SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19.

Background & Aims: Liver enzyme abnormalities are common in patients with coronavirus disease 2019 (COVID-19). Whether or not severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to liver damage per se remains unknown. Herein, we reported the clinical characteristics and liver pathological manifestations of COVID-19 patients with liver enzyme abnormalities. Methods: We analyzed 156 patients diagnosed with COVID-19 from 2 designated centers in China and compared clinical features between patients with or without elevated aminotransferases. Postmortem liver biopsies were obtained from 2 cases who had elevated aminotransferases. We investigated the patterns of liver impairment by electron microscopy, immunohistochemistry, TUNEL assay and pathological studies. Results: Sixty-four out of 156 (41.0%) patients with COVID-19 had elevated aminotransferases. The median levels of alanine aminotransferase were 50 U/L vs. 19 U/L, respectively, aspartate aminotransferase were 45.5 U/L vs. 24 U/L, respectively in abnormal and normal aminotransferase groups. Liver enzyme abnormalities were associated with disease severity, as well as a series of laboratory tests including higher alveolar-arterial oxygen partial pressure difference, higher gamma-glutamyltransferase, lower albumin, decreased CD4+ T cells and B lymphocytes. Ultrastructural examination identified typical coronavirus particles, characterized by spike structures, in the cytoplasm of hepatocytes in 2 COVID-19 cases. SARS-CoV-2-infected hepatocytes displayed conspicuous mitochondrial swelling, endoplasmic reticulum dilatation and glycogen granule decrease. Histologically, massive hepatic apoptosis and some binuclear hepatocytes were observed. Taken together, both ultrastructural and histological evidence indicated a typical lesion of viral infection. Immunohistochemical results showed scarce CD4+ and CD8+ lymphocytes. No obvious eosinophil infiltration, cholestasis, fibrin deposition, granuloma, massive central necrosis, or interface hepatitis were observed. Conclusions: SARS-CoV-2 infection in the liver directly contributes to hepatic impairment in patients with COVID-19. Hence, a surveillance of viral clearance in liver and long-term outcome of COVID-19 is required. Lay summary: Liver enzyme abnormalities are common in patients with coronavirus disease 2019 (COVID-19). We reported the clinical characteristics and liver pathological manifestations of COVID-19 patients with elevated liver enzymes. Our findings suggested that SARS-CoV-2 infection of the liver is a crucial factor contributing to hepatic impairment in patients with COVID-19.

Effect of TIPE1 on Immune Function of Dendritic Cells and Its Signaling Pathway in Septic Mice.

Dendritic cell (DC) dysfunction plays a pivotal role in sepsis-induced immunosuppression. Tumor necrosis factor α (TNF-α)-induced protein 8 like-1 (TIPE1), a new member of the tumor necrosis factor α-induced protein 8 family, may be related to cell death. The aim of the present study was to elucidate the effect of TIPE1 on the immune function of DCs and its regulatory mechanism via PD-L1/PD-1 signaling in mice. Sepsis was induced in adult C57BL/6 male mice via cecal ligation and puncture. In vitro, we found that expression of CD80, CD86, and major histocompatibility complex class II in DCs and levels of cytokines, including tumor necrosis factor α and interleukin 12p40, were elevated; similarly, T-cell proliferation and differentiation were promoted when the gene expressing TIPE1 was silenced. Next, we examined the in vivo role of TIPE1 in a cecal ligation and puncture animal model system. Flow cytometry of the immune functional status in DCs revealed negative regulation of TIPE1 on DC maturation, as well as activation. Moreover, changes in PD-L1/PD-1 levels confirmed the negative effect of TIPE1 in DCs. Collectively, we report that TIPE1 might exert negative regulation in sepsis, at least in part by inhibiting DC maturation and subsequent T-cell-mediated immunity via PD-L1/PD-1 signaling.

Adoptive transfer of bone marrow-derived dendritic cells decreases inhibitory and regulatory T-cell differentiation and improves survival in murine polymicrobial sepsis.

