Development and Validation of an MRI Radiomics-Based Signature to Predict Histological Grade in Patients with Invasive Breast Cancer.

Background: Histological grade is an important factor in the overall prognosis of patients with invasive breast cancer. Therefore, the non-invasive assessment of histological grade in breast cancer patients is an increasing concern for clinicians. We aimed to establish an MRI-based radiomics model for preoperative prediction of invasive breast cancer histological grade. Methods: We enrolled 901 patients with invasive breast cancer and pre-operative MRI. Patients were randomly divided into the training cohort (n=630) and validation cohort (n=271) with a ratio of 7:3. A radiomics signature was constructed by extracting radiomics features from MRI images and was developed according to multivariate logistic regression analysis. The diagnostic performance of the radiomics model was assessed using receiver operating characteristic (ROC) curve analysis. Results: Of the 901 patients, 618 (68.6%) were histological grade 1 or 2 while 283 (31.4%) were histological grade 3. The area under the ROC curve (AUC) of the radiomics model for the prediction of the histological grade was 0.761 (95% CI 0.728-0.794) in the training cohort and 0.722 (95% CI 0.664-0.777) in the validation cohort. The decision curve analysis (DCA) demonstrated the radiomics model's clinical application value. Conclusion: This is a preliminary study suggesting that the development of an MRI-based radiomics model can predict the histological grade of invasive breast cancer.

Association Between Overweight and Renal Transplant Outcomes: A Meta-Analysis.

OBJECTIVES: As the demand for kidney transplant allografts has increased, many centers are expanding the upper limit of acceptable body mass index for kidney donors. However, obesity is a risk factor for developing renal disease. Our goal was to quantify body mass index trends in donor nephrectomy patients and to institute nutrition counseling to promote sustainable weight loss to reduce the risk of metabolic syndrome-derived renal dysfunction. MATERIALS AND METHODS: Ninety patients who underwent donor nephrectomy between 2007 and 2012 consented to having height and weight data collected at multiple time points. After data collection, each patient underwent a standardized nutrition counseling session. One year later, body mass index was reassessed. RESULTS: Preoperatively, 52% of the patients were overweight or obese. The percentage of overweight and obese patients remained stable for 2 years after surgery. However, at 3, 4, and 5 years after surgery, these rates increased to 59%, 69%, and 91%. Each patient was counseled about obesity-related comorbidities and provided information about lifestyle modification. One year later, 94% of previously overweight patients and 82% of previously obese patients had a decrease in mean body mass index from 27.2 ± 4.0 kg/m2 to 25.1 ± 3.6 kg/m2. CONCLUSIONS: Living-donor nephrectomy patients are at risk of developing obesity, similar to the adult population. Nutrition counseling may be beneficial to help normalize body mass index in patients who have become overweight or obese to potentially prevent obesity-related comorbidities. All patients were evaluated by a nutrition specialist after surgery to review our donor nephrectomy nutrition brochure. Body mass index monitoring and primary care follow-up appear to be appropriate surveillance methods.

[Cytomegalovirus infection down-regulates the expressions of CD226 and CD16 in blood NK cells of the renal transplant recipient].

