BackgroundNeutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes.ResultsTo better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected.ConclusionsDANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0072-y) contains supplementary material, which is available to authorized users.

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. We investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution.

The process of friction stir welding involves high tool forces and requires robust machinery; the forces involved make tool wear a predominant problem. As a result, many alternatives have been proposed in decreasing tool forces such as laser assisted friction stir welding and ultra-sound assisted friction stir welding. However, these alternatives are not commercially successful on a large scale due to scalability and capital/maintenance costs. In an attempt to reduce forces in a cost-feasible manner, electrically-assisted friction stir welding (EAFSW) is studied in this work. EAFSW is a result of applying the concept of electrically-assisted manufacturing (i.e., passing high direct electrical current through a workpiece during processing) to the conventional friction stir welding process. The concept of EAFSW is a relatively new adaptation of conventional frictional stir welding, which is well established. The expected benefits are reduction in the feed force and torque, which allow for improved processing productivity as well as the possibility for deeper penetration of the weld.

B cells have been long studied for their role and function in the humoral immune system. Apart from generating antibodies and an antibody-mediated memory response against pathogens, B cells are also capable of generating cell-mediated immunity. It has been demonstrated by several groups that B cells can activate antigen-specific CD4 and CD8 T cells, and can have regulatory and cytotoxic effects. The function of B cells as professional antigen presenting cells (APCs) to activate T cells has been largely understudied. This, however, requires attention as several recent reports have demonstrated the importance of B cells within the tumor microenvironment, and B cells are increasingly being evaluated as cellular therapies. Antigen presentation through B cells can be through antigen-specific (B cell receptor (BCR) dependent) or antigen non-specific (BCR independent) mechanisms and can be modulated by a variety of intrinsic and external factors. This review will discuss the pathways and mechanisms by which B cells present antigens, and how B cells differ from other professional APCs.

BackgroundSystemic radiation treatments that preferentially irradiate cancer cells over normal tissue, known as targeted radionuclide therapy (TRT), have shown significant potential for treating metastatic prostate cancer. Preclinical studies have demonstrated the ability of external beam radiation therapy (EBRT) to sensitize tumors to T cell checkpoint blockade. Combining TRT approaches with immunotherapy may be more feasible than combining with EBRT to treat widely metastatic disease, however the effects of TRT on the prostate tumor microenvironment alone and in combinfation with checkpoint blockade have not yet been studied.MethodsC57BL/6 mice-bearing TRAMP-C1 tumors and FVB/NJ mice-bearing Myc-CaP tumors were treated with a single intravenous administration of either low-dose or high-dose 90Y-NM600 TRT, and with or without anti-PD-1 therapy. Groups of mice were followed for tumor growth while others were used for tissue collection and immunophenotyping of the tumors via flow cytometry.Results90Y-NM600 TRT was safe at doses that elicited a moderate antitumor response. TRT had multiple effects on the tumor microenvironment including increasing CD8 +T cell infiltration, increasing checkpoint molecule expression on CD8 +T cells, and increasing PD-L1 expression on myeloid cells. However, PD-1 blockade with TRT treatment did not improve antitumor efficacy. Tregs remained functional up to 1 week following TRT, but CD8 +T cells were not, and the suppressive function of Tregs increased when anti-PD-1 was present in in vitro studies. The combination of anti-PD-1 and TRT was only effective in vivo when Tregs were depleted.ConclusionsOur data suggest that the combination of 90Y-NM600 TRT and PD-1 blockade therapy is ineffective in these prostate cancer models due to the activating effect of anti-PD-1 on Tregs. This finding underscores the importance of thorough understanding of the effects of TRT and immunotherapy combinations on the tumor immune microenvironment prior to clinical investigation.

Metastatic castration-resistant prostate cancer (mCRPC) is a challenging disease to treat, with poor outcomes for patients. One antitumor vaccine, sipuleucel-T, has been approved as a treatment for mCRPC. DNA vaccines are another form of immunotherapy under investigation. DNA immunizations elicit antigen-specific T cells that cause tumor cell lysis, which should translate to meaningful clinical responses. They are easily amenable to design alterations, scalable for large-scale manufacturing, and thermo-stable for easy transport and distribution. Hence, they offer advantages over other vaccine formulations. However, clinical trials with DNA vaccines as a monotherapy have shown only modest clinical effects against tumors. Standard therapies for CRPC including androgen-targeted therapies, radiation therapy and chemotherapy all have immunomodulatory effects, which combined with immunotherapies such as DNA vaccines, could potentially improve treatment. In addition, many investigational drugs are being developed which can augment antitumor immunity, and together with DNA vaccines can further enhance antitumor responses in preclinical models. We reviewed the literature available prior to July 2020 exploring the use of DNA vaccines in the treatment of prostate cancer. We also examined various approved and experimental therapies that could be combined with DNA vaccines to potentially improve their antitumor efficacy as treatments for mCRPC.

BackgroundPrevious studies of prostate cancer autoantibodies have largely focused on diagnostic applications. So far, there have been no reports attempting to more comprehensively profile the landscape of prostate cancer-associated antibodies. Specifically, it is unknown whether the quantity of antibodies or the types of proteins recognized change with disease progression.MethodsA peptide microarray spanning the amino acid sequences of the gene products of 1611 prostate cancer-associated genes was synthesized. Serum samples from healthy male volunteers (n=15) and patients with prostate cancer (n=85) were used to probe the array. These samples included patients with various clinical stages of disease: newly diagnosed localized prostate cancer (n=15), castration-sensitive non-metastatic prostate cancer (nmCSPC, n=40), castration-resistant non-metastatic prostate cancer (n=15) and castration-resistant metastatic disease (n=15). The patients with nmCSPC received treatment with either standard androgen deprivation therapy (ADT) or an antitumor DNA vaccine encoding prostatic acid phosphatase. Serial sera samples from these individuals were also used to probe the array, to secondarily determine whether this approach could be used to detect treatment-related changes.ResultsWe demonstrated that this peptide array yielded highly reproducible measurements of serum IgG levels. We found that the overall number of antibody responses did not increase with disease burden. However, the composition of recognized proteins shifted with clinical stage of disease. Our analysis revealed that the largest difference was between patients with castration-sensitive and castration-resistant disease. Patients with castration-resistant disease recognized more proteins associated with nucleic acid binding and gene regulation compared with men in other groups. Our longitudinal data showed that treatments can elicit antibodies detectable by this array, and notably vaccine-treated patients developed increased responses to more proteins over the course of treatment than did ADT-treated patients.ConclusionsThis study represents the largest survey of prostate cancer-associated antibodies to date. We have been able to characterize the classes of proteins recognized by patients and determine how they change with disease burden. Our findings further demonstrate the potential of this platform for measuring antigen spread and studying responses to immunomodulatory therapies.

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