S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation.

Testicular invasion and persistence are features of Zika virus (ZIKV), but their mechanisms are still unknown. Here, we showed that S100A4+ macrophages, a myeloid macrophage subpopulation with susceptibility to ZIKV infection, facilitated ZIKV invasion and persistence in the seminiferous tubules. In ZIKV-infected mice, S100A4+ macrophages were specifically recruited into the interstitial space of testes and differentiated into interferon-γ-expressing M1 macrophages. With interferon-γ mediation, S100A4+ macrophages down-regulated Claudin-1 expression and induced its redistribution from the cytosol to nucleus, thus increasing the permeability of the blood-testis barrier which facilitated S100A4+ macrophages invasion into the seminiferous tubules. Intraluminal S100A4+ macrophages were segregated from CD8+ T cells and consequently helped ZIKV evade cellular immunity. As a result, ZIKV continued to replicate in intraluminal S100A4+ macrophages even when the spermatogenic cells disappeared. Deficiencies in S100A4 or interferon-γ signaling both reduced ZIKV infection in the seminiferous tubules. These results demonstrated crucial roles of S100A4+ macrophages in ZIKV infection in testes.

TNF-α Differentially Regulates Synaptic Plasticity in the Hippocampus and Spinal Cord by Microglia-Dependent Mechanisms after Peripheral Nerve Injury.

Clinical studies show that chronic pain is accompanied by memory deficits and reduction in hippocampal volume. Experimental studies show that spared nerve injury (SNI) of the sciatic nerve induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, but impairs LTP in the hippocampus. The opposite changes may contribute to neuropathic pain and memory deficits, respectively. However, the cellular and molecular mechanisms underlying the functional synaptic changes are unclear. Here, we show that the dendrite lengths and spine densities are reduced significantly in hippocampal CA1 pyramidal neurons, but increased in spinal neurokinin-1-positive neurons in mice after SNI, indicating that the excitatory synaptic connectivity is reduced in hippocampus but enhanced in spinal dorsal horn in this neuropathic pain model. Mechanistically, tumor necrosis factor-alpha (TNF-α) is upregulated in bilateral hippocampus and in ipsilateral spinal dorsal horn, whereas brain-derived neurotrophic factor (BDNF) is decreased in the hippocampus but increased in the ipsilateral spinal dorsal horn after SNI. Importantly, the SNI-induced opposite changes in synaptic connectivity and BDNF expression are prevented by genetic deletion of TNF receptor 1 in vivo and are mimicked by TNF-α in cultured slices. Furthermore, SNI activated microglia in both spinal dorsal horn and hippocampus; pharmacological inhibition or genetic ablation of microglia prevented the region-dependent synaptic changes, neuropathic pain, and memory deficits induced by SNI. The data suggest that neuropathic pain involves different structural synaptic alterations in spinal and hippocampal neurons that are mediated by overproduction of TNF-α and microglial activation and may underlie chronic pain and memory deficits. SIGNIFICANCE STATEMENT: Chronic pain is often accompanied by memory deficits. Previous studies have shown that peripheral nerve injury produces both neuropathic pain and memory deficits and induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn (SDH) but inhibits LTP in hippocampus. The opposite changes in synaptic plasticity may contribute to chronic pain and memory deficits, respectively. However, the structural and molecular bases of these alterations of synaptic plasticity are unclear. Here, we show that the complexity of excitatory synaptic connectivity and brain-derived neurotrophic factor (BDNF) expression are enhanced in SDH but reduced in the hippocampus in neuropathic pain and the opposite changes depend on tumor necrosis factor-alpha/tumor necrosis factor receptor 1 signaling and microglial activation. The region-dependent synaptic alterations may underlie chronic neuropathic pain and memory deficits induced by peripheral nerve injury.

Polysaccharides from Polygonatum Inhibit the Proliferation of Prostate Cancer-Associated Fibroblasts.

