Clinical isolates of Escherichia coli are resistant both to antibiotics and organotin compounds.

Antibiotic-resistant Escherichia coli are common causative agents of human urinary tract infections. Organotin compounds (OTCs) are man-made chemicals that may affect the renal function of exposed humans and rodents. OTCs are widely recognized as bactericides. However, many environmental and a few clinically relevant bacteria have been found resistant to high concentrations of some OTCs. We examined the susceptibility from 47 E. coli clinical isolates to 12 antibiotics and 5 OTCs. Minimum inhibitory concentrations were determined by the fully automated Sensititre™ ARIS™ 2X system, and E. coli strains were classified as resistant, intermediate resistant, or sensitive, according to the M07-A10 and M100-S26 criteria from the National Committee for Clinical Laboratory Standards. All 47 E. coli strains were susceptible to amikacin but resistant to imipenem and intermediate resistant to ampicillin, cefuroxime, and chloramphenicol. In addition, 26 strains were resistant and 21 intermediate resistant to aztreonam, 24 strains were resistant and 23 intermediate resistant to ceftazidime, 44 strains were intermediate resistant and 3 sensitive to cephalothin, and 43 strains were intermediate resistant and 4 sensitive to ciprofloxacin. Approximately half of the strains were susceptible to cefepime, cefotaxime, and gentamicin. E. coli strains were also found resistant to triphenyltin, tributyltin, dibutyltin, trimethyltin, or dimethyltin at final concentration between 10 µmol/L and 1 mmol/L, during 72-h in vitro culture. However, higher in vitro growth inhibition was induced by these OTCs in the presence of the efflux pump inhibitor carbonyl cyanide-m-chlorophenyl hydrazone, which suggests that efflux pumps contribute to making antibiotic-resistant E. coli also resistant to OTCs.

Adjuvant treatment with a dialyzable leukocytes extract contributes to maintain HPV-infected women free of low-grade cervical lesions.

PURPOSE OF INVESTIGATION: To investigate if adjuvant treatment with a dialyzable extract of leukocytes (DLE), may help HPV-infected patients with low-grade intraepithelial squamous cervical lesions (LIS) to get free of HPV infection and cervical lesions. MATERIALS AND METHODS: Patients with untreated, low-grade cervical lesions were treated either with surgery (Group A) or with DLE (Group B). Pa- tients with low-grade but recurrent cervical lesions were newly treated with surgery plus DLE (Group C). RESULTS: A decreased or ab- sent cervical lesion correlated with a diminished or absent HPV viral load at one year of treatment (r = 0.6,p <0.05). Seventy-nine percent of Group B but only 50 % of Group C and 38 % of Group A patients were free of cervical lesion after 24 months of treatment (p < 0.05). CONCLUSION: The present data support the benefit of adding DLE as adjuvant for treating HPV-infected women with LIS.

Sensitization to TRAIL-induced apoptosis and modulation of FLICE-inhibitory protein in B chronic lymphocytic leukemia by actinomycin D.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of the cell death pathway and exerts tumoricidal activity in vivo with minimal toxicity. In order to investigate the therapeutic potential of TRAIL in B chronic lymphocytic leukemia (B-CLL) we have analyzed the expression of TRAIL receptors (TRAIL-Rs) in leukemic cells from B-CLL patients and their in vitro sensitivity to apoptosis induced by recombinant human TRAIL. We have found TRAIL-R1 and -R2 death receptor, and TRAIL-R3 and -R4 decoy receptor mRNA expression in most of the 57 B-CLL patients studied (R1 82%, R2 100%, R3 96% and R4 82%). TRAIL-R1 and R2 proteins were expressed on the surface and within the cells, whereas R3 and R4 decoy receptors were almost exclusively expressed in the cytoplasm. Despite TRAIL death receptor expression, B-CLL cells were relatively resistant to induction of apoptosis by recombinant human TRAIL (300 ng/ml). However, the susceptibility to TRAIL-induced apoptosis was increased by treatment of B-CLL cells with actinomycin D (Act D). Western blot analysis showed higher constitutive expression of the long form of FLICE-inhibitory protein (FLIP(L)) in B-CLL as compared to normal tonsillar B cells. Act D treatment down-regulated both long and short FLIP expression, which was correlated with the increase in B-CLL sensitivity to TRAIL. Although the surface TRAIL death receptor expression was up-regulated both by cell culture and by Act D treatment, the changes were not correlated with a gain in susceptibility to TRAIL. In addition, neither decoy receptors nor Bcl-2 expression were affected by Act D. Our findings suggest the possible involvement of FLIP in regulating TRAIL-mediated apoptosis in B-CLL.

Nitric oxide induces apoptosis in NALM-6, a leukaemia cell line with low cyclin E protein levels.

Intracellular nitric oxide levels may differ in resting and stimulated cells and contribute to the regulation of cell survival and proliferation through a variety of mechanisms and effects. We exposed two B-cell lines to a range of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) concentrations in order to examine their susceptibility to exogenous nitric oxide and the participation of nitric oxide as modulator of cell proliferation. Although both FLEB and NALM-6 decreased their levels of thymidine incorporation, only NALM-6 cells were induced to undergo G1 arrest, phosphatidyl serine exposure and DNA fragmentation when cultured in the presence of 250 microm SNAP. This higher sensitivity of NALM-6 coincided with initially low cyclin E protein levels which were increased 7.8-fold after culture for 24 h with 250 microm SNAP. In contrast, there was no difference in cyclins A and D3, Bcl-2 and actin levels, neither at the beginning nor at the end of the 24 h culture. Our study reveals that FLEB and NALM-6 exhibit different response to the same concentration of nitric oxide, that nitric oxide can simultaneously induce cell cycle alterations and apoptosis, and further suggests an association between these two processes, with the involvement of cell cycle regulatory molecules.

Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms.

Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg(11,13)]-dynorphin A(-13)-Gly-NH-(CH(2))(5)-NH(2), a selective kappa-opioid receptor agonist; and poly-l-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.

Increased serum levels of soluble Fas in progressive B-CLL.

Clinical progression of B-cell chronic lymphocytic leukemia (B-CLL) depends on survival and accumulation of leukemic cells, regulated in part by physical cell contact and soluble molecules. Here we have studied the Fas/FasL system in relation to clinical progression in B-CLL. Serum levels of soluble Fas (sFas) and FasL (sFasL) were determined by ELISA in 43 progressive and 40 non-progressive B-CLL patients and in 21 control individuals. Correlation between sFas serum levels and clinical progression, stage and survival were statistically analyzed. We found high levels of sFas in B-CLL sera correlated with disease progression (p<0.01). In addition, higher sFas levels were found in patients in stages II, III and IV in comparison to patients in stage 0 (p<0.05, p<0.01, p<0.03, respectively). Survival was significantly shorter for patients with > or =6 ng/ml sFas serum levels, although a multivariate analysis did not show sFas to be a significant independent prognostic factor. Fresh B-CLL cells showed only low levels of membrane expression, which were not correlated to sFas levels in serum. In vitro activation of B-CLL cells increased Fas expression, as reported earlier, and induced cells to release sFas into the supernatant. In conclusion, our results indicate that sFas in serum may be a useful parameter for the prediction of clinical progression in B-CLL.

IGF-II and IL-2 act synergistically to alter HDAC1 expression following treatments with trichostatin a.

Histone deacetylases play key roles in the regulation of gene transcription. Studies have shown that expression of interleukins IL-2 and IL-8, and insulin-like growth factor 2 (IGF2) are affected by treatment with histone deacetylase inhibitors. We have previously shown that the gene for histone deacetylase 1 (HDAC1) is upregulated following treatment with TSA. The murine homologue of this gene has been reported to be inducible by IL-2. In this study, we have examined the effects IL-2, IGF-II and TSA have on HDAC1 expression in the human hepatocellular carcinoma derived cell line Hep3B. Our results indicate that in contrast to the mouse, HDAC1 is not inducible by IL-2. However, in TSA treated cells, IL-2 and IGF-II were found to act synergistically to reduce TSA induced HDAC1 mRNA levels almost to normal.

