[Investigation of the immune profile of multiple myeloma patients achieving long-term survival after autologous stem cell transplantation].

Objective: To identify the characteristics of the bone marrow immune microenvironment associated with long-term survival in multiple myeloma (MM) patients. Methods: In the follow-up cohort of patients with newly diagnosed MM and who received "novel agent induction therapy and subsequent autologous stem cell transplantation and immunomodulator maintenance therapy" in the First Affiliated Hospital of Sun Yat-sen University, a cross-sectional study was carried out between August 2019 and May 2020. Using NanoString technology, the RNA expression of 770 bone marrow immune-related markers was compared between 16 patients who had progression-free survival ≥5 years and 5 patients with progressive disease. Among the 16 patients who achieved long-term survival, 9 achieved persistent minimal residual disease (MRD) negative while the other 7 had persistent positive MRD. The functional scores of each kind of immune cells were calculated based on the expression level of characteristic genes, so as to indirectly obtained the proportion of each immune cell subset. The Mann-Whitney U test and the Kruskal Wallis test were used for statistical analysis. Results: The proportion of neutrophils was significantly higher in long-surviving MM patients than in patients with progressive disease [functional scores, 13.61 (13.33, 14.25) vs. 12.93 (12.58, 13.38); Z=2.31, P=0.021]. Among long-surviving patients, those who were MRD-positive had a significantly greater number of mast cells compared with those who were MRD-negative [functional scores, 7.09 (6.49, 8.57) vs. 6.03 (5.18, 6.69); H=2.18, P=0.029]. Compared with patients with progressive disease, four genes (CTSG, IFIT2, S100B, and CHIT1) were significantly downregulated and six (C4B, TNFRSF17, CD70, IRF4, C2, and GAGE1) were upregulated in long-surviving patients. Among long-surviving patients, only gene CMA1 was significantly upgraded, 10 genes (ISG15, OAS3, MX1, IFIT2, DDX58, SIGLEC1, CXCL10, IL1RN, SERPING and TNFSF10) were significantly downregulated in the MRD-positive group compared with that in the MRD-negative group, the first 5 of which are related to the interferon response pathway. Conclusions: The increased neutrophil and mast cell numbers may be related to long-term survival in MM. Interferon signaling activation may be a key bone marrow immune profiling feature for MRD-negative, long-surviving patients with MM.

[The role and mechanism of regulatory T cells in indirect acute lung injury].

Objective: To study the role and mechanism of CD(4)(+) CD(25)(+) FoxP3(+) regulatory T cells (Tregs) in the pathophysiological process of indirect acute lung injury (iALI) in mice. Methods: The iALI model was successfully induced by shock/cecal ligation and puncture method. Sham (n=8), cecal ligation and puncture (CLP, n=10), and hemorrhage (Hem, n=12) groups were established as controls. Two experimental groups were established: CLP+Hem (n=15) without Tregs adoptive transfer (AT), and CLP+Hem with Tregs adoptive transfer (CLP+Hem+AT, n=14). The number of Tregs, subsets of lymphocytes, neutrophil activity, apoptosis, cytokine levels and histopathological changes were measured in the lung tissue of each group. The protein exudation and the expression of IL-10 in bronchoalveolar lavage fluid (BALF) were also detected. After in vitro cell co-culture, the proliferation of activated T cells and the expression of IL-10, INF-γ and iNOS protein were detected. Results: The percentage and the absolute cell number of CD(4)(+) CD(25)(+) FoxP3(+) Tregs in lung tissue of iALI mice were (2.530±0.086)%, and (1.441±0.090)×10(4)/ml, respectively, which were significantly higher than the control groups (P<0.05). Adoptive transfer of Tregs could significantly decrease CD3-positive T lymphocytes, myeloperoxidase (MPO) activity, caspase-3 activity in lung tissue as well as protein leakage in BALF (P<0.05). Meanwhile interleukin-10 (IL-10) levels in lung tissue and BALF were up-regulated from (121.4±43.76) pg/ml to (201.0±61.96) pg/ml (t=2.776, P<0.05) and (206.2±90.88) pg/ml to (339.4±109.5) pg/ml (t=2.477, P<0.05), respectively. Histopathology was also significantly improved. The proliferation of activated T lymphocytes in the adoptive transfer Treg (AT-Treg) group (n=5) was significantly lower than that in the natural regulatory T cell (N-Treg) group (n=5, t=7.485, P<0.01) and the negative control group (n=5, t=16.66, P<0.01). However, iNOS enzyme inhibitor L-NMMA could significantly reduce the T cell proliferation (P<0.05). Conclusion: CD(4)(+)CD(25)(+)FoxP3(+) Tregs could reduce inflammatory reaction in mice with iALI, and the iNOS signaling pathway may be involved in this process.