OBJECTIVE: To investigate the effect of cytomegalovirus (CMV) infection after renal transplantation on the expressions of the activation receptors CD226 and sCD16 in natural killer (NK) cells. METHODS: Peripheral blood samples were obtained from these kidney transplant recipients with CMV infection, stable recipients, and normal healthy controls, and the expressions of CD3, CD56, CD16 and CD226 of all the samples were analyzed by flow cytometry. RESULTS: Compared with patients recovered from CMV infection, normal healthy controls and the stable recipients, the percentage of NK population in lymphocytes did not vary among these groups (P=0.18). However, the percentage of peripheral blood CD226⁺NK cells in NK population of the CMV infection group [(75.06 ± 13.65)%] was significantly lower than that of the healthy controls [(88.28 ± 11.98)%] and stable recipients [(87.53±6.43)%]. While the percentage was recovered during CMV infection recovery stage [(88.37±8.91)%], which was not different from that of normal healthy controls and the stable recipients. The mean fluorescence intensity (MFI) of CD226 was not significantly different among the health controls, stable recipients and CMV infection patients, while it was significantly higher in the patients recovered from CMV infection than in the above three groups. A decrease in the percentage of CD16(+)NK cells in NK population was observed in the group of CMV infection [(75.06 ± 13.65)%], which was significantly lower than that of the stable recipients [(88.28 ± 11.98)%] and health controls [(90.35 ± 10.07)%]. The CD16 expression on NK cells was elevated to (86.30 ± 14.01)% when the infection was controlled, which was not different from the stable recipients and healthy controls. The MFI of CD16MFI in NK cells was similar in all groups. CONCLUSION: CMV infection down-regulates the expressions of CD226 and CD16 in NK cells of patients after kidney transplantation. When the infection was controlled, the expressions of CD226 and CD16 on NK cells were recovered. These results indicated that the membrane CD226 and CD16 molecules were involved in NK cell-mediated anti-CMV infection.

Interleukin-6 first plays pro- then anti-inflammatory role in early versus late acute renal allograft rejection.

This study aimed to investigate the potential role of IL-6 in acute T-cell-mediated renal rejection (ACR) during different periods post-transplantation. Fifty-three patients with ACR (32 of whom developed ACR within the first month; 12, between 2 and 6 months; and 9, between 7 and 12 months post-transplantation), 31 patients with delayed graft function (DGF), and 38 recipients with stable renal allograft function were recruited. Luminex analysis was used to monitor levels of IL-6, sIL-6R, IL-1α, IL-1ß, and IL-1 receptor antagonist (IL-1Ra) in 228 serum samples from 122 patients, including ACR patients before and during rejection, and after rejection reversal, DGF patients, and stable controls. The associations between IL-6 levels and sIL-6R, IL-1α, IL-1ß, and IL-1Ra levels were analyzed using Spearman correlation analysis. In patients who developed ACR within the first month post-transplantation, serum IL-6 concentrations increased significantly compared to the stable control group, but decreased in patients who developed ACR between 2 and 12 months post-transplantation. Concomitantly, levels of sIL-6R gradually increased when ACR occurred between 2 and 12 months post-transplantation. IL-6 levels correlated with IL-1ß levels in early stage ACR and with levels of IL-1Ra in late stage ACR. Our results suggest that IL-6 acts as a pro-inflammatory cytokine during early-stage ACR, and plays an anti-inflammatory role during later stages post-transplantation.

Construction and characterization of a CTLA-4-targeted scFv-melittin fusion protein as a potential immunosuppressive agent for organ transplant.

Immunotoxins with selective cytotoxicity are frequently used as therapeutic immunosuppressive agents in solid-organ transplantation because of their efficiency and high specificity. In this study, we present a new recombinant immunotoxin termed anti-CTLA-4-scFv-melittin prepared from Escherichia coli aimed at clearing activated T cells at the same time avoiding all-round decline in systematic immunity. This fusion protein is composed of anti-CTLA-4-scFv unit and melittin analog unit with properties of low immunogenicity and selective cytotoxicity to CTLA-4-positive T cells. In preliminary biological activity assays, our results confirmed the feasibility of activated T cell clearance strategy and there were significant differences in cell survival rates between CTLA-4-positive group and control group at all experimental concentrations of the immunotoxin. The selective cytotoxicity, low immunogenicity, and low production cost make it an attractive alternate to traditional immunosuppressants.

CD4(+)CD25(-)Nrp1(+) T cells synergize with rapamycin to prevent murine cardiac allorejection in immunocompetent recipients.