Inhibition of cancer-associated broblasts (CAFs) may improve the efficacy of cancer therapy. Polysaccharide extracted from polygonatum can selectively inhibit the growth of prostate-CAFs (<.001) without inhibiting the growth of normal broblasts (NAFs). Polysaccharides from polygonatum stimulate autophagy of prostate-CAFs. 3-methyl-adenine(3-MA) is an autophagy inhibitor. 3-MA was added to prostate-CAFs with polysaccharide from polygonatum to determine whether autophagy plays an important role in the restrained effect. Finally, polysaccharide from polygonatum treatment significantly increased the activation of Beclin-1 and LC3, key autophagy proteins. Polysaccharides from polygonatum stimulate autophagy of prostate-CAFs and inhibits prostate-CAF growth, indicating that a novel anti-cancer strategy involves inhibiting the growth of prostate- CAFs.

Downregulation of ClC-3 in dorsal root ganglia neurons contributes to mechanical hypersensitivity following peripheral nerve injury.

ClC-3 chloride channel/antiporter has been demonstrated to play an important role in synaptic transmission in central nervous system. However, its expression and function in sensory neurons is poorly understood. In present work, we found that ClC-3 is expressed at high levels in dorsal root ganglia (DRG). Co-immunofluorescent data showed that ClC-3 is mainly distributed in A- and C-type nociceptive neurons. ClC-3 expression in DRG is decreased in the spared nerve injury (SNI) model of neuropathic pain. Knockdown of local ClC-3 in DRG neurons with siRNA increased mechanical sensitivity in naïve rats, while overexpression of ClC-3 reversed the hypersensitivity to mechanical stimuli after peripheral nerve injury. In addition, genetic deletion of ClC-3 enhances mouse mechanical sensitivity but did not affect thermal and cold threshold. Restoration of ClC-3 expression in ClC-3 deficient mice reversed the mechanical sensitivity. Mechanistically, loss of ClC-3 enhanced mechanical sensitivity through increasing the excitability of DRG neurons. These data indicate that ClC-3 is an endogenous inhibitor of neuropathic pain development. Downregulation of ClC-3 by peripheral nerve injury is critical for mechanical hypersensitivity. Our findings suggest that ClC-3 is a novel therapeutic target for treating neuropathic pain.

DH332, a synthetic ß-carboline alkaloid, inhibits B cell lymphoma growth by activation of the caspase family.

AIM: The purpose of this study was to investigate anti-tumor effects and safety of DH332, a new ß-carboline alkaloids derivatives in vitro and in vivo. MATERIALS AND METHODS: The effects of DH332 on human (RAMOS RA.1) and mouse (J558) B lymphoma cell lines were detected using a CCK-8 kit (Cell Counting Kit-8), and apoptosis was detected by flow cytometry with PI/annexinV staining. Western blotting was used to detected caspase-3 and caspase-8. Neurotoxic and anti-tumor effects were evaluated in animal experiments. RESULTS: DH332 exerts a lower neurotoxicity compared with harmine. It also possesses strong antitumor effects against two B cell lymphoma cell lines with low IC50s. Moreover, DH332 could inhibit the proliferation and induce the apoptosis of RAMOS RA.1 and J558 cell lines in a dose-dependent manner. Our results suggest that DH332 triggers apoptosis by mainly activating the caspase signaling pathway. In vivo studies of tumor-bearing BALB/c mice showed that DH332 significantly inhibited growth of J558 xenograft tumors. CONCLUSIONS: DH332 exerts effective antitumor activity in vitro and in vivo, and has the potential to be a promising drug candidate for lymphoma therapy.

Peripheral nerve injury leads to working memory deficits and dysfunction of the hippocampus by upregulation of TNF-α in rodents.

