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Brain metastases (BrM) remain an intractable, deadly complication for advanced melanoma patients and efficient therapeutic strategies are desperately needed. The tumor microenvironment (TME) plays an important role in response to therapy. However, studies addressing the contribution of the TME to therapy efficacy for BrM are lacking, mostly due to limited access to human samples and scarcity of appropriate preclinical models. Here, we describe two novel isogenic immunocompetent BrM models generated by intracardiac injection of UV-induced mouse melanoma cell lines, representative of mutant-RAS human melanoma subtypes. We used these models to test immune checkpoint blockade (ICB) therapy and to interrogate the role of the TME in therapeutic efficacy. To evaluate response, we developed and applied a new machine-learning method to quantify metastatic burden. We investigated the TME by high-parametric flow cytometry and single-cell RNA sequencing (scRNA-seq). We showed that the models have distinct metastatic behaviors, with BR1 being mostly brain tropic and BR3 displaying widespread metastases. Notably, BR1 BrM were sensitive to ICB with a better response to anti-PD-L1/anti-CTLA-4 combination therapy as compared to monotherapies. In contrast, BR3 BrM were resistant to both mono- and combination therapies. Interestingly, we found that ICB efficacy on extracranial BR3 metastases is organ-dependent. Characterization of the BrM immune microenvironment before and after treatment revealed dramatic differences between the models. Untreated BR1 BrM showed significant recruitment of T cells, dendritic cells, and natural killer cells, while neutrophils were enriched in untreated ICB-resistant BR3 BrM. Moreover, we uncovered phenotypically distinct microglia populations exclusively present in ICB-sensitive BR1 BrM that positively correlated with T cell infiltration. Consistent with this finding, scRNA-seq showed upregulation of genes encoding for T cell-attracting chemokines and antigen presentation uniquely in the BR1-associated microglia. Post-treatment analysis of the brain TME highlighted beneficial changes induced by ICB in the responsive BR1 model, including increased recruitment of CD8 T cells with an activated phenotype, while a mild recruitment of exhausted T cells was observed in the resistant BR3 model. Altogether, our data emphasize the importance of interrogating the BrM TME to understand therapeutic response. Our unique BrM models, mirroring the diversity of ICB response observed in patients, provide a robust platform for the much-needed mechanistic studies to optimize BrM therapy. Deciphering the contribution of the newly identified BR1 BrM-associated microglia to ICB efficacy will be crucial to the identification of novel therapeutic targets. Citation Format: Amélie Lopès, Jessica Rappaport, Eva Pérez Guijarro, Quanyi Chen, Emily Wu, Isabella Church, April Huang, Jessica Bridge, Sung Chin, Cari Smith, Charli Gruen, Khiem C. Lam, Romina E. Araya, Antonella Sassano, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid. Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5188.

Background Pressure dressings have been used after open hemorrhoidectomy to protect surgical wounds and manage postoperative bleeding for many years. However, pressure dressings may increase the incidence of postoperative complications, such as urinary retention, medical adhesive-related skin injury, and pain. A previous controlled trial included 67 patients who underwent Milligan-Morgan hemorrhoidectomy. The data indicated that the use of a nonpressure dressing after hemorrhoidectomy reduces the incidence of urinary retention and catheterization. However, the incidence of severe postoperative bleeding and other postoperative complications was not assessed. There is no consensus on whether it is necessary and beneficial to use a nonpressure dressing after hemorrhoidectomy. The results of this randomized clinical study will help answer this question. Methods In this study, we plan to include 186 patients who have undergone modified Milligan-Morgan hemorrhoidectomy, which only sutured external hemorrhoids to reduce the risk of bleeding. The purpose is to determine whether the use of nonpressure dressings after open hemorrhoidectomy is inferior to the use of pressure dressings in terms of severe postoperative bleeding and postoperative complications. The primary endpoints of the trial are the incidence of urinary retention within 24 h after surgery and the incidence of severe postoperative bleeding 1 h after dressing removal, which requires revision surgery within 24 h after the surgery. The secondary endpoints of the study are the pain score, anal distension score, postoperative use of analgesics, and incidence of medical adhesive-related skin injury, all of which will be assessed before removing the dressings. The length of hospitalization in days and hospitalization expenses will be recorded. Safety will be assessed with consideration of all adverse and severe adverse events related to the study treatment. Discussion The study received full ethics committee approval. The first patient was enrolled on 27 November 2020. The results of this trial will finally answer the question of whether a nonpressure dressing after open hemorrhoidectomy is necessary and beneficial. Trial registration Chinese Clinical Trial Registry ChiCTR2000040283. Registered on 28 November 2020.

