Retroviral-mediated gene transfer into human myeloma cells

SUMMARY. SW-480 cells, derived from a primary human colon adenocarcinoma, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma. SW-480 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E-64 (10 μM) but was limited by cell pretreatment with phospholipase A₂. SW-480 cell suspension caused marked dose-dependent decreases in plasma recalcification times using normal, factor VIII-deficient and factor IX-deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW-480 cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW-480 TCIPA. Taken together, these data suggest that SW-480 TCIPA arises from SW-480 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW-480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotien IIb/IIIa and Ib prevent SW-480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC₅₀ 0.09 μM) and rhodostomin (IC₅₀ 0.03 μM) were about 6000 and 18000 times, respectively, more potent than GRGDS (IC₅₀ 0.56mM).     

Antileishmanial and immunomodulatory activity of Xylopia discreta

This study aimed at determining the in vitro antileishmanial activity of the essential oil and eight extracts obtained from Xylopia discreta . J774 and U937 macrophages were exposed to the different substances to establish the median lethal concentration (LC₅₀). The median effective concentration (EC₅₀) was obtained by determining the reduction of Leishmania panamensis - infected cells. A selectivity index (SI) (LC₅₀/EC₅₀) ≥ 20 defined a specific activity for one Xylopia discreta leaf extracts and for the essential oil, being these the two that showed the highest activity (SI = 64·8 and 110, respectively in J774 cells). To assess the substances' immunomodulatory activity, pro- and anti-inflammatory soluble mediators produced after treating infected macrophages were quantified by flow cytometry. The leaf methanol extract and the essential oil induced a differential production of monocyte chemoattractant protein-1, a chemokine associated with a Leishmania -resistant phenotype (Th1).     

Induction of tissue factor expression in human monocyte/endothelium cocultures

Summary. Induction of tissue factor (TF) expression on monocyctes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 × 10⁴/well) and endothelial cells (2 × 10⁴/well) produced 35.3± 8.5 mU of PCA/well, representing a 5-fold increase over the combined PCA of each cell type cultured alone (7.1 ± 1.5 mU, n = 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN-γ. When endothelium was prestimulated with 500U/ml IFN-γ there was 142 ±11% increase over unstimulated cocultures (n = 5, P< 0.01). TF induction was inhibited by 32 ± 6% in the presence of anti-ICAM-1 mAb (n = 5, P < 001). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.     

GM-CSF: modulation of biochemical and cytotoxic effects of tiazofurin in HL-60 cells

Summary. Cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) recruit quiescent cells into the cell cycle and sensitize these cells towards cell cycle specific chemotherapeutic agents. We examined the in vitro effects of GM-CSF on HL-60 cells and tested its modulatory influence on biochemical and cytotoxic effects seen with tiazofurin, a potent and specific inhibitor of IMP dehydrogenase. Incubation of HL-60 cells with 500 U/ml GM-CSF for 4 d enhanced cell proliferation, which was accompanied by a significant increase in IMP dehydrogenase activity (from 2·22 in control cells to 3·70 nmol/mg/h in cells pretreated with GM-CSF). When HL-60 cells were incubated with 100 μm tiazofurin for 2 h, intracellular GTP decreased to 46% of untreated control cells. In HL-60 cells pretreated with GM-CSF, GTP pools decreased to 38% of control after incubation with tiazofurin which is 69% of the predicted value for additive effect. The MTT chemosensitivity assay yielded significantly decreased IC₅₀ values for tiazofurin in HL-60 cells, preincubated with GM-CSF (IC₅₀ decreased from 13 μ m to 10 μ m ). Therefore our results suggest that combination therapy with GM-CSF and tiazofurin may be beneficial for the treatment of refractory leukaemia patients.     

Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression

Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell-induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D(3)-differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti-P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator-stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti-P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.     