Patients with chronic pain usually suffer from working memory deficits, which may decrease their intellectual ability significantly. Despite intensive clinical studies, the mechanism underlying this form of memory impairment remains elusive. In this study, we investigated this issue in the spared nerve injury (SNI) model of neuropathic pain, a most common form of chronic pain. We found that SNI impaired working memory and short-term memory in rats and mice. To explore the potential mechanisms, we studied synaptic transmission/plasticity in hippocampus, a brain region critically involved in memory function. We found that frequency facilitation, a presynaptic form of short-term plasticity, and long-term potentiation at CA3-CA1 synapses were impaired after SNI. Structurally, density of presynaptic boutons in hippocampal CA1 synapses was reduced significantly. At the molecular level, we found that tumor necrosis factor-α (TNF-α) increased in cerebrospinal fluid, in hippocampal tissue and in plasma after SNI. Intracerebroventricular or intrahippocampal injection of recombinant rat TNF mimicked the effects of SNI in naive rats, whereas inhibition of TNF-α or genetic deletion of TNF receptor 1 prevented both memory deficits and synaptic dysfunction induced by SNI. As TNF-α is critical for development of neuropathic pain, we suggested that the over-production of TNF-α following peripheral nerve injury might lead to neuropathic pain and memory deficits, simultaneously.

TNF-α contributes to up-regulation of Nav1.3 and Nav1.8 in DRG neurons following motor fiber injury.

A large body of evidence has demonstrated that the ectopic discharges of action potentials in primary afferents, resulted from the abnormal expression of voltage gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons following peripheral nerve injury are important for the development of neuropathic pain. However, how nerve injury affects the expression of VGSCs is largely unknown. Here, we reported that selective injury of motor fibers by L5 ventral root transection (L5-VRT) up-regulated Nav1.3 and Nav1.8 at both mRNA and protein level and increased current densities of TTX-S and TTX-R channels in DRG neurons, suggesting that nerve injury may up-regulate functional VGSCs in sensory neurons indirectly. As the up-regulated Nav1.3 and Nav1.8 were highly co-localized with TNF-α, we tested the hypothesis that the increased TNF-α may lead to over-expression of the sodium channels. Indeed, we found that peri-sciatic administration of recombinant rat TNF-α (rrTNF) without any nerve injury, which produced lasting mechanical allodynia, also up-regulated Nav1.3 and Nav1.8 in DRG neurons in vivo and that rrTNF enhanced the expression of Nav1.3 and Nav1.8 in cultured adult rat DRG neurons in a dose-dependent manner. Furthermore, inhibition of TNF-α synthesis, which prevented neuropathic pain, strongly inhibited the up-regulation of Nav1.3 and Nav1.8. The up-regulation of the both channels following L5-VRT was significantly lower in TNF receptor 1 knockout mice than that in wild type mice. These data suggest that increased TNF-α may be responsible for up-regulation of Nav1.3 and Nav1.8 in uninjured DRG neurons following nerve injury.

[The significance and function of IFN-gamma on the changes of peripheral blood platelet count during tumor-rejection induced by a low dose of melphalan in C57BL/6 mice].

