Pulmonary tumor thrombotic microangiopathy: Two case reports and literature review.

RATIONALE: Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare but serious complication in patients with malignancy; its main manifestation includes acute pulmonary hypertension with severe respiratory distress. More than 200 cases have been reported since it was first identified in 1990. PTTM accounts for approximately 0.9% to 3.3% of deaths due to malignancy, but only a minority of patients are diagnosed ante-mortem, with most patients having a definitive diagnosis after autopsy. PATIENT CONCERNS: Two middle-aged women both died within a short period of time due to progressive dyspnea and severe pulmonary hypertension. DIAGNOSES: One patient was definitively confirmed as a gastrointestinal malignant tumor by liver puncture biopsy pathology. Ultimately, the clinical diagnosis was pulmonary tumor thrombotic microangiopathy. INTERVENTIONS: The patient was treated symptomatically with oxygen, diuresis, and anticoagulation, while a liver puncture was perfected to clarify the cause. OUTCOMES: Two cases of middle-aged female patients with rapidly progressive pulmonary hypertension and respiratory failure resulted in death with malignant neoplasm. LESSONS: PTTM has a rapid onset and a high morbidity and mortality rate. Our clinicians need to be more aware of the need for timely diagnosis through a targeted clinical approach, leading to more targeted treatment and a better prognosis.

Poly I:C-based rHBVvac therapeutic vaccine eliminates HBV via generation of HBV-specific CD8+ effector memory T cells.

OBJECTIVE: Chronic hepatitis B (CHB) virus infection is a global health problem. Finding a cure for CHB remains a challenging task. DESIGN: In this study, poly I:C was employed as an adjuvant for HBV therapeutic vaccine (referred to as pHBV-vaccine) and the feasibility and efficiency of pHBV-vaccine in CHB treatment were evaluated in HBV-carrier mice. RESULTS: We found that pHBV-vaccine decreased HBsAg and HBV DNA efficiently and safely in HBV-carrier mice. Further investigation showed that pHBV-vaccine promoted maturation and antigen presentation ability of dendritic cells in vivo and in vitro. This vaccine successfully restored the exhaustion of antigen-specific CD8+ T cells and partly broke the immune tolerance established in HBV-carrier mice. pHBV-vaccine also enhanced the proliferation and polyfunctionality of HBV-specific CD11ahi CD8αlo cells. Importantly, we observed that T cell activation molecule KLRG1 was only expressed on HBV specific CD11ahi CD8αlo cells. Furthermore, pHBV-vaccine reduced the expression of Eomes and increased the serum IL-12 levels, which in turn promoted the generation of effector memory short-lived effector cells (SLECs) to exhibit a critical role in HBV clearance. SLECs induced by pHBV-vaccine might play a crucial role in protecting from HBV reinfection. CONCLUSIONS: Findings from this study provide a new basis for the development of therapeutic pHBV-vaccine, which might be a potential candidate for clinical CHB therapy.

Chimeric antigen receptor (CAR)-transduced natural killer cells in tumor immunotherapy.

Natural killer (NK) cells are potential effector cells in cell-based cancer immunotherapy, particularly in the control of hematological malignancies. The chimeric antigen receptor (CAR) is an artificially modified fusion protein that consists of an extracellular antigen recognition domain fused to an intracellular signaling domain. T cells genetically modified with a CAR have demonstrated remarkable success in the treatment of hematological cancers. Compared to T cells, CAR-transduced NK cells (CAR-NK) exhibit several advantages, such as safety in clinical use, the mechanisms by which they recognize cancer cells, and their abundance in clinical samples. Human primary NK cells and the NK-92 cell line have been successfully transduced to express CARs against both hematological cancers and solid tumors in pre-clinical and clinical trials. However, many challenges and obstacles remain, such as the ex vivo expansion of CAR-modified primary NK cells and the low transduction efficiency of NK cells. Many strategies and technologies have been developed to improve the safety and therapeutic efficacy in CAR-based immunotherapy. Moreover, NK cells express a variety of activating receptors (NKRs), such as CD16, NKG2D, CD226 and NKp30, which might specifically recognize the ligands expressed on tumor cells. Based on the principle of NKR recognition, a strategy that targets NKRs is rapidly emerging. Given the promising clinical progress described in this review, CAR- and NKR-NK cell-based immunotherapy are likely promising new strategies for cancer therapy.

Natural killer cell dysfunction in hepatocellular carcinoma and NK cell-based immunotherapy.

The mechanisms linking hepatitis B virus (HBV) and hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) remain largely unknown. Natural killer (NK) cells account for 25%-50% of the total number of liver lymphocytes, suggesting that NK cells play an important role in liver immunity. The number of NK cells in the blood and tumor tissues of HCC patients is positively correlated with their survival and prognosis. Furthermore, a group of NK cell-associated genes in HCC tissues is positively associated with the prolonged survival. These facts suggest that NK cells and HCC progression are strongly associated. In this review, we describe the abnormal NK cells and their functional impairment in patients with chronic HBV and HCV infection, which contribute to the progression of HCC. Then, we summarize the association of NK cells with HCC based on the abnormalities in the numbers and phenotypes of blood and liver NK cells in HCC patients. In particular, the exhaustion of NK cells that represents lower cytotoxicity and impaired cytokine production may serve as a predictor for the occurrence of HCC. Finally, we present the current achievements in NK cell immunotherapy conducted in mouse models of liver cancer and in clinical trials, highlighting how chemoimmunotherapy, NK cell transfer, gene therapy, cytokine therapy and mAb therapy improve NK cell function in HCC treatment. It is conceivable that NK cell-based anti-HCC therapeutic strategies alone or in combination with other therapies will be great promise for HCC treatment.

Characteristics and clinical outcome of nonsteroidal anti-inflammatory drug-induced acute hepato-nephrotoxicity among Chinese patients.

AIM: To determine the clinicopathological characteristics of nonsteroidal anti-inflammatory drug (NSAID)-induced acute hepato-nephrotoxicity among Chinese patients. METHODS: We conducted a retrospective chart review of patients using the International Classification of Diseases, Ninth Revision diagnosis code for acute kidney injury (AKI) (584.5 or 584.9) and for acute liver injury (ALI) (570.0 or 573.3) from January 2004 to December 2013. Medical records were reviewed to confirm the diagnosis of AKI and ALI and to quantify NSAID administration. RESULTS: Seven of 59 patients (11.8%) were identified with acute hepato-nephrotoxicity induced by NSAIDs. Five patients (71.4%) received over the recommended NSAIDs dose. Compared with NSAIDs-associated mere AKI, the risk factors of NSAIDs-induced acute hepato-nephrotoxicity are age older than 60 years (57.1%), a high prevalence of alcohol use (71.4%) and positive hepatitis B virus (HBV) markers (85.7%). Compared with NSAIDs-associated mere ALI, the risk factors of NSAIDs-induced acute hepato-nephrotoxicity are age older than 60 years (57.1%), increased extracellular volume depletion (71.4%), and renin-angiotensin-aldosterone system (RAAS) inhibitor combined use (57.1%). Acute interstitial nephritis and acute tubulointerstitial disease were apparent in three out of six (42.9%) kidney biopsy patients, respectively. Acute hepatitis was found in four out of six (66.7%) liver biopsy patients. Overall complete recovery occurred in four patients within a mean of 118.25 ± 55.42 d. CONCLUSION: The injury typically occurred after an overdose of NSAIDs. The risk factors include age older than 60 years, alcohol use, positive HBV markers, extracellular volume depletion and RAAS inhibitor combined use.

Increased susceptibility to experimental steatohepatitis induced by methionine-choline deficiency in HBs-Tg mice.

BACKGROUND: Worldwide, about 25% of individuals with chronic hepatitis B have fatty liver disease. Lipogenic diets that are completely devoid of methionine and choline induce nonalcoholic fatty liver disease. However, no animal model of nonalcoholic steatohepatitis associated with HBV infection is available, and the influence of viral infection on nutritional hepatic steatosis is unclear. METHODS: We used HBV surface antigen transgenic mice (HBs-Tg mice), which mimic healthy human carriers with hepatitis B surface antigen. The mice were fed with a high-fat methionine-choline-deficient diet (MCD) to build a reliable rodent nutritional model of nonalcoholic steatohepatitis associated with HBV infection, and the changes in body weight and serum triglycerides were measured. Hepatocyte ballooning changes were determined by hematoxylin and eosin staining. The extent of hepatic fat accumulation was evaluated by oil red O staining. Immunohistochemical assays were performed to detect proliferating cell nuclear antigen as an index of cell proliferation. RESULTS: MCD feeding provoked systemic weight loss and liver injury. MCD feeding caused more macrovesicular fat droplets and fat accumulation in the livers of HBs-Tg mice than in wild-type C57BL/6 mice. In addition, within 30 days of MCD exposure, more PCNA-positive nuclei were found in the livers of HBs-Tg mice. CONCLUSIONS: HBs-Tg mice fed with a lipogenic MCD form more macrovesicular fat droplets earlier, coincident with more hepatocyte proliferation, resulting in the appearance of increased susceptibility to experimental steatohepatitis in these mice.

[Clinical effects of subcutaneous tunnel hepatocholangioplasty on the treatment of hepatolithiasis].

OBJECTIVE: To evaluate the therapeutic effect of subcutaneous tunnel hepaticoplasty on the treatment of hepatolithiasis. METHODS: The early complications and clinical effects of 99 hepatolithiasis cases who underwent subcutaneous tunnel hepaticoplasty from January 1993 to August 2006 were analyzed retrospectively. The stones of 28 (28.3%) patients were in the left lobe, 24.2% (24/99) in the right, and 47.5% (47/99) in bilateral lobe. Sixty-six patients (66.7%) had both stones and biliary strictures. During the procedure, a portion of the liver habouring stone was resected if necessary. The hepatic duct and strictures were opened, the stones were removed, and the porta hepatis was repaired by one end of a segment of jejunum. The other end of the jejunum was set subcutaneously. The gall bladders of 27 patients (27.3%) were used as subcutaneous tunnel instead. RESULTS: Ninety-five out of ninety-nine cases were followed up with an average of 4.2 years (1 month to 13.5 years). The rates of residual stone, recurrent stone and cholangitis were 23.2% (23/99), 20.0% (19/95) and 14.7% (14/95) respectively. Postoperatively, 34 cases who had residual or recurrent stones were underwent lithotomy by choledochoscope through the subcutaneous blind loop and the achievement ratio was 91.2% (31/34). CONCLUSIONS: Subcutaneous tunnel hepatocholangioplasty decreases the relapsing cholangitis effectively, and makes an easy way to take out residual or recurrent stones.

Inhibition of human gastric carcinoma cell growth by treatment of N(3)-o-toluyl-fluorouracil as a precursor of 5-fluorouracil.

N(3)-o-toluyl-fluorouracil (TFU), the pro-drug of 5-fluorouracil (5-FU), is the metabolite of N(1)-acetyl-N(3)-o-toluyl-fluorouracil (atofluding). We aimed to evaluate the efficacy of TFU as a precursor of 5-FU on the growth inhibition of human gastric carcinoma cell lines SGC-7901 and MKN-45. Growth of SGC-7901 and MKN-45 cells was remarkably suppressed by treatment with TFU in the presence of liver microsomal enzymes in vitro, suggesting that TFU may be converted to 5-FU by the enzymes. Similar treatment of TFU induced apoptosis of the cells, which was deduced from typical apoptotic features such as morphology, the formation of characteristic ladder pattern of DNA migration and the accumulation of sub-G1 phase. Cancer cells xenografts in nude mice were employed to evaluate the efficacy of TFU in vivo. Growth of human gastric carcinoma cells was significantly delayed by oral administration of TFU with low side effects. Apoptosis in xenografts was also observed by means of TUNEL staining method. These results suggest that the treatment of TFU in the presence of liver microsomal enzymes and the oral administration of TFU in mice induced anti-proliferation and apoptosis in gastric carcinoma cells. This suggests that TFU may be a promising pro-drug of 5-FU for cancer treatments.

Application of optical tweezers in the research of molecular interaction between lymphocyte function associated antigen-1 and its monoclonal antibody.

Lymphocyte function associated antigen-1 (CD11a/CD18, LFA-1) plays an important role in the structure of the immunological synapse and is required for efficient lysis of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. To study the activation mode of LFA-1 on the NK cell surface, optical tweezers were used in the work. As an emerging technology, optical tweezers are widely used to manipulate microscopic objects and measure the forces of molecular interactions in the field of biological research. In our study, a new platform was constructed to study the single molecular behavior of receptor on cell surface using optical tweezers. Based on the platform, the interaction between an NK cell and a polystyrene microsphere coated with anti-LFA-1 antibody was observed. The result confirmed that the adhesion forces between an NK cell and a polystyrene bead were time-dependent. According to our findings, we propose that anti-LFA-1 antibody may cause the clustering of LFA-1 on NK cell surface.

[Induction and mechanism study of anti-melanoma immune response by M. tuberculosis Ag85B].

AIM: To study the anti-melanoma immunity efficacy of Ag85B antigen gene therapy in vivo. METHODS: C57BL/6 mice were inoculated s.c. with B16 cells, and 8 days later the mice were inoculated s.c. again with B16 cells (control group 1), B16/pcDNA3 cells (control group 2) or B16/pcDNA3-Ag85B cells (experimental group), respectively. Tumor volume, survival time, serum IFN-gamma level and IL-4 level of 3 groups mice were observed. Antitumor activity of Ag85B was studied. RESULTS: From 12 to 23 day, the mean tumor volume of mice increased from 1.1058 cm(3) and 0.9123 cm(3) to 7.5983 cm(3) and 5.8746 cm(3) in the control group 1 and 2, respectively. But it increased from 0.5158 cm(3) to 1.5080 cm(3) in the experimental group. The mean survival time of mice was 24.1 days and 24.7 days in the control group 1 and 2, respectively. That was 27.8 days in the experimental group. Within 13 days after the last inoculation, the serum IFN-gamma level of all groups experienced increased (That increased to 26.3 ng/L, 23.0 ng/L and 25.2 ng/L in the control group 1, 2 and the experimental group, respectively). Subsequently, the serum IFN-gamma level in the two control groups decreased (That decreased to 19.3 ng/L and 18.3 ng/L in the control group 1 and 2) while it still augmented in the experimental group (That increased to 46.5 ng/L). IL-4 level was slightly but not significantly enhanced and then declined in all mice. CONCLUSION: Ag85B induced the increase of serum IFN-gamma level in the animals experiments, inhibited the tumor growth and prolonged the survival of the tumor-bearing mice.

Using caffeoyl pyrrolidine derivative LY52, a potential inhibitor of matrix metalloproteinase-2, to suppress tumor invasion and metastasis.

LY52 is a caffeoyl pyrrolidine derivative designed to fit the S'1 active pocket of gelatinases that act in tumor invasion and metastasis. Herein, we examined the effects of LY52 on expression of matrix metalloproteinase (MMP)-2 expression in human breast cancer MDA-MB-231 cells and on in vitro invasion and in vivo metastasis. LY52 significantly blocked MMP-2 activity as evidenced by a decrease in the degradation of succinylated gelatin. Gelatin zymography analysis showed that LY52 (0.1-200 microg/ml) inhibited expression of active MMP-2 in concanavalin A-stimulated MDA-MB-231 cells. Inhibition of MMP-2 expression was also observed in tissue of tumor xenografts in mice that were orally administered LY52 (25 or 100 mg/kg). Furthermore, LY52 displayed an inhibitory effect on in vitro invasion of MDA-MB-231 cells and pulmonary metastasis of B16F10 murine melanoma cells in mice without significant toxic effects. These results suggest that LY52 is a potential MMP-2 inhibitor that may effectively suppress tumor invasion and metastasis.

Analysis tissue expression of IFN-gamma in IL-12 and/or IL-18 gene ablated naïve mice.

Interleukin 12 (IL-12) and/or interleukin 18 (IL-18) gene ablated mice were applied for the investigation of the tissue expression of interferon gamma (IFN-gamma). For IL-12(-/-), IL-18(-/-), IL-12(-/-)/18(-/-) and wt mice, reproductive performance were recorded and IFN-gamma concentrations in heart, lung, liver, spleen, kidney and serum were quantified by ELISA. There were no significant differences of IFN-gamma in heart, lung and kidney between 4 strains although control group was higher. It was observed that for IL-12(-/-) mice, compared with other 3 groups, IFN-gamma in liver and spleen were decreased (p < 0.05) and reproductive performance appeared to be impaired. Serum IFN-gamma level of IL-12(-/-)/18(-/-) mice was significantly higher (p < 0.05). It was showed that IFN-gamma productions under the normal condition were independent upon IL-12 and IL-18, its expressions in various tissues were different, and optimal IFN-gamma is necessary for the normal growth and development of mammals. This study is helpful for clinical cytokines therapy.

[Effect of natural killer cell line NK-92 against human ovarian carcinoma cells in vitro and in vivo].

OBJECTIVE: To study the cytotoxic activity of NK-92 cells irradiated against human ovarian cancer. METHODS: NK-92 cells were exposed to different doses of radiation and assayed for proliferation by a standard (3)H-thymidine incorporation assay and cell count by using trypan blue exclusion. The cytotoxic activity of NK-92 cells against targets was measured in a standard (51)Cr-release assay in vitro. The effectiveness of irradiated NK-92 cells on ovarian cancer was compared with the control group of cancers (without injection of irradiated NK-92 cells). RESULTS: (1) In vitro:The proliferation of NK-92 cells was inhibited by radiation of 4, 8 and 16 Gy, respectively. From the (3)H-thymidine incorporation data, irradiation by 4 Gy reduced cell proliferation to 29% of control, while 8 Gy reduced proliferation to 6%. The cytotoxicity of NK-92 cells at 4 Gy 2 days following irradiation was approximately 42%-62% for ovarian cancer cell HO-8910, while it was 33%-58% at 8 Gy. (2) In vivo: Tumor size in treatment group was (0.047 +/- 0.019) cm(3) on day 30 after inoculation, and (0.167 +/- 0.021) cm(3) on day 40 and (0.343 +/- 0.022) cm(3) on day 50, while the sizes were smaller in treatment group (P < 0.01). In addition, the tumor group animals died between 74-82 days after injection of HO-8910 cells, while the treatment group animals were alive over 120 days (P < 0.01). CONCLUSION: Our study indicates that injection of irradiated NK-92 cells may be a potentially effective treatment for human ovarian carcinoma.

[The negative regulatory effect of IFN-gamma on cognitive function of human natural killer cells].

OBJECTIVE: To investigate the regulatory effect of IFN-gamma on recognition of target cells by human natural killer (NK) cells. METHODS: The cytotoxic activity of human NK cell lines (NK92, NKL) was detected by MTT method. Expression of NK cell receptors (NKG2D, NKG2A/B, KIR2DL1 and KIR2DS1) and MICA on target cells (the ligand of NKG2D) was measured by RT-PCR. RESULTS: Both NK92 and NKL cells exerted higher cytotoxicity to tumor cells with MICA expression, while tumors without MICA expression could resist NK cell lysis. IFN-gamma (> 1000 U/ml) inhibited NK lysis of tumor cells with MICA expression through down-regulating the expression of NKG2D, but up-regulating the expression of NKG2A/B and KIR2DL1. CONCLUSION: IFN-gamma has a negative effect on activation and cytotoxicity of human NK cells by altering the balance between the expression of activating and inhibitory receptors on NK cells in favor of inhibition. This may serve to limit NK cell over-activation in vivo.

Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1+ cells.

AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepatic lymphocytes. METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope. Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate ((percentage of post-digestion -percentage of pre-digestion)/percentage of pre-digestion). Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h (51)Cr release assay. RESULTS: NK1.1(+) cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%, respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and gammadelta cells in normal C57BL/6 mice. After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-gamma(+) (68%) or TNF-alpha(+) (15%) NK1.1(+) cells from poly (I:C)-treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells. CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1(+) cell.

Expression of prolactin receptor and response to prolactin stimulation of human NK cell lines.

We have previously shown a critical role of prolactin (PRL) during maturation and anti-tumor effects of murine natural killer (NK) cells in vitro and in vivo. We extended that study by exploring the ability of human NK cell lines (NK-92 and YT cell) to express PRL receptor (PRL-R) and to respond to PRL stimulation in vitro. Both human NK cell lines constitutively expressed PRL-R on membrane and mRNA transcripts, NK-92 cells contained higher level of PRL-R than YT cells, which correlated to the enhanced capacity of the cells to proliferate and to lyse target cells in response to PRL stimulation in the presence of trace amount of IL-2 or IL-15 in vitro. Two differences between IL-2 and IL-15 in functioning on human NK cells were for the first time observed. PRL synergized with IL-15 to improve proliferation of NK cells in a dose-dependent manner without double peak manifesting like IL-2. Although PRL enhanced the cytotoxicity of IL-2 or IL-15 activated NK cells, it exerted the function through up-regulating gene expression of perforin without influence of FasL in IL-2-stimulated NK cells, while in IL-15-stimulated NK cells, PRL did the function through up-regulating gene expression of both perforin and FasL but not IFN-gamma. PRL increased expressions of IL-2Ralpha on membrane and of IL-2 mRNA in cells, indicating that PRL up-regulated NK cell function by improving positive feedback between IL-2 and IL-2R. The similar results were also observed in network between IL-15 and IL-15R. These data indicate a potential role of PRL in human NK cell modulation.

[Negative Regulatory Effect of T-Lymphocytes on the Expansion of Human Bone Marrow Hematopoietic Cells In Vitro]

In this research, the regulatory effects of T-lymphocytes on the expansion of hematopoietic cells in human bone marrow were studied. Anti-CD3 McAb and anti-CD8 McAb were used to eliminate the T-lymphocytes in bone marrow MNCs. Cell surface antigens were analysed by flow cytometry. Hematopoietic cells expanded with hematopoietic growth factors (HGFs) in a liquid culture system and the number of CD34(+) cell, CFU-GM and CFU-GEMM were determined. After cultured with HGFs for 20 days the total number of cells expanded by 75.8, 79.6, 77.4 and 67.0 folds respectively in three experimental groups (anti-CD3 McAb, anti-CD8 McAb and anti-CD3 + anti-CD8 McAb) and control group (MNC of bone marrow). The number of CFU-GM in the four groups were 173.67 +/- 18.90, 165.33 +/- 26.58, 170.33 +/- 21.50 and 79.67 +/- 8.33 respectively. The number of CFU-GEMM in the four groups were 431.33 +/- 34.56, 370.33 +/- 42.10, 386.67 +/- 10.02 and 177.67 +/- 26.86 respectively. There were significant differences in the number of CFU-GM and CFU-GEMM between experimental groups and control group. The results showed that the T-lymphocytes in bone marrow could inhibit the expansion of hematopoietic cells in vitro and the formation of CFU-GM and CFU-GEMM. The regulatory mechanism was to be explored.