The signaling function of IDO1 incites the malignant progression of mouse B16 melanoma.

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene (Ido1WT ) or a mutated variant lacking the catalytic, but not signaling activity (Ido1H350A ). As compared to the Ido1WT -transfected counterpart (B16WT), B16-F10 cells expressing Ido1H350A (B16H350A) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16H350A cells, also detectable in vivo, were found to be accompanied by a reduction in tumor-infiltrating CD8+ T cells and an increase in Foxp3+ regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule.

Cross-sectional serosurvey of Coxiella burnetii in healthy cattle and sheep from extensive grazing system in central Italy.

A cross-sectional survey was carried out to estimate the seroprevalence of Coxiella burnetii in extensively grazed cattle and sheep from central Italy and to identify the related risk factors. Data on notified human Q fever cases in the area were also collected and described. A two-stage cluster sampling was performed. A total of 5083 animals (2210 cattle; 2873 sheep) belonging to 186 farms (92 herds; 94 flocks) were tested for the presence of antibodies against C. burnetii using a commercial enzyme-linked immunosorbent assay kit. The prevalence at the animal-level resulted three times higher in sheep compared to cattle (37.8% vs. 12.0%; χ2 = 270.10, P < 0.001). The prevalence at the herd-level was also higher in sheep than in cattle (87.2% vs. 68.5%; χ2 = 9.52, P < 0.01). The multivariate analysis showed a higher risk of seropositivity for cattle aged 67-107 months (OR 2.79, 95% CI 1.86-4.18), cattle >107 months of age (OR 2.07, 95% CI 1.36-3.14) and mixed breed cattle (OR 1.74, 95% CI 1.11-2.72). A herd size >92 animals was recognized as herd-level risk factor in cattle (OR 6.88, 95% CI 1.67-28.37). The risk of being seropositive was double in sheep belonging to flocks >600 animals (odds ratio (OR) 2.04, 95% CI 1.63-2.56). Sheep were confirmed to be the most exposed species. Nevertheless, the prevalence observed in cattle also suggests the potential involvement of this species in the circulation of the pathogen in the area. Seven confirmed human Q fever cases were reported. In five out of seven cases there was at least one exposed herd within a 5 km buffer. Even though the source of the infection was not identified, the possibility of C. burnetii circulating in the livestock and human population in the study area cannot be overlooked. The integration between veterinary and human surveillance will be crucial to understand the spread of this zoonosis and to support the adoption of appropriate control measures.

Identification of a 2-propanol analogue modulating the non-enzymatic function of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3 dioxygenase 1 (IDO1) is a metabolic enzyme that catalyzes the conversion of the essential amino acid tryptophan (Trp) into a series of immunoactive catabolites, collectively known as kynurenines. Through the depletion of Trp and the generation of kynurenines, IDO1 represents a key regulator of the immune responses involved in physiologic homeostasis as well as in neoplastic and autoimmune pathologies. The IDO1 enzyme has been described as an important immune checkpoint to be targeted by catalytic inhibitors in the treatment of cancer. In contrast, a defective expression/activity of the enzyme has been demonstrated in autoimmune diseases. Beside its catalytic activity, the IDO1 protein is endowed with an additional function associated with the presence of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which, once phosphorylated, bind SHP phosphatases and mediate a long-term immunoregulatory activity of IDO1. Herein, we report the screening of a focused library of molecules bearing a propanol core by a protocol combining microscale thermophoresis (MST) analysis and a cellular assay. As a result, the combined screening identified a 2-propanolol analogue, VIS351, as the first potent activator of the ITIM-mediated function of the IDO1 enzyme. VIS351 displayed a good dissociation constant (Kd = 1.90 µM) for IDO1 and a moderate cellular inhibitor activity (IC50 = 11.463 µM), although it did not show any catalytic inhibition of the recombinant IDO1 enzyme. Because we previously demonstrated that the enzymatic and non-enzymatic (i.e., ITIM-mediated) functions of IDO1 reside in different conformations of the protein, we hypothesized that in the cellular system VIS351 may shift the dynamic conformational balance towards the ITIM-favoring folding of IDO1, resulting in the activation of the signaling rather than catalytic activity of IDO1. We demonstrated that VIS351 activated the ITIM-mediated signaling of IDO1 also in mouse plasmacytoid dendritic cells, conferring those cells an immunosuppressive phenotype detectable in vivo. Thus the manuscript describes for the first time a small molecule as a positive modulator of IDO1 signaling function, paving the basis for an innovative approach to develop first-in-class drugs acting on the IDO1 target.

