High incidence and early onset of nivolumab-induced pneumonitis: four case reports and literature review.

BACKGROUND: Nivolumab, an anti-programmed cell death-1 (PD-1) monoclonal antibody used as an immune checkpoint inhibitor, is commonly employed for its anti-tumor effects against various types of malignant tumors. However, its administration is complicated by immune-related adverse events (irAEs), including pneumonitis. CASE PRESENTATION: We present a case series of four patients with malignant melanoma, non-small cell lung cancer, and hypopharyngeal carcinoma who demonstrated pneumonitis induced by nivolumab, and further review clinicopathological characteristics of these patients in comparison with those of previously reported patients with nivolumab-induced pneumonitis. In our series, 20% of patients who were treated with nivolumab developed pneumonitis, all of which occurred approximately 2 weeks after the initiation of nivolumab treatment. Prompt recognition of the nivolumab-induced pneumonitis allowed for successful resolution. Computed tomography scan images of the patients demonstrated predominantly cryptogenic organizing pneumonia patterns. All patients were males, who had been heavily treated with antitumor drugs prior to nivolumab. CONCLUSIONS: Our case series showed that nivolumab had a high incidence of drug-induced pneumonitis with early onset, supporting the need for renewed attention to nivolumab-induced pneumonitis, particularly in patients with a history of heavy antitumor treatments.

Individual differences in the distribution of sperm acrosome-associated 1 proteins among male patients of infertile couples; their possible impact on outcomes of conventional in vitro fertilization.

The aims of this study were to show the existence of individual differences in the distribution of sperm acrosome-associated 1 (SPACA1) among male patients of infertile couples and to examine their possible impact on the outcomes of conventional in vitro fertilization (IVF). The spermatozoa were collected from male patients of infertile couples, washed by centrifugation, collected by the swim-up method, and then used for clinical treatments of conventional IVF. The surplus sperm samples were fixed and stained with an anti-SPACA1 polyclonal antibody for the immunocytochemistry. In the clinical IVF treatments, fertilization rates and blastocyst development rates were evaluated. The immunocytochemical observations revealed that SPACA1 were localized definitely in the acrosomal equatorial segment and variedly in the acrosomal principal segment. Specifically, the detection patterns of SPACA1 in the acrosomal principal segment could be classified into three categories: (A) strong, (B) intermediate or faint, and (C) almost no immunofluorescence. The SPACA1 indexes were largely different among male patients with the wide range from 13 to 199 points. The SPACA1 indexes were significantly correlated with developmental rates of embryos to blastocysts (r = 0.829, P = 0.00162), although they were barely associated with fertilization rates at 19 h after insemination (r = 0.289, P = 0.389). These results suggest that the distribution of SPACA1 in sperm affects the outcomes of conventional IVF. In conclusion, this study provides initial data to promote large-scale clinical investigation to demonstrate that the SPACA1 indexes are valid as molecular biomarkers that can predict the effectiveness of conventional IVF of infertile couples.

Cultural adaptation, content, and protocol of a feasibility study of school-based "Let's learn about emotions" intervention for Finnish primary school children.

Introduction: Emotional awareness and emotion regulation are crucial for cognitive and socio-emotional development in children. School-based interventions on socio-emotional skills have the potential to prevent these problems and promote well-being of children. The Japanese school-based program, Universal Unified Prevention Program for Diverse Disorders (Up2-D2), has shown preventive effects on mental health of children in Japan. The aims of this protocol paper are to describe the unique process of adapting the Up2-D2 from Eastern to Western context, and to present a feasibility study of the intervention, conducted in Finland. Methods: The cultural adaptation process started with the linguistic translation of materials, followed by the modification of language to fit the Finnish context. While the Japanese ideology was saved, some content was adapted to fit Finnish school children. Further modifications were made based on feedback from pupils and teachers. The Finnish version of the program was named "Let's learn about emotions" and consisted of 12 sessions and targeted 8- to 12-year-old pupils. A teacher education plan was established to assist Finnish teachers with the intervention, including a workshop, teachers' manual, brief introductory videos, and online support sessions. A feasibility study involving 512 4th graders in the City of Hyvinkää, South of Finland, was conducted. It assessed emotional and behavioral problems, classroom climate, bullying, loneliness, perception of school environment, knowledge of emotional awareness, and program acceptability. Discussion: The originality of this study underlies in the East-West adaptation of a cognitive behavioral therapy-based program. If promising feasibility findings are replicated in Finland, it could pave the way for further research on implementing such programs in diverse contexts and cultures, promoting coping skills, awareness, social skills and early prevention of child mental health problems. Ethics: The ethical board of the University of Turku gave ethics approval for this research. The educational board of the City of Hyvinkää accepted this study.

