Recent advances in our understanding of the dynamics of cellular cross-talk have highlighted the significance of host-versus-tumor effect that can be harnessed with immune therapies. Tumors exploit immune checkpoints to evade adaptive immune responses. Cancer immunotherapy has witnessed a revolution in the past decade with the development of immune checkpoint inhibitors (ICIs), monoclonal antibodies against cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or their ligands, such as PD1 ligand 1 (PD-L1). ICIs have been reported to have activity against a broad range of tumor types, in both solid organ and hematologic malignancy contexts. However, less than one-third of the patients achieve a durable and meaningful treatment response. Expression of immune checkpoint ligands , mutational burden and tumor-infiltrating lymphocytes are currently used as biomarkers for predicting response to ICIs. However, they do not reliably predict which patients will benefit from these therapies. There is dire need to discover novel biomarkers to predict treatment efficacy and to identify areas for development of combination strategies to improve response rates. Emerging evidence suggests key roles of tumor extracellular matrix (ECM) components and their proteolytic remodeling products in regulating each step of the cancer-immunity cycle. Here we review tumor matrix dynamics and matrix remodeling in context of anti-tumor immune responses and immunotherapy and propose the exploration of matrix-based biomarkers to identify candidates for immune therapy. Initial antigen-mediated activation of T cells is modulated by several regulatory mechanisms, including engagement co-stimulatory signals like the binding of CD28 on T cells to CD80/B7-1 and/or CD86/B7-2 on antigen-presenting cells. Immune checkpoint pathways that have evolved as a mechanism to avoid auto-immunity, can be targeted with immune checkpoint inhibitors (ICIs). Immune checkpoints are inhibitory regulators that act as “breaks” on the immune response. Cytotoxic T lymphocyte antigen 4  competes with CD28 for the ligands CD80 and CD86, and antagonizes T cell receptor (TCR) signaling [The adaptive immune response protects against foreign threats, including infections and tumors. The therapeutic potential of host-versus-tumor effect can be harnessed with novel immune therapies. CD4ignaling . Programignaling .Tumor immunotherapy has witnessed a revolution in the past decade. The clinical successes of ICIs, monoclonal antibodies (mAb) against CTLA-4 and PD-1 pathways, was a breakthrough achievement. In 2010, a randomized phase III trial reported remarkable response to Ipilimumab, mAb against CTLA-4, in melanoma patients . IpilimuDespite the rapid progress of approvals for these classes of agents, the accumulated experience demonstrated that overall, only one-third of the patients achieve a durable and meaningful response. With CTLA-4 blockade by Ipilimumab or PD-1 inhibition with Nivolumab, response rates of 30-40% were observed in melanoma patients as monotherapies and combination therapy achieved a response rate of over 50% –18. In NHigh expression of PD-L1 is regarded as a marker of an active anti-tumor immune response and correlates with adaptive immune resistance in several tumor types, including melanoma, NSCLC, Merkel cell carcinoma, breast cancer, mismatch-repair deficient tumors, and Hodgkin's lymphoma , 28–34. Discordance between PD-L1 expression in metastatic sites and primary tumors has been noted in bladder cancer patients, suggesting the dynamic nature of TME . In contA systemic review of melanoma patients showed that responses to ICIs correlated with mutational load, neoantigen load, and immune-related gene expression . High muGenetic and immune heterogeneity has been observed in melanoma tumors responding to immunotherapy : individ+ cytotoxic T- lymphocytes (CTL), in the TME. These CTL are located at the invasive tumor margin and intratumorally, and are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance. In metastatic melanoma, the detection of CTL at the tumor margin predicted better response to ICI [+ effector memory cells that might explain durable response observed in many patients [The success of checkpoint blockade depends on prior recruitment of tumor-infiltrating lymphocytes, particularly CD8e to ICI , 40, 47.e to ICI . Chemokie to ICI . Higher e to ICI , 50–52. patients . Converspatients .+ T cells expressing interleukin-2 receptor chair-alpha  and transcription factor forkhead-box P3 (FOXP3), suppress CTL and contribute to a tumorigenic TME. They promote tumor growth by diverse mechanisms, including expression of immune checkpoints  as well as production of IL10 and transforming growth factor-beta (TGF-β) [Tumor-infiltrating Tregs, in particular, CD4 (TGF-β) , 56. CTL (TGF-β) . PD-1 bl (TGF-β) . An incr (TGF-β) , 59.Tumor-infiltrating myeloid cells, including myeloid-derived-suppressor cells (MDSCs), tumor-associated granulocytes, tumor-associated macrophages (TAMs) and dendritic cells (DCs), generate and promote both immunogenic and tolerogenic responses –63. MDSCTumor-associated neutrophils (TANs) and TAMs have been classified to have an anti-tumor (type 1) or pro-tumor (type 2) phenotype –77. Pro-+ cDCs  are associated with CTL activation and increased overall survival for patients with breast, head and neck or lung cancer [DCs, including classical (cDCs) and plasmacytoid DCs (pDCs), are antigen-presenting cells that prime and regulate CTL responses. Anti-viral immune responses rely heavily on pDC-derived type I interferons (IFN) ; howeverg cancer . β-cateng cancer . In lungg cancer .+ CTLs to FoxP3+CD4+ Tregs [Emerging evidence has suggested that the cross-talk between gut microbiota and immune cells plays a role in determining responses to ICI therapy . The com4+ Tregs . ModulatThe tumor microenvironment (TME) is an intricate milieu of cells hosting the tumor, including endothelial, mesenchymal and immune cells, along with the extracellular matrix (ECM) . Both ce+ T cells, and promotes an immunosuppressive TME by inhibition of DC maturation and induction of Tregs and MDSCs [The trafficking of CTLs, Tregs and immune-suppressive myeloid cells is dependent on several factors encountered in the ECM, including matrix components, vascular endothelial cells and cell surface glycoproteins , 109. Lend MDSCs . VEGF-A nd MDSCs . In meland MDSCs . Decreasnd MDSCs .ECM cytoskeleton remodeling, structural plasticity and mechanical forces are increasingly recognized as crucial factors in immune cell trafficking, activation and immunological synapse formation . Density. Tumor-associated MSCs promote tumor growth and differentiate into pericytes and CAFs in response to stromal growth factors, including platelet-derived growth factor-β (PDGF-β) and fibroblast growth factors (FGF) [Matrix components in the TME are produced by mesenchymal stem cells (MSCs), pericytes and cancer-associated fibroblasts (CAFs)rs (FGF) , 131. Pers (FGF) . In hepars (FGF) . In gliors (FGF) . In melars (FGF) .+ CAFs correlate with an immune suppressive phenotype, with increased CCL2 expression and decreased IFN-gamma and granzyme-B expression, promoting resistance to ICI therapy that is reversed by FAP+ CAF inhibition [CAFs regulate the stromal matrix and serve as a primary source of matrix-associated proteins , 136. CAhibition , 138. Hohibition , 140. Mahibition . TGF-β ihibition , 143. TGhibition .The extracellular matrix consists of hundreds of different components that together constitute the matrisome, including collagens, glycoproteins, and proteoglycans . About oCollagens provide tensile strength to the stroma and basement membrane. Collagen deposition is primarily mediated by fibroblasts and has a critical role in tumorigenesis and immune modulation. In colorectal cancer, tumor invasion and growth by increased collagen deposition and cross-linking has been observed . CollageThere are several matrisome glycoproteins that mediate cellular interactions and define the structure of a tissue along with collagens. Laminins form the basement membrane that is a potentially important barrier to infiltration of immune cells in the matrix. Laminins, especially laminin 411 (α4) and 511 (α5), modulate migration and polarization of the leukocytes . A higheGlycosaminoglycans, including hyaluronan (HA), heparin, heparan sulfate, and chondroitin sulfate, are key macromolecules that affect cell migration and growth by acting directly on cell receptors or via interactions with growth factors . HA is aProteoglycans contain repeating glycosaminoglycans that bind several cytokines and growth factors in the matrix. Heparan sulfate proteoglycans (HSPGs), including transmembrane (syndecan), glycosylphosphatidylinisotol (GPI)-anchored (glypican), secretory granule-derived (serglycin) and secreted HSPGs , are large heterogeneous molecules that interact with growth factors, chemokines and structural proteins of the ECM to influence cellular differentiation and tumor progression –164. EnzVersican (VCAN), a chondroitin sulfate proteoglycan, is normally present in small quantities in soft tissues but it accumulates in the inflamed cancerous and non-cancerous tissues . It inteCleavage of matrisome proteins by matrix-remodeling enzymes generates a wide variety of bioactive peptide fragments, the matrikines, which may act as chemokines or cytokines. Matrix metalloproteinases (MMPs) and adamalysins, including A disintegrin and metalloproteinases (ADAM) and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), are major families of the matrix enzymes that produce matrikines, many of which have unknown functions , 181.Matrikines have a critical role in the infiltration of immune cells in TME and interact with immune regulators like TLRs. Elastin-derived matrikines act as chemokines for fibroblasts and up-regulate collagenase in lung cancer cells . CollageMMPs have been associated with tumor progression and angiogenesis . MMP-2 hThe cancer-immunity cycle, proposed by Chen and Mellman, provides a critical framework to evaluate anti-tumor immune response. It progresses through the immune-mediated tumor cell death and release of tumor antigens, tumor antigen uptake and presentation, priming and activation of T cells, trafficking of T cells, tumor infiltration of T cells and recognition of tumor cells . The susMatrix-remodeling enzymes and matrikines, including versikine, exert direct effects on immune cell polarization and activation. They interact with immune receptors like TLRs and act as cytokines and chemokines to shape the direction and amplitude of the immune response. Recognition of tumor antigens and priming of CTLs is affected by matrikines via interactions with TLRs and DC function. Versikine appears to have a role in promoting local differentiation of the potent immunostimulator Batf3-cDC subset through IRF8 modulation , 186. EnThe detection of VCAN proteolysis in the TME may provide a convenient and reliable immune biomarker that can be utilized across tumor types. Its robust association with “T-cell inflammation” and likely, Batf3-cDC intratumoral density promises to select those patients most likely to respond to ICI and other immune-modulating therapies . The regThe development of novel immunotherapies, including ICIs, was the twenty-first century breakthrough in oncology. Six ICI drugs have been FDA approved and many are in the pipeline. Although there have been durable remissions with the use of ICIs, less than third of the patients derive a benefit from these therapies. An often overlooked facet of immune regulation is the tumor matrix: a diverse and highly dynamic contributor that plays a vital role in the generation and proliferation of the host immune response. Exploring transcriptional imprint and proteomic expression of stromal matrix components may identify promising predictive and prognostic biomarkers. VCAN proteolysis is one emerging paradigm of matrix remodeling and immune modulation. Matrix-derived immune biomarkers promise to generate novel approaches to improve patient stratification and optimize therapeutic strategies employing novel immunotherapies.