Novel nitrogen-doped carbon quantum dots/Ag 3 PO 4 (NCQDs/Ag 3 PO 4 ) complex photocatalysts were synthesized by a facile precipitation method at room temperature. The physical and chemical properties of Ag 3 PO 4 and NCQDs/Ag 3 PO 4 photocatalysts were detected through X-ray powder diffraction, field emission scanning electron microscopy, UV–vis diffuse reflectance spectroscopy, X-ray photoelectron spectroscopy and electron spin resonance techniques. The as-prepared 3-NCQDs/Ag 3 PO 4 composite exhibited much higher activity than the pure Ag 3 PO 4 for eliminating methyl orange and bisphenol A solution under visible light (λ > 420 nm). Moreover, in the cyclic experiments, the 3-NCQDs/Ag 3 PO 4 exhibited an excellent stability for the decolorization of methyl orange at some level. This suggested that NCQDs played an important role in the process of degradation. The function of NCQDs was discussed and a new mechanism was put forward for the degradation of methyl orange. The high activities and stability were attributed to the transfer of photogenerated charges through the vector of Ag 3 PO 4 → NCQDs → Ag in the photocatalytic process, leading to effective charge separation of Ag 3 PO 4 .

Interface engineering in organic solar cells (OSCs) plays an important role in improving electron extraction and suppressing carrier recombination. In this article, the improvement of device was achieved by doping POT2T into ZnO as a new electron transport layer (ETL). For PTB7-Th: PC71BM system, the device fabricated by using ZnO as ETL achieved a PCE of 9.03% with a JSC of 17.33 mA/cm2, a VOC of 0.783V and a FF of 64.17%. After doping 5wt% POT2T in ZnO, the JSC increased to 18.01 mA/cm2 and the FF increased slightly to 69.87%, as a result, the PCE increased to 9.84% with about 10% enhancement. The enhancement is attributed to the improved electron transport and conductivity, optimized ZnO surface morphology after adding POT2T, resulting in higher JSC and FF. Therefore, doping POT2T into ZnO as an ETL is a simple and effective method for obtaining high efficient OSCs.

Novel nitrogen-doped carbon quantum dots/Ag

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14–protein conjugate, are both dependent on CD4+ T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14–protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igha, but not Ighb, allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14–protein conjugate, avidity maturation of the 44.1-Id–dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.

IgG anti-polysaccharide (PS) responses to both intact Streptococcus pneumoniae (Pn) and PS conjugate vaccines are dependent on CD4+ T cells, B7-dependent costimulation, and CD40-CD40-ligand interactions. Nevertheless, the former response, in contrast to the latter, is mediated by an ICOS-independent, apoptosis-prone, extrafollicular pathway that fails to generate PS-specific memory. We show that pre-existing PS-specific Igs, the bacterial surface or particulation, selective recruitment of B cell subsets, or activation and recruitment of Pn protein-specific CD4+ T cells do not account for the failure of Pn to generate PS-specific IgG memory. Rather, the data suggest that the critical factor may be the lack of covalent attachment of PS to protein in intact Pn, highlighting the potential importance of the physicochemical relationship of PS capsule with the underlying bacterial structure for in vivo induction of PS-specific Igs.

During infections with extracellular bacteria, such as Streptococcus pneumoniae (Pn), the immune system likely encounters bacterial components in soluble form, as well as those associated with the intact bacterium. The potential cross-regulatory effects on humoral immunity in response to these two forms of Ag are unknown. We thus investigated the immunologic consequences of coimmunization with intact Pn and soluble conjugates of Pn-derived proteins and polysaccharides (PS) as a model. Coimmunization of mice with Pn and conjugate resulted in marked inhibition of conjugate-induced PS-specific memory, as well as primary and memory anti-protein Ig responses. Inhibition occurred with unencapsulated Pn, encapsulated Pn expressing different capsular types of PS than that present in the conjugate, and with conjugate containing protein not expressed by Pn, but not with 1-μm latex beads in adjuvant. Inhibition was long-lasting and occurred only during the early phase of the immune response, but it was not associated with tolerance. Pn inhibited the trafficking of conjugate from the splenic marginal zone to the B cell follicle and T cell area, strongly suggesting a potential mechanism for inhibition. These data suggest that during infection, bacterial-associated Ags are the preferential immunogen for antibacterial Ig responses.

