A Case of Minimal Change Nephrotic Syndrome Secondary to Methotrexate-associated Hodgkin Lymphoma.

The patient was a 64-year-old man who had been receiving methotrexate (MTX) for rheumatoid arthritis for 8 years. Computed tomography (CT) obtained one month prior to admission revealed numerous enlarged lymph nodes. Lower leg edema appeared two weeks prior to admission. Severe proteinuria was confirmed, and the patient was admitted. A renal biopsy revealed minimal changes in glomeruli. CT on day 14 revealed shrinking lymph nodes, and the patient was diagnosed with Hodgkin lymphoma by a neck lymph node biopsy. This is the first report of secondary minimal change nephrotic syndrome caused by an MTX-associated lymphoproliferative disorder.

Discovery of lipid profiles in plasma-derived extracellular vesicles as biomarkers for breast cancer diagnosis.

Lipids are a major component of extracellular vesicles; however, their significance in tumorigenesis and progression has not been well elucidated. As we previously found that lipid profiles drastically changed in breast tumors upon progression, we hypothesized that lipid profiles of plasma-derived extracellular vesicles could be utilized as breast cancer biomarkers. Here, we adopted modified sucrose cushion ultracentrifugation to isolate plasma-derived extracellular vesicles from breast cancer (n = 105), benign (n = 11), and healthy individuals (n = 43) in two independent cohorts (n = 126 and n = 33) and conducted targeted lipidomic analysis. We established a breast cancer diagnostic model comprising three lipids that showed favorable performance with the area under the receiver operating characteristic curve of 0.759, 0.743, and 0.804 in the training, internal validation, and external test sets, respectively. Moreover, we identified several lipids that could effectively discriminate breast cancer progression and subtypes: phosphatidylethanolamines and phosphatidylserines were relatively higher in Stage III, whereas phosphatidylcholines and sphingomyelins were higher in Stage IV; phosphatidylcholines and ceramides were correspondingly concentrated in HER2-positive patients, while lysophosphatidylcholines and polyunsaturated triglycerides were concentrated in the triple-negative breast cancer subtype. Lipid profiling of plasma-derived extracellular vesicles is a non-invasive and promising approach for diagnosing, staging, and subtyping breast cancer.

Tubulointerstitial nephritis with IgA kappa-positive plasma cells in a patient with primary Sjögren's syndrome and monoclonal gammopathy.

This is a case report of a 69-year-old Japanese man who has been undergoing treatment for primary Sjögren's syndrome (pSS) since he was 62 years. A renal biopsy, which revealed diffuse and severe mononuclear cell infiltration in the tubulointerstitium, was performed because of progressive renal dysfunction. Immunostaining demonstrated most of the infiltrating cells to be IgA, kappa, CD38, and CD138 positive. Immunofixation blood test revealed IgA kappa-type M protein; however, bone marrow abnormalities or lymph node enlargements on examination or imaging, respectively, were not observed. Tubulointerstitial nephritis caused by monotypic plasmacytic infiltration in pSS, accompanied with a monoclonal gammopathy of undetermined significance (MGUS), was diagnosed. A treatment of prednisolone 40 mg/day was initiated, promptly improving the patient's serum creatinine levels from 3.0 to 1.5 mg/dl. The infiltrating cells in pSS-associated tubulointerstitial nephritis are generally polytypic plasmacytes and lymphocytes, but in the present case, monotypic plasmacytes were predominant. This case is remarkable and rare and can be considered a complication of pSS or MGUS. Since it may become a new disease entity, it is important to accumulate similar cases.

Combination bezafibrate and nivolumab treatment of patients with advanced non-small cell lung cancer.

