Prognostic value of quantitative immune alterations in melanoma patients

Introducción y objetivos: Los pacientes con cáncer presentan una alteración de la respuesta inmune.La inmunosupresión en melanoma, juega un papel importante. El objetivo de este estudio fue valorar las alteracionescueantitativas de la inmunidad en pacienets con melanoma.• Pacientes y métodos: Se obtuvieron muestras de sangre periférica en EDTA de 86 pacientes con melanoma(63 pacientes libres de enfermedad y 23 pacientes con metástasis a distancia). Los niveles de leucocitostotales, linfocitos totales, linfocitos CD3, linfocitos CD4, linfocitos CD8, linfocitos B (CD19) y linfocitosNK (CD56) fueron valorados mediante marcadores de superficie por citometría de flujo usando un CoulterEpics Elite (Coulter Corp). Los niveles de IgA, IgG, IgE e IgM fueron valorados por nefelometría usando unnefelómetro Hyland PDQ laser.• Resultados: Hubo diferencias significativas entre pacienets metastásicos y pacientes libres de enfermedaden los niveles de linfocitos totales (mediana: 2251.57 vs 1783.04/mm3, p=.001), linfocitos B (CD19)(216.1 vs 108.36/mm3, p=.010), linfocitos NK (CD56) (149.54 vs 115.2/mm3, p=.016) y niveles de IgA(241.59 vs 300.55 mg dL, p=.044) al considerarlas como variables continuas. Al considerar cada parámetro deestudio como una variable cualitativa, sólo se observaron diferencias significativas en los niveles totales delinfocitos, existiendo un 73.9% d elos pacientes con enfermedad a distancia niveles d elinfocitos por debajo de2000 células/mm3 frente a un 36.5% de pacienets libres de enfermedad (χ2 Pearson = 9.476, df = 1, p = .002).La mediana de supervivencia para 46 pacientes con niveles totales de linfocitos por encima de 2000células/mm3 fue de 965 días (DF= 65.03, IC 95% = 792.72 - 1090.30) frente a 441 días (DF= 75.61, IC 95% =292.81 - 589.19) para 40 pacientes con niveles totales de linfocitos 2000 células / mm3 (log rank = 4.54, df=1,p= .0331).• Conclusiones: Existen diferencias significativas en los niveles de algunas subpoblaciones linfocitariasy en los niveles de IgA entre pacienets metastásicos y pacienets libres de enfermedad. Los pacientes con melanomacon niveles de linfocitos totales por encima de 2000 cells/mm3 tiene una mayor supervivencia que aquelloscon niveles por debajo de 2000 cells/mm3

Purpose: The immune response is altered in patients with neoplasms. Immunosuppression hasimportant consequences in patients with melanoma. The aim of this study was to assess quantitative immunealterations in melanoma patients..• Material and methods: We obtained a peripheral blood sample in EDTA from 86 melanoma patients(63 of them disease-free and 23 with distant disease). Total leukocytes and lymphocytes, B lymphocytes(CD19), types CD3, CD4, CD8 lymphocytes, and NK lymphocytes (CD56) were counted by determining thesurface markers by flow cytometry, using a Coulter Epics Elite (Coulter Corp.). IgA, IgG, IgE and IgM wereassayed by nephelometric methods employing a Hyland PDQ laser nephelometer.• Results: We found significant differences between disease-free patients and those with active diseasewith regard to lymphocytes total count (median: 2251.57 vs. 1783.04/mm3, p=0.010), NK lymphocytes(CD56) (149.54 vs. 115.2/mm3, p=0.016), and IgA levels (241.59 vs. 300.55 mg/dl, p=0.044), when taken ascontinuous variables. When considering each parameter as a discontinuous variable, only changes in absolutelymphocyte count retained an statistical difference depending on the presence or absence of active disease,73.9% of the patients with active metastatic disease having a lymphocyte count below 2000 cells/mm3 versusonly 36.5% of the disease-free patients (c2 Pearson=9.476, df=1, p=0.002). The median survival for the 46patients with absolute lymphocyte count above 2000 cells/mm3 was 965 days (DF=65.03, IC 95%=792.72-1090.30) versus 441 days (DF=75.61, IC 95%=292.81-589.19) for the 40 patients with absolute lymphocytecount below 2000 cells/mm3 (log rank=4.54, df=1, p=0.0331).• Conclusions: There are significant differences in some lymphocyte populations and IgA levels betweenpatients with metastases and disease-free patients. Melanoma patients with absolute lymphocyte levels above2000 cells/mm3 have a longer survival than those with a lymphocyte count below 2000 cells/mm3

Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) cells develop resistance to nucleoside analogs over time. This chemoresistance may be caused by selection for B-CLL cells with defects in the particular apoptosis pathway triggered by these drugs. Therefore, anticancer agents that induce apoptosis through alternative pathways might be useful in treating chemoresistant B-CLL. Farnesyltransferase inhibitors (FTIs) are a class of synthetic drugs with definite molecular targets, which have demonstrated cytotoxicity against leukemic cell lines. We have studied the ex vivo effect of the FTI BMS-214662 on cells from 18 patients with B-CLL. Low concentrations (<1 microM) of BMS-214662 prevented farnesylation of the chaperone marker HDJ-2 and had no effect on Akt activation. BMS-214662 induced apoptosis in B-CLL cells from all patients studied, including those showing resistance to cladribine and fludarabine ex vivo and in vivo. Treatment with BMS-214662 induced loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, proapoptotic conformational changes of Bax and Bak, reduction in Mcl-1 levels and activation of caspases 9 and 3. The general caspase inhibitor Z-VAD-fmk did not prevent BMS-214662-induced cell death. These results indicate that BMS-214662 may be a useful drug for treating B-CLL and, in particular, an alternative for the therapy of purine analog-resistant or relapsed B-CLL.

Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia.

We have evaluated the role of caspases and the mitochondrial apoptosis inducing-factor (AIF) in apoptosis induced by cladribine (2CdA), in vitro, in cells from patients of B-CLL and in peripheral blood lymphocytes from normal donors. In sensitive B-CLL cells, apoptosis was characterized by cell shrinking, loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, activation of caspases 3, 7, 8 and 9, reduction of Mcl-1 levels, translocation of AIF from mitochondria to nucleus and chromatin condensation. No significant variations in the levels of Bcl-2, Bax and Bak proteins were noticed upon treatment with 2CdA. Co-treatment of cells with the pan-caspase inhibitor Z-VAD-fmk attenuated some morphological and biochemical characteristics of apoptosis and delayed 2CdA-induced DeltaPsi(m) loss, but did not prevent cell death. Z-VAD-fmk did not prevent 2CdA-induced AIF translocation but in this case apoptotic cells displayed only peripheral chromatin condensation, characteristic of AIF action. Reduced or negligible caspase 3 expression did not prevent 2CdA toxicity in cells from four patients. Cells from three patients that responded poorly to 2CdA lacked expression of caspases 9 or 3. Cells from another patient resistant to 2CdA expressed caspases 3, 7, 8 and 9 but they were not activated by treatment. These results indicate that execution of apoptosis is carried out independently by AIF and caspases, which are responsible for the development of apoptotic phenotype in response to 2CdA. Although caspases can also collaborate in DeltaPsi(m) loss, proapoptotic proteins from the Bcl-2 superfamily may be the key inducers of DeltaPsi(m) loss and apoptosis in B-CLL cells sensitive to 2CdA.

Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells.

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments approximately 500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes deltapsim loss, caspase-9 activity, and apoptosis in Jurkat cells.

Doxorubicin induces caspase-3 activation and apoptosis in Jurkat cells but inhibition of this enzyme did not prevent cell death, suggesting that another caspase(s) is critically implicated. Western blot analysis of cell extracts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by doxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-CHO, Z-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpression of CrmA, prevented many morphological features of apoptosis but not loss of mitochondrial membrane potential (delta(psi)m), phospatidilserine exposure, and cell death. Western blot analysis of cells treated with doxorubicin in the presence of inhibitors allowed elucidation of the sequential order of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspase-9 activity, blocked the activation of all caspases studied, lamin B degradation, and the development of apoptotic morphology, but not cell death. All morphological and biochemical features of apoptosis, as well as cell death, were prevented by cotreatment of cells with the general caspase inhibitor Z-VAD-fmk or by overexpression of Bcl-2. Doxorubicin cytotoxicity was also blocked by the protein synthesis inhibitor cycloheximide. Delayed addition of Z-VAD-fmk after doxorubicin treatment, but prior to the appearance of cells displaying a low delta(psi)m, prevented cell death. These results, taken together, suggest that the key mediator of doxorubicin-induced apoptosis in Jurkat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X), which would cause delta(psi)m loss, release of apoptogenic factors from mitochondria, and cell death.

CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells.

Jurkat cells and the derived TCR / CD3-defective subline, J.RT3.T3.5 undergo activation induced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositol linked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross-linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA-stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti-Fas mAb SM1 / 23 and anti-APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti-CD59 stimulated cells was specifically prevented by the anti-APO2L blocking antibody. Anti-CD59 cross-linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti-APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.

