Antigen-Addicted T Cell Reserves Trickle Charge the Frontline Killers.

Highly active killer T cells mediate a stable standoff during controlled persistent infections. In this issue of Immunity, Robey and colleagues describe a unique antigen-addicted T cell population bearing characteristics of both effector and memory CD8(+) T cells that provides a continuous supply of potent killer T cells to curb Toxoplasma gondii growth during latency.

Interferon-gamma contributes to disease progression in the Ndufs4(-/-) model of Leigh syndrome.

AIM: Leigh syndrome (LS), the most common paediatric presentation of genetic mitochondrial dysfunction, is a multi-system disorder characterised by severe neurologic and metabolic abnormalities. Symmetric, bilateral, progressive necrotizing lesions in the brainstem are defining features of the disease. Patients are often symptom free in early life but typically develop symptoms by about 2 years of age. The mechanisms underlying disease onset and progression in LS remain obscure. Recent studies have shown that the immune system causally drives disease in the Ndufs4(-/-) mouse model of LS: treatment of Ndufs4(-/-) mice with the macrophage-depleting Csf1r inhibitor pexidartinib prevents disease. While the precise mechanisms leading to immune activation and immune factors involved in disease progression have not yet been determined, interferon-gamma (IFNγ) and interferon gamma-induced protein 10 (IP10) were found to be significantly elevated in Ndufs4(-/-) brainstem, implicating these factors in disease. Here, we aimed to explore the role of IFNγ and IP10 in LS. METHODS: To establish the role of IFNγ and IP10 in LS, we generated IFNγ and IP10 deficient Ndufs4(-/-)/Ifng(-/-) and Ndufs4(-/-)/IP10(-/-) double knockout animals, as well as IFNγ and IP10 heterozygous, Ndufs4(-/-)/Ifng(+/-) and Ndufs4(-/-)/IP10(+/-), animals. We monitored disease onset and progression to define the impact of heterozygous or homozygous loss of IFNγ and IP10 in LS. RESULTS: Loss of IP10 does not significantly impact the onset or progression of disease in the Ndufs4(-/-) model. IFNγ loss significantly extends survival and delays disease progression in a gene dosage-dependent manner, though the benefits are modest compared to Csf1r inhibition. CONCLUSIONS: IFNγ contributes to disease onset and progression in LS. Our findings suggest that IFNγ targeting therapies may provide some benefits in genetic mitochondrial disease, but targeting IFNγ alone would likely yield only modest benefits in LS.

Quiescence of Memory CD8(+) T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4.

Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory T cells remain inactive due to lack of T cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory T cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector T cells and drove transitioning as well as established memory CD8(+) T cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory T cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunity and improve T cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation.

Programming of CD8 T Cell Quantity and Polyfunctionality by Direct IL-1 Signals.

IL-1, generally considered an amplifier of adaptive immune responses, has been proposed for use as adjuvant during immunization with weak immunogens. However, its effects on memory T cell function remain largely undefined. Using the murine model of acute viral infection, in this paper, we show that in addition to augmenting the size of the Ag-specific pool, IL-1 signals act directly on CD8 T cells to promote the quality of effector and memory responses. Ablation of IL-1R1 or MyD88 signaling in T cells led to functional impairment; both the ability to produce multiple cytokines on a per cell basis (polyfunctionality) and the potential for recall proliferation in response to antigenic restimulation were compromised. IL-1 supplementation during priming augmented the expansion of Ag-specific CD8 T cells through the MyD88-IRAK1/4 axis, resulting in a larger memory pool capable of robust secondary expansion in response to rechallange. Together, these findings demonstrate a critical role of the IL-1-MyD88 axis in programming the quantity and quality of memory CD8 T cell responses and support the notion that IL-1 supplementation may be exploited to enhance adoptive T cell therapies against cancers and chronic infections.

Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo.