TLR2(-/-) mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4(+) T-cell-dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T-cell-dependent phosphorylcholine (PC)-specific IgG3 versus the T-cell-independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn-activated TLR2(-/-) BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti-PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4(+)T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2(-/-) mice for a normal CD4(+) T-cell IFN-gamma recall response. Notably, TLR2(-/-) B cells transferred into RAG-2(-/-) mice with WT CD4(+)T cells, or TLR2(-/-) CD4(+)T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti-PC response relative to WT cells. These data are the first to demonstrate a major role for B-cell and CD4(+) T-cell expression of TLR2 for eliciting an anti-bacterial humoral immune response.

Little is known regarding the role of Toll-like receptors (TLRs) in regulating protein- and polysaccharide-specific immunoglobulin (Ig) isotype production in response to an in vivo challenge with an extracellular bacterium. In this report we demonstrate that MyD88−/−, but not TLR2−/−, mice are markedly defective in their induction of multiple splenic proinflammatory cytokine- and chemokine-specific mRNAs after intraperitoneal (i.p.) challenge with heat-killedStreptococcus pneumoniaecapsular type 14 (S. pneumoniaetype 14). This is correlated with analogous responses in splenic cytokine protein release in vitro following addition ofS. pneumoniaetype 14. Consistent with these data, naïve MyD88−/−, but not TLR2−/−, mice are more sensitive to killing following i.p. challenge with liveS. pneumoniaetype 14, relative to responses in wild-type mice. However, prior immunization of MyD88−/−mice with heat-killedS. pneumoniaetype 14 protects against an otherwise-lethal challenge with liveS. pneumoniaetype 14. Surprisingly, both MyD88−/−and TLR2−/−mice exhibit striking and equivalent defects in elicitation of type 1 IgG isotypes (IgG3, IgG2b, and IgG2a), but not the type 2 IgG isotype, IgG1, specific for several protein and polysaccharide antigens, in response to i.p. challenge with heat-killedS. pneumoniaetype 14. Of note, the type 1 IgG isotype titers specific for pneumococcal surface protein A are reduced in MyD88−/−mice but not TLR2−/−mice. These data suggest that distinct TLRs may differentially regulate innate versus adaptive humoral immunity to intactS. pneumoniaeand are the first to implicate a role for TLR2 in shaping an in vivo type 1 IgG humoral immune response to a gram-positive extracellular bacterium.

Staphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intact Staphylococcus aureus elicits murine CD4 + T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responses in vivo . Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of live Staphylococcus aureus in vitro . Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremia in vivo relative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.

Polysaccharide (PS)- and protein-specific murine IgG responses to intact Streptococcus pneumoniae (Pn) are both dependent on CD4+ T cell help, B7-dependent costimulation, and CD40/CD40 ligand interactions. However, the primary PS-specific, relative to protein-specific, IgG response terminates more rapidly, requires a shorter period of T cell help and B7-dependent costimulation, and fails to generate memory. In light of the critical role for ICOS/ICOS ligand interactions in sustaining T cell-dependent Ig responses and promoting germinal center reactions, we hypothesized that this interaction was nonessential for PS-specific IgG responses to Pn. We now demonstrate that ICOS−/−, relative to wild-type, mice elicit a normal PS-specific IgG isotype response to Pn, despite marked inhibition of both the primary and secondary IgG anti-protein (i.e., PspA, PspC, and PsaA) response. A blocking anti-ICOS ligand mAb injected during primary Pn immunization inhibits both the primary anti-protein response and the generation of protein-specific memory, but has no effect when injected during secondary immunization. In contrast to Pn, both PS- and protein-specific IgG responses to a pneumococcal conjugate vaccine are inhibited in ICOS−/− mice. ICOS−/− mice immunized with intact Pn or conjugate exhibit nearly complete abrogation in germinal center formation. Finally, although mice that lack the adaptor molecule SAP (SLAM-associated protein) resemble ICOS−/− mice (and can exhibit decreased ICOS expression), we observe that the PS-specific, as well as protein-specific, IgG responses to both Pn and conjugate are markedly defective in SAP−/− mice. These data define a novel T cell-, SAP-, and B7-dependent, but ICOS-independent, extrafollicular pathway of Ig induction.