Despite the success of cancer immunotherapies such as programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) inhibitors, patients often develop resistance. New combination therapies with PD-1/PD-L1 inhibitors are needed to overcome this issue. Bezafibrate, a ligand of peroxisome proliferator-activated receptor-γ coactivator 1α/peroxisome proliferator-activated receptor complexes, has shown a synergistic antitumor effect with PD-1 blockade in mice that is mediated by activation of mitochondria in T cells. We have therefore now performed a phase 1 trial (UMIN000017854) of bezafibrate with nivolumab in previously treated patients with advanced non-small cell lung cancer. The primary end point was the percentage of patients who experience dose-limiting toxicity, and this combination regimen was found to be well tolerated. Preplanned comprehensive analysis of plasma metabolites and gene expression in peripheral cytotoxic T cells indicated that bezafibrate promoted T cell function through up-regulation of mitochondrial metabolism including fatty acid oxidation and may thereby have prolonged the duration of response. This combination strategy targeting T cell metabolism thus has the potential to maintain antitumor activity of immune checkpoint inhibitors and warrants further validation.

Spermidine activates mitochondrial trifunctional protein and improves antitumor immunity in mice.

Spermidine (SPD) delays age-related pathologies in various organisms. SPD supplementation overcame the impaired immunotherapy against tumors in aged mice by increasing mitochondrial function and activating CD8+ T cells. Treatment of naïve CD8+ T cells with SPD acutely enhanced fatty acid oxidation. SPD conjugated to beads bound to the mitochondrial trifunctional protein (MTP). In the MTP complex, synthesized and purified from Escherichia coli, SPD bound to the α and ß subunits of MTP with strong affinity and allosterically enhanced their enzymatic activities. T cell-specific deletion of the MTP α subunit abolished enhancement of programmed cell death protein 1 (PD-1) blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.

B cell-derived GABA elicits IL-10+ macrophages to limit anti-tumour immunity.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8+ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses.

Discovery of a Macropinocytosis-Inducing Peptide Potentiated by Medium-Mediated Intramolecular Disulfide Formation.

Macropinocytosis is a ubiquitous cellular uptake mechanism of peptide-based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8-residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

Combination of host immune metabolic biomarkers for the PD-1 blockade cancer immunotherapy.

BACKGROUNDCurrent clinical biomarkers for the programmed cell death 1 (PD-1) blockade therapy are insufficient because they rely only on the tumor properties, such as programmed cell death ligand 1 expression frequency and tumor mutation burden. Identifying reliable, responsive biomarkers based on the host immunity is necessary to improve the predictive values.METHODSWe investigated levels of plasma metabolites and T cell properties, including energy metabolism markers, in the blood of patients with non-small cell lung cancer before and after treatment with nivolumab (n = 55). Predictive values of combination markers statistically selected were evaluated by cross-validation and linear discriminant analysis on discovery and validation cohorts, respectively. Correlation between plasma metabolites and T cell markers was investigated.RESULTSThe 4 metabolites derived from the microbiome (hippuric acid), fatty acid oxidation (butyrylcarnitine), and redox (cystine and glutathione disulfide) provided high response probability (AUC = 0.91). Similarly, a combination of 4 T cell markers, those related to mitochondrial activation (PPARγ coactivator 1 expression and ROS), and the frequencies of CD8+PD-1hi and CD4+ T cells demonstrated even higher prediction value (AUC = 0.96). Among the pool of selected markers, the 4 T cell markers were exclusively selected as the highest predictive combination, probably because of their linkage to the abovementioned metabolite markers. In a prospective validation set (n = 24), these 4 cellular markers showed a high accuracy rate for clinical responses of patients (AUC = 0.92).CONCLUSIONCombination of biomarkers reflecting host immune activity is quite valuable for responder prediction.FUNDINGAMED under grant numbers 18cm0106302h0003, 18gm0710012h0105, and 18lk1403006h0002; the Tang Prize Foundation; and JSPS KAKENHI grant numbers JP16H06149, 17K19593, and 19K17673.

Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity.

Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor. These CTLs carry not only the activation of mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) but also an increment of their downstream transcription factors such as PPAR-gamma coactivator 1α (PGC-1α) and T-bet. Furthermore, direct activators of mTOR, AMPK, or PGC-1α also synergized the PD-1 blockade therapy whereas none of above-mentioned chemicals alone had any effects on tumor growth. These findings will pave a way to developing novel combinatorial therapies with PD-1 blockade.

