An investigation on the relationship between the occurrence of CMV DNAemia and the development of invasive aspergillosis in the allogeneic stem cell transplantation setting.

Cytomegalovirus (CMV) infection might increase the risk of fungal superinfection in allogeneic stem cell transplant patients. The potential association between the occurrence of CMV DNAemia and the development of invasive aspergillosis in this clinical setting was investigated. The current retrospective observational study included 167 patients undergoing T cell-replete allogeneic stem cell transplantation. Virological monitoring of active CMV infection was performed by the pp65 antigenemia assay and/or by a plasma real-time PCR assay. A total of 109 out of 167 patients developed CMV DNAemia. Twenty-three patients had proven (n = 4) or probable (n = 19) invasive aspergillosis. The occurrence of CMV DNAemia was not significantly associated with the subsequent development of invasive aspergillosis (P = 0.38). Overall, the duration of the episodes of active CMV infection and the peak level of CMV DNAemia within the episodes were comparable, irrespective of whether invasive aspergillosis developed subsequently or not (P = 0.99; P = 0.70, respectively). Peak CMV DNA load in patients with proven or probable invasive aspergillosis who died was higher (median, 5,461 copies/ml) than that in those who survived (median 1,179 copies/ml) (P = 0.41). The data argue against the existence of an association between the occurrence of CMV DNAemia and the development of invasive aspergillosis, however, CMV replication, particularly at high levels, might aggravate the prognosis of this disease.

Evaluation of cytomegalovirus (CMV)-specific T-cell immunity for the assessment of the risk of active CMV infection in non-immunosuppressed surgical and trauma intensive care unit patients.

The current study was designed to assess the predictive value of the evaluation of cytomegalovirus (CMV)-specific T-cell immunity early following admission to the intensive care unit for inferring the risk of active CMV infection in non-immunosuppressed surgical and trauma patients. A total of 31 CMV-seropositive patients were included. Patients were screened for the presence of CMV DNA in plasma and in tracheal aspirates by real-time PCR. Enumeration of CMV pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T cells was performed by flow cytometry for intracellular cytokine staining. Virological and immunological monitoring was conducted once or twice a week. Active CMV infection occurred in 17 out of 31 patients. Undetectable levels of pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T-cell subsets cells were observed in 10 patients who developed active CMV infection and in one who did not (at a median of 2 days following ICU admission). Peak CMV DNA loads in both tracheal aspirates and plasma were substantially higher (P = 0.018 and P = 0.091, respectively) in patients with undetectable IFN-γ T-cell responses than in patients with detectable responses. The expansion of both CMV-specific T-cell subsets following detection of active CMV infection was demonstrated in 9 out of 14 patients with active CMV infection. In conclusion, the evaluation of CMV pp65 and IE-1-specific IFN-γ-producing CD8(+) and CD4(+) T cells early following ICU admission may allow the identification of patients most at risk of either having or developing an episode of active CMV infection, particularly those associated with high-level virus replication.

Performance of the QuantiFERON-cytomegalovirus (CMV) assay for detection and estimation of the magnitude and functionality of the CMV-specific gamma interferon-producing CD8(+) T-cell response in allogeneic stem cell transplant recipients.

The performance of the QuantiFERON-cytomegalovirus (CMV) assay was compared to that of a flow cytometry intracellular cytokine staining (ICS) method for the detection of CMV-specific gamma interferon (IFN-γ)-producing CD8(+) T-cell responses in allogeneic stem cell transplant (allo-SCT) recipients and for estimations of their magnitude and functionality. A total of 90 whole-blood specimens from 23 allo-SCT recipients was analyzed by both methods. Overall, the percentage of specimens that yielded concordant results by both methods was 68.8% (κ = 0.691; 95% confidence interval [CI], 0.548 to 0.835), and the sensitivity of the QuantiFERON-CMV assay for the detection of positive IFN-γ T-cell responses (>0.2 IU/ml), taking the ICS method as the reference, was 76.3%. The magnitude of IFN-γ-producing CD8(+) T-cell responses to CMV-specific peptides measured with the QuantiFERON-CMV assay correlated significantly (σ = 0.695; P = <0.001) with that of the total IFN-γ-producing CD8(+) T cells and dual-functional (IFN-γ/tumor necrosis factor alpha [TNF-α] [σ = 0.652; P = <0.001] and IFN-γ/CD107a [σ = 0.690; P = <0.001]) and trifunctional (IFN-γ/TNF-α/CD107a [σ = 0.679; P = >0.001]) CMV-specific CD8(+) T-cell responses, as quantitated by ICS. In summary, the data indicated that the QuantiFERON-CMV assay is less sensitive than the ICS method for the detection of CMV-specific IFN-γ-producing CD8(+) T-cell responses in the allo-SCT setting. Nevertheless, it allowed the estimation of the total and polyfunctional CMV-specific IFN-γ-producing CD8(+) T-cell responses in specimens that tested positive by both methods.

