Photodynamic therapy (PDT) is a minimally invasive therapeutic strategy for cancer treatment, which can destroy local tumor cells and induce systemic antitumor immune response, whereas, focusing on improving direct cytotoxicity to tumor cells treated by PDT, there is growing interest in developing approaches to further explore the immune stimulatory properties of PDT. In this review we summarize the current knowledge of the innate and adaptive immune responses induced by PDT against tumors, providing evidence showing PDT facilitated-antitumor immunity. Various immunotherapeutic approaches on different cells are reviewed for their effectiveness in improving the treatment efficiency in concert with PDT. Future perspectives are discussed for further enhancing PDT efficiency via intracellular targetable drug delivery as well as optimized experimental model development associated with the study of antitumor immune response. Photodynamic therapy (PDT) has been developed as a minimally invasive therapeutic modality for the treatment of various cancers as well as nonmalignant diseases , 2. PDT  in vivo microenvironment of solid tumor can be essentially classified into (1) the bulk of solid tumor made up by tumor cells; (2) vasculature surrounding the tumor structure formed by endothelial cells; and (3) interstitial space comprised of stromal cells and immune cells [ in vivo involves three distinct mechanisms targeting all above three major components of solid tumor tissue: (1) PDT-generated oxidative stress can directly cause apoptosis and necrosis of tumor cells; (2) PDT can destruct tumor-associated vasculature, leading to tumor infarction; (3) PDT can activate an acute inflammatory and induce host-defense immune response that can consequently kill tumor cells [Thene cells , 10. It or cells , 12.Despite its multifaceted advantages, PDT has not yet become the mainstream of cancer intervention mainly due to its insufficient therapeutic efficacy, low targeting capability of currently available photosensitizers, and limited light penetration for deep tumor tissues , 14. In Besides above techniques, recognizing the importance of the immunological contribution to the effect of PDT, there are growing interests in developing approaches to induce and facilitate the antitumor immune response following PDT. In this review we will discuss the effect of PDT of cancer on the immune response, summarize recent progress on employing PDT-induced antitumor immunity to achieve better therapeutic outcomes, and propose future perspectives for better design of PDT protocols based on the understanding of tumor-immune system interaction.Although PDT is a local treatment, tumor structure destruction is accompanied by acute inflammatory response with infiltration of leukocytes into tumor site and production of proinflammatory factors and cytokines . MeanwhiThe innate arm of immune system reacts to pathogenic invaders by phagocytes , the complement cascade, and natural killer (NK) cells. Followed by an acute inflammatory response, PDT-induced activation of the innate immune system consists of cytokine release, complement activation, recruitment, and activation of innate immune cells , 26. AftThe adaptive arm of immune system involves antigen presenting cells (APC) that can trigger naive T cells to become cytotoxic tumor-specific T lymphocytes (CTLs) and antigen that can be recognized by B cells for eventual antibody production. PDT-induced immunogenic apoptosis of cancer cells  trigger an effective antitumor immune response through activation of DCs and consequent activation of specific T cell response . DCs areAs discussed above, in addition to direct effects on tumor cells through generating either apoptosis and/or necrosis and PDT-triggered vascular damage that lead to tumor destruction, PDT owns its unique features in stimulating the host immune system. PDT-induced immune response not only enhances the efficacy of PDT by systemically eliminating residual tumor lesions and thus controls primary tumor growth, but also owns it particular promise in fighting for cancer cells in metastatic stage and thus inhibiting secondary disease . TherefoIn order to elicit an immune response that is strong enough to achieve superior tumor cures, one of modalities is to introduce immunostimulators in combination with PDT. In a previous review, St Denis et al. summarized investigations about immunostimulant therapies combined with PDT to potentiate the already robust immune response by PDT and further enhance its capability to attack malignant tumors . The immWhen combining PDT with immunostimulant therapies, the design of applied immunostimulators with effective delivery dosage definitely worth careful consideration. Another critical issue regarding this approach is the administration route for delivering those immunostimulatory agents to targeted tumor areas. In general, additional immunostimulators involved in PDT-induced immune response can be introduced through traditional intravenous or intratumoral injection. However, systemic delivery through intravenous route may result in high concentrations of immunostimulating drugs through blood circulation. As a result, powerful activation of immune system could be stimulated and eventually boost severe systemic immune response that is very harmful or even fatal . TherefoDevelopment of systemic adaptive immune response after PDT involves the maturation of DCs, the presentation of TAAs by DCs, and the priming of CTL response. The ultimate therapeutic outcome strongly depends on the efficacy of antigen presentation and recognition of tumor antigens by immune cells. Various approaches have been examined to accelerate the priming cells towards expression of high level of TAAs. One of the approaches to increase immunogenicity of targeted tumor cells focused on their genetic modification. Wachowska et al. have shown that PDT can induce strong antigen-specific antitumor immunity towards tumors expressing P1A antigen, which is a type of TAA. They have further combined PDT with a clinically approved epigenetic agent 5-aza-20-deoxycytidine that can induce expression of P1A antigen in different cancer cells. From their results, following high expression level of P1A antigen after epigenetic modification, potentiated antitumor effects and long-term survival after PDT have been found in tumor-bearing mice as a result of strong specific antitumor immune responses –41.After the initial PDT-induced destruction of tumor structure, there will be either the direct interaction between immune cells and tumor cells or the influx and activation of immune cells caused by local inflammatory reaction. While the main focus of PDT is tumor cells, targeting immune cells by boosting their antitumor immunity or eliminating their role in processes such as immunosuppression could be an additional way to further increase the treatment effectiveness.DMAP expression in tumor cells plays significant role in activating DCs towards phenotypic and functional maturation. Evidences have shown that the DAMPs exposure follows PDT-induced ICD. Best characterized DAMPs induced by PDT include heat shock proteins 70 (HSP70) –45, calr in vitro and in vivo without significant cellular death. This strategy that can cause immature DCs towards effective vaccine adjuvants through photodynamic reaction is sufficient to initiate adaptive T cell responses in vivo [DCs play significant role in priming naive T cells to CTLs, which link innate and adaptive immune response. The activation of DCs followed by phenotypic and functional maturation is very crucial for TAA presentation and CTLs formation . Cheong  in vivo .Recent cell-based therapeutic strategy by introducing external DCs after PDT demonstrated an improvement in the outcome of PDT. Saji et al. have reported that tumor destruction with combination therapy of intratumoral injected DCs and ATXS10Na(II)-PDT against CT26 tumors in BALB/c mice was greater than that of individual therapies . Jalili'Monocytes can differentiate into two different macrophage phenotypes, M1 and M2. Whereas M1 macrophages are mostly involved in immune activation and invader attack, M2 macrophages play a role in promoting tumor progression and damage healing. M2 macrophages that function in suppressing antitumor immune response are termed as tumor-associated macrophage (TAM), which is actually a valid target for antitumor therapy –58. TherM2 macrophage-targeted photosensitizer delivery can be facilitated by the nanoparticles technology. Wen et al. utilized natural noninfectious nanoparticle cowpea mosaic virus (CPMV)/dendron hybrids as vehicle to specifically deliver Zn-EpPor photosensitizer to M2 macrophages as well as cancer cells. They observed differences in uptake of photosensitizer between the M1 and M2 populations and tested its efficiency in PDT of B16F10 melanoma cells . It is iT lymphocyte activation during cancer immunotherapy, especially cytotoxic CD8+ T effector cells, plays an important role in killing tumor cells. PDT-induced cancer cell death causes acute inflammation, which might augment immunity by forming more effective cytotoxic T lymphocytes . Gao et  Ex vivo activated human donor T cells were loaded with photosensitizer mTHPP and used as targeted delivery system for both adoptive T cell therapy and PDT. Bispecific antibody-redirected T lymphocytes possess dual specificity for recognizing both surface marker, such as TAA on the target cell, and a component of CD3 on T lymphocytes. When cocultivated with EpCAM-expressing human carcinoma cells, T lymphocytes induced the cytolytic activity, and at the same time photosensitizer was transferred to target cells. Consequently, significant improved cytotoxic capacity of photosensitizer boosted T cells was found after irradiation [Recognizing their crucial function in PDT-induced immunity, in addition to simulate DCs maturation and prime T lymphocytes, another alternative is to increase the number of T lymphocytes involved in PDT process. Blaudszun et al. reported their proof-of-concept study showing an innovative combination of adoptive transferred T lymphocyte mediated drug delivery, cellular immunotherapy, and PDT.adiation . in vivo is considered to offer a rationale to facilitate tumor eradication and enhances antitumor immunity [Besides cytotoxic T lymphocytes, tumor microenvironment may also induce immunosuppressive cytokine production and promote the proliferation of regulatory T cells (Tregs). Tregs can inhibit the process through which immature T cells became cytotoxic T cells, and unrestrained expansion of Treg may foster cancer progression . Therefoimmunity . Experimimmunity . And dueimmunity . Reginatimmunity .2+, which can also promote apoptosis [PDT-triggered cell death is closely associated with the intracellular localization and binding sites of photosensitizer . Photoacpoptosis , 72. PDTpoptosis , 74. TheER is at the center of a number