ITIH4 serum concentration increases during acute-phase processes in human patients and is up-regulated by interleukin-6 in hepatocarcinoma HepG2 cells.

The serum concentration of the inter-alpha trypsin inhibitor heavy chain 4 protein (ITIH4) increases (from 1.4-3 times) in male patients suffering of different acute-phase processes (myocardial infarction, unstable angina or programmed surgery). The concentration of C-reactive protein (CRP) in these samples ranged from 15 microg/ml to 133 microg/ml. Using the hepatocarcinoma HepG2 cell line we have observed up-regulation of ITIH4 mRNA expression upon dose-response treatments with interleukin-6 (IL-6). This effect correlates with the increase of radiolabeled ITIH4 in the cellular media of (35)S-labeled HepG2 cells treated with the cytokine. A similar effect was observed for haptoglobin mRNA, used as a control for acute-phase protein expression. IL-1beta, although up-regulating the expression of alpha(1)-acid glycoprotein in these cells, did not induce any effect in the expression of ITIH4. No changes were observed after TNF-alpha treatments. The results presented here indicate that ITIH4 is a type II acute-phase protein in humans.

Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (

Study of HLA as a predisposing factor and its possible influence on the outcome of multiple sclerosis in the sanitary district of Calatayud, northern Spain.

The relationship between multiple sclerosis (MS) and the HLA antigens DR2 and DQ1 is well recognised, but, in Spain, it has not been clearly defined. The aim of our study was to investigate the relationship between MS and HLA antigens in the sanitary district of Calatayud, northern Spain, and to correlate these antigens with the progression of the disease. Thirty-four patients were selected from a long-term (October 1990 to July 1996) prospective survey in the region where there was a prevalence rate of 58 per 100,000 population. The HLA antigens were determined in 31 patients. A control group of 895 people of Caucasian race was recruited from the same population. We performed serologic tests on all participants. Nucleotide typing was carried out in DR2-positive patients. The most frequent antigens in excess in MS were: A19 (odds ratio, OR: 2.29, p = 0.04), B5 (OR: 2.85, p = 0.02), B41 (OR: 7.65, p = 0.04), CW7 (OR: 3.4, p = 0.004), DR6 (OR: 6.18, p = 0.0001) and DR10 (OR: 3.4, p = 0. 004). The DR2 antigen was also more frequent in MS patients (39%) than in controls (19%; OR: 2.69, p = 0.01). All positive DR2 patients showed the DR15(2) split but not the DR16(2) split. The frequency of antigens CW4 and DR1 was lower in MS patients than in controls. The CW4 antigen was detected in 12% of the patients and in 33% of the controls (OR: 0.28, p = 0.04). The DR1 antigen was found in 20% of the controls and in none of the MS patients (OR: undefined, p = 0.01). The DQ1 antigen was observed in 68% of the patients and in 50% of the controls (OR: 2.1, p = 0.07). We did not find any relationship between HLA antigens and progression of the disease. Although we found that DR2 antigen is linked to MS, we also found other antigens related to the disease. This suggests a genetic heterogeneity in our geographic area. We also concluded that the DR1 antigen may play a protective role, as it was detected in 20% of the controls and in none of the MS cases.

[Clinical implications of hepatitis C virus genotype in human immunodeficiency virus infected patients with chronic liver disease]. / Implicaciones clínicas del genotipo del virus de la hepatitis C en infectados por el virus de la inmunodeficiencia humana con hepatopatía crónica.

OBJECTIVE: To know the prevalence of viral genotype in patients infected with hepatitis C virus (HCV) and coinfected with HIV and evaluate its clinical implications. PATIENTS AND METHODS: The genotype of the HVC was studied (INNO-LiPA HCV II, Imnogenetics, Belgium) in 40 patients coinfected with HIV; from 28 of these patients histologic data of chronic hepatitis were available. The most prevalent genotype was analyzed in this type of patients and its associations with different issues: risk behavior, histologic activity of liver disease and viremia level (quantitative PCR, Amplicor HCV, Roche Diagnostics). RESULTS: Genotype 1 was the most prevalent (55%), and subtype 1a predominated (36.3%). In most cases genotypes 1a and 3 were found (65%) and in four cases (10%) there was coinfection with two genotypes. The most common risk behavior was parenteral drug use (PDU) (34 cases), which might account for the higher prevalence of genotypes 1 and 3. A mild hepatic histologic activity was most frequently associated with genotype 3 compared with genotype 1 (63.6% versus 46.6%). The Knodell histologic activity index (HAI) was higher in the four patients with coinfection 1 + 3 versus the remaining patients (11.2 +/- 2.8 versus 7.8 +/- 3.6). The percentage of patients with genotype 1 with a viral load > 10(5) was higher than that of patients with genotype 3 (80% versus 7.6% [4]) (p < 0.05); in the two cases with subtype 1b viremia levels exceeded this limit. CONCLUSIONS: The prevalent HCV genotypes in patients coinfected with HIV in our environment seem to be 1a and 3, which is probably associated with the more common high risk behavior of PDU among these patients. Genotype 3 seems to be associated with a milder histologic liver damage and a lower viral load, and these two characteristics might be related. The HCV genotype should be considered in subjects coinfected with HIV to obtain a better clinical and prognostic evaluation of the chronic liver disease it causes.

