Enhanced neutrophil activity is associated with shorter time to tumor progression in glioblastoma patients.

Glioblastoma multiforme (GBM) is a highly malignant tumor with a poor outcome that is often positive for human cytomegalovirus (HCMV). GBM patients often have excessive numbers of neutrophils and macrophages near and within the tumor. Here, we characterized the cytokine patterns in the blood of GBM patients with and without Valganciclovir treatment. Furthermore, we determined whether neutrophil activation is related to HCMV status and patient outcome. Blood samples for analyses of cytokines and growth factors were collected from 42 GBM patients at the time of diagnosis (n = 42) and at weeks 12 and 24 after surgery. Blood neutrophils of 28 GBM patients were examined for CD11b expression. The levels of pro- and anti-inflammatory cytokines and chemokines-including interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-ß1, interferon-γ, interferon-α, tumor necrosis factor α, and monocyte chemoattractant protein (MCP)-1were analyzed with a bead-based flow cytometry assay. During the first six months after surgery, neutrophil activity was increased in 12 patients and was unchanged or decreased in 16. Patients with increased neutrophil activity had enhanced IL-12p70, high grade HCMV and a shorter time to tumor progression (TTP) than patients without or decreased neutrophil activity (median TTP; 5.4 vs. 12 months, 95% confidence interval; 1.6-10 vs. 0.1-0.6, hazard ratio = 3 vs. 0.4, p = 0.004). The levels of IL-12p70 were significantly decreased in Valganciclovir treated patients (n = 22, T 12W vs. T 24W, p = 0.03). In conclusion, our findings suggest that neutrophil activation is an early sign of tumor progression in GBM patients.

Poor survival in glioblastoma patients is associated with early signs of immunosenescence in the CD4 T-cell compartment after surgery.

Patients with glioblastoma multiforme (GBM) are immunosuppressed and have a broad range of immunological defects in both innate and adaptive immune responses. GBMs are frequently infected with human cytomegalovirus (HCMV), a virus capable of causing immunosuppression. In 42 HCMV-positive GBM patients in a clinical trial (VIGAS), we investigated T-cell phenotypes in the blood and assessed their relation to survival. Blood was collected before and 3, 12, and 24 weeks after surgery, and the frequency of T-cell subsets was compared with that in 26 age-matched healthy controls. GBM patients had lower levels of CD3 cells than the controls, but had significantly higher levels of CD4+CD28- T cells before and 3 and 12 weeks after surgery and increased levels of CD4+CD57+ and CD4+CD57+CD28+ T cells at all-time points. These T-cell subsets were associated with both immunosenescence and HCMV infection. GBM patients also had higher levels of γδ T cells at all-times after surgery and lower levels of CD4+CD25+ cells before and 3 weeks after surgery than healthy controls. Overall survival was significantly shorter in patients with higher levels of CD4+CD28- T cells (p = 0.025), CD4+CD57+ T (p = 0.025) cells, and CD4+CD28-CD57+CD28- T cells (p < 0.0004) at 3 weeks after surgery. Our findings indicate that signs of immunosenescence in the CD4+ compartment are associated with poor prognosis in patients with HCMV-positive GBMs and may reflect the HCMV activity in their tumors.

Discordant humoral and cellular immune responses to Cytomegalovirus (CMV) in glioblastoma patients whose tumors are positive for CMV.

Background. Glioblastoma (GBM) is the most common malignant brain tumor in adults and is nearly always fatal. Emerging evidence suggests that human Cytomegalovirus (HCMV) is present in 90-100% of GBMs and that add-on antiviral treatment for HCMV show promise to improve survival. Methods. In a randomized, placebo-controlled trial of valganciclovir in 42 GBM patients, blood samples were collected for analyses of HCMV DNA, RNA, reactivity against HCMV peptides, IgG, and IgM at baseline and at 3, 12, and 24 weeks of treatment. Results. All 42 tumors were positive for HCMV protein. All patients examined had at least one blood sample positive for HCMV DNA, 63% were HCMV RNA positive, and 21% were IgM positive. However, 29% of GBM patients were IgG negative for HCMV. Five of these samples were positive in an enzyme-linked immunosorbent assay (ELISA) that used antigens derived from a clinical isolate. Blood T cells from 11 of 13 (85%) HCMV IgG-negative GBM patients reacted against HCMV peptides. Valganciclovir did not affect IgG titers, DNA, or RNA levels of the HCMV immediate early (HCMV IE) gene in blood. Conclusion. In GBM patients, HCMV activity is higher than in healthy controls and serology is a poor test to define previous or active HCMV infection in these patients.

