Microglia Mediate HIV-1 gp120-Induced Synaptic Degeneration in Spinal Pain Neural Circuits.

HIV-1 infection of the nervous system causes various neurological diseases, and synaptic degeneration is likely a critical step in the neuropathogenesis. Our prior studies revealed a significant decrease of synaptic protein, specifically in the spinal dorsal horn of patients with HIV-1 in whom pain developed, suggesting a potential contribution of synaptic degeneration to the pathogenesis of HIV-associated pain. However, the mechanism by which HIV-1 causes the spinal synaptic degeneration is unclear. Here, we identified a critical role of microglia in the synaptic degeneration. In primary cortical cultures (day in vitro 14) and spinal cords of 3- to 5-month-old mice (both sexes), microglial ablation inhibited gp120-induced synapse decrease. Fractalkine (FKN), a microglia activation chemokine specifically expressed in neurons, was upregulated by gp120, and knockout of the FKN receptor CX3CR1, which is predominantly expressed in microglia, protected synapses from gp120-induced toxicity. These results indicate that the neuron-to-microglia intercellular FKN/CX3CR1 signaling plays a role in gp120-induced synaptic degeneration. To elucidate the mechanism controlling this intercellular signaling, we tested the role of the Wnt/ß-catenin pathway in regulating FKN expression. Inhibition of Wnt/ß-catenin signaling blocked both gp120-induced FKN upregulation and synaptic degeneration, and gp120 stimulated Wnt/ß-catenin-regulated FKN expression via NMDA receptors (NMDARs). Furthermore, NMDAR antagonist APV, Wnt/ß-catenin signaling suppressor DKK1, or knockout of CX3CR1 alleviated gp120-induced mechanical allodynia in mice, suggesting a critical contribution of the Wnt/ß-catenin/FKN/CX3R1 pathway to gp120-induced pain. These findings collectively suggest that HIV-1 gp120 induces synaptic degeneration in the spinal pain neural circuit by activating microglia via Wnt3a/ß-catenin-regulated FKN expression in neurons.SIGNIFICANCE STATEMENT Synaptic degeneration develops in the spinal cord dorsal horn of HIV patients with chronic pain, but the patients without the pain disorder do not show this neuropathology, indicating a pathogenic contribution of the synaptic degeneration to the development of HIV-associated pain. However, the mechanism underlying the synaptic degeneration is unclear. We report here that HIV-1 gp120, a neurotoxic protein that is specifically associated with the manifestation of pain in HIV patients, induces synapse loss via microglia. Further studies elucidate that gp120 activates microglia by stimulating Wnt/ß-catenin-regulated fractalkine in neuron. The results demonstrate a critical role of microglia in the pathogenesis of HIV-associated synaptic degeneration in the spinal pain neural circuit.

Neuron activity-induced Wnt signaling up-regulates expression of brain-derived neurotrophic factor in the pain neural circuit.

Brain-derived neurotrophic factor (BDNF) is a master regulator of synaptic plasticity in various neural circuits of the mammalian central nervous system. Neuron activity-induced BDNF gene expression is regulated through the Ca2+/CREB pathway, but other regulatory factors may also be involved in controlling BDNF levels. We report here that Wnt/ß-catenin signaling plays a key role in controlling neuron activity-regulated BDNF expression. Using primary cortical cultures, we show that blockade of Wnt/ß-catenin signaling inhibits the BDNF up-regulation that is induced by activation of the N-methyl-d-aspartic acid (NMDA) receptor and that activation of the Wnt/ß-catenin signaling pathway stimulates BDNF expression. In vivo, Wnt/ß-catenin signaling activated BDNF expression and was required for peripheral pain-induced up-regulation of BDNF in the mouse spine. We also found that conditional deletion of one copy of either Wntless (Wls) or ß-catenin by Nestin-Cre-mediated recombination is sufficient to inhibit the pain-induced up-regulation of BDNF. We further show that the Wnt/ß-catenin/BDNF axis in the spinal neural circuit plays an important role in regulating capsaicin-induced pain. These results indicate that neuron activity-induced Wnt signaling stimulates BDNF expression in the pain neural circuits. We propose that pain-induced Wnt secretion may provide an additional mechanism for intercellular coordination of BDNF expression in the neural circuit.

EXPRESS: Oligodendrocytes in HIV-associated pain pathogenesis.