A decrease in the number of dendritic cells (DCs) is a major cause of post-sepsis immunosuppression and opportunistic infection and is closely associated with poor prognosis. Increasing the number of DCs to replenish their numbers post sepsis can improve the condition. This therapeutic approach could improve recovery after sepsis. Eighty C57BL/6 mice were subjected to sham or caecal ligation and puncture (CLP) surgery. Mice were divided into four groups: (i) Sham + vehicle, (ii) Sham + DC, (iii) CLP + vehicle, and (iv) CLP + DC. Bone-marrow-derived DCs (BMDCs) were administered at 6, 12 and 24 hr after surgery. After 3 days, we assessed serum indices of organ function (alanine aminotransferase, aspartate aminotransferase, creatinine, amylase and lipase), organ tissue histopathology (haematoxylin and eosin staining), cytokine [interferon-γ (IFN-γ), tumour necrosis factor-α, interleukin-12p70 (IL-12p70), IL-6 and IL-10] levels in the serum, programmed death-1 (PD-1) expression on T cells, regulatory T-cell differentiation in the spleen, and the survival rate (monitored for 7 days). BMDC transfer resulted in the following changes: a significant reduction in damage to the liver, kidney and pancreas in the CLP-septic mice as well as in the pathological changes seen in the liver, lung, small intestine and pancreas; significantly elevated levels of the T helper type 1 (Th1) cytokines IFN-γ and IL-12p70 in the serum; decreased levels of the Th2 cytokines IL-6 and IL-10 in the serum; reduced expression of PD-1 molecules on CD4(+) T cells; reduced the proliferation and differentiation of splenic suppressor T cells and CD4(+)  CD25(+)  Foxp3(+) regulatory T cells, and a significant increase in the survival rate of the septic animals. These results show that administration of BMDCs may have modulated the differentiation and immune function of T cells and contributed to alleviate immunosuppression, hence reducing organ damage and mortality post sepsis. Hence, the immunoregulatory effect of BMDC treatment has potential for the treatment of sepsis.

RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells.

L929 fibroblastoma cells (L929-A) and L929 fibrosarcoma cells (L929-N) are different cell lines that are commonly used to study the cytotoxicity of tumor necrosis factor alpha (TNFα). TNFα has been reported to induce necrosis in both of these cell lines. However, comparing the TNFα-induced cell death in these two cell lines, we found that, unlike the L929-N cells that show typical RIP3-dependent necrosis, TNFα-induced cell death in L929-A cells is pan-caspase inhibitor Z-VAD-FMK (Z-VAD)-sensitive, which does not depend on RIP3. We also confirmed that the cell death signal in the L929-A cells was initiated through cytosol-preassembled ripoptosome and that the knockdown of either Caspase-8 or RIP1 protein blocked cell death. Compared with the L929-N cells, the L929-A cell line had lower levels of constitutive and inducible TNFα autocrine production, and the pan-caspase inhibitors Z-VAD or Q-VD did not kill the L929-A cells as they affect the L929-N cells. Moreover, the L929-A cells expressed less RIP3 protein than the L929-N cells; therefore, TNFα failed to induce RIP3-dependent necroptosis. In addition, the ripoptosome-mediated cell death signal was transduced to the mitochondria through Caspase-8-mediated and RIP1 kinase activity-dependent Bid cleavage. The RIP1 kinase inhibitor Necrostatin-1 (Nec-1) or Caspase-8 knockdown completely blocked Bid cleavage, and the knockdown of Bid or Bax/Bak prevented TNFα-induced cell death in the L929-A cells. Although the activation of Bax/Bak decreased the mitochondrial membrane potential, the levels of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-C) and Smac, declined, and western blotting and immunofluorescence staining analysis did not determine whether these proteins were redistributed to the cytosol. In addition, the mitochondrial outer membrane protein Tom20 was also reduced, indicating that the reduced mitochondria proteins may be induced by the reduced mitochondria numbers. No efficient cyt-C release was observed; therefore, the limited activation and cleavage of downstream caspases, including Caspase-9, Caspase-7, Caspase-6 and Caspase-3, was insufficient to kill the cells. The Caspase-9, Caspase-6 and Caspase-3/7 inhibitors or Caspase-9 and -3 knockdown also failed to block cell death, and the overexpression of Bcl-2 also did not abrogate cell death. Moreover, the dead cells showed necrotic-like but not apoptotic characteristics under transmission electronmicroscopy, and these features were significantly different from mitochondrial apoptosis, indicating that the effector caspases were not the executioners of cell death. These new discoveries show that TNFα-induced cell death in L929-A cells is different than typical RIP3-dependent necrosis and Caspase-8/Caspase-3-mediated apoptosis. These results highlight that caution is necessary when using different L929 cells as a model to investigate TNFα-induced cell death.

Upregulating nonneuronal cholinergic activity decreases TNF release from lipopolysaccharide-stimulated RAW264.7 cells.

