A p38 MAP kinase regulates the expression of the Aedes aegypti defensin gene in mosquito cells.

An Aedes aegypti p38 (Aap38) mitogen-activated protein kinase was isolated and characterized in this study. The 1761 bp long full-length Aap38 cDNA encodes an open reading frame of 358 amino acids, exhibiting characteristics of Thr/Tyr dual kinase specificities. We showed that bacteria activate both the kinase activity of Aap38 and the expression of the Aedes aegypti defensin A (AaDefA) gene, which is inhibited by a p38 kinase inhibitor SB203580 and dsRNA interference of Aap38. A similar result was obtained by a reporter construct containing the AaDefA regulatory region linked to Ds-Red. The lipopolysaccharide-activated reporter gene was inhibited by SB203580. In addition, Aap38 translocated to the nucleus after lipopolysaccharide induction. Our findings suggest that the p38 protein kinase pathway is involved in the antibacterial peptide synthesis in mosquitoes.

Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a serious cause of nosocomial infections. Rapid identification of this pathogen is required so that appropriate therapy can be given and outbreaks controlled. This study evaluated a multiplex PCR and an automated ribotyping system for the rapid identification of Acinetobacter baumannii. In total, 22 different reference strains and 138 clinical isolates of Acinetobacter spp., identified by 16S-23S rRNA intergenic spacer (ITS) sequence analysis, were evaluated. All A. baumannii isolates (82 clinical isolates and one reference strain) were identified by the multiplex PCR method (specificity 100%). The sensitivity and specificity of the ribotyping system for identification of A. baumannii were 85.5% (71/83) and 93.5% (72/77), respectively. An additional 100 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex were used to compare these two methods for identification of A. baumannii, and this comparison revealed a level of disagreement of 14% (14 isolates). The accuracy of the multiplex PCR was 100%, which was confirmed by sequence analysis of the ITS and recA gene of these isolates. Thus, the multiplex PCR method dramatically increased the efficiency and speed of A. baumannii identification.

An immune signalling kinase AaMEK3 from mosquitoes: cDNA cloning and characterization.

In mammals, the mitogen-activated protein (MAP) kinase pathway is one of the four major signalling systems that respond to stress and inflammatory stimuli. A full-length cDNA corresponding to Aedes aegypti MAP kinase kinase 3 (AaMEK3) was cloned and sequenced. It is 1.7 kb and contains an open reading frame of 334 amino acids and eleven conserved kinase domains, including signatures of a putative serine/threonine kinase active site and an ATP binding site. The messenger (mRNA) and protein expression levels of AaMEK3 are enhanced post bacterial inoculation. The in vitro kinase activity assay reveals that (1) AaMEK3 is not autophosphorylated but can phosphorylate myelin basic protein successfully, and (2) it is slightly enhanced by lipopolysaccharide stimulation. This suggests that AaMEK3 may be involved in mosquito immune signalling.

Current status of human parasitic infections in Taiwan.

The eradication of the 2 mosquito-borne parasitic diseases, malaria and lymphatic filariasis, is one of the greatest achievements of the parasite control campaigns in Taiwan. Most of the soil-transmitted nematode infections, with the exception of pinworm infection, are currently well controlled and limited to some aboriginal areas. Food-borne parasitic zoonosis such as infections with Angiostrongylus cantonensis, Clonorchis sinensis, and Taenia saginata asiatica are not rare, but the former is seasonal and the latter 2 are ethnically and geographically associated. Intestinal protozoal infections with Giardia lamblia and Cryptosporidium parvum are at low levels but may be widely distributed. Opportunistic protozoal infections among patients with acquired immunodeficiency syndrome, which included amebic colitis, Pneumocystis carinii pneumonia, and cerebral toxoplasmosis, are becoming increasingly important. The rapid increase in international travel and the introduction of large numbers of foreign workers from other countries in Southeast Asia may change the epidemiological patterns of parasitic infections in Taiwan.

High-level expression of recombinant dengue viral NS-1 protein and its potential use as a diagnostic antigen.