AIM: To investigate the significance and function of IFN-gamma on the changes of peripheral blood platelet count during tumor-rejection induced by a low dose of melphalan in C57BL/6 mice. METHODS: Mouse tumor rejection model induced by a single dose of melphalan was used in this experiment. Different gene-type tumor-bearing mice (IFN-gamma(+/-) and IFN-gamma(-/-)), which had the same genetic background of C57BL/6, were treated intraperitoneally with melphalan (7.5 mg/kg). Tumor size was observed and recorded every one to three days in these different gene-type mice subsequently. Blood samples were obtained from orbital venous sinus on different days before and after melphalan treatment, and then complete blood counts were performed. The function of IFN-gamma on the efficacy of chemotherapy and the changes of blood platelet count in IFN-gamma(+/-) and IFN-gamma(-/-) mice after melphalan treatment was analyzed. RESULTS: There was no significant difference in tumor sizes and blood platelet count between IFN-gamma(-/-) and IFN-gamma(+/-) mice (P>0.05). On the first day after melphalan (7.5 mg/kg) treatment, there were no significant changes in tumor sizes between mice in these two groups (P>0.05). Tumors shrank a little in IFN-gamma(-/-) mice and then grew gradually. Tumors relapsed in 2 w after melphalan injection in all IFN-gamma(-/-) mice, while tumor volumes decreased progressively and tumor cured at last in IFN-gamma(+/-) mice. The number of blood PLT in IFN-gamma(+/-) mice increased to (1935+/-378) x 10(9)/L 6 h after melphalan treatment, significantly higher than before (P<0.01); While in IFN-gamma(-/-) mice it was (1183+/-186) x 10(9)/L 6 h after melphalan treatment, no obvious increase than before. There was significant difference in blood PLT 6 h after melphalan treatment between IFN-gamma(+/-) and IFN-gamma(-/-) mice (P<0.01). Later, the numbers of blood PLT in IFN-gamma(+/-) mice decreased gradually and it dropped to normal (1158+/-270) x 10(9)/L on 11th day after melphalan treatment (P>0.05); While it sustained in normal range in IFN-gamma(-/-) mice. There was no significant difference in blood platelet count between IFN-gamma(-/-) and IFN-gamma(+/-) mice. CONCLUSION: Peripheral blood platelet count increased on the first day after melphalan treatment and tumors cured in IFN-gamma(+/-) mice; While tumors relapsed and there is no increase in blood platelet count on the first day after melphalan treatment in IFN-gamma(-/-) mice. These data indicated that the increase of blood PLT count was related to the function of IFN-gamma in tumor-bearing mice in vivo during tumor rejection induced by a low dose of melphalan.

[Changes and significance of peripheral blood count in tumor rejection induced by a low dose of melphalan in C57BL/6 mice].

AIM: To investigate the relationship between changes of peripheral blood counts and tumor rejection induced by a low dose of melphalan in C57BL/6 mice. METHODS: Mouse lymphoma EL4 cells were inoculated subcutaneously into wild type C57BL/6 mice. Twelve days later, 7.5 mg/kg melphalan were administered intraperitoneally and the same volume of Normal Saline as control. Tumor sizes were observed and recorded subsequently. Blood samples were obtained from orbital venous sinus on different days before and after melphalan treatment, and then complete blood counts were performed and the relationship between the alterations of blood counts and tumor shrinkage after melphalan treatment was analyzed. RESULTS: Tumor sizes decreased and tumors disappeared after 7.5 mg/kg melphalan treatment; while tumors grow continuously in control mice. The number of WBC was increased a little (10.6 + or - 2.3) x 10(9)/L 6 h after melphalan treatment, but there was no significant difference with mice before melphalan injection (9.8 + or - 0.32) x 10(9)/L (P>0.05); The number of WBC decreased significantly at 4(th) day after melphalan treatment (P<0.01); Later it increased a little, but at 28(th); day after melphalan it still obviously lower than that of the normal (P<0.01). Hemoglobin (Hb) concentration decreased from (132 + or - 7) g/L before melphalan treatment to (110 + or - 14) g/L at 6 h after melphalan treatment (P<0.05). Later, the amount of Hb was decreasing and at 7th day it got to its lowest point (96 + or - 5) g/L. It increased gradually back to normal in 2 weeks after melphalan treatment. The platelet count increased to (1502 + or - 142) x 10(9)/L 6 h after melphalan treatment, significantly higher that that (914 + or - 322) x 10(9)/L before melphalan injection (P<0.01). It maintained at a high level for one week and it recovered back to normal level at 28(th) day after melphalan treatment. CONCLUSION: Tumor shrinkage after melphalan treatment was not related to the decreased number of WBC or RBC, but correlated with the increased number of platelet in 10 days after melphalan treatment.

[Relationships between tumor shrinkage and peripheral blood cell changes during tumor-rejection induced by a high dose of 5-FU in C57BL/6 mice].

