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.

Effect of pharmacological treatment for urinary incontinence in the elderly and frail elderly: A systematic review.

AIM: The prevalence and severity of urinary incontinence (UI) increase with age and comorbidity. The benefits of pharmacotherapy for UI in the elderly are questionable. The aim of the present study was to systematically review the efficacy of pharmacological treatment for UI in the elderly and frail elderly. METHODS: We searched PubMed, EMBASE, Cochrane library and Cinahl databases through October 2013 to identify prospective controlled trials that evaluated pharmacological treatment for UI in persons aged ≥65 years. Elderly persons living in nursing homes were regarded as frail elderly. Outcomes were urinary leakage, quality of life and adverse events. RESULTS: We screened 1038 abstracts and assessed 309 full-text articles. We identified 13 trials of high or moderate quality; 11 evaluated anticholinergic drugs and two evaluated duloxetine. Oxybutynin, the only drug studied in the frail elderly population, had no effect on urinary leakage or quality of life in elderly with urgency UI (UUI). Seven trials evaluated the effects of darifenacin, fesoterodine, solifenacin, tolterodine or trospium. Urinary leakage decreased (standard mean difference: -0.24, 95% confidence interval -0.32-0.15), corresponding to a reduction of half a leakage per 24 h. Common side-effects of treatment were dry mouth and constipation. Data were insufficient for evaluation of the effect on quality of life or cognition. The evidence was insufficient to evaluate the effects of duloxetine. No eligible studies on mirabegron and estrogen were found. CONCLUSIONS: Anticholinergics have a small, but significant, effect on urinary leakage in older adults with UUI. Treatment with drugs for UUI in the frail elderly is not evidence based.

Surgery for urinary incontinence in women 65 years and older: a systematic review.

INTRODUCTION AND HYPOTHESIS: Urinary incontinence (UI) is common among the elderly, but the literature is sparse on the surgical treatment of UI among the elderly. This systematic review aims to assess the effectiveness of surgical interventions as treatment for urinary incontinence in the elderly population ≥65 years of age. METHODS: Randomized controlled trials (RCT) and prospective nonrandomized studies (NRS) were included. The databases PubMed (NLM), EMBASE (Elsevier), Cochrane Library (Wiley), and Cinahl (EBSCO) were searched for the period 1966 up to October 2013. The population had to be ≥65 years of age and had to have undergone urethral sling procedures, periurethral injection of bulking agents, artificial urinary sphincter surgery, bladder injection treatment with onabotulinumtoxin A or sacral neuromodulation treatment. Eligible outcomes were episodes of incontinence/urine leakage, adverse events, and quality of life. The studies included had to be at a moderate or low risk of bias. Mean difference (MD) or standard mean difference (SMD)as well as risk difference (RD) and the 95% CI were calculated. RESULTS: Five studies--all on the suburethral sling procedure in women-- that fulfilled the inclusion criteria were identified. The proportion of patients reporting persistent SUI after surgery ranged from 5.2 to 17.6%. One study evaluating quality of life (QoL) showed a significant improvement after surgery. The complication rates varied between 1 and 26%, mainly bladder perforation, bladder emptying disturbances, and de novo urge. CONCLUSION: The suburethral sling procedure improves continence as well as QoL among elderly women with SUI; however, evidence is limited.

Neuroprotective effects of human spinal cord-derived neural precursor cells after transplantation to the injured spinal cord.

To validate human neural precursor cells (NPCs) as potential donor cells for transplantation therapy after spinal cord injury (SCI), we investigated the effect of NPCs, transplanted as neurospheres, in two different rat SCI models. Human spinal cord-derived NPCs (SC-NPCs) transplanted 9 days after spinal contusion injury enhanced hindlimb recovery, assessed by the BBB locomotor test. In spinal compression injuries, SC-NPCs transplanted immediately or after 1 week, but not 7 weeks after injury, significantly improved hindlimb recovery compared to controls. We could not detect signs of mechanical allodynia in transplanted rats. Four months after transplantation, we found more human cells in the host spinal cord than were transplanted, irrespective of the time of transplantation. There was no focal tumor growth. In all groups the vast majority of NPCs differentiated into astrocytes. Importantly, the number of surviving rat spinal cord neurons was highest in groups transplanted acutely and subacutely, which also showed the best hindlimb function. This suggests that transplanted SC-NPCs improve the functional outcome by a neuroprotective effect. We conclude that SC-NPCs reliably enhance the functional outcome after SCI if transplanted acutely or subacutely, without causing allodynia. This therapeutic effect is mainly the consequence of a neuroprotective effect of the SC-NPCs.

Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target?

Neuroblastoma is the most common and deadly tumor of childhood, where new therapy options for patients with high-risk disease are highly warranted. Human cytomegalovirus (HCMV) is prevalent in the human population and has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumor cell immune evasion. Here we show that the majority of primary neuroblastomas and neuroblastoma cell lines are infected with HCMV. Our analysis show that HCMV immediate-early protein was expressed in 100% of 36 primary neuroblastoma samples, and HCMV late protein was expressed in 92%. However, no infectious virus was detected in primary neuroblastoma tissue extracts. Remarkably, all six human neuroblastoma cell lines investigated contained CMV DNA and expressed HCMV proteins. HCMV proteins were expressed in neuroblastoma cells expressing the proposed stem cell markers CD133 and CD44. When engrafted into NMRI nu/nu mice, human neuroblastoma cells expressed HCMV DNA, RNA and proteins but did not produce infectious virus. The HCMV-specific antiviral drug valganciclovir significantly reduced viral protein expression and cell growth both in vitro and in vivo. These findings indicate that HCMV is important for the pathogenesis of neuroblastoma and that anti-viral therapy may be a novel adjuvant treatment option for children with neuroblastoma.