Organotin compounds decrease in vitro survival, proliferation and differentiation of normal human B lymphocytes.

Organotin compounds (OTC) are organometallic compounds with vast industrial and agriculture applications that give rise to ubiquitous environmental contamination. OTC are immunotoxic, but most studies have been performed in rodents and almost exclusively focused on T cell immunity. Humans can be exposed to OTC by inhalation, absorption, and consumption of contaminated food and water. To analyse the effects of OTC in human immune tissue, we isolated B cells from tonsils and exposed them to five OTC at various concentrations, during in vitro culture. Non-stimulated B cells were killed by 100 nM of all tested OTC after 8 h in vitro culture, under sub-optimal conditions, except TET. OTC also decreased the proliferation of tonsillar B lymphocytes stimulated with Staphylococcus aureus Cowan 1 (SAC) and IL-2, when present at 100 nM and higher concentrations. IgM secretion was reduced in stimulated cell cultures exposed to 100 nM dibutyltin chloride (DBT). Accordingly, increased phosphatidylserine exposure demonstrated that 100 nM TPT and DBT induced B cells to die by apoptosis. These data indicate that human B cells are diminished in their capacity to survive, proliferate and differentiate in the presence of OTC in vitro.

Cytokine expression in B-CLL in relation to disease progression and in vitro activation.

Earlier, we reported an association between low in vitro and in vivo IL-1 and IL-6 production, decreased IL-1beta and IL-10 mRNA expression and B cell chronic lymphocytic leukemia (B-CLL) disease progression. We have now further investigated cytokine mRNA transcription in B-CLL cells and cytokine serum levels in B-CLL patients. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of tumor necrosis factor (TNFalpha), IFNgamma, IL-6 and BCGF was equally often seen in non-progressive and progressive patients. However, 4 out of 23 non-progressive cases expressed mRNA for IL-12 while no IL-12 expression was seen in 15 progressive patients. No IL-12 was found in sera or supernatants from in vitro stimulated B-CLL cells, whereas TNFalpha and IL-10 were detected in sera from 51 and 31 of 65 B-CLL patients, respectively. TNFalpha values were significantly high in sera from patients in stages III and IV with disease progression. TNFalpha and IL-10 were also detected in culture supernatants from in vitro stimulated B-CLL cells, whereas IFNgamma was undetectable in these cultures and rarely positive in serum. Although further investigations are required, our data and that from previous reports indicate that B-CLL-derived cytokines are involved in B-CLL disease progression.

Regulation of B-CLL apoptosis through membrane receptors and Bcl-2 family proteins.

The accumulation of monoclonal chronic lymphocytic leukemia B (B-CLL) cells may be due to excessive proliferation and longevity. Clinical progression may thus come from a constitutive but altered expression of a number of genes that results in extended B-CLL cells life span, increased proliferative capacity and diminished cell death. B-CLL cells express a number of surface markers that characterise the normal B-cells phenotype. However, B-CLL cells are CD5 positive and most of them also express CD6, surface receptors that are present in just a small subset of normal B-cells. When exploring CD6 function, we found out that cross-linking of CD6 protected B-CLL from anti-IgM-induced apoptosis. CD6 activation blocked anti-IgM- induced Bax(alpha) up-regulation and, by doing so, corrected an imbalance in the Bcl-2/Bax ratio that accompanies apoptosis. Here, we review all surface receptors and cytokines that have been described as participating in the induction or protection of B-CLL apoptosis together with data on chemosensitivity and gene modulation, data on the Fas receptor/Fas ligand system, and the implications of all the latter for B-CLL cell survival.

Apoptosis in B-chronic lymphocytic leukaemia.