of vital cellular processes, and ER functions have implications for various pathologies including cancer. ER stress is a key player of intracellular signaling pathways that govern ICD , 76. MosIt has been known that photosensitive drugs such as hypericin or mTHPC are predominantly localized in ER and can cause massive production of ROS-based ER stress upon light irradiation . Such ER in vivo treatment with PDT via hypericin (~590 nm) or mTHPC (~415 nm) can only be performed on superficial neoplasm tissue. To fully explore their capability in treating deep tumor tissue, it would be necessary to retain their ER localization and expand their light absorption wavelength to the near-infrared region through modification with derivatives.However, one critical issue that will possibly limit the applications of these photosensitizers remains to be their intrinsic absorbance wavelength of which light cannot penetrate into deep tissue. As a consequence,As the major cellular component where the photodynamic reaction occurs, localization of photosensitizers in mitochondria would provide more efficient cell killing in PDT. Mitochondria have been proposed to be the most effective subcellular targets for PDT , 82. In  ex vivo for potential immune response. They have found that mitochondria-targeted delivery of photosensitizer yielded significant high level of IL-18 secretion of breast cancer cells upon light stimulation, which contribute to remarkable production of interferon-gamma (IFN-γ) of activated DCs. Therefore, it suggests that targeting mitochondria for photosensitizer delivery could be a unique approach to generate effective tumor cell antigens and amplify the host immune responses following PDT [In addition to increase of ROS production, more interestingly, photodynamically induced mitochondrial apoptosis induces signal transduction pathways, which also participate in the development of immune responses . Marrachwing PDT .Nanomaterials refer to the materials that have at least one dimension less than 100 nm, which thus provide superior characteristics . In reorNanomaterials also represent an innovative strategy for delivering multiple immunostimulatory agents such as external antigens in a controllable and targeted manner. Or nanomaterial-based system can serve as inducers of apoptosis for release of internal antigens or inducers of cytokine production –102. AldOf particular interest, owing to their controllable size and large surface area allowing for multiple functionalization with various ligands, nanomaterials are ideal carrier of photosensitizers and immunostimulatory agents, while providing intracellular targeting capabilities. For instance, nanocarriers  consisting of photosensitizers and antigens can be further modified with a mitochondria-targeting moiety (TPP or MTS) and thus guide both therapeutic agents to mitochondria to achieve enhanced PDT effect and induce favorable immune response . Therefo in vivo experiments [In order to bring PDT-induced antitumor immunity closer to clinical application, it is crucial to perform preclinical tests to evaluate therapeutic efficacy of PDT based on a platform that can readily mimic human response. As traditional two-dimensional (2D) monolayer static cell culture fails to provide native 3D tissue structure, significant deviation has been noticed in transferring the results from 2D culture toeriments –109. Theeriments , 111. Al in vitro 3D culture systems, which represent a more valuable approach for improved preclinical tests, providing integrated analyses of the antitumor efficacy of PDT and PDT-induced immune response in situ. These techniques include tumor spheroid formation in scaffolds or hydrogel systems, tumor cell aggregates formation by hanging-drop [ in vitro 3D breast tumor tissue model by coculturing tumor cells with stromal cells of human origin in the microfluidic system and utilized this model to evaluate the efficiency of PDT under various conditions. By presenting structural, mechanical, and chemical cues, tumor tissues in our microfluidic device are able to mimic heterogeneous tumor microenvironment and emulate physiological conditions [Recently, many studies have been devoted to developing-drop  or biopring-drop , and tuming-drop . In our nditions . in vitro 3D tissue model is still in its very early developmental stage with focus on the regeneration of single tissue organ. However, more excitingly, this organ-on-a-chip concept can be further extended to body-on-a-chip concept in the near future, which allows for the integration of multiple functional units of human organs to mimic whole body physiology [ in vitro 3D tissue model by tissue engineering technology such as microfluidic technique could be a powerful alternative approach for the study of PDT-induced antitumor immunity.Currently,ysiology –118. Witysiology –121, in PDT is a clinically approved, minimally invasive therapeutic modality for types of tumors. In addition to local tumor destruction and vascular effects of PDT, the mechanisms whereby PDT activates antitumor immunity have been extensively researched. Recognizing the innate potentials of PDT in triggering host immune response, researchers have explored the possibility of various immunotherapeutic strategies in concert with improved efficiency in PDT. However, under what circumstances this PDT-induced immunity can be potentiated to overcome tumor immunosuppression and achieve sufficient antitumor response and to which extent this PDT-induced antitumor immunity will lead to complete tumor rejection still need to be fully elucidated. To answer these questions, a more thorough understanding of the pathways by which tumor cells evade from host immune response and a comprehensive knowledge of dose-dependent effects of different photosensitizers are thus required. While developing combination therapy of PDT and immunosimulating approaches may enhance therapeutic efficacy, intracellular delivery of photosensitizers with the assistance of nanotechnology may favor rational design of PDT regimen. A physiologically relevant tumor tissue model with human derived tumor architecture and heterotypic feature is necessary for the study of tumor mass and immune system interaction as well as PDT-induced antitumor immunity.


One of the most exciting scientific advances in recent decades has been the realization that the diverse and immensely active microbial communities are not only ‘passengers’ with plants, but instead play an important role in plant growth, development and resistance to biotic and abiotic stresses. A picture is emerging where plant roots act as ‘gatekeepers’ to screen soil bacteria from the rhizosphere and rhizoplane. This typically results in root endophytic microbiome dominated by Proteobacteria, Actinobacteria and to a lesser extent Bacteroidetes and Firmicutes, but Acidobacteria and Gemmatimonadetes being almost depleted. A synthesis of available data suggest that motility, plant cell-wall degradation ability and reactive oxygen species scavenging seem to be crucial traits for successful endophytic colonization and establishment of bacteria. Recent studies provide solid evidence that these bacteria serve host functions such as improving of plant nutrients through acquisition of nutrients from soil and nitrogen fixation in leaves. Additionally, some endophytes can engage ‘priming’ plants which elicit a faster and stronger plant defense once pathogens attack. Due to these plant growth-promoting effects, endophytic bacteria are being widely explored for their use in the improvement of crop performance. Updating the insights into the mechanism of endophytic bacterial colonization and interactions with plants is an important step in potentially manipulating endophytic bacteria/microbiome for viable strategies to improve agricultural production. It has been projected that the world’s population will increase to 9.1 billion by 2050 . IncreasAgave tequilana plants directly digested endophytic bacteria for a nitrogen (N) source for their growth  which bacteria live in plant endophytic habitats, (2) how do endosphere bacterial communities respond to plant stresses and environmental stimuli, (3) where exactly do endophytic bacteria colonize plants, (4) what are the traits that enable bacteria to successfully invade and persist into standing heterogeneous communities, (5) how do bacterial endophytes deal with the plant’s immune system, (6) how does the plant host influence endophytic colonization via hormone signaling pathways, and (7) what are the traits of endophytic bacteria that deliver plant phenotypes and therefore may hold promise for use in agriculture. We believe that understanding the interactions between endophytic bacteria and their plant hosts will assist in the design of new strategies for productive and sustainable practices in agriculture.4–108 per gram of root tissues, which is considerable less when compared with bulk soil  and rhizosphere   . This su tissue) . Root en tissue) . Other b tissue)  (Sessits tissue) . Archaea tissue) .Phaseolus vulgaris), which was revealed by a culture-based analysis.Understanding the differences, if any, between plant root and leaf/shoot endosphere microbiomes is the first step of many in developing a clear pathway to beneficial development of biofertilizers in agriculture. There is evidence that plant root bacterial endophytes are mainly recruited from soil, which then ascend to stems and leaves via the apoplast in xylem vessels . TherefoEnterobacter, Pseudomonas and Stenotrophomonas that constitute the core bacterial operational taxonomic units (OTUs) in root endosphere of both, the sweet potato IPB-137  (Pseudomonas-like OTU (34%) in the roots of Populus deltoids (Pseudomonas (52%) and Enterobacter (35.5%) in sugarcane stems   and riceia, 47%) . In somedeltoids  and two ne stems . Membersathogens . Metagenbidopsis  and wheabidopsis . Collect2]  infected potato (Solanum tuberosum) tubers, and this change occurred possibly due to oxygen depletion inside the tubers  and using reporter gene-  modified bacterial strains combined with microscopy. In plants, emerging lateral roots break through the epidermis, cortex, endodermis, casparian strip (band around endodermis) and pericycle, thereby naturally forming a ‘highway’ for bacteria to enter at these sites. From there, bacteria can further enter the phloem and xylem vessels that transport photosynthates (phloem), nutrients and water (xylem)   using cultivated model strains. Some of the most popular approaches to enumerate and visualize colonization of bacteria in plant tissues include fluorescence igure 1) . Bacteriigure 1) . Endophyigure 1) .Figure 1). For instance, PGPB Burkholderia sp. strain PsJN colonized root rhizodermis cells, internal tissues, particular internodes and leaves of grapevine . An unusual colonization strategy has been recently discovered for a facultative intracellular symbiont of Methylobacterium