Functional profile of cytomegalovirus (CMV)-specific CD8+ T cells and kinetics of NKG2C+ NK cells associated with the resolution of CMV DNAemia in allogeneic stem cell transplant recipients.

Immune mechanisms involved in control of cytomegalovirus (CMV) infection in the allogeneic stem cell transplantation setting have not been fully disclosed. CMV pp65 and IE-1-specific CD8(+) T cells expressing IFN-γ, TNF-α, and CD107a, alone or in combination, and NKG2C(+) NK cells were prospectively enumerated during 13 episodes of CMV DNAemia. The expansion of monofunctional and polyfunctional CD8(+) T cells was associated with CMV DNAemia clearance. The size and functional diversity of the expanding CD8(+) T-cell population was greater in self-resolved episodes than in episodes treated with antivirals. These differences were related to the magnitude of expansion of cognate antigen IFN-γ CD4(+) T cells. The resolution of CMV DNAemia was associated frequently with a marked expansion of both CD56(dim) /CD16(+) NK cells and NKG2C(+) CD56(bright) /CD16(-) NK cells. The data lend support to the role of polyfunctional CD8(+) T cells in controlling CMV replication in the allogeneic stem cell transplantation setting, and suggest that NKG2C(+) NK cells may be involved critically in the resolution of CMV DNAemia episodes.

Immunological insights into the pathogenesis of active CMV infection in non-immunosuppressed critically ill patients.

Dissociation of cytomegalovirus (CMV) DNA loads between the lower respiratory tract and blood, with high levels in the former compartment and low or undetectable levels in the latter, commonly occurs during active CMV infection in critically ill patients despite the presence of high frequencies of CMV-specific IFN-γ-producing CD8(+) and CD4(+) T cells in blood. Data presented in this case report suggest that inter-compartmental differences in interleukin-10 (IL-10) levels may, in part, explain the pathobiology of this phenomenon. In the absence of ganciclovir treatment, a significant correlation was observed between IL-10 levels and CMV DNA loads in lower respiratory tract specimens (P = 0.016), but not in plasma samples (P = 0.46). Comparable data were obtained during the course of active CMV infection episodes that developed in six CMV-seropositive critically ill patients with no canonical immunosuppression. The presence of higher levels of IL-10 in the lower respiratory tract than in plasma may result in increased impairment of CMV-specific T-cell effector responses in the lung compared to the systemic compartment, facilitating local CMV replication.

Dynamics of cytomegalovirus (CMV) plasma DNAemia in initial and recurrent episodes of active CMV infection in the allogeneic stem cell transplantation setting: implications for designing preemptive antiviral therapy strategies.

Preemptive antiviral therapy strategies for active cytomegalovirus (CMV) infection occurring in allogeneic stem cell transplant recipients should be optimized to avoid overtreatment. The current study was aimed at determining whether the analysis of the kinetics of CMV DNA load in plasma may provide useful information for the therapeutic management of active CMV infection in this setting. A total of 59 consecutive patients were included in the study, of which 40 (67.8%) developed 1 (n = 21) or more (n = 19) episodes of CMV DNAemia. The need for antiviral therapy for initial or secondary episodes of CMV DNAemia could not be predicted on the basis of the CMV DNA load value in the first plasma testing positive by polymerase chain reaction (PCR). In contrast, in the absence of antiviral therapy, an increase of ≥3-fold between the baseline CMV DNA load and that measured a median of 6 days later discriminated between initial episodes eventually requiring antiviral treatment and those resolving spontaneously (sensitivity, 76.4%; specificity, 89.4%; positive predictive value, 86.6%; negative predictive value, 80.9%). This criterion was not useful for identifying recurrent episodes of CMV DNAemia that required antiviral therapy. The CMV doubling time and CMV DNA loads at the time of the first positive PCR and at initiation of preemptive therapy did not differ significantly between episodes that responded immediately to antiviral therapy from those showing a delayed response. The analysis of the dynamics of CMV DNA load in plasma in the absence of antiviral therapy allowed early recognition of episodes of CMV DNAemia that eventually needed to be treated, but did not permit prediction of the kinetics of CMV DNA clearance in response to antiviral therapy.