Although a number of tactics towards the fabrication and biomedical exploration of stimuli-responsive polymeric assemblies being responsive and adaptive to various factors have appeared, the controlled preparation of assemblies with well-defined physicochemical properties and tailor-made functions are still challenges. These responsive polymeric assemblies, which are triggered by stimuli, always exhibited reversible or irreversible changes in chemical structures and physical properties. However, simple drug/polymer nanocomplexes cannot deliver or release drugs into the diseased sites and cells on-demand due to the inevitable biological barriers. Hence, utilizing therapeutic or imaging agents-loaded stimuli-responsive block copolymer assemblies that are responsive to tumor internal microenvironments  or external stimuli  have emerged to be an important solution to improve therapeutic efficacy and imaging sensitivity through rationally designing as well as self-assembling approaches. In this review, we summarize a portion of recent progress in tumor and intracellular microenvironment responsive block copolymer assemblies and their applications in anticancer drug delivery and triggered release and enhanced imaging sensitivity. The outlook on future developments is also discussed. We hope that this review can stimulate more revolutionary ideas and novel concepts and meet the significant interest to diverse readers. With the increasing numbers of cancerous types and patients, cancer diagnosis and therapy have received a growing attention of chemists and biologists. A range of treatment protocols has been developed, including surgery, radiotherapy, and chemotherapy. However, conventional chemotherapy mainly relies on small molecule anticancer drugs, leading to indiscriminate killing of both cancerous and healthy tissues/cells ,2. In adTo overcome these limitations, polymeric materials based multifunctional delivery systems capable of targeting transport and follow-up site-specific controlled drug release have been developed ,10,11,12Nanocarriers can be able to accumulate at tumor sites either through passive targeting by the enhanced permeability and retention (EPR) effect ,20 or thFurthermore, the tunable sizes and diverse assembly morphologies are another two key factors for polymeric assemblies that influences blood circulation time and tumor accumulation ,24. It hUp to now, extensive polymer assemblies have been developed for the delivery of small molecule drugs and imaging reagents, not only protected them from degradation or inactivation in vivo, but also used to optimize their doses and working efficacy. Small molecule drugs can either be physically encapsulated into polymeric assemblies or covalently conjugated onto polymer chains with high drug-loading content and minimal adverse effects on normal organs. In the case of ordinary physical encapsulation, these assemblies can be achieved generally through in situ drug loading by inner hydrophobic core during assembly formation, but the accurately controlled drug release within target tissues or specific cells is difficult to carry out. The major problems are the undesirable drug leaking during blood circulation due to the non-covalent linkage and the inefficient assembly accumulation in the targeting disease site. An efficient and implemented program to solve the drug leaking defect is either attaching drug molecules to the main/side chains of biocompatible amphiphilic block copolymers to form “polymer-drug conjugates”  or polymAside from the drug protection from wrapped polymers, the introduction of stimuli-responsive polymers, groups, or linkages into nanocarriers will endow additional routes for triggered release of incorporated therapeutic agents into specific tissues by pH, temperature, enzyme concentration, oxidation-reduction surrounding, electromagnetic field, light, or specific bioactive molecules ,36,37,38Due to the urgent requirement of accurate diagnosis, imaging technology guided theranostic has become a newly presented research hotspot. Several kinds of imaging approaches like fluorescence imaging, magnetic resonance imaging (MRI), computed tomography (CT), and radionuclide imaging have been developed in turn ,45,46,47In this article, some critical achievements and rising trend in stimuli-responsive block copolymer assemblies, which are responsive to tumor and intracellular microenvironments and their applications in the area of bioimaging, drug delivery, and therapeutics are summarized. Different from the traditional focus, herein, aside from those frequently appeared factors , the novel chemical structures and tailor-made functions, such as polyprodrug amphiphiles and responsive linker incorporated self-immolative system, are systematically discussed. Moreover, block copolymer nanocarriers responsive to more than one type of stimuli signal for sequential or synergistic microstructural evolution and programmed drug release will also be discussed. Finally, we present an outlook and perspective concerning the potential usage of structural stable hydrophilic helical poly(phenyl isocyanide) (PPI) contained copolymers in this emergent field of bio-relevant nanocarriers. The inherent helical structure of PPI is an excellent mimic for cell-penetrating peptides (CPPs), which performed as an efficient medium to facilitate intracellular delivery of various cargos. The selection of papers was definitely not easy due to the limited page space. We hope this review will be suitable for diverse readers in the field of chemical, biological, and medical science.The abnormal temperature gradients and sensitivity in tumor tissues are critical physiochemical features as compared with that of normal tissues. For example, the local temperature of breast cancer is obviously higher than that of the surrounding tissues . Thus, tN-isopropylacrylamide) (PNIPAM), with a typical lower critical solution temperature (LCST) of ~32 °C [MWs) or copolymerization with hydrophobic or hydrophilic monomers to form PNIPAM incorporated block copolymers. For example, Yang and Discher et al. described the synthesis and preparation of thermoresponsive polymersomes from diblock copolymers of PEO-b-PNIPAM [Representatively, poly (PNIPAM-PS), PNIPAM-b-poly(n-butyl methacrylate) (PNIPAM-PBMA), and stearoyl-terminated PNIPAM-C18 were reported [c-PNIPAM)-b-PCL, via ring-opening polymerization directly initiating from cyclic precursors [c-PNIPAM)-b-PCL copolymers self-assembled into spherical micelles consisting of hydrophobic PCL cores and well-solvated coronas of c-PNIPAM in aqueous solution. As the Dox-loaded micelle dispersion was placed in a pH 7.4 PBS buffer at 20 °C, ~55% of the loaded Dox was released after 50 h. When the buffer temperature increased to 37 °C (above the LCST of PNIPAM coronas), enhanced release rate of Dox can be clearly observed, and ~84% of the loaded Dox can be released after 50 h. The results confirmed that the rate of drug release from micelles fabricated from PNIPAM contained amphiphilic diblock copolymers can be accelerated at elevated temperatures. For comparison, linear diblock copolymer with comparable molecular weight and composition, (l-PNIPAM)-b-PCL, was also synthesized. The effect of chain topologies on the thermal phase transition properties and their functions as controlled release drug nanocarriers were systematically investigated. Similar strategy can be also utilized for constructing core-thermoresponsive micelles [Aside from temperature-sensitive DHBCs, a series of other PNIPAM containing thermoresponsive diblock copolymers, including PNIPAM-reported . All of reported . For exaecursors . At 20 °micelles ,62.N-(2-hydroxypropyl)methacrylamide lactate) (P(HPMA-Lacn)) [2MA with two EO units and POEGMA475 with nine EO units possess LCST values of ~26 and ~90 °C [In addition to PNIPAM, many other thermoresponsive polymers were also developed, including poly(2-isopropyl-2-oxazoline) (PIPO) , oligo(eA-Lacn)) , and elaA-Lacn)) , etc. Thd ~90 °C , respectd ~90 °C . The LCSd ~90 °C . Moreoved ~90 °C . They arb-Phis and PHEMA-b-Phis) or triblock (PLA-b-PEG-b-Phis) copolymers were synthesized and used to construct polymeric assemblies serving as a tumor acidic pH-specific anticancer drug carrier. Under tumor acidic microenvironment, the protonation of PHis blocks might lead to reorganization and deformation of assembly structures, resulting in acidic pH-triggered drug release. Similarly, Bellomo and Wyrsta et al. reported the preparation of amphiphilic diblock copolypeptides containing Nε-2-(2-(2-methoxyethoxy)ethoxy)acetyl-L-lysine and L = poly(L-leucine) [Generally, the extracellular pH of most tumor tissues is weakly acidic (pH 6.5–6.8), which is obviously lower than that of normal tissues (pH 7.2–7.4). The pH discrepancy is also usually used as the trigger factor for ultrasensitive pH-responsive delivery of therapeutic and imaging agents. In the past decades, a series of ultrasensitive pH-responsive polymers have been explored ,70. Amphleucine) . These cleucine) . The remSince the early pioneering work reported by Shen and Ryser et al. the concept of pH-triggered hydrolysis of the linkage between polymer scaffold and pendent group in polymeric assemblies was emerged to release specific molecules to targeted areas . Many pHDlinkm-PDLLA, for improved cancer therapy [Dlinkm linkage, leading to the release of the PEG corona as well as the increase of zeta potential of the nanoparticle, both of which in turn facilitated cellular uptake. With loading of docetaxel (DTXL), the enhanced internalization would also improve in vitro and in vivo therapeutic efficacy. Following the same concept, the same group reported the preparation of Dlinkm linked PEG-Dlinkm-PLGA block copolymers [dPEGNPPLGA/siRNA composite nanoparticles could not only prolong circulation in the blood and accumulate preferentially in tumor sites, but also facilitated cellular uptake by detachment of the PEG corona in response to tumor pH and rapid release of siRNA within tumor cells by replacing the PLA block with PLGA copolymer.The immaculate nanoparticle-based delivery systems should be capable of fitting the demands of a series of stages during delivery procedure, such as prolonged circulation, enhanced accumulation in the tumor, facilitated cellular internalization, and rapid release of the active drug molecules in the tumor cells. However, the optimal strategy for improving one aspect often conflicts with the other, especially the conflict between enhanced cellular uptake and prolonged blood circulation. To get a comprehensive effect, Wang et al. reported a biocompatible and tumor-pH responsive polymeric nanoparticles based on an acid-degradable amide bond (Dlinkm) bridged PEG and poly (PDLLA) block copolymers, denoted as PEG- therapy . The amppolymers . After eTo enhance the stability of polymeric micelles, cross-linked micellar structure is an excellent solution. Recently, Hu et al. reported the fabrication of core cross-linked (CCL) polymeric micelles, which covalently labeled with DOTA(Gd) and green-emitting fluorophores within pH-responsive cores , servingl-lysine) (PEG-PMPA-PLL), consisting of three distinctive functional segments for constructing gene delivery systems [Ka of 6.2 possessed the capacity of buffering, and PLL segment was used to condense DNA due to the much higher pKa (~9.4). The PEG-PMPA-PLL triblock copolymers could form three-layered polyplex micelles consisting of a pDNA/PLL core, an intermediate layer of PMPA, and an outer shell of PEG. A significantly enhanced transfection activity through the buffering capacity of the PMPA segment and efficiently compacting pDNA by the PLL segment were achieved. Furthermore, a newly designed system of polyplex micelles as a multifunctional nanocarrier platform for light-induced gene transfer was reported by the same group [Another category of pH-sensitive assemblies is the polyplex micelles that fabricated from DHBCs through charge neutralization of the oppositely charged polyion blocks ,87. The  systems . PEG wasme group . A threeb-PCL and a targeting ligand c(RGDfK) modified PCL-b-PAE (PCL-b-PAE-c(RGDfK)) in water [Similarly, the hydrophobic poly(β-amino ester) (PAE) could exhibit pH-responsive hydrophobic-hydrophilic transition characteristics due to the presence of tertiary amine moieties. Micelles composed of PAE blocks were also exploited to encapsulate drugs, showing pH-dependent micellar stability ,91,92. Fin water . The micfolate-PEG-b-polyaspartamide)-cholic acid (FA-PEG-b-PAsp(DIP)-CA) for active tumor targeting based on the fact that folate receptor is over-expressed in many cancer cells [It should be noted that the acidic microenvironment of intracellular endosomes and lysosomes is not unique to tumor cells, normal cells can be also response to acid responsive delivery systems. Thus, the therapeutic efficacy of drug nanocarriers based on pH-responsive assemblies mainly relies on the targeting capacity to tumor cells over normal cells . To achier cells . In aqueAnother powerful tool to cleave the linkers in polymer-drug conjugates for drug release is enzyme ,97,98,99N-(2-hydroxypropyl) methacrylamide) that have progressed to clinical trials, GPLG is a tetrapeptide linker Gly-Phe-Leu-Gly that known to be cleaved by lysosomal thiol-dependent protease cathepsin B, the first-generation aromatase inhibitor  is chosen as the endocrine model [Drug molecules covalently linked to polymer scaffold via a peptide linker are always responsive to specific enzyme, which are always present with much higher concentrations and abnormally activity in the tumor tissue compared to that in normal tissues due to the over expression ,102. Thune model . In the ne model . The ProKa: ~6.9) was used to cover the surface of CPX1 to form CPX2, which was stable under normal biological milieu and could avoid undesirable accumulation in the liver. Once exposure to the acidic microenvironment (pH < 7) in tumor tissue, pH-sensitive His-alginate-PEG firstly left the CPX2 due to electrostatic repulsion. The naked CPX1 was further degraded in the presence of gelatinase and deoxyribonuclease (DNase), resulting in the effective release of encapsulated Dox  scaffolds were formed  and intracellular (reduction) microenvironments endow the drug delivery systems a new mechanism. Disulfide bonds, the most frequently studied reduction-responsive groups, favor the oxidizing environment in the extracellular microenvironment and can maintain the structure and functions ,113,114.Although encapsulation of small-molecule drugs into copolymer assemblies has been extensively used for preparing polymeric nanocarriers, the inevitable low drug loading efficiency tends to be a major obstacle to limit the therapy efficiency. To address this challenge, a dimeric drug conjugate bearing a trigger responsive domain was designed by Cheng et al.  and usedb-PCPTM, where PEG is poly(ethylene glycol) and PCPTM is polymerized block of reduction-cleavable CPT prodrug monomer [polyprodrug amphiphiles, can self-assemble into four types. Among them, the unprecedented staggered lamellae assemblies outperform the other three nanostructure types. The controlled hierarchical organization of polyprodrug amphiphiles and shape-tunable biological performance opens up new horizons for exploring next-generation block copolymers based drug delivery systems with improved efficacy. This work represents the typical example of polyprodrug amphiphiles which are amenable to the fabrication of multiple hierarchical nanostructures for the investigation of shape-tunable biological performance. Based on this remarkable excogitation, a new class of theranostic nanoparticles constructed from hyperbranched polyprodrug amphiphiles consisting of hyperbranched cores conjugated with reduction-activatable CPT prodrugs and MRI contrast agent (Gd complex), and hydrophilic coronas functionalized with guanidine residues was designed by the same group [Furthermore, Liu et al. described the synthesis, hierarchical self-assembly, and relevant biological functions of amphiphilic block copolymers, PEG- monomer . These dme group . The hyp2O2), hydroxyl radicals (•OH), superoxide radicals (O2−), hypochlorite (OCl−), and singlet oxygen (1O2), play a really important role in both healthy and diseased biological processes [Reactive oxygen species (ROS), such as hydrogen peroxide  at pH 7.4, suggesting great promise for inflammation specific drug delivery. Similarly, another kind of phenylboronic ester functionalized amphiphilic block copolymers was reported by Liu et al. [2O2 could trigger the transformation of amphiphilic charge-generation polymers from initially uncharged state into a cationic polyelectrolyte on demand by taking advantage of the chemo-selective cleavage of phenylboronic-carbamate protecting moieties. The charge-generation process was then coupled with the induced aggregation of Au nanoparticles to design colorimetric probes.A variety of block copolymer-based oxidation-responsive systems have been developed and utilized for different purpose, take Ht groups . The copdrolysis . Moreoveu et al. . The pre2O2) or reductant (GSH)  bonds (bond energy: 172 KJ/mol) can be either oxidized to seleninic acid in the presence of oxidants or reduced to selenol in a reducing environment . For exant (GSH) , leadingLight, with variable wavelength and intensity, is a cheap and easily manipulated clean resource and can be used in a spatiotemporal precision manner. Cargo-loaded ultraviolet (UV) light-responsive polymeric micelles and vesicles have gradually received much attention due to their potential applications in the diagnosis and treatment, such as quick and localized drug release and high-efficiency on-demand disease treatment compared with other formulations ,129,130.trans-azobenzene groups were transformed to the more polar nonplanar cis-forms, which significantly increased the hydrophilicity of the original hydrophobic blocks. This process can return back under irradiation at 420–490 nm or place in the darkness. Therefore, the assemblies could be dissociated with UV light and reassembled using visible light in a reversible manner. For example, Oriol and coworkers reported an AB3 type miktoarm copolymers possessing one polymethacrylate (PAZO) arm with 4-isobutyloxyazobenzene side chain and three PEG arms (PEG), denoted as (PAZO)(PEG)3, which could form stable polymeric vesicles in water [λ = 350–400 nm), causing the disruption of the vesicles and cargos release  ,132 and in water . After e release .b-PSP  to a hydrophilic charged opened-ring merocyanine form  under UV (365 nm) and visible light irradiation (620 nm). Compared to AZO moieties, the difference in polarity between hydrophobic SP and hydrophilic ME units is greater, hence, the SP-incorporated polymers with reversible behavior have been widely studied for various applications such as light-actuated nanovalves and nanoswitches. For example, PEO block SP-containing methacrylate monomers were reported by Matyjaszewski group , which tb-PSP a. In aqub-PSP b. When aο-nitrobenzyl (NB) derivatives, for example, have been incorporated into amphiphilic block copolymers and proved to be an efficient method for constructing photo-triggered cleavage or degradation nanocarriers [ο-nitrobenzyloxy-l-carbonyl protecting group was removed, the PT could undergo a 1,4-elimination followed by a 1,8-elimination, leading to chain shattering and the release of the constituent drug molecules (block-poly(l-lactide) (PEL) in water, the CSPTs/PEL nanoparticles (d < 150 nm) with extremely high drug loading (>48 wt %) and loading efficiency (>92%) were obtained  diblock copolymers, where SPA is SP-based monomer containing a unique carbamate linkage, were prepared by the same group [b-PSPA were crucial for polymersome stabilization in the zwitterionic MC state, and the reversible phototriggered SP-to-MC polymersome transition was accompanied by membrane polarity and permeability switching from being nonimpermeable to selectively permeable toward noncharged, charged, and zwitterionic small molecule species below critical molar masses. The photoswitchable spatiotemporal release of 4’,6-diamidino-2-phenylindole  within living HeLa cells was further demonstrated.Recently, Liu et al. chanced upon the solution of a long-standing dilemma concerning the synchronized stability and permeability of vesicles (polymersomes) by employing a light-regulated “traceless” crosslinking strategy . Brieflyr chains . This feme group . Upon seco-AA)-b-PCL, with appropriate response to pathological environment and high payload of anticancer drug  was fabricated [Although great progress has been made in the field of stimuli-responsive drug carriers, more intricate designs and innovative strategies need to be developed to adapt to the more complex situations. For example, most of the investigated stimuli-responsive block copolymer delivery systems are only responsive to a single stimulus, which are not capable of integrating more complicated delivery and release needs. Thus, engineering sequential or simultaneous multi-stimuli responsive delivery systems will facilitate more intricate drug delivery and controlled release processes and imaging ability ,144,145.bricated . The nan2 polycondensation methodology and sequential postfunctionalization [2O2 with a high detection limit, and colorimetric H2O2 assay by integrating triggered dispersion of gold nanoparticle aggregates with enzyme-mediated cycle amplification. The modular design, facile synthesis, multifunctional construction, water-dispersibility, high intrinsic chemical amplification, and potency of integrating with external cycle amplification modules of the reported hSIPs platform opens a new avenue to fabricate novel stimuli responsive materials with high selectivity, sensitivity, and specificity, a prerequisite for functions in complex biological media. Moreover, the fabrication of self-immolative polymersomes via self-assembly from amphiphilic block copolymers consisting of a hydrophobic multi-triggered self-immolative block and a hydrophilic poly (PDMA) block was also reported [Recently, a library of water-dispersible hyperbranched self-immolative polymers (hSIPs) with structurally and functionally diversity were fabricated by utilizing one-pot ABlization . The sporeported . The selWith the rapid development of polymer synthetic and nano techniques, stimuli-responsive block copolymers and corresponding assemblies were created one after another and used for targeted delivery of drugs and imaging/contrast agents to tumor sites and realized controlled/triggered release. In this review, recently emerged tumor and intracellular microenvironment-responsive block copolymer assemblies with diverse functions, structures, and self-assembling morphologies have been discussed. Typical tumor microenvironments, such as weak acidic pH, abnormal temperature gradients, a variety of specifically over-expressed enzymes, and redox species are available to be utilized to construct responsive block copolymer based integrated platform, allowing for triggered payload release and enhanced imaging sensitivity. Although great progress in this field has been achieved, not all of the therapeutic effects are performed as well as expected. The materials that respond to physiological relevant concentrations within micromolar or nanomolar regime are still lag behind requirement.The common feature of the above mentioned examples is that all the selective and controlled modulation processes were based on the stimuli-responsiveness of polymeric building blocks, and the accumulation in tumor sites is assisted by either passive or active targeting. However, the nonnegligible low percent (sometimes only 0.7% ) of injeHowever, the artificial noncharged polymer mimics exhibiting cell-penetrating properties are rarely emerged. Poly(phenyl isocyanide) (PPI) is a class of interesting synthetic polymers because it can maintain stable helical conformation in solution and in solid state. Yashima et al. reported that PPI possessing a stable rigid rod-helical conformation with a long persistence length . These h


Pulmonary arterial hypertension, categorized as group 1 pulmonary hypertension by the World Health Organization classification system, represents a major complication of systemic sclerosis resulting from pulmonary vascular involvement of the disease. The high mortality seen in systemic sclerosis-associated pulmonary arterial hypertension is likely due to the impairment of right ventricular systolic function and the coexistence of other non-group-1 pulmonary hypertension phenotypes that may negatively impact clinical response to pulmonary arterial hypertension-targeted therapy. This review highlights two areas of recent advances regarding the management of systemic sclerosis patients with pulmonary hypertension: the tolerability of pulmonary arterial hypertension-targeted therapy in the presence of mild to moderate interstitial lung disease and the potential clinical significance of the antifibrotic effect of soluble guanylate cyclase stimulators demonstrated in preclinical studies. Based on the extent of skin fibrosis, SSc has been classified as either limited cutaneous or diffuse cutaneous phenotype3, with diffuse cutaneous SSc characterized by earlier and more frequent internal organ involvement4. The term “systemic sclerosis sine scleroderma” is sometimes used to refer to SSc with visceral organ involvement in the absence of cutaneous manifestation6.Systemic sclerosis (SSc) is a systemic rheumatic disease characterized by autoimmunity, vascular injury, and unchecked collagen synthesis leading to fibrosis of the skin and internal organs11. PAH represents group 1 pulmonary hypertension (PH) in the World Health Organization (WHO) classification system, characterized by elevated mean pulmonary arterial pressure (mPAP) ≥25 mmHg with elevated pulmonary vascular resistance (PVR) ≥3 Wood units and normal pulmonary capillary wedge pressure (PCWP) ≤15 mmHg in the absence of etiologies known to cause other groups (2–5) of PH12. More recently, mPAP >20 mmHg has been proposed as a new definition of precapillary PH, which includes PAH13. Approximately 10% of SSc patients  are affected by PAH16, and a similar prevalence was re-demonstrated in a recent study17. While SSc-associated PAH (SSc-PAH) comprises a major proportion of PAH registries18, less is known about its incidence, with one study reporting 0.61 cases per 100 patient-years19.One major complication of SSc is pulmonary arterial hypertension (PAH). Historically, PAH has been quoted as one of the leading causes of death in patients with SSc20, suffers from a particularly poor prognosis. The presence of PAH predicts early death in SSc22, and mortality associated with SSc-PAH is higher than that seen in other PH groups and even other subtypes of PAH23. Multiple studies demonstrated poorer survival in SSc-PAH compared to idiopathic PAH (IPAH) despite less pronounced hemodynamic derangement29. A recent retrospective study of 375 patients showed similar findings , and the difference in mortality was attributed to older age and more pronounced gas exchange impairment in SSc-PAH30. Another plausible explanation for the high mortality in SSc-PAH is reduced contractility of the right ventricle (RV). A number of retrospective studies have consistently documented more impaired RV systolic function in SSc-PAH compared to that in IPAH for a given RV afterload32, and the RV function appears to improve with PAH-targeted therapy in IPAH but not in SSc-PAH33. SSc-PAH is associated with worse outcomes and higher mortality, even when compared to other connective tissue disease-related PAH (CTD-PAH), suggesting that SSc-PAH represents a unique phenotype that perhaps should be managed differently to other subgroups of CTD-PAH34.SSc-PAH, which can complicate both limited and diffuse cutaneous subtypes of SSc35. Clinical failure  was observed in 18% of those receiving the combination treatment compared to 31% of monotherapy recipients (hazard ratio of 0.50). After 24 weeks of intervention, the combination therapy was associated with improvements in secondary outcomes, including the 6-minute walk distance and N-terminal pro-brain natriuretic peptide levels, compared to pooled monotherapy. Other recent studies suggest that PAH-targeted therapy, such as the ambrisentan and tadalafil combination treatment, riociguat, and selexipag, likely confers clinical benefit in the subgroup of CTD-PAH patients38. The optimal medical therapy for the subset of SSc-PAH patients, however, is less defined39, as demonstrated by the SSc-PAH-associated mortality that remains high40 and largely unchanged over time42. For instance, a recent study from the REVEAL registry  demonstrated that 5-year mortality in subjects newly diagnosed with SSc-PAH can be as high as 60%41. Despite improved understanding of predictors of mortality in SSc-PAH44 and the development of new prognostic models specifically validated in SSc-PAH cohorts47, clinical outcomes of SSc-PAH remain rather disappointing48.The Ambrisentan and Tadalafil in Patients with Pulmonary Arterial Hypertension (AMBITION) trial was a recently published randomized double-blind multicenter study that compared upfront ambrisentan and tadalafil combination therapy to monotherapy in 500 treatment-naïve participants with WHO functional class II–III PAH49. SSc is often complicated by fibrotic interstitial lung disease (ILD)52, more commonly in diffuse cutaneous SSc than in limited cutaneous SSc53. The fibrotic ILD can be further complicated by neuromuscular weakness and chronic aspiration54, contributing to the development of hypoxemia-induced (WHO group 3) PH. Unselected use of PAH-specific therapy with vasodilatory effect in the presence of ILD can exacerbate ventilation–perfusion (V/Q) mismatch, resulting in aggravation of hypoxemia and secondary PH55.The poor clinical outcome and suboptimal response to treatment observed in SSc-PAH likely reflect the various phenotypes of PH that can coexist in SSc, precluding straightforward identification of those SSc patients with isolated PAH 49. SSc 58. Although smoking history places SSc patients at a higher risk of developing CPFE59, it is also observed in never-smokers and those with minimal tobacco history57. A significant portion of SSc patients with CPFE go on to develop PH60, illustrated by a recent study demonstrating a higher incidence of precapillary PH and higher mortality in 36 SSc-CPFE patients compared to 72 SSc-ILD patients without emphysema59. Emphysema in CPFE can cause an additional reduction in carbon monoxide diffusing capacity (DLCO) and lung volume pseudo-normalization, and these physiologic consequences of CPFE can potentially compromise the diagnostic accuracy of PH in SSc patients with fibrotic ILD57.One form of ILD increasingly recognized in SSc is combined pulmonary fibrosis and emphysema (CPFE)62. In patients with SSc-PAH, the presence of computed tomography findings suggestive of PVOD has been associated with pulmonary edema in response to vasodilator therapy and worse clinical outcomes64. Even more rarely, patients with SSc can suffer from pulmonary capillary hemangiomatosis (PCH), a disease that is now thought to be related to PVOD in its etiology65. Other unrelated mechanisms by which SSc causes post-capillary PH and precipitates pulmonary edema include myocardial fibrosis68 and ischemic heart disease due to accelerated atherosclerosis (WHO group 2)70. Subtle left ventricular diastolic dysfunction due to occult fibrosis of the myocardium may not be apparent and become unmasked with fluid challenge during cardiac catheterization71.Under-recognized pulmonary venous hypertension in SSc is also believed to underlie the poor response to treatment and high mortality seen in SSc-PAH. Pathologic studies of lung tissues from SSc-PAH patients demonstrate fibrosis of veins and venules resulting in pulmonary veno-occlusive disease (PVOD)-like changes (WHO group 1)74. There is an increased prevalence of antiphospholipid antibodies in SSc, although it remains unclear whether these antibodies represent the autoimmune nature of SSc-PAH, under-recognition of CTEPH, or both78. SSc is also associated with primary biliary cholangitis, which may subsequently produce portopulmonary hypertension80. These various phenotypes of PH often coexist in SSc, and they underscore the difficulty in selecting the right patient population who will benefit from PAH-targeted therapy or even anticoagulation.Many studies have also documented the increased risk of venous thromboembolism (VTE) in SSc, which predisposes the patient to developing chronic thromboembolic PH 53, and the presence of concurrent ILD is associated with increased mortality in SSc patients with precapillary PH83. While PAH-specific drugs may alleviate the increased PVR resulting from pulmonary arteriopathy of SSc, the often-coexisting ILD in this patient population has raised the concern of exacerbating V/Q mismatch as more blood flows to the non-ventilated parts of the lungs55. As a result, there has been uncertainty regarding the tolerability of PAH-targeted therapy in SSc-associated precapillary PH owing to the high prevalence of concurrent ILD. Contrary to this historical perspective, however, two recent studies illustrate that PAH-targeted therapy may be well tolerated in the presence of ILD.The role of PAH-targeted therapy in SSc patients with both ILD and precapillary PH represents a major area of uncertainty and interest to both clinicians and researchers. SSc often involves ILD35. A recentpost hoc analysis of the AMBITION trial showed that the combination PAH-targeted therapy conferred similar clinical benefit in the subgroup of SSc-PAH patients36. Of the 118 subjects with SSc-PAH, clinical failure  was seen in 21% of those receiving the combination treatment compared to 40% of monotherapy recipients (hazard ratio of 0.44 with 56% risk reduction). In the SSc-PAH subgroup, the combination therapy led to significantly improved outcomes compared to what had been previously reported in the general SSc-PAH patient population. While the AMBITION trial specifically targeted subjects with PAH, it is notable that the participants were allowed to have mild to moderate lung disease 35. Therefore, it is probable that a substantial portion of the SSc-PAH subgroup had some degree of ILD or even SSc-ILD-PH, although the presence of severe ILD would have been unlikely given that the average TLC was 90% of predicted36. The subgroup analysis did not investigate the effect of the combination therapy on hypoxemia and gas exchange; however, the study suggests PAH-targeted therapy with vasodilatory effect can be well tolerated in SSc-PAH patients with concurrent non-severe ILD, and it may be unnecessary to exclude a modest degree of ILD prior to trialing PAH-targeted therapy.As previously mentioned, the AMBITION trial was a randomized double-blind study that demonstrated that the upfront combination therapy with ambrisentan and tadalafil protected treatment-naïve PAH patients from clinical worsening compared to monotherapy84. The ILDs in these patients were physiologically mild , followed either nonspecific interstitial pneumonia (NSIP) or usual interstitial pneumonia (UIP) pattern, and affected >20% of the lungs in 65.5% (19 of 29) of cases. The majority of the participants had mild (mPAP 25–35 mmHg) or moderate (mPAP 35–45 mmHg) PH, and six of the 29 had post-capillary PH with PCWP >15 mmHg. A total of 24 of the 29 patients were treated with PAH-targeted therapy, and they tolerated a single, dual, or triple-agent PAH regimen well. Importantly, the PAH-targeted treatment did not worsen V/Q mismatching, again supporting the notion that PAH-specific therapy can be safely used in SSc patients with non-severe ILD. Given the non-randomized nature of this evidence, however, more robust, large-scale, randomized studies are needed before firm conclusions and generalized clinical applications can be made.A more recent single-center cohort study evaluated response to PAH-targeted therapy in 29 SSc patients who had both right heart catheterization-proven PH and ILD visualized on high-resolution computed tomography of the chest85. In mice, the sGC stimulator BAY 41-2272 prevented and reversed skin fibrosis86. Similarly, riociguat, another sGC stimulator in clinical use, was shown to be effective against skin and gastrointestinal tract fibrosis in multiple mouse models, with its antifibrotic effect more pronounced than that of sildenafil87.Soluble guanylate cyclase (sGC) stimulators, another class of PAH-targeted drugs, have recently gained interest for their potential use in SSc-PAH. In addition to vasodilation, sGC stimulation and a resultant increase in the cyclic guanosine monophosphate level produce an antifibrotic effect by inhibiting non-canonical TGF-β signaling pathways89. A recent subgroup analysis of the two trials demonstrated in SSc-PAH patients that riociguat was associated with improvement in cardiac index and reduction in PVR at 12 weeks, and possibly with an increase in the 6-minute walk distance compared to placebo37. Another case series described three patients whose SSc-PAH improved after phosphodiesterase-5 inhibitors were replaced with riociguat90. It remains uncertain, however, whether the antifibrotic effect of riociguat confers additional clinical benefit specific to SSc-PAH.Since SSc is characterized by fibrosis as well as vasculopathy, sGC stimulators with both antifibrotic and vasodilatory effects are considered promising candidates for the treatment of SSc-PAH. Pulmonary Arterial Hypertension Soluble Guanylate Cyclase-Stimulator Trial (PATENT)-1 was a double-blind randomized international study that established the efficacy of riociguat in PAH patients, which was extended to PATENT-2 for the assessment of long-term effects of riociguat91. However, in a recently published multicenter randomized trial, Riociguat for Idiopathic Interstitial Pneumonia-Associated Pulmonary Hypertension (RISE-IIP), riociguat was associated with harm and increased mortality in patients with precapillary PH and idiopathic (therefore non-CTD) ILD (mean TLC 66% of predicted)92. While RISE-IIP did not involve SSc or other CTD patients, the demonstrated negative impact of riociguat in patients with significant lung disease will likely deter future attempts to prospectively investigate sGC stimulation in SSc-ILD-PH, barring new, convincing observational data favoring its use in this patient population. Lastly, given that riociguat is effective in CTEPH93 and SSc patients are predisposed to VTE, another interesting question regards the potential role of sGC stimulation in SSc patients affected by CTEPH.Despite these seemingly favorable outcomes, the safety and tolerability of riociguat in SSc-PAH patients with concurrent ILD are unclear. In a pilot study of 22 patients with concurrent ILD (TLC >30% of predicted) and treatment-naïve precapillary PH, the use of riociguat was associated with lower PVR and higher cardiac output, suggesting riociguat can safely provide similar hemodynamic benefit in patients with SSc-ILD-PHPAH-targeted therapy appears to be well tolerated and beneficial in SSc-PAH patients with its safety further questioned in the coexistence of ILD. Challenges still remain in choosing the patient population best suited for PAH-specific medications. The distinction between SSc-PAH with ILD (WHO group 1) and SSc-ILD-PH with increased PVR (WHO group 3) is arbitrary and remains undefined. Similarly, the severity of ILD beyond which PAH-targeted therapy becomes harmful is unknown, and this threshold may in part depend on the type of ILD, patient demographics, and other comorbidities. Each SSc patient with PAH or ILD-PH may develop varying degrees of other confounding complications that can independently cause PH  either before or even during PAH-targeted treatment, precluding reliable patient selection and accurate response assessment. For example, it is likely prudent to avoid indiscriminate use of PAH-targeted therapy in the presence of significant left-sided heart failure in order to prevent the potential precipitation of pulmonary edema. Furthermore, how rigorously and frequently SSc patients with established PH should be screened for other associated causes of PH is unclear. High-quality, randomized trial-based data are lacking, partially owing to the rarity of the disease.96. While we await answers to the aforementioned questions, the decision to implement PAH-targeted therapy in SSc will have to remain individualized, incorporating both the clinician’s experience and the patient’s values.Novel therapeutic approaches may clarify the unresolved treatment strategies in this patient population. Inhaled PAH-targeted therapy has the theoretical advantage of improving perfusion only in the well-ventilated areas of the lungs, thereby preventing worsened V/Q mismatch that may be seen with systemic administration of the medication. Based on this hypothesis, an ongoing clinical trial (NCT02630316) looks at the role of inhaled treprostinil in ILD-PH patients, the results of which may be applicable to SSc patients with concurrent precapillary PH and ILD. Early evidence suggests the possibility of using gene expression profiling to predict clinical course and monitor response to PAH-targeted therapy in SSc patients, although more data are needed until clinical applicability can be reachedAMBITION, Ambrisentan and Tadalafil in Patients with Pulmonary Arterial Hypertension; CPFE, combined pulmonary fibrosis and emphysema; CTD, connective tissue disease; CTD-PAH, connective tissue disease-related pulmonary arterial hypertension; CTEPH, chronic thromboembolic pulmonary hypertension; DLCO, carbon monoxide diffusing capacity; ILD, interstitial lung disease; IPAH, idiopathic pulmonary arterial hypertension; mPAP, mean pulmonary artery pressure; PAH, pulmonary arterial hypertension; PATENT, Pulmonary Arterial Hypertension Soluble Guanylate Cyclase-Stimulator Trial; PCWP, pulmonary capillary wedge pressure; PH, pulmonary hypertension; PVOD, pulmonary veno-occlusive disease; PVR, pulmonary vascular resistance; RISE-IIP, Riociguat for Idiopathic Interstitial Pneumonia-Associated Pulmonary Hypertension; RV, right ventricle; sGC, soluble guanylate cyclase; SSc, systemic sclerosis; SSc-PAH, systemic sclerosis-associated pulmonary arterial hypertension; TLC, total lung capacity; V/Q, ventilation–perfusion; VTE, venous thromboembolism; WHO, World Health Organization.


Chronic obstructive pulmonary disease (COPD) is a leading cause of disability and death world-wide, where chronic inflammation accelerates lung function decline. Pathological inflammation is worsened by chronic bacterial lung infections and susceptibility to recurrent acute exacerbations of COPD (AECOPD), typically caused by viral and/or bacterial respiratory pathogens. Despite ongoing efforts to reduce AECOPD rates with inhaled corticosteroids, COPD patients remain at heightened risk of developing serious lung infections/AECOPD, frequently leading to hospitalization and infection-dependent delirium. Here, we review emerging mechanisms into why COPD patients are susceptible to chronic bacterial infections and highlight dysregulated inflammation and production of reactive oxygen species (ROS) as central causes. This underlying chronic infection leaves COPD patients particularly vulnerable to acute viral infections, which further destabilize host immunity to bacteria. The pathogeneses of bacterial and viral exacerbations are significant as clinical symptoms are more severe and there is a marked increase in neutrophilic inflammation and tissue damage. AECOPD triggered by a bacterial and viral co-infection increases circulating levels of the systemic inflammatory marker, serum amyloid A (SAA). SAA is a functional agonist for formyl peptide receptor 2 (FPR2/ALX), where it promotes chemotaxis and survival of neutrophils. Excessive levels of SAA can antagonize the protective actions of FPR2/ALX that involve engagement of specialized pro-resolving mediators, such as resolvin-D1. We propose that the anti-microbial and anti-inflammatory actions of specialized pro-resolving mediators, such as resolvin-D1 should be harnessed for the treatment of AECOPD that are complicated by the co-pathogenesis of viruses and bacteria. Chronic obstructive pulmonary disease (COPD) is an umbrella term encompassing multiple lung pathologies  that manifest into persistent and poorly reversible airflow limitation. It is a major chronic disease that is predicted to become the third leading cause of death world-wide by 2030  and has Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis ; accounting for up to 40–50% of bacteriologically examined cases. COPD is a common underlying chronic comorbid condition in CAP  and influenza , 27. ExpH. Influenzae from the pre-existing lung microbiota, which persisted for over 40 days  as an acute phase reactant that is markedly increased during severe episodes . Further4) to pro-resolving mediators during the course of infection and acute inflammation (4 (LXA4) is a specialized pro-resolving mediator that directly interacts with FPR2/ALX to initiate resolution pathways. This eicosanoid opposes leukocyte migration and activation by suppressing transendothelial  superfamily characterized by seven putative trans-membrane domains that displays diverse ligand affinities, interacting with over 30 different ligands . By bindammation . Lipoxinothelial  and tranothelial  migratioothelial . Furthermulation . LXA4 all injury . In addiammation , 59.4, RvD1 and its more stable aspirin-triggered derivative (AT-RvD1) display potent anti-inflammatory actions in a number of disease models including cigarette smoke exposure and acute lung injury  and enhance neutrophil efferocytosis to prevent collateral lung injury , which belong to the D series resolvins derived from the omega-3 fatty acid, docosahexaenoic acid . Like LXg injury , 61. Thig injury , 63. Morung load . We alsoung load . Our appung load . The redung load . AT-RvD1ung load .S. pneumoniae can enter and proliferate in the brain, with the ensuing inflammatory response causing bacterial meningitis in severe cases (in vitro conditions SAA proved to be a potent stimulus for primary mouse microglia activation, and this response was markedly suppressed by the anti-inflammatory actions of AT-RvD1 (In addition to reducing the degree of localized pneumonia, specialized pro-resolving mediators, such as AT-RvD1 may also treat the central consequences of co-infection or bacterial super-infection. In the classic bacterial super-infection setting, re cases . Whilst re cases . Brain ire cases , 68 and re cases . The cirre cases  and excere cases . Consistre cases . Further AT-RvD1 .The likelihood of developing a co-infection or secondary bacterial infection is increased in COPD largely due to the ROS-dependent suppression of macrophage phagocytic function. Suppressed macrophage phagocytosis contributes to the establishment of chronic bacterial infection of the lower airways. Consequently, acute infection with a newly acquired respiratory virus, such as influenza A can promote uncontrolled bacterial outgrowth in the lung. This is observed clinically, as the detection of a respiratory virus and bacteria during AECOPD occurs frequently and is associated with an increase in exacerbation severity and neutrophilic inflammation. We have shown that the exuberant inflammatory response during co-infections are associated with elevated levels of SAA, which is an agonist for FPR2/ALX that promotes neutrophil migration and survival. Specialized pro-resolving mediators, such as lipoxins and D-series resolvins can counteract the pro-inflammatory actions of SAA. We provide compelling pre-clinical data to demonstrate that AT-RvD1 is a very effective therapeutic in the co-infection setting, where it potently suppressed leukocyte tissue accumulation in the lungs and concurrently improved pneumococcal clearance. Furthermore, co-infections markedly increase brain inflammation and AT-RvD1 can suppress the release of inflammatory cytokines from activated microglia. In summary, specialized pro-resolving mediators have great potential in the co-infection setting as they not only reduce local tissue inflammation and improve bacterial clearance at the site of infection, but they can also dampen central microglial inflammation that prolongs sickness behavior and cognitive dysfunction (Figure SB performed the literature review. HW, DA, and SB designed the figures. SB, SS, HW, DA, and RV organized, wrote and edited the 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.


Selective removal of initially tumor-positive axillary lymph nodes in breast cancer patients who underwent neoadjuvant systemic treatment (NST) improves the accuracy of nodal staging and provides the opportunity for more tailored axillary treatment. This study evaluated whether radioguided occult lesion localization (ROLL) of clip-marked lymph nodes is feasible in clinical practice.Prior to NST, a clip marker was placed inside a proven tumor-positive lymph node in all breast cancer patients (cTis-4N1-3 M0). After NST, technetium-99m-labeled macroaggregated albumin was injected in the clip-marked lymph nodes. The next day, these ROLL-marked nodes were selectively removed at surgery to evaluate the pathological response of the axilla.Thirty-seven patients (38 axillae) underwent clip insertion. After NST, the clip was visible by ultrasound in 36 procedures (95%). In the other two patients, the ROLL-node injection was performed in a sonographically suspicious unclipped node (1), and near the clip under computed tomography guidance (1). Initial surgery successfully identified the ROLL-marked node with clip in 33 procedures (87%). Removed specimens in the other five procedures contained only the sonographically suspicious tumor-positive unclipped node (1), a node with signs of complete response but no clip (2), a clip without node (1), and tissue without node nor clip, and a second successful ROLL-node procedure was performed (1). Overall, 10 ROLL-marked nodes had no residual disease.This study demonstrates that the ROLL procedure to identify clip-marked lymph nodes is feasible. This facilitates selective removal at surgery and may tailor axillary treatment in patients treated with NST. Neoadjuvant systemic treatment (NST) is applied for tumor downstaging to facilitate breast-conserving therapy in patients with node-negative or node-positive disease . PatholoAlthough sentinel lymph node (SLN) biopsy has become the standard of care for nodal staging of clinically node-negative breast cancer patients , SLN bioCaudle et al. developed the targeted axillary dissection (TAD) technique which resulted in a FNR below 5% . The TADSimultaneously, a technique solely based on removal of the pre-NST marked node without SLN biopsy was developed, the so-called MARI (marking of the axilla with radioactive iodine-125 seeds) procedure .Although iodine-125 seeds are increasingly being used for breast-conserving surgery and provide excellent surgical guidance in locating axillary lymph nodes , there aTo overcome these limitations, we propose a technique for locating clip-marked lymph nodes without SLN biopsy by using an injection of technetium-99m(Tc-99m)-labeled macroaggregated albumin (MAA) instead of iodine-125 seeds. This is a modification of the MARI procedure by using a two-step node marking procedure. The concept is based on two case series studying the radioguided occult lesion localization (ROLL) procedure for identifying axillary lymph nodes. In these studies, Tc-99m-MAA is injected directly into sonographically suspicious axillary lymph nodes , 18. TheThe present study assessed the feasibility of ROLL injections into clip-marked lymph nodes that were tumor-positive prior to NST and selectively remove them at surgery after NST in breast cancer patients.Since February 2015, all female patients with clip-marked cytology-proven node-positive breast cancer in the Slotervaart hospital (SVH) and referred to the Nuclear Medicine Department of the Netherlands Cancer Institute (NCI) were prospectively entered into a database. These patients underwent a Tc-99m-MAA injection in the clip-marked lymph node for response evaluation after NST. This procedure was part of the patient’s standard clinical workup as replacement of the MARI procedure as standard clinical practice, since there was no authorization to work with iodine-125 seeds in the SVH. This dual-center retrospective study was approved by the Institutional Review Board of the NCI.Once breast cancer was confirmed, the size and extent of the primary tumor were routinely assessed by mammography, ultrasound, and magnetic resonance imaging (MRI). All patients underwent ultrasound of the breast and axilla. Whole-body 18F]fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) was performed before NST for the detection of regional and distant metastases. In addition, PET/CT of the thorax was performed in prone position and with hanging breasts. Fine-needle aspiration was performed in case of suspicious lymph nodes identified by physical examination, ultrasound, MRI, or PET/CT. When cytological evaluation demonstrated metastasis, a clip marker  was placed within the largest tumor-positive lymph node under ultrasound guidance by a breast radiologist at the SVH , highly hormone-sensitive tumors, and co-morbidity.yiN status. Suspicious lymph nodes were defined by a cortex of ≥ 2.3 mm. A 21-G needle was placed percutaneously with the tip in the center of the node  was used to localize the area of maximal radioactivity allowing the appropriate location of the incision. In patients undergoing a mastectomy without primary breast reconstruction, the incision of the mastectomy was used to dissect towards the ROLL-marked node. A separate incision was made in the other patients. Careful dissection was carried out following the area of maximum radioactivity until the ROLL-marked node was identified and excised. Measurements of the ex vivo specimen and background were performed to confirm the removal of the ROLL-marked node. The excised specimens in patients with four or more 18F-FDG-avid axillary lymph nodes on pretreatment PET/CT were sent to the pathologist for intraoperative frozen section analysis. An ALND was performed when the frozen section came back tumor-positive.Histopathological examination was performed on all harvested lymph nodes. All lymph nodes were fixed in formalin, bisected, sliced at 2-mm intervals, and embedded in paraffin, followed by hematoxylin-eosin and immunohistochemical staining . The pathologist confirmed the presence of the clip and lymph nodes in the excised specimens.Tailored axillary treatment was performed after adapting the previously described protocol in which axillary treatment was based on pretreatment PET/CT prior to NST and pathological status of the clipped node after NST , 20. In In total, 1 bilateral (left and right axilla) and 36 unilateral procedures met the inclusion criteria. The clinicopathologic and treatment characteristics of these 37 patients with 38 ROLL-node procedures are listed in Table In 26 procedures (68%), one to three 18F-FDG-avid axillary nodes were detected on PET/CT, and in 12 procedures (32%), four or more nodes were detected.In 36 procedures (95%), the ROLL-node injection was performed in close proximity to the clip using ultrasound guidance. The clip could not be visualized sonographically in two patients. Therefore, a sonographically suspicious enlarged unclipped lymph node was injected in one patient and the other patient proceeded to a CT-guided injection of a deeply located clip-marked node Fig. .Fig. 2FScintigraphic images were performed at on average 24 min (range 11–130 min) post-injection. Three different scintigraphic patterns were observed . Histopathology confirmed that in 95% (36/38) of the procedures, a lymph node was removed. Initial surgery successfully identified the ROLL-marked with clip in 33 of the 37 procedures (89%) in which the Tc-99m-MAA was injected near the clip. A tumor-positive ROLL-marked node was found in the procedure with the injection in the sonographically suspicious unclipped node.In two patients, only a tumor-negative lymph node was found with signs of response on histopathological assessment. These patients were considered as axillary responders in further tailored axillary treatment despite the missing clip. Post-operative CT confirmed that the clip was still in situ in these two patients.Both cases in which no lymph node was found were cN<4 patients. The removed specimen of one patient contained only the clip with fatty tissue. This patient was treated as a non-responder and received adjuvant axillary radiotherapy, because the primary tumor showed partial response and pretreatment PET/CT detected also 18F-FDG-avid internal mammary chain nodes. The specimen of the other patient consisted of an old hematoma surrounded by fat without clip. This patient underwent a second ROLL-node procedure of the clip-marked node 3 weeks after the first surgery. This second procedure successfully identified the ROLL-marked node with a clip. Histopathological assessment showed no residual disease, so further axillary treatment was omitted.Frozen section of the ROLL-marked node was performed in 10 of the 12 axillary cN4+ patients. Additional ALND was performed for 8 of these 10 patients because of a positive frozen section. For two patients, ALND was performed without frozen section since these patients had highly suspicious palpable axillary lymph nodes intraoperatively. In both cases, histopathological results were positive. A median of 7 tumor-positive nodes was found in the 10 ALND specimens (range 2–20).yiN+ and 22 procedures (85%) with yiN0 status, the ROLL-marked node with clip was successfully removed during initial surgery.Overall, 27% (10/37) of the procedures demonstrated a pCR of the ROLL-marked node .This is the first study that investigated the use of ROLL-node injections into clip-marked lymph nodes that were tumor-positive prior to NST and selectively remove them at surgery after NST. Identifying the ROLL-marked node with clip both preoperatively and at surgery was successful in 87% (33/38) of the procedures. We used a combination of pretreatment 18F-FDG PET/CT and assessment of pathologic complete response of axillary lymph nodes by selectively removing the ROLL-marked node to further tailor axillary treatment, and hereby we have omitted ALND in 74% of our procedures.There is an emerging interest in selective identification of axillary lymph nodes, that were tumor-positive before NST, to assess pathological response after NST since the SLN procedure after NST in patients with tumor-positive nodes before NST is only successful in a selected subgroup of patients . Since tIt is desirable to use definitive markers prior to NST to allow node resection without any other step. In this regard, iodine-125 seeds are still the most suitable markers and currently used in the NCI. A disadvantage of the two-step protocol is that the clip can be difficult to find with ultrasound, especially in nodes that respond well to NST. In our study, the clip was not localized by ultrasound in 2 procedures (5%). Earlier studies have reported that the clip could not be found by ultrasound in 4–28% of the procedures –26. TherIn two patients, the removed ROLL-marked node showed signs of response but did not contain the clip, although the ROLL-node injection was placed near the clip. Thus, a strict identification of the removed ROLL-marked node being the clip-marked tumor-positive lymph node prior to NST on initial surgery was hampered in these two patients. In a third patient, the removed specimen only contained the clip without any node. These findings suggest that nodal shrinkage during NST may cause clip displacement into the perinodal fatty tissue as was reported by Hartmann et al. .Two limitations of this study were identified. First, the FNR was not evaluated, because ALND was only performed in cN4+;ypN+(ROLL-marked node) patients. Previous studies have proven that marking tumor-positive axillary nodes prior to NST and selectively removing them after NST results in low FNRs , 13. AltPotentially, the ROLL-node procedure can be combined with the SLN procedure. Combining a targeted lymph node procedure with the SLN procedure will reduce the false-negative rate . LymphosIt is technically feasible to perform ROLL procedures of clip-marked lymph nodes and selectively remove them at surgery. The ROLL-node injection can be performed the day before surgery. This is a promising method for hospitals that cannot use iodine-125 seeds for target lymph node procedures to tailor axillary treatment after NST.


Syncerus caffer) of Hluhluwe-iMfolozi Park (HiP) and Kruger National Park, South Africa. In HiP, the disease has been actively managed since 1999 through a test-and-cull procedure targeting BTB-positive buffalo. Prior studies in Kruger showed associations between microsatellite alleles, BTB and body condition. A sex chromosomal meiotic drive, a form of natural gene drive, was hypothesized to be ultimately responsible. These associations indicate high-frequency occurrence of two types of male-deleterious alleles . One type negatively affects body condition and BTB resistance in both sexes. The other type has sexually antagonistic effects: negative in males but positive in females. Here, we investigate whether a similar gene drive system is present in HiP buffalo, using 17 autosomal microsatellites and microsatellite-derived Y-chromosomal haplotypes from 401 individuals, culled in 2002–2004. We show that the association between autosomal microsatellite alleles and BTB susceptibility detected in Kruger, is also present in HiP. Further, Y-haplotype frequency dynamics indicated that a sex chromosomal meiotic drive also occurred in HiP. BTB was associated with negative selection of male-deleterious alleles in HiP, unlike positive selection in Kruger. Birth sex ratios were female-biased. We attribute negative selection and female-biased sex ratios in HiP to the absence of a Y-chromosomal sex-ratio distorter. This distorter has been hypothesized to contribute to positive selection of male-deleterious alleles and male-biased birth sex ratios in Kruger. As previously shown in Kruger, microsatellite alleles were only associated with male-deleterious effects in individuals born after wet pre-birth years; a phenomenon attributed to epigenetic modification. We identified two additional allele types: male-specific deleterious and beneficial alleles, with no discernible effect on females. Finally, we discuss how our findings may be used for breeding disease-free buffalo and implementing BTB test-and-cull programs.Bovine tuberculosis (BTB) is endemic to the African buffalo ( Zea mays), insects (Drosophila melanogaster), fish (Actinopterygii), mammals (Mus musculus) and fungi (Neurospora) [Sexual diploid organisms are vulnerable to meiotic drivers. Meiotic drivers are selfish genetic elements that violate Mendel’s law of equal segregation by favouring their own transmission, or that of the chromosome on which they reside. They do this by distorting meiosis, or by impairing the function or increasing the mortality of the opposite-sex gametes . Moleculrospora) ,5.Dicrostonyx torquatus, Myopus schisticolor and Akodon azarae) [Sex-ratio distorters constitute a specific class of meiotic drivers that distort the primary sex ratio because they are located on one of the sex chromosomes. They have been observed in insects , and also in mammals ( azarae) . Many me azarae) . However azarae) .Syncerus caffer) of southern Africa may provide an opportunity to address these outstanding issues. A complex gene drive system involving sex-ratio distorters and suppressors was recently hypothesized for the buffalo population of Kruger National Park [2 area vs. 37,000 individuals in a 20,000 km2 area), receives 50% more rain , and has a markedly different BTB disease management strategy in place, viz., a yearly test-and-cull policy in which BTB-infected animals are removed from the population [The African buffalo  cause a negative effect in males on body condition and resistance to bovine tuberculosis (BTB) ,9. One gFurther, microsatellite alleles were mainly associated with deleterious traits in animals born after relatively wet 3yr-pre-birth-rainfall periods  . The assThe high frequencies of the male-deleterious alleles have been attributed to interactions with a Y-chromosomal sex-ratio distorter-suppressor pair, whose activity also seems to be influenced by body condition and health status –9. The sDespite the complexity of the underlying mechanism and its dependency on multiple hypothesises, we think it constitutes the most plausible explanation for a wide range of observations . In partThe male-deleterious alleles were expected to have a negative effect on population viability in Kruger by decreasing male body condition and by increasing disease occurrence; the latter directly in males and indirectly in females by contacting infected males . The malBy way of background information, it is worth noting that the HiP buffalo population has the lowest genetic diversity  of any buffalo population studied so far ,17–19, eP = 0.05 [Random genetic drift caused by the founder event may by itself explain the relatively low frequencies of the DE and SAE alleles in HiP. However, low frequencies may also be caused by negative selection of male-deleterious alleles as Y-haplotype 112 was not observed among 180 HiP males typed previously . In P = 0.05 . As a reP = 0.05 ,17. In KINFG gene and significant differences in number and frequency of rare alleles at closely linked microsatellites between BTB-positive and BTB-negative buffalo.Disease-focussed culling may contribute to negative selection by decreasing lifetime mating success of BTB-positive males . BTB wasHere we report on the extent to which male-deleterious alleles are active in HiP buffalo. We assess whether these alleles are under negative selection and, if so, to what extent negative selection can be attributed to BTB, facilitated by the absence of an active Y distorter. More specifically we addressed the following four research questions.Are DE and SAE allele frequency differences between HiP and Kruger correlated with genotype-phenotype association strength in Kruger ? We hypothesized decreasing DE and SAE allele frequencies in HiP compared to Kruger with increasing association strength in Kruger. This would indicate that selection on the linked male-deleterious alleles is negative in HiP. Additionally, we tested for an influence of parental body condition on selection by correlating birth-year cohort allele frequencies with 3yr-pre-birth rainfall in HiP.Does pre-birth rainfall have an influence on genotype-phenotype association strength in HiP ? We hypothesized the same statistical interaction between 3yr-pre-birth rainfall and genotype-phenotype association strength as in Kruger: i.e., no or weak association among individuals born after three dry years, but a strong positive association among individuals born after three wet years, consistent with epigenetic modification .Is the frequency of Y-haplotype 557 (linked to a Y suppressor in Kruger) among male birth-year cohorts correlated with 3yr-pre-birth rainfall? We hypothesized a negative correlation, as observed in Kruger ,7, whichDoes female-biased primary sex-ratio distortion occur in HiP? In Kruger, both male- and female-biased sex-ratio distortion occurred among foetuses, which were attributed to respectively a Y distorter and a postulated X distorter ,9. All eEthical approval was not considered necessary, because BTB diagnosis and blood sample collection were conducted under the directive of the provincial parks authority, KwaZulu-Natal Wildlife, for other purposes. Thus the BTB test results and the use of blood samples in the present study are incidental. Full details of collection and sampling methods have been published before . Briefly2 (28°S and 31–32°E) Hluhluwe-iMfolozi Park  contains around 4000 African buffalo. Between 2002–2004, blood samples of 387 individual buffalo  were genotyped from 13 breeding herds across all management regions of the park: Manzibomvu , Nqumeni , Masinda , Makhamasi  and Mbhuzane  (The 960 kmay 2002) . BTB tes10.5061/dryad.s4cc3) [The same seventeen autosomal microsatellite loci as in two earlier Kruger buffalo genetics studies ,9 were aBM3517, BM4028, ETH010, ETH225, INRA006, INRA128, TGLA227 and TGLA263). Their average frequency per individual  was associated with low body condition, thereby indicating linkage to genes expressing a deleterious allele [BM1824, BM3205, BM0719, CSSM019, DIK020, ILSTS026, SPS115, TGLA057 and TGLA159). These pooled alleles were also associated with high body condition and decreased BTB infection risk in females, thereby indicating linkage to genes expressing a sexually antagonistic allele [majority alleles) at the DE loci, and to the nine pooled-threesomes as the sexually-antagonistic-effect associated alleles  at the SAE loci , calculated with FSTAT 2.9.3.2, indicate little population structure in HiP . Further, median pairwise opposite-sex relatedness r . As in Kruger, linkage disequilibrium  was observed in HiP between locus pairs CSSM019-BM1824, CSSM019-BM3205, BM1824-BM3205 and INRA006-ILSTS026 . LD was probably due to physical linkage [Wright’s  CI: -0.0, +0.025)Annual rainfall data, with years running from October to September, were obtained from eight rainfall stations throughout HiP. These are updated rainfall data compared to an earlier genetic study on HiP buffalo , after dWe analysed selection at the level of individual microsatellite alleles using non-parametric randomization tests. A randomization approach was chosen, because observations based on multiple alleles per locus and from different loci in LD cannot be assumed to be independent from one another. We randomized multilocus microsatellite genotypes , which guaranteed the same statistical dependence among alleles in the randomized data sets as in the observed data. We included only those alleles observed ≥ 15 times in southern Kruger (frequency ≈ 0.05) to reduce the number of statistical outliers.Nmales = 35, Nfemales = 37) and high-body-condition BTB-negative individuals . We compared low-body-condition BTB-positive and high-body-condition BTB-negative individuals from southern Kruger, because these two groups were characterized by considerably larger allele frequency differences than pairs of groups based on differences in body-condition status (low body condition vs. high body condition) alone or disease status (BTB-positive vs. BTB-negative) alone [For each individual allele in southern Kruger, where the strongest male-deleterious effects were observed , we estiAsex-indep and Asex-anta respectively. In estimating these ratios, we define with respect to individual allele frequency a in males (subscript m) or females (subscript f) that are in high (subscript h) or low (subscript l) body condition and are BTB-positive (+) or BTB-negative (-) using the notation asex,condition,BTB state. Thus, for example, af,h,- represents the frequency of the allele in question in high-body-condition BTB-negative females, while am,l,+ represents the frequency of the allele in question in low-body-condition, BTB-positive males. From this it follows thatWe denoted the sex-independent and sexually-antagonistic association strength of microsatellite alleles by the ratios Asex-indep could be attributed to one of the two sexes we also estimated the sex-specific association strength, denoted by the ratios Amale-spec and Afemale-spec, as:To analyse whether effects of A values > 1 indicate association with deleterious effects , while A values < 1 indicate association with beneficial effects. Larger deviations from 1 indicate stronger associations. We only included the individual DE alleles with Amale-spec and Afemale-spec either both > 1 or both < 1  with either Amale-spec > 1 and Afemale-spec < 1 or vice versa .As a negative control, we also analysed the SAENindividuals = 401, northern Kruger: Nindividuals = 138), were associated with Asex-indep and Asex-anta. A negative association with Asex-indep in case of DEindv alleles and with Asex-anta in case of SAEindvO alleles would be indicative of negative selection in HiP (research question 1). Allele frequencies from northern Kruger, rather than southern Kruger, were used to estimate the HiP/northern-Kruger frequency ratio in order to make this variable as independent as possible from Asex-indep and Asex-anta. We additionally explored associations between the HiP/northern-Kruger frequency ratio of SAEindvN alleles on the one hand and Amale-spec and Afemale-spec on the other .We performed two tests to see whether allele frequency differences between HiP and Kruger, estimated as the ratio of allele frequency in HiP to allele frequency in northern Kruger  and Asex-indep  significantly differed between alleles observed in both populations and alleles observed only in Kruger. This test could only be conducted for the SAEindvO and SAEindvN alleles, because only two DEindv alleles were unique to Kruger. In the second test , which resulted in six allele types: DEA>1indv,, DEA<1indv,, SAEA>1indvO,, SAEA<1indvO,, SAEA>1indvN, and SAEA<1indvN, alleles , using 100,000 randomizations implemented using Excel 2016. The statistical value was difference in average rank of A in Test 2 and Spearman ρ in Tests 3 and 4. In Test 4, the combined probability of various allele types was estimated using average |ρ| as the statistical value.Significance of Tests 2 and 3 was estimated by randomizing multilocus microsatellite genotypes from southern Kruger among groups of buffalo based on body-condition and disease status . Randomizations were not performed among individuals from different populations because LD strength between loci can vary among populations, resulting in different LD strengths in the randomized data sets as compared to the observed data. The classification as DEWe performed three logistic regression analyses to study: 1) statistical interactions between individual genetic background and 3yr-pre-birth rainfall, 2) the effect of 3yr-pre-birth rainfall on Y-haplotype status and 3) the effect of age and 3yr-pre-birth rainfall on sex. The logistic regression analyses were conducted using the ‘lme4’ package (version 1.1.13) in R. Herd affiliation and sampling year were incorporated as random intercepts in a mixed modelling approach.majority and SAEpooled alleles per individual: HomDEmajority, HomSAEpooled, HetDEmajority and HetSAEpooled. Heterozygosity was not calculated as 1-homozygosity, but as the fraction of heterozygous alleles among the loci that were not homozygous for DEmajority or SAEpooled alleles [m and f subscripts to denote that quantities were estimated separately among the male and female subgroups, male-deleterious load (MDL) was estimated in males as:Identical to the methodology in Van Hooft et al. 2018 , male-de alleles . This ap alleles . Using mm and MDLf did not significantly deviate from normality (Shapiro-Wilk test using SPSS 23: P > 0.8). Further, we explored whether the average total per-locus frequency of the SAEA<1indvN, alleles . The SAEA>1indvN, alleles (m and MDLf were treated as one independent variable: standardized MDL (MDLs).MDL alleles  were nots and their interaction were included as fixed independent variables with BTB-infection status as dependent variable , sex, three-way interaction between SAEA<1indvN, allele frequency, sex and 3yr-pre-birth rainfall, and all lower order two-way interactions .p = probability of BTB infection, MDLs = standardized MDL, PBRs = standardized 3yr-pre-birth rainfall, SAEsindvN, = standardized total per-locus frequency of the SAEA<1indvN, alleles, sex = categorical variable .In the first regression model 3yr-pre-birth rainfall, MDLvariable . A signiractions . The thrNindividuals with Y-haplotype 557 = 53, Nindividuals with other Y-haplotypes = 104, EPV = 53; p = probability of carrying Y-haplotype 557, PBR = 3yr-pre-birth rainfall.We conducted a logistic regression between Y-haplotype status and 3yr-pre-birth rainfall  and 3yr-pre-birth rainfall as the independent variables (http://doi.org/10.5061/dryad.xxx (http://datadryad.org).The raw data set supporting the results of this article can be found in the Dryad Digital Repository: XXX, Our results reveal a complex set of interconnected genetic factors. To assist the reader in making these connections, we have provided a table , next tomajority and SAEpooled allele frequencies were on average lower in HiP than in northern Kruger, but non-significantly so  showed significantly larger Asex-anta values than those also observed in HiP ; indvO alleles in Kruger showed only small differences between those observed and not observed in HiP . This selection occurred on the linked sexually antagonistic alleles or haplotypes of male-deleterious and female-beneficial alleles. The similar results with respect to the Asex-indep values of the SAEindvN alleles, the negative-control analyses, were not expected . Rather than considering these to be false positive results, they may indicate negative selection of another type of male-deleterious allele.The relatively large indvN alleles in HiP was much more pronounced in relation to Amale-spec . These results indicate linkage to alleles that are predominantly deleterious to males  and SAEindvN  alleles, but not the DEindv alleles (P > 0.4), correlated with 3yr-pre-birth rainfall. This observation indicates that selection of SAEindv alleles was influenced by parental body condition  and SAEA<1indvN, alleles , and negative for SAEA<1indvO, alleles  and SAEA>1indvN, alleles  of both the SAEondition , Table 3A<1indvO, and SAEA<1indvN, alleles, originally hypothesized not to be linked to any particular protein-coding allele, could not entirely be an artefact of the correlations of the SAEA>1indvO, and SAEA>1indvN, alleles, as the total frequencies of the latter two groups combined did not show any meaningful correlation with 3yr-pre-birth rainfall . Thus, these correlations indicate linkage to at least three types of alleles or haplotypes , male-specific deleterious alleles  and male-specific beneficial alleles . The last allele type is supported by an effect of 3yr-pre-birth rainfall on its association with BTB susceptibility . Although the SAEA<1indvO, alleles may be linked to female-beneficial alleles, their negative correlation with pre-birth rainfall may also be an artefact of the significant positive correlation involving the SAEA<1indvN, alleles.The correlations of the SAEplotypes : sexualls and 3yr-pre-birth rainfall on BTB-infection risk when only MDLs and 3yr-pre-birth rainfall were included as main factors . The interaction became near-significant  and mainly after relatively dry pre-birth years  when, as a control, we replaced 3yr-pre-birth rainfall with age or 3yr-post-birth rainfall  in the regression equations.The two- and three-way interactions involving the male-beneficial-linked SAE alleles  indicateP = 0.19), there was a significant Spearman correlation between Y-haplotype-557 frequency and 3yr-pre-birth rainfall (ρ = -0.51 (weighted by sample size per birth-year cohort), P = 0.043; Although the logistic regression between Y-haplotype status and 3yr-pre-birth rainfall was not significant . The likely occurrence of a Y suppressor at Y-haplotype 557 (Question 3) together with female-biased birth sex ratios (Question 4) support the mechanism depicted in The main goal of this study was to find out whether absence of a Y distorter in the HiP buffalo could facilitate BTB-driven negative selection of male-deleterious alleles. We found statistical support to affirmatively answer our four research questions and their underlying hypotheses. We found strong support for negative selection of male-deleterious alleles (Question 1). BTB was probably one of the selective agents, considering that the frequency decrease of microsatellite alleles in HiP relative to northern Kruger was related to their association strength with BTB infection . This was further supported by the association between MDLs  when Amale-spec > 1, and beneficial and under positive selection (high frequencies in HiP) when Amale-spec < 1. The male-beneficial effects were supported by the logistic regression analysis  , and conr in HiP  suggestss in logistic regression analysis indicates epigenetic suppression of male-deleterious alleles among animals born after three dry pre-birth years, as was earlier also observed in Kruger [P = 0.46, The interaction between 3yr-pre-birth rainfall and MDLn Kruger . A possiA<1indvN, allele frequencies, indicative of epigenetic suppression of male-specific beneficial alleles, not after three dry but after three wet pre-birth years. This indicates that males born after extended wet multiyear periods have reduced body condition not only because of active deleterious alleles but also because of suppressed beneficial alleles . The alternating temporal pattern of epigenetic suppression between male-deleterious and male-beneficial alleles may possibly have evolved to prevent fixation of either the Y distorter or the Y suppressor by reinforcing the epigenetic-driven negative feedback. Such a feedback was earlier hypothesized to be responsible for negative frequency-dependent selection of the Y distorter-suppressor pair [Surprisingly, there was also an interaction between 3yr-pre-birth rainfall and SAEsor pair .s individuals with low frequencies of male-beneficial alleles. Further, targeting males carrying one of the hypothesized sex-ratio distorters would allow removal of the underlying selective agents from the population.The population viability of the HiP buffalo may have been reduced by the high genetic load , despite negative selection of male-deleterious alleles and positive selection of male-beneficial alleles . Howevers and male-beneficial allele frequencies, genotyping of sex chromosomes and screening of DNA methylation variation may also be applied in the commercial breeding of African buffalo (which has become a lucrative business in South Africa), especially with respect to producing disease-free buffalo [Further improvement of the culling program may be possible by additionally targeting males with active male-deleterious alleles and suppressed male-beneficial alleles, thereby eliminating weak males from the population. Identifying such males may be done by screening of DNA methylation variation . Estimat buffalo .Further studies of the gene drive system in the African buffalo may provide new insights for synthetic gene drive systems and their risks. These are being developed to control vector populations of human diseases ,35. ThisS1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S1 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.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.