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.

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.

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.

Leukemic cells from progressive B-CLL respond strongly to growth stimulation in vitro.

Isolated leukemic B cells from patients with B-chronic lymphocytic leukemia (B-CLL) were tested for proliferative response in vitro to Staphylococcus Aureus strain Cowan 1 (SAC), IL-2, and low molecular weight (MW) BCGF. Patients were classified according to clinical stage and progressiveness. Ten of eighteen cell populations from patients with progressive B-CLL responded in vitro with a stimulation index (SI) > 20. Only 1/16 non-progressive patients had a proliferative but low response. Normal unfractionated tonsillar B cells responded to SAC and BCGF, whereas normal high buoyant density B cells were unresponsive. After 3 days of stimulation, responding B-CLL cells had multiplied and the B cells expressed CD5, CD19, and weakly CD21. No cells in the responding cultures exhibited CD3 or the EBV nuclear antigen EBNA-1. Cell maturation, measured as IgM secretion, was found in some, but not in all responding B-CLL cultures. Thus, B-CLL cells from patients with progressive disease have the capacity to respond to signaling through surface Ig receptors and to certain T-cell factors which was not the case for B-CLL cells from non-progressive patients. The pattern of in vitro response may be related to disease progression, reflecting a dependency of normal immunoregulatory mechanisms and/or a dysregulation of the growth control in the leukemic cells.

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.

Membrane expression of IL-1 alpha in chronic B lymphocytic leukemia.

Patients with B chronic lymphocytic leukemia (B-CLL) demonstrate a high variability in disease activity, from a benign monoclonal lymphocytosis to a rapidly fatal condition. Progressive B-CLL is related to a high expression of different growth factor receptors on the leukemic cells and to a high proliferative in vitro response to Staphylococcus aureus strain Cowan 1 (SAC). As the expression of membrane IL-1 alpha (mIL-1 alpha) indicates B lymphocyte activation, we have investigated mIL-1 alpha on cells from patients at different stages of the disease. Total cellular levels of IL-1 alpha were measured by flow cytometry of permeabilized cells and compared with CD5, CD19, CD25 and IgM expression on the cell surface. mIL-1 alpha is upregulated, both in leukemic and normal lymphocytes, in response to sIgM cross-linking with SAC or phorbol ester activation. A significantly higher expression of mIL-1 alpha was found in cells from patients with a clinically benign form of the disease.

Low IL-1 beta production in leukemic cells from progressive B cell chronic leukemia (B-CLL).

In vitro production of IL-1 beta by cells from 32 patients with benign monoclonal lymphocytosis of undetermined significance (MLUS) and B cell chronic lymphocytic leukemia (B-CLL) was investigated. Normal B lymphocytes (2 x 10(6)) secreted approximately 5 ng/ml of IL-1 beta during 24 h and approximately ten times more after stimulation with Staphylococcus aureus, strain Cowan 1 (SAC). When patients were studied, a loss of IL-1 beta production was found in leukemic cells from progressive disease. Cells from MLUS patients secreted near normal levels of IL-1 beta and responded to SAC stimulation, whereas cells from patients with progressive B-CLL produced no, or little IL-1 beta, and did not respond to SAC. Loss of IL-1 beta production in progressively growing B-CLL may be related to an increased malignant character of these cells. This is discussed in relation to the immunogenicity of the leukemic cells and their capacity to differentiate.

Serum levels of helper factors (IL-1 alpha, IL-1 beta and IL-6), T-cell products (sCD4 and sCD8), sIL-2R and beta 2-microglobulin in patients with B-CLL and benign B lymphocytosis.

Chronic B-lymphocytic leukemia (B-CLL) cells may be regulated by immune functions. In an attempt to analyze such functions, helper factors (IL-1 alpha, IL-1 beta and IL-6), T-cell products (sCD4 and sCD8) and sIL-2R and beta 2-microglobulin were measured in serum of patients at different stages of the disease. Patients were classified as having monoclonal lymphocytosis of undetermined significance (MLUS), stable or progressive B-CLL respectively. A significant, but modest, increase of IL-1 alpha was found in B-CLL as well as in MLUS patients whereas IL-6 levels were increased in MLUS only. sCD8 levels were increased both in MLUS and B-CLL but augmented sCD4 concentrations were found statistically significant only in progressive B-CLL. beta 2-microglobulin and sIL-2R were related to the extent of the monoclonal B-cell fraction. The data indicate an increased T-suppressor activity in both MLUS and B-CLL patients and a selective increase of helper T-cell activity in progressive B-CLL. A possible immunoregulatory influence of helper T cells on disease progression is discussed.

