Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

Pivotal role for a tail subunit of the RNA polymerase II mediator complex CgMed2 in azole tolerance and adherence in Candida glabrata.

Antifungal therapy failure can be associated with increased resistance to the employed antifungal agents. Candida glabrata, the second most common cause of invasive candidiasis, is intrinsically less susceptible to the azole class of antifungals and accounts for 15% of all Candida bloodstream infections. Here, we show that C. glabrata MED2 (CgMED2), which codes for a tail subunit of the RNA polymerase II Mediator complex, is required for resistance to azole antifungal drugs in C. glabrata. An inability to transcriptionally activate genes encoding a zinc finger transcriptional factor, CgPdr1, and multidrug efflux pump, CgCdr1, primarily contributes to the elevated susceptibility of the Cgmed2Δ mutant toward azole antifungals. We also report for the first time that the Cgmed2Δ mutant exhibits sensitivity to caspofungin, a constitutively activated protein kinase C-mediated cell wall integrity pathway, and elevated adherence to epithelial cells. The increased adherence of the Cgmed2Δ mutant was attributed to the elevated expression of the EPA1 and EPA7 genes. Further, our data demonstrate that CgMED2 is required for intracellular proliferation in human macrophages and modulates survival in a murine model of disseminated candidiasis. Lastly, we show an essential requirement for CgMed2, along with the Mediator middle subunit CgNut1 and the Mediator cyclin-dependent kinase/cyclin subunit CgSrb8, for the high-level fluconazole resistance conferred by the hyperactive allele of CgPdr1. Together, our findings underscore a pivotal role for CgMed2 in basal tolerance and acquired resistance to azole antifungals.

Importance of genotypic and phenotypic tolerance in the treatment of experimental endocarditis due to Streptococcus gordonii.

Genotypic and phenotypic tolerance was studied in penicillin treatment of experimental endocarditis due to nontolerant and tolerant Streptococcus gordonii and to their backcross transformants. The organisms were matched for in vitro and in vivo growth rates. Rats with aortic endocarditis were treated for 3 or 5 days, starting 12, 24, or 48 h after inoculation. When started at 12 h, during fast intravegetation growth, 3 days of treatment cured 80% of the nontolerant parent compared with <30% of the tolerant derivative (P < .005). When started at 24 or 48 h and if intravegetation growth had reached a plateau, 3 days of treatment failed against both bacteria. However, a significant difference between the 2 organisms was restored when treatment was extended to 5 days. Thus, genotypic tolerance conferred a survival advantage in both fast- and slow-growing bacteria, demonstrating that the in vitro-defined tolerant phenotype also carried the risk of treatment failure in vivo.

Error and attack tolerance of layered complex networks.

Many complex systems may be described by not one but a number of complex networks mapped on each other in a multi-layer structure. Because of the interactions and dependencies between these layers, the state of a single layer does not necessarily reflect well the state of the entire system. In this paper we study the robustness of five examples of two-layer complex systems: three real-life data sets in the fields of communication (the Internet), transportation (the European railway system), and biology (the human brain), and two models based on random graphs. In order to cover the whole range of features specific to these systems, we focus on two extreme policies of system's response to failures, no rerouting and full rerouting. Our main finding is that multi-layer systems are much more vulnerable to errors and intentional attacks than they appear from a single layer perspective.

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

Tolerance of whitefish embryos to Pseudomonas fluorescens linked to genetic and maternal effects, and reduced by previous exposure.

Juvenile or adult fish can alter their behaviour and rely on an innate and adaptive immune system to avoid/counteract pathogens, while fish embryos have to depend on egg characteristics and may be partly protected by their developing immune system that is building up from a certain age on. We developed an infection protocol that allows testing the reaction of individual whitefish embryos (Coregonus palaea) to repeated exposures to Pseudomonas fluorescens, an opportunistic bacterial fish pathogen. We used a full-factorial in vitro breeding design to separately test the effects of paternal and maternal contributions to the embryos' susceptibility to different kinds of pathogen exposure. We found that a first non-lethal exposure had immunosuppressive effects: pre-exposed embryos were more susceptible to future challenges with the same pathogen. At intermediate and high levels of pathogen intensity, maternal effects turned out to be crucial for the embryos' tolerance to infection. Paternal (i.e. genetic) effects played a significant role at the strongest level of infection, i.e. the embryos' own genetics already explained some of the variation in embryo susceptibility. Our findings suggest that whitefish embryos are largely protected by maternally transmitted substances, but build up some own innate immunocompetence several days before hatching.

Disentangling human tolerance and resistance against HIV.