Insulin-mediated regulation of decidual protein induced by progesterone (DEPP) in adipose tissue and liver.

We analyzed the profile of the genes expressed in human adipose tissue and identified the fat-derived molecules, adiponectin and aquaporin 7, which modulate glucose and lipid metabolism. The same Bodymap analysis revealed abundant expression of the decidual protein induced by progesterone (DEPP) in the white adipose tissue. Northern blot analysis confirmed that human DEPP mRNA was highly expressed in white adipose tissue. Mouse DEPP mRNA was detected in heart, lung, skeletal muscle, and white adipose tissue under feeding state. In contrast, under fasting state, mouse DEPP mRNA was enhanced in lung, skeletal muscle, and white adipose tissue and it appeared also in the liver and kidney, suggesting up regulation of DEPP by fasting. Because fasting-induced DEPP expression was observed in insulin-sensitive organs, we investigated the regulation of DEPP in white adipose tissue and liver. During adipogenesis of mouse 3T3-L1 cells, DEPP mRNA increased in a differentiation-dependent manner similar to adiponectin and aquaporin 7. Treatment of cultured 3T3-L1 mature adipocytes, rat H4IIE, and human HepG2 hepatoma cells with insulin significantly decreased DEPP mRNA levels in dose- and time-dependent manners. IN VIVO experiments showed significant decrease of hepatic and adipose DEPP mRNA levels in refed mice, compared to fasted animals, and also showed significant increase in DEPP mRNA in streptozotocin-induced insulin-deficient diabetic mice. These results indicate that DEPP is a novel insulin-regulatory molecule expressed abundantly in insulin-sensitive tissues including white adipose tissue and liver.

Amino acids and the spikes from the retinal ganglion cells.

The effects of amino acids or mixtures of amino acids on the spike discharges from the optic ganglion cells were studied. Mixtures of amino acids have different effects than single amino acids. Proper composition of excitatory and inhibitory amino acids can enhance only the light-induced spikes without giving rise to any spontaneous discharges. It is implied that amino acids may play an important role in the regulation of the general sensitivity of cells in the central nervous system.

Improved differentiation of Mycobacterium tuberculosis strains, including many Beijing genotype strains, using a new combination of variable number of tandem repeats loci.