Resistance to apoptosis correlates with a highly proliferative phenotype and loss of Fas and CPP32 (caspase-3) expression in human leukemia cells.

Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T-cell leukemia Jurkat is sensitive to Fas-induced apoptosis and to activation-induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas- and TNF-induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub-lines derived from Jurkat and U937 cells. These hp sub-lines were resistant to Fas- and TNF-induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat-derived sub-lines, also of CD3, CD2 and CD59 molecules. The sub-lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub-lines were no longer sensitive to doxorubicin-induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z-VAD-fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias.

[Viremia levels of the chronic hepatitis c virus in HIV infected patients, clinical implications]. / Niveles de viremia del virus de la hepatitis crónica C en infectados por el virus de inmunodeficiencia humana. Implicaciones clínicas.

BACKGROUND: To know the clinical implications of the viremia level and its evolution in time of the hepatitis C virus (HCV) in patients with chronic hepatitis and infected with the Human Immunodeficiency Virus (HIV). PATIENTS AND METHODS: We have studied the viremia level of the HCV in a a 38 patients group with active chronic hepatitis and infected with the HIV, using a quantitative PCR technic (Amplicor HCV, Roche Diagnostics); we had histological data in 33 of these patients. In 20 patients was analyzed the evolution in time of the viremia level with two or three serialized measurements (20 and 10 patients respectively), throughout 7.5 and 14.8 months on the average. We have analyzed some aspects like the risky behaviors associated with transmission, the estimated time from the contagious, the degree of histological damage and the immunitary impairment. RESULTS: We have observed a tendency to present a higher viremia level (logarithmic expression) with longer evolution time from the infection (p = 0.08). The viral load had an inverse relation with the degree of histological fibrosis (Light fibrosis: 4.5 +/- 0.8 log vs Severe fibrosis: 3.7 +/- 0.8 log) (p < 0.01) and a direct relation with the Knodell histological activity index (HAI), only with those patients with a lower fibrosis degree (p < 0.01). There was no relation between the viremia level of the HCV and the degree of immunosuppression measured by the CD4 lymphocyte count, at least in those patients in which it was higher than 200/mm3. We have not observed relations between the viral load and the age or the transaminases level. The evolution in time of the viremia tended to rise from 3.7 +/- 1.3 to 4.5 +/- 0.9 log in 14.8 months on the average, although there were some cases with tendency to decrease. We have not observed relation between its increase/month and the degree of histological damage or the CD4 lymphocyte count. CONCLUSIONS: The viral load of the HCV in HIV-infected patients seems to have an inverse relation with the degree of liver fibrosis and direct relation with the histological activity when the fibrosis light and so it could indirectly inform us about the liver aggression. The degree of immunosuppression measured by the CD4 lymphocyte count, when these are > 200/mm3, doesn't seem to influence the viremia level of the HCV. The evolution of the viral load in time tend to rise although there could be some cases with intermittent or descending evolution, without these tendencies have any clinical implications.

Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way.

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T-leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti-Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase-3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co-incubation with blocking anti-Fas antibodies prevented Fas-induced but not DOX-induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti-Fas or DOX can be prevented by Z-VAD-fmk, a general caspase inhibitor. DEVD-cho, a specific inhibitor of CPP32-like caspases which completely blocks Fas-mediated apoptosis, prevented drug-induced nuclear apoptosis but not cell death. We conclude that: (i) DOX-induced apoptosis in human T-leukemia/lymphoma is Fas-independent and (ii) caspase-3 is responsible of DOX-induced nuclear apoptosis but other Z-VAD-sensitive caspases are implicated in cell death.