The kinase Pyk2 is involved in renal fibrosis by means of mechanical stretch-induced growth factor expression in renal tubules.

Unilateral ureteral obstruction is a well-established experimental model of progressive renal fibrosis. We tested whether mechanical stretch and subsequent renal tubular distension might lead to renal fibrosis by first studying renal tubular epithelial cells in culture. We found that mechanical stretch induced reactive oxygen species that in turn activated the cytoplasmic proline-rich tyrosine kinase-2 (Pyk2). This kinase is abundantly expressed in tubular epithelial cells where it is activated by several stimuli. Using mice with deletion of Pyk2 we found that the expression of transforming growth factor-ß1 induced by mechanical stretch in renal tubular epithelial cells was significantly reduced. The expression of connective tissue growth factor was also reduced in the Pyk2(-/-) mice. We also found that expression of connective tissue growth factor was independent of transforming growth factor-ß1, but dependent on the Rho-associated coiled-coil forming protein kinase pathway. Thus, Pyk2 may be an important initiating factor in renal fibrosis and might be a new therapeutic target for ameliorating renal fibrosis.

Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment.

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.

A method for terminus proteomics: selective isolation and labeling of N-terminal peptide from protein through transamination reaction.

A novel method for selectively labeling and isolating N-terminal peptide from protein has been developed. An N(alpha)-amino group of protein was converted to a carbonyl group through transamination reaction and the resulting carbonyl group was modified with O-(4-nitrobenzyl)hydroxylamine (NBHA). After proteolytic digestion using Grifola frondosa metalloendopeptidase (LysN), the modified N-terminal peptide remained unbound in the following treatment using amino-reactive p-phenylenediisothiocyanate (DITC) glass, whereas peptides other than the N-terminal peptide were effectively scavenged from the supernatant solution. The modified N-terminal peptide was thus successfully isolated and sequenced by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.

Peritoneal mesothelial cells as a target of local aldosterone action: upregulation of connective tissue growth factor expression via serum- and glucocorticoid-inducible protein kinase 1.

BACKGROUND/AIMS: Peritoneal fibrosis can lead to the discontinuation of continuous ambulatory peritoneal dialysis. The present study investigated the direct effect of aldosterone, which influences tissue fibrosis, and its cellular mechanism using cultured rat peritoneal mesothelial cells (RPMCs). MATERIALS AND METHODS: The expression of aldosterone synthase (CYP11B2), mineralocorticoid receptors, 11beta-hydroxysteroid dehydrogenase 2, serum- and glucocorticoid-inducible protein kinase 1 (SGK1) and connective tissue growth factor (CTGF) was evaluated using reverse transcriptase-polymerase chain reaction and Western blot. The ability of RPMCs to produce aldosterone was examined by enzyme immunoassay. Small interfering RNA of SGK1 was transfected to determine the role of SGK1. RESULTS: CYP11B2, mineralocorticoid receptors and 11beta-hydroxysteroid dehydrogenase 2 were expressed in RPMCs. The release of aldosterone from RPMCs into the culture medium was confirmed. Stimulation of RPMCs with the addition of aldosterone significantly increased SGK1 expression and phosphorylation and CTGF upregulation, and these effects were completely inhibited by the mineralocorticoid receptor antagonist spironolactone. SGK1 gene silencing abrogated aldosterone-induced CTGF expression. CONCLUSION: The local aldosterone system exists and acts directly as a profibrotic factor in the peritoneal mesothelium.

The specific isolation of C-terminal peptides of proteins through a transamination reaction and its advantage for introducing functional groups into the peptide.