Besides CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), other immunosuppressive T cells also participated in the regulation of immune tolerance. Reportedly, neuropilin-1 (Nrp1) might be one of the molecules by which regulatory cells exert their suppressive effects. Indeed, CD4(+)CD25(-)Nrp1(+) T cells exhibit potent suppressive function in autoimmune inflammatory responses. Here we investigated the specific role of CD4(+)CD25(-)Nrp1(+) T cells in the setting of the transplant immune response. Through MLR assays, we found that CD4(+)CD25(-)Nrp1(+) T cells suppressed the proliferation of naive CD4(+)CD25(-) T cells activated by allogeneic antigen-stimulation. Adoptive transfer of CD4(+)CD25(-)Nrp1(+) T cells synergized with rapamycin to induce long-term graft survival in fully MHC-mismatched murine heart transplantation, which was associated with decreased IFN-γ, IL-17 and increased IL-10, TGF-ß, Foxp3 and Nrp1 expression in the grafts. Importantly, our data indicated that CD4(+)CD25(-)Nrp1(+) T cell transfer augments the accumulation of Tregs in the recipient, and creates conditions that favored induction of hyporesponsiveness of the T effector cells. In conclusion, this translational study indicates the possible therapeutic potential of CD4(+)CD25(-)Nrp1(+) T cells in preventing allorejection. CD4(+)Nrp1(+) T cells might therefore be used in bulk as a population of immunosuppressive cells with more beneficial properties concerning ex vivo isolation as compared to Foxp3(+) Tregs.

Down regulation of TRAIL and FasL on NK cells by Cyclosporin A in renal transplantation patients.

TNF-related apoptosis-inducing ligand (TRAIL) and FasL can participate in cell mediated cytotoxicity via their death domain-mediated apoptotic signaling in the host-versus-graft disease occurred after renal transplantation. However, the effect of Cyclosporin A (CsA) commonly used as a drug to prevent and to treat renal transplant rejection, on these molecules have not been fully determined. In the present study, we found that with CsA administration, the expression of TRAIL and FasL predominantly on NK cells from renal transplantation patients was increased at day 5 after operation and went down to normal level on day 13. While, the levels of soluble TRAIL (sTRAIL) and sFasL in the serum increased within 25 days and went down to normal level three month later. In addition, we showed that a remarkable increase of TRAIL and FasL expression both on the surface of activated lymphocytes especially on NK cells and in the supernatants generated from mixed lymphocytes culture (MLC). Furthermore, the enhancement of these two molecules was greatly decreased by adding 500 ng/mL CsA at the beginning of MLC. We conclude that CsA may inhibit the transplant rejection partially by down-regulating the expression of TRAIL and FasL on NK cells.

Combination of IL-1 receptor antagonist, IL-20 and CD40 ligand for the prediction of acute cellular renal allograft rejection.

PURPOSE: The outcome of renal transplantation is difficult to predict, even with an allograft biopsy. The aim of this study was to develop a sensitive, specific and noninvasive method for prediction of acute cellular rejection (ACR). METHODS: Luminex analysis was used to determine the levels of 95 cytokines/chemokines and their soluble receptors in sera from recipients with: ACR (in the first month post-transplantation, before and during rejection, and after rejection reversal); stable allograft function; delayed graft function (DGF); pulmonary infection. Evaluation of significant differential protein expression in ACR patients compared with stable allograft controls revealed a three-analyte combination as a marker of renal transplantation outcome. The predictive value of this combination was further validated in DGF and infection groups and in a blind binary code study of 24 additional serum samples. RESULTS: Significant differential expression was detected in 26 proteins expressed in patients during the period preceding an ACR episode compared with stable controls. A blood test for discrimination of such patients was developed based on the simultaneous quantification of three analytes (IL-1 receptor antagonist, IL-20 and sCD40 ligand). This test exhibited 90.9 % sensitivity, 96 % specificity, a positive predictive value (PPV) of 95.2 % and a negative predictive value (NPV) of 92.3 %. Moreover, this combination allowed discrimination between patients with ACR and DGF and pulmonary infection. CONCLUSIONS: With further development and validation, this blood test can be used to predict ACR and direct the treatment of transplant patients in the clinic.