BACKGROUND: Although the contributions of microglia and astrocytes to chronic pain pathogenesis have been a focal point of investigation in recent years, the potential role of oligodendrocytes, another major type of glial cells in the CNS that generates myelin, remains largely unknown. RESULTS: We report here that cell markers of the oligodendrocyte lineage, including NG2, PDGFRa, and Olig2, are significantly increased in the spinal dorsal horn of HIV patients who developed chronic pain. The levels of myelin proteins myelin basic protein and proteolipid protein are also aberrant in the spinal dorsal horn of "pain-positive" HIV patients. Similarly, the oligodendrocyte and myelin markers are up-regulated in the spinal dorsal horn of a mouse model of HIV-1 gp120-induced pain. Surprisingly, the expression of gp120-induced mechanical allodynia appears intact up to 4 h after myelin basic protein is knocked down or knocked out. CONCLUSION: These findings suggest that oligodendrocytes are reactive during the pathogenesis of HIV-associated pain. However, interfering with myelination does not alter the induction of gp120-induced pain.

Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.

OBJECTIVE: Chronic pain is a common neurological comorbidity of human immunodeficiency virus (HIV)-1 infection, but the etiological cause remains elusive. The objective of this study was to identify the HIV-1 causal factor that critically contributes to the pathogenesis of HIV-associated pain. METHODS: We first compared the levels of HIV-1 proteins in postmortem tissues of the spinal cord dorsal horn (SDH) from HIV-1/acquired immunodeficiency syndrome patients who developed chronic pain (pain-positive HIV-1 patients) and HIV-1 patients who did not develop chronic pain (pain-negative HIV-1 patients). Then we used the HIV-1 protein that was specifically increased in the pain-positive patients to generate mouse models. Finally, we performed comparative analyses on the pathological changes in the models and the HIV-1 patients. RESULTS: We found that HIV-1 gp120 was significantly higher in pain-positive HIV-1 patients (vs pain-negative HIV-1 patients). This finding suggested that gp120 was a potential causal factor of the HIV-associated pain. To test this hypothesis, we used a mouse model generated by intrathecal injection of gp120 and compared the pathologies of the model and the pain-positive human HIV-1 patients. The results showed that the mouse model and pain-positive human HIV-1 patients developed extensive similarities in their pathological phenotypes, including pain behaviors, peripheral neuropathy, glial reactivation, synapse degeneration, and aberrant activation of pain-related signaling pathways in the SDH. INTERPRETATION: Our findings suggest that gp120 may critically contribute to the pathogenesis of HIV-associated pain.

Wingless-type mammary tumor virus integration site family, member 5A (Wnt5a) regulates human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120)-induced expression of pro-inflammatory cytokines via the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK) signaling pathways.

BACKGROUND: HIV-1 infection causes chronic neuroinflammation in the central nervous system (CNS). RESULTS: The spinal cytokine up-regulation induced by HIV-1 gp120 protein depends on Wnt5a/CaMKII and/or Wnt5a/JNK pathways. CONCLUSION: gp120 stimulates cytokine expression in the spinal cord dorsal horn by activating Wnt5a signaling. SIGNIFICANCE: The finding reveals Wnt signaling-mediated novel mechanisms by which HIV-1 may cause neuroinflammation. Chronic expression of pro-inflammatory cytokines critically contributes to the pathogenesis of HIV-associated neurological disorders (HANDs), but the host mechanism that regulates the HIV-induced cytokine expression in the CNS remains elusive. Here, we present evidence for a crucial role of Wnt5a signaling in the expression of pro-inflammatory cytokines in the spinal cord induced by a major HIV-envelope protein, gp120. Wnt5a is mainly expressed in spinal neurons, and rapidly up-regulated by intrathecal injection (i.t.) of gp120. We show that inhibition of Wnt5a by specific antagonists blocks gp120-induced up-regulation of IL-1ß, IL-6, and TNF-α in the spinal cord. Conversely, injection (i.t.) of purified recombinant Wnt5a stimulates the expression of these cytokines. To elucidate the role of the Wnt5a-regulated signaling pathways in gp120-induced cytokine expression, we have focused on CaMKII and JNKs, the well characterized down-stream targets of Wnt5a signaling. We find that Wnt5a is required for gp120 to activate CaMKII and JNK signaling. Furthermore, we demonstrate that the Wnt5a/CaMKII pathway is critical for the gp120-induced expression of IL-1ß, whereas the Wnt5a/JNK pathway is for TNF-α expression. Meanwhile, the expression of IL-6 is co-regulated by both pathways. These results collectively suggest that Wnt5a signaling cascades play a crucial role in the regulation of gp120-induced expression of pro-inflammatory cytokines in the CNS.

