Hydrogen therapy promotes macrophage polarization to the M2 subtype by inhibiting the NF-κB signaling pathway / 中国辐射卫生

Objective To investigate the role of hydrogen therapy in reducing radiation-induced lung injury and the specific mechanism. Methods Forty C57BL/6 mice were randomly divided into four groups: normal control group, model group, hydrogen therapy group I, and hydrogen therapy group II. A mouse model of radiation-induced lung injury was established. The pathological changes in the lung tissue of the mice were examined with HE staining. Immunofluorescence staining was used to detect the expression of surface markers of M1 and M2 macrophages to observe macrophage polarization. The expression of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and IL-10 in the lung tissue was measured by immunohistochemistry. The expression of nuclear factor-kappa B (NF-κB) p65 and phosphorylated NF-κB (P-NF-κB) p65 was measured by Western blot. Results HE staining showed that compared with the control group, the model group exhibited alveolar septal swelling and thickening, vascular dilatation and congestion, and inflammatory cell infiltration in the lung tissue; the hydrogen groups had significantly reduced pathological damage and inflammatory response than the model group, with more improvements in hydrogen group II than in hydrogen group I. Immunohistochemical results showed that compared with those in the control group, the levels of the inflammatory cytokines IL-6 and TNF-α were significantly increased in the model group; the hydrogen groups showed significantly decreased IL-6 and TNF-α levels and a significantly increased level of the anti-inflammatory factor IL-10 than the model group, which were more marked in hydrogen group II than in hydrogen group I. Immunofluorescence results showed that compared with the control group, the expression of the surface marker of M1 macrophages in the model group was significantly upregulated; the hydrogen groups showed significantly downregulated M1 marker and significantly upregulated M2 marker, and hydrogen group II showed significantly increased M2 marker compared with hydrogen group I. Western blot results showed that compared with that in the control group, the ratio of P-NF-κB p65/NF-κB p65 in the model group was significantly increased; the P-NF-κB p65/NF-κB p65 ratio was significantly reduced in the hydrogen groups than in the model group, and was significantly lower in hydrogen group II than in hydrogen group I. Conclusion Hydrogen inhalation therapy may reduce the inflammatory response of radiation-induced lung injury by inhibiting the NF-κB signaling pathway to promote the polarization of the macrophage M1 subtype to the M2 subtype.

BMSCs promote M2 macrophage polarization to attenuate acute radiation-induced lung injury / 中国辐射卫生

Objective To investigate the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) for radiation-induced lung injury (RILI) and the underlying mechanism. Methods Forty-five healthy adult male C57BL/6 mice were randomly divided into control, model, and BMSCs groups. The model and BMSCs groups received a single irradiation dose of 20 Gy to the chest, while the control group did not receive X-ray irradiation. For the BMSCs group, an injection of 1 × 106 BMSCs cells was administered via the tail vein within 6 h after irradiation. In the 5th week, the lung tissue was taken to observe pathological changes with HE staining; examine the expression of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) with immunohistochemical staining; observe the polarization of macrophages with immunofluorescence staining; and measure the expression of the epithelial-mesenchymal transition markers E-cadherin, N-cadherin, and vimentin proteins by Western blot. Results After radiation, the model group developed pulmonary vasodilation and congestion with septal thickening and inflammatory cell infiltration, and these changes were markedly reduced in the BMSCs group. The model group showed significantly down-regulated expression of IL-6 and TNF-α compared with significantly increased levels in the model group (P < 0.01, P < 0.05). Treatment with BMSCs significantly increased the polarization of lung macrophages towards the M2 type, while significantly decreasing the abnormally increased N-cadherin and vimentin levels in RILI mice (P < 0.05, P < 0.01). Conclusion BMSCs have therapeutic effects for RILI mice, which may be through promoting macrophage polarization from M1 to M2.

Spatiotemporal landscape of SARS-CoV-2 pulmonary infection reveals Slamf9+Spp1+ macrophages promoting viral clearance and inflammation resolution

While SARS-CoV-2 pathogenesis has been intensively investigated, the host mechanisms of viral clearance and inflammation resolution are still elusive because of the ethical limitation of human studies based on COVID-19 convalescents. Here we infected Syrian hamsters by authentic SARS-CoV-2 and built an ideal model to simulate the natural recovery process of SARS-CoV-2 infection from severe pneumonia1,2. We developed and applied a spatial transcriptomic sequencing technique with subcellular resolution and tissue-scale extensibility, i.e., Stereo-seq3, together with single-cell RNA sequencing (scRNA-seq), to the entire lung lobes of 45 hamsters and obtained an elaborate map of the pulmonary spatiotemporal changes from acute infection, severe pneumonia to the late viral clearance and inflammation resolution. While SARS-CoV-2 infection caused massive damages to the hamster lungs, including naive T cell infection and deaths related to lymphopenia, we identified a group of monocyte-derived proliferating Slamf9+Spp1+ macrophages, which were SARS-CoV-2 infection-inducible and cell death-resistant, recruiting neutrophils to clear viruses together. After viral clearance, the Slamf9+Spp1+ macrophages differentiated into Trem2+ and Fbp1+ macrophages, both responsible for inflammation resolution and replenishment of alveolar macrophages. The existence of this specific macrophage subpopulation and its descendants were validated by RNAscope in hamsters, immunofluorescence in hACE2 mice, and public human autopsy scRNA-seq data of COVID-19 patients. The spatiotemporal landscape of SARS-CoV-2 infection in hamster lungs and the identification of Slamf9+Spp1+ macrophages that is pivotal to viral clearance and inflammation resolution are important to better understand the critical molecular and cellular players of COVID-19 host defense and also develop potential interventions of COVID-19 immunopathology.

The function and regulation of TET2 in innate immunity and inflammation

TET2, a member of ten-eleven translocation (TET) family as α-ketoglutarate- and Fe

Polycomb chromobox Cbx2 enhances antiviral innate immunity by promoting Jmjd3-mediated demethylation of H3K27 at the Ifnb promoter

Polycomb chromobox (CBX) proteins regulate gene transcription by maintaining chromatin states, which guide a variety of biological processes. Now, epigenetic regulation of innate immune response is an emerging field. However, the role of CBX proteins in innate immunity remains unclear. We confirmed that the expression of CBX family proteins, especially Cbx2, was decreased in macrophages upon viral infection, and then we investigated the role of Cbx2 in the antiviral immune response. Silencing or knockdown of Cbx2 in macrophages inhibited virus-induced production of IFN-β. Furthermore, heterozygous Cbx2 knockout were susceptible to VSV challenge. Mechanistically, Cbx2 binds to and recruits Jmjd3 to the Ifnb promoter, leading to demethylation of H3K27me3 and increased transcription of IFN-β. Together, our study reveals a non-traditional function of a Cbx protein and adds new insight into the epigenetic regulation of antiviral innate immunity.

Roles of regulatory T cell in tumor immunity and tumor immunotherapy / 中国肿瘤生物治疗杂志

There are multiple types of inhibitory immune cells in tumor. Among these cells, Treg (regulatory T cell) plays an extremely important role in tumor development and progression. Treg exihibits potent inhibitory effects on effector cells by a variety of mechanisms, which might be the the key factor for tumor immune escape. These mechanisms include inhibiting the effector cell function by inhibitory cytokines, killing effector cells by granzyme and profrin, interfering effector cell metabolism, and affecting Treg differentiation and proliferation by regulating the function of dendrtic cells, etc. The research on Treg has provided new strategies for tumor immunotherapy. Tumor immunotherapies targeting Treg and related immunosuppressive factors, such as deleting Treg nonsepcificlly or sepcificlly controling the numbers and functions of Treg, might have a bright future in clinical application.keyword regulatory T cell(Treg); neoplasms; immune escape; immunotherapy

Advances in DNA recognition receptors / 中国肿瘤生物治疗杂志

DNA, as the material basis of all living cells, triggers innate immune responses through TLR9 and other cytosolic recognition receptors. In recent years, the research progress of TLR9 is mainly manifested by the following four aspects: (1) the determinants of TLR9 interacting with its ligands; (2) the mechanisms and the importance of TLR9 translocation from the endoplasmic reticulum to the endosome; (3) the roles of the endosomal acidification and maturation, and subsequent TLR9 cleavage in TLR9 signal transduction pathway; and (4) the possible mechanisms by which the organism distinguish self DNA from microbial DNA. Meanwhile, a series of experiments on TLR9 antagonists and TLR9 deficient mice confirmed the presence of TLR9-independent cytosolic DNA sensors. So far, three TLR9-independent DNA sensors have been found, and they are DAI, AIM2, and RNA polymerase Ⅲ.

Antitumor effects of interleukin-18 gene-modified hepatocyte cell line on implanted liver carcinoma / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma.</p><p><b>METHODS</b>Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines, BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line), tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2). Two weeks later, the serum levels of IL-18, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed, the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured, and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach.</p><p><b>RESULTS</b>In the treatment group, the serum levels of IL-18, IFN-gamma, TNF-alpha and NO increased significantly. The splenic CTL activity increased markedly (P < 0.01), accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice.</p><p><b>CONCLUSIONS</b>In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.</p>

Enhancement of antigen presenting function of dendritic cells by IL-2 gene modification and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of IL-2 gene modification enhancement of the antigen-presenting function of the mouse bone marrow derived dendritic cells and on the activation of CTL induced by MHC class I molecule restricted antigen peptides as well as the related immunological mechanisms.</p><p><b>METHODS</b>DCs were prepared from mouse bone marrow and modified by recombinant IL-2 adenovirus (DC-IL-2). The IL-12 and IFN-gamma levels in culture supernatant of DC and CTL were examined by ELISA, the expression of costimulatory molecules and fluorescent intensity of endocytosis of OVA-FITC in DC by FACS, the capacity of presenting 3LL cell tumor antigen by (3)H-TdR incorporation method, the MHC class I-restricted tumor-antigen-peptide Mut1 of 3LL cells pulsed DC-IL-2 to induce CTL cytotoxicity by (51)Cr 4-hr releasing assay.</p><p><b>RESULTS</b>After IL-2 gene modification, DC-IL-2 could produce high level of IL-12 [(78.4 +/- 6.6) pg.(1 x 10(6) cells)(-1).ml(-1)]. The expression of costimulatory molecules on DC-IL-2 was increased, the fluorescent intensity of DC captured OVA-FITC was enhanced, and the proliferation of allo-T cells from 3LL bearing mouse pulsed with Mut1 was also enhanced. Mut1 antigen peptide pulsed DC-IL-2 could induce more potent antigen-specific CTL cytotoxicity and excrete high concentration of IFN-gamma [(1 168 +/- 58.4) pg/ml] in vivo.</p><p><b>CONCLUSION</b>IL-2 gene modification of DC can activate second signal for DC presenting antigen, and enhance the function for capturing and presenting tumor antigen. DC-IL-2 pulsed with MHC class I restricted tumor-antigen-peptide can induce specific anti-tumor immune response more effectively. Owing to IL-2 gene modification, the functions of IL-12 excretion and T cell activation of DC were promoted, so that the capacity of CTL excreting IFN-gamma was enhanced, which are relevant to the immune mechanism.</p>

The affection to lymphocyte subsets in the draining lymphoid node and immune pro tected effect from the MHC class I antigen peptides pulsed DCs with IL-2 gene m odified immunized mice / 中国免疫学杂志

Objective:To investigate the specific immune act ivated effects in the mouse immunized by MHC I-restriced tumor antigen peptide pulsed dendritic cells(DC) with IL-2 gene modified.Methods:DC were transfected with IL-2 gene via adenovirus vector(DC-IL-2) the mRNA of mIL-2 was examined by RT-PCR.Syngeneic mice were immunized by DC-IL-2 pul sed with MHC-I-restricted Mutl tumor antigen peptiede of 3LL Lewis lung carcin oma (DC-IL-2-Mutl),the effects of lymphocyte subsets in the draining lymphoid node from the tumor antigen pulsed DCs with gene modified immunized mice by flo w-cytometric analysis(FACS) analysis.Results:The mRNA of mIL-2 was expressed in the DCs.The ratio of CD8+T cells was hosted in the dra ining lymphoid node from Mutl pulsed DCs(DC-Mutl) immunized mice.The ratio of C D8+T cells and NK cells were all hosted obviously in the draining lymphoid nod e from DC-IL-2-Mutl immunized mice .Conclusion:Immunized mice with DC-IL-2-Mutl can induce the mouse specific antitumor immunized affect in vivo and can activate a variety of immunized effect in the mouse.

The number and function analysis of T cell from mice immunized with MIF gene-modified tumor vaccine / 第二军医大学学报

Objective: To investigate the role of T cell in the antitumor immune responses induced by MIF gene-modified tumor vaccine. Methods: MIF gene was transferred into FBL3 erythroleukemia cel l by adenovirus carrier and a new type of tumor vaccine was prepared. The chang es of the number and the function of T cell in spleen and lymph node was observe d. Results: After the mice were immunized with MIF gene-m odified FBL3 vaccine, the number of lymphocyte in spleens and lymph nodes increa sed markedly and the specific CTL activities of splenocytes also increased great ly. FACS analysis showed that the CD3+,　CD4+,　CD8+ T cells and CD28 posi tive cells in draining lymph nodes of MIF-FBL3 group mice increased more marked ly than that of control groups. When the wild type FBL3 cells were injected into the mice immunized with MIF gene-modified FBL3 vaccine, the growth of tumors w ere obviously inhibited and the survival rate of the mice was increased. Conclusion: It is suggested that MIF gene-modified tumor vaccine can induce specific antitumor immune responses mediated by T cells and may be a candidate for gene therapy of tumor.

The biocharacterization of intrasplenic transplantation of gene modified mouse fetal liver cells / 中华消化杂志

Objective To develop a genetically modified fetal liver cells (FLC) based transplantation system that can release therapeutic levels of hematopoietic growth factors into the system circulation which can facilitate treatment of patient receiving cytokine therapy following chemotherapy. Method Examine adeno virus mediated gene transfer to isolated murine FLC and evaluate the biocharacterization of intrasplenic transplantation of gene modified murine FLC. Results Substantial transfection rate of 80%～85% were achieved at a ratio of 50 for 2 hr of exposure. Gene modified FLC (FLC GM) labeled with 111 In were injected into the allogenic mice, spleen, the %ID/g of liver was 20%～25% at 24 hr and 50%～55% at 48 hr after transplantation. In addition, serum concentration of GM CSF in mice with intrasplenic transplantation reached its maximum at 48 hr [(356 ?58 ) pg/ml]. Conclusion Intrasplenic transplantation of FLC GM can be predominantly localized in liver and spleen, and engraft rapidly and maintain normal function, which represent a critical step toward successfully accomplishing liver directed gene therapy.

The in vivo gene transfer efficacy and expression patterns by hepatic artery administration of recombinant adenovirus / 中华放射学杂志

Objective To investigate the gene transfer efficiency and lasted time in rat organs by hepatic artery injection with LacZ reporter gene recombinant adenoviruses. Methods Seven groups of rats were injected with Ad.LacZ (2?10 9 pfu/ml) and two groups of rats were injected with PBS 1 ml as control separately through gastra intestinal artery, and liver, spleen, lung, and kidney were gotten at 12 hrs, 18 hrs, 72 hrs, 7 day, 14 day, 21 day, and 28day, respectively. X gal staining was used to check up expression of LacZ gene. Results Expression of LacZ gene was detected in liver 12 hrs after injection, but none were done in spleen, lung, and kidney. Up to 21days, LacZ gene expressed in liver, but the gene expression lasted for only 14 days in spleen, lung, and kidney LacZ gene was not detected in the two control groups in all organs at 7 day. Conclusion When recombinant adenovirus was administrated through hepatic artery, the introduced gene expressed preferentially in liver. This result was the basis of intraarterial administration of cytokines gene to treat liver tumor.

Treatment of metastatic liver cancer in rat by hepatic artery injection of cytokine recombinant adenoviruses / 中华放射学杂志

Objective To investigate the therapeutic effects of TNF and IL 2 recombinant adenoviruses via intra arterial injection on metastatic liver cancer in rat model. Methods Recombinant adenoviruses harboring hTNF ? or hIL 2 gene were amplified in 293 cells and subjected to titration by the pathogenetic effects on 293 cell. The rats bearing metastatic liver cancer of Walker 256 breast carcinoma were randomly grouped and administered via gastra intestinal artery with hTNF ? recombinant adenoviruses alone, or hIL 2 recombinant adenoviruses alone, or at the dose of 1.0?10 9 pfu/rat. The therapeutic effects were observed including their survival time. Results The prepared recombinant adenoviruses of hTNF ? and hIL 2 were with the titers of 2.0?10 9 pfu/ml and 2.1?10 9 pfu/ml, respectively. 1.0 ?10 9 pfu hTNF was the proper dose. Administration of hTNF ? or hIL 2 recombinant adenoviruses via hepatic artery could extend the survival time of metastatic liver cancer bearing rats, with the better therapeutic effects achieved by combinatorial administration of these two adenoviruses. Conclusion Arterial administration of adenoviruses may be an effective approach to targeted immunogene therapy for cancer.

Myeloid-derived suppressor cells and tumor immune escape / 中国肿瘤生物治疗杂志

Myeloid-derived suppressor cells(MDSCs) are heterogeneous cells derived from myeloid progenitor cells and immature myeloid cells(IMCs) in bone marrow;they are the progenitors of dendritic cells(DCs),macrophages and granulocytes.MDSCs proliferate in the blood,spleen,and tumor tissues in tumor-bearing mice and in the peripheral blood and tumor tissues in patients with cancer.MDSCs prevent tumors from attacks by body immunosurveillance and promote tumors progression through inhibiting both innate and adaptive antitumor immunity by a variety of pathways;they are recruited to the peripheral tissues from bone marrow and exert their inhibitory effects on antitumor immunity after activation in peripheral tissues.Chronic inflammation-related cytokines produced by tumors play crucial roles in the recruitment and activation of MDSCs.Progress has been made in antitumor therapies targeting MDSCs.But it has only been 10 years since the discovery of MDSCs,and many questions remain to be answered through experimental and clinical investigations.This review focuses on progress in MDSCs and its subsets,the recruitment and activation of MDSCs,the mechanisms of MDSCs-mediated immunosurveillance and antitumor treatment targeting MDSCs.

Prokaryotic Expression, Purification and Characterization of Recombinant Human Interleukin-17 / 中国肿瘤生物治疗杂志

Objective: To select suitable conditions for prokaryotic expression and purification of rhIL-17. Methods: rhIL-17 was expressed in E. coli host under heat induction. After compared among the expression amounts in different media under different heat induction time, the most suitable conditions was selected. The target protein was present in the form of inclusion body. The precipitate of inclusion was obtained and purified after 6M guanidine solublization or 2% SDS solublization. Results: Either protocol could yield rhIL-17 with high purity and stable activity. The SDS solublization mehthod gives rise to much more higher productivity than the guanidine solublization method. Conclusion: rhIL-17 were expression in E. coli system and purified to homogenicity by SDS solublization methods with high productivity.

Roles of T Cells and Related Immunological Molecules in the Enhancement of Cancer CD Suicide Gene Therapy by Lymphotactin / 中国肿瘤生物治疗杂志

We previously showed that adenvirus-mediated lymphotactin (Ltn) gene transfer in vivo could improve the an-titumor efficacy of cytosine deaminase (CD) gene therapy significantly. In the precent study, we investigated the im-munological mechanisms involved in the enhanced antitumor efficacy. Upregulation of CD80 and CD54 on murine CT26 colon carcinoma cells was observed after combined transfection with adenovirus encoding CD (AdCD) and adenovirus encoding murine Ltn ( AdLtn) followed by administration of 5-PC in vitro. IL-2 and IFN-? level secreted by splenocytes increased significantly after the combination therapy. In vivo depletion analysis showed that both CD4~+ and CD8~+ T cells participated in the antitumor effect of the host with CD8~+ T cells being the main T cell subset responsible for the enhanced antitumor immune response. These data suggested that increased irnmunogenicity and efficient induction of antitumor immunity of the host might contribute to the enhanced antitumor effects of the combined Ltn and CD suicide therapy.

Generation of Dendritic Cells from Human Peripheral Blood / 中国肿瘤生物治疗杂志

Dendritic cells (DC) are antigen presenting cells (APC) that play critical roles in the initiation of T cell response and development of T cell-dependent antibodies in vivo. CD34_+ hematopoietic progenitor cells of bone marrow and peripheral blood can differentiate into DC when cultured with GM-CSF and TNF-? in vitro. In the present study, we cultured monocytes isolated from human peripheral blood with 100ng/ml hGM-CSF and 500U/ml hIL-4 for one week, and then found that a large number of DC with high purity were gengerated. DC expressed MHC I , MHC II and costimuladng moleculers highly on cell surface and cound stimulate proliferation of allogeneic T lymphocytes. Self serum or fetal calf serum are best for generation of DC. When hGM-CSF was used alone, the monocytes differentiated into macrophages but not to DC. TNF-? could induce further maturation of DC when added in late period of the culture. Generation of DC from human peripheral blood may facilitate further studies on DC and their clinical applications.

xperimental Study on Adenovirus-Mediated Suicide Gene Therapy for Human Hepatucellular Carcinoma Using Escherichia Coli Cytosine Deaminase / 中国肿瘤生物治疗杂志

To evaluate whether in vitro and in vivo transfer of E. Coli cytosine deaminase gene will confer sensitivity of a solid tumor to prodrug 5-fluorocytosine(5FC), we used an adenovirus vector(AdexCMV. CD) carrying the cytosine deaminase gene driven by the CMV promoter, infected SMMC-7721 or HepG2 cells hepatocellular carcinoma cells in vitro, and found AdexCMV. CD vector could effectively suppressed the growth of SMMC-7721 and HepG2 cells. When the two cells were infected with AdexAFP. CD vector in which the CD gene was driven by the AFP gene 5'-flanking region, only HepG2 cells were conferred sensitivity to 5FC. (Infection with AdexCMV.CD, when as few as 20% of cells transfected the CD gene, SMMC-7721 cells were associated with a bystander effect when combined with 5FC in cell mixing studies.) Consistent with these in vitro observations, AdexCMV. CD was directly injected into established subcutaneous SMMC-7721 tumors in nude mice receiving 5FC,there was a 60% reduction in tumor size at day 8, 70% reduction at day 24. Our results suggested that adenovirus-mediated tumor-specific gene transfer of CD gene and concomitant administration of 5 FC may have potential as a strategy for local control of tumor growth.

In vitro Biological Characteristic Changes and in vivo Tumorigenicity of mlL-4 Gene Modified G422 Cells by Adenovirus-Mediated Gene Transfer / 中国肿瘤生物治疗杂志

Murine interleukin-4 gene(mIL-4) was transfected into mice glioblastoma cell line G422 by recombinant adenovirus vector. We detected mRNA transcription of target gene in gene modified tumor cells(G422-mIL4). High level of mouse IL-4 could be detected in the culture supernatant of G422-mIL4. When inoculated subcuatneously, the tumor growth of G422-mIL4 was significantly inhibited as compared to wild type G422 and LacZ gene modified G422( G422-LacZ) . The period of survival of mice inoculated with G422-mlL4 was significantly prolongated(p