Effective and stable gene transduction in rhesus macaque iPSCs capable of T-lineage differentiation utilizing the piggyBac system.

Introduction: Genetically modified human induced pluripotent stem cell (iPSC)-based regenerative medicine has substantial potential in the treatment of refractory human diseases. Thus, preclinical studies on the safety and efficacy of these products are essential. Non-human primate (NHP) models such as the rhesus macaque are highly similar to humans in terms of size, lifespan, and immune system, rendering them superior models. However, effective gene transduction in rhesus macaque iPSCs (Rh-iPSCs) remains challenging. In this study, we investigated the effective gene transduction into Rh-iPSCs and its effect on differentiation efficiency. Methods: We established a gene transduction method using the piggyBac transposon vector system. Gene transduced Rh-iPSCs were analyzed for undifferentiated markers. We did teratoma assay to check pluripotency. Gene transduced Rh-iPSCs were differentiated into hematopoietic stem and progenitor cells (HSPCs) and T-cell lineage cells. Additionally, gene transduced Rh-iPSCs were compared the differentiation efficiency with parental Rh-iPSCs. Results: We could establish a gene transduction method using the piggyBac transposon vector system, demonstrating high efficiency and stable transgene expression in Rh-iPSCs. These Rh-iPSCs maintained long-term gene expression while expressing undifferentiated markers. Teratoma assay indicated that these Rh-iPSCs had pluripotency. These Rh-iPSCs could differentiate into HPSCs and T cells that express transgenes. These Rh-iPSCs can differentiate into hematopoietic stem cells and T cells that express transgenes. No significant differences in efficiency of differentiation were observed between parental Rh-iPSCs and these Rh-iPSCs. Conclusions: These results indicate that the piggyBac transposon vector is an excellent gene transfer tool for rhesus macaque iPSCs and could contribute to the advancement of preclinical studies using rhesus macaque iPSCs.

Evaluation of a Webinar for Pharmacists Learning Basic Clinical-Oncology during COVID-19 Pandemic in Japan.

It is essential for oncology pharmacists to update their knowledge, skills, and ethical attitudes. The Japanese Society of Pharmaceutical Oncology is an academic society for healthcare professionals involved in cancer treatment. It has conducted in-person seminars every year to cultivate the knowledge necessary for practicing advanced cancer medicine. Owing to the coronavirus disease (COVID-19) pandemic, the society was obligated to conduct a web-based seminar this year. A questionnaire survey was conducted before and after the webinar to explain how it works and to assess the learning attitudes of beginner and moderately skilled pharmacists in the field of oncology. Questionnaire surveys were conducted with the participants before and after watching the webinar. The questionnaires sought to determine participants' perspectives on the webinar and their knowledge of the seven modules. Of the 1756 webinar attendees, 1661 (94.6%) answered the pre-webinar survey and 1586 (90.3%) answered the post-webinar survey. Results indicate that the median post-webinar knowledge score was significantly higher than the median pre-webinar score (p < 0.001) in all modules. Principal component analysis of the degree of knowledge of seven modules revealed that the improved score group consisted of those from younger age groups, with less experience as pharmacists, non-society members, and those with less experience in past society seminars. Moreover, the web-based seminar provided a uniform learning effect throughout the country without distinguishing between urban and rural learners. The web-based educational program was an acceptable educational tool for Japanese oncology pharmacists.

Radiological and clinical features of twig-like middle cerebral artery in comparison with moyamoya angiopathy: a multicenter retrospective study.

OBJECTIVE: Twig-like middle cerebral artery (T-MCA) is a rare congenital anomaly that is difficult to distinguish from moyamoya angiopathy (MMA), given the similarity of the angioarchitectures. The aim of this study was to gain insights into the radiological and clinical features of T-MCA and to distinguish this condition from MMA. METHODS: A multicenter retrospective study was conducted in 29 patients with T-MCA and 57 patients with MMA. Demographic, radiological, and clinical data were compared between the patients with T-MCA and those with MMA. RESULTS: The T-MCA group tended to be older than the MMA group (mean age 47 ± 18 vs 39 ± 22 years). Twenty patients with T-MCA (69%) were initially diagnosed with MMA. All T-MCA cases had twig-like networks and steno-occlusive changes involving the MCA. The T-MCA group had a higher incidence of intracranial aneurysms (35% vs 11%) and coexisting arterial anomalies (48% vs 12%). T-MCA and MMA cases had significant differences in involvement of the internal carotid artery terminus (0% vs 100%) and posterior cerebral artery (0% vs 23%), and in transdural anastomosis (0% vs 51%). T-MCA cases were less likely to present with stroke (59% vs 86%) and more likely to be asymptomatic (28% vs 12%). Of the patients with stroke, those with T-MCA had more hemorrhagic strokes (41% vs 29%) and fewer ischemic strokes (59% vs 71%) compared to those with MMA. CONCLUSIONS: This study suggests that T-MCA is a different disease entity from MMA based on significant differences in the radiological and clinical features. Neurosurgeons should recognize this anomaly and understand the key features that differentiate T-MCA from MMA.

Evaluation of a web-based educational programme for pharmacists during the COVID-19 pandemic in Japan.

WHAT IS KNOWN AND OBJECTIVE: Continuing education is essential for pharmacists to acquire and maintain the knowledge, skills, and ethical attitudes necessary for clinical practice. However, with the emergence of COVID-19, the social circumstances and face-to-face learning environments have changed. The objectives of this study were to determine Japanese pharmacists' perception of a web-based educational programme in oncology, and assess changes in their understanding of pharmaceutical care in oncology before and after their participation in the webinar. METHODS: Questionnaire-based surveys were conducted for the participants of the web-based educational programme to determine their perspectives on the webinar, and their degree of comprehension of the five cancer types covered before and after watching the webinar. RESULTS AND DISCUSSION: Of the 1936 pharmacists taking the programme, all participated in the pre-webinar survey, and 1861 (96.1%) in the post-webinar survey. Compared with previous seminars that were held in the offline mode before the COVID-19 pandemic, 76.8% of respondents were significantly satisfied with the web-based educational programme. The median post-webinar comprehension scores in all modules were significantly higher than the median pre-webinar scores (p < 0.0001). A majority of the participants agreed that a web-based educational programme was satisfactory in acquiring knowledge. WHAT IS NEW AND CONCLUSION: This web-based educational programme was effective for Japanese pharmacists for postgraduate education in pharmaceutical care in oncology. To the best of our knowledge, our study is the first to report the effectiveness of a web-based educational programme for oncology pharmacists using a large population.

Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease.

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.

Lumbar Subdural Hematoma Detected After Surgical Treatment of Chronic Intracranial Subdural Hematoma.

BACKGROUND: Spinal subdural hematoma (SSDH), which can cause lower back pain, leg pain, and leg weakness, is rare and will usually be associated with a bleeding tendency, trauma, spinal vascular malformation, intraspinal tumor, or iatrogenic invasion. Only a few cases of SSDH after intracranial chronic subdural hematoma (CSDH) have been reported. We report a case of lumbar SSDH in the absence of predisposing factors after reoperation for recurrent intracranial CSDH, which improved with conservative treatment. CASE DESCRIPTION: Approximately 1 month after falling, a 63-year-old woman was experiencing left hemiparesis and impaired orientation that was diagnosed as right intracranial CSDH using computed tomography. Surgical treatment of the CSDH led to immediate improvement of her symptoms. On postoperative day 29, the right CSDH had recurred with left hemiparesis, and successful reoperation relieved the symptoms within a few hours postoperatively. However, 1 day after the second operation, very small acute subdural hematomas in regions along the left tentorium cerebelli and left falx cerebri were found on computed tomography. On day 31, she complained of sitting-induced bilateral radiating lower limb pain. Magnetic resonance imaging on day 34 showed an acute SSDH at the L4-L5 level and a sacral perineural cyst filled with hematoma, although her radiating pain was showing improvement. She was treated conservatively and was discharged without symptoms on day 44. CONCLUSIONS: Although SSDH is rare, it is important for neurosurgeons and physicians to consider the possibility of a SSDH when lower limb pain or paresis occurs after procedures that will result in rapid intracranial pressure alterations such as drainage of an intracranial CSDH.