B-chronic lymphocytic leukaemia (B-CLL) is characterised by the progressive accumulation of monoclonal B cells, which may be the result of several factors leading to extended B-CLL cell lifespan, increased proliferative capacity and diminished cell death. Here we review the implications of several signals mediated by receptors, such as surface IgM, CD6 and CD40, for the B-CLL cell survival, together with data on gene modulation in relation to the apoptosis process in B-CLL cells. We also describe some features of the Fas/FasL system in B-CLL that hypothetically might contribute to the accumulation of leukaemic cells and the progression of the disease, by downregulating the apoptotic response or avoiding the autologous immune response.

Inhibitors of arachidonic acid metabolism reduce DNA and nuclear fragmentation induced by TNF plus cycloheximide in U937 cells.

U937 human myeloid leukemia cells are induced to apoptosis by tumour necrosis factor (TNF) plus cycloheximide (CHX). We have analysed the effect of various inhibitors of the arachidonic acid (AA) metabolism on several features of this process. The formation of high molecular weight and oligonucleosomal DNA fragments as well as nuclear fragmentation were reduced by inhibitors of 5-lipoxygenase (BWA4C and BWB70C), 5-LO activating protein (MK-886), and cytosolic PLA2 (AACOCF3). None of these agents blocked the morphological changes detected by microscopy or flow cytometry, phosphatidylserine exposure on the cell surface or Caspase 3-like activation. AA also induced nuclear fragmentation at a concentration of 1-20 microM. However, the mechanisms by which these inhibitors act, remain unexplained since there was no 5-LO expression in the U937 cells and no AA release followed their stimulation with TNF plus CHX.

CD6 ligation modulates the Bcl-2/Bax ratio and protects chronic lymphocytic leukemia B cells from apoptosis induced by anti-IgM.

CD6 and CD5 belong to a scavenger-receptor cysteine-rich (SRCR) super family of membrane glycoproteins that are expressed on chronic lymphocytic leukemia B (B-CLL) cells, normal T cells, and a small subset of normal B cells. CD6 configures in the membrane in relation to the cellular activation level and can act as a coreceptor for T-cell activation. We have examined a group of progressive and nonprogressive B-CLL cells. Most B-CLL cells were positive for CD6 and the expression of CD6 was increased after activation with Staphylococcus aureus Cowan I plus interleukin-2 or 12-O-tetradecanoylphorbol 13-acetate, although anti-CD6 antibodies did not increase proliferative responses to these stimuli. However, anti-CD6 stimulation was found to protect against anti-IgM-induced apoptosis in B-CLL. bax(alpha) upregulation and bcl-2 downregulation were found in anti-IgM- and glucocorticoid (GCC)-induced apoptotic cells, respectively. Furthermore, CD6 cross-linking downregulated bax(alpha) mRNA levels in anti-IgM-treated cells, resulting in an increased bcl-2/bax(alpha) ratio. CD6 activation also prevented bcl-2 mRNA downregulation and apoptosis induced by GCC in one of six GCC-sensitive patients. These data suggest that an interaction between CD6 and its ligand might contribute to B-CLL survival through the modulation of the Bcl-2/Bax ratio.

Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis.

Platelets are physiologically anucleated cells, derived from megakaryocytes, that undergo vesiculation and transformation into small particles when they are stimulated in vitro by ionomycin and other agents. Electron microscopy images suggest a similarity to apoptosis in cells with nuclei, which ends with cell disintegration and formation of apoptotic bodies. By PCR, we have demonstrated mRNA expression of bcl-2, bax, and p53 in highly purified non-stimulated platelets. A side-scatter shift and a decrease in the Bcl-2/Bax protein ratio were observed by flow cytometry analysis after stimulation with ionomycin. The ionomycin-induced modifications were inhibited by the calpain I inhibitor calpeptin and, less effectively, by VAD-cmk, a broad-spectrum caspase inhibitor. However, caspase 3-like activity was very low, with only a twofold increase after ionomycin stimulation, as measured by the cleavage of the fluorogenic peptide substrate DEVD-AMC. Our data indicate that platelets may constitute a natural model for the analysis of cytoplasmic events in apoptosis.

BCL-2 delay apoptosis and PARP cleavage induced by NO donors in GT1-7 cells.