White blood cell subtypes after STEMI: temporal evolution, association with cardiovascular magnetic resonance--derived infarct size and impact on outcome.

The evolution of white blood cells after ST elevation myocardial infarction (STEMI) and their association with infarct size and major adverse cardiac events (MACE) remains unclear. Two hundred eleven patients underwent CMR after STEMI. Infarct mass (grams) was determined. Neutrophil, lymphocyte, and monocyte counts (×1,000 cells/ml) were measured upon arrival and at 12, 24, 48, 72, and 96 h. Patients with large infarctions (3rd tertile ≥ 28.5 g vs. 1st and 2nd tertiles < 28.5 g) showed a larger neutrophil count at 12 h (14.8 ± 4.8 vs. 11.4 ± 3.3, p < 0.0001) and an increased monocyte count (maximum at 24 h (0.65[0.50-0.91] vs. 0.55[0.42-0.71], p = 0.004)) but no difference in lymphocyte count. Neutrophil count at 12 h independently predicted large infarctions (OR 1.14, 95%CI [1.04-1.26], p = 0.008). During follow-up (median 504 days), 25 MACE occurred. Neutrophil count at 96 h independently predicted MACE (HR 1.2, 95%CI [1.1-1.4], p = 0.003). Large infarctions show a marked neutrophil peak and an increasing monocyte count. Neutrophil count independently predicts large infarctions and MACE.

Kinetics of cytomegalovirus (CMV) pp65 and IE-1-specific IFNgamma CD8+ and CD4+ T cells during episodes of viral DNAemia in allogeneic stem cell transplant recipients: potential implications for the management of active CMV infection.

The dynamics of CMV pp65 and IE-1-specific IFNgamma-producing CD8(+) (IFNgamma CD8(+)) and CD4(+) (IFNgamma CD4(+)) T cells and CMV DNAemia were assessed in 19 pre-emptively treated episodes of active CMV infection. Peripheral counts of IFNgamma CD8(+) and IFNgamma CD4(+) T cells inversely correlated with CMV DNAemia levels (P = <0.001 and P = 0.003, respectively). A threshold value of 1.3 cells/microl predicting CMV DNAemia clearance was established for IFNgamma CD8(+) T cells (PPV, 100%; NPV, 93%) and for IFNgamma CD4(+) T cells (PPV, 100%; NPV, 75%). Undetectable T-cell responses were usually observed at the time of initiation of pre-emptive therapy. Either a rapid (within 7 days) or a delayed (median 31 days) expansion of both T-cell populations concomitant with CMV DNAemia clearance was observed in 5 and 8 episodes, respectively. An inconsistent or a lack of expansion of both T-cell subsets was related to a persistent CMV DNAemia. Robust and maintained CMV-specific T-cell responses after CMV DNAemia clearance and cessation of antiviral therapy were associated with a null incidence of relapsing infections at least during the following month. Data obtained in the present study may be helpful in the design of therapeutic strategies for the management of active CMV infections in the allo-SCT recipient.

Virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit.