extorquens strain DSM13060, which aggregated around the nucleus of the living cells of Scots pine (Pinus sylvestris L.) shoot tips . Bacteria have also been detected in plant reproductive organs, such as flowers, fruits and seeds, but in small numbers . Current evidence reveals that some bacteria live in symbiosis with plant endophytic fungi . For instance, the endofungal bacterium Rhizobium radiobacter F4 hosted by the fungus Piriformospora indica colonizes plant roots and forms aggregates of attached cells and dense biofilms at the root surface  defense signaling pathway targets biotrophic pathogens, while the jasmonic acid (JA) pathway suppresses the SA pathway playing a role in defense against necrotrophic pathogens, but also insects and beneficial plant-microbe interactions , which generally do not elicit significant plant immune responses, such as the expressions of pathogenesis-related (PR) proteins . This thmpestris . This sumpestris . Eight . Azoarcus sp. strain BH72 and described it as ‘disarmed’ due to the lack of both T3SS and T4SS as well as other important cell surface components that are usually present in pathogens. Similarly, the genomic inventory of five grass endophytic bacterial strains, including Herbaspirillum frisingense GSF30(T), Gluconacetobacter diazotrophicus PAI5, Azoarcus sp. BH72, Klebsilla pneumoniae 342 and Azospirillum sp. B510 characterized in biomass grasses completely lack T3SS (Herbaspirillum strains examined so far lack the T4SS that functions in virulence (Bradyrhizobium sp. SUTN9-2 (isolated from the leguminous grass Aeschynomene americana L.) to colonize the roots of rice seedlings .Among the SSs, T3SS and T4SS are pivotal for pathogens to deliver effector proteins into the plant, which can induce effector-triggered immunity . T3SS anack T3SS . Furtherack T3SS . All theirulence . Howevereedlings . With reeedlings . Taken aAtractylodes lancea . A high number and diversity of genes encoding ROS-scavenging enzymes such as superoxide dismutase (SOD) and glutathione reductase (GR) are represented in the metagenome of the endophytic bacterial communities in rice roots  is typically a non-specific tactic for plant defense to induce HR and PCD against biotrophic pathogens . Interess lancea . To detoce roots . Genes e sp. 638 . ROS-scace roots , 2013. Tinterior , 2013. IMedicago truncatula by the PGPB Klebsiella pneumoniae 342 (Kp342) and the human enteric pathogen Salmonella enterica serovar Typhimurium . The suppression of bacterial colonization may be a strategy of the plant’s immune system to control the abundance of hosted bacteria and to maintain the most ‘plant-favorable’ bacterial density in the inner tissues. The potential use of plant hormones for the suppression of specific plant endophytic bacteria warrants further investigation .Arka Abha than that of the susceptible cultivar Arka Vikas  or perception (35S-etr1) was lower than that of wild-type plants .The diversity of bacterial communities in the endosphere may correlate to plant defense capabilities. This is supported by the higher bacterial diversity in the root endosphere of wilt-resistant tomato cultivar ka Vikas . Moreoveka Vikas . These fka Vikas . Similar pathway . A recene plants . OverallFusarium, Pythium and Phytophthora ssp . Bacterial strains with plant growth-promoting functions continue to be discovered but as of yet a clear path to developing PGPTs for agricultural purposes has not been developed  possessed efficient ACC deaminase activity that were able to increase seed germination, root and shoot length, leaf area and numbers of L. sinese seedlings under salinity stress (The production of ET in stressed plants may lead to decreased plant growth or even cell death when present at high concentrations . Some miy stress . While dPseudomonas vancouverensis OB155 and P. frederiksbergensis OS261 were able to better cope with cold stress (10–12°C) (LeCBF1 and LeCBF3) were induced in bacteria-inoculated plants  . Less med plants . Similarsistance . Endophyo plants . TranscrStreptomyces sp. GMKU 3100 .Although iron is essential for all living organisms, its bioavailability in soil is limited. The production of siderophores by microbes assists plant growth, since these compounds chelate iron in the soil and generate soluble complexes that can be absorbed by plants . We prevMKU 3100 . Its benMKU 3100   and denitrification genes . Direct inhibition of pathogens is mainly mediated by the synthesis of inhibitory allelochemicals such as antibiotics, hydrogen cyanide (HCN), iron-chelating siderophores and antifungal metabolites . For instance, the study by Enterobacter radicincitans DSM 16656, a highly competitive colonizer of the endophytic environment of various crops, is able to induce priming in Arabidopsis via SA- and JA/ET- dependent pathways. Similarly, the endofungal bacterium R. radiobacter F4 is able to colonize plant roots without specificity and it is able to increase plant resistance against the bacterial leaf pathogens Xanthomonas translucens pv. translucens and Pseudomonas syringae pv. tomato DC3000. Mutational analysis indicated that the resistance was mediated by ISR via a JA-dependent pathway . For instance, a series of isoforms of iturins were purified from Bacillus amyloliquefaciens  showed enhanced resistance against the northern corn leaf blight whose causative agent is the fungus Setosphaeria turcica . For instance, certain endophytic bacterial strains in Cannabis sativa L. disrupt cell-to-cell communication of the biosensor strain Chromobacterium violaceum via quenching its QS signals  would allow the elucidation of the importance of the endosphere microbiota in plant growth and health. Additionally, it will be of great interest in the future to reveal the mechanisms and ecological rationales behind the rare presence of Acidobacteria, Gemmatimonadetes and Archaea in the plant’s endosphere. These microorganisms are still prohibitively difficult to be cultured. Besides taxonomic surveys, recently emerging techniques like single-cell isolation and sequencing should provide alternatives that circumvent the necessity for cultivation and thereby give steps forward to obtain more comprehensive pictures about their lifestyles and interactions with plants . Furtherin vitro mechanisms for plant growth promotion, reports on successful use of endophytic bacteria in plants under field conditions are extremely scarce. Nevertheless, the effect of the Burkholderia phytofirmans strain PsJN has been demonstrated to increase biomass and promote growth in switchgrass (Panicum virgatum L.) especially in low fertility soils  in the field by an additive effect of the endophyte Bacillus pumilus INR7 combined with the chemical inducer benzothiadiazole . As mentioned, research efforts have been made on manipulating plant microbiomes by inducing the plant’s signaling defense pathways using exogenous phytohormone treatments. Nevertheless, the contradicting results obtained from different studies suggest that it is still challenging to manipulate the plant’s endosphere microbiome. Future studies need to be conducted to bridge the knowledge gaps of how microbial function of the endosphere is affected by plant immunity. A combination of multi ‘omics’ such as metagenomics, proteomics and metabolomics and the advancing computational data-mining approaches should be able to reveal a more comprehensive picture of the endosphere microbiome, therefore transforming the way we understand bacterial endophytes and their interactions with plant hosts. It must, however, be addressed that culture-dependent methods are still important because they provide the indispensable materials for identifying bacterial physiological characteristics and allow the predication of the metabolic potential and biogeochemical function of a lineage by using genomic surveys. Promising areas to develop are efforts to breed for endophyte-optimized crops, endophytic microbiomes engineering and a better understanding how key endophytes can be attracted, maintained and adapted to benefit plants at various growth stages. While substantial basic and applied work remains to be done, it is envisioned that in the not too distant future bacterial endophytes can be at least partial substitutes for chemical fertilizers and pesticides and their targeted applications on crops may push forward a paradigm shift in agriculture.HL did the writing and drew the graphs. LC, MC, ES, PD, CP, and PS revised this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TM) based on monoclonal antibody sandwich immunoassays using biotin-streptavidin conjugate chemistry were selected with reagents from R&D Systems. The two platforms differed in ease and time of completion, number of samples per assay, and dynamic concentration range. Intra-assay variability assessed using a coefficient of variation (CV%) was 2.2–3.4 for Luminex® and 1.2–2.9 for EllaTM. Correlations for biomarker concentrations within dynamic range of both platforms were best for IL-6 , IP-10  and sFlt-1 . Agreement between concentrations obtained by both methods assessed by the Bland-Altman test varied, with best agreement for CHI3L1. Our data suggest that biomarkers of endothelial and immune activation can be readily measured with multiplex platforms. Luminex® and EllaTM produced reliable results with excellent CV% values. The EllaTM platform was more automated and completed in 75 minutes, potentially compatible with near-patient use. Trends in concentrations obtained by these methods were highly correlated, although absolute values varied, suggesting caution is required when comparing data from different multiplex platforms.Biomarkers can prognosticate outcome and enable risk-stratification. In severe infection, focusing on multiple markers reflecting pathophysiological mechanisms of organ injury could enhance management and pathway-directed therapeutics. Limited data exist on the performance of multiplex biomarker platforms. Our goal was to compare endothelial and immune activation biomarkers in severe pediatric infections using two multiplex platforms. Frozen plasma from 410 children presenting to the Jinja Regional Hospital in Uganda with suspected infection was used to measure biomarkers of endothelial  and immune  activation. Two multiplex platforms (Luminex®, Ella The search for novel biological markers to predict response to therapies, prognosticate outcome, or assist in patient enrollment in clinical therapeutic trials is quickly evolving . In the The third iteration of the international consensus definitions for sepsis and septic shock  acknowleOf the many pathophysiologic pathways that may contribute to the high morbidity and mortality of severe infections, endothelial , 8 and iTM -[Loge Ella]) across the mean of concentration values (([Loge Luminex]+[Loge Ella])/2) was calculated using the Pitman's test [e-transformed biomarker concentrations (bias), correcting for random