Comparative study of the expression of cholinergic system components in the CNS of experimental autoimmune encephalomyelitis mice: Acute vs remitting phase.

Acetylcholine (ACh) is involved in the modulation of the inflammatory response. ACh levels are regulated by its synthesizing enzyme, choline acetyltransferase (ChAT), and by its hydrolyzing enzymes, mainly acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A more comprehensive understanding of the cholinergic system in experimental autoimmune encephalomyelitis (EAE) disease progression could pave the path for the development of therapies to ameliorate multiple sclerosis (MS). In this work, we analyzed possible alterations of the CNS cholinergic system in the neuroinflammation process by using a MOG-induced EAE mice model. MOG- and vehicle-treated animals were studied at acute and remitting phases. We examined neuropathology and analyzed mRNA expression of ChAT, AChE and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR), as well as AChE and BuChE enzyme activities, in brain and spinal cord sections during disease progression. The mRNA expression and enzyme activities of these cholinergic markers were up- or down-regulated in many cholinergic areas and other brain areas of EAE mice in the acute and remitting phases of the disease. BuChE was present in a higher proportion of astroglia and microglia/macrophage cells in the EAE remitting group. The observed changes in cholinergic markers expression and cellular localization in the CNS during EAE disease progression suggests their potential involvement in the development of the neuroinflammatory process and may lay the ground to consider cholinergic system components as putative anti-inflammatory therapeutic targets for MS.

Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation.

Glioblastoma (GBM) is the most aggressive human brain tumor. The high growth potential and decreased susceptibility to apoptosis of the glioma cells is mainly dependent on genetic amplifications or mutations of oncogenic or pro-apoptotic genes, respectively. We have previously shown that the activation of the M2 acetylcholine muscarinic receptors inhibited cell proliferation and induced apoptosis in two GBM cell lines and cancer stem cells. The aim of this study was to delve into the molecular mechanisms underlying the M2-mediated cell proliferation arrest. Exploiting U87MG and U251MG cell lines as model systems, we evaluated the ability of M2 receptors to interfere with Notch-1 and EGFR pathways, whose activation promotes GBM proliferation. We demonstrated that the activation of M2 receptors, by agonist treatment, counteracted Notch and EGFR signaling, through different regulatory cascades depending, at least in part, on p53 status. Only in U87MG cells, which mimic p53-wild type GBMs, did M2 activation trigger a molecular circuitry involving p53, Notch-1, and the tumor suppressor mir-34a-5p. This regulatory module negatively controls Notch-1, which affects cell proliferation mainly through the Notch-1/EGFR axis. Our data highlighted, for the first time, a molecular circuitry that is deregulated in the p53 wild type GBM, based on the cross-talk between M2 receptor and the Notch-1/EGFR pathways, mediated by mir-34a-5p.

Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients.

Multiple sclerosis (MS) is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh) contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS) patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD), is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis.

Tenofovir/emtricitabine/efavirenz plus rosuvastatin decrease serum levels of inflammatory markers more than antiretroviral drugs alone in antiretroviral therapy-naive HIV-infected patients.

OBJECTIVES: Statins are lipid-lowering drugs that exhibit anti-Inflammatory and immune-modulatory properties, leading to a reduction of serum levels of C-reactive protein (CRP) in the general population. DESIGN: To assess the anti-inflammatory effects of statins in HIV-infected patients, because very limited data are available today. METHODS: Longitudinal, observational study of HIV-infected adult patients naive to antiretroviral therapy who started tenofovir/emtricitabine/efavirenz and were followed-up for 48 weeks. Patients with baseline normal cholesterol level and taking only antiretroviral drugs (group A) were compared to those with baseline hypercholesterolemia who received rosuvastatin (10 mg daily) in association with antiretroviral treatment (group B). The primary observation was change in serum markers of inflammation (high-sensitivity C-reactive protein [hsCRP], interleukin-6 [IL-6], interleukin-8 [IL-8]) and tumor necrosis factor-α [TNF- α]) in both groups, whereas secondary observations include variations in CD4 lymphocyte count, HIV viral load, and occurrence of adverse events. RESULTS: Eighty-six patients were enrolled into the study: 46 in group A and 40 in group B. After 48 weeks, patients treated with antiretroviral therapy plus rosuvastatin had significantly greater decreases in serum concentrations of all Inflammatory markers than those taking antiretroviral therapy only. Changes in mean levels of hsCRP and TNF-α were -35.1% and -22.4% in group B and -8.2% and 5.4% in group A, respectively (P < .001, for both parameters). No significant differences in immunovirological parameters and safety profile were reported across the compared groups. CONCLUSIONS: Our findings suggest that tenofovir/emtricitabine/efavirenz plus rosuvastatin has a greater antiInflammatory effect than antiretroviral drugs only.