Cytomegalovirus (CMV) reactivation occurs frequently in critically ill patients. The natural course of CMV infection and the interaction between CMV and the adaptive immune system in this setting remain poorly defined. Fifty-three CMV-seropositive patients in a surgical and trauma intensive care unit were included in this study. The CMV DNA load in tracheal aspirates (TA) and plasma (PL) was monitored by qPCR. CMV-specific T-cell immunity was assessed by intracellular cytokine staining. Plasma TNF-alpha levels were determined by ELISA. CMV reactivation occurred in 39.7% of patients (23% had CMV DNA detected only in TA). The analysis of TA allowed an earlier diagnosis in 28% of patients. Clearance of CMV DNAemia preceded that of CMV DNA in TA in some episodes. Peak CMV DNA levels were significantly higher in TA than in PL (P = 0.02). CMV reactivation developed in the presence of CMV-specific T cells. Termination of CMV reactivation was associated with an expansion of functional CMV-specific T cells. Plasma levels of TNF-alpha did not allow for the prediction of the occurrence of CMV reactivation. CMV-specific T-cell immunity is preserved in most critically ill patients experiencing CMV reactivation. Analysis of respiratory specimens is imperative for an optimal monitoring of CMV reactivation in this setting.

An assessment of the effect of human herpesvirus-6 replication on active cytomegalovirus infection after allogeneic stem cell transplantation.

Human herpesvirus-6 (HHV-6) may enhance cytomegalovirus (CMV) replication in allogeneic stem cell transplant (allo-SCT) recipients either through direct or indirect mechanisms. Definitive evidence supporting this hypothesis are lacking. We investigated the effect of HHV-6 replication on active CMV infection in 68 allo-SCT recipients. Analysis of plasma HHV-6 and CMV DNAemia was performed by real-time PCR. Enumeration of pp65 and IE-1 CMV-specific IFNgamma CD8(+) and CD4(+)T cells was performed by intracellular cytokine staining. HHV-6 DNAemia occurred in 39.8% of patients, and was significantly associated with subsequent CMV DNAemia in univariate (P=.01), but not in multivariate analysis (P=.65). The peak of HHV-6 DNAemia was not predictive of the development of CMV DNAemia. Timing and kinetics of active CMV infection were comparable in patients either with or without a preceding episode of HHV-6 DNAemia. The occurrence of HHV-6 DNAemia had no impact on CMV-specific T cell immunity reconstitution early after transplant. The receipt of a graft from an HLA-mismatched donor was independently associated with HHV-6 (P=.009) and CMV reactivation (P=.04). The data favor the hypothesis that a state of severe immunosuppression leads to HHV-6 and CMV coactivation, but argue against a role of HHV-6 in predisposing to the development of CMV DNAemia or influencing the course of active CMV infection.

Analysis of chronic lymphotic leukemia transcriptomic profile: differences between molecular subgroups.

B cell chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder with a variable clinical course. Patients with unmutated IgV(H) gene show a shorter progression-free and overall survival than patients with immunoglobulin heavy chain variable regions (IgV(H)) gene mutated. In addition, BCL6 mutations identify a subgroup of patients with high risk of progression. Gene expression was analysed in 36 early-stage patients using high-density microarrays. Around 150 genes differentially expressed were found according to IgV(H) mutations, whereas no difference was found according to BCL6 mutations. Functional profiling methods allowed us to distinguish KEGG and gene ontology terms showing coordinated gene expression changes across subgroups of CLL. We validated a set of differentially expressed genes according to IgV(H) status, scoring them as putative prognostic markers in CLL. Among them, CRY1, LPL, CD82 and DUSP22 are the ones with at least equal or superior performance to ZAP70 which is actually the most used surrogate marker of IgV(H) status.

Uncontrolled immune response in acute myocardial infarction: unraveling the thread.

Recently, the theory that hyperinflammation is the body's primary response to potent stimulus has been challenged. Indeed, a deregulation of the immune system could be the cause of multiple organ failure. So far, clinicians have focused on the last steps of the inflammatory cascade. However, little attention has been paid to lymphocytes, which play an important role as strategists of the inflammatory response. Experimental evidence suggests a crucial role of T lymphocytes in the pathophysiology of atherosclerosis and acute myocardial infarction (AMI). In summary, from the bottom of an imaginary inverted pyramid, a few regulatory T-cells control the upper parts represented by the wide spectrum of the inflammatory cascade. In AMI, a loss of regulation of the inflammatory system occurs in patients with a decreased activity of regulatory T-cells. As a consequence, aggressive T-cells boost and anti-inflammatory T-cells drop. A pleiotropic proinflammatory imbalance with damaging effects in terms of left ventricular performance and patient outcome is the result of this uncontrolled immune response. It is needed to unravel the thread of the inflammatory cells to better understand the pathophysiology as well as to open innovative therapeutic options in AMI.