Expression of tissue factor and factor VIIa/tissue factor inhibitor activity in endotoxin or phorbol ester stimulated U937 monocyte-like cells

Previously, unstimulated cells of the human monocytic tumor cell line U937 have been shown to possess a negligible cell-surface tissue factor (TF) activity, and to secrete a small amount of factor VIIa/tissue factor (VIIa/TF) inhibitor activity. On stimulation with endotoxin or with phorbol myristate acetate (PMA), TF of these cells is known to be increased approximately fourfold. In this report, we demonstrate that VIIa/TF inhibitor is also increased on stimulation of U937 cells with endotoxin (approximately equal to threefold) or with PMA (approximately equal to 20-fold). Notably, the secretion of the inhibitor persisted after the cell surface TF had started to decline. Further, when serum- free media from PMA stimulated cells was electrophoresed on a sodium dodecyl sulfate (SDS) gel, we eluted two inhibitor activity peaks corresponding to Mr approximately equal to 47,000 and Mr approximately equal to 36,000. The molecular weights of these peaks are similar to those obtained earlier from human plasma for this inhibitor(s).     

Prostaglandin E counteracts the gamma interferon induction of major histocompatibility complex class-II antigens on U937 cells and induction of responsiveness of U937 colony-forming cells to suppression by lactoferrin, transferrin, acidic isoferritins, and prostaglandin E

The established human monoblast or early monocyte cell line, U937, was evaluated for modulating influences of prostaglandin E2 (PGE2) on human gamma interferon (HuIFN gamma) induction of MHC class-II (Ia) antigens on U937 cells and the HuIFN gamma induction of responsiveness of U937 colony-forming cells (CFC) to inhibition by lactoferrin (LF), transferrin (TF), acidic isoferritins (AIF), and prostaglandin E (PGE). U937 CFC were induced to a state of responsiveness to the suppressive influences of PGE by HuIFN gamma. When MHC class-II antigens were induced on U937 cells and the cells sorted on the fluorescence activated cell sorter (FACS) IV into positive and negative cells, colony formation by the MHC class-II antigen+ population of cells was suppressed by LF, TF, AIF, and PGE2. Colony formation by the sorted population of MHC class-II antigen- cells was not influenced significantly by LF, TF, AIF, or PGE2. When PGE was present in the suspension culture for 72 h with U937 cells exposed to HuIFN gamma plus indomethacin, it blocked the induction of MHC class-II antigens as well as the associated inhibition of U937 CFC by LF, TF, AIF, and PGE2.     

All-trans retinoic acid upregulates thrombomodulin and downregulates tissue-factor expression in acute promyelocytic leukemia cells: distinct expression of thrombomodulin and tissue factor in human leukemic cells

The expressions of thrombomodulin (TM) and tissue factor (TF) by all- trans retinoic acid (ATRA) were studied in human leukemic cell lines including NB4 (acute promyelocytic leukemia) and U937 (monoblastic leukemia). ATRA remarkably upregulated TM antigen expression in cell lysates as well as TM cofactor activity on the cell surfaces of NB4. The level of TM mRNA in NB4 cells was increased by ATRA. Inherently procoagulant NB4 cells contained markedly higher content of TF, which was efficiently reduced by ATRA. Modest increase of TM and decrease of TF were observed when NB4 cells were treated with dibutyryl cyclic adenosine monophosphate (dbcAMP). On the other hand, both ATRA and dbcAMP showed dramatic increase of TM antigen level and modest decrease of TF antigen in U937 cells. These results suggest that ATRA regulates expressions of TM and TF antigens and activity in NB4 and U937 cell lines, and provide evidence for a potential efficiency of ATRA as a preventive and therapeutic agent for disseminated intravascular coagulation in promyelocytic and monocytic leukemia.     