The prevalence of NS1 Ab response in patients with dengue viral infection and the potential of using recombinant NS1 protein as a diagnostic antigen for dengue viral infection were investigated. In this study, the full-length and C-terminal half of NS1 proteins (rNS1, rNS1-C) were highly expressed (10-30 mg/l) and further purified and refolded. The good antigenicity of the full-length rNS1 protein was confirmed by interaction with 19 dengue NS1-specific monoclonal antibodies (MAbs) in ELISA; however, the antigenicity of rNS1-C was relatively lower. The full-length rNS1 antigen also differentiated reliably between sera from dengue virus-infected patients and sera from normal controls. When rNS1 was used as an antigen to detect human anti-NS1 IgM and IgG Ab, the anti-NS1 Ab response was found in 15 of 17 patients (88%) with primary dengue infection and all 16 patients (100%) with secondary dengue infection. These results indicated that using the full-length rNS1 whose antigenicity is restored as ELISA antigen, a high anti-NS1 antibody prevalence could be detected in patients with either primary or secondary dengue infection. This finding suggested that the anti-NS1 antibody appeared not only in secondary and severe dengue virus infection and might not correlate the pathogenesis of dengue hemorrhagic fever. The study also verified that our purified rNS1 protein showed similar immunological properties as native dengue viral proteins. Genetic engineering production of recombinant NS1 antigen could provide a safe and valuable resource for dengue virus serodiagnosis.

Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.

Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.

Western and Chinese antirheumatic drug-induced T cell apoptotic DNA damage uses different caspase cascades and is independent of Fas/Fas ligand interaction.

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR.

AIMS: An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). METHODS AND RESULTS: Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. CONCLUSION: Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD.

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.

Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients.

Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine(111) (His(111)) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine(111) (Leu(111)). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.

Antibody-dependent enhancement of heterotypic dengue infections involved in suppression of IFNgamma production.

Antibody-dependent enhancement has been implicated in some outbreaks of epidemic dengue hemorrhagic fever, however, the mechanism of antibody-dependent enhancement is not well known. This study was conducted to investigate the cross-protection and cross-enhancement of dengue-2 virus infections by dengue-1 immune sera. It was found that dengue-1 immune sera at 1:5 dilution (n = 12) could neutralize dengue-2 infections in BHK-21 cells, as assessed by a standard plaque-reduction neutralization assay. Two-thirds of the dengue-1 immune sera at 1:25 dilution demonstrated neutralizing effects for dengue-2 infections, whereas, non-immune sera revealed no neutralization for dengue-2 infections in BHK-21 cells. Human mononuclear leukocytes in response to dengue-2 infections elicited a T cell helper 1 (Th1) response revealing induction of IFNgamma but not IL-4 production. Dengue-1 immune sera did not neutralize dengue-2 infections in mononuclear leukocytes. Subneutralizing titers of dengue-1 immune sera at 1:250, but not at 1:10 dilution, enhanced dengue-2 infections in mononuclear leukocytes (1.2 +/- 0.7 x 10(4) vs. 2.8 +/- 0.3 x 10(2) PFU/ml). The enhancement of dengue-2 infections in mononuclear leukocytes by dengue-1 immune sera at 1:250 was associated with an increase in the lymphocyte proliferation index, and a decrease in IFNgamma production (56 +/- 24 vs. 12 +/- 3 pg/ml). The addition of IFNgamma (0.1 microg/ml) suppressed significantly the antibody-dependent enhancement induced by dengue-1 immune sera, whereas the presence of anti-IFNgamma F(ab)2 antibody augmented the antibody-dependent enhancement effect. Results from this study suggest that suppression of Th1 response may be involved in the antibody-dependent enhancement of heterotypic dengue infections. Better regulation of Th1/Th2 reactions may be useful for prevention of heterotypic immune enhancement of dengue infections.

Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production.

Dengue virus (DV) infection is a major problem in public health. It can cause fatal diseases such as Dengue hemorrhagic fever and Dengue shock syndrome. Dendritic cells (DC) are professional APCs required for establishing a primary immune response. Here, we investigated the role of human PBMC-derived DC in DV infection. Using different techniques, including plaque assay, flow cytometry analysis, nested RT-PCR, and confocal microscope and electron microscope examinations, we show that DV can enter cultured human DC and produce virus particles. After entrance, DV could be visualized in cystic vesicles, vacuoles, and the endoplasmic reticulum. The DV-infected DC also showed proliferation and hypertrophy of the endoplasmic reticulum as well as the swollen mitochondria. In addition, the DV-stimulated DC could express maturation markers such as B7-1, B7-2, HLA-DR, CD11b, and CD83. Furthermore, the infection of DC by DV induced production of TNF-alpha and IFN-alpha, but not IL-6 and IL-12. Although DC underwent spontaneous apoptosis in the absence of feeding cytokines, this process appeared to be delayed after DV infection. Our observations provide important information in understanding the pathogenesis of DV infection.

A model to study antioxidant regulation of endotoxemia-modulated neonatal granulopoiesis and granulocyte apoptosis.