Nonneuronal cholinergic system plays a primary role in maintaining homeostasis. It has been proved that endogenous neuronal acetylcholine (ACh) could play an anti-inflammatory role, and exogenous cholinergic agonists could weaken macrophages inflammatory response to lipopolysaccharide (LPS) stimulation through activation of α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR). We assumed that nonneuronal cholinergic system existing in macrophages could modulate inflammation through autocrine ACh and expressed α7nAChR on the cells. Therefore, we explored whether LPS continuous stimulation could upregulate the nonneuronal cholinergic activity in macrophages and whether increasing autocrine ACh could decrease TNF release from the macrophages. The results showed that, in RAW264.7 cells incubated with LPS for 20 hours, the secretion of ACh was significantly decreased at 4 h and then gradually increased, accompanied with the enhancement of α7nAChR expression level. The release of TNF was greatly increased from RAW264.7 cells at 4 h and 8 h exposure to LPS; however, it was suppressed at 20 h. Upregulating choline acetyltransferase (ChAT) expression through ChAT gene transfection could enhance ACh secretion and reduce TNF release from the infected RAW264. 7cells. The results indicated that LPS stimulation could modulate the activity of nonneuronal cholinergic system of RAW264.7 cells. Enhancing autocrine ACh production could attenuate TNF release from RAW264.7 cells.

N-acetylcysteine administration is associated with reduced activation of NF-kB and preserves lung dendritic cells function in a zymosan-induced generalized inflammation model.

PURPOSE: In severe sepsis, functional impairment and decreased numbers of dendritic cells (DCs) are essential reasons for immune function paralysis, secondary organ infection, and organ failure. We investigated the effects of N-acetylcysteine (NAC) administration on protecting lung DCs function in a zymosan-induced generalized inflammation (ZIGI) model. METHODS: ZIGI was initiated in 80 Balb/c mice by intraperitoneal injection of zymosan (ZYM; 900 mg/kg). Mice were divided into 4 groups: (1) SHAM+Vehicle; (2) SHAM+NAC; (3) ZYM+Vehicle; and (4) ZYM+NAC. NAC (100 mg/kg) was administered at different time after ZYM injection. After 48 h, we assessed: lung tissue pathological changes; arterial blood gas values; purified lung DCs surface expressions of MHC-II/I-A(d) and co-stimulatory molecules CD80, CD83, and CD86; lung DCs mRNA levels of chemokine receptors CCR5 and CCR7; lung DCs apoptosis; lung DCs ultrastructure by transmission electron microscopy; lung DCs NF-kB transcription factor activity; and LPS-stimulated lung DCs in vitro production of IL-12 and IL-10 were examined. RESULTS: NAC treatment resulted in: significant improvements in ZYM-induced lung tissue damage and impaired lung function; inhibited lung DCs ZYM-induced increased expression of MHC-II/I-A(d), CD83, and CD86, but not CD80; reduced lung DCs ZYM-induced CCR5 and CCR7 mRNA levels; suppressed ZYM-induced lung DCs apoptosis; ameliorated ZYM-induced lung DCs ultrastructural abnormalities; inhibited ZYM-induced lung DCs NF-κB activity; and enhanced lung DCs production of IL-12 and inhibited their production of IL-10. CONCLUSIONS: Repeated injections of NAC during the early stage of severe sepsis effectively inhibited lung DCs activation and their apoptosis, which could preserve DCs function.

Microvascular damage is involved in the pathogenesis of heroin induced spongiform leukoencephalopathy.

OBJECTIVE: To investigate whether microvascular damage is involved in the pathogenesis of heroin induced spongiform leukoencephalopathy (HSLE). METHODS: The brain tissues were collected from 4 HSLE patients and 5 controls and then fixed in 4% paraformaldehyde. The frontal lobe, corpus callosum and cerebellum were separated. The expressions of myelin base protein (MBP) and CD34 were detected by immunohistochemistry. TUNEL staining was applied to detect cell apoptosis. The correlation between microvascular changes and pathological vacuoles was evaluated. RESULTS: No obvious abnormalities were found in the brain of controls. Immunohistochemistry for MBP showed the collapse and fracture of myelin sheath and vacuole formation in the subcortical white matter, corpus callosum, and cerebellar white matter of HSLE patients. TUNEL staining showed the number of apoptotic cells in the cerebellar white matter and corpus callosum of HSLE patients was significantly higher than that in controls (F = 389.451, P < 0.001). Masson's trichrome staining revealed vacuolar degeneration in the cerebral white matter of HSLE patients, and the vacuoles were distributed around the microvessels. Immunohistochemistry revealed CD34 positive cells were seldom found besides the vessels in the cerebellar white matter and corpus callosum of HSLE patients, but a variety of CD34 positive cells was found in the vascular wall of controls (F = 838.500, P < 0.001). CONCLUSION: Apoptosis of oligodendrocytes may be related to the HSLE. Cerebral vascular injury and microcirculation dysfunction are involved in the pathogenesis of HSLE. The interrelation between apoptosis of oligodendrocytes and the microvascular damage are required to be studied in future investigations.

Differential expression of ERCC-1 in the primary tumors and metastatic lymph nodes of patients with non-small cell lung cancer adenocarcinoma.