Ratio of peripheral blood absolute lymphocyte count to absolute monocyte count at diagnosis is associated with progression-free survival in follicular lymphoma.

The prognosis of follicular lymphoma (FL) is significantly associated with host immunity and tumor microenvironment. Lymphopenia has been identified as a negative prognostic factor for FL. The association between monocytosis and progression-free survival (PFS) in FL remains controversial. It is unknown whether the ratio of peripheral blood absolute lymphocyte count to absolute monocyte count (ALC/AMC) at diagnosis is associated with FL prognosis. We studied 99 consecutive patients with FL who were treated with rituximab-containing chemotherapy at Kitano Hospital or Kyoto University Hospital between 2000 and 2012. We analyzed individual variables associated with the ALC/AMC ratio before treatment, as well as known prognostic factors of FL, and found that an ALC/AMC ratio of 4.7 was the best cut-off value for PFS. Kaplan-Meier analysis showed that a decreased ALC/AMC ratio was associated with inferior PFS (P = 0.022). Multivariate analysis showed that a decreased ALC/AMC ratio was a significant poor prognostic factor independent of other variables (hazard ratio, 2.714; 95 % confidence interval, 1.060-6.948; P = 0.037). The ALC/AMC ratio before treatment may be a significant prognostic factor predicting PFS of FL.

Effective use of sertraline for pathological laughing after severe vasospasm due to aneurysmal subarachnoid hemorrhage: case report.

Pathological laughing, one subgroup of psuedobulbar affect, is known as laughter inappropriate to the patient's external circumstances and unrelated to the patient's internal emotional state. The authors present the case of a 76-year-old woman with no significant medical history who experienced pathological laughing after subarachnoid hemorrhage (SAH) due to rupture of an aneurysm, which was successfully treated with craniotomy for aneurysm clipping. In the acute stage after the operation she suffered from severe vasospasm and resulting middle cerebral artery territory infarction and conscious disturbance. As she regained consciousness she was afflicted by pathological laughing 6 months after the onset of SAH. Her involuntary laughter was inappropriate to the situation and was incongruent with the emotional state, and she could not control by herself. Finally the diagnosis of pathological laughing was made and treatment with sertraline, a selective serotonin reuptake inhibitor (SSRI), effectively cured the symptoms. Her pathological laughing was estimated to be consequence of infarction in the right prefrontal cortex and/or corona radiata, resulting from vasospasm. To the authors' knowledge, this is the first report of pathological laughing after aneurysmal SAH. The authors offer insight into the pathophysiology of this rare phenomenon. Effectiveness of sertraline would widen the treatment modality against pathological laughing.

Sunitinib treatment for multiple brain metastases from jejunal gastrointestinal stromal tumor: case report.

Gastrointestinal stromal tumors (GISTs) are rare malignant tumors and only a few reported cases of brain metastases can be found. Introduction of a new molecular targeted agent, imatinib mesylate in the last decade has dramatically changed the treatment strategy and prognosis. However, imatinib is usually ineffective for brain metastasis from GISTs. The authors present the case of multiple brain metastases from jejunal GIST. The brain metastasis in the right prefrontal gyrus was detected 20 months after resection of the primary lesion when left hemiparesis began although the patient was on imatinib. Then the patient began taking sunitinib instead of imatinib, and the lesion shrunk and the symptom improved. However, after the dose reduction due to side effects, a new brain metastasis was found and this time, stereotactic radiation was effectively done. Sunitinib is one of the promising receptor tyrosine kinase inhibitors used for metastatic renal cell carcinomas or imatinib-refractory GISTs. Sunitinib is thought to penetrate blood-brain barrier, and recent reports indicate effectiveness to brain metastasis. To the authors' knowledge, this is the first report of brain metastases from jejunal GIST responding to sunitinib therapy.

Impact of high-dose chemotherapy and autologous transplantation as first-line therapy on the survival of high-risk diffuse large B cell lymphoma patients: a single-center study in Japan.

High-dose chemotherapy (HDT), together with autologous stem cell transplantation (ASCT), plays an important role in the treatment of diffuse large B cell lymphoma (DLBCL), especially as second-line therapy. However, its significance in up-front settings remains to be elucidated. In our institute, patients with DLBCL in both the high-intermediate and high international prognostic index (IPI) groups initially underwent CHOP/R-CHOP treatment followed by HDT/ASCT at upfront settings between 2002 and 2011. We retrospectively analyzed 25 patients who were all treated with upfront HDT/ASCT. We excluded one patient who failed to undergo transplantation because of primary refractory disease from the analysis. The median follow-up was 77 months (range 17-110 months). Five-year overall survival (OS) and progression-free survival (PFS) were 91.7 and 79.2 %, respectively, which were higher than the equivalents in previous studies. The OS and PFS in the high-risk group were lower than those in the high-intermediate group. Treatment-related mortalities or fatal complication were not observed. Our results confirm that HDT/ASCT for high-risk aggressive lymphoma is a feasible and promising therapy, but patients with high IPI continued to have poor prognoses; improvements in treatment strategy are clearly needed. Since HDT/ASCT is an aggressive treatment option associated with long-term complications, we need to identify patient groups that will gain the maximum benefit from HDT/ASCT in the upfront setting.

Immune reconstitution inflammatory syndrome mimics a relapse of AIDS-related Burkitt lymphoma.

Immune reconstitution inflammatory syndrome (IRIS) is associated with clinical manifestations that can overlap with the patients with acquired immunodeficiency disease (AIDS)-related non-Hodgkin's lymphoma. We herein report a case of AIDS-related Burkitt lymphoma which was successfully treated with dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisone (EPOCH). However, the patient developed a lymphoma-like clinical presentation shortly after the conclusion of chemotherapy. The patient's symptoms were identical to the initial symptoms characteristic of lymphoma; however, the laboratory data revealed no evidence of a relapse of Burkitt lymphoma. A bone marrow examination showed T-cell clonality, even though there were no signs of any progression of the lymphoma. The patient was diagnosed with IRIS, and the clinical manifestations rapidly improved following treatment.

[Saccular aneurysm arising in the non-branching segment of the distal anterior cerebral artery with stalk-like narrow neck: a case report].

An aneurysm arising in the distal anterior cerebral artery (DACA) is relatively rare. Among them, those in the non-branching segment are seldom reported so far. The authors present the case of an 87-year-old woman who developed intracerebral hemorrhage and acute hydrocephalus due to rupture of an aneurysm arising in the non-branching site of DACA. External ventricular drainage followed by aneurysm clipping by bifrontal craniotomy was performed as treatment. Microscopic observations revealed that the aneurysm was saccular-shaped, located at the non-branching site of the A3 portion of the anterior cerebral artery, and had significant atherosclerosis neither on its parent artery nor neck. Histopathological examinations of the aneurysm wall denied traumatic aneurysm or mycotic aneurysm, and showed partial disruption of the internal elastic lamina, suggesting a difference from common aneurysms arising at arterial branchings. Profound knowledge of this type of aneurysms would be useful in dealing with them at surgery.

Abrupt evolution of Philadelphia chromosome-positive acute myeloid leukemia in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder arising from an alteration in multipotent stem cells, which lose the ability of normal proliferation and differentiation. Disease progression occurs in approximately 30% MDS cases. Specific chromosomal alterations seem responsible for each step in the evolution of acute myeloid leukemia (AML). Multiple genetic aberrations occur during the clonal evolution of MDS; however, few studies report the presence of the Philadelphia (Ph) chromosome. We report a rare case of Ph-positive AML, which evolved during the course of low-risk MDS. The patient, a 76-year-old man with mild leukocytopenia, was diagnosed with MDS, refractory neutropenia (RN). After 1.5 yr, his peripheral blood and bone marrow were suddenly occupied by immature basophils and myeloblasts, indicating the onset of AML. A bone marrow smear showed multilineage dysplasia, consistent with MDS evolution. Chromosomal analysis showed an additional t(9;22)(q34;q11) translocation. Because progression occurred concurrently with emergence of the Ph chromosome, we diagnosed this case as Ph-positive AML with basophilia arising from the clonal evolution of MDS. The patient was initially treated with nilotinib. A hematological response was soon achieved with disappearance of the Ph chromosome in the bone marrow. Emergence of Ph-positive AML in the course of low-risk MDS has rarely been reported. We report this case as a rare clinical course of MDS.

Entadosides A-D, triterpene saponins and a glucoside of the sulphur-containing amide from the kernel nuts of Entada phaseoloides (L.) Merrill.

From a MeOH extract of kernel nuts of Entada phaseoloides (L.) Merrill, one new and one known sulphur-containing glucoside were isolated. From the 1-BuOH-soluble fraction of a H(2)O extract, four new triterpene saponins containing N-acetylglucosamine in their sugar chains were isolated. The antiproliferative activities of the triterpene saponins were assayed.

Chronic periodontitis with multiple risk factor syndrome: a case report.

OBJECTIVE: Multiple risk factor syndrome is a clustering of cardiovascular risk factors, such as diabetes, dyslipidemia, hypertension, and obesity associated epidemiologically with insulin resistance. This report describes the clinical course of a patient suffering from severe periodontitis with multiple risk factor syndrome, and discusses the association between periodontal infection and systemic health. METHODS: The patient had a history of type 2 diabetes, dyslipidemia, and hypertension for over 10 years. At baseline, her hemoglobin A1 c was 8.1%. However, she had no diabetic complications except periodontitis. The IgG antibody titers against Porphyromonas gingivalis FDC 381 and SU63 were elevated above the mean of healthy subjects +2 standard deviations. Intensive periodontal treatment, including periodontal surgery, was performed to reduce periodontal infection and bacteremia. Her systemic and periodontal conditions were evaluated longitudinally for 10 years. RESULTS: Following periodontal treatment, antibody titers against Porphyromonas gingivalis and hemoglobin A1c values were significantly improved. The other clinical data and medication for her systemic condition also remained stable during supportive periodontal therapy. However, she developed myocardial infarction, and showed continuous deterioration of hemoglobin A1 c level and periodontitis. CONCLUSION: The long-term clustering of risk factors, such as diabetes, dyslipidemia, hypertension, and periodontitis, are associated with the development of myocardial infarction. Treatment of systemic conditions in combination with comprehensive periodontal treatment is important in management of patients with multiple risk factor syndrome.

ß-Adrenergic receptor gene polymorphism is a genetic risk factor for cardiovascular disease: a cohort study with hypertensive patients.

Single-nucleotide polymorphisms (SNPs) of the ß-adrenergic receptor (ßADR) subtypes are related to hypertension and obesity. This hospital-based cohort study with hypertensive patients evaluated five ßADR SNPs in association with cardiovascular events. The cohort included 357 hypertensive patients (male = 181; mean age = 61.5 ± 11.8 years) seen between January 1998 and June 2004. The SNPs (Ser49Gly and Arg389Gly for ß(1)ADR; Gly16Arg and Glu27Gln for ß(2)ADR; Trp64Arg for ß(3)ADR) were identified by PCR. We used Kaplan-Meier curves to assess the prognostic effect of these SNPs on cardiovascular disease (CVD). The SNP frequencies were Ser/Ser:Ser/Gly:Gly/Gly = 243:104:10; Arg/Arg:Arg/Gly:Gly/Gly = 256:95:6; Gly/Gly:Gly/Arg:Arg/Arg = 71:201:85; Gln/Gln:Glu/Gln = 308:49; and Trp/Trp:Trp/Arg:Arg/Arg = 265:89:3. A total of 17 stroke and 15 coronary artery disease cases were recorded. By Kaplan-Meier analysis, the Ser/Ser SNP in Ser49Gly (P = 0.0398), the Glu/Gln SNP in Glu27Gln (P = 0.0390) and the Trp/Trp SNP in Trp64Arg (P = 0.0132) were associated with lower event-free CVD survival (log-rank, Mantel-Cox model). A Cox proportional hazards model revealed that only the Trp/Trp SNP (P = 0.0321) and age (P = 0.0186) were independently related to lower event-free survival for CVD, adjusted for gender, diabetes, dyslipidemia, blood pressure, body mass index, medication and hypertensive complications. Combination Kaplan-Meier analysis of these three positive SNPs indicated a higher frequency of CVD among patients with the combination of Ser/Ser in Ser49Gly of ß(1), Glu/Gln in Glu27Gln of ß(2) and Trp/Trp in Trp64Arg of ß(3) (P = 0.0209). These three SNPs, especially the Trp64Arg SNP of ß(3)ADR, might be risk factors for CVD in hypertensive patients.

The counterregulating role of ACE2 and ACE2-mediated angiotensin 1-7 signaling against angiotensin II stimulation in vascular cells.

To clarify the role of endogenous angiotensin (Ang)-converting enzyme 2 (ACE2) and its cleavage product, Ang 1-7, in the atherogenic stimulation of vascular cells, we investigated the effect of pharmacological inhibition of ACE2 and Mas, an Ang 1-7 receptor, on cellular responses against Ang II stimulation. We measured extracellular signal-regulated kinase (ERK) 1/2 phosphorylation by western blot, smooth muscle cell (SMC) proliferation by WST assay and the adhesion of monocytes labeled with PKH67 to endothelial cells (ECs) by fluorescence microplate reader. Cells were pretreated with Ang 1-7, olmesartan (Ang II type 1 receptor (AT1) blocker), DX600 (ACE2 inhibitor), -Ala7-Ang1-7 (D-Ala; Mas antagonist), or combinations of treatments before the application of Ang II. Treatment with Ang II increased phosphorylated ERK 1/2 of SMC and EC, proliferation of SMC and adhesion of monocyte to EC, which were blocked by olmesartan. Pretreatment with DX600 either did not accelerate or only slightly accelerated these cellular responses. However, when Ang II signaling through AT1 was reduced by olmesartan, the additional treatment with DX600 significantly blunted some of the effect of olmesartan. Similarly, pretreatment with D-Ala reduced the inhibitory effect of olmesartan in response to Ang II stimulation. Endogenous ACE2 in vascular cells may contribute to counteracting the Ang II-mediated cellular response partly by upregulating the Ang 1-7 signaling through Mas.

Loss of ACE2 accelerates time-dependent glomerular and tubulointerstitial damage in streptozotocin-induced diabetic mice.

As angiotensin-converting enzyme-2 (ACE2) was identified as a negative regulator of the renin-angiotensin system, there have been many reports concerning its role in several tissues, including the kidney. However, the role of ACE2 during the development of diabetic nephropathy remains undetermined, as previous reports did not necessarily support a protective role against renal injury. Thus, we performed detailed observations of kidneys in ACE2-knockout (ACE2-KO) mice at early (4 weeks) and advanced (18 weeks) stages of diabetes. ACE2-KO and wild-type C57BL/6 mice were rendered diabetic by intraperitoneal injection of streptozotocin. Diabetic ACE2-KO mice showed earlier onset and more severe progression of albuminuria than those did wild-type mice. The elevation of serum creatinine and urea nitrogen levels at 18 weeks of diabetes was more prominent in ACE2-KO mice. Periodic acid-Schiff-stained cross-section of diabetic ACE2-KO mice showed a more severe time-dependent increase in glomerular/tubulointerstitial damage than did that of wild-type mice, confirmed by the immunostaining of alpha-smooth muscle actin, collagen IV and F4-80 antigen. Glomeruli of diabetic ACE2-KO mice showed earlier and more severe decrease in the expression of nephrin, whose degradation is involved in the onset of albuminuria, and more potent increase of vascular endothelial growth factor expression. In addition, treatment with AT1 receptor blocker olmesartan significantly, but not totally, ameliorated the functional and morphological deterioration of diabetic nephropathy in ACE2-KO mice. These results suggest that ACE2 might continuously protect from both glomerular and tubulointerstitial injury during the development of diabetic nephropathy. The renal-protective effect of ACE2 might involve more than just suppressing angiotensin II-mediated AT1 receptor signaling.

Macrophage inflammatory protein-1beta induced cell adhesion with increased intracellular reactive oxygen species.

UNLABELLED: To investigate the role of macrophage inflammatory protein-1 beta (MIP-1beta) in the development of atherosclerosis, we designed an in vitro study to elucidate the mechanisms of monocyte-endothelium adhesion via intracellular reactive oxygen species (ROS). Angiotensin II (AngII) was used as a positive control. Furthermore, we examined the efficacy of MIP-1beta as a predictor of stroke and cardiovascular events in hypertensive patients. MIP-1beta or AngII stimulation significantly increased ROS production and adhesion of THP-1 cells to inflamed human umbilical vein endothelial cells. Cell adhesion and ROS production were inhibited in stimulated THP-1 cells by: inhibition of ROS signaling with N-acetylcysteine, diphenyleneiodonium, or PEG-Catalase; inhibition of PI3Kgamma with siRNA or LY294002; and by Rac1 siRNA. The MIP-1 beta or AngII stimulation did not increase surface expression of integrins, very late antigen 4 (VLA-4) and lymphocyte function-associated antigen 1 (LFA-1), but cell adhesion was reduced by using an antiVLA-4 or an antiLFA-1 antibody. Moreover, cell adhesion and ROS production stimulated with MIP-1beta or AngII were completely inhibited by fluvastatin. In our clinical study, patients with the highest quartile of MIP-1beta showed a higher risk of stroke and cardiovascular events by a Cox proportional-hazards model. In conclusion, MIP-1beta directly induced cell adhesion to endothelial cells through oxidative stress via PI3k-Rac1 cascades. Serum MIP-1beta level might be a useful predictor for cerebro-cardiovascular events in hypertensive patients. CONDENSED ABSTRACT: We designed an in vitro investigation to examine the role of MIP-1beta on the development of atherosclerosis, including cell adhesion involving CAMs and ROS production, compared with angiotensin II. Furthermore, we investigated the prognostic impact of MIP-1beta on stroke and cardiovascular events in hypertensive patients in a small cohort study.

Macrophage-adipocyte interaction: marked interleukin-6 production by lipopolysaccharide.

OBJECTIVE: Recent studies suggested macrophages were integrated in adipose tissues, interacting with adipocytes, thereby exacerbating inflammatory responses. Persistent low-grade infection by gram-negative bacteria appears to promote atherogenesis. We hypothesized a ligand for toll-like receptor 4 (TLR4), bacterial lipopolysaccharide (LPS), would further exaggerate macrophage-adipocyte interaction. RESEARCH METHODS AND PROCEDURES: RAW264.7 macrophage cell line and differentiated 3T3-L1 preadipocytes were co-cultured using transwell system. As a control, each cell was cultured independently. After incubation of the cells with or without Escherichia coli LPS, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production was evaluated. RESULTS: Co-culture of macrophages and adipocytes with low concentration of Escherichia coli LPS (1 ng/mL) markedly up-regulated IL-6 production (nearly 100-fold higher than that of adipocyte culture alone, p < 0.01), whereas TNF-alpha production was not significantly influenced. This increase was partially inhibited by anti-TNF-alpha neutralizing antibody. Recombinant TNF-alpha and LPS synergistically up-regulated IL-6 production in adipocytes. However, this increase did not reach the level of production observed in co-cultures stimulated with LPS. DISCUSSION: A ligand for TLR-4 stimulates macrophages to produce TNF-alpha. TNF-alpha, thus produced, cooperatively up-regulates IL-6 production with other soluble factors secreted either from adipocytes or macrophages in these cells. Markedly up-regulated IL-6 would greatly influence the pathophysiology of diabetes and its vascular complications.