. The results indicate that CHS 828 induced active and delayed cell death with a non-apoptotic morphology.

In vivo activity of CHS 828 on hollow-fibre cultures of primary human tumour cells from patients.

Fresh human tumour cells from patients with ovarian cancer and chronic lymphocytic leukaemia were cultured in semipermeable hollow fibres. The fibres were implanted on immunocompetent rats, which were treated with the cyanoguanidine N-(4-chlorophenoxyhexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828). CHS 828 showed high antitumour activity against all eight tumour samples; the fibres from control animals had a mean net growth of 73% while CHS 828 treatment induced a 37% mean reduction of cell density, without observable haematological toxicity. The results show a feasibility of using tumour cells directly from patients in the hollow-fibre rat model.

FAB M4 and high CD14 surface expression is associated with high cellular resistance to Ara-C and daunorubicin: implications for clinical outcome in acute myeloid leukaemia.

In 145 adult patients diagnosed with non-M3 acute myeloid leukaemia (AML) the relevance of FAB-subtype and immunophenotype to in vitro cellular drug resistance towards the anthracyclines aclarubicin (Acla) and daunorubicin (Dau), and the nucleoside analogue cytarabine (Ara-C), as well as other antileukaemic drugs, was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. We demonstrate that high CD14 expression is highly significantly associated with high cellular Ara-C and Dau resistance in univariate as well as multivariate analyses. FAB subtypes with highest and lowest cellular Ara-C resistance were M4 and M5, respectively (P < 0.01, one-way anova), whereas FAB subtypes with highest and lowest cellular Dau resistance were M4 and M1, respectively (P < 0.01, one-way anova). By contrast, no significant differences in cellular drug resistance towards Acla could be demonstrated among FAB subtypes. Furthermore, in two cohorts of AML patients treated by two different regimens for remission induction over a period of 15 yr (1985-94, n = 159 and 1995-99, n = 76, respectively) we demonstrate in univariate analyses a significance of CD14 expression with respect to clinical outcome. With the exception of significance to probability of obtaining complete remission in the first cohort (P = 0.03, logistic regression), this significance was, however, lost in multivariate analyses. It was demonstrated that FAB-M4 patients were older than M5 patients and that high CD14 expression was associated with the presence of secondary AML and older age. We conclude that although cases with high blast cell CD14 expression (and FAB-M4 cases) were more resistant to Ara-C as well as Dau in vitro, the clinical and biological significance of this may be debatable because of interactions with major prognostic factors in AML.

Identification of acute myeloid leukemia patients with diminished expression of CD13 myeloid transcripts by competitive reverse transcription polymerase chain reaction (RT-PCR).

Normal myeloid cells of monocytic and granulocytic origin express the metallopeptidase cluster of differentiation 13 (CD13) on the surface just as leukemic blasts in most acute myeloid leukemias (AML). A minor percentage of AML patients, however, lack the surface expression of CD13 antigen. To study this difference in CD13 surface expression, specific CD13 mRNA from 44 individuals were quantified by competitive reverse transcription polymerase chain reaction (RT-PCR). Absolute values for CD13 transcripts were normalised against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels to control for variations in sample preparation and mRNA degradation. By correlating normalised CD13 transcript levels and CD13 surface expression, a subgroup of AML patients was identified, having simultaneous diminished levels of myeloid CD13 transcripts and surface expression of the corresponding antigen. For this subgroup we suggest CD13/aminopeptidase N (APN) gene expression to be restricted primarily by limited amounts of transcripts. For the majority of AML patients determinants in addition to transcript levels must be involved in regulating CD13/APN gene expression.