Variable number of tandem repeats (VNTR) typing was done on 230 Mycobacterium tuberculosis strains, including 41 strains isolated from 17 groups of epidemiologically linked patients. By PCR amplification, 185 (80.4%) of the 230 strains were Beijing genotype strains. VNTR typing was performed using the 15 loci proposed as a standard set by Supply et al. [Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., Rusch-Gerdes, S., Willery, E., Savine, E., de Haas, P., van Deutekom, H., Roring, S., Bifani, P., Kurepina, N., Kreiswirth, B., Sola, C., Rastogi, N., Vatin, V., Gutierrez, M.C., Fauville, M., Niemann, S., Skuce, R., Kremer, K., Locht, C., van Soolingen, D., 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol. 44, 4498-4510], and cluster analyses of these data were done. By the VNTR typing with the proposed 15 loci, strains having low similarity values by restriction fragment length polymorphism (RFLP) analysis were clustered. Use of a supplemental9 loci, proposed as a high-resolution tool, with the 15 loci showed that strains with low similarity by RFLP analysis were still clustered. Twelve VNTR loci were selected based on previously reported discriminatory index (DI) values and used with the proposed 15 loci for better differentiation by VNTR typing. When eight loci with higher DI values were used with the 15 loci, there were no clusters, including strains with low RFLP similarity. The15 loci and eight additional loci decreased the numbers of clustered strains isolated from epidemiologically unlinked patients significantly compared to using only the 15 loci. Among all tested loci, obvious differences of DI values were observed for 8 loci (miru10, miru16, miru39, Mtub29, Mtub30, QUB11a, QUB26, and QUB1895) of RD105 lineage strains compared to those of other lineage strains. These results suggest that the proposed VNTR typing method cannot be used as a routine epidemiological tool in areas where Beijing genotype strains are prevalent. Several VNTR loci should be added to the proposed method based on differences in polymorphism of VNTR loci among Beijing genotype lineages.

In vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody.

In studies on antitumor antibody:drug conjugates as potential antitumor agents, methotrexate (MTX) was conjugated with a murine monoclonal antibody (aMM46) to an antigen on ascitic mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary. MTX was linked to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA by dithiothreitol treatment followed by oxidation on standing at 4 degrees C. The MTX linking was performed, without protection of the thiol group of HSA, by using MTX N-succinimidyl ester prepared via MTX intramolecular anhydride. The resulting HSA:MTX was reacted with the immunoglobulin with the maleimide group introduced. The aMM46:HSA:MTX obtained retained both antibody binding and drug activities. The cytotoxicity of aMM46:HSA:MTX against MM antigen-positive MM46 cells was greater than that of control 96.5 (anti-human melanoma-associated antigen, p97):HSA:MTX and was inhibited by unconjugated aMM46. No different cytotoxicity of aMM46:HSA:MTX compared with that of 96.5:HSA:MTX was observed against MM antigen-negative mouse mammary tumor MM48 cells. The presence of ammonium chloride or leupeptin abrogated the selective cytotoxicity against MM46 cells of aMM46 conjugate but did not affect the nonspecific cytotoxicity of 96.5:HSA:MTX. These results support the idea that the selective cytotoxicity of aMM46:HSA:MTX is antibody directed and exhibited through lysosomal degradation of the conjugate.

Nature of linkage and mode of action of methotrexate conjugated with antitumor antibodies: implications for future preparation of conjugates.

The binding of methotrexate (MTX) to IgG in conjugates was examined by studies on a direct MTX conjugate with a monoclonal antibody (aMM46) to mouse mammary tumor MM46 cells and corresponding irrelevant antibody and normal gamma-globulin conjugates, all prepared by the active ester method with MTX N-succinimidyl ester (MTX-OSu). The binding was examined in terms of effects on the potency and selectivity of the cytotoxic activity of the aMM46 conjugate. The results obtained supported the following conclusions: (a) MTX-OSu reacts not only with the amino group of IgG to give an amide bond, but also with another group(s) to give a less stable bond(s) such as an ester bond; (b) in contrast to the amide bond-linked MTX, which is taken up by the cells by endocytic internalization, a substantial portion of the MTX linked by an ester or other less stable bond(s) is released from the conjugates extracellularly and enters the cells by the MTX active transport system, as revealed by the inhibitory effect of thiamine pyrophosphate on the cytotoxicity; (c) this MTX linked by a less stable bond(s) that causes nonspecific cytotoxicity can be removed by treatment with hydroxylamine; (d) the overall cytotoxicity of aMM46-MTX decreased on removal of this less stably linked MTX, suggesting that the lysosomal degradation of the conjugate carrying amide bond-linked MTX to liberate MTX derivatives of low molecular weight is insufficient; (e) the liberation of low-molecular-weight substances in the lysosomes is probably more important for efficient entry of active substances into the cytosol, than for inhibition of the activity of dihydrofolate reductase, because after hydroxylamine treatment, the amide bond-linked MTX showed greater decrease in drug cytotoxicity than in inhibitory activity against dihydrofolate reductase; (f) in combination with hydroxylamine treatment, insertion between MTX and IgG of a linkage capable of ready cleavage in lysosomes deserves exploitation as a method for making potent conjugates with less nonspecific activity.

Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages.

BACKGROUND: Excessive lipid accumulation in macrophages plays an important role in the development of atherosclerosis. Recently, we discovered an adipocyte-specific plasma protein, adiponectin, that is decreased in patients with coronary artery disease. We previously demonstrated that adiponectin acts as a modulator for proinflammatory stimuli and inhibits monocyte adhesion to endothelial cells. The present study investigated the effects of adiponectin on lipid accumulation in human monocyte-derived macrophages. METHODS AND RESULTS: Human monocytes were differentiated into macrophages by incubation in human type AB serum for 7 days, and the effects of adiponectin were investigated at different time intervals. Treatment with physiological concentrations of adiponectin reduced intracellular cholesteryl ester content, as determined using the enzymatic, fluorometric method. The adiponectin-treated macrophages contained fewer lipid droplets stained by oil red O. Adiponectin suppressed the expression of the class A macrophage scavenger receptor (MSR) at both mRNA and protein levels by Northern and immunoblot analyses, respectively, without affecting the expression of CD36, which was quantified by flow cytometry. Adiponectin reduced the class A MSR promoter activity, as measured by luciferase reporter assay. Adiponectin treatment dose-dependently decreased class A MSR ligand binding and uptake activities. The mRNA level of lipoprotein lipase as a marker of macrophage differentiation was decreased by adiponectin treatment, but that of apolipoprotein E was not altered. Adiponectin was detected around macrophages in the human injured aorta by immunohistochemistry. CONCLUSIONS: The adipocyte-derived plasma protein adiponectin suppressed macrophage-to-foam cell transformation, suggesting that adiponectin may act as a modulator for macrophage-to-foam cell transformation.

PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein.

Insulin resistance and its dreaded consequence, type 2 diabetes, are major causes of atherosclerosis. Adiponectin is an adipose-specific plasma protein that possesses anti-atherogenic properties, such as the suppression of adhesion molecule expression in vascular endothelial cells and cytokine production from macrophages. Plasma adiponectin concentrations are decreased in obese and type 2 diabetic subjects with insulin resistance. A regimen that normalizes or increases the plasma adiponectin might prevent atherosclerosis in patients with insulin resistance. In this study, we demonstrate the inducing effects of thiazolidinediones (TZDs), which are synthetic PPARgamma ligands, on the expression and secretion of adiponectin in humans and rodents in vivo and in vitro. The administration of TZDs significantly increased the plasma adiponectin concentrations in insulin resistant humans and rodents without affecting their body weight. Adiponectin mRNA expression was normalized or increased by TZDs in the adipose tissues of obese mice. In cultured 3T3-L1 adipocytes, TZD derivatives enhanced the mRNA expression and secretion of adiponectin in a dose- and time-dependent manner. Furthermore, these effects were mediated through the activation of the promoter by the TZDs. On the other hand, TNF-alpha, which is produced more in an insulin-resistant condition, dose-dependently reduced the expression of adiponectin in adipocytes by suppressing its promoter activity. TZDs restored this inhibitory effect by TNF-alpha. TZDs might prevent atherosclerotic vascular disease in insulin-resistant patients by inducing the production of adiponectin through direct effect on its promoter and antagonizing the effect of TNF-alpha on the adiponectin promoter.

Visceral fat is a major contributor for multiple risk factor clustering in Japanese men with impaired glucose tolerance.