Negative hyper-selection of metastatic colorectal cancer patients for anti-EGFR monoclonal antibodies: the PRESSING case-control study.

BACKGROUND: Refining the selection of metastatic colorectal cancer patients candidates for anti-epidermal growth factor receptor (EGFR) monoclonal antibodies beyond RAS and BRAF testing is a challenge of precision oncology. Several uncommon genomic mechanisms of primary resistance, leading to activation of tyrosine kinase receptors other than EGFR or downstream signalling pathways, have been suggested by preclinical and retrospective studies. PATIENTS AND METHODS: We conducted this multicentre, prospective, case-control study to demonstrate the negative predictive impact of a panel of rare genomic alterations [PRESSING (PRimary rESiStance IN RAS and BRAF wild-type metastatic colorectal cancer patients treated with anti-eGfr monoclonal antibodies) panel], including HER2/MET amplifications, ALK/ROS1/NTRK1-3/RET fusions and PIK3CA mutations. Hypothesizing a prevalence of candidate alterations of 15% and 0% in resistant and sensitive RAS and BRAF wild-type patients, respectively, with two-sided α and ß errors of 0.05 and 0.20, 47 patients per group were needed. RESULTS: Forty-seven patients per group were included. PRESSING panel alterations were significantly more frequent in resistant (24 out of 47, 51.1%) than in sensitive (1 out of 47, 2.1%) patients (P < 0.001) and in right- (12 out of 29, 41.4%) than left-sided (13 out of 65, 20.0%) tumours (P = 0.03). The predictive accuracy of PRESSING panel and sidedness was 75.3% and 70.2%, respectively. Among hyper-selected patients, right-sidedness was still associated with resistance (P = 0.002). The predictive accuracy of the combined evaluation of PRESSING panel and sidedness was 80.4%. As a secondary analysis, 8 (17.0%) resistant and 0 sensitive patients showed microsatellite instability (P < 0.001). CONCLUSION: The investigated panel of genomic alterations allows refining the selection of RAS and BRAF wild-type metastatic colorectal cancer patients candidates for anti-EGFRs, partially explaining and further corroborating the predictive ability of primary tumour sidedness.

HER2 loss in HER2-positive gastric or gastroesophageal cancer after trastuzumab therapy: Implication for further clinical research.

Mechanisms of acquired resistance to trastuzumab-based treatment in gastric cancer are largely unknown. In this study, we analyzed 22 pairs of tumor samples taken at baseline and post-progression in patients receiving chemotherapy and trastuzumab for advanced HER2-positive [immunohistochemistry (IHC) 3+ or 2+ with in-situ hybridization (ISH) amplification] gastric or gastroesophageal cancers. Strict clinical criteria for defining acquired trastuzumab resistance were adopted. Loss of HER2 positivity and loss of HER2 over-expression were defined as post-trastuzumab IHC score <3+ and absence of ISH amplification, and IHC "downscoring" from 2+/3+ to 0/1+, respectively. HER2 IHC was always performed, while ISH was missing in 3 post-progression samples. Patients with initial HER2 IHC score 3+ and 2+ were 14 (64%) and 8 (36%), respectively. Loss of HER2 positivity and HER2 over-expression was observed in 32 and 32% samples, respectively. The chance of HER2 loss was not associated with any of the baseline clinicopathological variables. The only exception was in patients with initial IHC score 2+ versus 3+, for both endpoints of HER2 positivity (80 vs. 14%; p = 0.008) and HER2 over-expression (63 vs. 14%; p = 0.025). As already shown in breast cancer, loss of HER2 may be observed also in gastric cancers patients treated with trastuzumab-based chemotherapy in the clinical practice. This phenomenon may be one of the biological reasons explaining the failure of anti-HER2 second-line strategies in initially HER2-positive disease.

Functional characterisation of wheat Pgip genes reveals their involvement in the local response to wounding.