CD25, the high-affinity interleukin-2 (IL-2) receptor alpha chain, is rapidly upregulated by antigen-specific CD8(+) T cells after T cell receptor stimulation. Here, we demonstrate that during an acute viral infection, CD25 expression is quite dynamic-after initial upregulation, a subset of virus-specific T cells sustains CD25 expression longer than the rest. At this time when there is distinct heterogeneity in CD25 expression, examination of the in vivo fate of effector cells revealed that CD25(lo) cells, which are relatively less sensitive to IL-2, preferentially upregulate CD127 and CD62L and give rise to functional long-lived memory cells. In contrast, CD25(hi) cells perceiving prolonged IL-2 signals proliferate more rapidly, are prone to apoptosis, exhibit a more pronounced effector phenotype, and appear to be terminally differentiated. Consistent with this, sustained IL-2 receptor signaling during expansion drove terminal-effector differentiation. These data support the hypothesis that prolonged IL-2 signals during priming promote terminal-effector differentiation.

Role of vitamin D in cytotoxic T lymphocyte immunity to pathogens and cancer.

The discovery of vitamin D receptor (VDR) expression in immune cells has opened up a new area of research into immunoregulation by vitamin D, a niche that is distinct from its classical role in skeletal health. Today, about three decades since this discovery, numerous cellular and molecular targets of vitamin D in the immune system have been delineated. Moreover, strong clinical associations between vitamin D status and the incidence/severity of many immune-regulated disorders (e.g. infectious diseases, cancers and autoimmunity) have prompted the idea of using vitamin D supplementation to manipulate disease outcome. While much is known about the effects of vitamin D on innate immune responses and helper T (T(H)) cell immunity, there has been relatively limited progress on the frontier of cytotoxic T lymphocyte (CTL) immunity--an arm of host cellular adaptive immunity that is crucial for the control of such intracellular pathogens as human immunodeficiency virus (HIV), tuberculosis (TB), malaria, and hepatitis C virus (HCV). In this review, we discuss the strong historical and clinical link between vitamin D and infectious diseases that involves cytotoxic T lymphocyte (CTL) immunity, present our current understanding as well as critical knowledge gaps in the realm of vitamin D regulation of host CTL responses, and highlight potential regulatory connections between vitamin D and effector and memory CD8 T cell differentiation events during infections.

In Vitro and In Vivo Activities of a Bi-Aryl Oxazolidinone, RBx 11760, against Gram-Positive Bacteria.

RBx 11760, a bi-aryl oxazolidinone, was investigated for antibacterial activity against Gram-positive bacteria. The MIC90s of RBx 11760 and linezolid against Staphylococcus aureus were 2 and 4 mg/liter, against Staphylococcus epidermidis were 0.5 and 2 mg/liter, and against Enterococcus were 1 and 4 mg/liter, respectively. Similarly, against Streptococcus pneumoniae the MIC90s of RBx 11760 and linezolid were 0.5 and 2 mg/liter, respectively. In time-kill studies, RBx 11760, tedizolid, and linezolid exhibited bacteriostatic effect against all tested strains except S. pneumoniae RBx 11760 showed 2-log10 kill at 4× MIC while tedizolid and linezolid showed 2-log10 and 1.4-log10 kill at 16× MIC, respectively, against methicillin-resistant S. aureus (MRSA) H-29. Against S. pneumoniae 5051, RBx 11760 showed bactericidal activity, with 4.6-log10 kill at 4× MIC compared to 2.42-log10 and 1.95-log10 kill for tedizolid and linezolid, respectively, at 16× MIC. RBx 11760 showed postantibiotic effects (PAE) at 3 h at 4 mg/liter against MRSA H-29, and linezolid showed the same effect at 16 mg/liter. RBx 11760 inhibited biofilm production against methicillin-resistant S. epidermidis (MRSE) ATCC 35984 in a concentration-dependent manner. In a foreign-body model, linezolid and rifampin resulted in no advantage over stasis, while the same dose of RBx 11760 demonstrated a significant killing compared to the initial control against S. aureus (P < 0.05) and MRSE (P < 0.01). The difference in killing was statistically significant for the lower dose of RBx 11760 (P < 0.05) versus the higher dose of linezolid (P > 0.05 [not significant]) in a groin abscess model. In neutropenic mouse thigh infection, RBx 11760 showed stasis at 20 mg/kg of body weight, whereas tedizolid showed the same effect at 40 mg/kg. These data support RBx 11760 as a promising investigational candidate.