About 80 % of lung cancers are carcinomas that are classified histologically as non-small-cell lung carcinoma (NSCLC) and targeted chemotherapy of this cancer is currently based on sensitivity of the primary tumor to specific drugs. The purpose of this study was to compare the levels of four serum markers of cancer and the levels of six molecular markers which are possibly associated with drug selection in the primary tumors and metastatic lymph nodes of 39 consecutive NSCLC patients who were admitted to a single institution in China. Serum markers of cancer (neuron-specific enolase, carcinoembryonic antigen (CEA), cancer antigen 125, cytokeratin fragment 21-1) were measured by an automated electrochemiluminescence system and molecular markers (multidrug resistance protein 1, LDL receptor-related protein, ribonucleotide reductase M1, epidermal growth factor receptor, excision repair cross-complementing gene 1, and breast cancer 1) were measured by immunohistochemistry of the primary tumors and metastatic lymph nodes. The results indicate that the serum level of CEA was higher in NSCLC patients with adenocarcinoma relative to those with squamous cell carcinoma, but no significant differences in the other serum markers. Expression of excision repair cross-complementing gene 1 was significantly different in the primary tumors and metastatic sites of NSCLC patients with adenocarcinoma, but there were no other significant differences. This study provides an initial step toward the development of individualized chemotherapy of NSCLC based on measurement of molecular markers in the primary tumors and metastatic lymph nodes.

[Expression of ubiquitin in the spleen and its influence on the dendritic cell immunomodulation function in multiple organ dysfunction syndrome in mice].

OBJECTIVE: To explore the expression change in ubiquitin (Ub) in the spleen and its significance in multiple organ dysfunction syndrome (MODS) in mice, and to study the effects of ubiquitination of major histocompatibility complex II (MHCII) on the activity and immunomodulation function of splenic dendritic cell (DC). METHODS: Two hundred and ten mice were divided into the normal control group (n = 30) and MODS group (n = 180) according to the method of random digital table, and MODS model was replicated by intraperitoneal injection of zymosan. The MODS group mice were further divided evenly into 6, 12, 24, 48-hour and 5-7-day and 10-12-day groups. Ub protein and expression of CD11c⁺DC and MHCII molecule I-A(b) were examined using immunohistochemistry and immunofluorescence methods. Ub mRNA expression in the spleen was measured by real-time quantitative reverse transcription--polymerase chain reaction (RT-PCR). RESULTS: (1)Immunohistochemistry results showed: the number of Ub positive cells in the spleen increased significantly at 6 hours in MODS group compared with that of the normal control group, and it reached the peak at 24 hours [(16.83 ± 0.38)% vs. (8.60 ± 0.86)%, P < 0.05] and then decreased gradually. At 10-12 days, the number of Ub positive cells decreased significantly compared with that of the normal control group [(4.66 ± 0.34)% vs. (8.60 ± 0.86)%, P < 0.05]. (2)RT-PCR results displayed: compared with normal control group, Ub mRNA expression in spleen increased at 6 hours in MODS group, and it reached the peak at 24 hours (2.17 ± 0.20 vs. 1.00 ± 0.00, P < 0.01). Then, it decreased gradually. At 10-12 days, Ub mRNA decreased significantly as compared with that of normal control group (0.72 ± 0.08 vs. 1.00 ± 0.00, P < 0.05). (3)Immunofluorescence results displayed: compared with normal control group, CD11c⁺DC increased significantly at 6 hours in MODS group and reached the peak at 24 hours [(7.55 ± 0.04)% vs. (2.08 ± 0.13)%, P < 0.05], and then it decreased gradually. At 10-12 days, it was close to that of the normal control group [(2.28 ± 0.06)% vs. (2.08 ± 0.13)%, P>0.05]. Compared with the normal control group, I-A(b) positive cells in the spleen was significantly increased at 6 hours in MODS group [(10.90 ± 1.40)% vs. (5.78 ± 0.47)%, P < 0.01], but it decreased at 24 hours [(3.32 ± 0.91)% vs. (5.78 ± 0.47)%, P < 0.05]. I-A(b) positive cells were restored to the normal level at 48 hours and 5--7 days, and decreased significantly again at 10-12 days [(2.20 ± 0.97)% vs. (5.78 ± 0.47)%, P < 0.05]. The number of Ub positive cells correlated positively to the expression of I-A(b) and the CD11c [r1 = 0.899, r2=0.987, both P < 0.05]. CONCLUSIONS: Ub might influence the maturation and activation of DC via ubiquitination of the MHCII molecule on DC, thereby influencing the immune response at different stages of MODS. The result might provide a new way to recognize immune response and also a new monitoring index for immune response regulation.

[Advanced dermatofibrosarcoma protuberans treated with imatinib mesylate].

OBJECTIVE: To evaluate the efficacy, side effects and toxicity of imatinib mesylate in the treatment of patients with locally advanced and/or metastatic dermatofibrosarcoma protuberans (DFSP). METHODS: Twenty-four cases of advanced DFSP diagnosed by pathology and treated in our hospital from Nov. 2004 to Oct. 2009 were included in this study. The patients were treated with imatinib mesylate (dosage: 400 mg, po, qd) and carefully observed for treatment efficacy, side effects and survival time. There were 2 patients taking the drug as second line therapy, and other 22 patients as third or more than third line therapy. RESULTS: The 24 patients were evaluable for the efficacy. There were 8 patients (33.3%) with CR, 10 pts (41.7%) PR, 2 patients (8.3%) SD, and 4 patients (16.7%) PD. The disease control rate (DCR = CR+PR+SD) was 83.3%. The median response time in 18 cases with CR and PR was 5.6 months. The median survival time in 20 cases with disease control was 30 months, however, that in nonresponse (PD) cases was only 10 months. Side reactions related to imatinib mesylate included nausea and vomiting (20.8%), neutropenia (12.5%), and edema (8.3%). CONCLUSIONS: Our results are consistent with previous reports in the literature. Imatinib is a safe and effective moleucular target drug used for Chinese. Only mild adverse reactions occur in the treated patients. It is worth using imatinib in the treatment of advanced DFSP patients.

[The study of follow up of percutaneous microwave ablation for uterine fibroids treatment].

OBJECTIVE: To study the prognosis of fibroid after ultrasound-guidance percutaneous microwave ablation (PMAUF). METHODS: From Mar. 2007 to Jul.2010 forty uterine fibroids in forty patients with symptoms which were diagnosed in our hospital accepted PMAUF. One day after treatment blood supply within the fibroid was evaluated, by enhanced MRI. The size of fibroid was measured by ultrasonography in one year with 3 months interval, the monthly menstrual of patient was followed and the mass discharge through vagina were collected for pathological examination. RESULTS: Among the 40 fibroid nodules, 22 are intramural fibroids, 15 subserosal fibroids and 3 submucosal fibroids. The baseline mean diameter of the fibroids ranged from (3.7 to 9.0) cm, with an average of (6.4 ± 1.5) cm. The mean volume ranged from (14.6 - 341.1) cm(3), with an average of (140.1 ± 87.4) cm(3). Three months after treatment, the anechoic zone was observed within the ablated nodules and disappeared in six to seven months. the echo of ablated zone keep higher than the surrounding tissue, whereas the size of fibroid shrank significantly. Meat tissue was discharged from vagina in 8 patients (2 cases of submucosal fibroids, 6 cases intramural fibroids) in the period of menstrual in 1 - 8 months after ablation. The largest dimension of the discharge was 2.1 cm. Pathological examination confirmed the discharges as necrotic fibroid. The patients with subserosal fibroid had no discharge of necrotic tissue. The fibroid shrink rate was higher in the patients with vaginal discharges than that without vaginal discharges (P < 0.01). CONCLUSIONS: After PMAUF necrotic tissue of submucosal and intramural fibroids can be discharged through vagina, liquefaction in the center of subserosal and intramural fibroids may occur and be absorbed gradually, that may be the main reasons for fibroids reduced significantly or disappearance.

[Changes in peripheral dendritic cells in serious burn patients and its clinical significance].

OBJECTIVE: To explore the changes in peripheral dendritic cells (DCs) in serious burn patients and its relationship with the burn severity and pathogenesis of sepsis. METHODS: Twenty-two serious burn patients were divided into the burn group (n = 10) and the burn sepsis group (n = 12) according to diagnostic criteria of sepsis, they were stratified according to the total burn surface area (TBSA), into the TBSA I group (TBSA 30%-50%, n = 14) and the TBSA II group (TBSA 51%-80%, n = 8). Peripheral blood of all patients was collected on 1, 3, 7 ,14 ,20 day after burn. The number of two subtypes of peripheral DC i. e myeloid dendritic cells [mDC, Lineage1(-)HLA-DR(+)CD11c(+)] and plasmacytoid dendritic cells [pDC, Lineage1(-)HLA-DR(+)CD123(+)] were quantified by flow cytometer. Ten healthy volunteers served as normal controls at the same time. RESULTS: In the healthy control group, mDC in the peripheral blood was (0.450 ± 0.150)% and pDC was (0.241 ± 0.084)%. Compared with the healthy control group, in the burn group both mDC [(0.257 ± 0.116)%, (0.274 ± 0.086)%, (0.317 ± 0.056)%] and pDC [(0.122 ± 0.058)%, (0.165 ± 0.051)%, (0.177 ± 0.024)%] decreased significantly on 1, 3, 7 day after burn (all P < 0.05), and the number returned to the normal level on 14 day and 20 day. Compared with the burn group, the number of mDC [(0.230 ± 0.090)%] and pDC [(0.114 ± 0.071)%] in patients of the burn sepsis group were significantly lower (both P < 0.05) on 1 day after burn. Both cells [mDC (0.246 ± 0.076)% vs. (0.412 ± 0.097)% and pDC (0.097 ± 0.032)% vs. (0.203 ± 0.039)%] were still significantly lower (both P < 0.05) on 20 day. Both mDC [(0.266 ± 0.062)%, (0.289 ± 0.071)%, (0.351 ± 0.054)%] and pDC [(0.131 ± 0.025)%, (0.163 ± 0.037)%, (0.178 ± 0.038)%] in the patients in the TBSA I group decreased significantly on 1, 3, 7 day after burn as compared with those of the healthy control group(all P < 0.05), and they returned to the normal level on 14 day and 20 day. Compared with the TBSA I group, mDC [(0.227 ± 0.070)%] and pDC [(0.112 ± 0.047)%] in patients of the TBSA II group decreased significantly(both P < 0.05)on 1 day after burn, and both cells [mDC (0.297 ± 0.072)% vs. (0.423 ± 0.046)% and pDC (0.107 ± 0.061)% vs.(0.197 ± 0.042)%] were still significantly lower (both P < 0.05) on 20 day. CONCLUSION: Both the number of mDC and pDC decrease in peripheral blood in early stage in serious burn patients, and those who have more serious burn have lower number of mDC or pDC. Deficiency in mDCs and pDC subsets may contribute to immunosuppression in burn victims, and those who suffered obvious loss of mDC and pDC are susceptible to sepsis following severe burn. It indicates that the percentage of mDC and pDC can be a predictive index of sepsis after burn.

The circRNA_102911/miR-129-5p/SOX6 axis is involved with T lymphocyte immune function in elderly patients with laparoscopic left hepatectomy for hepatolithiasis.

The aim of the present study was to investigate the impact of laparoscopic left hepatectomy (LLH) for hepatolithiasis on the T lymphocyte immune changes of elderly patients and to analyze underlying mechanisms of action behind these changes. A total of 164 patients who underwent LLH due to left-sided hepatolithiasis were recruited. In terms of T lymphocyte immune changes, it was found that firstly, the basic quantity of peripheral lymphocytes in the elderly group was significantly lower than that in a younger preoperative group. Secondly, after surgical trauma, the immune function of T lymphocytes had a significant decline and lasted longer when compared with younger patients, which was reflected by the perioperative changes in the T lymphocyte proliferative ability, levels of IL-2 secreted by T lymphocytes and the percentage of CD3+/CD4+ T lymphocytes in the peripheral blood. Circular RNA (circRNA) 102911 (102911) was upregulated and microRNA (miR)-129-5p was downregulated in CD3+/CD4+ T lymphocytes from elderly patients with LLH for hepatolithiasis. Furthermore, the overexpression of 102911 inhibited the proliferation of CD3+/CD4+ T lymphocytes as well as promoting cell apoptosis, with the opposite effects being observed on knockdown of 102911. miR-129-5p is involved in the proliferation and apoptosis of CD3+/CD4+ T lymphocytes and may be a promising target of 102911. Moreover, SOX6 is a downstream molecule of miR-129-5p. Immune function and number of T lymphocytes decreased significantly after surgical trauma compared to younger patients, and this decline lasted longer in older patients treated with LLH for hepatolithiasis. The 102911/miR-129-5p/SOX6 axis was found to be involved in T lymphocytes immune function, which provided a novel insight for the treatment of elderly patients with hepatolithiasis.

Clinical and pathological investigation of patients with severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a pandemic. This study addresses the clinical and immunopathological characteristics of severe COVID-19. METHODS: Sixty-nine patients with COVID-19 were classified into severe and nonsevere groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from 2 deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations. RESULTS: Circulating cytokines, including IL-8, IL-6, TNF-α, IP10, MCP1, and RANTES, were significantly elevated in patients with severe COVID-19. Dynamic IL-6 and IL-8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II and type I pneumocytes and endothelial cells, leading to severe lung damage through cell pyroptosis and apoptosis. In severe cases, lymphopenia, neutrophilia, depletion of CD4+ and CD8+ T lymphocytes, and massive macrophage and neutrophil infiltrates were observed in both blood and lung tissues. CONCLUSIONS: A panel of circulating cytokines could be used to predict disease deterioration and inform clinical interventions. Severe pulmonary damage was predominantly attributed to both cytopathy caused by SARS-CoV-2 and immunopathologic damage. Strategies that prohibit pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of patients with severe COVID-19.

Relationship between HMGB1 content and MHC-II expression in circulating monocytes and spleen of mice challenged with zymosan.

OBJECTIVE: To observe the regularity of change in high mobility group protein box 1 (HMGB1) content in serum and spleen of mice with multiple organ dysfunction syndrome (MODS), to analyze the correlation between HMGB1 content and major histocompatibility complex (MHC)-II---I-A(b) expression on monocytes in blood and spleen, and to explore the effect of HMGB1 on immune function of circulating monocytes and splenocytes. METHODS: One hundred 8-week-old male 57BL/6 mice were randomly divided into normal group and experimental group subdivided into 8 subgroups: 3, 8, 12 hours, 1, 2, 3, 5-7 days and 10-12 days post zymosan injection (PZI). MODS model was replicated by injecting zymosan into the peritoneal cavity. At each time point, blood and spleen were collected to detect HMGB1 content and the rate of I-A(b) positive monocytes. RESULTS: In normal and PZI 3-hour, 8-hour mice, serum HMGB1 was not detected, but it significantly increased at PZI 12 hours. In spleen of normal mice, there was low level of HMGB1 expression. In zymosan-treated mice, HMGB1 started to rise in spleen at PZI 3 hours. Subsequently, HMGB1 content in both serum and spleen significantly increased, and it reached the peak level in 1-2 days, decreased in 5 days, and then increased in 10-12 days. The number of I-A(b) positive monocytes in circulating blood and spleen decreased at 1-2 days (t equal to 9.589, 4.432, P <0.01) and 10-12 days following the challenge, forming a two trough like decrease, just corresponding with two-peak increase of HMGB1. However, at 3 hours after zymosan challenge, I-A(b) expression on circulating monocytes was downregulated (t =5.977, P less than 0.01), while that in spleen upregulated (t equal to 4.814, P less than 0.01). CONCLUSION: In mice with MODS, up-regulated HMGB1 expression can regulate I-A(b)expression on monocytes to depress their ability of presenting antigen, which results in immune disturbance contributing development of MODS.

[Effect of change in high mobility group protein box 1 expression on activity of immunocytes in spleen of mice with multiple organ dysfunction syndrome].

OBJECTIVE: To explore the regularity of high mobility group protein box 1 (HMGB1) expression and major histocompatibility complex II I-A(b) in spleen of mice with multiple organ dysfunction syndrome (MODS) , and its effect on the activity of immunocytes and relationship with pathogenesis of MODS. METHODS: MODS model was replicated by injecting zymosan into abdominal cavity of mice. The mice were randomly divided into normal control group, and 3, 8, 12 hours, and 1, 2, 3, 5, 10-12 days after injection groups. The expression levels of HMGB1, I-A(b) and apoptosis rate in the spleen were detected. RESULTS: There was a little HMGB1 expression in the spleen of control mice. After zymosan administration, HMGB1 expression was increased, it reached the highest level on 1-2 days (P<0.01), decreased on day 5, then elevated on 10-12 days (P<0.05). Change in HMGB1 mRNA expression was in accordance with that of the protein. At 8 hours following injury, the I-A(b) expressed on the splenocytes was enhanced similar to that of HMGB1, and then it reversed. The occurrence of splenocyte apoptosis was also consistent with change in HMGB1 content in the spleen. CONCLUSION: The increased apoptosis of splenocytes in mice with MODS is closely related with up-regulation of HMGB1 expression, which inhibits the expression level of I-A(b) on antigen presenting cells, thus weakens the capability of immune responses and affected the development of MODS.

MicroRNA­140 suppresses Helicobacter pylori­positive gastric cancer growth by enhancing the antitumor immune response.

Immune checkpoint blockade is a promising therapeutic strategy against various human malignancies. MicroRNAs (miRNAs/miRs) regulate gene expression, by repressing mRNA translation or promoting its degradation. The aim of the current study was to investigate the role and molecular mechanisms of miR­140 in Helicobacter pylori (Hp)­associated gastric cancer, and to examine its relationship with immune function in gastric cancer. Gastritis tissue samples from gastritis patients, and gastric cancer tissue samples from gastric cancer patients were collected for miR­140 expression detection. miR­140 expression was detected using reverse transcription­quantitative polymerase chain reaction, and protein expression was measured by western blotting. TargetScan and dual luciferase reporter assays were used to reveal the association between miR­140 and programmed cell death­ligand 1 (PD­L1). BGC823 cell proliferation was detected by MTT assays. Ex vivo immune analysis by flow cytometry and ELISA were used to analyze immune function. It was demonstrated that miR­140 expression was significantly reduced in Hp­positive gastric cancer. PD­L1 was confirmed as a direct target of miR­140 in gastric cancer cells. In addition, PD­L1 expression was significantly increased in Hp­positive gastric cancer. Overexpression of miR­140 significantly suppressed gastric cancer cell proliferation through regulating PD­L1 expression. In vivo experiments also revealed that miR­140 markedly repressed tumor growth in the C57BL/6 mice. Furthermore, it was determined that the tumor­suppressive role of miR­140 in gastric cancer was associated with increased cytotoxic CD8+ T cell and reduced myeloid­derived suppressive and regulatory T cell infiltration. miR­140 significantly prevented mammalian target of rapamycin signaling in gastric cancer cells. Notably, these miR­140 overexpression­induced alterations were inhibited by PD­L1 plasmid. These findings indicated that miR­140 exerted an anti­gastric cancer effect by targeting immune checkpoint molecule PD­L1. Thus, miR­140 may be a promising and novel immunotherapeutic target for gastric cancer treatment.

[Roles of bcl-2/bax expression and oligodendrocyte apoptosis in the pathogenesis of heroin-induced spongiform leucoencephalopathy].

OBJECTIVE: To investigate the role of oligodendrocyte apoptosis under the regulation of the bcl-2/bax protein expression in brain white matter in the pathogenesis of heroin-induced spongiform leucoencephalopathy (HSLE). METHODS: Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE cases and 5 normal controls and underwent light microscopy and electron microscopy. Immunocytochemistry was used to detect the expression of myelin basic protein (MBP), caspase-3, bcl-2 protein, and bax protein. RESULTS: Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum and corpus callosum of the HSLE cases, most severely in the cerebellum. The levels of caspase-3 and bax expression of the HSLE group were significantly higher than those of the control group (both P <0.05) , however, the bcl-2 level of the HSLE group was no significantly different from that of the control group (P > 0.05). CONCLUSION: Widespread demyelination in the white matter is a prevailed pathological change of HSLE. Oligodendrocyte apoptosis under induced by the decrease of bcl-2/bax ratio may contribute to the pathogenesis.

[Roles of cyclooxygenase 2/2', 3'-cyclic nucleotide3' phosphohydrolase in the oligodendrocyte apoptosis in heroin-induced spongiform leucoencephalopathy].

OBJECTIVE: To investigate the regulation of cyclooxygenase (Cox)-2/2', 3'-cyclic nucleotide3' phosphohydrolase (CNPase) on the oligodendrocyte apoptosis in the pathogenesis of the heroin-induced spongiform leucoencephalopathy (HSLE). METHODS: Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE patients and 5 patients who died of diseases other than cerebral diseases (controls) and underwent light microscopy and electron microscopy. Immunocytochemistry was carried out to detect the expression of myelin basic protein (MBP), caspase-3, COX-2, and CNPase protein. Apoptosis was examined by TUNEL staining. RESULTS: Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum, and corpus callosum of the HSLE cases, most severely in cerebellum. In he HSLE group, the levels of caspase-3 and COX-2 expression were significantly higher, and the level of CNPase was significantly lower than those of the control group (all P < 0.05). CONCLUSION: Widespread demyelination in the white matter is a prevailing pathological change of HSLE. Oligodendrocyte apoptosis is one of the causes of HSLE. The upregulation of COX-2 and downregulation of CNPase may contribute to the pathogenesis.

[Flt3 ligand in recovering the function of the splenic dendritic cells and the immune system in mice with multiple organ dysfunction syndrome].

OBJECTIVE: To study the immunoprotective effect of fms-like tyrosine kinase receptor 3 ligand (FL) in multiple organ dysfunction syndrome(MODS) in mice. METHODS: Ninety mice were randomized into three groups: normal, MODS and MODS+FL (each n=30). The MODS model of mice was reproduced. Splenic dendritic cell (DC), I-Ab of peripheral blood mononuclear cells (PBMCs) and T cell subpopulation of peripheral blood of mice were analyzed by flow cytometry. The histomorphology of spleen and DC was studied by light microscope and electron microscope. RESULTS: In MODS group, the number of splenic immature DC increased (P<0.05), the number of CD4+ T lymphocytes reduced, while the number of CD8(+)T lymphocytes increased, thus CD4/CD8 ratio was reduced (P<0.05). White pulp of the spleen dispersed, with decrease in splenic bodies. There was apoptosis of DC. The expression of I-Ab of the mononuclear cells decreased remarkably (P<0.01). The mortality of mice before treatment was 18%. In FL treatment groups, the number of splenic mature DC was markedly increased (P<0.05). The number of CD4+ T lymphocytes and CD4/CD8 ratio was also markedly elevated. The expression of I-Ab of the mononuclear cells became normal. The pathological changes in spleen were alleviated. The mortality rate was significantly lowered (7%). CONCLUSION: FL can not only stimulate the recovery the DC activity, but also improve the immune function in the late phases of MODS.