Bortezomib induces different apoptotic rates in B-CLL cells according to IgVH and BCL-6 mutations.

BACKGROUND: B-cell chronic lymphocytic leukemia (B-CLL) is a remarkably heterogeneous disorder. Some patients have an indolent disease whereas others undergo a more agressive presentation needing treatment. New therapeutics approaches are necessary for the treatment of B-CLL. Bortezomib (Btz), is a proteasome inhibitor, currently undergoing clinical trials whose function, at least in part, by stabilizing the IkappaBalpha protein and inhibiting NFkappaB activation. OBJECTIVE: The objective of this work was to study the effects of Btz on isolated human B-CLL cells, in vitro, and to correlate the differential rates of apoptosis induction with biological variables. MATERIAL AND METHODS: 31 B-CLL samples, from patients in stage A of Binet were used for this study, and the apoptotic effect of Btz on these cells was measured. RESULTS: Our data show that Btz treatment of B-CLL cells induces apoptosis in a time and dose-dependent manner. The apoptosis induction is mediated in part by inhibition of NFkappaB and is dependent on caspases activation. Interesting, in IgVH mutated cells, Btz have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status. CONCLUSIONS: Btz is a promising pharmacologic agent for the treatment of B-CLL, but its efficacy seems to be related to IgVH and BCL-6 mutational status, therefore, it could be interesting to further investigate the mechanisms involved in the different behavior of the cells in response to apoptosis induction by this drug.

Human cytomegalovirus (HCMV)-specific CD4+ T lymphocyte response in AIDS patients with no past or current HCMV disease following HAART.

BACKGROUND: The incidence of Human Cytomegalovirus (HCMV) end-organ disease has dramatically decreased since the implementation of highly active antiretroviral therapies (HAARTs), but the precise immune mechanism whereby HCMV is controlled remains to be elucidated. OBJECTIVES: To investigate the effect of (HAART) on CD4+ T-cell immunity to HCMV in AIDS patients with no past or current HCMV disease. STUDY DESIGN: Seventeen patients were prospectively examined for CD4+ (CD45RO+ and CD45 RA+) T-cell counts (flow cytometry), HIV RNA load (Amplicor HIV test), HCMV leukoDNAemia and HCMV DNA in urine (nested PCR), lymphoproliferative response (LPR) to HCMV, phytohemagglutinin (PHA) and purified protein derived from Mycobacterium tuberculosis (PPD) by measurement of 5-bromo-2'-deoxyuridine incorporation to DNA (ELISA) and cytokine secretion (IFN-gamma, IL-4 and IL-10) by HCMV-stimulated peripheral blood mononuclear cell (PBMC) cultures (ELISA). RESULTS: Fifteen patients responded favorably to HAART (virologically, immunologically, or both). Of these, six patients presented LPR to HCMV at least once during follow-up, whereas most displayed detectable LPRs to PHA. IFN-gamma was detected at least once in supernatants of HCMV-stimulated PBMC cultures from 14 of the 17 patients. All but one patient tested negative for HCMV leukoDNAemia and HCMV DNA in urine, and none developed HCMV disease during the observation period. CONCLUSIONS: Control of HCMV replication and the absence of HCMV disease are not consistently associated with recovery and/or maintenance of LPR to HCMV in AIDS patients under HAART and with no prior HCMV disease. Whether detection of IFN-gamma by PBMCs upon HCMV antigenic stimulation may serve as a surrogate marker for protection against HCMV disease requires further investigation.

Peripheral T-cell lymphoma associated consecutively with hemophagocytic lymphohistiocytosis and hypereosinophilic syndrome.