Vitamin D Control of Hematopoietic Cell Differentiation and Leukemia.

It is now well known that in the mammalian body vitamin D is converted by successive hydroxylations to 1,25-dihydroxyvitamin D (1,25D), a steroid-like hormone with pleiotropic properties. These include important contributions to the control of cell proliferation, survival and differentiation, as well as the regulation of immune responses in disease. Here, we present recent advances in current understanding of the role of 1,25D in myelopoiesis and lymphopoiesis, and the potential of 1,25D and analogs (vitamin D derivatives; VDDs) for the control of hematopoietic malignancies. The reasons for the unimpressive results of most clinical studies of the therapeutic effects of VDDs in leukemia and related diseases may include the lack of a precise rationale for the conduct of these studies. Further, clinical trials to date have generally used extremely heterogeneous patient populations and, in many cases, small numbers of patients, generally without controls. Although low calcemic VDDs have been used and combined with agents that can increase the leukemia cell killing or differentiation effects in acute leukemias, the sequencing of agents used for combination therapy should to be more clearly delineated. Most importantly, it is recommended that in future clinical trials the rationale for the basis of the enhancing action of drug combinations should be clearly articulated and the effects on anticancer immunity should also be evaluated.

MicroRNA-17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections.

The precise microRNAs and their target cellular processes involved in generation of durable T-cell immunity remain undefined. Here we show a dynamic regulation of microRNAs as CD8 T cells differentiate from naïve to effector and memory states, with short-lived effectors transiently expressing higher levels of oncogenic miR-17-92 compared with the relatively less proliferating memory-fated effectors. Conditional CD8 T-cell-intrinsic gain or loss of expression of miR-17-92 in mature cells after activation resulted in striking reciprocal effects compared with wild-type counterparts in the same infection milieu-miR-17-92 deletion resulted in lesser proliferation of antigen-specific cells during primary expansion while favoring enhanced IL-7Rα and Bcl-2 expression and multicytokine polyfunctionality; in contrast, constitutive expression of miR-17-92 promoted terminal effector differentiation, with decreased formation of polyfunctional lymphoid memory cells. Increased proliferation upon miR-17-92 overexpression correlated with decreased expression of tumor suppressor PTEN and increased PI3K-AKT-mTOR signaling. Thus, these studies identify miR17-92 as a critical regulator of CD8 T-cell expansion and effector and memory lineages in the physiological context of acute infection, and present miR-17-92 as a potential target for modulating immunologic outcome after vaccination or immunotherapeutic treatments of cancer, chronic infections, or autoimmune disorders.

CCR10 regulates balanced maintenance and function of resident regulatory and effector T cells to promote immune homeostasis in the skin.

BACKGROUND: CCR10 and CCL27 make up the most skin-specific chemokine receptor/ligand pair implicated in skin allergy and inflammatory diseases, including atopic dermatitis and psoriasis. This pair is thought to regulate the migration, maintenance, or both of skin T cells and is suggested to be therapeutic targets for treatment of skin diseases. However, the functional importance of CCR10/CCL27 in vivo remains elusive. OBJECTIVE: We sought to determine the expression and function of CCR10 in different subsets of skin T cells under both homeostatic and inflammatory conditions to gain a mechanistic insight into the potential roles of CCR10 during skin inflammation. METHODS: Using heterozygous and homozygous CCR10 knockout/enhanced green fluorescent protein knockin mice, we assessed the expression of CCR10 on regulatory and effector T cells of healthy and inflamed skin induced by chemicals, pathogens, and autoreactive T cells. In addition, we assessed the effect of CCR10 knockout on the maintenance and functions of different T cells and inflammatory status in the skin during different phases of the immune response. RESULTS: CCR10 expression is preferentially induced on memory-like skin-resident T cells and their progenitors for their maintenance in homeostatic skin but not expressed on most skin-infiltrating effector T cells during inflammation. In CCR10 knockout mice the imbalanced presence and dysregulated function of resident regulatory and effector T cells result in over-reactive and prolonged innate and memory responses in the skin, leading to increased clearance of Leishmania species infection in the skin. CONCLUSION: CCR10 is a critical regulator of skin immune homeostasis.

