Isolation and preliminary characterization of amino acid substitution mutations that increase the activity of the osmoregulated ProP protein of Salmonella enterica serovar Typhimurium.

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease.

Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape.

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored.

Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3.

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells.

PD-1/PD-L1 interactions contribute to functional T-cell impairment in patients who relapse with cancer after allogeneic stem cell transplantation.

Tumor relapses remain a serious problem after allogeneic stem cell transplantation (alloSCT), despite the long-term persistence of minor histocompatibility antigen (MiHA)-specific memory CD8(+) T cells specific for the tumor. We hypothesized that these memory T cells may lose their function over time in transplanted patients. Here, we offer functional and mechanistic support for this hypothesis, based on immune inhibition by programmed death-1 (PD-1) expressed on MiHA-specific CD8(+) T cells and the associated role of the PD-1 ligand PD-L1 on myeloid leukemia cells, especially under inflammatory conditions.

Development of ipilimumab: contribution to a new paradigm for cancer immunotherapy.

Identification of cytotoxic T-lymphocyte antigen-4 (CTLA-4) as a key negative regulator of T-cell activity led to development of the fully human, monoclonal antibody ipilimumab to block CTLA-4 and potentiate antitumor T-cell responses. Animal studies first provided insight into the ability of an anti-CTLA-4 antibody to cause tumor regression, particularly in combination regimens. Early clinical studies defined ipilimumab pharmacokinetics and possibilities for combinability.

Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.

Purpose: Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, may suppress antitumor immunity. This phase I study sought to determine the safety and tolerability of anti-PD-1 blockade in patients with treatment-refractory solid tumors and to preliminarily assess antitumor activity, pharmacodynamics, and immunologic correlates.

Patients And Methods: Thirty-nine patients with advanced metastatic melanoma, colorectal cancer (CRC), castrate-resistant prostate cancer, non-small-cell lung cancer (NSCLC), or renal cell carcinoma (RCC) received a single intravenous infusion of anti-PD-1 (MDX-1106) in dose-escalating six-patient cohorts at 0.

Enhanced tumor eradication by combining CTLA-4 or PD-1 blockade with CpG therapy.

Tumor immunotherapy aims to break effector T-cell anergy and to block suppressive cell types and ligands allowing effector cells to exert tumor eradication. Previous reports demonstrate that cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibodies promote T-cell activation and render T effector cells resistant to T regulatory cells (Tregs) whereas programmed death receptor-1 (PD-1)/PD-L1 blockade results in loss of peripheral tolerance. Herein, we explored single or combined antibody blockade of CTLA-4 and PD-1 alone or combined with the toll-like receptor agonists CpG or bacillus Calmette-Guérin for treatment of murine experimental bladder cancer.

An internet-based simulation system for training and development of regional-healthcare-centers managers.

RHCMS (Regional Healthcare Center Management System) is an Internet-based simulation training system for Regional Healthcare-Centers (RHCs) managers. The system is based on an integrative model, designed and developed by an interdisciplinary team of experts, for the purpose of training and developing RHCs' managers. This model involves the study of the following fields: healthcare management, business administration, organizational behavior, health economics, management science, and information technologies.

PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+ CD25(Hi) regulatory T cells.

Regulatory CD4(+)CD25(Hi) T cells (Treg) and programmed death-1 (PD-1) molecule have emerged as pivotal players in immune regulation. However, the underlying mechanisms by which they impact antigen-specific CD8(+) immune responses in cancer patients and how they interact with each other under physiologic conditions remain unclear. Herein, we examined the relationship of PD-1 and its abrogation to the function of Treg in patients with melanoma using short-term in vitro assays to generate melanoma-specific T cells.

Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical negative regulator of immune responses. Uniquely among known inhibitory receptors, its genetic ablation results in a fulminating and fatal lymphoproliferative disorder. This central regulatory role led to the development of antibodies designed to block CTLA-4 activity in vivo, aiming to enhance immune responses against cancer.

Anti-programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor--secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors.

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1 blockade, which has been shown to augment cellular immune responses.

Experimental Design: Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model.

High-programmed death-1 levels on hepatitis C virus-specific T cells during acute infection are associated with viral persistence and require preservation of cognate antigen during chronic infection.

Hepatitis C virus (HCV) is an important human pathogen that represents a model for chronic infection given that the majority of infected individuals fail to clear the infection despite generation of virus-specific T cell responses during the period of acute infection. Although viral sequence evolution at targeted MHC class I-restricted epitopes represents one mechanism for immune escape in HCV, many targeted epitopes remain intact under circumstances of viral persistence. To explore alternative mechanisms of HCV immune evasion, we analyzed patterns of expression of a major inhibitory receptor on T cells, programmed death-1 (PD-1), from the time of initial infection and correlated these with HCV RNA levels, outcome of infection, and sequence escape within the targeted epitope.

Biological effectiveness of (12)C and (20)Ne ions with very high LET.

Purpose: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of (12)C and (20)Ne ions.

Materials And Methods: Chinese hamster ovary (CHO-K1) cells were exposed to high LET (12)C (33.2 MeV, 20.

Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions.

Chinese hamster ovary CHO-K1 cells were exposed to high LET (12)C-beam (LET: 830 keV/microm) in the dose range of 0-6 Gy and to (60)Co irradiation and the RBE value was obtained. Effects of (12)C-beam exposure on cell survival and chromosomal aberrations were calculated. The chromosomal aberration data were fitted with linear equation.

Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. The administration of fully human anti-CTLA-4 blocking monoclonal antibodies to advanced-cancer patients increases immune-mediated tumor destruction in some subjects. Nonetheless, patients that respond also frequently manifest serious inflammatory pathologies, raising the possibility that the therapeutic and toxic effects of CTLA-4 blockade might be linked.

Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs.

Negative co-stimulatory signaling mediated via cell surface programmed death (PD)-1 expression modulates T and B cell activation and is involved in maintaining peripheral tolerance. In this study, we examined the effects of a fully human PD-1-abrogating antibody on the in vitro expansion and function of human vaccine-induced CD8+ T cells (CTLs) specific for the melanoma-associated antigens glycoprotein 100 (gp100) and melanoma antigen recognized by T cells (MART)-1. PD-1 blockade during peptide stimulation augmented the absolute numbers of CD3+, CD4+, CD8+ and gp100/MART-1 MHC:peptide tetramer+ CTLs.

Enhanced protein production by engineered zinc finger proteins.

Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings.

An irradiation facility with a horizontal beam for radiobiological studies.

A facility with a horizontal beam for radiobiological experiments with heavy ions has been designed and constructed at the Heavy Ion Laboratory in Warsaw University. The facility is optimal to investigate the radiobiological effects of charged heavy particles on a cellular or molecular level as in the region of the Bragg peak.

Checkpoint blockade in cancer immunotherapy.

The progression of a productive immune response requires that a number of immunological checkpoints be passed. Passage may require the presence of excitatory costimulatory signals or the avoidance of negative or coinhibitory signals, which act to dampen or terminate immune activity. The immunoglobulin superfamily occupies a central importance in this coordination of immune responses, and the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4):B7.

Principles and use of anti-CTLA4 antibody in human cancer immunotherapy.

Cytotoxic T lymphocyte antigen-4 has become recognized as one of the key negative regulators of adaptive immune responses, having a central role in the maintenance of peripheral tolerance and in shaping the repertoire of emergent T cell responses. Concurrent recognition of the potential importance of inhibitory immune regulators in limiting antitumor responses, either as a result of chronic antigenic stimulation or the self-nature of many tumor-selective target antigens, has led to the development of cytotoxic T lymphocyte antigen-4-blocking antibodies as therapeutic anticancer agents. Following extensive preclinical modeling, these agents have entered clinical trials, where they are showing encouraging activity in heavily pretreated patients with advanced-stage disease, particularly with melanoma or renal carcinoma.

Tumor immunotherapy: preclinical and clinical activity of anti-CTLA4 antibodies.

Cancer immunotherapy utilizing vaccines has relied upon the patients' pre-existing immune activation capabilities, augmented by existing adjuvants, to promote tumor-antigen specific immune responses. Generating effective antitumor responses in this way requires overcoming multiple mechanisms of tumor evasion of the immune system. In addition, the generation of tumor immunity must overcome tolerance to tumor antigens, which in most cases are self-antigens.

Impact of an electronic antibiotic advice and approval system on antibiotic prescribing in an Australian teaching hospital.

The impact of a computer-based infectious diseases electronic antibiotic advice and approval system ("IDEA(3)S") was assessed as an alternative to a labour-intensive, phone-based approval system. IDEA(3)S-based approvals replaced 48% of all approvals for the most frequently requested antimicrobial agents (ceftriaxone/cefotaxime, vancomycin) and were associated with stable overall rates of antimicrobial use. Antibiotic prescribing for community-acquired pneumonia was 76% concordant with IDEA(3)S recommendations, and clinical acceptance of IDEA(3)S was excellent.

Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques.

The immune modulatory molecule CTLA-4 (CD152), through interactions with the B7 costimulatory molecules, has been shown to be a negative regulator of T cell activation in various murine model systems. Abs that block CTLA-4 function can enhance immune responses that mediate potent antitumor activity. However, CTLA-4 blockade can also exacerbate autoimmune disease.

Spotted fever group rickettsiae in ticks from the Masai Mara region of Kenya.

We have identified for the first time Rickettsia africae, and the ticks that harbored them, in Kenya. A total of 5,325 ticks were collected from vegetation, livestock, and wild animals during two field trips to southwestern Kenya. Most were immature forms (85.

Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients.

A large number of cancer-associated gene products evoke immune recognition, but host reactions rarely impede disease progression. The weak immunogenicity of nascent tumors contributes to this failure in host defense. Therapeutic vaccines that enhance dendritic cell presentation of cancer antigens increase specific cellular and humoral responses, thereby effectuating tumor destruction in some cases.