AIM: To investigate the relationship between the alterations of complete blood counts and tumor shrinkage during tumor rejection induced by a high dose of 5-FU in C57BL/6 mice. METHODS: Wild type C57BL/6 tumor-bearing mice were treated with different doses of 5-FU intraperitoneally. 75 mg/kg 5-FU was the minimal effective dose of 5-FU that could cure the tumor-bearing mice. Then another 6 tumor-bearing mice were treated intraperitoneally with 5-FU (75 mg/kg). Blood samples were obtained from orbital venous sinus on different days before and after 5-FU treatment, and then complete blood counts were performed and the relationship between the alterations of blood counts and tumor shrinkage after 5-FU treatment was analyzed. RESULTS: Tumor sizes decreased steadily and tumors disappeared within the first week after 5-FU treatment; and at the same time 75 mg/kg 5-FU also had side effects on peripherial blood cells. The number of WBC significantly decreased from the first day after 5-FU treatment (P<0.001). But during the 7 th to 15 th day the number of WBC rebounced back to normal level (P>0.05). Later it decreased again and it couldn't recover back to normal level at the 28th day after 5-FU treatment (P<0.01). The concentration of Hb decreased at the first day and lasted for 2 weeks (P<0.01). It increased gradually back to normal in 2 weeks after 5-FU treatment. The inhibitory effect of 5-FU on platelets count was not obvious. The platelet count increased significantly at the first and at the 11th day after 5-FU treatment respectively (P<0.01). CONCLUSION: Tumor shrinkage after 5-FU treatment is not related to the decreased number of WBC or RBC, but correlated with the increased number of platelet at the first day after 5-FU treatment.

[Study of the immunological mechanism of anti-tumor effects of 5-FU by establishing EL4 tumor-bearing mouse models].

AIM: To investigate the immunological mechanism of anti-tumor effect of 5-FU by establishing lymphoma EL4 tumor-bearing mouse models in wild type C57BL/6 mice and nude C57BL/6 mice, respectively. METHODS: The mouse lymphoma EL4 cells were inoculated subcutaneously into wild type C57BL/6 mice (immune-competent mice). Twelve days later, 5-FU of different doses was administered intraperitoneally to treat these wild type C57BL/6 tumor-bearing mice. The size of tumors in the wild type C57BL/6 mice was observed and recorded to explore the minimal dose of 5-FU that could cure the tumor-bearing mice. Then the same amount of EL4 tumor cells was inoculated subcutaneously into wild type C57BL/6 mice and nude C57BL/6 mice (T cell-deficient mice) simultaneously, which had the same genetic background of C57BL/6. Twelve days later, 5-FU of the minimal dose was given intraperitoneally to treat both the wild type and nude C57BL/6 tumor-bearing mice. The size of tumors in the two different types of mice was observed and recorded. RESULTS: A single dose of 5-FU (75 mg/kg) cured both the EL4 tumor-bearing wild type C57BL/6 mice and the EL4 tumor-bearing nude C57BL/6 mice in the first week. Two weeks after 5-FU treatment, all of the nude mice died of tumor relapse while most of the wild type C57BL/6 mice were fully recovered. CONCLUSION: A single dose of 5-FU has marked anti-tumor effects on lymphoma EL4 tumor-bearing C57BL/6 mice with or without T lymphocytes. The relapse of tumors after 5-FU treatment might be related to the function of T lymphocytes.

[Relationship between TNFalpha and tumor rejection induced by a single dose of melphalan in C57BL/6 mice].