A novel ketolide, RBx 14255, with activity against multidrug-resistant Streptococcus pneumoniae.

We present here the novel ketolide RBx 14255, a semisynthetic macrolide derivative obtained by the derivatization of clarithromycin, for its in vitro and in vivo activities against sensitive and macrolide-resistant Streptococcus pneumoniae. RBx 14255 showed excellent in vitro activity against macrolide-resistant S. pneumoniae, including an in-house-generated telithromycin-resistant strain (S. pneumoniae 3390 NDDR). RBx 14255 also showed potent protein synthesis inhibition against telithromycin-resistant S. pneumoniae 3390 NDDR. The binding affinity of RBx 14255 toward ribosomes was found to be more than that for other tested drugs. The in vivo efficacy of RBx 14255 was determined in murine pulmonary infection induced by intranasal inoculation of S. pneumoniae ATCC 6303 and systemic infection with S. pneumoniae 3390 NDDR strains. The 50% effective dose (ED50) of RBx 14255 against S. pneumoniae ATCC 6303 in a murine pulmonary infection model was 3.12 mg/kg of body weight. In addition, RBx 14255 resulted in 100% survival of mice with systemic infection caused by macrolide-resistant S. pneumoniae 3390 NDDR at 100 mg/kg four times daily (QID) and at 50 mg/kg QID. RBx 14255 showed favorable pharmacokinetic properties that were comparable to those of telithromycin.

Interleukin-21 is a critical cytokine for the generation of virus-specific long-lived plasma cells.

Long-lived plasma cells that reside in the bone marrow constitutively produce antibody in the absence of antigen and are the cellular basis of durable humoral immunity. The generation of these long-lived plasma cells depends upon a series of highly orchestrated interactions between antigen-specific CD4 T cells and B cells and the formation of germinal centers (GCs). In this study, we have examined the role of the cytokine interleukin-21 (IL-21) in regulating humoral immunity during acute viral infections. Using IL-21 receptor-deficient (IL-21R(-/-)) mice, we found that virus-specific CD4 T cells were generated after infection with lymphocytic choriomeningitis virus (LCMV) and that these CD4 T cells differentiated into T follicular helper (TFH)-like cells in the absence of IL-21 signaling. There was also no defect in the formation of GCs, although after day 15 these GCs disappeared faster in IL-21R(-/-) mice than in wild-type mice. Isotype switching and the initial LCMV-specific IgG response were normal in IL-21R(-/-) mice. However, these mice exhibited a profound defect in generating long-lived plasma cells and in sustaining antibody levels over time. Similar results were seen after infection of IL-21R(-/-) mice with vesicular stomatitis virus and influenza virus. Using chimeric mice containing wild-type or IL-21R(-/-) CD4 T cells and B cells, we showed that both B and CD4 T cells need IL-21 signaling for generating long-term humoral immunity. Taken together, our results highlight the importance of IL-21 in humoral immunity to viruses.

Vitamin D receptor signals regulate effector and memory CD8 T cell responses to infections in mice.

BACKGROUND: Vitamin D insufficiency is associated with broad-ranging human disease sequelae such as bone disease, cancer, cardiovascular disease, allergy, autoimmune disorders, diabetes, and infectious diseases. Disease risk and severity of a large proportion of the nonskeletal disorders heavily involve the cytotoxic cluster of differentiation (CD) 8 T lymphocyte (CTL) arm of cellular adaptive immunity. Considering the importance of vitamin D in CTL-dependent diseases, there is a critical need for systematic in-depth explorations into the role of vitamin D deficiency in generation and maintenance of CTL immunity during infections and vaccinations. OBJECTIVE: With the use of wild-type (WT) vitamin D-sufficient mice and the vitamin D receptor knockout (Vdr(-/-)) mouse model of in vivo deficiency of vitamin D signaling, we systematically analyzed the impact of vitamin D deficiency on antigen-specific effector and memory CD8 T cell responses to acute viral and bacterial infections. METHODS: WT and Vdr(-/-) mice were infected with lymphocytic choriomeningitis virus, a natural mouse pathogen, and antigen-specific CTL responses were analyzed during priming, expansion, contraction, and memory phases. Magnitude, breadth, cytokine production, and localization of antiviral effector and memory CTLs to lymphoid and nonlymphoid tissues were specifically assessed. RESULTS: The absence of vitamin D signals led to 1) aberrant CD8 T cell effector differentiation (∼2-fold lower granzyme B and reduced B cell lymphoma 2; P ≤ 0.05) and enhanced contraction (∼15% increase; P ≤ 0.05) in antigen-specific CTLs; 2) a significantly restricted (P ≤ 0.05) breadth of the antigen-specific CD8 T cell effector and memory repertoire; and 3) preferential localization of effector (∼2.5-fold increase; P ≤ 0.01) and memory (∼5-fold increase; P ≤ 0.001) CD8 T cells to the lymph nodes compared to nonlymphoid tissues. CONCLUSION: Our data show a previously unrecognized impact of vitamin D deficiency on the quantity, quality, breadth, and location of CD8 T cell immunity to acute viral and bacterial infections.

Peripheral macrophages drive CNS disease in the Ndufs4(-/-) model of Leigh syndrome.

Subacute necrotizing encephalopathy, or Leigh syndrome (LS), is the most common pediatric presentation of genetic mitochondrial disease. LS is a multi-system disorder with severe neurologic, metabolic, and musculoskeletal symptoms. The presence of progressive, symmetric, and necrotizing lesions in the brainstem are a defining feature of the disease, and the major cause of morbidity and mortality, but the mechanisms underlying their pathogenesis have been elusive. Recently, we demonstrated that high-dose pexidartinib, a CSF1R inhibitor, prevents LS CNS lesions and systemic disease in the Ndufs4(-/-) mouse model of LS. While the dose-response in this study implicated peripheral immune cells, the immune populations involved have not yet been elucidated. Here, we used a targeted genetic tool, deletion of the colony-stimulating Factor 1 receptor (CSF1R) macrophage super-enhancer FIRE (Csf1rΔFIRE), to specifically deplete microglia and define the role of microglia in the pathogenesis of LS. Homozygosity for the Csf1rΔFIRE allele ablates microglia in both control and Ndufs4(-/-) animals, but onset of CNS lesions and sequalae in the Ndufs4(-/-), including mortality, are only marginally impacted by microglia depletion. The overall development of necrotizing CNS lesions is not altered, though microglia remain absent. Finally, histologic analysis of brainstem lesions provides direct evidence of a causal role for peripheral macrophages in the characteristic CNS lesions. These data demonstrate that peripheral macrophages play a key role in the pathogenesis of disease in the Ndufs4(-/-) model.

Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics.

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs.

Novel fluorobenzothiazole as a dual inhibitor of gyrase B and topoisomerase IV against Gram-positive pathogens.

Aim: The development of a novel inhibitor targeting gyrase B and topoisomerase IV offers an opportunity to combat multidrug resistance. Methods: We investigated the activity of RBx 10080758 against Gram-positive bacteria in vitro and in vivo. Results: RBx 10080758 showed a potent 50% inhibitory concentration of 0.13 µM and 0.25 µM against gyrase B and topoisomerase IV, respectively, and exhibited strong whole-cell in vitro activity with MIC ranges of 0.015-0.06 and 0.015-0.03 µg/ml against Staphylococcus aureus and Streptococcus pneumoniae, respectively. In a rat thigh infection model with methicillin-resistant S. aureus, RBx 10080758 at 45 mg/kg exhibited a >3 log10 CFU reduction in thigh muscles. Conclusion: RBx 10080758 displayed potent activity against multiple multidrug-resistant Gram-positive bacteria with a dual-targeting mechanism of action.