Luminex batch effects for values within dynamic range of both assays.Platform comparisons were performed for values within dynamic range of both assays using three statistical tests , 37. Spen's test . Linear Statistical analyses were performed using STATA v14.1 (StataCorp 2015). Graphical presentation was done using Excel (Microsoft 2016) and Prism v5.0a (GraphPad 2007).TM platforms differed in ease of assay performance, number of samples analyzed per run, and time to perform each assay. Both platforms required optimization of plasma dilutions to ensure biomarker concentrations fell within the dynamic range of the assay.The Luminex® and EllaThe time to complete a Luminex assay was approximately 5 hours, accommodating up to 80 samples and up to 49 analytes per plate. The assay required manual preparation of serial dilutions of analyte standards, addition of pre-diluted samples, micro-particles, biotin-labeled antibodies, streptavidin substrate, three triplicate wash steps, and loading the plate into a MAGPIX instrument.TM platform requires loading the pre-diluted samples into a cartridge and inserting it into the instrument. No further user interface is required since the Ella instrument is fully automated, performs all assays in triplicate and uses internal microfluidics for completion of all immunoassay wash and detection steps.The time to complete an Ella assay was 1.25 hours, accommodating up to 16 samples and up to 4 analytes per plate. The EllaTM platforms differed in lower limit of quantification (assay sensitivity) and biomarker dynamic range (Table 1). For samples measured using the Luminex® platform at an appropriate plasma dilution, 63.7–99.5% were within assay dynamic range. Majority of values outside of dynamic range were below the lower limit of quantification, suggesting lower assay sensitivity. For samples measured using the Ella platform at an appropriate plasma dilution, 94.6–100% were within assay dynamic range. The EllaTM dynamic range was superior compared to Luminex® platform using reagents from R&D System especially, especially at the lower limits of quantification, and the only biomarker with values below the lower limit of assay quantification was IL-6. sICAM-1 values were not included in analyses as the sample dilution was inappropriate for reliable concentration quantification of values above the upper limit of the standard curve.The Luminex® and EllaFig 1.Intra-assay performance was calculated using coefficient of variation (CV%) values, to determine the precision of the concentration values obtained by both platforms . The resTM platform, all samples were run as internal triplicates and 2.7–12.3% of biomarkers were analyzed in duplicate due to an occlusion in one of three glass nanorods. For the 7 biomarkers analyzed at appropriate dilutions, the CV% varied between 1.2–2.9.For the Luminex® platform, 156 random samples were assayed in duplicate. The CV% for the 8 biomarkers varied between 2.2–3.4. For the EllaS2 Table).Inter-assay variability was determined by quantifying the same healthy control sample on each day of analysis . The same sample was frozen in multiple aliquots and each aliquot was thawed once on the day of the assay. The quantified biomarkers for the healthy control sample were at the lower end or below the limit of quantification for both multiplex platforms. Therefore, inter-assay variability was greater than if a sample with biomarker concentration within the mid-zone of the standard curve was selected . The absolute median values for individual biomarkers quantified using the Luminex® and EllaTM platforms varied (Table 2). For biomarker concentrations within the dynamic range of both multiplex platforms, there was no difference between the median CHI3L1 concentrations  but other biomarker concentrations varied significantly between the two multiplex platforms.Biomarker concentrations within dynamic range of both assays were compared using the Spearman correlation and paired Wilcoxon signed rank sum test. The strength of association for biomarker concentrations obtained using both platforms was excellent. For biomarkers quantified at appropriate dilutions, the strength of association varied between Spearman’s rho correlation coefficient (ρ) values of 0.79–0.97 . Using the Pitman's test of difference in variance, the bias for most biomarkers was consistent across the range of concentrations of both platforms, .As shown in TM) using reagents from a single manufacturer (R&D Systems) to test over 400 well-annotated plasma samples from febrile children presenting with suspected severe infections. We found that the correlations between values for biomarkers of endothelial and immune activation tested within assay dynamic range were excellent. Moreover, using two different statistical methods, we identified consistent differences between absolute biomarker concentrations obtained using the two different multiplex platforms.In the past decade, the use of multiplex immunoassays has made the quantification of multiple analytes obtained from clinical samples more feasible. However the variety of potential platforms and reagents has created challenges in selecting optimal assays for large-scale projects that demand consistent results and ultimately has hindered the use of these platforms in clinical research and their translation into clinical practice , 27, 40.TM [TM platform has the advantage of being fully automated with internal triplicate sample testing, decreasing the sample and reagent quantity, and time required for assay performance. Although the per analyte cost was 5.8-fold greater for the Ella relative to the Luminex platform, the expense related to staff time for assay performance and data analysis could decrease this difference.The selection of a multiplex immunoassay platform for a specific clinical project is based on many factors. When considering the optimal platform, we evaluated the availability of reagents for analytes of interest, the dilution factors to enable inclusion of multiple biomarkers in a single assay, the ease of assay performance, the time required to complete the assay with potential for translation to near-patient-care settings, as well as cost. Based on these criteria, the Luminex®  and EllaTM  platformTM . The EllAll of the biomarkers included in this study have been associated with severity of illness in severe infection –19. TherTM platforms, however, was uniform across the concentration ranges for most biomarkers as identified by the non-significant Pitman test p-values. This suggests that values of the biomarker concentrations were not different between the platforms at a different concentration range, making the relative values between the two tests comparable. Also worth noting is that although the absolute biomarker concentration values were statistically significantly different, the visual representation of the Loge-transformed biomarker concentrations displayed on Bland-Altman plots suggest that these concentrations were in a similar pg/mL range. The differences in absolute concentrations could be attributed to differences in the capture and/or detection monoclonal antibody specificity and/or affinity as described in S3 Table. Alternatively, competition between multiple monoclonal antibodies for antigen detection in the multiplex Luminex® assay could have been overcome with the EllaTM platform multiplex design which uses independent microfluidic channels for each analyte. Finally, although both methods used reagents from R&D Systems and fluorescence was used as the indicator, the fluorochrome conjugated to streptavidin differed (Phycoerythrin (PE) for Luminex® and DyLight650 conjugate for EllaTM), affecting how the fluorochrome was excited, and what emission wavelength was detected by each platform, possibly contributing to a difference in concentration ranges on both lower and upper limits of quantification.Of note, there were significant differences observed in absolute biomarker concentrations between these two platforms. The bias towards higher concentrations with either the Luminex® or EllaTM platform offers the required characteristics. Our data provide evidence that concentrations obtained by both platforms are highly correlated. Therefore, it is possible to use either platform for clinical research examining a relationship between biomarker level and clinical end-point. However, given the statistically significant differences in absolute values, direct comparison of concentration values between platforms is problematic. These observations have implications for future point-of-care novel biomarker research as direct comparisons of data will likely only be possible if assays are performed using the same methodology with reagents obtained from the same manufacturer.Selecting a platform to analyze large number of samples in a short time frame is best performed with an assay that can be done in multi-well format, enabling high throughput. Of the two platforms we tested, the Luminex® platform was better suited to this purpose. However, when near-patient or point-of-care data is required, with fast turn-around and with the capacity to test fewer samples, the EllaTM microfluidics-based system [TM platform was a superior dynamic range compared to Luminex using reagents from R&D Systems.The strengths of this study include inclusion of biomarkers of current clinical interest in the field of critical illness and life-threatening infections. The large sample size of more than 400 patients provides this study with higher analytical power. Additionally, the selection of the Luminex® microsphere-based system, an established multiplex platform , as welld system  allowed TM platform for sICAM-1 due to inappropriate sample dilution. This limitation highlights the limitation of multiplex assays which require all analytes tested to be at dilution suitable for the assay dynamic range. Also, up to 34% of concentration values quantified by the Luminex platform were below the limit of quantification . This limited the ability to compare the complete sample set, likely diminishing the accuracy of the comparison between the two platforms. This also limits the generalizability of the results to more critically ill patients, with higher biomarker levels.This study had some limitations. First, frozen versus fresh plasma was used. Future studies are planned to validate biomarker concentrations quantified in fresh blood samples. Second, we could not analyze data obtained from the EllaFuture clinical studies evaluating the potential of plasma markers as predictive and prognostic tools will require reliable, robust, easy to use multiplex platforms, providing data in real time at the point-of-care. Ultimately this approach could enable precision medicine via rapid triage of febrile patients and stratification for pathway-directed therapeutics for severe infections that may be applicable in critically ill patients identified in an emergency department or an intensive care unit.TM platform. The intra-assay variability is excellent for both multiplex platforms. The trends in concentrations obtained by the Luminex and Ella instruments are highly correlated but the absolute value of analyte concentrations vary between these platforms. Novel multiplex assays that produce rapid and reliable results, with minimal user interface and enable near-patient application, could transform prediction of response to therapies, prognostication of clinical outcome, and risk-stratification for enrollment in clinical trials.In conclusion, plasma biomarkers of endothelial and immune activation can be reliably quantified using multiplex immunoassay-based platforms. The dynamic range of biomarker concentrations is greater for the EllaS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Plasmodium species, the causative agents of malaria. Efficient elimination of Plasmodium liver stages prior to blood infection is one of the most promising malaria vaccine strategies. Here, we describe different genetically arrested parasites (GAPs) that have been engineered in Plasmodium berghei, P. yoelii and P. falciparum and compare their vaccine potential. A better understanding of the immunological mechanisms of prime and boost by arrested sporozoites and experimental strategies to enhance vaccine efficacy by further engineering existing GAPs into a more immunogenic form hold promise for continuous improvements of GAP-based vaccines. A critical hurdle for vaccines that elicit long-lasting protection against malaria, such as GAPs, is safety and efficacy in vulnerable populations. Vaccine research should focus on solutions toward turning malaria into a vaccine-preventable disease, which would offer an exciting new path of malaria control.Continuous stage conversion and swift changes in the antigenic repertoire in response to acquired immunity are hallmarks of complex eukaryotic pathogens, including  Plasmodium falciparum, P. vivax, and three other human-infecting Plasmodium parasites, at different life cycle stages, which together reduces morbidity and mortality in endemic regions. Attacking the parasite at its vector stage with long-lasting insecticide treated bed nets and insecticide-indoor residual spraying combined with access to rapid diagnosis and artemisinin-based combination therapy for clinical malaria episodes are recommended by the WHO  elicit sterile protection against Liver stage developmental arrest can also be achieved by an alternative approach, termed chemical attenuation of sporozoites (CAS), which is based on simultaneous administration of normal sporozoites and anti-malarial drugs  as tailored whole parasite vaccines against malaria infections  family  and UIS4, fulfilled three principal criteria: (i) stage-specific gene expression in pre-erythrocytic stages, thereby allowing recombinant knockout strains to be selected during blood stage transfection; (ii) abundant expression in pre-erythrocytic stages, indicating likely vital roles during this phase of the life cycle; and (iii) genes that are unique to Plasmodium and related haemosporidian parasites, which all share the hallmark of the first obligate population expansion phase in the host liver , a member of the Plasmodium-specific 6-Cys protein family  or its paralog, P36, arrest again early during liver stage development after completion of sporozoite transformation . Although, they start to mature into liver stage trophozoites, ΔP36p parasites suddenly abort this development, most likely because maintenance and maturation of the PVM are critically impaired  could not be detected, and the mechanism of breakthrough infections remains unsolved  and P36 was selected for a first phaseI/IIa clinical trial  genes , which is critical for sporozoite latency, and stage-specific knockout of the corresponding eIF2α-P protein phosphatase 1 (PP1/UIS2), which is a regulator of protein translation after hepatocyte invasion, led to incomplete early arrests before and immediately after hepatocyte invasion , in P. berghei revealed a specific defect during liver stage maturation  pathway produces saturated fatty acids in the apicoplast. It includes a cyclic reaction that catalyzes fatty acid elongation and a large pyruvate dehydrogenase (PDH) complex that forms acetyl-CoA for the elongation cycles  and β-hydroxyacyl-ACP dehydratase (FabZ) , a protein of unknown function. Targeted deletion of PALM did not affect parasite growth or apicoplast morphology, but resulted in an even later arrest after completion of liver stage development prior to merozoite release  in pre-erythryocytic development , which is shared across different life cycle stages and already essential during blood infection, as it plays prominent roles in merozoite egress and gametogenesis. Since generation of a ΔDevelopment of parasite lines that harbor multiple gene deletions is likely to increase safety; however, whether synergistic or antagonistic effects modulate immunogenicity is less straightforward to predict and likely depends on the selected knockout combination.UIS3 and ΔUIS4, was engineered in P. berghei  and P36, which are neighboring genes and likely arose through gene duplication, safety was not improved  knockout provides any additional benefit beyond perception of additional gene deletions. An important investigation with combinatorial knockouts will be the systematic expression profiling of liver stage-specific genes, as was previously done for ΔSLARP parasites  knockout mice and recognition of a CSP-specific T cell clone by hepatocytes with a matching MHCI haplotype revealed that liver stage antigens are presented to the CD8+ T cells via MHCI on the surface of infected hepatocytes -secreting CD8+ T cell-dependent elimination most likely is the immune effector mechanism in GAP-immunized animals. Indeed, a study employing P. berghei ΔUIS3 parasites showed that immunizations of B and T cell-deficient rag1−/− and IFNγ−/− knockout mice failed to induce protection, confirming the central role of adaptive immune responses leading to IFNγ production in vaccine-induced immunity  and is most likely a combination of DC-mediated antigen presentation in different organs, including skin-draining lymph nodes, spleen, and liver  sterile immunity. Second, confirmation of safety and immunogenicity of the P. berghei GAP line in G. dolichurus immunization and challenge study provide an evidence-based rationale for translation to P. falciparum GAP trials.Together, comparative studies on vaccine efficacy should include the Intravenous injections are the preferential route of vaccine administration in mouse models; however, in real life this is inapplicable. Pediatric vaccines are exclusively administered either by intramuscular syringe injection or orally. It is evident that a malaria vaccine must adhere to the same safe routes of administration. Alternative methods that are used in drug delivery, such as intradermal, intravenous, or intraperitoneal injections, are unreasonable for a malaria vaccine to be delivered in resource-poor health infrastructures, because of the risks associated with these routes of administration, except under physician's care.Plasmodium sporozoites apparently lack the ability to transmigrate through muscle or fat tissue. Accordingly, after intradermal, intramuscular, subcutaneous, or intraperitoneal injection only a very small proportion of sporozoites reach a blood vessel, resulting in reduced liver infection. This was confirmed in human volunteer studies, where intramuscular or intradermal syringe injections of cryopreserved sporozoites showed substantial delays in blood infections and high doses were required to consistently induce blood infections (Shekalaghe et al., via these routes was negligible both in the P. berghei model and in volunteer studies with P. falciparum sporozoites (Epstein et al., + T cell response toward a sustained effector-memory T cell response will be essential. It is conceivable that timing of antigen expression is important, and the temporal dynamics of expansion and contraction of antigen-specific T cells need to be analyzed to inform vaccine protocols (Hafalla et al., The necessity for booster immunizations remains another critical limitation, which is unlikely to be removed. However, a reduction of the vaccine doses would facilitate the distribution of a malaria vaccine considerably. A better understanding of how to best amplify an initial protective CD8Plasmodium sporozoites are particularly sensitive to environmental conditions, and it remains entirely speculative whether an appropriate preservation process can be implemented. A GAP vaccine formulation might be further improved by addition of an adjuvant; however, no examples exist yet for live attenuated Plasmodium vaccines.Cryopreservation of sporozoites is so far the only way of efficiently preserving the infectivity of attenuated sporozoites, posing huge logistic constraints. Thus, development of a vaccine formulation, which is stable under cooled conditions or, ideally, at ambient temperature and induces long-term protection, remains a critical bioengineering milestone. P. falciparum and P. vivax vaccines involves major research investments in preclinical murine malaria models before translation to the human parasite is warranted. Proper design of human clinical trials with predictive power for vaccine safety, negligible adverse events, and vaccine efficacy in young children living in very diverse tropical countries remains very challenging.Building on the success of live attenuated, metabolically active RAS, genetic engineering of liver stage-arrested parasites offers unprecedented opportunities to develop a precision malaria vaccine. Comparative studies with GAPs that display distinct temporal arrests during liver stage maturation provide a foundation for systems immunology approaches, which might in turn lead to a better mechanistic understanding of immune effector mechanisms that contribute to lasting protection against re-infection. Ultimately, evidence-based design of safe and effective whole sporozoite All authors contributed equally to this work and approved the manuscript for publication.KMa is listed as inventor on international and national patents “Live genetically attenuated malaria vaccine” and “Live genetically engineered protozoan vaccine.” These patents were filed by the authors' non-profit institutions to promote the development and distribution of malaria vaccines to people in need worldwide, in accordance with a global access strategy. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genetic variants that underlie microbial environmental adaptation are key components of models of microbial diversification. Characterizing adaptive variants and the pangenomic context in which they evolve remains a frontier in understanding how microbial diversity is generated. The genomics of rhizobium adaptation to contrasting soil environments is ecologically and agriculturally important because these bacteria are responsible for half of all current biologically fixed nitrogen, yet they live the majority of their lives in soil. Our study uses whole-genome sequencing to describe the pan-genome of a focal clade of wild mesorhizobia that show contrasting levels of nickel adaptation despite high relatedness (99.8% identity at 16S). We observe ecotypic specialization within an otherwise genomically cohesive population, rather than finding distinct specialized bacterial lineages in contrasting soil types. This finding supports recent reports that heterogeneous environments impose selection that maintains differentiation only at a small fraction of the genome. Our work further uses a genome-wide association study to propose candidate genes for nickel adaptation. Several candidates show homology to genetic systems involved in nickel tolerance and one cluster of candidates correlates perfectly with soil origin, which validates our approach of ascribing genomic variation to adaptive divergence. A central challenge in microbial evolutionary ecology is identifying genomic variation responsible for environmental adaptation. Local adaptation results when environment-specific selection is stronger than migration and builds local reservoirs of genetic diversity within a population's biogeographic structure . CurrentRhizobium leguminosarum  and are readily culturable in the laboratory , and from spatially adjacent non-serpentine soils containing lower levels of nickel (0.8–6.85 mg Ni kg−1) at each of three Californian reserves . Next, we identified ‘de novo' genes in the wild strains that were absent in M. huakuii bv. loti. Regions of the draft assemblies that did not contain reference genes were aligned using BLASTX to the non-redundant GenBank CDS translations+PDB+Swiss-Prot+PIR+PRF (nr) database using the same BLASTX parameters. Orthology among de novo genes was determined using reciprocal BLAST . ‘Accessory' genes are absent from one or more strain in each subset. Delineation of core and accessory was calculated for each of the three subsets. For genes in the core genome across all 53 strains, we performed multiple sequence alignments to identify single-nucleotide polymorphisms (SNPs) , and the biological process, DNA metabolism . The accessory genome is also strongly enriched in DNA recombination , with all but one gene annotated with this term located in the accessory genome . Finally, the accessory genome is enriched in cellular compartment terms related to the cell membrane, including membrane , intrinsic to membrane  and membrane part   .W=4 337 428, P<2.2E-16; Compared with the core genome, the accessory genome showed elevated rates of protein sequence evolution relative to genome sequence change, reflected in the ratio of non-synonymous to synonymous substitutions  and the pattern was marginally significant for the FDR 10% genes .In the focal clade, nickel-adaptation candidates from the accessory genome are shared with lineages that are more phylogenetically distant to the focal clade than expected. This pattern was strongest for nickel-adaptation candidates at 1% FDR—of these 43 genes, 33 were found in bacterial lineages with existing 16S sequence data. On average, this set was 3.3% more divergent in terms of 16S base pair similarity (0.033 average nucleotide identity) from Mesorhizobium from California shows that adaptive differentiation across contrasting soil nickel levels occurs within an otherwise cohesive genome and gives insight into the biogeography of the pan-genome, the identity of adaptive genomic variants and the process of environmental adaptation in bacteria.Understanding rhizobial adaptation to soil environments is important because symbiotic rhizobia are responsible for half of all current biologically fixed nitrogen  yet liveMesorhizobium clade in this study is a genomically cohesive population that is widely dispersed across habitat boundaries yet harbors ecotypic variation for adaptation to high-nickel soils. Phylogenomic analysis of this population (99.8% 16S similarity) yields a star-like topology for both variation in the core and accessory genomes, with relatively long terminal branches that reflect strain-specific mutations similar to those observed in other rhizobial populations . Contrasting patterns of functional enrichment and molecular evolution in core vs accessory Mesorhizobium genomic compartments demonstrate that they experience different evolutionary processes. The accessory genome is strongly enriched in genes related to DNA recombination and metabolism, and many appear to reside in the symbiosis island region. These characteristics accord with the concept that horizontally transferred genes occur at sites of recombination -family metal exporter in Mycobacterium tuberculosis (2+ and Co2+, is controlled by the high-affinity Ni2+/Co2+ sensor KmtR and may use Ni2+ as a substrate (Methylophaga aminisulfidivorans (Mesorhizobium huakuii bv. loti and is homologous to NccN, a nickel–cadmium–cobalt resistance protein that contributes to nickel efflux in Alcaligenes xylosoxydans (Bradyrhizobium from serpentine soil (Methlyophaga thiooxydans DMS010 (Nickel efflux systems prevent toxic intracellular nickel concentrations  and are sistance . Candidasoxydans . Genes iC. metallidurans (Escherichia coli (Three other candidates are annotated with functions that suggest involvement in symbiosis and/or environmental stress. Candidate ‘denovo003250' is homologous to iron superoxide dismutase protein ChrC, which contributes to nickel tolerance in hia coli . Candidahia coli . Opines,hia coli .Mesorhizobium population exhibits environment-specific adaptive variants, yet lacks strong genome-wide differentiation between environments, suggesting that gene flow across environments may maintain genomic cohesion within this population. This is consistent with a population classification of ‘stage 2' in The focal Our findings support previous work showing that the accessory genome acts as a reservoir that harbors adaptive variation resulting from selection that impacts microbial populations at local ecological scales . While mMesorhizobium strains (Our focal population shows adaptation to high-nickel serpentine soil, but does not show the pattern of reciprocal local adaptation found in the larger set of 292  strains . In the  strains , which c strains  and coul strains . This laWe observe habitat specialization within an otherwise genomically cohesive population of wild mesorhizobia, rather than finding distinct, specialized bacterial lineages in contrasting soil types. Thus, heterogeneous environments can drive selection maintaining adaptive variants at a small fraction of the genome. Our work proposes candidate genes for nickel adaptation, several of which show homology to already-characterized genetic systems for nickel tolerance, which validates the general approach of ascribing habitat-assorting genomic variation to the processes of ecotypic adaptation.


Intrauterine transfusion is the most common and successful intrauterine procedure for the treatment of fetal anemia due to red cell alloimmunization. Fetal intracranial hemorrhage is a very rare complication of intrauterine transfusion in patients with Rh(D) alloimmunization and it has been demonstrated only in a few case reports in the literature. Herein, we described a case of grade IV intraventricular hemorrhage that was diagnosed following the first intrauterine transfusion and reviewed the literature about the fetal intracranial hemorrhage that occurred after intrauterine intravascular transfusion procedure. Intrauterine transfusion (IUT) is the most common and successful intrauterine procedure for the treatment of fetal anemia due to red cell alloimmunization . The benHerein, we described a case of grade IV intraventricular hemorrhage that was diagnosed following the first IUT and reviewed the literature about the fetal intracranial hemorrhage that occurred after intrauterine intravascular transfusion procedure in patients with Rh(D) alloimmunization. We searched PubMed, Scopus, Embase, and Google Scholar databases using the keywords Rh isoimmunization “OR” intrauterine transfusion “AND” intracranial hemorrhage “OR” brain injury “OR” brain damage. We found only two papers that define three cases of intracranial hemorrhage associated with intrauterine transfusion due to Rh alloimmunization. The initial platelet value was not noted in the third case. Author, case number, patient's age, gestational age, pretransfusion hemoglobin value, pretransfusion platelet value, neurosonogram after the first intrauterine transfusion, and the outcome were summarized in μl and 163000/μl, resp.). A few days later, ultrasound examination revealed the presence of an echogenic collection involving right lateral ventricle and extending to the surrounding cerebral parenchyma compatible with grade IV intraventricular hemorrhage , and there was no sign of increasing bleeding time such as excessive bleeding from the umbilical cord after withdrawal of the needle. Furthermore, we hypothesized that preservative-anticoagulant system such as additive solution-1 (AS-1), AS-3, AS-5, citrate-phosphate-dextrose-adenine-1 (CPDA-1), citrate-phosphate-dextrose (CPD), and citrate-phosphate-dextrose-dextrose (CP2D) that were used in red blood cell preparation might predispose to intracranial hemorrhage by altering the coagulation system of the fetus. Thus, we proposed that removing these anticoagulants from the transfusion aliquots before intrauterine transfusion by centrifugation and volume reduction could have a beneficial effect on preventing the hemorrhagic complication of this treatment.Intrauterine transfusion has been reported as the most successful fetal therapy procedure with 95% perinatal survival rate . The oveSimonazzi et al. demonstrated the risk of cerebellar damage in fetuses with severe anemia due to RhD alloimmunization after intrauterine intravascular transfusion procedure . They reIn conclusion, in this study, a case of intracranial hemorrhage in a fetus with hydrops fetalis due to Rh alloimmunization after intrauterine intravascular transfusion has been presented and a comprehensive, up-to-date review has been performed. Although intracranial hemorrhage is a rare complication of IUT, clinicians should be aware of increased risk of brain damage in fetuses with Rh(D) alloimmunization undergoing this procedure. Detailed sonographic examination of the fetal central nervous system before and after the treatment should be performed in patients that are planning IUT. Also, communicating with blood bank to decrease the additive anticoagulant agents as minimal as possible in the preparation process of red blood cells and reduction of first transfusion volume to avoid sudden fluctuations in cerebral blood flow may be helpful to prevent this complication.


Viruses infect host cells releasing their genome (DNA or RNA) containing all information needed to replicate themselves. The viral genome takes control of the cells and helps the virus to evade the host immune system. Some viruses alter the functions of infected cells without killing them. In some cases infected cells lose control over normal cell proliferation and becomes cancerous. Viruses, such as HCMV and HIV-1, may leave their viral genome in the host cells for a certain period (latency) and begin to replicate when the cells are stressed causing diseases. HCMV and HIV-1 have developed multiple strategies to avoid recognition and elimination by the host’s immune system. These strategies rely on viral products that mimic specific components of the host cells to prevent immune recognition of virally infected cells. In addition to viral proteins, viruses encode short non-coding RNAs (vmiRNAs) that regulate both viral and host cellular transcripts to favor viral infection and actively curtail the host’s antiviral immune response. In this review, we will give an overview of the general functions of microRNAs generated by HCMV and HIV-1, their processing and interaction with the host’s immune system. MicroRNAs (miRNAs) are short (∼22 nucleotides) single-stranded non-coding RNA molecules that negatively regulate gene expression at post-transcriptional level. The miRNA maturation machinery involves several steps and multiple proteins in both nucleus and cytoplasm. miRNAs can be transcribed as part of independent primary transcripts (pri-miRNAs), which are mainly generated by RNA polymerase II , and dis∗)-strand] is usually degraded. The RISC complex contains Dicer and many associated proteins such as Argonaute (Ago) protein family and the RNA-binding protein TRBP [human immunodeficiency virus transactivating response RNA (TAR) binding protein]  for the annealing of miRNAs to the target mRNA, whereas the other strand [the star (protein] .seed region) at the 5′ end of the miRNA. Complete complementarity between the miRNA and target mRNA sequence directs mRNA degradation, while absent of perfect complementarity will silence the gene target by preventing its translation  of Herpesviridae family .Viruses have developed multiple strategies to avoid recognition and elimination by the host’s immune system. These strategies rely on viral products that mimic specific components of the host cells to prevent immune recognition of virally infected cells. In addition to viral proteins, viruses encode miRNAs (vmiRNA) that regulate both viral and host cellular transcripts during viral infection. The first vmiRNAs was identified in a cell line latently infected with Epstein–Barr virus (EBV) , a membearcinoma . Current vmiRNAs . To dateiRNAs , which is trimmed by the host RNase III endonuclease Drosha microprocessor into approximately 80 nt long hairpin structures, known as viral pre-miRNAs (vpre-miRNAs). Vpre-miRNAs are rapidly exported to the cytoplasm where a specialized multi-domain ribonuclease III enzyme, known as Dicer, removes the loop structure leaving the vmiRNA duplex. Some functional vmiRNAs, such as murine γ-herpesvirus 68 (MHV68) miRNAs, are produced by host RNA polymerase III and tRNase Z, independently from the microprocessor-Drosha component , whereasIn general, virus and the host-cell’s miRNAs use different mechanisms to interfere each other. Viruses can either block or impair the host cells miRNA pathway by interacting with key proteins , synthesin utero, HCMV can cause different clinical syndromes the severity of which depends on the degree of immunosuppression . HCMV miRNAs are differently expressed during latent and lytic infection. Of note, only a subset of vmiRNAs are produced during HCMV latency, most of which originate from the unique long (UL) region of the HCMV genome . Since this subject has been recently reviewed by HCMV miRNAs are known to target several cellular genes to evade immune system, control cell cycle and vesicle trafficking  and downregulates MICB expression during viral infection, therefore, leading to decreased binding of NKG2D and reduced killing by NK cells . This miIn addition, the HCMV miR-UL148D, one of the most highly expressed vmiRNAs during latent infection, contribute to immune evasion by directly targeting the chemokine (C-C Motif) ligand 5 (CCL5), a chemokine known to attract immune cells to sites of inflammation and tissue damage . This doFinally, the HCMV miR-US4-1 was shown to directly target the endoplasmic reticulum aminopeptidase 1 (ERAP1), a key peptidase that trims peptide precursors to their optimal length to bind MHC class I molecules . The redAll these mechanisms focus on the possibility to enhance viral replication by hindering viral clearance by NK cells and T cells for the period of time necessary to virus to replicate.+ T-cell loss making individuals susceptible to get infections and develop a wide range of immunological abnormalities until oncological complications. Although HIV infections are able to induce vigorous antiviral immune responses, HIV-1 replication is not fully controlled by the innate and adaptive immune system. Like many other viruses, HIV-1 has evolved a number of strategies to evade host immune responses, most notably by using viral accessory proteins and RNA.HIV-1 infection cause progressive CD4Table 1). Despite so, the presence of HIV-1 derived vmiRNAs has been highly controversial and further studies are necessary to better understand their potential role in viral infection and pathogenesis. The importance of viral and cell host miRNAs in the context of HIV-1 infection was first suggested by silencing of Drosha and Dicer leading to significant enhancement of HIV-1 replication  may provide a new approach to control disease progression. Finally, understanding the mechanism of vmiRNA-mediated immune evasion will allow us to develop novel strategies to prevent and cure viral infections. Targeting vmiRNAs with molecules such as antagomirs may represent a novel therapeutic strategy to limit chronic immune activation and the progression of viral infection.DF and AG analyzed the literature, wrote the manuscript, and approved the final version for publication. RR analyzed the literature, revised the manuscript and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thyroid carcinoma is the most common endocrine malignancy in the United States with increasing incidence and diagnosis but stable mortality. Differentiated thyroid cancer rarely presents with distant metastases and is associated with a low risk of morbidity and mortality. Despite this, current protocols recommend remnant ablation with radioactive iodine and evaluation for local and distant metastasis in some patients with higher risk disease. There are several case reports of false positive results of metastatic surveys that are either normal physiologic variants or other pathological findings. Most false positive findings are associated with tissue that has physiologic increased uptake of I-131, such as breast tissue or lung tissue; pathological findings such as thymic cysts are also known to have increased uptake. Our case describes a rare finding of a thymic cyst found on a false positive I-131 metastatic survey. The patient was taken for surgical excision and the final pathology was a benign thymic cyst. Given that pulmonary metastases of differentiated thyroid cancer are rare, thymic cysts, though also rare, must be part of the differential diagnosis for false positive findings on an I-131 survey.  A 61-year-old female was found to have a palpable thyroid nodule on routine physical examination by her primary care physician. The patient was then referred for a thyroid ultrasound which showed a 2.1 cm, left-sided thyroid nodule. The patient underwent an ultrasound guided fine needle aspiration (FNA) biopsy which revealed a papillary thyroid carcinoma. The patient was referred to ENT and subsequently had a total thyroidectomy. She was referred to our endocrine clinic for postsurgical management of her T1b N1a MX stage 3 papillary thyroid carcinoma 4 weeks after total thyroidectomy. The tumor size was 1.7 cm in maximum diameter and was unifocal. At the time of surgery, the surgical margins were positive and focal lymphovascular invasion was observed with 1 out of 6 dissected lymph nodes positive for metastatic disease. The patient was prepared for radioactive iodine remnant ablation with a low iodine diet and thyrotropin alfa injections. An initial diagnostic SPECT/CT (Philips Precedence 16P) scan was performed 24 hours after administration of 5.3 mCi of Iodine-123 (I-123). The scan showed uptake in the left posterior thyroid bed and anteriorly at the level of the hyoid bone, both likely representing residual thyroid tissue. Physiologic uptake was also noted in the salivary glands, nasopharyngeal mucosa, and gastrointestinal tract. Additionally, a large (8 cm × 9 cm × 8 cm) heterogeneous left anterior mediastinal mass with mixed solid and cystic architecture was noted. This mass did not demonstrate uptake of the I-123 tracer Figures . A CT-guSurgical pathology sections demonstrated a multiloculated mass composed of cystic spaces lined by a variety of epithelial types, including areas of low-cuboidal , mucinouThyroid carcinoma is a disease of significant interest in American public health given the increasing trend of diagnosis and incidence with relatively stable mortality . DiffereUptake of radioiodine by nonthyroid structures can result from normal expression of the sodium-iodide symporter (NIS), presence of metabolized or physiologically retained radioiodine in body fluids, uptake of radioiodine by inflamed tissues, or contamination. Major pathological mechanisms of increased I-131 uptake not related to metastases from thyroid carcinoma include those of pathological transudates, inflammation (acute or chronic), and neoplasms of nonthyroid origin ; nonpathIn our case, initial findings on the I-131 posttherapy metastatic survey indicated the presence of possible metastases. However, given the patient's low thyroglobulin tumor marker level following surgery, alternative diagnoses were explored. The initial CT-guided biopsy of the patient's mediastinal mass was an FNA sample. No core biopsies were obtained. In addition, the washout from the FNA was not analyzed for thyroglobulin or PTH. This is a potential limitation of our initial work-up of the patient's abnormal finding on imaging. The patient's serum corrected calcium levels were normal at initial evaluation. The differential diagnoses ranged from acute or chronic inflammatory processes to benign or malignant lesions. Inflammatory lesions localized to the thorax include acute respiratory infections, pulmonary tuberculosis, pulmonary aspergilloma, myocardial infarction, and bronchiectasis. Benign and malignant lesions include mesothelioma, struma cordis, primary lung cancer, and breast cancer . It is pFunctional NIS-mediated radioiodine uptake has been documented in breast tissue, salivary glands, the gastrointestinal tract, and the thymus. There is an association with uptake into serous cavities or cysts such as scrotal hydroceles, lymphoepithelial cysts, ovarian cysts, renal cysts, and pleuropericardial cysts located in the anterior mediastinum .Concentration of radioiodine by the thymus is not commonly observed by whole body scintigraphy, with estimates from a case series in a cohort of 175 female patients suggesting that approximately 1% to 2% of scans may demonstrate thymic uptake . The norHere we present the rare finding of prominent radioactive iodine uptake by a thymic cyst following total thyroidectomy for papillary thyroid cancer. This patient raises a number of important concepts. First, there are multiple natural sites of radioiodine uptake beyond the thyroid. Second, following excision of a radioiodine avid neoplasm, secondary sites of uptake could represent metastatic disease. Third, there are anatomic variants with iodine avidity that should be considered in the differential diagnosis in cases of aberrant radioiodine uptake. Finally, one must consider the relatively low incidence of pulmonary metastases in DTC. In conclusion, clinicians should consider the array of differential diagnoses for lesions resembling pulmonary metastases from primary differentiated thyroid cancer.


The specific allorecognition pathway used by T cells is dictated by intrinsic and extrinsic factors to the allograft and can influence the nature and magnitude of the alloresponse and rejection process. Consequently, various organs and tissues such as skin, cornea, and solid organ transplants are recognized differently by pro-inflammatory T cells through these distinct pathways, which may explain why these grafts are rejected in a different fashion. On the other hand, the mechanisms by which anti-inflammatory regulatory T cells (Tregs) recognize alloantigen and promote transplantation tolerance are still unclear. It is likely that thymic Tregs are activated through indirect allorecognition, while peripheral Tregs recognize alloantigens in a direct fashion. As we gain insights into the mechanisms underlying allorecognition by pro-inflammatory and Treg cells, novel strategies are being designed to prevent allograft rejection in the absence of ongoing immunosuppressive drug treatment in patients.Recognition of donor antigens by recipient T cells in secondary lymphoid organs initiates the adaptive inflammatory immune response leading to the rejection of allogeneic transplants. Allospecific T cells become activated through interaction of their T cell receptors with intact allogeneic major histocompatibility complex (MHC) molecules on donor cells (direct pathway) and/or donor peptides presented by self-MHC molecules on recipient antigen-presenting cells (APCs) (indirect pathway). In addition, recent studies show that alloreactive T cells can also be stimulated through recognition of allogeneic MHC molecules displayed on recipient APCs (MHC cross-dressing) after their transfer  Allorecognition relates to the detection of genetically encoded polymorphisms between individual organisms of the same species by the immune system. Allorecognition has been described in nearly all multicellular phyla, including invertebrates that are devoid of an adaptive immune system . Indeed,The following section describes the three known pathways  by which recipient T cells recognize donor alloantigens [major histocompatibility complex (MHC) and minor antigens] after allotransplantation.high determinant density and the multiple binary complex models ] is believed to rely on the high frequency of precursor T cells capable of recognizing allogeneic MHC molecules. It is also possible, however, that the MLR may reflect the presence of alloreactive memory T cells generated after infections through cross-reactive recognition of self-MHC molecules bound to microbial peptides mimicking an allogeneic MHC–peptide complex, a phenomenon called heterologous immunity  (passenger leukocytes) migrate through lymphatics to host regional lymph nodes (LNs) , 7. Naïvx models –16. The immunity , 18. Forimmunity . Consequarasites , 20, 21.+ cytotoxic T cells in vitro, a phenomenon referred to as cross-presentation  and trigger the activation of some CD8entation . Most imly 1980s , 24. It ly 1980s , 24. Basted mice . The relted mice , 27. Sub article –30. Dete article , 32. How article . Cryptic article . However article . Seconda article , 36 and  article .via transfer of donor antigens through cell–cell contact or phagocytosis of extracellular vesicles secreted by donor cells.In addition to its role in allo-MHC recognition, indirect T cell recognition is considered as the main driving force being T cell responses to minor antigens (mH), which are peptides usually derived from housekeeping proteins displaying some degree of polymorphism . The convia cell–cell contact (trogocytosis), nanotubes, or through the release of extracellular vesicles such as exosomes  through cell–cell contact in a temperature- and energy-dependent manner. In these studies, allo-MHC cross-dressed cells induced proliferation of Ag-specific T cells in vitro  secretion] for the direct activation of other CD4+ T cells (three-cell cluster model) or the differentiation of CD8+ cytotoxic T cells recognizing donor MHC class I peptides in a direct fashion (four-cell cluster model)  (trans-costimulation)  Figure . Likewis) Figure . At the + and CD8+ pro-inflammatory T cells and the rejection of allogeneic skin, corneal, and heart grafts.Many factors either intrinsic or extrinsic to the graft influence the nature and magnitude of the T cell response induced by a defined pathway of allorecognition. Consequently, the contribution of each T cell allorecognition pathway (direct or indirect) to the rejection process varies upon the nature of the tissue or organ transplanted, the site of the body where it is placed, and the immunological status of the recipient. This section describes some of the factors governing the initiation of direct and indirect alloresponses by CD4+ T cells are induced after transplantation of fully MHC-mismatched skin allografts  they are placed in the eye that is an immune-privileged site of the body  responses  or after peripheral recognition of antigens   , 104. Inin vitro  and are  in mice . tTRegs  in mice . While t in mice . Toleranhis view –113. In his view –117. Alt+ and CD8+, activated directly and indirectly, are highly immunogenic and thereby resistant to tolerance induction. In contrast, corneal allografts that elicit only indirect alloresponses by CD4+ T cells are tolerogenic and often spontaneously accepted. On the other hand, early acute rejection of solid organ allografts such as hearts and kidneys is mediated essentially by T cells activated directly. While this immune response results in a potent inflammatory reaction, it is readily inhibited by calcineurin inhibitors. This explains why these drugs have been effective at achieving prolonged survival of organ allografts in patients. These treatments do not, however, efficiently suppress alloreactive memory T cells, thus precluding transplantation in patients sensitized to their potential donors (10% of patients). Most importantly, many transplanted organs are progressively lost due to chronic rejection, a process presumably initiated by indirectly activated T cells and subsequent production of cytotoxic anti-donor antibodies. For reasons that are still unclear, this response is not always efficiently suppressed by current immunosuppressive drugs. Therefore, future challenges in clinical transplantation will be to suppress or eliminate allospecific memory T cells and to prevent the development of indirect alloresponses.It is now firmly established that the mechanisms by which T cell recognize and respond to alloantigens greatly vary upon the nature of the transplanted organ or tissue, the site of anatomical placement, and the immunological status of the host. This explains why certain transplants, such as skin allografts, which induce potent inflammatory responses by both CD4All the authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.