Both hemophagocytic lymphohistiocytosis and hypereosinophilic syndrome have been associated with hematologic neoplasms and are respectively related to an overproduction of the cytokines Thelper 1 (Th1) and Th2 by tumor cells or reactive cells. To our knowledge, this is the first time a case of a peripheral T-cell lymphoma consecutively associated with both paraneoplastic conditions has been reported. Importantly, in this case when the lymphoma exclusively involved the bone marrow, severe paraneoplastic systemic damage, a CD8+ suppressor/cytotoxic phenotype and a hypereosinophilia not related to high levels of interleukin (IL)-5 was found. Interestingly, progression of the lymphoma coincided with an increase in the serum levels of several Th2 cytokines and IL-2, a decrease in tumor necrosis factor and granulocyte-macrophage colony-stimulating factor levels and the onset of a hypereosinophilic syndrome.

Reconstitution of lymphocyte populations and cytomegalovirus viremia or disease after allogeneic peripheral blood stem cell transplantation.

Early reconstitution of lymphoid populations was monitored by immunophenotyping in 57 allogeneic peripheral blood stem cell (allo-PBSC) transplant patients either with or without cytomegalovirus (CMV) viremia or disease. Cell counts for total lymphocytes and CD4(+) T cells above the percentile 60th at day 14 postransplant were associated significantly with CMV viremia-free survival within 120 days after transplant. Recovery of total lymphocyte, CD3(+), and CD8(+) T-cell counts proceeded at a more rapid rate in CMV viremic patients than in nonviremic patients, irrespective of whether preemptive treatment with ganciclovir had been prescribed. Significant expansion of CD8(+) and CD8(+) CD57(+) T-cell subsets was associated with recovery from viremia and no progression to CMV disease. Immunophenotyping may provide useful information for the clinical management of CMV infection in allo-PBSC transplant recipients.

Genomic abnormalities acquired in the blastic transformation of splenic marginal zone B-cell lymphoma.

Among 20 cases of typical splenic marginal zone lymphoma (SMZL), two cases had blastic transformation. The genetic mechanisms underlying the morphologic transformation were investigated by comparing genetic changes in initial and blastic phases. A complex karyotype including trisomy of 3q and genomic gain of 17q22-q24 was seen in both cases at diagnosis. However, the extra copy of 3q was lost during the transformation process in both tumors. Additionally, the Karpas 1718 cell line, which was derived from a patient with transformed SMZL and carried a trisomy of 3q, also evidenced the spontaneous loss of the extra 3q during the culturing process. Other acquired abnormalities observed exclusively in the transformation phase included amplification and/or translocation of bands 7p22-q22 and 19p13. These findings suggest that the loss of + 3q and the acquisition of other genomic imbalances may represent unique markers for the transformation process of SMZL. We hypothesize that the trisomy of 3q may correlate with the indolent nature of SMZL, and that the loss of this acquired abnormality leads to or accompanies the development of blastic tumors.

Targeted oligonucleotide delivery in human lymphoma cell lines using a polyethyleneimine based immunopolyplex.

The efficacy of antisense gene therapy depends on efficient delivery of oligonucleotides into targeted cells. Although polyethyleneimine based polyplexes have been reported as good transfection reagents, they are inefficient in lymphoid cell transfection. We report the construction of an immunopolyplex, a targeted nonviral vector based on a polyplex backbone and its application for oligonucleotide transfer on human lymphoma cell lines. The salient characteristic of immunopolyplex lies in the possibility of easily replacing the targeting element (antibody), leaving the polyplex backbone intact. Furthermore, a study was made of the influence of endocytosis inhibitors on immunopolyplex activity. The capacity of the immunopolyplex for oligonucleotide transfer was studied in vitro using FITC-labeled oligonucleotides as fluorescent reporters, an anti-CD3 antibody as targeting element, and a CD3-positive cell line (Jurkat) as a target cell line, in the absence and presence of endocytosis inhibitors. A CD3-negative Jurkat-derived mutant cell line (J.RT3-T3.5) was used as control. A nine-fold increase in fluorescence in the CD3-positive above that in the CD3-negative cell line was observed, indicating that oligonucleotide transfer is mainly specific. Low fluorescence values were obtained in the presence of endocytosis inhibitor or with untargeted polyplexes. We conclude that the immunopolyplex is a good nonviral vector for specific oligonucleotide delivery. Abolition of immunopolyplex activity in the presence of endocytosis inhibitor suggests that targeted oligonucleotide transfer occurs through an endocytic pathway.