In ecology, "disease tolerance" is defined as an evolutionary strategy of hosts against pathogens, characterized by reduced or absent pathogenesis despite high pathogen load. To our knowledge, tolerance has to date not been quantified and disentangled from host resistance to disease in any clinically relevant human infection. Using data from the Swiss HIV Cohort Study, we investigated if there is variation in tolerance to HIV in humans and if this variation is associated with polymorphisms in the human genome. In particular, we tested for associations between tolerance and alleles of the Human Leukocyte Antigen (HLA) genes, the CC chemokine receptor 5 (CCR5), the age at which individuals were infected, and their sex. We found that HLA-B alleles associated with better HIV control do not confer tolerance. The slower disease progression associated with these alleles can be fully attributed to the extent of viral load reduction in carriers. However, we observed that tolerance significantly varies across HLA-B genotypes with a relative standard deviation of 34%. Furthermore, we found that HLA-B homozygotes are less tolerant than heterozygotes. Lastly, tolerance was observed to decrease with age, resulting in a 1.7-fold difference in disease progression between 20 and 60-y-old individuals with the same viral load. Thus, disease tolerance is a feature of infection with HIV, and the identification of the mechanisms involved may pave the way to a better understanding of pathogenesis.

One-sided tolerance interval in a two-way balanced nested model with mixed effects

In many research areas (such as public health, environmental contamination, and others) one deals with the necessity of using data to infer whether some proportion (%) of a population of interest is (or one wants it to be) below and/or over some threshold, through the computation of tolerance interval. The idea is, once a threshold is given, one computes the tolerance interval or limit (which might be one or two - sided bounded) and then to check if it satisfies the given threshold. Since in this work we deal with the computation of one - sided tolerance interval, for the two-sided case we recomend, for instance, Krishnamoorthy and Mathew [5]. Krishnamoorthy and Mathew [4] performed the computation of upper tolerance limit in balanced and unbalanced one-way random effects models, whereas Fonseca et al [3] performed it based in a similar ideas but in a tow-way nested mixed or random effects model. In case of random effects model, Fonseca et al [3] performed the computation of such interval only for the balanced data, whereas in the mixed effects case they dit it only for the unbalanced data. For the computation of twosided tolerance interval in models with mixed and/or random effects we recomend, for instance, Sharma and Mathew [7]. The purpose of this paper is the computation of upper and lower tolerance interval in a two-way nested mixed effects models in balanced data. For the case of unbalanced data, as mentioned above, Fonseca et al [3] have already computed upper tolerance interval. Hence, using the notions persented in Fonseca et al [3] and Krishnamoorthy and Mathew [4], we present some results on the construction of one-sided tolerance interval for the balanced case. Thus, in order to do so at first instance we perform the construction for the upper case, and then the construction for the lower case.

Tolerance and reactivity of NK cells: two sides of the same coin?

NK cells can kill transformed, infected and stressed cells while most normal cells are spared. NK cells are activated by various endogenous self-ligands, some of which are actually expressed by normal cells. Thus, NK cells are inherently self-reactive and consequently, potentially auto-aggressive. How these cells are prevented from attacking normal cells while ensuring reactivity to diseased cells is a major unresolved question for NK-cell biologists.

Lung microbiota promotes tolerance to allergens in neonates via PD-L1.

Epidemiological data point toward a critical period in early life during which environmental cues can set an individual on a trajectory toward respiratory health or disease. The neonatal immune system matures during this period, although little is known about the signals that lead to its maturation. Here we report that the formation of the lung microbiota is a key parameter in this process. Immediately following birth, neonatal mice were prone to develop exaggerated airway eosinophilia, release type 2 helper T cell cytokines and exhibit airway hyper-responsiveness following exposure to house dust mite allergens, even though their lungs harbored high numbers of natural CD4(+)Foxp3(+)CD25(+)Helios(+) regulatory T (Treg) cells. During the first 2 weeks after birth, the bacterial load in the lungs increased, and representation of the bacterial phyla shifts from a predominance of Gammaproteobacteria and Firmicutes towards Bacteroidetes. The changes in the microbiota were associated with decreased aeroallergen responsiveness and the emergence of a Helios(-) Treg cell subset that required interaction with programmed death ligand 1 (PD-L1) for development. Absence of microbial colonization(10) or blockade of PD-L1 during the first 2 weeks postpartum maintained exaggerated responsiveness to allergens through to adulthood. Adoptive transfer of Treg cells from adult mice to neonates before aeroallergen exposure ameliorated disease. Thus, formation of the airway microbiota induces regulatory cells early in life, which, when dysregulated, can lead to sustained susceptibility to allergic airway inflammation in adulthood.

Longer Intervals Between Hematopoietic Stem Cell Transplantation and Subsequent 90Y-Ibritumomab Radioimmunotherapy May Correlate With Better Tolerance.