Higher proliferative response in B-chronic lymphocytic leukemia (B-CLL) as compared to B-monoclonal lymphocytosis of undetermined significance (B-MLUS) after stimulation with Staphylococcus aureus and anti-CD40 monoclonal antibodies.

B-CLL is a malignant monoclonal B-cell disorder and B-MLUS is the benign counterpart. The proliferative response and the capacity to secrete IgM was measured in B-CLL and B-MLUS, respectively, and compared to normal B-cells. SAC and a mAb against CD40 were used as stimulatory agents. No cell population responded to anti-CD40 mAb alone. SAC only induced a high DNA synthesis rate in normal B-cells as well as in B-CLL cells, although the magnitude was three-fold lower and delayed for about 48 h in B-CLL. B-MLUS cells did not proliferate in response to SAC. The combination of anti-CD40 and SAC enhanced the proliferative capacity of normal B-cells and produced a more rapid response in B-CLL. B-MLUS cells were not activated. Normal B-cells and B-MLUS did not secrete IgM after SAC stimulation, while B-CLL cells had a continuous increase in the IgM production during a 6-day culture period. The higher proliferative capacity of B-CLL cells compared with B-MLUS cells may be explained by an increased expression of activation molecules e.g. receptors for various cytokines and growth factors. Moreover, the inertness and inability of B-MLUS cells in comparison to normal B- and B-CLL cells to respond to powerful activation signals might indicate an intrinsic defect of B-MLUS cells in the signal transduction leading to a block of mitosis and a benign course of the disease.

Higher T-cell imbalance and growth factor receptor expression in B-cell chronic lymphocytic leukemia (B-CLL) as compared to monoclonal B-cell lymphocytosis of undetermined significance (B-MLUS).

The surface marker phenotype of lymphocytes derived from 12 patients with B-CLL was compared to that of lymphocytes from 10 patients with an other monoclonal but clinical benign form of B-cell proliferative disorder termed monoclonal B-cell lymphocytosis of undetermined significance (B-MLUS). A panel of well characterized monoclonal antibodies was used for the surface marker determinations. The mean total number of B cells (CD20) was 8.5 x 10(9)/1 in B-MLUS as compared to 44 x 10(9)/1 in B-CLL (p less than 0.001). B-CLL had a greater imbalance in T-cell subpopulations than B-MLUS and healthy controls. Total numbers of CD3+, CD8+ cells as well as cells expressing the NK-related antigens (CD16, Leu-7) and IL-2 receptor (CD25) bearing lymphocytes were statistically significant higher in B-CLL than in B-MLUS. Analyses of B-cell enriched populations showed that B-CLL represented B cells of an early maturation stage, whereas B cells from B-MLUS were more mature as judged by the loss of the CD21 surface marker. A larger fraction of B cells in B-CLL compared to B-MLUS exhibited a higher activation stage as revealed by the expression of the CD21, CD25 and CD35 structures as well as the FMC7 antigen.

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.

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.

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.

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.

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.

Monoclonal antibodies against leucoagglutinin-reactive human T-lymphocyte surface components. I. Characterization of cellular binding sites.

The binding specificities of three biologically active anti-lymphocyte monoclonal antibodies (MoAb) (K46M, K3G, and 3-19-2) produced against human T-cell surface components reactive with the mitogenic lectin leucoagglutinin from Phaseolus vulgaris (La) were analysed. K46M is a strong T-cell mitogen, while K3G and 3-19-2 inhibited cell-mediated cytotoxicity. Resting peripheral blood lymphocytes (PBL) contained 4-16% K46M+ cells, 8-35% K3G+ cells, and less than 0.3-4% 3-19-2+ cells. After stimulation with T-cell mitogens the proportion of K46M+ and 3-19-2+ cells increased markedly (mean 59 and 30% positive cells, respectively), while the increase in K3G+ cells was less prominent (38%). K46M-reactive structures were expressed on mature T cells and probably also on B cells. K3G reacted with B and T cells while 3-19-2 showed a broader specificity reacting also with erythrocytes. All three MoAb reacted with lipid extracts of resting and activated PBL as well as with purified neutral glycolipids of lymphoid origin. In addition 3-19-2 reacted with lipid extracts of erythrocytes. K46M immuno-precipitated four surface peptides from lectin-stimulated PBL. Their apparent molecular weights were 53,000, 42,000, and 16,000 (doublet). The 53,000 and 42,000 MW peptides were identified as the alpha and beta chains of the T-cell antigen receptor. The identity of the 16,000 MW peptides is presently unknown. K3G and 3-19-2 did not specifically precipitate any lymphocyte surface peptide.

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.