Neonates with septicemia tend to develop granulocytopenia, which may, in part, be due to septic mediators such as oxygen free radicals and tumor necrosis factor alpha (TNF-alpha). Granulocytopenia may be caused by a decrease in granulocyte growth and/or an increase in granulocyte destruction. In the present study, we investigated antioxidant regulation of endotoxin-modulated neonatal granulopoiesis and granulocyte apoptosis. Using human umbilical cord blood (HUCB), we found that simulating endotoxemia in vitro elicited significant superoxide production within a few minutes. Endotoxin exposure suppressed colony-forming unit-granulocyte and monocyte formation in a dose-dependent fashion. Addition of antioxidants such as N-acetyl-cysteine could reverse the endotoxin suppression of colony-forming unit-granulocyte and monocyte formation (13 +/- 5 versus 75 +/- 5 colony-forming units/mL). Spontaneous in vitro granulocyte apoptosis in 6 h, as reflected by phosphatidylserine expression on the cell surface, was higher in granulocytes from HUCB than in those from adult blood (10.8 +/- 1.0% versus 5.6 +/- 1.2%). The addition of endotoxin or IL-8 to the cells in the in vitro model did not promote granulocyte apoptosis, but TNF-alpha, a major mediator of the effects of endotoxin, significantly induced granulocyte apoptosis in HUCB (control versus TNF-alpha: 8.9 +/- 1.2% versus 35.9 +/- 2.9%). Addition of the antioxidant N-acetyl-cysteine effectively blocked TNF-alpha-induced granulocyte apoptosis as demonstrated by DNA fragmentation. Results from these studies indicate that oxygen radicals are directly involved in endotoxin suppression of granulopoiesis, and indirectly promote granulocyte apoptosis, presumably through TNF-alpha-mediated action. Thus, under certain conditions, modulation of oxygen radical production in the blood may benefit neonates with granulocytopenia.

Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains.

PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis. VrrA-typing divided the B. anthracis into four groups. Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B. anthracis were separated into three groups, which were different from those of the B. cereus strains. Ribotyping separated the B. anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B. anthracis strains into two groups. VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B. cereus strains. The results suggest that a combination of all three methods provides a high resolution typing method for B. anthracis and B. cereus. Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B. anthracis strains from other B. cereus group bacteria.

Failure of dengue-2 virus antibody to interfere with the isolation of dengue-2 virus from Aedes aegypti (Diptera: Culicidae).

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure using Aedes aegypti (L.) that we infected with the 16,681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37 degrees C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collected Ae. aegypti from endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated into Ae. aegypti, but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.

Potential dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and NF-kappaB are sequentially involved.

Direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. In this study, we demonstrated that type 2 dengue (DEN-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line SK-N-SH. Phospholipase A(2) (PLA(2)) was activated by DEN-2 infection, which led to the generation of arachidonic acid (AA). Inhibition of PLA(2) activity by the PLA(2) inhibitors, AACOCF(3) and ONO-RS-082, diminished DEN-2 virus-induced apoptosis. In contrast, the cyclooxygenase inhibitors aspirin and indomethacin, thought to increase AA accumulation by blocking AA catabolism, enhanced apoptosis. Exogenous AA induced apoptosis in a dose-dependent manner. Superoxide anion, which is thought to be generated through the AA-activated NADPH oxidase, was increased after infection. Pretreatment with superoxide dismutase (SOD) protected cells against DEN-2 virus-induced apoptosis. Furthermore, generation of superoxide anion was blocked by AACOCF(3). In addition, the transcription factors, NF-kappaB and c-Jun, were found to be activated after DEN-2 virus infection. However, pretreatment of cells with oligodeoxynucleotides containing NF-kappaB, but not c-Jun, binding sites (transcription factor decoy) strongly prevented dengue virus-induced apoptosis. The finding that AACOCF(3) and SOD significantly block activation of NF-kappaB suggests that this activation is derived from the AA-superoxide anion pathway. Our results indicate that DEN-2 virus infection of human neuroblastoma cells triggers an apoptotic pathway through PLA(2) activation to superoxide anion generation and subsequently to NF-kappaB activation. This apoptotic effect can be either directly derived from the action of AA and superoxide anion on mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-kappaB.

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice.

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.

Effect of glutamine on Th1 and Th2 cytokine responses of human peripheral blood mononuclear cells.

Decreased glutamine concentrations are found in patients with catabolic stress and are related to susceptibility to infections. In this study, we evaluated the role of glutamine in Th1/Th2 cytokine responses. Peripheral blood mononuclear cells were stimulated with phytohemagglutinin (PHA), live attenuated bacillus Calmette-Guérin (BCG), or measles virus in the presence of different glutamine concentrations. We found that glutamine at an optimal concentration (0.6 mM) significantly enhanced PHA-stimulated lymphocyte proliferation as well as Th1 [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] and Th2 cytokine (IL-4 and IL-10) production. In the absence of glutamine, BCG and measles virus elicited minimal lymphocyte proliferation, whereas BCG enhanced Th1 cytokine response and measles virus promoted Th2 cytokine response. Interestingly, addition of glutamine promoted the BCG-elicited Th1 cytokine response (IFN-gamma), but suppressed the measles-induced Th2 cytokine response (IL-10). These results suggest that appropriate glutamine levels may influence host responses to different antigens and microorganisms. Furthermore, predominately Th1, but not Th2, cytokine responses required the presence of optimal concentrations of glutamine.

Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction.

Decreased glutamine concentrations are found during catabolic stress and are related to susceptibility to infections. However, little is known about the mechanism of glutamine modulation of lymphocyte functions. Glutamine is not only an important energy source in mitochondria, but is also a precursor of glutamate, which is used for cellular glutathione (GSH) biosynthesis in lymphocytes. In this study, we investigated the effects of glutamine on the redox reaction during lymphocyte proliferation. Peripheral blood mononuclear cells, obtained from healthy adult volunteers, were cultured and stimulated by phytohaemagglutinin (PHA) in the presence of different glutamine concentrations. Cells were harvested and prepared for analysis of lymphocyte proliferation, cell cycle propagation, intracellular glutathione levels and reactive oxygen species (ROS) production. We found that glutamine supplementation significantly enhanced PHA-stimulated lymphocyte proliferation and propagation of the cell cycle from the G1 to S and G2/M phases. Glutamine also enhanced production of both intracellular ROS and GSH levels in PHA-stimulated lymphocytes. Flow cytometric analysis by the mercury orange staining method showed that glutamine significantly enhanced intracellular non-protein thiols in PHA-stimulated CD4+, but not CD8+ lymphocyte subsets. Furthermore, intracellular GSH detected by monochlorobimane dye probe showed that glutamine enhanced GSH both in PHA-stimulated CD4+ and CD8+ lymphocyte subsets. Inadequate glutamine supplementation resulted in decreased lymphocyte proliferation in association with decreased levels of intracellular GSH. Addition of exogenous GSH significantly enhanced lymphocyte proliferation, whereas blockade of GSH synthesis enhanced ROS production and suppressed lymphocyte proliferation. These results suggest that the modulation of PHA-stimulated lymphocyte proliferation by glutamine is closely related to the maintenance of appropriate intracellular redox status.

Pentoxifylline synergizes with all-trans retinoic acid to induce differentiation of HL-60 myelocytic cells, but suppresses tRA-augmented clonal growth of normal CFU-GM.

All-trans retinoic acid (tRA) has been shown to promote terminal differentiation of promyelocytic leukemia cells, but frequently induce hyperleukocytosis and pulmonary leakage syndrome. Employing pentoxifylline (PTX), a phosphodiesterase inhibitor which could raise intracellular cAMP and modulate leukocyte activation, we sought to investigate if PTX could enhance tRA-induced promyelocytic leukemic cell differentiation but suppress tRA-augmented growth and activation of human granulocytes. tRA could significantly suppress clonal growth of U937 and HL-60 leukemic cells but enhanced the CFU-GM formation of normal bone marrow cells (22 +/- 6 vs. 90 +/- 16 CFU/well). PTX significantly augmented tRA suppression of clonal growth of U937 and HL-60 leukemic cells but suppressed tRA-augmented CFU-GM formation of normal bone marrow cells (90 +/- 16 vs. 25 +/- 9 CFU/well). In addition, PTX enhanced tRA-induced growth inhibition and differentiation of promyelocytic HL-60 leukemic cells, but suppressed respiratory burst activation by the immature granulocytic HL-60 cells and suppressed CD11b adhesion molecule expression by mature granulocytes. PTX similar to dibutyric cAMP promoted HL-60 myelocytic leukemic cell differentiation and growth inhibition, whereas PTX, in contrast to dibutyric cAMP which could augment phorbol myristate acetate (PMA)-elicited respiratory burst activity by immature granulocytes, suppressed the PMA-elicited respiratory burst activity by immature and mature granulocytes. PTX did not raise the intracellular cAMP level of HL-60 cells, but partly suppressed the dibutyric cAMP-elicited elevation of intracellular cAMP level. Results from these studies suggest that PTX might act through different signaling pathways to enhance tRA-induced myelocytic leukemic cell differentiation but prevent from hyperreactive normal granulopoiesis and granulocyte activation.