We previously demonstrated that induction of splenic cytokine and chemokine secretion in response to Streptococcus pneumoniae (Pn) is MyD88-, but not critically TLR2-dependent, suggesting a role for additional TLRs. In this study, we investigated the role of TLR2, TLR4, and/or TLR9 in mediating this response. We show that a single deficiency in TLR2, TLR4, or TLR9 has only modest, selective effects on cytokine and chemokine secretion, whereas substantial defects were observed in TLR2(-/-)xTLR9(-/-) and TLR2(-/-)xTLR4(-/-) mice, though not as severe as in MyD88(-/-) mice. Chloroquine, which inhibits the function of intracellular TLRs, including TLR9, completely abrogated detectable cytokine and chemokine release in spleen cells from TLR2(-/-)xTLR4(-/-) mice, similar to what is observed for mice deficient in MyD88. These data demonstrate significant synergy between TLR2 and both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Pn.

MyD88−/− mice exhibit defective innate, diminished CD4+ T cell-dependent (TD) type 1, but enhanced type 2, humoral immunity in response to intact Streptococcus pneumoniae (Pn). Because type 1 IL-1R (IL-1R1) signaling is MyD88 dependent, a role for endogenous IL-1 was determined. IL-1R1−/−, in contrast to MyD88−/−, mice exhibited relatively intact innate splenic cytokine expression in response to Pn. Nevertheless, IL-1R1−/−, like MyD88−/−, mice were more sensitive to killing with live Pn relative to wild-type controls. Although IL-1R1−/− mice elicited a normal T cell-independent IgM antipolysaccharide (PS) response to heat-killed Pn, the induction of PS- and protein-specific cognate, but not noncognate, TD type 1 and type 2 IgG isotypes were markedly reduced. Additionally, CD4+ T cells from Pn-primed IL-1R1−/− mice failed to elicit IFN-γ, IL-5, or IL-13 secretion upon restimulation with Pn in vitro, whereas MyD88−/− mice secreted normal levels of IFN-γ and enhanced levels of IL-5 and IL-13. In contrast, IgG responses to a soluble, pneumococcal protein-PS conjugate, with or without adjuvant, showed little dependence on IL-1R1 and normal CD4+ T cell priming. These data are the first to demonstrate a nonredundant role for endogenous IL-1 in TD induction of humoral immune responses to an intact pathogen, although not a pathogen-derived soluble conjugate, suggesting that antigenic context is a key determinant for IL-1 dependence. These data further suggest that IL-1 may be critical for preserving CD4+ Th2 function in the presence, but not absence, of MyD88-dependent signaling via TLRs.

We determined whether T cell-independent Ig isotype responses to isolated pneumococcal polysaccharides (PPS) required TLR signaling in vivo. IgG anti-PPS responses to PPS3, PPS14, and C-polysaccharide (C-PS) were virtually undetectable in TLR2−/− mice, whereas specific IgM induction was variably reduced compared with wild-type mice. All PPS-containing preparations induced IL-6 and TNF-α from wild-type, but not TLR2−/−, macrophages. TLR2 activity was distinct from that of PPS, in that it was phenol extractable. Immunization of wild-type mice with phenol-extracted PPS14 also resulted in a marked reduction in the IgG, although not the IgM-anti-PPS14, response compared with untreated PPS14. The commercial 23-valent PPS vaccine, Pneumovax-23 also contained TLR ligands (TLR2 and TLR4), which were absolutely critical for the IgG-inducing activity of the vaccine in mice. Finally, the commercial pneumococcal conjugate vaccine, Prevnar, contained a TLR2 ligand(s) that substantially enhanced both the primary and secondary anti-PPS responses in mice, especially the type 1 IgG isotypes. These data strongly suggest the absolute need for a distinct, TLR-dependent second signal for inducing in vivo IgG T cell-independent humoral immune responses to isolated pneumococcal polysaccharide Ags and highlight the potential importance of previously unappreciated copurified and/or contaminating TLR ligands in PPS vaccine preparations.