A novel method for isolating C-terminal peptides from proteolytic digests of proteins was developed. Proteins were digested with lysyl endopeptidase (LysC) and applied to metal-ion-catalyzed transamination reactions. This reaction enabled the selective conversion of an Nalpha-amino group to a carbonyl group. Subsequent incubation with p-phenylenediisothiocyanate (DITC) glass effectively scavenged the lysine-containing N-terminus and internal peptides. The obtained C-terminal peptide is open to modification with reagents having virtually any type of functionality owing to the reactive alpha-ketocarbonyl group. In this report, 2,4-dinitrophenylhydrazine (DNPH) was used as an example of a nucleophile to the carbonyl group. The isolated C-terminal peptide was modified with DNPH, which exhibited signal enhancement, and was sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

An improved method for de novo sequencing of arginine-containing, Nalpha-tris(2,4,6-trimethoxyphenyl)phosphonium-acetylated peptides.

An improved method for de novo sequencing of arginine-containing peptides modified with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) is reported. A tagging reagent, TMPP-Ac-OSu, was introduced to improve the sequence analysis of peptides owing to the simplified fragmentation pattern. However, peptides containing arginine residues did not fragment efficiently even after TMPP-Ac modification at their N-termini. This report describes how fragmentation efficiency of TMPP-Ac-modified arginine-containing peptides was significantly improved by modifying the guanidino group on the side chain of arginine with acetylacetone.

A case report of Mycobacterium abscessus peritonitis in a peritoneal dialysis patient.

Peritonitis due to nontuberculous mycobacterium in peritoneal dialysis (PD) patients is rare. However, when it occurs, PD catheter removal is required in most cases because of resistance to antibiotic therapy. We report a case of Mycobacterium abscessus peritonitis subsequent to tunnel infection after PD catheter-replacement surgery. The patient underwent this surgery as her tunnel infection had not resolved following the usual 3 month course of antibiotic therapy. After surgery, tunnel infection of the second catheter and peritonitis occurred. Nontuberculous mycobacteria were detected on acid-fast stain from both the old and new exit-site drainage and the peritoneal effluent. The mycobacteria were identified as M. abscessus. Removal of the new catheter and surgical excision of the previous catheter tunnel were performed and multiple antibiotics were started. After 3 months the postsurgical wounds had healed completely. This case demonstrates the importance of further evaluation of unidentified PD catheter-related infections, including an examination for nontuberculous mycobacterium.

Transmission of extramembrane conformational change into current: construction of metal-gated ion channel.

Interaction with Fe(III) induces the reversible conformational switch of the extramembrane segment in the artificial receptor channel, which is transmitted into membranes as an increase in channel current (ion flux).

Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement.

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

Acute tubulointerstitial nephritis associated with autoimmune-related pancreatitis.

Various etiologic factors have been identified in tubulointerstitial nephritis (TIN), including allergic drug reaction, infection, and immune-mediated disease. Immune-mediated TIN without significant glomerular involvement has been reported to occur as a renal complication secondary to Sjögren's syndrome, lupus nephritis, and antitubular basement membrane antibody-related disease. We present a first case of acute TIN associated with autoimmune-related pancreatitis (AIP). A 64-year-old man was referred to our division from a surgeon for the close examination of renal dysfunction. The pancreatic and biliary imaging showed segmental narrowing of the pancreatic duct with localized swelling of the pancreatic head, suggesting the carcinoma of the pancreatic head at that time. However, the laboratory findings also showed renal dysfunction with high level of serum immunoglobulin G and hypocomplementemia. Renal biopsy was performed to investigate the etiology of the renal dysfunction. The renal biopsy specimen showed acute TIN. The patient had no drug history, which may cause TIN. Oral corticosteroid therapy improved the renal function as well as histological damage, the pancreatic imaging study, and the laboratory tests of pancreatic and hepatobiliary enzyme. Although the pancreatic biopsy has not performed in our patient, his clinical course confirmed us that AIP was the final diagnosis for his pancreatic lesion. Despite further examination, there was no evidence of other autoimmune-related diseases such as Sjögren's syndrome. To our knowledge, this is the first report of acute TIN associated with AIP. We suggest that AIP may be an etiologic factor in some cases of TIN.