Outcomes and risk factors for hepatitis B virus (HBV) reactivation after kidney transplantation in occult HBV carriers.

PURPOSE: The purpose of this study was to explore the outcomes and risk factors for hepatitis B virus (HBV) reactivation after kidney transplantation in occult HBV carriers, who are hepatitis B surface antigen (HBsAg) seronegative and hepatitis B core antibody (HBcAb) seropositive before kidney transplantation. METHODS: We retrospectively analyzed 322 occult HBV carriers who received kidney transplantation in our hospital from January 1998 to June 2008. HBsAg and HBV DNA were routinely checked for diagnosis of HBV reactivation. RESULTS: Our results showed that 15 cases (4.7%) of occult HBV carriers had HBV reactivation after kidney transplantation. Kaplan-Meier analysis showed that 1-, 3-, 5-, and 10-year patient survival was 86.7%, 79.4%, 72.2%, and 65.0%, respectively, in the HBV reactivation group, and was 96.1%, 93.8%, 91.5%, and 84.5%, respectively, in the non-HBV reactivation group (log-rank 4.12, P = 0.042). Graft survival showed no difference between these 2 groups (P > 0.05). The incidences of impairment of liver function, liver function failure, hepatocellular carcinoma, and acute rejection were significantly higher in the HBV reactivation group compared with the non-HBV reactivation group (P < 0.05). Logistic multivariate analysis showed that older age (>60 years) and using anti-T-cell antibodies were independent risk factors for HBV reactivation after kidney transplantation, while being hepatitis B surface antibody (HBsAb) seropositive and using lamivudine prophylaxis could protect occult HBV carriers from HBV reactivation after kidney transplantation (P < 0.05). CONCLUSION: In conclusion, our data showed that HBV reactivation may diminish the patient survival but not graft survival. Older age and anti-T-cell antibodies may increase the risk of HBV reactivation, whereas lamivudine prophylaxis may prevent HBV reactivation after kidney transplantation.

[Efficacy and safety of autologous hematopoietic stem cell transplantation in elderly multiple myeloma patients: a single center retrospective study].

Objective: To evaluate the efficacy and safety of autologous hematopoietic stem cell transplantation (auto-HSCT) in elderly patients (≥65 years old) with multiple myeloma (MM) . Methods: From June 1, 2006 to July 31, 2020, 22 MM patients (≥65 years old) who were diagnosed in the First Affiliated Hospital, Sun Yat-sen University and received novel drug induction followed by auto-HSCT were analyzed retrospectively. These patients were evaluated for important organ functions before transplantation, and the International Myeloma Working Group frail score was used in 2016 to screen out transplant-eligible patients. Results: The median (interquartile range, IQR) age at the time of transplantation of the 22 patients was 66.75 (IQR 4.50) years. A total of 20 patients received stem cell mobilization. The median number of mononuclear cells collected was 4.53×10(8)/kg, that of CD34(+) cells was 3.37×10(6)/kg, and the median number of apheresis procedures performed was 2. After stem cell transfusion, the median time of neutrophil implantation was 11 days, that of platelet implantation was 13 days, and the treatment-related mortality was 0 at 100 days after transplantation. The median follow-up was 48.7 months. The median time to progression time was not reached, and the median overall survival time was 111.8 months. Conclusion: Auto-HSCT is a safe and effective treatment for selected elderly patients of 65 years or older with MM.