Chronic-pain-associated astrocytic reaction in the spinal cord dorsal horn of human immunodeficiency virus-infected patients.

Studies with animal models have suggested that reaction of glia, including microglia and astrocytes, critically contributes to the development and maintenance of chronic pain. However, the involvement of glial reaction in human chronic pain is unclear. We performed analyses to compare the glial reaction profiles in the spinal dorsal horn (SDH) from three cohorts of sex- and age-matched human postmortem tissues: (1) HIV-negative patients, (2) HIV-positive patients without chronic pain, and (3) HIV patients with chronic pain. Our results indicate that the expression levels of CD11b and Iba1, commonly used for labeling microglial cells, did not differ in the three patient groups. However, GFAP and S100ß, often used for labeling astrocytes, were specifically upregulated in the SDH of the "pain-positive" HIV patients but not in the "pain-negative" HIV patients. In addition, proinflammatory cytokines, TNFα and IL-1ß, were specifically increased in the SDH of pain-positive HIV patients. Furthermore, proteins in the MAPK signaling pathway, including pERK, pCREB and c-Fos, were also upregulated in the SDH of pain-positive HIV patients. Our findings suggest that reaction of astrocytes in the SDH may play a role during the maintenance phase of HIV-associated chronic pain.

Research on cloud manufacturing service recommendation based on graph neural network.

There are an increasing number of manufacturing service resources appeared on the cloud manufacturing (CMfg) service platform recently, which leads to a serious information overloading problem to the enterprises that need these resources. To tackle this problem, a graph neural network-based recommendation method for CMfg service resources is proposed, which effectively overcomes some limitations of the traditional recommendation methods. Specifically, we first use different similarity calculation methods (e.g., Cosine similarity, Pearson correlation coefficient, etc.) to calculate the similarities between different resources based on the feature information of CMfg service resources. A resource graph dataset is accordingly established. A graph neural network is then used to perform representation learning of nodes in these graphs, obtaining the vector representations of these nodes. Finally, new links that may appear in a graph are predicted by performing dot product calculations on these nodes' vector representations. And these links can be used to recommend suitable resources. Experiments mainly show that (i) the proposed method obtains better link prediction accuracy compared with that of other link prediction algorithms; (ii) when the network density used for training is relatively high, the predictive performance of the proposed method is improved significantly. Our method can shed light on how to choose suitable CMfg service resources from the CMfg service platform.

Single-Position Complete Retroperitoneoscopic Radical Nephroureterectomy with Bladder Cuff Excision for Upper Urinary Tract Urothelial Carcinoma.

Purpose: We proposed a new technique, single-position complete retroperitoneoscopic radical nephroureterectomy (SCRNU), which proved to be efficient for the treatment of upper urinary tract urothelial carcinoma (UTUC). Materials and Methods: In this study, we retrospectively evaluated 86 patients diagnosed with UTUC at our hospital from June 2013 to June 2021. The patients who underwent traditional retroperitoneoscopic nephroureterectomy (TRNU) (n = 28) and SCRNU (n = 58) were consecutively enrolled. Demographic characteristics, perioperative parameters, and follow-up data were collected and compared between the two groups. Results: Both procedures were performed effectively in 86 patients without converting to open surgery. The mean follow-up time was 45.4 months for the SCRNU group and 39 months for the TRNU group. All follow-up patients survived without incidence of bladder incision tumor. Further, the follow-up results showed that there was no significant difference in the recurrence rate of bladder tumor between the two methods. SCRNU group was superior to TRNU group because of shorter operating time, fewer perioperative complications, less postoperative pain, lower recurrence rate, and cheaper medical expenditure. Conclusions: The SCRNU technique is less invasive, have fewer complications, and has a better cosmetic outcome.

RCC2 promotes prostate cancer cell proliferation and migration through Hh/GLI1 signaling pathway and cancer stem-like cells.

BACKGROUND: Regulator of chromosome condensation 2 (RCC2) was a telophase disk-binding protein on mitosis, and functions as an oncogene in many human cancers. However, its role on prostate cancer (PCa) was unknown. The goal of this study is to explore the function of RCC 2 on PCa development. METHODS: The expression of RCC2 and its methylation level, its correlation with lymph node metastasis or disease-free survival (DFS) was analyzed using TCGA database. The effect of RCC2 on PCa cell proliferation, migration and invasion were detected using CCK-8, cell colony formation, Transwell and wood healing assays. RNA-seq and GSEA analysis were used to search the downstream genes and pathways of RCC2 in mediated PCa progression. Western blot was used to detect the proteins in PCa cells transfected with indicated siRNAs or plasmids. RESULTS: RCC2 had high expression and low promoter methylation level in PCa, and its expression was correlated with regional node metastasis and disease-free survival. Cell proliferation, migration, invasion and EMT of PCa cells in vitro were greatly enhanced after RCC2 overexpression, while the RCC2 knockdown suppressed these processes. RNA-seq and GSEA results showed the Hedgehog signaling regulator Gli1 and Gli3 were involved in RCC2 knockdown DU145 cells. Gli1 was also a marker of cancer stem-like cells (CSCs). Mechanistically, RCC2 induced cell growth, EMT, CSCs markers through Gli1; inhibiting Gli1 expression using siGli1 or GLI inhibitor suppressed cell progression in vitro and tumor growth in vivo. CONCLUSION: In summary, RCC2 promoted PCa development through Hh/Gli1 signaling pathway via regulating EMT and CSCs.

Structural Optimization of Fibroblast Growth Factor Receptor Inhibitors for Treating Solid Tumors.

Small-molecule fibroblast growth factor receptor (FGFR) inhibitors have emerged as a promising antitumor therapy. Herein, by further optimizing the lead compound 1 under the guidance of molecular docking, we obtained a series of novel covalent FGFR inhibitors. After careful structure-activity relationship analysis, several compounds were identified to exhibit strong FGFR inhibitory activity and relatively better physicochemical and pharmacokinetic properties compared with those of 1. Among them, 2e potently and selectively inhibited the kinase activity of FGFR1-3 wildtype and high-incidence FGFR2-N549H/K-resistant mutant kinase. Furthermore, it suppressed cellular FGFR signaling, exhibiting considerable antiproliferative activity in FGFR-aberrant cancer cell lines. In addition, the oral administration of 2e in the FGFR1-amplified H1581, FGFR2-amplified NCI-H716, and SNU-16 tumor xenograft models demonstrated potent antitumor efficacy, inducing tumor stasis or even tumor regression.

SARS-CoV-2 restructures host chromatin architecture.

Some viruses restructure host chromatin, influencing gene expression, with implications for disease outcome. Whether this occurs for SARS-CoV-2, the virus causing COVID-19, is largely unknown. Here we characterized the 3D genome and epigenome of human cells after SARS-CoV-2 infection, finding widespread host chromatin restructuring that features widespread compartment A weakening, A-B mixing, reduced intra-TAD contacts and decreased H3K27ac euchromatin modification levels. Such changes were not found following common-cold-virus HCoV-OC43 infection. Intriguingly, the cohesin complex was notably depleted from intra-TAD regions, indicating that SARS-CoV-2 disrupts cohesin loop extrusion. These altered 3D genome/epigenome structures correlated with transcriptional suppression of interferon response genes by the virus, while increased H3K4me3 was found in the promoters of pro-inflammatory genes highly induced during severe COVID-19. These findings show that SARS-CoV-2 acutely rewires host chromatin, facilitating future studies of the long-term epigenomic impacts of its infection.

Regulation of Wnt signaling by nociceptive input in animal models.

BACKGROUND: Central sensitization-associated synaptic plasticity in the spinal cord dorsal horn (SCDH) critically contributes to the development of chronic pain, but understanding of the underlying molecular pathways is still incomplete. Emerging evidence suggests that Wnt signaling plays a crucial role in regulation of synaptic plasticity. Little is known about the potential function of the Wnt signaling cascades in chronic pain development. RESULTS: Fluorescent immunostaining results indicate that ß-catenin, an essential protein in the canonical Wnt signaling pathway, is expressed in the superficial layers of the mouse SCDH with enrichment at synapses in lamina II. In addition, Wnt3a, a prototypic Wnt ligand that activates the canonical pathway, is also enriched in the superficial layers. Immunoblotting analysis indicates that both Wnt3a a ß-catenin are up-regulated in the SCDH of various mouse pain models created by hind-paw injection of capsaicin, intrathecal (i.t.) injection of HIV-gp120 protein or spinal nerve ligation (SNL). Furthermore, Wnt5a, a prototypic Wnt ligand for non-canonical pathways, and its receptor Ror2 are also up-regulated in the SCDH of these models. CONCLUSION: Our results suggest that Wnt signaling pathways are regulated by nociceptive input. The activation of Wnt signaling may regulate the expression of spinal central sensitization during the development of acute and chronic pain.

Single-cell RNA sequencing reveals intratumoral heterogeneity and potential mechanisms of malignant progression in prostate cancer with perineural invasion.

Background: Prostate cancer (PCa) is the second most common cancer among men worldwide. Perineural invasion (PNI) was a prominent characteristic of PCa, which was recognized as a key factor in promoting PCa progression. As a complex and heterogeneous disease, its true condition is difficult to explain thoroughly with conventional bulk RNA sequencing. Thus, an improved understanding of PNI-PCa progression at the single-cell level is needed. Methods: In this study, we performed scRNAseq on tumor tissues of three PNI-PCa patients. Principal component analysis (PCA) and Uniform manifold approximation and projection (UMAP) were used to reduce dimensionality and visualize the cellular composition of tumor tissues. The differently expressed genes among each cluster were identified by EdgeR. GO enrichment analysis was used to understand the roles of genes within the clusters. Pseudotime cell trajectory was used to reveal the molecular pathways underlying cell fate decisions and identify genes whose expression changed as the cells underwent transition. We applied CellPhoneDB to identify cell-cell interactions among the epithelial and neural cells in PNI-PCa. Results: Analysis of the ∼17,000 single-cell transcriptomes in three PNI prostate cancer tissues, we identified 12 major cell clusters, including neural cells and two epithelial subtypes with different expression profiles. We found that basal/intermediate epithelial cell subtypes highly expressed PCa progression-related genes, including PIGR, MMP7, and AGR2. Pseudotime trajectory analysis showed that luminal epithelial cells could be the initiating cells and transition to based/intermediate cells. Gene ontology (GO) enrichment analysis showed that pathways related to cancer progressions, such as lipid catabolic and fatty acid metabolic processes, were significantly enriched in basal/intermediate cells. Our analysis also suggested that basal/intermediate cells communicate closely with neural cells played a potential role in PNI-PCa progression. Conclusion: These results provide our understanding of PNI-PCa cellular heterogeneity and characterize the potential role of basal/intermediate cells in the PNI-PCa progression.

Reactive Oxygen Species (ROS) are Critical for Morphine Exacerbation of HIV-1 gp120-Induced Pain.

Many HIV patients develop chronic pain and use opioid-derived medicine as primary analgesics. Emerging clinical evidence suggests that chronic use of opioid analgesics paradoxically heightens pain states in patients. This side effect of opioid analgesics has a significant negative impact on clinical practice, but the underlying pathogenic mechanism remains elusive. Using a mouse model of HIV-associated pain, we simulated the development of morphine exacerbation on pain and investigated potential underlying cellular and molecular pathways. We found that repeated morphine treatment promoted astrocyte activation in the spinal dorsal horn (SDH) and up-regulation of pro-inflammatory cytokines IL-1ß and TNF-α. Furthermore, we observed that morphine administration potentiated mitochondrial reactive oxygen species (ROS) in the SDH of the HIV pain model, especially on astrocytes. Systemic application of the ROS scavenger phenyl-N-t-butyl nitrone (PBN) not only blocked the enhancement of gp120-induced hyperalgesia by morphine but also astrocytic activation and cytokine up-regulation. These findings suggest a critical role of ROS in mediating the exacerbation of gp120-induced pain by morphine. Graphical abstract.

Neuron Type-Dependent Synaptic Activity in the Spinal Dorsal Horn of Opioid-Induced Hyperalgesia Mouse Model.

Opioids are widely used for pain relief; however, chronic opioid use causes a paradoxical state of enhanced pain sensitivity, termed "Opioid-induced hyperalgesia (OIH)." Despite the clinical importance of OIH, the detailed mechanism by which it enhances pain sensitivity remains unclear. In this study, we tested whether repeated morphine induces a neuronal circuit polarization in the mouse spinal dorsal horn (SDH). Transgenic mice expressing GFP to neurokinin 1 receptor-expressing neurons (sNK1Rn) and GABAergic interneurons (sGABAn) that received morphine [20 mg/kg, once daily for four consecutive days (i.p.)] developed mechanical hypersensitivity. Repeated morphine altered synaptic strengths in the SDH as a specific cell-type but not in a gender-dependent manner. In sNK1Rn and non-tonic firing neurons, repeated morphine treatment significantly increased frequency of spontaneous excitatory postsynaptic current (sEPSC) and evoked EPSC (eEPSC). In addition, repeated morphine treatment significantly decreased evoked inhibitory postsynaptic current (eIPSC) in sNK1Rn. Conversely, in sGABAn and tonic firing neurons, repeated morphine treatment significantly decreased sEPSC frequency and eEPSC, but had no change of eIPSC in sGABAn. Interestingly, repeated morphine treatment significantly decreased neuronal rheobase of sNK1Rn but had no effect on sGABAn. These findings suggest that spinal neuronal circuit polarization maybe the mechanism of OIH and identify a potential therapeutic mechanism to prevent or treat opioid-induced pain.

Identification of Novel Fused Heteroaromatics-Based MALT1 Inhibitors by High-Throughput Screening to Treat B Cell Lymphoma.

Development of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors is of great value and significance in the treatment of neoplastic disorders and inflammatory and autoimmune diseases. However, there is a lack of effective MALT1 inhibitors in clinic. Herein, a novel class of potent 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-based MALT1 inhibitors and their covalent derivatives were first identified and designed through high-throughput screening. We demonstrated that compounds 15c, 15e, and 20c effectively inhibited the MALT1 protease and displayed selective cytotoxicity to activated B cell-like diffuse large B cell lymphoma with low single-digit micromolar potency. Furthermore, compound 20c specifically repressed NF-κB signaling and induced cell apoptosis in MALT1-dependent TMD8 cells in a dose-dependent manner. More importantly, 20c showed good pharmacokinetic properties and antitumor efficacy with no significant toxicity in the TMD8 xenograft tumor model. Collectively, this study provides valuable lead compounds of MALT1 inhibitors for further structural optimization and antitumor mechanism study.

Discovery of novel ceramide analogs with favorable pharmacokinetic properties and combination with AKT inhibitor against colon cancer.

Ceramides have emerged as potential therapeutic option with novel mechanism to affect the proliferation, differentiation, senescence, and apoptosis of cancer cells through regulation of multiple signal transduction. Aiming at the improvement of the apoptosis activity and pharmacokinetic profiles of ceramides, a novel series of ceramide analogs were developed through structure simplification and conformation restriction. Among them, compound 12 bearing an alkoxyl naphthyl motif, with favorable rat pharmacokinetic properties, showed better anti-proliferative activity against various colon cancer cells (IC50 ∼20 µM) than other ceramide analogues, as well as the synergistic effect combined with AKT inhibitor MK2206. Additionally, we demonstrated that this combination therapy promoted caspase 3-dependent apoptotic pathway and intensified cell cycle arrest in the G0/G1 phase. Furthermore, the combination of compound 12 and MK2206 displayed synergistic anti-tumor effect in vivo.

Increased Tim-3 expression on TILs during treatment with the Anchored GM-CSF vaccine and anti-PD-1 antibodies is inversely correlated with response in prostate cancer.

Programmed death receptor-1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) play important roles in tumor immune evasion. PD-1 blockade could produce an effective antitumor effect in many solid tumors except prostate cancer (PCa) because of rare programmed death ligand-1 (PD-L1) expression on PCa cells. Streptavidin (SA)-GM-CSF surface-anchored tumor cell (Anchored GM-CSF) vaccines could increase the number of tumor-infiltrating lymphocytes (TILs) and induce specific antitumor immune responses. The Anchored-GM-CSF vaccine and anti-PD-1 antibodies exerted synergistic effects in mouse models of PCa metastasis. However, the response rate was low due to the presence of other negative regulatory pathways. Tim-3 expression could be upregulated at resistance to combination therapy with anti-PD-1 antibodies and the Anchored GM-CSF vaccine. Sequential administration of anti-PD-1 and anti-Tim-3 antibodies could further improve the efficacy of the Anchored GM-CSF vaccine therapy, and tumor regression was noted in over 60% of animals. This triple therapy improved the specific cytotoxic activity, proliferation and secretion of CD8+ TILs and reduced the production of tumor-promoting cytokines. These findings indicated that this triple therapy could induce a robust antitumor immune response in mouse models of PCa.

Male germ cell-specific protein Trs4 binds to multiple proteins.

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.