OBJECTIVE: The significance of abdominal visceral fat accumulation was evaluated in Japanese men with impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS: The IGT subjects (n = 123) were aged 55 +/- 9 years with a BMI of 24 +/- 3 kg/m(2). The 148 control subjects with normal glucose tolerance (NGT) were matched for age and BMI. IGT and NGT were classified according to the 1985 World Health Organization criteria. Abdominal fat distribution was analyzed by computed tomography at umbilical level. Plasma lipid, glucose, and insulin concentrations and blood pressure (BP) were measured. RESULTS: In subjects with IGT, the average visceral fat area (VFA) was significantly greater than in subjects with NGT. Fasting insulin, the sum of insulin concentrations during an oral glucose tolerance test, insulin resistance according to a homeostasis model assessment for insulin resistance (HOMA-IR), systolic BP, and serum triglyceride were significantly higher, whereas the DeltaI(30-0)/DeltaG(30-0) was significantly lower, in subjects with IGT. Subjects with IGT and NGT were then divided into three subgroups according to the number of risk factors they possessed (dyslipidemia, hypertension, neither, or both). In both IGT and NGT subjects, BMI, VFA, subcutaneous fat area, fasting insulin, HOMA-IR, and insulin secretion of the homeostasis model assessment were significantly higher in the double-risk factor subgroup than in the no-risk factor subgroup, and VFA was a potent and independent variable in association with the presence of a double risk factor. CONCLUSIONS: Visceral fat accumulation is a major contributor for multiple risk factor clustering in Japanese men with IGT and NGT.

Some properties of inorganic pyrophosphatase from Bacillus subtilis.

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1; PPase) from Bacillus subtilis was purified to a homogeneous state electrophoretically when analysed by SDS-PAGE. The enzyme consists of six identical subunits; the molecular weight of the native enzyme estimated by gel filtration was approx. 120,000, and denaturing polyacrylamide gel electrophoresis gave a single band corresponding to 24,000. The enzyme absolutely required a divalent cation for its activity. Mg2+ was most effective, showing two steps of concentration-dependent activation. Mg2+ could be partially replaced by Mn2+ and Co2+. The enzyme was thermostable in the presence of Mg2+, and no loss of activity was observed on the incubation at 55 degrees C for an hour.

Measurement of retinal permeability to sodium fluorescein in vitro.

Fluorescein permeabilities of isolated dog retinal pigment epithelium (RPE)-choroid and sensory retina were measured individually. Retina to choroid (outward) permeability of sodium fluorescein was significantly larger than choroid to retina (inward) permeability at RPE-choroid under both open- and short-circuit conditions. Outward permeability was decreased by the addition of 10(-3) M KCN and of 10(-4) M probeneside but was not affected by 10(-6) M ouabain. These drugs decreased transepithelial potential and short-circuit current. No directional difference of fluorescein permeability for sensory retina was found. Active transport of fluorescein across RPE from retina to choroid is suggested. Fluorescein permeability of sensory retina was larger than inward but less than outward permeability of RPE-choroid. The result indicates that the sensory retina acts as a diffusion barrier for sodium fluorescein.

Resistance of H1 histone to proteolytic attack in chromatin from rat-ascites hepatoma.

From rat-liver and ascites-hepatoma chromatins, NaCl-soluble fractions were prepared. The 0.35 M NaCl-soluble fraction from the hepatoma (AH) chromatin contained much non-histone protein of high-molecular weight, compared with the fraction from the rat-liver (RL) chromatin. The 0.35 M NaCl-insoluble, but 2 M NaCl/5 M urea-soluble fraction was composed mainly of 5 classes of histones. These histones were quantitatively not different between AH and RL chromatins. However, H1 histone was rather protease-resistant in AH chromatin, but not in RL chromatin. The proteolytic capacity was also lower in AH chromatin. In addition, in the micrococcal-nuclease digest of AH nuclei, the oligonucleosomes were considerably retained even by long-time digestion, but not in that of RL nuclei.

Cerebral myxopapillary ependymoma.

