Segmentation, retardation and mass approximation of birefringent particles on a standard light microscope.

This study presents a simple technique for the approximation of retardation, thickness and mass of birefringent particles with a retardation from 8 to 231 nm retardation. Tuning of the imaging system (standard light microscope equipped with a left and a right circular polarizer) to match grey values of polymer retarder films of known retardation with rendered grey values allows for a robust calibration and accurate approximation of retardation. In addition, a technique for accurate particle segmentation using a Canny-Deriche algorithm was used to minimize the bias on mass estimated from different thresholding techniques. The technique was tested using microscopic calcitic plates called coccoliths produced by the marine algal group coccolithophores, and the results compare well with published coccolith mass estimates obtained from volumetric analysis. LAY DESCRIPTION: Material with certain optical properties display interference colours when observed in a light microscope under circular polarized light. This study presents a simple technique for measuring the thickness and retardation of small particles within the 8 to 231 nm retardation range based on the grey values of their interference colours. Retardation is a measure of the distance between waves of two mutually perpendicular polarized light waves after passing through material. The technique involves the tuning of a standard light microscope system equipped with a left and a right circular polarizer and a digital camera to match grey values of polymer retarder films with a known retardation with grey values of a digitially rendered Michel-Lévy chart. A technique for accurate isolation of particles from the image background using a Canny-Deriche algorithm is also described, which avoids possible biased results from thresholding. The techniques were tested using microscopic calcitic plates called coccoliths produced by the marine algal group coccolithophores, and the results compare well with published estimates obtained from volumetric analysis.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

OBJECTIVE: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. DESIGN: Gemcitabine metabolites were analysed in LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. RESULTS: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. CONCLUSIONS: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.

Accurate representation of interference colours (Michel-Lévy chart): from rendering to image colour correction.

Here a work flow towards an accurate representation of interference colours (Michel-Lévy chart) digitally captured on a polarised light microscope using dry and oil immersion objectives is presented. The work flow includes accurate rendering of interference colours considering the colour temperature of the light source of the microscope and chromatic adaptation to white points of RGB colour spaces as well as the colour correction of the camera using readily available colour targets. The quality of different colour correction profiles was tested independently on an IT8.7/1 target. The best performing profile was using the XYZ cLUT algorithm and it revealed a ΔE00 of 1.9 (6.4 no profile) at 5× and 1.1 (8.4 no profile) at 100× magnification, respectively. The overall performance of the workflow was tested by comparing rendered interference colours with colour-corrected images of a quartz wedge captured over a retardation range from 80-2500 nm at 5× magnification. Uncorrected images of the quartz wedge in sRGB colour space revealed a mean ΔE00 of 12.3, which could be reduced to a mean of 4.9 by applying a camera correction profile based on an IT8.7/1 target and the Matrix only algorithm (ΔE00 < 1.0 signifies colour differences imperceptible by the human eye). ΔE00 varied significantly over the retardation range of 80-2500 nm of the quartz wedge, but the reasons for this variation is not well understood and the quality of colour correction might be further improved in future by using custom made colour targets specifically designed for the analysis of high-order interference colours.

No Significant Cytotoxic Effect of the EZH2 Inhibitor Tazemetostat (EPZ-6438) on Pediatric Glioma Cells with Wildtype Histone 3 or Mutated Histone 3.3.

BACKGROUND: Glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG) belong to the most aggressive cancers in children with poor prognosis and limited therapeutic options. Therapeutic targeting of epigenetic proteins may offer new treatment options. Preclinical studies identified Enhancer of Zeste Homolog 2 (EZH2) within polycomb repressor complex 2 (PRC2) as a potential epigenetic anti-tumor target in adult GBM cells but similar inhibition studies in pediatric GBM/DIPG were still missing. Moreover, approximately 30% of pediatric high grade gliomas (pedHGG) including GBM and DIPG harbor a lysine 27 mutation (K27M) in histone 3.3 (H3.3) which is correlated with poor outcome and was shown to influence EZH2 function. PATIENTS, MATERIALS AND METHODS: The present study investigated the correlation of expression of EZH2 and other PRC2 genes (EZH1, SUZ12, EED) with overall survival of pediatric GBM patients and the cytotoxic impact of EZH2 inhibition by the novel agent Tazemetostat in pediatric GBM/DIPG cells harboring either a H3.3 mutation or a H3 wildtype. RESULTS: EZH2 gene expression does not correlate with survival of pedHGG patients, and EZH2 inhibition does not induce significant cytotoxicity in pedHGG cells independently of H3.3 mutations. DISCUSSION AND CONCLUSION: We suggest that EZH2 inhibition might not offer an effective single agent treatment option for paedHGG patients. However, the therapeutic efficacy in combination with cytotoxic and/or other epigenetically active agents still has to be elucidated.