Prevalence of renal disease within an urban HIV-infected cohort in northern Italy.

BACKGROUND: Renal disease is an increasingly recognized noninfectious comorbidity associated with human immunodeficiency virus (HIV) infection. METHODS: Our retrospective, cross-sectional study evaluated prevalence of nephropathy among HIV-infected patients followed up in our outpatient clinic during the year 2011. Renal dysfunction and chronic kidney disease (CKD) were defined as estimated glomerular filtration rate (eGFR) <90 ml/min per 1.73 m(2) and as renal damage or eGFR <60 ml/min per 1.73 m(2) over a 3-month or greater period, respectively. RESULTS: We enrolled 894 HIV-infected patients with a mean age of 44.2 years and a mean current CD4 lymphocyte count of 508 cells/mm(3). The prevalence of renal dysfunction and CKD was 27.4 and 21.3 %, respectively. Older age, male gender, hypertension, diabetes, proteinuria, hypertriglyceridemia, lower nadir CD4 cell count, current use of tenofovir or tenofovir plus a ritonavir-boosted protease inhibitor were independently associated with renal dysfunction. CONCLUSION: Renal dysfunction is a frequent comorbidity among HIV-infected persons and requires a careful clinical and laboratory monitoring of renal function.

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells.

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.

Incidence of renal toxicity in HIV-infected, antiretroviral-naïve patients starting tenofovir/emtricitabine associated with efavirenz, atazanavir/ritonavir, or lopinavir/ritonavir.

OBJECTIVES: We performed a retrospective cohort study of HIV-infected antiretroviral-naïve patients starting a first antiretroviral therapy with tenofovir/emtricitabine plus efavirenz (EFV), atazanavir/ritonavir (ATV/r), or lopinavir/ritonavir (LPV/r). METHODS: The incidence of renal impairment or proximal tubular dysfunction was evaluated during a 12-month follow-up. Renal impairment was diagnosed by a reduced estimated glomerular filtration rate (eGFR) calculated using the Modification of Diet in Renal Disease (MDRD) formula, and tubular dysfunction was diagnosed when ≥ 2 among proteinuria, glucosuria, hypouricaemia, hypophosphataemia, and hypokalaemia, were identified. RESULTS: A total of 235 patients were enrolled: 82 taking EFV, 78 ATV/r, and 75 LPV/r. The mean decline in eGFR after the 12-month follow-up was significantly greater in subjects treated with ATV/r (-10.4 ml/min/1.73 m(2)) than in those receiving EFV (- 5.1; p = 0.002) or LPV/r (-4.8; p = 0.003). Similarly, a significantly higher incidence of proximal tubulopathy was observed among ATV/r-treated patients (14.1%) compared with patients receiving EFV (4.9%) or LPV/r (5.3%). CONCLUSIONS: In our retrospective study, naïve patients receiving tenofovir/emtricitabine and ATV/r for 12 months showed a significantly higher decline in eGFR and a significantly higher incidence of proximal tubulopathy than those receiving tenofovir/emtricitabine plus EFV or LPV/r, even though clinically evident renal toxicity associated with tenofovir-based treatment is a very uncommon event.