PURPOSE: This study aimed to evaluate the efficacy and toxicity of radioimmunotherapy (RIT) in recurrent lymphoma after hematopoietic stem cell transplantation (HSCT). METHODS: We reviewed 9 patients, 7 with follicular lymphoma (DLBCL), 1 with mantle cell lymphoma (MCL), and 1 with diffuse large B-cell lymphoma treated with Y-ibritumomab tiuxetan 6 to 140 months after HSCT. Patients underwent In-ibritumomab scintigraphy and were treated 1 week later with standard 14.8 MBq/kg (n = 4) or 11.1 MBq/kg (n = 4) Y-ibritumomab. One patient who had allo-HSCT had reduced activity (70%) treatment. RESULTS: Among the 7 FL patients, we observed complete response (CR) in 2 patients and partial response (PR) in 5 patients. One patient with CR relapsed after 15 months; the other persisted 43.5 months after RIT. Of 5 patients with PR, 3 relapsed between 13 and 17 months; 1 persisted until unrelated death at 11.5 months. The fifth patient with PR received adoptive immunotherapy and improved to metabolic (FDG-PET) CR that persists 45.5 and 41 months after Y-ibritumomab and immunotherapy, respectively. Patients with MCL and DLBCL progressed or experienced stabilization (5 months), respectively. Six patients had grade 1 to 3 bone marrow (BM) toxicity and recovered within 3 months. Three patients having Y-ibritumomab 6, 14, and 24 months after HSCT experienced grade 4 BM toxicity. One of them (RIT 24 months after HSCT) recovered after 3 months, another delayed after 9 months, and the third patient only partially recovered, eventually developed myelodysplasia, and was allografted. CONCLUSIONS: Radioimmunotherapy after HSCT is an effective rescue therapy in FL. However, BM toxicity may be important; 3 of 8 patients treated with standard Y-ibritumomab activity experienced grade 4 BM toxicity, with incomplete recovery 3 months after RIT in 2 patients, both treated early (6 and 14 months) after HSCT.

Selectively expanding subsets of T cells in mice by injection of interleukin-2/antibody complexes: implications for transplantation tolerance.

The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti-IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti-IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.

Sedation and analgesia for colonoscopy: patient tolerance, pain, and cardiorespiratory parameters.

BACKGROUND: Colonoscopy is generally performed with the patient sedated and receiving analgesics. However, the benefit of the most often used combination of intravenous midazolam and pethidine on patient tolerance and pain and its cardiorespiratory risk have not been fully defined. METHODS: In this double-blind prospective study, 150 outpatients undergoing routine colonoscopy were randomly assigned to receive either (1) low-dose midazolam (35 micrograms/kg) and pethidine (700 micrograms/kg in 48 patients, 500 micrograms/kg in 102 patients), (2) midazolam and placebo pethidine, or (3) pethidine and placebo midazolam. RESULTS: Tolerance (visual analog scale, 0 to 100 points: 0 = excellent; 100 = unbearable) did not improve significantly more in group 1 compared with group 2 (7 points; 95% confidence interval [-2-17]) and group 3 (2 points; 95% confidence interval [-7-12]). Similarly, pain was not significantly improved in group 1 as compared with the other groups. Male gender (p < 0.001) and shorter duration of the procedure (p = 0.004), but not amnesia, were associated with better patient tolerance and less pain. Patient satisfaction was similar in all groups. Oxygen desaturation and hypotension occurred in 33% and 11%, respectively, with a similar frequency in all three groups. CONCLUSIONS: In this study, the combination of low-dose midazolam and pethidine does not improve patient tolerance and lessen pain during colonoscopy as compared with either drug given alone. When applying low-dose midazolam, oxygen desaturation and hypotension do not occur more often after combined use of both drugs. For the individual patient, sedation and analgesia should be based on the endoscopist's clinical judgement.

In vivo mechanisms leading to transplantation tolerance induced by regulatory T cells

Purpose: The mechanisms by which CD4+CD25+Foxp3+ T cells (Tregs) regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer and skin transplantation model, we analyzed the in vivo expansion, effector function and trafficking of effector T cells and donor-specific Tregs, in response to an allograft. Methods and materials: Antigen-specific Tregs were generated and expanded in vitro by culturing freshly isolated Tregs from BALB/c mice (H2d) with syngeneic dendritic cells pulsed with an allopeptide (here the Kb peptide derived from the MHC class I molecule of allogeneic H2b mice). Fluorescent-labelled CD4+CD25- naive T cells and donor-antigen-specific Tregs were transferred alone or coinjected into syngeneic BALB/c-Nude recipients transplanted with allogeneic C57BL/6xBALB/c donor skin. Results: As opposed to their in vitro hyporesponsiveness, Tregs divided in vivo, migrated and accumulated in the allograft draining lymph nodes (drLN) and within the graft. The co-transfer of Tregs did not modify the early proliferation and homing of CD4+CD25- T cells to secondary lymphoid organs. But, in the presence of Tregs, effector T cells produced significantly less IFN- and IL-2 effector cytokines, while higher amounts of IL-10 were detected in the spleen and drLN of these mice. Furthermore, time-course studies showed that Tregs were recruited into the allograft at a very early stage posttransplantation and prevented infiltration by effector T cells. Conclusion: Overall, our results suggest that suppression of graft rejection involves the early recruitment of donor-specific Tregs at the sites of antigenic challenge and that Tregs mainly regulate the effector arm of T cell alloresponses.