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Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming-TAM-populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, Fc gamma RIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment.

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells(1,2). CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels)(2). DSBs may also be repaired by homology-directed repair (HDR)(1,2) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations(3). Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient(3-8) and can be corrupted by additional indels(9), preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono-and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Casblocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe))(10) and presenilin 1 (PSEN1M146V)(11) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

Clinical disease caused by weakly pathogenic mycobacterial species, which is known as Mendelian susceptibility to mycobacterial disease (MSMD), is a rare entity. IFN-gamma and IL-17 production are defective due to insufficient response to IL-2 and IL-23 in IL-12R beta 1 deficiency; so this also causes tendency to intracellular microorganisms and candidal diseases. Here, we present a patient who suffers IL-12R beta 1 deficiency caused by a novel bi-allelic mutation with recurrent salmonellosis, mycobacterial, fungal infections and remained asymptomatic during 13 months of follow-up after hIFN-gamma treatment. In addition she had hemolytic anemia and midline defects like cleft lip and palate which have not been reported in a patient with MSMD in the literature prior to this case report. In conclusion, diagnosis of MSMD should be kept in mind in patients with recurrent salmonellosis, mycobacterial and fungal infections especially in countries with a high consanguinity rate.

Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pT(reg) cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure for the 'instruction' of naive CD4(+) T cells to differentiate into pT(reg) cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs were dispensable, while classical dendritic cells (cDCs) were critical, for pT(reg) cell induction and oral tolerance. CD11b(-) cDCs from the gut-draining lymph nodes efficiently induced pT(reg) cells and, conversely, loss of transcription factor IRF8-dependent CD11b(-) cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in the induction of pT(reg) cells and their redundancy during the development of oral tolerance.

The elevation of cancer antigen 125 (CA125) levels in the serum of asymptomatic patients precedes the radiologic detection of high-grade serous ovarian cancer by at least 2 mo and the final clinical diagnosis by 5 mo. PET imaging of CA125 expression by ovarian cancer cells may enhance the evaluation of the extent of disease and provide a roadmap to surgery as well as detect recurrence and metastases. Methods: Zr-89-labeled mAb-B43.13 was synthesized to target CA125 and evaluated via PET imaging and biodistribution studies in mice bearing OVCAR3 human ovarian adenocarcinoma xenografts. Ex vivo analysis of tumors and lymph nodes was performed via autoradiography, histopathology, and immunohistochemistry. Results: PET imaging using Zr-89-DFO-mAb-B43.13 (DFO is desferrioxamine) clearly delineated CA125-positive OVCAR3 xenografts as early as 24 h after the administration of the radioimmunoconjugate. Biodistribution studies revealed accretion of Zr-89-DFO-mAb-B43.13 in the OVCAR3 tumors, ultimately reaching 22.3 +/- 6.3 percentage injected dose per gram (%ID/g) at 72 h after injection. Most interestingly, activity concentrations greater than 50 %ID/g were observed in the ipsilateral lymph nodes of the xenograft-bearing mice. Histopathologic analysis of the immuno-PET positive lymph nodes revealed the presence of grossly metastasized ovarian cancer cells within the lymphoid tissues. In control experiments, only low-level, non-specific uptake of 89Zr-labeled isotype IgG was observed in OVCAR3 tumors; similarly, low-activity concentrations of Zr-89-DFO-mAb-B43.13 accumulated in CA125-negative SKOV3 tumors. Conclusion: Immuno-PET with Zr-89-labeled mAb-B43.13 is a potential strategy for the noninvasive delineation of extent of disease and may add value in treatment planning and treatment monitoring of high-grade serous ovarian cancer.

L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na+ channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na+ current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na+ channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na+ channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na+ channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity.

Background: Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. Objectives:We sought to report longer-term tofacitinib efficacy and safety in patients with moderate to severe psoriasis. Methods: Data from 2 identical phase-III studies, Oral-treatment Psoriasis Trial Pivotal 1 and 2, were pooled with data from these patients in an ongoing open-label long-term extension study. Patients (n = 1861) were randomized 2:2:1 to tofacitinib 5 mg, 10 mg, or placebo twice daily (BID). At week 16, placebo patients were rerandomized to tofacitinib. Pivotal study participants could enroll into the long-term extension where they received tofacitinib at 10 mg BID for 3 months, after which dosing could be 5 or 10 mg BID. Results: At week 28, the proportions of patients randomized to tofacitinib 5 and 10 mg BID achieving 75% or greater reduction in Psoriasis Area and Severity Index score from baseline were 55.6% and 68.8%, and achieving Physician Global Assessment of clear or almost clear were 54.7% and 65.9%. Efficacy was maintained in most patients through 24 months. Serious adverse events and discontinuations because of adverse events were reported in less than 11% of patients over 33 months of tofacitinib exposure. Limitations: There was no dose comparison beyond week 52. Conclusions: Oral tofacitinib demonstrated sustained efficacy in patients with psoriasis through 2 years, with 10 mg BID providing greater efficacy than 5 mg BID. No unexpected safety findings were observed.

Certain components and functions of the immune system, most notably cytokine production and immune cell migration, are under circadian regulation. Such regulation suggests that circadian rhythms may have an effect on disease onset, progression, and resolution. In the vesicular stomatitis virus (VSV)-induced encephalitis model, the replication, caudal penetration, and survivability of intranasally applied VSV depends on both innate and adaptive immune mechanisms. In the current study, we investigated the effect of circadian time of infection on the progression and outcome of VSV-induced encephalitis and demonstrated a significant decrease in the survival rate in mice infected at the start of the rest cycle, zeitgeber time 0 (ZT0). The lower survival rate in these mice was associated with higher levels of circulating chemokine (C-C motif) ligand 2 (CCL2), a greater number of peripherally derived immune cells accumulating in the olfactory bulb (OB), and increased production of proinflammatory cytokines, indicating an immune-mediated pathology. We also found that the acrophase of molecular circadian clock component REV-ERB alpha mRNA expression in the OB coincides with the start of the active cycle, ZT12, when VSV infection results in a more favorable outcome. This result led us to hypothesize that REV-ERB alpha may mediate the circadian effect on survival following VSV infection. Blocking REV-ERB alpha activity before VSV administration resulted in a significant increase in the expression of CCL2 and decreased survival in mice infected at the start of the active cycle. These data demonstrate that REV-ERB alpha-mediated inhibition of CCL2 expression during viral- induced encephalitis may have a protective effect.

Background: beta-Amyloid (A beta) is a key pathologic element in Alzheimer's disease (AD), but the mechanisms by which it disrupts neuronal function in vivo are not completely understood. AD is characterized by a prothrombotic state, which could contribute to neuronal dysfunction by affecting cerebral blood flow and inducing inflammation. The plasma protein factor XII triggers clot formation via the intrinsic coagulation cascade, and has been implicated in thrombosis. Objectives: To investigate the potential for A beta to contribute to a prothrombotic state. Methods and results: We show that A beta activates FXII, resulting in FXI activation and thrombin generation in human plasma, thereby establishing A beta as a possible driver of prothrombotic states. We provide evidence for this process in AD by demonstrating decreased levels of FXI and its inhibitor C1 esterase inhibitor in AD patient plasma, suggesting chronic activation, inhibition and clearance of FXI in AD. Activation of the intrinsic coagulation pathway in AD is further supported by elevated fibrin levels in AD patient plasma. Conclusions: The ability of A beta to promote coagulation via the FXII-driven contact system identifies new mechanisms by which it could contribute to neuronal dysfunction and suggests potential new therapeutic targets in AD.