M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

Effect of a small molecule BCL-2 inhibitor on immune function and use with a recombinant vaccine.

Small molecule BCL-2 inhibitors are being examined as monotherapy in phase I/II clinical trials for several types of tumors. However, few data are available about the effect of BCL-2 inhibitors on immune function. The aims of our study were to investigate the effect of a small molecule BCL-2 inhibitor on immune function and determine the most effective way of combining this inhibitor with a recombinant vaccine to treat tumors. The in vitro effect of the pan-BCL-2 inhibitor GX15-070 was assessed in mouse CD8 T lymphocytes at 2 different stages of activation as well as regulatory T lymphocytes (Treg). The in vivo effect of GX15-070 after recombinant vaccinia/fowlpox CEA-TRICOM vaccination was analyzed in tumor-infiltrating lymphocytes, and in splenocytes of mice bearing subcutaneous tumors. The therapeutic efficacy of such sequential therapy was measured as a reduction of pulmonary tumor nodules. Activated mature CD8 T lymphocytes were more resistant to GX15-070 as compared to early-activated cells. Treg function was significantly decreased after treatment with the BCL-2 inhibitor. In vivo, GX15-070 was given after vaccination so as to not negatively impact the induction of vaccine-mediated immunity, resulting in increased intratumoral activated CD8:Treg ratio and significant reduction of pulmonary tumor nodules. Our study is the first to show the effect of a small molecule BCL-2 inhibitor on the immune system and following a vaccine. It is also the first to demonstrate the efficacy of this sequence in reducing tumors in mouse models, providing a rationale for the design of combinational clinical studies.

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype.

Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.

TGF-beta modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression.

This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-beta contributed markedly to the tumor-infiltrating CD8+ T cells' (TILs) reduced functionality, which could be reversed using a small molecule TGF-beta inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-beta inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-beta activity. Spred-1 was upregulated in CD8+ TILs and TGF-beta enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-beta mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies.

Butyrylcholinesterase and Acetylcholinesterase polymorphisms in Multiple Sclerosis patients: implication in peripheral inflammation.

Multiple Sclerosis (MS) is an autoimmune disease, having not fully understood aetiology, and both genetic and environmental factors contribute to the pathogenesis of the disease. The cholinergic system has been indicated as a mediator of neuro-immune interactions, as well as an internal regulator of immune responses. The aim of the present research was to assess the associations between BChE and AChE genetic variations and serum cholinergic and inflammatory profiles in 102 Relapsing Remitting-MS patients and 117 healthy controls. An increased frequency of the BChE K-allele in MS patients as compared to controls was found. In addition, data showed that patients had higher BChE enzymatic activity, which is increased by the presence of the polymorphic allele and reduced amounts of circulating ACh. AChE polymorphism was significantly associated to reduced activity in both patients and controls. We propose that serum BChE and AChE activity may be used as a secondary markers to assess the role of non-neuronal cholinergic system in regulating peripheral inflammation via ACh regulation. This pilot study shed light on the role of the non-neuronal cholinergic system in immune cells to better understand MS pathogenesis. The cross-talk between the periphery and the CNS could have a new undescribed crucial role for MS, regarded as a systemic disease.

Cholinergic System and Neuroinflammation: Implication in Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system (CNS) characterized by leucocytes infiltration, demyelination, axonal degeneration and neuronal death. Although the etiology of MS is still unkwon, inflammation and autoimmunity are considered to be key players of the disease. Nervous System: The severe alterations affecting the nervous system contribute to the motor and cognitive disabilities and are in large part dependent on severe inflammatory processes active in both central nervous system and immune system. Acetylcholine (ACh) appears to be involved in the modulation of central and peripheral inflammation. Immune cells as well as astrocytes and microglia respond to ACh stimuli by activation of cholinergic receptors. Muscarinic and nicotinic receptors differently contribute to the modulation of immunological and inflammatory processes stimulating pro- and anti-inflammatory cytokines respectively. The role played by ACh in MS is not yet fully understood, although some results point to its involvement in different neurological disorders such as Alzheimer's disease and schizophrenia. CONCLUSION: In the present review we summarize the evidence indicating the correlation between nervous system dysfunction in MS, with inflammation and cholinergic system alterations. Experiments performed in MS animal models and analyses on biological fluids from MS patients such as blood, serum and cerebrospinal fluid suggest that cholinergic alterations may contribute to the dysregulated inflammatory processes of MS. Many current therapeutic approaches in MS are based on anti-inflammatory drugs. We also discuss how the use of cholinesterase inhibitors or ACh mimetics may represent a new interesting therapeutic approach in MS.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human glioblastoma cancer stem cells.

The involvement of muscarinic receptors in cancer has been reported. Recently we have demonstrated that the activation of M2 muscarinic receptors, through arecaidine propargyl ester, arrests cell proliferation and induces apoptosis in primary and established glioblastoma cell lines. Considering the inability of conventional drugs to completely counteract the growth of glioblastoma cancer stem cells (GSCs), we have investigated the effect produced by arecaidine on GSC growth and survival. The expression of M2 receptors has been analyzed in GSC cell lines derived from human biopsies. Based on the M2 receptor expression levels, we have selected two gliolastoma cell lines (GB7 and GB8). In both cell lines the treatment with arecaidine decreased GCS cell growth. GB7 cells exhibited a time- and dose-dependent decrease of cell proliferation. Moreover arecaidine caused a reduced cell survival in particular in GB8 cell line. These effects appear to be mediated by M2 receptor activation as suggested by pharmacological experiments performed in the presence of M1 and M3 preferring antagonists (pirenzepine and 4-DAMP respectively) and M2/M4 antagonist methoctramine. M2 receptor silencing by siRNA has further confirmed that the inhibition of cell growth arecaidine-induced was mediated by the M2 receptor activation. These results suggest that the M2 receptors may represent a new interesting therapeutic tool to counteract glioblastoma cancer stem cell growth and survival.

Cytotoxic and genotoxic effects mediated by M2 muscarinic receptor activation in human glioblastoma cells.

Glioblastomas are the most common brain tumors in humans. Previously, we demonstrated that the muscarinic receptor agonist, arecaidine propargyl ester, via M2 receptors, inhibits cell proliferation in a time and dose-dependent manner and induces a severe apoptosis in human U251 and U87 glioblastoma cell lines. In order to clarify the mechanisms causing apoptosis after arecaidine treatment, we analyzed the ability of arecaidine to induce oxidative stress. By dichloro-dihydro-fluorescein diacetate (DCFDA) staining, we demonstrated that arecaidine increased the intracellular ROS levels. ROS accumulation was completely counteracted by the ROS scavenger, N-acetyl-l-cysteine (NAC). Apoptotic cell death appeared directly correlated to ROS production since NAC was able to counteract this effect. Although there was an up-regulation of some detoxifying enzyme expression such as superoxide dismutase (MnSOD) and sirtuin-1 (SIRT1), the cytotoxic effect caused by arecaidine treatment caused DNA damage, as demonstrated by the increase of histone γ-H2AX positive cells, and chromosomal aberrations. These effects were mediated by M2 receptor activation; in fact after silencing of M2 receptors by siRNA, the increase of γ-H2AX positive cells was abolished. In conclusion, in addition to a cytostatic effect previously described, in the present study we have better characterized the mechanisms causing the cytotoxic effects and the apoptotic cell death in glioblastoma cells after M2 receptor activation. These data allow to consider this receptor a new interesting therapeutic tool for the glioblastoma treatment.

Nicotinic receptor activation negatively modulates pro-inflammatory cytokine production in multiple sclerosis patients.

Acetylcholine (ACh) and its receptors of muscarinic and nicotinic types are involved in the modulation of immune and inflammatory responses. In present work we have characterized the nicotinic receptors expression in PBMC of RR-MS patients and healthy donors (HD) and their ability to modulate pro-inflammatory cytokines. Here we report that the IL-1ß e IL-17 levels are significantly increased in serum of RR-MS patients in respect to HD and that the PBMC stimulation with PHA caused a significant increase in pro-inflammatory cytokine levels both in RR-MS and HD subjects, with higher increase of protein release in RR-MS patients than in HD. The PBMC treatment with PHA plus nicotine produced a significant decrease of IL-1ß e IL-17 both as transcript and as protein, confirming that the PBMC of the patients respond to the cholinergic stimulation more than PBMC of HD. By real time PCR and western blot analysis we have also demonstrated that in particular α7 receptor subtype appeared expressed at comparable levels both in RR-MS patients and HD. The PHA stimulation results to inhibit the α7 subunit expression while the nicotine causes a significant increase in α7 transcripts but only in MS patients. The data obtained highlight the role of α7 receptor subtype in the modulation of anti-inflammatory cytokines also in MS. Moreover the ability of nicotine to up-regulate the expression of α7 receptor subtype in RR-MS patients, indicates that nicotinic receptor stimulation may contribute to down-modulate the inflammation occurred in MS by a positive feedback control of its expression.

M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells.

The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 µM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target.

Peripheral CD45RO, PD-1, and TLR4 expression in metastatic colorectal cancer patients treated with bevacizumab, fluorouracil, and irinotecan (FOLFIRI-B).

CD45RO, PD-1, and TLR4 immune pathways have proven pivotal in regulating antitumor response and correlate with survival for localized colorectal cancer (CRC). We evaluated if their peripheral expression was associated with outcome in metastatic CRC (mCRC). Thirty-one mCRC patients were eligible for this prospective study ( clinicaltrial.gov NCT01533740) and treated with first-line FOLFIRI-B. Blood was drawn before the first and third cycle and analyzed by flow cytometry for frequency (%) of CD4+, CD8+, CD45RO+, and PD1+ mononuclear cells and for TLR4 expression on neutrophils. Two cycles of chemotherapy determined changes in immune variables that were prognostically meaningful. Pre-third-cycle (ptc) CD45RO+CD8+cell% displayed a statistically significant association with progression-free survival (PFS) (median PFS 22.4 vs. 9.4 months for patients with CD45RO+CD8+cell%> vs.

Effects of conventional therapeutic interventions on the number and function of regulatory T cells.

Several lines of investigation have revealed the apparent interplay between the immune system of the host and many conventional, "standard-of-care" anticancer therapies, including chemotherapy and small molecule targeted therapeutics. In particular, preclinical and clinical studies have demonstrated the important role of regulatory T cells (Tregs) in inhibiting immune responses elicited by immunotherapeutic regimens such as those based on anticancer vaccines or checkpoint inhibitors. However, how the number and immunosuppressive function of Tregs change in cancer patients undergoing treatment with non-immune anticancer therapies remains to be precisely elucidated. To determine whether immunostimulatory therapies can be employed successfully in combination with conventional anticancer regimens, we have investigated both the number and function of Tregs obtained from the peripheral blood of carcinoma patients before the initiation and during the course of chemotherapeutic and targeted agent regimens. Our studies show that the treatment of breast cancer patients with tamoxifen plus leuprolide, a gonadotropin releasing hormone agonist, has minimal effects on Tregs, while sunitinib appears to exert differential effects on Tregs among patients with metastatic renal carcinoma. However, the administration of docetaxel to patients with metastatic prostate or breast cancer, as well as that of cisplatin plus vinorelbine to non-small cell lung cancer patients, appears to significantly increase the ratio between effector T cells and Tregs and to reduce the immunosuppressive activity of the latter in the majority of patients. These studies provide the rationale for the selective use of active immunotherapy regimens in combination with specific standard-of-care therapies to achieve the most beneficial clinical outcome among carcinoma patients.