BCL-2 is a negative regulator of cell death in several systems. Here we report that bcl-2 expression protects against apoptosis induced by nitric oxide (NO) donors in GT1-7 hypothalamic cells. BCL-2 significantly inhibited neuronal death caused by 200 microM S-nitroso-cysteine (SNOC), 200 microM S-nitroso-N-acetyl-penicillamine (SNAP), or 1 mM 3-morpholinosydnonimine (SIN-1). To explore further the protective mechanism(s) elicited by bcl-2 expression, we investigated whether BCL-2 could prevent NO-induced cleavage of poly-ADP-ribose-polymerase (PARP), which is a substrate for interleukin-1 beta converting enzyme (ICE)-like proteases in apoptosis. Formation of 85 and 25 kDa PARP fragments elicited by NO donors was inhibited in cells over-expressing bcl-2.

Bcl-2, Bax and p53 expression in B-CLL in relation to in vitro survival and clinical progression.

Our previous data have shown that isolated leukemic cells from progressive chronic lymphocytic leukemia (B-CLL) patients respond to growth stimulation in vitro and express high levels of p53, immunoreactive with the configuration-specific antibody PAb 240. We have now analyzed the in vitro survival of B-CLL cells in relation to Bcl-2, Bax alpha and p53 expression and compared this with the clinical progression of the disease. Leukemic cells from patients with progressive disease demonstrated higher in vitro survival, compared with non-progressive B-CLL and normal B cells. All cells were sensitive to treatment with a combination of glucocorticoid and cAMP. Bcl-2 protein levels were not related to clinical progression, as measured by flow cytometry. Competitive PCR showed that Bcl-2 mRNA was over-expressed in most of the B-CLL samples and that p53 mRNA expression was similar between B-CLL groups and normal values and thus not related to clinical progression. However, since Bax alpha expression was lower in progressive than in non-progressive patients, the Bcl-2/Bax alpha ratio at the mRNA level was significantly higher in the progressive group. Our data suggest that the Bcl-2/Bax alpha ratio is important for the regulation of B-CLL cell survival, that p53 over-expression in progressive B-CLL is the result of post-transcriptional modifications and that a directed PKA activation may potentiate the cytolytic effect of glucocorticoids in vivo.

Interleukin-10 mRNA expression in B-cell chronic lymphocytic leukaemia inversely correlates with progression of disease.

Interleukin-10 (IL-10) has been shown in vitro to inhibit survival and spontaneous DNA synthesis in B-cell chronic lymphocytic leukaemia (B-CELL) cells by induction of programmed cell death. We have analysed the presence of mRNA transcripts for IL-10 in purified B-CLL cells from 35 patients by RT-PCR. Transcripts for IL-10 were detected in 11/20 patients with non-progressive disease. In cell preparations from patients with progressive B-CLL IL-10 mRNA were detected in only 2/15 samples (P < or = 0.01). The Epstein-Barr virus status of the cells did not account for the difference in IL-10 mRNA expression observed between the two groups of patients. Thus, IL-10 mRNA expression in leukaemic cells from patients with B-CLL was strongly associated with non-progressive disease. This finding may support other observations suggesting that IL-10 might be a candidate for immune therapy of progressive B-CLL.

Studies on the regulation and localization of 5-lipoxygenase in human B-lymphocytes.

Stimulated B-lymphocytes, isolated from patients with chronic lymphocytic leukemia of B-cell type (B-CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B-lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5-lipoxygenase products. Several thiol-reactive compounds such as N-ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bis[dimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)-PCR analysis demonstrated the expression of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase mRNA in B-CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B-CLL cell membrane. Furthermore, MK886, the FLAP-binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5-HETE formation. Immunocytochemistry showed that 5-lipoxygenase was mainly localized in the nuclei of non-activated B-CLL cells, tonsillar B-lymphocytes and monoclonal B-cells. In contrast, neither human peripheral T-lymphocytes nor Jurkat cells were stained. These results suggest that 5-lipoxygenase and its products function in the nucleus of B-lymphocytes.