[New agents-based induction chemotherapy followed by autologous stem cell transplantation and maintenance treatment strategy for multiple myeloma: a single center retrospective study of 300 cases].

Objective: To examine the survival and influential factors of an integrated approach of novel agents, autologous hematopoietic stem cell (auto-HSCT) , and maintenance therapy in patients with multiple myeloma (MM) patients from a single center over the past 15 years. Methods: In our center, 300 MM patients who received an integrated strategy of new agents, auto-HSCT, and maintenance therapy over 15 years were retrospectively and prospectively analyzed. Results: The complete remission rates (CR) and ≥very good partial remission rates (VGPR) following induction therapy, transplantation, and maintenance therapy were respectively 35.3% and 55.2% , 72.4% and 80.0% , 89.2% , and 93.4% . When compared to patients receiving double-drug induction, the ≥VGPR and ORR of patients receiving triple-drug induction were improved. No difference existed in CR, ≥VGPR, and ORR between the PAD (bortezomib + liposome doxorubicin+ dexamethasone) and RAD (lenalidomide + liposome doxorubicin + dexamethasone) regimens, but the benefits speed differed. The negative rate of flow minimal residual disease following induction, transplantation, and maintenance was 18.8% (54 cases) , 41.4% (109 cases) , and 58.7% (142 cases) , respectively. The median time to progress (TTP) was 78.7 months and the median overall survival (OS) was 109 months. The median TTP for RISS-Ⅰ-Ⅲ patients were 111.8 months, 77.4 months, and 30.6 months, and the median OS was 118.8 months, 91.4 months, and 48.5 months, respectively. At various points during treatment, the TTP and OS of patients obtaining CR and MRD negative were longer than those of patients who did not obtain CR and MRD negative. TTP was noticeably shorter in high-risk cytogenetic patients compared to standard-risk patients even when CR was acquired during induction. There was no difference in TTP between patients with high-risk cytogenetics and those with standard-risk cytogenetics if MRD negative was acquired during induction. According to a multivariate analysis, the R-ISS stage was a poor predictor of TTP and OS at various treatment intervals. Therapeutic effectiveness was a newly independent prognostic factor following treatment. Conclusion: A median survival of almost 10 years is possible for MM patients who receive an integrated strategy of induction regimens followed by auto-HSCT and maintenance therapy, which significantly improves prognosis. However, this approach did not significantly benefit high-risk cytogenetic MM patients.

[High dose melphalan (HDM) is superior to cyclophosphamide plus etoposide and busulfan (CVB) as the conditioning regimen in autologous stem cell transplantation for multiple myeloma].

Objective: To compare the efficacy, response and survival between high-dose melphalan (HDM) and cyclophosphamide+ etoposide+ busulfan (CVB) as the conditioning regimen in autologous stem cell transplantation (ASCT) for newly diagnosed multiple myeloma (NDMM) . Methods: Retrospectively enrolled 123 consecutive NDMM patients who had received PAD induction with subsequent ASCT from Jan 2011 to Aug 2017. The CVB group and HDM group had 82 and 41 patients respectively. Results: ①No differences existed between these 2 groups in non-hematological side effects. ②Patients of CVB group had faster neutrophil and platelet engraftment time, with the median neutrophil engraftment time of 10 (9-35) day vs 11 (9-12) day for patients of HDM group (z=-3.433, P=0.001) , and with median platelet engraftment time of 11 (7-55) day vs 13 (10-35) day for patients of HDM group (z=-3.506, P<0.001) . CVB group entered neutropenia and severe thrombocytopenia more earlier than the HDM group, resulting similar neutropenia duration and severe thrombocytopenia duration between the CVB group and HDM group. However, patients of CVB group had significantly longer fever persistent time and antibiotic administration time. ③The response rate was significantly lower in patients of CVB group vs. patients of HDM group (9/46 vs 14/28, P=0.021) . Further, the minimal residual disease (MRD) negative rate at 3(rd) month post-transplantation seemed to be lower in CVB group than that in HDM group (31.7%vs 48.8%, P=0.065) . ④Both the univariate and multivariate analysis showed that HDM and CVB groups had similar duration to progression (TTP) (P=0.619) and overall survival (OS) (P=0.295) . Conclusion: HDM conditioning regimen is superior to CVB regimen in hematological side effects, tumor burden reduction and administration convenience. However, these two regimen had similar TTP and OS in MM patients receiving ASCT.