[Preliminary study on prefabricated urethra in expander capsule].

OBJECTIVE: To investigate the feasibility of prefabricating urethra in the expander capsule with gelatin sponge and micro-mucosa compound transplantation. METHODS: Eight 8-week-old Guizhou miniature pigs (male and/or female) weighing 20-25 kg were used. Six expanders (15 mL) were placed subcutaneously on the dorsal thorax of each miniature pig. Autologous oral mucosa of every pig was harvested 2 weeks later to prepare micro-mucosa with a diameter less than 1 mm. Gelatin sponge 3 cm x 2 cm in size was transplanted to the expander capsule after being coated by the autologous micro-mucosa at the area expansion ratio of 4 : 1 (group A), 8 : 1 (group B), and 16 : 1 (group C), respectively (n = 2 per group). The implantation of gelatin sponge served as the blank control (group D, n = 2). Physiological saline was injected into the expander immediately after operation, and the pressure in the expander was 40 mm Hg (1 mm Hg = 0.133 kPa). The postoperative general condition of the animals was observed. At 1, 2, and 3 weeks after operation, the animals were killed to receive general, HE staining, and immunohistochemistry staining observations. RESULTS: All animals survived till the end of the experiment. The wounds healed well. General observation: in groups A, B, and C at 1 week after operation, there was no obvious degeneration of gelatin, the mucous was survived partially, and there were significant differences among three groups in terms of mucosa healing rate (P < 0.05), groups A and B were better than group C, and group A was better than group B; at 2 weeks, the gelatin sponge was partly absorbed, most of the mucosa survived, and the mucosa healing rate of groups A and B was better than that of group C (P < 0.05); at 3 weeks, the gelatin sponge was still not absorbed completely, the wound reached epithelialization approximately, and there were no significant differences among three groups in terms of mucosa healing rate (P > 0.05). No neo-mucosa was evident in group D at each time point. Histology and immunohistochemistry staining observation: at each time point, the mucosa epithelium survival, inflammatory cell infiltration, and pan-cytokeratin were evident in groups A, B, and C; at 3 weeks after operation, the stratified squamous epithelium presented obvious polarity and the submucous neovascularization was abundant in groups A, B, and C. There was no mucosa epithelium and positive stained pan-cytokeratin in group D. For the percentage of positive pan-cytokeratin stained area, there were significant differences among groups A, B, and C 1 week after operation (P < 0.05); at 2 and 3 weeks after operation, there was significant difference between group A and group C, and between group B and group C (P < 0.05); but no significant difference was evident between group A and group B (P > 0.05). CONCLUSION: Micro-mucosa and gelatin spongy compound transplantation on the expander capsule can form mucosal lining, achieve complete epithelialization in 2 weeks, and contribute to maintain the normal function of prefabricated urethra.

[Role of the activation of interleukin-6/STAT3 in cholangiocyte proliferation induced by cold ischemia reperfusion and injury].

OBJECTIVE: To investigate the role of IL-6/STAT3 pathway in the proliferation of cholangiocyte induced by cold ischemia and reperfusion injury. METHODS: Rats were randomized into CP 1 h and CP 12 h groups (supplied livers were preserved for 1 or 12 h), anti-IL-6R (rats in CP 12 h group were administrated with anti-rat soluble IL-6 receptor antibody), and control group. At 1, 3, 7, 14 d postoperative, IL-6 concentration in liver homogenate and cholangiocyte proliferation were detected by enzyme linked immunosorbent assay and histochemistry respectively. Expressions of IL-6 mRNA, phosphorylated-STAT3 and cyclin D1 protein in cholangiocytes were determined by real-time PCR or Western blot analysis. Serum concentrations of ALP and GGT and histology analysis were also performed. RESULTS: Minimal expressions of IL-6, p-STAT3 and cyclin D1 were detected in CP 1 h group, with a slight cholangiocytes proliferation. Cholangiocytes responded to extended cold preservation with severe bile duct injures and marked increase in IL-6 secretion, p-STAT3 and cyclin D1 protein expression, followed by compensatory cholangiocytes regeneration. Parallel to this observation, biochemical index and morphology indicated that bile duct injury was recovery at 14 d postoperative. However, anti-sIL-6R inhibited cholangiocytes proliferation and reduced the expressions of IL-6, STAT3 and cyclin D, with the cellular injury and increase of serum ALP or GGT. CONCLUSIONS: IL-6/STAT3 pathway might participate to initiate cholangiocytes regeneration after cold ischemia and preservation injury, which might benefit biliary recovery after liver transplantation.

[Role of IL-6/STAT3 in rat cholangiocyte proliferation induced by lipopolysaccharide].

OBJECTIVE: To investigate whether lipopolysaccharide (LPS) stimulates cholangiocyte proliferation via the IL-6/STAT3 pathway in vivo. METHODS: Rats were randomized into three groups: LPS group (injected intravenously with LPS 2.5 mg/kg), anti-IL-6 group (injected intravenously with anti-IL-6 0.5 mg/kg 1hr after LPS injection), and control group. At 6, 12, 24, 48 and 72 h after LPS injection, LPS concentration in plasma was detected by kinetic turbidimetric limulus test. IL-6 concentrations in liver homogenate was determinded by ELISA, cholangiocyte proliferation was checked by immunohistochemistry, expression of IL-6 mRNA was quantified by real-time RT-PCR, the level of phophorylated-STAT3 (P-STAT3) protein was analyzed by western blotting. RESULTS: Cholangiocytes responded to LPS by a marked increase in cell proliferation, IL-6 secretion and P-STAT3 expression. Anti-IL-6 neutralizing antibody inhibited LPS-induced cholangiocytes proliferation, and decreased levels of IL-6 and p-STAT3. CONCLUSIONS: LPS promotes cholangiocyte proliferation through the IL-6/STAT3 pathway.

[Long-term observation of prefabricated urethra with buccal mucosa in expanded capsule].

OBJECTIVE: To investigate the histological and keratinous variation of prefabricated urethra in the capsule with micro-mucosa and gelatin sponge compound graft. METHODS: Five 8-week-old Guizhou miniature pigs (2 females and 3 males) weighing 20-25 kg were used. Eight tissue expanders were bilaterally inserted into subcutaneous position on the dorsal thorax of each pig. Forty inserted expanders were randomized into two groups (n=20 per group). For the experimental group, the free buccal mucosa was cut into particles less than 1 mm in diameter, spread onto the gelatin sponge (3 cm x 2 cm) and then transplanted to the capsule; the area expansion ratio of autogenous micro-mucosa was 8 : 1. For the control group, soft tissue expander without mucosa graft was implanted. The pressure in inserted expander was about 40 mm Hg (1 mm Hg=0.133 kPa). Inflation should be stopped when the injected saline volume reached 15 mL. The animals were killed 1 and 2 weeks and 1, 2, and 4 months after the implant to receive examination. Microscope, histology, and immunohistochemistry changes were observed. RESULTS: All the animals survived to the end of the experiment and the wounds healed by first intention. There was no obvious degeneration of gelatin sponge, and some of the mucosa survived 1 week after implant. The gelatin sponge was partly absorbed, most of the mucosa survived 2 weeks after implant. Visual examination showed complete epithelialization of the entire cavity 1 month after implant. The experimental group at 2 and 4 months were similar to that of at 1 month in gross observations. The neo-mucosa was not found in the control group at different time points after implant. Histology examination revealed that compound implant was mainly infiltrated by inflammatory cells and the micro-mucosa survived well 1 week after implant in the experimental group. The stratified squamous epithelium presented obvious polarity and the submucous neovascularization was abundant 2 weeks after implant. The compound implant achieved complete epithelialization 1 month after implant. The epithelium degeneration occurred 2 months after implant. The stratified squamous epithelium presented no abovious polarity 4 months after implant. No neo-mucosa was evident in control group at different time points. The experimental group was positive for the pan-cytokeratin staining at 1, 2 weeks, and 1, 2 months after implant, but negative at 4 months after implant. The pan-cytokeratin staining was negative in the control group at different time points. CONCLUSION: The buccal micromucosa and gelatin sponge compound graft can grow well on the expanded capsule 1 month after implant and the epithelium degeneration is evident 2 months after implant. Environment of implanted mucosa has great influence on epithelium mucosa.

Activation of interleukin-6/STAT3 in rat cholangiocyte proliferation induced by lipopolysaccharide.

BACKGROUND: Cholangiocytes are exposed to endotoxins (lipopolysaccharide, LPS) in a variety of biliary inflammations. It is known that LPS enhances the release of interleukin (IL)-6, a potent cholangiocyte mitogen. However, the role of LPS in cholangiocyte proliferation in vivo is unknown. Aims To investigate whether LPS stimulates cholangiocyte proliferation in vivo via the IL-6/STAT3 pathway. METHODS: Rats were randomized into four groups: the LPS group (injected intravenously with LPS 2.5 mg/kg), anti-IL-6 group (injected intravenously with anti-IL-6 0.5 mg/kg 1 h after LPS injection), RPM group (treated with RPM 0.4 mg/kg intraperitoneally 30 min before LPS injection), and control group. At 6, 12, 24, 48, and 72 h after LPS injection, LPS in plasma was detected by kinetic turbidimetric limulus test. IL-6 concentrations in liver homogenate and cholangiocyte proliferation were determined by ELISA or immunohistochemistry, respectively. Expression of IL-6 mRNA and phosphorylated-STAT3 (P-STAT3) protein in cholangiocytes was analyzed by real-time RT-PCR and western blotting. RESULTS: Cholangiocytes responded to LPS by a marked increase in cell proliferation, IL-6 secretion, and P-STAT3 expression. Anti-IL-6 neutralizing antibody inhibited LPS-induced proliferation of cholangiocytes and decreased levels of IL-6 and STAT3. Furthermore, after being treated with RPM, STAT3 activation was also depressed, which resulted a decreased proliferation of cholangiocytes. CONCLUSIONS: LPS promotes cholangiocyte proliferation through the IL-6/STAT3 pathway, while RPM shows a depressive effect in this pathway.

Establishment of an ELISA system for determining soluble LAIR-1 levels in sera of patients with HFRS and kidney transplant.

LAIR-1, the leukocyte-associated Ig-like receptor-1, is a trans-membrane molecule that functions as an inhibitory receptor on natural killer cells, T lymphocytes and monocytes. It has been well known that many trans-membrane receptors can shed from the cell surface and be released into the circulation in soluble form when lymphocytes, endothelials and other immune cells are activated. In many cases, the levels of soluble receptors in the circulation can be used as markers of lymphocyte activation in transplant patients and virus infection patients. To investigate whether LAIR-1 is able to be released into the sera, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system based on two anti-LAIR-1 monoclonal antibodies (MAb) with different epitope specificities. Using this ELISA, we found that sLAIR-1 existed in the supernatants collected from PMA, PHA or CD3 MAb-stimulated lymphocytes cultures in vitro for the first time. Moreover, we found that LAIR-1 level in serum samples from healthy individuals was 6.2 +/- 3.3 ng/ml, whereas the levels in sera of patients with hemorrhagic fever with renal syndrome (HFRS) and patients 3-7 days after kidney transplant increased to 47.2 +/- 35.9 and 24.4 +/- 16.0 ng/ml, respectively. Furthermore, HFRS patients in oliguric phase showed higher serum sLAIR-1 levels than those in other phases, and transplant patients with rejection showed higher serum sLAIR-1 level than those without rejection. These findings demonstrated that LAIR-1 can be released when lymphocytes are activated, suggesting sLAIR-1 may be used as a predictor for monitoring immune reaction in some virus infections and organ transplants which may be useful in clinical treatment of these diseases.