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall leucine-rich repeat (LRR) proteins involved in plant defence. The hexaploid wheat (Triticum aestivum, genome AABBDD) genome contains one Pgip gene per genome. Tapgip1 (B genome) and Tapgip2 (D genome) are expressed in all tissues, whereas Tapgip3 (A genome) is inactive because of a long terminal repeat, Copia retrotransposon insertion within the coding region. To verify whether Tapgip1 and Tapgip2 encode active PGIPs and are involved in the wheat defence response, we expressed them transiently and analysed their expression under stress conditions. Neither TaPGIP1 nor TaPGIP2 showed inhibition activity in vitro against fungal polygalacturonases. Moreover, a wheat genotype (T. turgidum ssp. dicoccoides) lacking active homologues of Tapgip1 or Tapgip2 possesses PGIP activity. At transcript level, Tapgip1 and Tapgip2 were both up-regulated after fungal infection and strongly induced following wounding. This latter result has been confirmed in transgenic wheat plants expressing the ß-glucuronidase (GUS) gene under control of the 5'-flanking region of Tdpgip1, a homologue of Tapgip1 with an identical sequence. Strong and transient GUS staining was mainly restricted to the damaged tissues and was not observed in adjacent tissues. Taken together, these results suggest that Tapgips and their homologues are involved in the wheat defence response by acting at the site of the lesion caused by pathogen infection.

Cytotoxic T lymphocyte antigen 4-immunoglobulin G is a potent adjuvant for experimental allergen immunotherapy.

Allergen-specific immunotherapy (SIT) is the only treatment for allergic diseases that targets allergen-specific T helper type 2 (Th2) cells, which are the cause of the disease. There is an unmet requirement for adjuvants that increase the clinical efficacy of SIT allowing application of lower doses of the allergen, thereby reducing the risk of anaphylactic reactions. Cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA-4-Ig) has been shown to induce immunological tolerance in autoimmunity and allograft transplantation by blocking T cell co-stimulation and induction of the immunoregulatory enzyme indoleamine 2,3 dioxygenase (IDO). Previously, we showed that CTLA-4-Ig treatment at the time of allergen inhalation induced tolerance to subsequent allergen exposure in a mouse model of asthma. In this study, we test the hypothesis that CTLA-4-Ig acts as an adjuvant for experimental SIT. We evaluated the adjuvant effects of CTLA-4-Ig on SIT in a mouse model of ovalbumin-driven asthma. We used both wild-type and IDO-deficient mice to assess the role of IDO in the adjuvant effects of CTLA-4-Ig. Co-administration of CTLA-4-Ig strongly increased SIT-induced suppression of airway hyperreactivity (AHR), specific IgE in serum, airway eosinophilia and Th2 cytokine levels. Moreover, we found that CTLA-4-Ig, as an adjuvant for SIT, is equally effective in IDO-deficient and wild-type mice, demonstrating that the effect of CTLA-4-Ig is independent of IDO expression. We show that CTLA-4-Ig acts as a potent adjuvant to augment the therapeutic effects of SIT. As the adjuvant activity of CTLA-4-Ig is independent of IDO, we conclude that it acts by blocking CD28-mediated T cell co-stimulation.

Targeting metabotropic glutamate receptors in neuroimmune communication.

L-Glutamate (L-Glu) is the principal excitatory neurotransmitter in the Central Nervous System (CNS), where it regulates cellular and synaptic activity, neuronal plasticity, cell survival and other relevant functions. Glutamatergic neurotransmission is complex and involves both ionotropic (ligand-gated ion channels; iGluRs) and metabotropic receptors (G-protein coupled receptors). Recent evidence suggests that glutamatergic receptors are also expressed by immune cells, regulating the degree of cell activation. In this review we primarily focus on mGluRs and their role in the crosstalk between the central nervous and immune systems during neuroinflammation.

Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans.