[Bortezomib-based induction chemotherapy followed by autologous hematopoietic stem cell transplantation and maintenance in 200 patients with multiple myeloma: long-term follow-up results from single center].

Objective: To study the efficacy, safety and long-term outcomes of integrated strategy of bortezomib-based induction regimens followed by autologous hematopoietic stem cell (ASCT) and maintenance therapy in Chinese multiple myeloma (MM) patients. Methods: 200 MM patients receiving integrated strategy of bortezomib--based induction regimens followed by ASCT and maintenance therapy were retrospectively and prospectively analyzed from December 1. 2006 to April 30. 2018. Results: The complete remission rates (CR) and better than very good partial remission rates (VGPR) after induction therapy, transplantation and maintenance therapy were respectively 31% and 75.5%, 51.8% and 87.7%,73.6% and 93.4%. There was no difference between 4 cycles and more than 5 cycles induction chemotherapy. The negative rate of MRD detection by flow cytometry was 17.6% and 38.2% respectively after induction and 3 months after transplantation. The negative rate of MRD gradually increased during the maintenance therapy. The success rate of high dose CTX combined with G-CSF mobilization was 95.5% and transplantation related mortality (TRM) was zero. The median time to progress (TTP) was 75.3 months and the median overall survival (OS) was 99.5 months. TTP of patients obtaining CR and negative MRD after induction were longer that those of no CR and positive MRD. TTP and OS of patients receiving triple-drug induction and ASCT in early stage were longer than those of double-drug induction and ASCT in late stage. LDH≥240 U/L, high risk cytogenetics, ISS II+III stage and HBsAg positive were prognostic factors at diagnosis. However, only MRD and high risk cytogenetics were independent prognostic factors after transplantation and maintenance therapy. The clinical characteristics of patients of TTP ≥6 years were listed below: light-chain type M protein, ISS I stage, normal level of hemoglobin and platelet, normal LDH, HBsAg negative, chromosome 17p-negative, good response and sustained good response. Conclusions: Integrated strategy of bortezomib-based induction regimens followed by ASCT and maintenance therapy can significantly improve the short-term and long-term efficacy. The prognostic factors of TTP in different disease stages were different. Response to treatment, especially MRD, played a more important role in prognostic factors.

Adenoviral delivery of a leukocyte-type 12 lipoxygenase ribozyme inhibits effects of glucose and platelet-derived growth factor in vascular endothelial and smooth muscle cells.

The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.

[Epidemiology of febrile neutropenia in patients with hematological disease-a prospective multicentre survey in China].