Effects of valganciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma: a randomized, double-blind, hypothesis-generating study.

Cytomegalovirus is highly prevalent in glioblastomas. In 2006, we initiated a randomized, double-blind, placebo-controlled, hypothesis-generating study to examine the safety and potential efficacy of Valganciclovir as an add-on therapy for glioblastoma. Forty-two glioblastoma patients were randomized in double-blind fashion to receive Valganciclovir or placebo in addition to standard therapy for 6 months. Magnetic resonance images were obtained before and immediately and 3 and 6 months after surgery to evaluate treatment efficacy by measuring contrast enhancing tumor volume (primary end point). Survival data were analyzed for patients and controls in explorative analyses to aid the design of future randomized trials. Trends but no significant differences were observed in tumor volumes in Valganciclovir and placebo patients at 3 (3.58 vs. 7.44 cm3, respectively, p = 0.2881) and 6 (3.31 vs. 13.75 cm3, p = 0.2120) months. Median overall survival (OS) was similar in both groups (17.9 vs. 17.4 months, p = 0.430). Patients could take Valganciclovir for compassionate use after the study phase. Explorative analyses showed an OS of 24.1 months (95% CI, 17.4-40.3) in patients receiving >6 months of Valganciclovir (Val > 6M) versus 13.1 months (95% CI, 7.9-17.7, p < 0.0001) in patients receiving Valganciclovir for 0 or <6 months, and 13.7 months (95% CI, 6.9-17.3, p = 0.0031) in contemporary controls. OS at 4 years was 27.3% in Val>6M patients versus 5.9% in controls (p = 0.0466). Prolonged OS in Val>6M patients suggest that future randomized trials are warranted and should evaluate whether continuous antiviral treatment can improve outcome in glioblastoma patients.

High TNF-alpha and IL-8 levels predict low blood dendritic cell counts in primary cytomegalovirus infection.

BACKGROUND: In vitro studies suggest that human cytomegalovirus (CMV) modulates the functions of dendritic cells (DCs). However, there are limited data on DC homeostasis in CMV-infected patients. OBJECTIVES: The aim of this study was to characterize circulating DCs and plasma cytokine levels in immunocompetent patients with primary, symptomatic CMV infections. STUDY DESIGN: The study population consisted of 14 patients suffering of CMV mononucleosis and 14 healthy volunteers (11 CMV-seropositive and 3 CMV-seronegative subjects) included as controls. Peripheral blood mononuclear cells were isolated and used to characterize DCs and to quantify CMV in the blood. Plasma levels of pro-inflammatory and anti-inflammatory cytokines were also measured. RESULTS: We observed that patients who were developing CMV mononucleosis presented lower myeloid and plasmacytoid DC counts in peripheral blood compared with healthy controls. We also noted elevated levels of inflammatory mediators, of which tumor necrosis factor-α (TNF-α)-which activates DCs and endothelial cells-was the highest. Notably, the decrease in blood DCs correlated with high TNF-α and IL-8 levels by a hyperbolic function. CONCLUSIONS: Our results suggest that increased levels of inflammatory factors facilitate alterations in DC homeostasis during primary CMV infection, which may contribute to viral-induced modulation of host immunity.

The novel anti-rheumatic compound Rabeximod impairs differentiation and function of human pro-inflammatory dendritic cells and macrophages.

Rabeximod (9-chloro-2,3-dimethyl-6-(N,N-dimethylaminoethylamino-2-oxoethyl)-6H-indolo[2,3-b]quinoxaline) is a synthetic compound that is currently being developed for the treatment of rheumatoid arthritis (RA). Here, we investigated the effects of Rabeximod on the functionality of human antigen-presenting cells (APCs) of myeloid origin. Different subsets of professional APCs were generated from human monocytes in vitro and simultaneously treated with different doses of Rabeximod. Although Rabeximod had no effect on the differentiation of monocytes into anti-inflammatory macrophages (AI-Mϕs), this compound impaired monocyte differentiation into monocyte-derived dendritic cells (MDCs) and pro-inflammatory allostimulated macrophages (Allo-Mϕs). MDCs that were treated with Rabeximod resulted in a significant decrease in their ability to pinocytose antigens, while no effect was exerted by the drug on the ability of Allo-Mϕs and AI-Mϕs to phagocytose. Furthermore, we observed a significant reduction in the allostimulatory ability of MDCs and Allo-Mϕs after treatment with Rabeximod, although this compound did not affect the low immunostimulatory capacity of AI-Mϕs. Conversely, the effect of Rabeximod in influencing cytokine secretion by APCs appeared to be limited. In conclusion, Rabeximod impairs differentiation of monocytes into different pro-inflammatory APCs, leading to impaired immunostimulatory abilities of these cells. Our observations shed light on the cellular mode of action and the immunomodulatory effect of Rabeximod.

Generalized Wegener's granulomatosis in an immunocompetent adult after cytomegalovirus mononucleosis and bacterial urinary tract infection.

Human cytomegalovirus (HCMV) is frequently detected in autoimmune diseases, but its role in such disorders is poorly understood. Herein we describe the case of a young woman who developed generalized Wegener's granulomatosis (WG) after HCMV mononucleosis and urinary tract infection. During mononucleosis, the patient had extraordinarily high plasma levels of proinflammatory cytokines such as interleukin-5 and lymphotoxin alpha, autoantibodies, and a higher blood level of viral DNA than were found in other immunocompetent patients infected with HCMV or healthy controls. Active HCMV replication was detected after the onset of vasculitis, and HCMV genomes or antigens were found in blood, urine, and inflammatory lesions on the kidney. Thus, HCMV may have triggered or exacerbated inflammation and autoimmunity in this case of WG.

Effect of combined T- and B-cell depletion of allogeneic HLA-mismatched bone marrow graft on the magnitude and kinetics of Epstein-Barr virus load in the peripheral blood of bone marrow transplant recipients.

Recipients of T-cell-depleted bone marrow (BM) transplants (BMT) frequently develop Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) preceded by a rapid and prominent increase of EBV load in the peripheral blood. The level of this increase positively correlates with the incidence of PTLD. Using a semiquantitative PCR assay we compared the blood levels of EBV-DNA in patients transplanted with either T-cell or T- and B-cell-depleted human leukocyte antigen (HLA)-mismatched BM grafts. Combined T- and B-cell depletion correlated with significantly lower maximal levels of EBV load, which were reached with slower kinetics. These data indicate that B-cell depletion of BM can be used for prophylaxis of PTLD in BM transplant recipients and can affect the long-term balance between EBV and its host.

Immunologic properties of human fetal mesenchymal stem cells.

OBJECTIVE: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow and fetal liver. We investigated the immunologic properties of undifferentiated and differentiated human fetal MSCs. STUDY DESIGN: Expression of HLA class I and II was investigated by flow cytometry and Western blot on undifferentiated fetal MSC and after in vitro differentiation to adipocytes and osteocytes. Alloreactivity was studied after adding fetal MSCs to allogeneic lymphocytes in mixed lymphocyte cultures. RESULTS: Fetal MSCs expressed HLA class I but not HLA class II. The presence of interferon gamma (IFN-gamma) in the growth medium for 2 days initiated the intracellular synthesis of HLA class II, but 7 days of exposure was required for cell surface expression. Neither undifferentiated nor differentiated fetal MSCs induced proliferation of allogenic lymphocytes. Fetal MSCs treated with IFN-gamma suppressed alloreactive lymphocytes. CONCLUSION: Undifferentiated and differentiated fetal MSCs do not elicit alloreactive lymphocyte proliferation. The results suggest that fetal MSCs have potentials for allogenic transplantation.

Functional characterization of human mesenchymal stem cell-derived adipocytes.

The function of adipocytes derived from human mesenchymal stem cells (hMSC) was investigated for the first time in hMSC from fetal liver (FL) and adult bone marrow (BM) and compared with preadipocytes from human subcutaneous adipose tissue differentiated according to adipocyte-specific protocols. FL- and BM-derived adipocytes displayed both morphological and functional characteristics of mature adipocytes including specific intracellular signaling pathways for tumor necrosis factor-alpha, catecholamine-regulated lipolysis as well as secretion of adiponectin and leptin. Similar to differentiated preadipocytes, hMSC adipocytes displayed lipolytic effects mediated by beta-adrenoceptors and antilipolytic effects mediated by the alpha 2A-adrenoceptor (alpha 2A-AR) and expressed proteins with a pivotal role in human lipolysis, including beta 2-AR, alpha 2A-AR, and hormone-sensitive lipase. We conclude that hMSC-derived adipocytes are morphologically and functionally similar to preadipocytes and display an intact lipolytic signaling pathway and endocrine function. These systems could be of great value in adipocyte research as a renewable source of adipocytes.

HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.

OBJECTIVE: Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. METHODS AND RESULTS: Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated with IFN-gamma. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-gamma for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-gamma. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-gamma. CONCLUSIONS: Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals.