Newly diagnosed follicular lymphoma during pembrolizumab treatment for lung cancer.

Malignant lymphoma developing during anti-PD-1 antibody treatment is extremely rare. A 74-year-old female was admitted with left hypochondrial pain. She was diagnosed with squamous cell carcinoma of the right upper lobe of the lung, and had undergone surgery and postoperative chemotherapy three years prior. Needle biopsy of a mediastinal lymph node revealed recurrent lung cancer (LC). Pembrolizumab (PEM) monotherapy was started as salvage treatment. Although her lymphadenopathy improved, thrombocytopenia and splenomegaly developed during treatment with nine doses of PEM. Laboratory findings included anemia, increased lactate dehydrogenase, and soluble interleukin-2 receptor levels of 6379 U/mL. Flow cytometry of peripheral blood and bone marrow showed CD20+, κ ≪ λ cell populations. IGH-BCL2 fusion was detected by fluorescence in situ hybridization in bone marrow. Positron emission tomography showed abnormal uptake in tonsils, both cervical lymph nodes, mediastinum (different location from the recurrent LC), spleen, and abdominal cavity. Follicular lymphoma (FL) grade 1/2 was histologically diagnosed by tonsillar biopsy. She achieved a complete metabolic response (CMR) after rituximab monotherapy on PEM discontinuation. Relapsed FL was diagnosed by submandibular gland biopsy four months after restarting PEM and she achieved a second CMR after rituximab-containing chemotherapy. We describe the first case of newly diagnosed FL during PEM treatment.

Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiency.

Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II-induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II-induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II-induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A2 analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.

Low-dose L-NAME induces salt sensitivity associated with sustained increased blood volume and sodium-chloride cotransporter activity in rodents.

To investigate the cause of salt sensitivity in a normotensive animal model, we treated rats with a low-dose of the nitric oxide synthase inhibitor, L-NAME, that does not elevate blood pressure per se or induce kidney fibrosis. A high salt diet increased the circulating blood volume both in L-NAME-treated and nontreated animals for the first 24 hours. Thereafter, the blood volume increase persisted only in the L-NAME-treated rats. Blood pressure was higher in the L-NAME-treated group from the start of high salt diet exposure. Within the first 24 hours of salt loading, the L-NAME treated animals failed to show vasodilation and maintained high systemic vascular resistance in response to blood volume expansion. After four weeks on the high salt diet, the slope of the pressure-natriuresis curve was blunted in the L-NAME-treated group. An increase in natriuresis was observed after treatment with hydrochlorothiazide, but not amiloride, a change observed in parallel with increased phosphorylated sodium-chloride cotransporter (NCC). In contrast, a change in blood pressure was not observed in L-NAME-treated NCC-deficient mice fed a high salt diet. Moreover, direct L-NAME-induced NCC activation was demonstrated in cells of the mouse distal convoluted tubule. The vasodilatator, sodium nitroprusside, downregulated phosphorylated NCC expression. The effect of L-NAME on phosphorylated NCC was blocked by both the SPAK inhibitor STOCK2S-26016 and the superoxide dismutase mimetic TEMPO which also attenuated salt-induced hypertension. These results suggest that the initiation of salt sensitivity in normotensive rodents could be due to hyporeactivity of the vasculature and that maintaining blood pressure could result in a high circulating volume due to inappropriate NCC activity in the low-dose L-NAME model. Thus, even slightly impaired nitric oxide production may be important in salt sensitivity regulation in healthy rodents.

Mineralocorticoid receptor blockade suppresses dietary salt-induced ACEI/ARB-resistant albuminuria in non-diabetic hypertension: a sub-analysis of evaluate study.

Excessive dietary salt intake can counteract the renoprotective effects of renin-angiotensin system (RAS) blockade in hypertensive patients with chronic kidney disease (CKD). In rodents, salt loading induces hypertension and renal damage by activating the mineralocorticoid receptor (MR) independently of plasma aldosterone levels. Thus, high salt-induced resistance to RAS blockade may be mediated by MR activation. To test this, a post hoc analysis of the Eplerenone Combination Versus Conventional Agents to Lower Blood Pressure on Urinary Antialbuminuric Treatment Effect (EVALUATE) trial was conducted. Thus, 304 non-diabetic hypertensive patients on RAS-blocking therapy were divided into tertiles according to salt intake (estimated 24-h urinary sodium excretion at baseline) and compared in terms of percent reduction in urinary albumin-to-creatinine ratio (UACR) at 52 weeks relative to baseline. The eplerenone-treated patients in the highest sodium excretion tertile exhibited significantly greater reduction in UACR than the placebo subjects in the same tertile (-22.5% vs. +21.8%, p = 0.02). This disparity was not observed in the lowest (-10.2% vs. -0.84%, p = 0.65) or middle (-19.5% vs. +9.5%, p = 0.22) tertiles. Similar systolic blood pressure changes were observed. In the whole cohort, reduction in UACR correlated positively with reduction in systolic blood pressure (r2 = 0.04, p = 0.02). These results support the hypothesis that excessive salt intake can enhance resistance to RAS blockade by activating MR. They also suggest that eplerenone plus RAS blockade may be effective for CKD in hypertensive patients, especially those with excessive salt intake.

Aberrant DNA methylation of hypothalamic angiotensin receptor in prenatal programmed hypertension.

Maternal malnutrition, which causes prenatal exposure to excessive glucocorticoid, induces adverse metabolic programming, leading to hypertension in offspring. In offspring of pregnant rats receiving a low-protein diet or dexamethasone, a synthetic glucocorticoid, mRNA expression of angiotensin receptor type 1a (Agtr1a) in the paraventricular nucleus (PVN) of the hypothalamus was upregulated, concurrent with reduced expression of DNA methyltransferase 3a (Dnmt3a), reduced binding of DNMT3a to the Agtr1a gene, and DNA demethylation. Salt loading increased BP in both types of offspring, suggesting that elevated hypothalamic Agtr1a expression is epigenetically modulated by excessive glucocorticoid and leads to adult-onset salt-sensitive hypertension. Consistent with this, dexamethasone treatment of PVN cells upregulated Agtr1a, while downregulating Dnmt3a, and decreased DNMT3a binding and DNA demethylation at the Agtr1a locus. In addition, Dnmt3a knockdown upregulated Agtr1a independently of dexamethasone. Hypothalamic neuron-specific Dnmt3a-deficient mice exhibited upregulation of Agtr1a in the PVN and salt-induced BP elevation without dexamethasone treatment. By contrast, dexamethasone-treated Agtr1a-deficient mice failed to show salt-induced BP elevation, despite reduced expression of Dnmt3a. Thus, epigenetic modulation of hypothalamic angiotensin signaling contributes to salt-sensitive hypertension induced by prenatal glucocorticoid excess in offspring of mothers that are malnourished during pregnancy.

Aberrant DNA methylation of Tgfb1 in diabetic kidney mesangial cells.

Epigenetic modulation may underlie the progression of diabetic nephropathy (DN). Involvement of TGFB1 in mesangial fibrosis of DN led us to hypothesize that Tgfb1 DNA demethylation contributes to progression of DN. In primary mesangial cells from diabetic (db/db) mouse kidneys, demethylation of Tgfb1 DNA and upregulation of Tgfb1 mRNA progressed simultaneously. USF1 binding site in Tgfb1 promoter region were demethylated, and binding of USF1 increased, with decreased binding of DNMT1 in db/db compared with control. Given downregulation of Tgfb1 expression by folic acid, antioxidant Tempol reversed DNA demethylation, with increased and decreased recruitment of DNMT1 and USF1 to the promoter, resulting in decreased Tgfb1 expression in db/db mice. Addition of H2O2 to mesangial cells induced DNA demethylation and upregulated Tgfb1 expression. Finally, Tempol attenuated mesangial fibrosis in db/db mice. We conclude that aberrant DNA methylation of Tgfb1 due to ROS overproduction play a key to mesangial fibrosis during DN progression.

Labial salivary gland biopsy for diagnosing immunoglobulin light chain amyloidosis: a retrospective analysis.

Our goal was to evaluate the usefulness of labial salivary gland (LSG) biopsy for diagnosing immunoglobulin light chain (AL) amyloidosis, by comparing bone marrow and skin biopsies in the same patient population. This retrospective study included 34 consecutive patients who showed evidence of monoclonal proteins and symptoms considered to be due to amyloidosis, and who underwent a tissue biopsy from LSG between January 2005 and December 2012 at Nagoya City University Hospital. All samples of superficial tissues, including LSG, bone marrow, and skin, were independently evaluated as having amyloid deposits by a central review, which was blind to clinical information. An AL amyloidosis diagnosis was based on evidence of amyloid deposition in any biopsied tissue. Eighteen patients were diagnosed with AL amyloidosis. The sensitivity for detecting amyloid deposition was highest in biopsies of LSG at 89 %, followed by 77 % for bone marrow, and 72 % for skin. Amyloid deposition was detected in at least one superficial tissue of all the 18 patients. An LSG biopsy may be appropriate as a first-choice procedure to diagnose AL amyloidosis. Multiple biopsies of superficial tissues, including LSG, bone marrow, and skin, are recommended to increase the sensitivity for diagnosing AL amyloidosis.

Serum soluble (pro)renin receptor levels in patients with essential hypertension.

The (pro)renin receptor ((P)RR) is expressed in several tissues including kidney, heart and brain, and is thought to regulate the tissue renin-angiotensin system (RAS) through the non-proteolytic activation of prorenin. (P)RR is cleaved by furin to generate soluble (P)RR (s(P)RR), which is secreted into the extracellular space. s(P)RR is a candidate biomarker reflecting the status of the tissue RAS. Here, we investigated the relationship between background factors and serum s(P)RR levels. We measured s(P)RR levels in 122 patients with essential hypertension (EH) and assessed the relationships between background factors and s(P)RR levels. Serum s(P)RR levels were 19.0±4.9 ng ml(-1). Single regression analyses showed that age (r=0.251, P<0.01), serum creatinine levels (r=0.229, P<0.05) and urinary angiotensinogen excretion (r=0.196, P<0.05) were positively correlated with s(P)RR levels, whereas estimated glomerular filtration rate (eGFR; r=-0.337, P<0.001) were negatively correlated. Multiple regression analyses of age, blood pressure (BP), hemoglobin A1c (HbA1c) and s(P)RR levels revealed that age and s(P)RR levels were negatively correlated with the eGFR (P<0.05). In patients with EH, serum s(P)RR levels correlated positively with renal function independent of age, BP and HbA1c. These findings support s(P)RR as a useful biomarker that reflects the status of the tissue RAS.

Amelanotic malignant melanoma of unknown primary origin metastasizing to the bone marrow: a case report and review of the literature.

We herein describe the case of a 77-year-old Japanese man who presented with progressive thrombocytopenia. No lymphadenopathies, bone lesions, hepatosplenomegaly or masses within any internal organs were detectable. Bone marrow smears revealed diffuse infiltration of large atypical cells morphologically resembling mature lymphoid neoplasms. A flow cytometric analysis showed that the tumor cells strongly expressed CD56 without myeloid or lymphoid antigens, suggesting that they were non-hematologic in origin. Ultimately, amelanotic malignant melanoma of unknown primary origin was diagnosed based on positive immunostaining for S100 proteins, HMB-45 and Melan-A. This case illustrates the usefulness of flow cytometric analyses for making such diagnoses. We also review the available literature on similar cases.

HTLV-1 bZIP factor-specific CD4 T cell responses in adult T cell leukemia/lymphoma patients after allogeneic hematopoietic stem cell transplantation.

We document human T lymphotropic virus type 1 (HTLV-1) bZIP factor (HBZ)-specific CD4 T cell responses in an adult T cell leukemia/lymphoma (ATL) patient after allogeneic hematopoietic stem cell transplantation (HCT) and identified a novel HLA-DRB1*15:01-restricted HBZ-derived naturally presented minimum epitope sequence, RRRAEKKAADVA (HBZ114-125). This peptide was also presented on HLA-DRB1*15:02, recognized by CD4 T cells. Notably, HBZ-specific CD4 T cell responses were only observed in ATL patients after allogeneic HCT (4 of 9 patients) and not in nontransplanted ATL patients (0 of 10 patients) or in asymptomatic HTLV-1 carriers (0 of 10 carriers). In addition, in one acute-type patient, HBZ-specific CD4 T cell responses were absent in complete remission before HCT, but they became detectable after allogeneic HCT. We surmise that HTLV-1 transmission from mothers to infants through breast milk in early life induces tolerance to HBZ and results in insufficient HBZ-specific T cell responses in HTLV-1 asymptomatic carriers or ATL patients. In contrast, after allogeneic HCT, the reconstituted immune system from donor-derived cells can recognize virus protein HBZ as foreign, and HBZ-specific immune responses are provoked that contribute to the graft-versus-HTLV-1 effect.

Fibroblast growth factor 23 accelerates phosphate-induced vascular calcification in the absence of Klotho deficiency.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating hormone that acts primarily on the kidney and parathyroid. With declining kidney function there is an increase in circulating FGF23 levels, which is associated with vascular calcification and mortality in chronic kidney disease. Whether FGF23 exerts direct effects on vasculature is unclear. We evaluated the expression of Klotho and FGF receptors in rat aortic rings and rat aorta vascular smooth muscle cells maintained in culture by reverse transcription-PCR, western blotting, and immunostaining. Signaling pathways underlying FGF23 effects were assessed by western blotting, and effects of FGF23 on osteogenic markers and phosphate transporters were assessed by real-time reverse transcription-PCR. We detected Klotho and FGFR1 in total aorta but not in vascular smooth muscle cells. FGF23 augmented phosphate-induced vascular calcification in the aortic rings from uremic rats and dose dependently increased ERK1/2 phosphorylation in Klotho-overexpressing but not naive vascular smooth muscle cells. FGF23-induced ERK1/2 phosphorylation was inhibited by SU5402 (FGFR1 inhibitor) and U0126 (MEK inhibitor). FGF23 enhanced phosphate-induced calcification in Klotho-overexpressing vascular smooth muscle cells and increased osteoblastic marker expression, which was inhibited by U0126. In contrast, phosphate transporter expression was not affected by phosphate or FGF23. Thus, FGF23 enhances phosphate-induced vascular calcification by promoting osteoblastic differentiation involving the ERK1/2 pathway.

Antitumor effects of bevacizumab in a microenvironment-dependent human adult T-cell leukemia/lymphoma mouse model.

OBJECTIVE: The objective of this study was to evaluate the therapeutic potential of bevacizumab with or without systemic chemotherapy for adult T-cell leukemia/lymphoma (ATL) and clarify the significance of angiogenesis for ATL pathogenesis. METHODS: NOD/Shi-scid, IL-2Rγ(null) (NOG) mice were used as recipients of tumor cells from a patient with ATL, which engraft and proliferate in a microenvironment-dependent manner. The ATL cells could be serially transplanted in NOG mice, but could not be maintained in in vitro cultures. RESULTS: Injection of bevacizumab alone significantly increased necrosis and decreased vascularization in the tumor tissue. Levels of human soluble interleukin two receptor in the serum (reflecting the ATL tumor burden) of bevacizumab-treated mice were significantly lower than in untreated mice. Although bevacizumab monotherapy showed these clear anti-angiogenesis effects, it did not prolong survival. In contrast, injection of bevacizumab together with cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP) led to a significant prolongation of survival of the ATL mice relative to CHOP alone. CONCLUSIONS: This is the first report to evaluate the efficacy of bevacizumab for ATL in a tumor microenvironment-dependent model. Bevacizumab therapy combined with chemotherapy could be a valuable treatment strategy for that subgroup of ATL probably depending to a large extent on angiogenesis via vascular endothelial growth factor.

Global real-time quantitative reverse transcription-polymerase chain reaction detecting proto-oncogenes associated with 14q32 chromosomal translocation as a valuable marker for predicting survival in multiple myeloma.

CCND1, FGFR3 and c-MAF mRNA expression of tumor samples from 123 multiple myeloma patients were analyzed by global RQ/RT-PCR. CCND1, FGFR3 and c-MAF were positive in 44 (36%), 28 (23%) and 16 (13%) of patients, respectively. In 7 patients, both FGFR3 and c-MAF were positive. The expression of c-MAF was independent unfavorable prognostic factors for overall survival (OS). Autologous stem cell transplantation improved progression-free survival of CCND1-positive patients. Bortezomib, thalidomide or lenalidomide extended OS of FGFR3 and/or c-MAF-positive patients. Thus, CCND1, FGFR3 and c-MAF mRNA expression can predict survival and is useful for planning stratified treatment strategies for myeloma patients.

Autologous Tax-specific CTL therapy in a primary adult T cell leukemia/lymphoma cell-bearing NOD/Shi-scid, IL-2Rγnull mouse model.