Human cytomegalovirus particles directly suppress CD4 T-lymphocyte activation and proliferation.

CD4 T cells are important regulators of the immune system and are vital for mounting a strong immune response against viral infections. Human cytomegalovirus (HCMV) is known to be a strong modulator of the innate as well as adaptive immune responses. In this study, we found that HCMV directly inhibited proliferation of CD4 T cells and rendered them unresponsive to immunological stimuli. This effect was not observed when CD4 T cells were treated with herpes simplex virus-1/2 or measles virus. When stimulated with phytohemagglutinin, concanavalin A, or phorbol myristate acetate, HCMV-treated T cells were unable to proliferate, revealing an ability of HCMV to inhibit CD4 T cell response. Furthermore, HCMV also prevented proliferation of leukemic T-cell lines. HCMV-treated CD4 T cells expressed the activation markers CD45RO and CD69, were not apoptotic and produced decreased levels of the cytokines IL-4, IFN-γ and TNF-α, compared to untreated controls. The inhibitory effect of HCMV on CD4 T cell proliferation was not mediated by HCMV gH, gB or other immunogenic glycoproteins, since intravenous immunoglobulins or gB- or gH-specific neutralizing antibodies did not prevent the suppression of T-cell proliferation. Our observations show that HCMV inhibits CD4 T cell function with potential clinical consequences for both humoral and cell-mediated immune responses.

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target.

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.

Human neural stem cells and astrocytes, but not neurons, suppress an allogeneic lymphocyte response.

Transplantation of human neural stem cells (NSCs) and their derivatives is a promising future treatment for neurodegenerative disease and traumatic nervous system lesions. An important issue is what kind of immunological reaction the cellular transplant and host interaction will result in. Previously, we reported that human NSCs, despite expressing MHC class I and class II molecules, do not trigger an allogeneic T cell response. Here, the immunocompetence of human NSCs, as well as differentiated neural cells, was further studied. Astrocytes expressed both MHC class I and class II molecules to a degree equivalent to that of the NSCs, whereas neurons expressed only MHC class I molecules. Neither the NSCs nor the differentiated cells triggered an allogeneic lymphocyte response. Instead, these potential donor NSCs and astrocytes, but not the neurons, exhibited a suppressive effect on an allogeneic immune response. The suppressive effect mediated by NSCs most likely involves cell-cell interaction. When the immunogenicity of human NSCs was tested in an acute spinal cord injury model in rodent, a xenogeneic rejection response was triggered. Thus, human NSCs and their derived astrocytes do not initiate, but instead suppress, an allogeneic response, while they cannot block a graft rejection in a xenogeneic setting.

HCMV infection of PDCs deviates the NK cell response into cytokine-producing cells unable to perform cytotoxicity.

Plasmacytoid dendritic cells (PDCs) are thought to induce natural killer (NK) cell CD69 expression, cytotoxicity, and cytokine secretion. Since human cytomegalovirus (HCMV) interferes with multiple functions of infected cells, we investigated whether the HCMV infection of PDCs affects NK cell activation. Human PDCs infected with HCMV strain VR1814 at multiplicity of infection (MOI) 10 or stimulated with control CpG-A were cocultured with human NK cells in an autologous system. As expected, CpG-stimulation of PDCs increased expression of the NK cell activation marker CD69, enhanced cytotoxicity and stimulated secretion of tumor necrosis factor (TNF)-alpha and IFN-alpha, but not IFN-gamma, and induced NK cell migration. In contrast, incubation with HCMV-infected PDCs induced CD69 expression, migration and elevated production of both TNF-alpha and IFN-gamma by NK cells, but these cells did not exhibit enhanced cytotoxicity. Also, HCMV-infected PDCs were unable to induce increased intracellular perforin levels. Thus, HCMV infection of PDCs induce NK cells to increase CD69 expression and produce inflammatory cytokines, but infected PDCs are unable to induce NK cell cytotoxicity. This NK cell phenotype with impaired killing abilities, but enhanced production of inflammatory cytokines may instead facilitate reactivation and replication of HCMV. This data indicate that HCMV can target PDCs through novel dual strategies that may result in evasion of the innate immune response at the same time as facilitating virus reactivation and replication early in the infection, through enhanced inflammation.

Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures.

In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long-term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first-trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra-1-60, Tra-1-81, and SSEA-4, were present in approximately 0.5% of the cells in donor tissues and neurospheres. The proportions of nestin- and proliferating cell nuclear antigen-immunoreactive (IR) cells were also maintained, whereas the CD133-IR population increased in vitro. Glial fibrillary acidic protein-IR cells increased in number, and in contrast the fraction of beta-tubulin III-IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro-differentiated forebrain- and spinal cord-derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long-term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.

Effects of human cytomegalovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein (ULBP)1 and ULBP2 is counteracted by the viral UL16 protein.

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins.

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.

Human cytomegalovirus protein pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR alpha-chain.

Human cytomegalovirus (HCMV) has developed multiple strategies to escape immune recognition. Here, we demonstrate that HCMV down-regulates HLA-DR expression in infected interferon gamma (IFN-gamma)-stimulated fibroblasts at 1 day after infection. Decreased HLA-DR expression was not observed on cells infected with an HCMV strain lacking the pp65 gene (RVAD65), but was observed on cells transfected with the pp65 gene. HLA-DR expression accumulated in vacuoles near the nucleus in HCMV-infected, but not in uninfected or RVAD65-infected cells. In addition, the HLA-DR alpha-chain, but not the beta-chain or HLA-DM, was degraded in HCMV-infected but not in RVAD65-infected cells. Thus, the HCMV protein pp65 mediates decreased expression of HLA-DR, by mediating an accumulation of HLA class II molecules in lysosomes that results in degradation of the HLA-DR alpha-chain.