AIM: To investigate the relationship between TNFalpha and tumor rejection induced by a single dose of melphalan in C57BL/6 mice. METHODS: Different gene type mice (TNFR1(+/+), TNFR1(+/-) and TNFR1(-/-)) with the same genetic background of C57BL/6 were used in this experiment. Murine lymphoma EL4 cells were inoculated subcutaneously into the different gene type mice simultaneously. Twelve days later, 7.5 mg/kg melphalan was used intraperitoneally to treat the tumor-bearing mice with TNFR1(+/+), TNFR1(+/-) and TNFR1(-/-). The tumors in the different gene type mice were observed and recorded every one to three day. RESULTS: After the treatment of 7.5 mg/kg melphalan during the first week, the tumors in the different gene type mice shrank at a similar rate. In the following 2 months, the tumors in the TNFR1(+/+) and TNFR1(+/-) C57BL/6 mice gradually shrank and were cured but most tumors in the TNFR1(-/-) C57BL/6 mice relapsed after melphalan treatment. CONCLUSION: TNFalpha plays an important role in melphalan-induced tumor rejection. The anti-tumor effect of melphalan has no relationship with the expression of tumor necrosis factor 1 in tumor-bearing mice. TNFR1 is required to prevent or avoid the relapse of tumors in mice instead of tumor cells.

[Relationship between anti-tumor effects of melphalan and IFN-gamma].

AIM: To investigate the relationship between IFN-gamma and anti-tumor effects of melphalan in vivo. METHODS: Different gene-type mice (IFN-gamma(+/+), IFN-gamma(+/-) and IFN-gamma(-/-)), which had the same genetic background of C57BL/6, were used in this experiment. Murine lymphoma EL4 cells were inoculated subcutaneously into different gene-type mice simultaneously. Twelve days later, 7.5 mg/kg melphalan was used intraperitoneally to treat all these IFN-gamma(+/+), IFN-gamma(+/-) and IFN-gamma(-/-) tumor-bearing mice. Tumor size was observed and recorded every one to three days in these different gene-type mice subsequently. RESULTS: After melphalan (7.5 mg/kg) treatment, tumor size decreased at a similar rate in IFN-gamma(-/-), IFN-gamma(+/-) and IFN-gamma(+/+) mice within the first 3 d. Tumors shrank further or completely disappeared in most of IFN-gamma(+/-) and IFN-gamma(+/+) mice, whereas tumors grew gradually in all IFN-gamma(-/-) mice. CONCLUSION: 7.5 mg/kg melphalan has obvious anti-tumor effects on tumor-bearing IFN-gamma(+/+) and IFN-gamma(+/-) mice, but little effects on IFN-gamma(-/-) mice. These data suggest that IFN-gamma is required for the anti-tumor effects of melphalan on tumor-bearing mice in vivo.

[Establishment of EL4 tumor-bearing mouse models and investigation on immunological mechanisms of anti-tumor effect of melphalan].

AIM: To establish mouse lymphoma EL4 tumor-bearing mouse models in wild type C57BL/6 mice and nude C57BL/6 mice respectively, and to further investigate the immunological mechanisms of anti-tumor effect of melphalan. METHODS: Mouse lymphoma EL4 cells were inoculated subcutaneously into wild type C57BL/6 mice (immune-competent mice). Twelve days later, melphalan of different doses were administered intraperitoneally to treat these wild type C57BL/6 tuomr-bearing mice. Tumor sizes were observed and recorded subsequently to find out the minimal dose of melphalan that could cure the tuomr-bearing mice. Then the same amount of EL4 tumor cells were inoculated subcutaneously into wild type C57BL/6 mice and nude C57BL/6 mice (T cell-deficient mice) simultaneously, which had the same genetic background of C57BL/6. Twelve days later, melphalan of the minimal dose was given intraperitoneally to treat both the wild type and nude C57BL/6 tuomr-bearing mice. Tumor sizes were observed and recorded in these two different types of mice subsequently. RESULTS: A single dose of melphalan (7.5 mg/kg) could cure EL4 tumor-bearing wild type C57BL/6 mice, but could not induce tumor regression in EL4 tumor-bearing nude C57BL/6 mice. CONCLUSION: A single dose of melphalan has obvious anti-tumor effect on mouse lymphoma EL4 tumor-bearing wild type C57BL/6mice, which requires the involvement of T lymphocytes in the host probably related to their killing functions.