SUPT6H controls estrogen receptor activity and cellular differentiation by multiple epigenomic mechanisms.

The estrogen receptor alpha (ERα) is the central transcriptional regulator of ductal mammary epithelial lineage specification and is an important prognostic marker in human breast cancer. Although antiestrogen therapies are initially highly effective at treating ERα-positive tumors, a large number of tumors progress to a refractory, more poorly differentiated phenotype accompanied by reduced survival. A better understanding of the molecular mechanisms involved in the progression from estrogen-dependent to hormone-resistant breast cancer may uncover new targets for treatment and the discovery of new predictive markers. Recent studies have uncovered an important role for transcriptional elongation and chromatin modifications in controlling ERα activity and estrogen responsiveness. The human Suppressor of Ty Homologue-6 (SUPT6H) is a histone chaperone that links transcriptional elongation to changes in chromatin structure. We show that SUPT6H is required for estrogen-regulated transcription and the maintenance of chromatin structure in breast cancer cells, possibly in part through interaction with RNF40 and regulation of histone H2B monoubiquitination (H2Bub1). Moreover, we demonstrate that SUPT6H protein levels decrease with malignancy in breast cancer. Consistently, SUPT6H, similar to H2Bub1, is required for cellular differentiation and suppression of the repressive histone mark H3K27me3 on lineage-specific genes. Together, these data identify SUPT6H as a new epigenetic regulator of ERα activity and cellular differentiation.

Induced G1 cell-cycle arrest controls replication-dependent histone mRNA 3' end processing through p21, NPAT and CDK9.

Proper cell cycle-dependent expression of replication-dependent histones is essential for packaging of DNA into chromatin during replication. We previously showed that cyclin-dependent kinase-9 (CDK9) controls histone H2B monoubiquitination (H2Bub1) to direct the recruitment of specific mRNA 3' end processing proteins to replication-dependent histone genes and promote proper pre-mRNA 3' end processing. We now show that p53 decreases the expression of the histone-specific transcriptional regulator Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) by inducing a G1 cell-cycle arrest, thereby affecting E2F-dependent transcription of the NPAT gene. Furthermore, NPAT is essential for histone mRNA 3' end processing and recruits CDK9 to replication-dependent histone genes. Reduced NPAT expression following p53 activation or small interfering RNA knockdown decreases CDK9 recruitment and replication-dependent histone gene transcription but increases the polyadenylation of remaining histone mRNAs. Thus, we present evidence that the induction of a G1 cell-cycle arrest (for example, following p53 accumulation) alters histone mRNA 3' end processing and uncover the first mechanism of a regulated switch in the mode of pre-mRNA 3' end processing during a normal cellular process, which may be altered during tumorigenesis.

Interfering with the dynamics of estrogen receptor-regulated transcription.

In recent years, there has been a growing realization that a static two-dimensional model of gene activation by transcription factors is inadequate. Based on the work from a number of groups (Kang et al. 2002; Liu and Bagchi 2004; Metivier et al. 2003; Park et al. 2005; Reid et al. 2003; Shang et al. 2000; Sharma and Fondell 2002; Vaisanen et al. 2005), it is becoming clear that transcriptional regulation by nuclear receptors is a dynamic and cyclical process (Metivier et al. 2006). There are significant consequences that arise from this shift in understanding, from nuclear receptors as ligand activated factors that bind to a response element to activate expression of a target gene to a process where the receptor repeatedly binds in order to achieve transcription. New insights that arise from viewing the activation process as cyclical and the consequences of this for developing new strategies that modulate the activity of the estrogen receptor are outlined in this chapter.

Mutual antagonism of estrogen receptors alpha and beta and their preferred interactions with steroid receptor coactivators in human osteoblastic cell lines.

Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skeleton. Two isoforms of the estrogen receptor (ERalpha and ERbeta) mediate the transcriptional effects of estrogen. Although both isoforms of ER are present and functional in some human osteoblast (OB) cell lines, there is minimal information on the differential regulation of transcription by ERalpha and ERbeta homo- or heterodimers. This report demonstrates that ERalpha and ERbeta coexpression decreases the transcriptional capacity (relative to each ER isoform alone) on an estrogen response element-dependent reporter gene in OBs but not in other non-osteoblastic cell lines. These data suggest that ERalpha and ERbeta coexpression can differentially influence the degree of transcriptional activation in certain cell types. Interestingly, the overexpression of the steroid hormone receptor coactivator-1 (SRC1) resulted in preferential transcriptional enhancement by ERbeta as well as coexpressed ERalpha and ERbeta, whereas SRC2 overexpression appeared to preferentially enhance ERalpha transactivation. SRC3 overexpression failed to enhance estrogen-dependent transcription of any ER combination in OBs. Similar overexpression experiments in COS7 cells exhibited preferential enhancement of ERalpha function with all SRCs, including SRC3. Our data also demonstrated that SRC3 mRNA is reduced in osteoblastic cells, suggesting that SRC3 may have only a minor role in these cells. These data suggest that the transactivation capacity of various ER isoforms is both SRC species and cell type dependent.

Overexpression of a nuclear protein, TIEG, mimics transforming growth factor-beta action in human osteoblast cells.

Although transforming growth factor-beta (TGF-beta) is a growth factor with many known regulatory activities in many different cell types, its intracellular signaling pathway is still not fully understood. A TGF-beta-inducible early gene (TIEG) was discovered and shown by this laboratory to be a 3-zinc finger transcription factor family member; its expression is rapidly induced in cells treated with TGF-beta. To ascertain whether TIEG plays a major role in the TGF-beta pathway, human osteosarcoma MG-63 cells were stably transfected either with an expression vector containing a TIEG cDNA or with the vector alone. Clones that contain only the vector express normal levels of TIEG mRNA and protein and display the same patterns of gene expression and levels of cell proliferation as the nontransfected, non-TGF-beta-treated parental cells. However, transfected cells that overexpress TIEG mRNA and protein (TIEG-6 and TIEG-7) display changes that mimic those of MG-63 cells treated with TGF-beta, i.e. increased alkaline phosphatase activity, decreased levels of osteocalcin mRNA and protein, and decreased cell proliferation. The degree of these changes correlated with the level of TIEG expressed in the cell lines. TGF-beta treatment of the overexpressed cells showed no added effects. These findings and other published reports support a primary role of TIEG as a transcription factor in the TGF-beta signaling pathway.

PGE2 production after angiotensin-converting enzyme inhibition.

Using OKT3 monoclonal antibody driven T-lymphocyte proliferation, we investigated the effects of plasma 10, 20 and 30% in cell cultures on the proliferation ex vivo after exposure to captopril or enalapril taken orally by healthy volunteers. We also studied the effects of captopril, angiotensin II and bradykinin in vitro. We observed a plasma dependent dual effect of ACE inhibition both ex vivo and in vitro and of bradykinin in vitro being a stimulated proliferation at low (10% plasma) and a suppression of proliferation at high (30% plasma). The suppression was shown to be PGE2 mediated but the nature of the stimulatory signal is unknown. Proliferation was also suppressed by angiotensin II mediated by PGE2, but angiotensin II had no stimulatory effect. The results indicate that the effects of ACE inhibition on OKT3 mAb driven T-lymphocyte proliferation is plasma dependent, class specific for ACE inhibitors and mediated by both the ACE inhibitor itself and by bradykinin. Furthermore, it was shown that indomethacin in combination with an ACE inhibitor or bradykinin converted a suppressive response into proliferation indicating an immunostimulatory activity by indomethacin.

Tetrahydroaminoacridine-induced ribosomal changes and inhibition of protein synthesis in rat hepatocyte suspensions.

Tacrine (tetrahydroaminoacridine) is currently the only drug approved for the treatment of Alzheimer's disease. Unfortunately, tetrahydroaminoacridine therapy is often limited by this drug's propensity to induce reversible hepatotoxicity. Using suspensions of freshly isolated rat hepatocytes, we investigated the mechanism of tetrahydroaminoacridine cytotoxicity by examining the effect of tetrahydroaminoacridine on hepatocyte viability, protein synthesis, protein, DNA and RNA levels and ultrastructure. Our experimental findings support the explanation that tetrahydroaminoacridine-induced hepatotoxicity results from tetrahydroaminoacridine's adverse effect on protein synthesis and ribosomal structure and function. We found that viable, tetrahydroaminoacridine-treated hepatocytes (1.0 to 2.0 mmol/L or 118 to 235 micrograms/10(6) cells) demonstrated a dose-dependent and dramatic aggregation of ribosomes on endoplasmic reticulum as well as the aggregation of other nucleic acids found in the nucleus (chromatin) and in mitochondria. These electron microscopy data suggest that tetrahydroaminoacridine treatment results in severe ribosomal dysfunction. This was confirmed by the observed rapid loss of cellular RNA content (but not DNA or protein) and the rapid and complete inhibition of protein synthesis in tetrahydroaminoacridine-treated cells (lowest concentration tested was 0.5 mmol/L or 58 micrograms/10(6) cells). Thus tetrahydroaminoacridine treatment appears to aggregate hepatocellular nucleic acids, and in doing so adversely affects ribosomal function and protein synthesis. We propose that these adverse effects of exposure to tetrahydroaminoacridine are responsible for tetrahydroaminoacridine-induced hepatotoxicity.

Long-term enalapril treatment in diabetic nephropathy.

A randomized comparison of enalapril and metoprolol in patients with type 1 diabetes and nephropathy showed that the decline in kidney function was 5.6 +/- 5.9 ml/min/year in the metoprolol-treated and 2.0 +/- 3.2 ml/min/year in the enalapril-treated patients (P = 0.02). In the present study, the enalapril treated patients have been studied for two additional years. In the metoprolol-treated group, only the endpoints of death or uremia have been recorded, and six of the patients have reached end-stage renal failure and three are dead, compared to three and two, respectively in the enalapril treated group. The mean fall in glomerular filtration rate in 18 enalapril-treated patients is 8.4 +/- 9.4 ml/min/1.73 m2 after four years; 7.5 +/- 9.8 ml/min/1.73 m2, occurred during the first six months treatment. The mean decline in kidney function was 1.7 +/- 2.4 ml/min/year over the whole study period and 0.3 +/- 3.9 ml/min/year after exclusion of the first six months. In this study, long-term enalapril treatment in diabetic nephropathy was associated with a low rate of decline in kidney function.

Cholesterol: a renal risk factor in diabetic nephropathy?

In a prospective follow-up of 30 patients with type 1 diabetes and nephropathy, serum cholesterol, triglycerides, apolipoprotein Al and B, and lipoprotein(a) were determined to study their relationship to the rate of decline in glomerular filtration rate. The patients had proteinuria and advanced nephropathy with a mean +/- SD glomerular filtration rate of 39 mL/min/1.73 m2. The decline in glomerular filtration rate was determined during 2.5 +/- 0.5 years. High serum cholesterol, triglycerides, and apolipoprotein B were correlated to a more rapid deterioration in kidney function. The rate of decline in glomerular filtration rate was 1.0 +/- 2.5 mL/min/yr in the 10 patients with the lowest cholesterol level, compared with 4.5 +/- 3.2 mL/min/yr in the patients with the highest serum cholesterol (P = 0.015). The combined effect of the measured lipids, blood pressure, type of antihypertensive treatment, protein intake, proteinuria, and hemoglobin A1C on the rate of decline in glomerular filtration rate was assessed by multiple regression analysis. The measured factors together had a high explanatory power for the rate of decline in glomerular filtration rate. In this model, 73% of the variation in decline in glomerular filtration rate was explained by the measured variables (multiple r2 = 0.73). Low cholesterol and treatment with an angiotensin-converting enzyme inhibitor were the strongest predictors of a favorable renal prognosis. This suggests that hypercholesterolemia is an important risk factor for diabetic nephropathy.

Renal protective effect of enalapril in diabetic nephropathy.

OBJECTIVE: To determine whether inhibition of angiotensin converting enzyme can reduce the rate of decline in kidney function more than reducing blood pressure with other antihypertensive treatment. DESIGN: Prospective, open randomised study lasting a mean of 2.2 years in patients with diabetic nephropathy. SETTING: Three outpatient nephrology clinics. PATIENTS: 40 patients with insulin dependent diabetes and diabetic nephropathy with reduced renal function. INTERVENTION: Antihypertensive treatment with enalapril or metoprolol, usually combined with frusemide. MAIN OUTCOME MEASURE: Rate of decline in glomerular filtration rate measured as chromium-51 edetic acid clearance. RESULTS: Glomerular filtration rate declined a mean of 2.0 (SD 3.2) ml/min/year in the group given enalapril and 5.6 (5.9) ml/min/year in the control group. The mean arterial blood pressure during the study was 102 (5) mm Hg in the patients given enalapril and 103 (5) mm Hg in the patients given metoprolol. Urinary albumin excretion during treatment with enalapril was 60% lower than during treatment with metoprolol. CONCLUSIONS: Enalapril has an antiproteinuric effect independent of the effect on systemic blood pressure. Treatment with enalapril can reduce the rate of decline in kidney function in patients with diabetic nephropathy more than equally effective antihypertensive treatment with metoprolol. This points to a specific renal protective effect of angiotensin converting enzyme inhibitors in diabetic nephropathy.

Contrasting effects of enalapril and metoprolol on proteinuria in diabetic nephropathy.

OBJECTIVE: To assess whether angiotensin converting enzyme inhibition reduces proteinuria in diabetic nephropathy more than blood pressure reduction with other antihypertensive treatment. DESIGN: Prospective, open randomised study lasting eight weeks in patients with diabetic nephropathy. SETTING: Outpatient nephrology clinics. PATIENTS: 40 Patients with type I diabetes and diabetic nephropathy with reduced renal function. INTERVENTION: Antihypertensive treatment with enalapril or metoprolol, usually combined with frusemide. MAIN OUTCOME MEASURES: Arterial blood pressure and urinary excretion of albumin and protein. RESULTS: Arterial blood pressure after eight weeks was 135/82 (SD 13/7) mm Hg in the group given enalapril and 136/86 (16/12) mm Hg in the group given metoprolol. Proteinuria and albuminuria were similar in both groups before randomisation. After eight weeks' treatment, the geometric mean albumin excretion was 0.7 (95% confidence interval 0.5 to 1.2) g/24 h in the patients given enalapril and 1.6 (1.1 to 2.5) g/24 h in the patients given metoprolol (p less than 0.02). The proteinuria was 1.1 (0.7 to 1.7) and 2.4 (1.6 to 3.6) g/24 h respectively (p less than 0.02). CONCLUSIONS: Antihypertensive treatment with enalapril reduced proteinuria in patients with diabetic nephropathy more than an equally effective antihypertensive treatment with metoprolol. This points to a specific antiproteinuric effect of the angiotensin converting enzyme inhibitor independent of the effect on systemic blood pressure.

Differential sensitivity to cAMP among human cord and maternal/adult peripheral lymphocytes discloses differences between PHA- and OKT3-induced activation pathways.

Previous studies have demonstrated that human cord blood lymphocytes are resistant to the anti-proliferative action of prostaglandin E2 (PGE2) in phytohaemagglutinin (PHA)-induced mitogenesis, whereas the same cells activated by OKT3 are at least as sensitive to PGE2 as the corresponding cells from their mothers or other adults. In the present investigation it was found that: (i) PHA-induced proliferation of cord peripheral blood mononuclear leucocytes (PBML) is less sensitive to inhibition by forskolin--a direct activator of adenylate cyclase (AC)--and dibutyryl cAMP--a permeant cAMP analogue--than the proliferation of the corresponding maternal cells; (ii) OKT3-induced proliferation of cord as well as maternal PBML is highly sensitive to inhibition by forskolin and dibutyryl cAMP; (iii) cord PBML show an overall lower rate of AC activity compared with maternal PBML, in response to PGE2 and other autacoids as well as to receptor-independent stimulation; (iv) cord PBML also display a significantly lower rate of degradation of cAMP through cAMP-phosphodiesterase compared with maternal cells; (v) unbroken cord and maternal PBML show comparable rates of cAMP accumulation after stimulation with PGE2. The results suggest a lower sensitivity to the effect of cAMP in cord compared with maternal/adult lymphocytes in PHA-induced proliferation. Moreover, the data illustrate differences between PHA- and OKT3-mediated activation pathways, as well as differences in cell regulation mechanisms between cord and maternal PBML.