We expanded human T-lymphotropic virus type 1 Tax-specific CTL in vitro from PBMC of three individual adult T cell leukemia/lymphoma (ATL) patients and assessed their therapeutic potential in an in vivo model using NOG mice bearing primary ATL cells from the respective three patients (ATL/NOG). In these mice established with cells from a chronic-type patient, treatment by i.p. injection of autologous Tax-CTL resulted in greater infiltration of CD8-positive T cells into each ATL lesion. This was associated with a significant decrease of ATL cell infiltration into blood, spleen, and liver. Tax-CTL treatment also significantly decreased human soluble IL-2R concentrations in the sera. In another group of ATL/NOG mice, Tax-CTL treatment led to a significant prolongation of survival time. These findings show that Tax-CTL can infiltrate the tumor site, recognize, and kill autologous ATL cells in mice in vivo. In ATL/NOG mice with cells from an acute-type patient, whose postchemotherapeutic remission continued for >18 mo, antitumor efficacy of adoptive Tax-CTL therapy was also observed. However, in ATL/NOG mice from a different acute-type patient, whose ATL relapsed after 6 mo of remission, no efficacy was observed. Thus, although the therapeutic effects were different for different ATL patients, to the best of our knowledge, this is the first report that adoptive therapy with Ag-specific CTL expanded from a cancer patient confers antitumor effects, leading to significant survival benefit for autologous primary cancer cell-bearing mice in vivo. The present study contributes to research on adoptive CTL therapy, which should be applicable to several types of cancer.

Oxidative stress augments pulmonary hypertension in chronically hypoxic mice overexpressing the oxidized LDL receptor.

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

Prediction of gestational diabetes mellitus by soluble (pro)renin receptor during the first trimester.

CONTEXT: There are currently no factors that have been shown to predict gestational diabetes mellitus (GDM) during early pregnancy. The soluble (pro)renin receptor [s(P)RR] may contribute to the development of GDM. OBJECTIVE: The objective of the study was to determine whether plasma s(P)RR concentrations during early pregnancy are associated with the development of GDM later in pregnancy. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study was conducted at a referral birth center. Pregnant women who first visited our hospital during the first trimester (<14 weeks of gestation) between 2010 and 2011 were enrolled. Inclusion criteria included singleton pregnancy and the absence of preexisting diabetes mellitus. A total of 716 women participated in this study. MAIN OUTCOME MEASURE: The association of plasma s(P)RR concentrations with the onset of GDM later in pregnancy was measured. RESULTS: Among 716 participants, 44 (6.1%) had GDM and 672 (93.9%) did not. There were 176 participants in the first plasma s(P)RR concentration quartile (Q1: < 25.8 ng/mL), 179 in the second (Q2: 25.8-30.2 ng/mL), 181 in the third (Q3: 30.2-34.2 ng/mL), and 180 in the fourth (Q4: > 34.2 ng/mL). GDM distribution was 7 (4.0%) in Q1, 5 (2.8%) in Q2, 13 (7.2%) in Q3, and 19 (10.6%) in Q4. A multivariate model adjusted for baseline characteristics, medical complications, and gestational characteristics revealed that the risk of developing GDM among women in Q4 compared with Q1 was 2.90 (95% confidence interval 1.11-7.49). CONCLUSION: Increased s(P)RR concentrations during the first trimester may predict the development of GDM later in pregnancy.

Angioimmunoblastic T-cell lymphoma mice model.

We established an angioimmunoblastic T-cell lymphoma (AITL) mouse model using NOD/Shi-scid, IL-2Rγ(null) mice as recipients. The immunohistological findings of the AITL mice were almost identical to those of patients with AITL. In addition, substantial amounts of human immunoglobulin G/A/M were detected in the sera of the AITL mice. This result indicates that AITL tumor cells helped antibody production by B cells or plasma cells. This is the first report of reconstituting follicular helper T (TFH) function in AITL cells in an experimental model, and this is consistent with the theory that TFH cell is the cell of origin of AITL tumor cells.

Heterogeneous electrophysiological and morphological properties of neurons in the mouse medial amygdala in vitro.

Neurons in the medial nucleus of the amygdala (MeA) play a key role in the innate maternal, reproductive, defensive, and social behaviors. However, it is unclear how activation of the vomeronasal system leads to the behavioral outputs that are associated with pheromones. Here, we characterized the electrophysiological and morphological properties of MeA neurons using whole-cell recordings in mice slice preparations. Biocytin labeling revealed that MeA neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. In 70% of recorded MeA neurons, monosynaptic excitatory postsynaptic currents (EPSCs) were evoked from the accessory olfactory bulb afferent in which the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate component was dominant and was rarely followed by the N-methyl-d-aspartic acid component. Norepinephrine increased the frequency of spontaneous inhibitory postsynaptic currents in some neurons, whereas α-methyl-5-hydroxytryptamine increased spontaneous EPSCs in other neurons. Morphologically and physiologically, heterogeneous MeA neurons appear likely to produce multiplex outputs of instinctive behaviors.