Upregulation of thrombomodulin antigen levels in U937 cells by combined stimulation with estradiol-17beta and vitamin K2 (menaquinone 4)

The expression of thrombomodulin (TM) and tissue factor (TF) antigens by estradiol and vitamin K2 were studied in human leukemic cell lines including U937 (monoblastic leukemia), NB4 (acute promyelocytic leukemia), and HL60 (acute myeloblastic leukemia). Combined stimulation with estradiol-17beta and menaquinone 4 (MK4), homologue of vitamin K2, showed a remarkable increase of total TM antigen level only in U937 cells among these leukemic cell lines, whereas a single treatment of each agent showed a modest or a moderate increase. A synergistic effect of cotreatment with estradiol-17beta and MK4 was observed in an optimum concentration of 1.0 micromol of estradiol-17beta and 1.0 micromol of MK4. Estrogen receptors were detected only in U937 cells among these cell lines, and the competitive assay with an antiestrogenic agent showed a suppression on TM expression in a dose-dependent manner. In the mean time, concerning expression of TF antigens, if at all, only a very slight decrease was observed by costimulation with estradiol-17beta and MK4 in U937 and NB4 cells, whereas all-trans-retinoic acid (ATRA) showed a remarkable decrease in surface TF antigen levels in NB4 cells and also a modest decrease in U937 cells. These findings suggest that estradiol-17beta would up-regulate TM antigen expression via estrogen receptors and in cooperation with MK4, showing a different mechanism from ATRA.     

Induction of differentiation in U-937 and NB4 cells is associated with inhibition of tissue factor production.

Tissue factor (TF) production is under strict control in mature monocytic cells. However, constitutive expression of TF can be found in myelomonocytic cells and in haematopoietic cells arrested at an early stage of differentiation. In this paper we show that TF expression is down-regulated during the monocyte/granulocyte differentiation process, using the human monoblastic U-937 and the acute promyelocytic leukaemia NB4 cell lines as models. Expression of TF mRNA, protein and procoagulant activity (PCA) was constitutively high in untreated cells. Exposure of U-937 cells to 1alpha,25-dihydroxycholecalciferol (VitD3) and all-trans retinoic acid (ATRA) resulted in down-regulation of TF expression and PCA. In NB4 cells induction by ATRA, but not VitD3, resulted in the down-regulation of TF expression and PCA. Consistent with this, induction of terminal differentiation, as confirmed by the expression of differentiation associated antigens and cell cycle arrest, was inversely correlated to TF expression in U-937 and NB4 cells. Moreover, terminally differentiated U-937 cells retained the capacity to respond to inflammatory mediators, i.e. lipopolysaccharide and interferon-gamma, by a rapid increase in TF expression. In conclusion, we show that not only ATRA but also VitD3 is a potent suppressor of monocytic TF expression and thus might have potential clinical use for the treatment of coagulopathies.     

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells

The vitamin D₃ derived hormone 1,25 (OH)₂ vitamin D₃ (1,25 D₃) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1,25 D₃ reduced proliferation as measured by ³H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D₃ 65% inhibition was achieved at 48 n M . The MC 1288 stereoisomer of 1,25 D₃ was more potent and had the same activity at 48 n M .
The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4.
The antiproliferative effect of liposomal 1,25 D₃ was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33.
When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)₂ vitamin D₃ may offer a novel approach to treatment of myelomonocytic leukaemia.     

Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells

Abstract:  Hepatocellular carcinoma (HCC) is one of the most common cancers and its incidence is increasing worldwide. Melatonin, an indoleamine hormone, exerts anti-oxidant, immunomodulatory, anti-aging, and antitumor effects. Previous studies have shown that melatonin can act through specific receptors, including MT₁, MT₂, MT₃ receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Recently, we have described their role in the oncostatic and pro-apoptotic effects of melatonin on HepG2 human HCC cells. However, the potential role of the different melatonin cellular receptors on its antiproliferative effects remains unknown. In the present study, we examined the effect of melatonin treatment on HepG2 human HCC cells, analyzing cell cycle arrest and melatonin receptor expression. Melatonin was administered for 2, 4, and 6 days at 1000 or 2500 μ m . Melatonin induced a dose- and time-dependent inhibition on cell proliferation. This treatment caused an alteration in the cell cycle, with an increase in the number of cells in G₂/M phase at both 1000 and 2500 μ m melatonin concentrations, and a significant increase on S phase cell percentage by the highest dose. Furthermore, increases in protein expression of MT₁, MT₃, and retinoic acid-related orphan receptor-α were found after melatonin treatments. These increases were coincident with a significant induction in the expression of p21 protein, which negatively regulates cell cycle progression. Our results confirm the antitumor effect of melatonin in HCC cells, suggesting that its oncostatic properties are related, at least in part, to changes on the expression of their different subtypes of receptors.     

The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to Endotoxin

Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-α (TNF-α) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-α should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca²⁺]_i) is required for LPS-induced TNF-α production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca²⁺]_i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin.
Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 μmol/liter). After addition of LPS (10 μg/ml) to culture media, [Ca²⁺]_i was measured using a fluorescent indicator, fura-2.
Results: LPS increased [Ca²⁺]_i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 μg/ml)-induced TNF-α production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05).
Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin.     

Potentiating Effect of Acetylcholine on Stimulation by Isoproterenol of L-Type Ca2+ Current and Arrhythmogenic Triggered Activity in Guinea Pig Ventricular Myocytes

ACh Facilitation of Triggered Activity. Introduction : The objective of this study was to determine whether the effect of isoproterenol (Iso) to increase L-type Ca²⁺ current [I_ca(L)] and action potential duration (APD) was potentiated in ventricular myocytes following termination of an exposure of these cells to acetylcholine (ACh), and whether this potentiating effect of Ach could he arrhythmogenic.
Methods and Results : Transmembrane currents and potentials of guinea pig Isolated ventricular myocytes were measured using the whole cell, patch clamp technique. Stimulation of I_ca(L) and prolongation of APD caused by Iso (10 nmol/L) were attenuated in the presence of Ach (10 μ mol/L), but were transiently enhanced by 111%± 20% and 214%± 44%, respectively, following termination of a 2- to 4-minute exposure of myocytes to ACh. No changes were observed in the absence of Iso. Both the amplitude and incidence of isoinduced transient inward current, afterdepolarizations, and sustained triggered activity were greater immediately after termination of exposure to ACh than before application of ACh.
Conclusion : Stimulation by Iso of I_ca(L) is transiently enhanced in guinea pig ventricular myocytes following termination of exposure of these cells to ACh. The rebound increase of Iso-stimulated I_ca(L) is associated with an increase of APD and induction of arrhythmogenic triggered activity.     

Red Cell Hydrolases

Abstract. Human erythrocyte plasma membranes solubilized in 0.1% Triton X-100, with tritium-labeled 5-acetamido-3,5-dideoxy- l -arabino-2-heptulosonic acid fetuin as substrate, were found to contain considerable neuraminidase activity. The enzyme was purified 470-fold, with 17% recovery, by centrifugation and gel chromatography on Sephadex G-100 and Sephadex G-150. The highest activity of the purified enzyme was at pH 4.2, but the neuraminidase was active over a broad pH range. No cofactors were necessary for neuraminidase activity; the enzyme was inhibited by 0.05 m Ca acetate, 0.1 m CuSO₄, 0.01 m FeCl₃, 0.01 m FeCl₂, 0.01 m HgCl₂, and 0.005 m p -chloromercuribenzene sulfonate. The purified enzyme had maximal activity at 30–40 °C, and a linear relationship was demonstrated for activity versus time and for activity versus amount of enzyme.
Using the Eadie-Hofstee plot and a fetuin molecular weight of 48,000, a V_max of 4.5 × 10⁴ cpm ċ h⁻¹ ċ mg⁻¹, and a Km of 9 × 10⁻⁶ m were calculated. On whole-cell electrophoresis, it was demonstrated that purified human erythrocyte plasma membrane neuraminidase, like Clostridium perfringens neuraminidase, could cause release of terminal sialic acid residues from the external surfaces of intact human erythrocytes.     

Lamivudine, adefovir and tenofovir exhibit long-lasting anti-hepatitis B virus activity in cell culture

In this work, we investigated the anti-hepatitis B virus (HBV) activity of lamivudine, adefovir, tenofovir, penciclovir and lobucavir after short-term (i.e. 24 or 48 h) or continuous (9 days) exposure of the HBV-containing cell line, HepG2 2.2.15, to these drugs. Lamivudine maintained significant anti-HBV activity when added for only 24 or 48 h to the cell cultures compared to when the drug was present for the whole period (9 days) on the cells, i.e. 50% effective concentration (EC₅₀) values for the inhibition of HBV DNA synthesis were 0.07 ± 0.02 μg ml⁻¹ after 24 h of incubation, 0.02 ± 0.01 μg ml⁻¹ after 48 h of incubation and 0.0016 ± 0.001 μg ml⁻¹ after 9 days of incubation. Similarly, the nucleoside phosphonate analogues, adefovir and tenofovir, retained significant anti-HBV activity when added for only a short period of time to the cells. The EC₅₀ values were 12 ± 1 μg ml⁻¹ (24 h) and 1.0 ± 0.2 μg ml⁻¹ (48 h) vs 0.003 ± 0.001 μg ml⁻¹ (9 days) for adefovir, and 6.5 ± 1.1 μg ml⁻¹ (24 h) and 0.8 ± 0.1 μg ml⁻¹ (48 h) vs 0.03 ± 0.02 μg ml⁻¹ (9 days) for tenofovir. In contrast, penciclovir and lobucavir lost most of their anti-viral activity when present on the cells for 48 h or less.     

Serum factor from patients with cirrhosis and hepatocellular carcinoma enhances production of prostaglandin E2 by U937 cells.

The effect of serum from patients with cirrhosis and hepatocellular carcinoma on the release of prostaglandin E2 by the human histiocytic lymphoma cell line U937 was investigated to explain the mechanism underlying the immunoregulatory dysfunction of monocytes in cirrhosis and hepatocellular carcinoma. Prostaglandin E2 production by U937 cells cultured with serum from cirrhosis patients (5.9 +/- 2.7 ng/ml, p less than 0.01) and hepatocellular carcinoma patients (5.4 +/- 2.6 ng/ml, p less than 0.01) was significantly higher than that of control cultures (2.0 +/- 1.0 ng/ml). This activity was decreased after heating and after freezing and thawing. By size exclusion fast protein liquid chromatography, the probable factor was eluted in the fraction with a molecular weight of 150 kD. By anion exchange chromatography with a stepwise increase of the NaCl concentration, the peak activity augmenting prostaglandin E2 production by U937 cells was eluted in the 0.05 to 0.1 mol/L NaCl fraction. The high level of this factor (monocyte-regulating factor) in patient serum might be one cause of abnormal monocyte immunoregulatory function in cirrhosis and hepatocellular carcinoma.     

Diethyldithiocarbamate Methyl Ester Sulfoxide, an Inhibitor of Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase and Putative Metabolite of Disulfiram

S-methyl N,N -diethylthiolcarbamate sulfoxide (DETC-MeSO) is a potent inhibitor of rat liver mitochondrial low K _m aldehyde dehydrogenase (ALDH₂) both in vivo and in vitro, and has been proposed to be the metabolite responsible for ALDH₂ inhibition by disulfiram. Diethyldithiocarbamate methyl ester (DDTC-Me), a key intermediate in the metabolism of disulfiram, has been shown to be bioactivated by microsomal monooxygenases to diethyldithiocarbamate methyl ester sulfoxide (DDTC-Me sulfoxide). Studies were conducted to determine if DDTC-Me sulfoxide was also an active metabolite of disulfiram and inhibitor of ALDH₂. DDTC-Me sulfoxide inhibited ALDH₂ in vitro with an IC₅₀ of 10 μm, and in vivo with an ID₅₀ of 31 mg/kg (170 μmol/kg). Maximal ALDH₂ inhibition in vivo was observed 8 hr after the administration of 45.2 mg/kg DDTC-Me sulfoxide, with ALDH₂ activity returning to control levels after 48 hr. Although DDTC-Me sulfoxide inhibited ALDH₂ in vivo, DDTC-Me sulfoxide was not detected in plasma from rats treated with either disulfiram (75 mg/kg), DDTC-Me (122.25 mg/kg), or DDTC-Me sulfoxide (45.2 mg/kg). However, DDTC-Me and S -methyl N,N -diethylthiolcarbamate (DETC-Me) were detected in plasma from rats treated with DDTC-Me sulfoxide. In rats treated with DDTC-Me sulfoxide and challenged with ethanol, a small increase of ∼9 μm in blood acetaldehyde and an inconsistent drop in blood pressure was observed. In conclusion, DDTC-Me sulfoxide inhibited ALDH₂ in vitro and in vivo, was less potent than DETC- MeSO, and was not detected after disulfiram administration.     

Cholecystokinin-Stimulated Monocytes Produce Inflammatory Cytokines and Eicosanoids

Objectives : Plasma cholecystokinin increases with enteral feeding. Cholecystokinin increases intracellular calcium in lymphocytes/monocytes and is a lymphocyte co-mitogen. We hypothesize that decreased cholecystokinin production with "bowel rest" and parenteral nutrition may be beneficial in inflammatory bowel disease by down-regulating gut immune/inflammatory mechanisms. The majority of cells observed in mucosa of inflammatory bowel disease are monocytes and neutrophils. Cholecystokinin effect was therefore measured on monocyte production of proinflammatory mediators (tumor necrosis factor α, interleukin-1 β, intcrteukin-6) and neutrophil chemotaxins/activators (interleukin-8, granulocyte-macrophage colony stimulating factor, and leukotriene B₄). Methods : Peripheral blood monocytes (0.5 ± 10⁶) from healthy donors in 1 mL of RPMI 1640 plus 5% fetal calf serum were cultured for 24 h in 5% CO₂ at 37±C with 5 μg/mL endotoxin, 1 ± 10-⁻⁷ M cholecystokinin, or no agonist. Supernatants were analyzed by ELISA for cytokines and leukotriene B₄. Results : Endotoxin-stimulated monocytes produced 1130 pg/mL tumor necrosis factor versus 81 pg/mL for cholecystokinin, 612 pg/mL interleukin-1 versus 10 pg/mL, 694 pg/mL interIeukin-6 versus 30 pg/mL, 4531 pg/mL of interleukin-8 versus 3848 pg/mL, 21 pg/mL granulocytemacrophage colony stimulating factor versus 9 pg/mL, and 21 pg/mL leukotriene B₄ versus 12 pg/mL. Controls produced no cytokines/eicosanoids (N = 8, p < 0.001). Conclusion : Cholecystokinin increase with enteral feeding may up-regulate gut immune response. Cholecystokinin suppression with parenteral alimentation may decrease inflammatory mediator production.     

Anti-IgG Autoantibodies in HIV-infected Hemophilia Patients

Sera of 76 HIV-negative hemophilia patients, 103 HIV-positive (HIV+) hemophilia patients free of AIDS or AIDS related complex (ARC), and 32 HIV+ hemophilia patients with AIDS/ARC were tested for four different anti-IgG activities. IgG-anti-F(ab')_2γ, IgM-anti-F(ab')_2γ, and IgG-anti-Fc_γ serum activities were significantly associated with the clinical stage of HIV infection, whereas IgM-anti-Fc_γ was not. IgG-anti-F(ab')_2γ activity was found to be caused by cross-reaction of anti-HIV antibody with an epitope within the constant CHI domain of human IgG. HIV+ hemophilia patients with severe thrombocytopenia (< 50,000/μl platelet counts) had significantly higher IgM-anti-IgG activity than patients with > 50,000/μl platelets. Because anti-IgG antibodies possess immunoregulatory properties, our results may serve as a possible explanation for the frequent B cell disorders encountered in HIV-infected patients.