Distinct mitogens reveal different mechanisms of suppressor activity in human cord blood.

We have studied the suppressive ability of human cord blood lymphoid cells in a three-days mixed lymphocyte culture proliferation assay stimulated by mitogen. Sex chromosomes served as markers for dividing cord (male) or maternal cells. Three distinct mitogenic agents were used in the co-cultures: the mitogenic lectin PHA, the anti-CD3 monoclonal antibody OKT3, and 12-0-tetradecanoyl-13-acetate (TPA), a direct activator of protein kinase C. With all mitogens we observed significant, non-specific suppression of maternal/adult cell division. However, three separate levels of suppression were evident. PHA-stimulated co-cultures always showed the highest amount of cold suppressor activity (mean +/- SEM: 64.9 +/- 3.9). The mean suppression in OKT3- and TPA-stimulated co-cultures was 34.7 +/- 6.0 and 22.0 +/- 4.1%, respectively. Furthermore, indomethacin, a prostaglandin (PG) synthetase inhibitor, reduced by 41% the suppression in PHA-driven co-cultures, whereas having no significant effect on the corresponding OKT3-driven co-cultures. Our results indicate the existence of an indomethacine-sensitive, PG-dependent mechanism and a separate, indomethacine-resistant, mechanism of cord cell suppression.

Mitogenic action of phorbol ester TPA and calcium ionophore A23187 on human cord and maternal/adult peripheral lymphocytes: regulation by prostaglandin E2.

We have investigated the mitogenic action of the phorbol ester 12-O-tetrade-canoylphorbol-13-acetate (TPA) and the calcium ionophore A23187 on peripheral blood mononuclear leucocytes (PBML) from human neonates (cord), their mothers and other unrelated adults. TPA induced similar proliferation among maternal and unrelated adult PBML. In contrast, cord PBML regularly gave lower responses, on average 40-45% at optimal TPA concentration, than either maternal or other adult cells. A23187 had only a weak mitogenic effect on cord and maternal/adult cells. However, A23187 added together with TPA induced a strong proliferative response at low, non-mitogenic concentrations of either agent. Furthermore, TPA and the mitogenic OKT3 antibody showed a marked synergism (5- to 6-fold, on average) among cord PBML, whereas this effect was weaker (up to 2-fold) among maternal/adult cells. Prostaglandin E2 (PGE2), at 1.4 x 10(-6) M, inhibited cord and maternal/adult PBML proliferation induced by A23187 (an average 50% inhibition at optimal ionophore concentration). In contrast, PBML cultures stimulated by TPA or by TPA combined with A23187, OKT3 or PHA were virtually insensitive to PGE2-mediated suppression. Our results suggest that PGE2 can down-regulate ionophore-induced, receptor-mediated lymphocyte responses, whereas post-protein kinase C activation events (induced by TPA) are insensitive to PG-mediated inhibition.

A novel arachidonic acid metabolite with stimulatory effect on PHA-induced mitogenesis of lymphocytes.

The isolation of a novel arachidonic acid (Aa) metabolite from the supernatant of unstimulated human cord blood mononuclear leucocytes is reported. The metabolite, arbitrarily named 'compound 4' is neither a known lipoxygenase nor a cyclooxygenase product. 'Compound 4' was added to PHA-stimulated peripheral blood mononuclear leucocytes (PBML) from healthy blood donors, from mothers at term and from patients with immunodeficiency. 'Compound 4' induced an increase in the 3H-TdR incorporation by the maternal PBML and by the PBML from patients with various immunodeficiencies such as Wiskott-Aldrich syndrome and common variable immunodeficiency, whereas it had no effect on the proliferation of PBML from blood donors.