In vitro and in vivo activities of the novel Ketolide RBx 14255 against Clostridium difficile.

The MIC(90) of RBx 14255, a novel ketolide, against Clostridium difficile was 4 µg/ml (MIC range, 0.125 to 8 µg/ml), and this drug was found to be more potent than comparator drugs. An in vitro time-kill kinetics study of RBx 14255 showed time-dependent bacterial killing for C. difficile. Furthermore, in the hamster model of C. difficile infection, RBx 14255 demonstrated greater efficacy than metronidazole and vancomycin, making it a promising candidate for C. difficile treatment.

Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory.

Differential interleukin-2 (IL-2) signaling and production are associated with disparate effector and memory fates. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of IL-2 signaling and their differential impact on CD8 T cell responses remain unclear. Using distinct models of germline and conditional IL-2 ablation in post-thymic CD8 T cells, this study shows that paracrine IL-2 is sufficient to drive optimal primary expansion, effector and memory differentiation, and metabolic function. In contrast, autocrine IL-2 is uniquely required during primary expansion to program robust secondary expansion potential in memory-fated cells. This study further shows that IL-2 production by antigen-specific CD8 T cells is largely independent of CD4 licensing of dendritic cells (DCs) in inflammatory infections with robust DC activation. These findings bear implications for immunizations and adoptive T cell immunotherapies, where effector and memory functions may be commandeered through IL-2 programming.

Induction of thymic atrophy and loss of thymic output by type-I interferons during chronic viral infection.

Type-I interferon (IFN-I) signals exert a critical role in disease progression during viral infections. However, the immunomodulatory mechanisms by which IFN-I dictates disease outcomes remain to be fully defined. Here we report that IFN-I signals mediate thymic atrophy in viral infections, with more severe and prolonged loss of thymic output and unique kinetics and subtypes of IFN-α/ß expression in chronic infection compared to acute infection. Loss of thymic output was linked to inhibition of early stages of thymopoiesis (DN1-DN2 transition, and DN3 proliferation) and pronounced apoptosis during the late DP stage. Notably, infection-associated thymic defects were largely abrogated upon ablation of IFNαßR and partially mitigated in the absence of CD8 T cells, thus implicating direct as well as indirect effects of IFN-I on thymocytes. These findings provide mechanistic underpinnings for immunotherapeutic strategies targeting IFN-1 signals to manipulate disease outcomes during chronic infections and cancers.

Activity of RBx 11760, a novel biaryl oxazolidinone, against Clostridium difficile.

OBJECTIVES: RBx 11760, a novel oxazolidinone, was investigated for in vitro and in vivo activity against Clostridium difficile. METHODS: The in vitro activity of RBx 11760 and three other agents against 50 diverse C. difficile clinical isolates and other obligate anaerobic bacteria was determined. The effect of RBx 11760 on sporulation and toxin production was determined against different C. difficile isolates. We used a hamster infection model to investigate the efficacy of RBx 11760, vancomycin and metronidazole. The mechanism of action of RBx 11760 against C. difficile ATCC 43255 was determined by macromolecular synthesis inhibition. RESULTS: RBx 11760 MICs were in the range of 0.5-1 mg/L for C. difficile isolates, and it demonstrated concentration-dependent killing of C. difficile ATCC 43255 and C. difficile 6387 up to 2-4× MIC (1-2 mg/L). RBx 11760, at concentrations as low as 0.25-0.5 mg/L, resulted in a significant reduction in de novo toxin production as well as sporulation in different C. difficile isolates. In contrast, vancomycin, metronidazole and linezolid had little or no effect on toxin production and appeared to promote the formation of spores. In the hamster infection model, treatment with RBx 11760 resulted in prolonged survival of animals as compared with vancomycin or metronidazole, which correlated well with the histopathology results. Macromolecular labelling results suggest that RBx 11760 is a potent inhibitor of bacterial protein synthesis. CONCLUSIONS: RBx 11760 showed excellent in vitro and in vivo activity against C. difficile, and it could be a promising novel candidate for future drug development against C. difficile infection.