We analyzed the contribution of intracellular signaling to the functional plasticity of dendritic cells (DCs) presenting Candida albicans, a human commensal associated with severe diseases. Distinct intracellular pathways were activated by recognition of different fungal morphotypes in distinct DC subsets and in Peyer's patches DCs. Inflammatory DCs initiated Th17/Th2 responses to yeasts through the adaptor myeloid differentiation factor-88 (MyD88), whereas tolerogenic DCs activate Th1/T regulatory cell (Treg) differentiation programs to hyphae involving Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) as an intermediary of signaling. In addition, signal transducer and activator of transcription 3 (STAT3), affecting the balance between canonical and non-canonical activation of nuclear factor-kappaB (NF-kappaB) and 2,3 indoleamine dioxygenase (IDO), pivotally contributed to DC plasticity and functional specialization. As Candida-induced tolerogenic DCs ameliorated experimental colitis, our data qualify Candida as a commensal with immunoregulatory activity, resulting from the orchestrated usage of multiple, yet functionally distinct, receptor-signaling pathways in DCs. Ultimately, affecting the local Th17/Treg balance might likely be exploited by the fungus for either commensalism or pathogenicity.

Early posttransplant lymphoproliferative disease in pediatric liver transplant recipients.

Among 23 pediatric patients who underwent orthotopic liver transplant (OLT), we report two (11 and 26 months old) with posttransplant lymphoproliferative disease (PTLD) that occurred in the early posttransplantation period. They were Epstein-Barr Virus (EBV)-negative and received graft from EBV-positive donors. The surveillance for EBV viremia using serial EBV polymerase chain reaction determinations in the peripheral blood was positive at 10 and 90 days after OLT concomitant with symptoms of primary infection, both patients were treated with gancyclovir. The patients should progression to a Burkitt's and a non-Hodgkin's lymphoma that appeared 3 months posttransplantation. They were treated by withdrawal of immunosuppression and six courses of cyclophosphamide as well as anti-CD20 monoclonal antibody (Rituximab) every 21 days. One patient experienced acute graft rejection, which resolved with steroids and low doses of tacrolimus, she is free of disease at 24 months after the end of treatment. The other patient relapsed with a cerebral lymphoma, receiving aggressive chemotherapy, but died due to sepsis. In conclusion, PTLD occurred among in 2/23 patients who underwent OLT and appeared in the first quarter post OLT. The risk factors associated with early PTLD were primary EBV infection after OLT, young age, and EBV-negative recipient receiving a transplant from an EBV-positive donor. Antiviral treatment alone was inefficient; withdrawal of immunosuppression and courses of Rituximab and cyclophosphamide were well tolerated and controlled PTLD. The risk of graft rejection was increased by withdrawal of immunosuppression. One patient died.

Hydroxyurea chemotherapy for meningiomas: enlarged cohort with extended follow-up.

Meningiomas account for 18-20% of all intracranial tumours and often recur despite surgical resection. Hydroxyurea is under evaluation as adjuvant therapy of meningiomas. In the authors' initial report of 17 patients with meningioma, hydroxyurea demonstrated modest efficacy, with a median time to progression (TTP) of 80 weeks. In the current study, 21 patients with meningioma have been placed on hydroxyurea (20 mg/kg/day orally), with extended follow-up of the original cohort. Eighteen of 20 evaluable patients (90%) responded with stable disease ranging from 20 to 328 + weeks (median TTP 176 weeks; 11 patients censored). Five of the stabilized patients progressed after 20, 56, 36, 216 and 56 weeks, respectively. Two patients had progressive disease after 10 weeks. Toxicity was mainly haematological. Hydroxyurea has modest activity against meningiomas and should be considered for patients who are poor surgical candidates, have unresectable or large residual meningiomas, or have progressed after surgical resection or irradiation, or both.

Morphologic evolution of CAH with and without therapy. A two year follow-up.

The morphologic evolution of chronic active hepatitis (CAH) over a two-year period in 57 post-puberty patients with homogeneous characteristics was studied retrospectively. Of these, 29 received therapy with steroids and immunosuppressive drugs while 28 received no therapy. At the end of the 2-year period, the statistically assessed morphologic results showed that the therapy had had a positive effect on the initial morphologic pattern.

Chenodeoxycholic acid (CDCA) versus ursodeoxycholic acid (UDCA): a comparison of their effects in pregnant rats.

Treatment of pregnant rats with ursodeoxycholic acid or chenodeoxycholic acid at 3 dose levels resulted in no dismorphogenic effects although embryotoxicity and fatty infiltration in maternal liver were more frequent after CDCA. Light microscopic examination showed no evidence of derangement in fetal liver. Electron microscopic study, carried out in maternal liver, showed that changes were present only in dams treated with the highest doses of both bile acids.