A rare case of myxopapillary ependymoma is reported. The tumor occurred in the cerebral hemisphere of an 8-year-old girl and had no relationship to the lateral ventricles. Microscopically, it showed abundant mucin production around papillary or reticular structures. Immunohistochemically, these tumor cells were weakly positive, with glial fibrillary acidic protein demonstrated in part of the tumor and vimentin strongly demonstrated throughout the tumor. The results may indicate the poorly differentiated nature of this tumor. This is the second reported case of intracranial myxopapillary ependymoma.

Importance of the antigen-binding valency and the nature of the cross-linking bond in ricin A-chain conjugates with antibody.

As a continuation of our work on toxin A-chain conjugates with antitumor antibodies for selective delivery of the toxin to the target cells, four ricin A-chain conjugates were prepared by linking A-chain to Fab' or F(ab')2 of rabbit IgG against L1210 with or without employing a cross-linking agent, N,N'-o-phenylenedimaleimide (PDM), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N-succinimidyl m-(N-maleimido)benzoate (SMB), and the effects of antigen-binding valency and of the nature of the cross-linking bond on their in vitro cytotoxicity were studied. The relative potencies of the conjugates in terms of IC94's were as follows: F(ab')2-SPDP-A-chain, 100; Fab'-S-S-A-chain, 21; F(ab')2-SMB-A-chain, 1.3; Fab'-PDM-A-chain 0.38. Among the four conjugates, F(ab')2-SPDP-A-chain and Fab'-S-S-A-chain can be cleaved into the homing and the cytotoxic components with 2 mM 2-mercaptoethanol. These results suggest that divalency in antigen-binding and susceptibility of the cross-linking bond to cleavage by mercapto reagent are desirable for high potency. Protein synthesis in a cell-free system of rabbit reticulocyte lysate was inhibited by Fab'-S-S-A-chain and by Fab'-PDM-A-chain as effectively as by free A-chain, indicating that the liberation of A-chain is not important, at least on ribosomes, but it is important for the A-chain to reach a ribosome after binding of the conjugates to the cell-surface.

Matrix solid-phase dispersion extraction and high-performance liquid chromatographic determination of residual sulfonamides in chicken.

Simultaneous determination of the six sulfonamides (SAs) sulfadiazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline in chicken using matrix solid-phase dispersion (MSPD) with neutral aluminium oxide as an MSPD sorbent and high-performance liquid chromatography (HPLC) is presented. In the present MSPD, six SAs could be isolated by only one step, elution with a 70% (v/v) aqueous ethanol solution, without the sorbent conditioning and the sorbent-tissue matrix washing. For the HPLC determination, a LiChrospher 100 RP-8 and a mixture of 1% acetic acid solution (pH 3.0, in water)-acetonitrile-N,N-dimethylformamide (78:22:5, v/v/v) as the mobile phase with a photodiode array detector were used. Average recoveries were greater than 87.6% with relative standard deviations between 0.5 and 8.6%. The total time and amount of solvent required for the analysis of one sample were <1.5 h and <12 ml, respectively.

Glutathione conjugation of methazolamide and subsequent reactions in the ciliary body in vitro.

Conjugation reaction of methazolamide with glutathione and its subsequent reactions were studied in vitro. Glutathione, cysteinylglycine, and cysteine conjugates of methazolamide were chemically synthesized. All of the three compounds showed absorbance below 330 nm, with maximal absorbance at approximately 300 nm. At the wavelengths below 220 nm, absorbance was proportional to the number of the amino acids each compound had. Amino acid analysis of the glutathione conjugate showed that the conjugation reaction involved the cysteine residue of glutathione. In order to identify the chemical structure of the reaction product, cysteine conjugate was subjected to infrared, proton nuclear magnetic resonance, and mass spectral analyses. These studies indicated that the cysteine conjugate was S-(5-acetylimino-4-methyl-delta 2-1,3,4-thiadiazolinyl)cysteine. The reaction with glutathione was not catalyzed by glutathione S-transferases, but proceeded in the absence of the enzyme. The glutathione conjugate was degraded by bovine ciliary body homogenate to the cysteinylglycine conjugate and then to the cysteine conjugate.