OBJECTIVE: To investigate the incidence, clinical and microbiological features of febrile, and risk factors during neutropenia periods in patients with hematological diseases. METHODS: From October 20, 2014 to March 20, 2015, consecutive patients who had hematological diseases and developed neutropenia during hospitalization were enrolled in the prospective, multicenter and observational study. RESULTS: A total of 784 episodes of febrile occurred in 1 139 neutropenic patients with hematological diseases. The cumulative incidence of febrile was 81.9% at 21 days after neutropenia. Multivariate analysis suggested that central venous catheterization (P<0.001, HR=3.407, 95% CI 2.276-4.496), gastrointestinal mucositis (P<0.001, HR=10.548, 95% CI 3.245-28.576), previous exposure to broad-spectrum antibiotics within 90 days (P<0.001, HR=3.582, 95% CI 2.387-5.770) and duration of neutropenia >7 days (P<0.001,HR=4.194, 95% CI 2.572-5.618) were correlated with higher incidence of febrile during neutropenia. With the increase of the risk factors, the incidence of febrile increased gradually (35.4%, 69.2%, 86.1%, 95.6%, P<0.001). Of 784 febrile cases, 253 (32.3%) were unknown origin, 429 (54.7% )of clinical documented infections and 102(13.0%) of microbiological documented infections. The most common sites of infection were pulmonary (49.5%), upper respiratory (16.0%), crissum (9.8%), blood stream (7.7%). The most common pathogens were gram-negative bacteria (44.54%), followed by gram-positive bacteria (37.99% ) and fungi (17.47% ). There was no significant difference in mortality rates between cases with febrile and cases without febrile (9.2% vs 4.8%, P=0.099). Multivariate analysis also suggested that >40 years old (P=0.047, HR=5.000, 95% CI 0.853-28.013), hemodynamic instability (P=0.001, HR=13.185, 95% CI 2.983-54.915), prior colonization or infection by resistant pathogens (P=0.005, HR=28.734, 95% CI 2.921-313.744), blood stream infection (P=0.038, HR=9.715, 95% CI 1.110-81.969) and pulmonary infection (P=0.031, HR=25.905, 95% CI 1.381-507.006) were correlated with higher mortality rate in cases with febrile. CONCLUSIONS: Febrile was the common complication during neutropenia periods in patients with hematological disease. There was different distribution of organisms in different sites of infection. Moreove, the duration of neutropenia >7 days, central venous catheterization, gastrointestinal mucositis and previous exposure to broad-spectrum antibiotics within 90 days were the risk factors for the higher incidence of febrile.

Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries.

BACKGROUND: 12-Lipoxygenase (12-LO) products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells. We have also recently demonstrated increased 12-LO mRNA and protein expression in the neointima of balloon-injured rat carotid arteries. In this study, we evaluated whether 12-LO activation plays a role in neointimal thickening in this rat model by using a specific ribozyme (Rz) directed to rat 12-LO. METHODS AND RESULTS: We designed a chimeric DNA-RNA hammerhead Rz to cleave rat leukocyte-type 12-LO mRNA. This Rz dose-dependently cleaved a 166-nucleotide target 12-LO mRNA substrate in vitro and reduced 12-LO mRNA and protein expression in rat vascular smooth muscle cells. A control mutant Rz (MRz) with a point mutation in the catalytic site was inactive. To test the in vivo efficacy of the 12-LO Rz, the left common carotid arteries of rats were injured with a balloon catheter. The distal half of the injured arteries was treated with Rz or MRz mixed with lipofectin. The proximal half received only lipofectin. Twelve days after injury, intima-to-media ratios were significantly lower in the Rz-treated sections than in untreated sections from the same rat (0.742+/-0.16 versus 1.749+/-0.12, P:<0.001). In contrast, the MRz had no significant effect. CONCLUSIONS: These results indicate the important role of the leukocyte-type 12-LO pathway in restenosis in response to injury.

Selective estrogen receptor modulator idoxifene inhibits smooth muscle cell proliferation, enhances reendothelialization, and inhibits neointimal formation in vivo after vascular injury.

BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.

In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex.

The steroid 11 beta-hydroxylase (P450c11) enzyme is responsible for the conversion of 11-deoxycortisol to cortisol in the zona fasciculata of the adrenal cortex. Animal studies have suggested that this enzyme or a closely related isozyme is also responsible for the successive 11 beta- and 18-hydroxylation and 18-oxidation of deoxycorticosterone required for aldosterone synthesis in the zona glomerulosa. There are two distinct 11 beta-hydroxylase genes in man, CYP11B1 and CYP11B2, which are predicted to encode proteins with 93% amino acid identity. We used a sensitive assay based on the polymerase chain reaction to analyze the expression of the CYP11B1 and B2 genes. Transcripts of CYP11B1 were detected at high levels in surgical specimens of normal adrenals and also in an aldosterone-secreting adrenal tumor. Transcripts of CYP11B2 were found at low levels in normal adrenals, but at a much higher level in the aldosterone-secreting tumor. CYP11B2 mRNA levels were increased in cultured zona glomerulosa cells by physiological levels of angiotensin-II. The entire coding regions of both CYP11B1 and B2 cDNAs were cloned from the tumor mRNA. Expression of these cDNAs in cultured COS-1 cells demonstrated that the CYP11B1 product could only 11 beta-hydroxylate 11-deoxycortisol or deoxycorticosterone, whereas the CYP11B2 product could also 18-hydroxylate cortisol or corticosterone. A small amount of aldosterone was synthesized from deoxycorticosterone only in cells expressing CYP11B2 cDNA. These data demonstrate that the product of CYP11B2 is required for the final steps in the synthesis of aldosterone.

Identification of a ligand-binding region of the human insulin receptor encoded by the second exon of the gene.

Structure-function studies of the insulin molecule indicate that an insulin B chain domain comprising residues 22-26 is involved both in binding to the insulin receptor (INSR) and in insulin dimer formation, suggesting that this domain might also interact with a structure resembling the insulin dimer interface in the INSR. Expression of a mutant INSR cDNA with a deletion of the region corresponding to exon 2 of the INSR gene produces a protein devoid of insulin-binding activity, although the mutant protein is processed appropriately to alpha- and beta-subunits, suggesting that the insulin-binding domain is encoded at least in part by exon 2. Within this region of the INSR molecule, the sequence 83-103 fulfills the structural criteria for a dimer interface. Studies of mutant INSRs with substitutions for phenylalanine 88 or 89 show that the presence of phenylalanine at position 89 is essential for full binding affinity.

Evidence for 12-lipoxygenase induction in the vessel wall following balloon injury.

OBJECTIVE: Vascular smooth muscle cell (VSMC) migration and proliferation are key events in the development of atherosclerosis and restenosis following angioplasty. These events are mediated by several growth factors and cytokines whose cellular effects include activation of phospholipases and arachidonic acid metabolism via the lipoxygenase (LO) pathway. Since 12-LO products have potent growth and chemotactic effects, we have examined if 12-LO is upregulated in the neointima of injured rat carotid arteries and also if LO inhibition could attenuate neointimal thickening. METHODS: The left common carotid arteries of male Sprague Dawley rats were injured using a 1.8 F PTCA balloon catheter. Four-fourteen days after injury, injured and uninjured tissue samples were processed for histology, and immunohistochemistry or polymerase chain reaction (PCR) to examine 12-LO expression. RESULTS: Twelve days after injury, immunohistochemical staining with a 12-LO antibody revealed intense staining in injured left carotid arteries, mainly in neointimal VSMCs and inflammatory cells, but not in the uninjured right arteries. There was also a marked upregulation of 12-LO mRNA (over five-fold by competitive PCR) in the injured arteries. Treatment of the arteries with a LO inhibitor, phenidone, soon after injury resulted in significant inhibition of neointimal thickening. In contrast, a cyclooxygenase inhibitor, ibuprofen, had no effect. CONCLUSIONS: These results indicate for the first time that balloon injury results in marked induction of 12-LO mRNA and protein expression in the vessel wall. Furthermore, LO pathway activation may mediate, at least in part, the development of the lesion or plaque instability, suggesting a novel target for therapeutic intervention to block these pathological events.

Carvedilol, a new vasodilating beta adrenoceptor blocker antihypertensive drug, protects endothelial cells from damage initiated by xanthine-xanthine oxidase and neutrophils.

OBJECTIVE: Oxygen radical mediated endothelial injury plays an important role in cardiovascular disease. Carvedilol, a new beta blocker and antihypertensive agent, has been shown to have antioxidant activity. The aim of this study was to determine whether carvedilol protects oxygen radical induced endothelial injury. METHODS: Cultured bovine pulmonary artery (BPAEC) and human umbilical vein endothelial cells (HUVEC) were used and oxygen radicals were generated by xanthine-xanthine oxidase or phorbol myristate acetate (PMA) activated human neutrophils. Cell injury was assessed by lactate dehydrogenase (LDH) release and cell death, or 51 Cr release from prelabelled BPAEC. The electron paramagnetic resonance (EPR) spin trapping technique was used to detect the amount of radical spin adducts formed in cell lipids. RESULTS: Carvedilol dose dependently inhibited xanthine-xanthine oxidase induced LDH release from BPAEC and HUVEC, with IC50 values of 3.8 microM and 2.6 microM, respectively, and significantly reduced cell death by xanthine-xanthine oxidase. Other beta blockers tested (propranolol, labetalol, pindolol, and celiprolol) showed a mild effect or no effect at all. Increasing the time of pretreatment with carvedilol enhanced its cell protective effect against oxidative stress. Carvedilol also protected BPAEC dose dependently from PMA activated, neutrophil induced cell injury. Carvedilol had no effect on xanthine oxidase activity. EPR study confirmed that xanthine-xanthine oxidase induced the formation of lipid derived radicals in cell lipids and carvedilol scavenged free radicals, as indicated by the decreased EPR signal. CONCLUSIONS: Carvedilol protects endothelial cells against oxygen radical mediated cell injury and death by scavenging free radicals. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent.

Interleukin-1 beta regulates the expression of a leukocyte type of 12-lipoxygenase in rat islets and RIN m5F cells.

The leukocyte type of 12-lipoxygenase (12-LO) may play a role in inflammatory reactions in many cell types through the conversion of arachidonic acid to proinflammatory eicosanoids that include 12-hydroperoxyeicosatetraenoic acid and 12-hydroeicosatetraenoic acid. Previous studies demonstrating the presence of a functional 12-LO pathway in rat and human pancreatic beta-cells plus the recent cloning of a rat leukocyte type of 12-LO allowed us to evaluate whether inflammatory cytokines such as interleukin-1 beta (IL-1 beta) can regulate the beta-cell 12-LO enzyme pathway, thus providing a potential link between the cytotoxic effects of cytokines on pancreatic beta-cells and the proinflammatory effects of 12-LO products. We demonstrate that IL-1 beta induces 12-LO protein and messenger RNA (mRNA) expression in RIN m5F cells and 12-LO mRNA expression in rat islets. RIN m5F cells treated for 16 h with IL-1 beta (25, 50, and 100 ng/liter) showed a maximal 2-fold increase in the expression of a leukocyte form of 12-LO demonstrated by Western blots. A concomitant increase in 12-LO mRNA expression was seen at this time point using a highly sensitive competitive polymerase chain reaction assay. The increase in mRNA and protein expression was preceded by increased 12-LO pathway activity measured by a RIA for 12-S-HETE. Separate experiments using purified Sprague-Dawley rat islets also showed increased expression of 12-LO mRNA and enzyme activity in response to IL-1 beta. These results demonstrate that IL-1 beta can up-regulate 12-LO expression and activity in rat beta-cells.

Evidence that a leukocyte type of 12-lipoxygenase is expressed and regulated by angiotensin II in human adrenal glomerulosa cells.

The 12-lipoxygenase (LO) pathway of arachidonic acid plays an important role in angiotensin II (AII)-mediated aldosterone synthesis. Several distinct isoforms of 12-LO have been cloned. However, in humans only the platelet form of 12-LO has been reported to be present. Western immunoblotting analysis in cultured human adrenal glomerulosa cells using polyclonal antibodies to porcine leukocyte 12-LO enzyme or peptide showed a specific 72-kilodalton band, which is identical to the molecular size of the porcine leukocyte form of 12-LO. In addition, AII (10(-7)) increased the intensity of the 72-kilodalton band nearly 2-fold over basal. In situ hybridization analysis indicated a strong positive reaction with the porcine leukocyte type of 12-LO antisense riboprobe in the zona glomerulosa of the adrenal cortex. The 12-LO probe also recognized a near 4-kilobase messenger RNA (mRNA) from human glomerulosa cells in Northern blots. Since the leukocyte type of 12-LO is highly homologous to human 15-LO, a reverse transcriptase and polymerase chain reaction was used to analyze the specific type of 12-LO mRNA in human cells. The mRNA for a porcine leukocyte type of 12-LO was detected in human adrenal glomerulosa cells, and the level of 12-LO transcripts was increased approximately 60-fold by AII (10(-7) M). The leukocyte type of 12-LO also was detected in human monocyte-like U937 cells, but not in IM-9 lymphocytes or human erythroleukemia cells. These results suggest that human adrenal glomerulosa cells and human monocyte-like U937 cells express a 12-LO which has immunological and molecular biological characteristics similar to the porcine leukocyte 12-LO.

Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor.

The interaction of growth factors, such as epidermal growth factor (EGF) with their receptors, on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acids, such as arachidonic acid, which can be further metabolized by the lipoxygenase (LO) pathway. Several LO products have been shown to stimulate oncogenes and have mitogenic and chemotactic effects. In this study, we have evaluated the regulation of 12-LO activity and expression in breast cancer cells and tissues. Leukocyte-type 12-LO messenger RNA (mRNA) expression was studied by a specific RT-PCR method in matched, normal, uninvolved and cancer-involved breast tissue RNA samples from six patients. In each of these six patients, the cancer-involved section showed a much higher level of 12-LO mRNA than the corresponding normal section. 12-LO mRNA levels also were greater in two breast cancer cell lines, MCF-7 and COH-BR1, compared with the nontumorigenic breast epithelial cell line, MCF-10F. The growth of the MCF-7 cells was significantly inhibited by two specific LO blockers but not by a cyclooxygenase blocker. Treatment of serum-starved MCF-7 cells with EGF for 4 h led to a dose-dependent increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid. EGF treatment also increased the levels of the leukocyte-type 12-LO protein expression at 24 h. These results suggest that activation of the 12-LO pathway may play a key role in basal and EGF-induced breast cancer cell growth.

Carvedilol, a new antihypertensive agent, prevents lipid peroxidation and oxidative injury to endothelial cells.

The protective effects of carvedilol, a new beta-adrenergic receptor blocker and vasodilating antihypertensive agent, against oxygen free radical-mediated injury were studied in cultured bovine endothelial cells and compared with five other beta-blockers. Carvedilol dose-dependently inhibited oxygen radical-induced lipid peroxidation (50% inhibition at 2.6 mumol/L) and glutathione depletion (50% inhibition at 1.8 mumol/L) in the cells. Under the same conditions, other beta-blockers--propranolol, labetalol, pindolol, atenolol, and celiprolol--had only mild or no effect. Moreover, carvedilol protected against oxygen radical--mediated cell damage, as assessed by cellular lactate dehydrogenase release, with a 50% inhibition at 4.1 mumol/L and increased the cell survival in a dose-dependent manner, whereas other beta-blockers had mild or no effects. Pretreatment of the cells with carvedilol for 7 days significantly enhanced the protective effects of carvedilol. Using 2-methyl-2-nitrosopropane as a trapping agent, the spin adduct in cell lipids was monitored by electron paramagnetic resonance. Carvedilol dose-dependently decreased the intensity of the free radical signals, indicating its free radical-scavenging ability. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent.