Risk factors for CAR-T cell manufacturing failure among DLBCL patients: A nationwide survey in Japan

CAR-T細胞製造を成功させるためのレシピ --アフェレーシス前の下ごしらえでの工夫--. 京都大学プレスリリース. 2023-04-27.For successful chimeric antigen receptor T (CAR-T) cell therapy, CAR-T cells must be manufactured without failure caused by suboptimal expansion. In order to determine risk factors for CAR-T cell manufacturing failure, we performed a nationwide cohort study in Japan and analysed patients with diffuse large B-cell lymphoma (DLBCL) who underwent tisagenlecleucel production. We compared clinical factors between 30 cases that failed (7.4%) with those that succeeded (n = 378). Among the failures, the proportion of patients previously treated with bendamustine (43.3% vs. 14.8%; p < 0.001) was significantly higher, and their platelet counts (12.0 vs. 17.0 × 10⁴/μL; p = 0.01) and CD4/CD8 T-cell ratio (0.30 vs. 0.56; p < 0.01) in peripheral blood at apheresis were significantly lower than in the successful group. Multivariate analysis revealed that repeated bendamustine use with short washout periods prior to apheresis (odds ratio [OR], 5.52; p = 0.013 for ≥6 cycles with washout period of 3–24 months; OR, 57.09; p = 0.005 for ≥3 cycles with washout period of <3 months), low platelet counts (OR, 0.495 per 105/μL; p = 0.022) or low CD4/CD8 ratios (<one third) (OR, 3.249; p = 0.011) in peripheral blood at apheresis increased the risk of manufacturing failure. Manufacturing failure remains an obstacle to CAR-T cell therapy for DLBCL patients. Avoiding risk factors, such as repeated bendamustine administration without sufficient washout, and risk-adapted strategies may help to optimize CAR-T cell therapy for DLBCL patients

Analysis of the Nutrient Intake and the Nutritional and Health State of the people in a Nepalese Mountainous Village:Dithal VDC

【目的】世界辺境地区の一つであるネパール山間地方の住民の摂取栄養と健康状態について解析した。【方法】神戸常盤大学を中心に共同調査プロジェクト(JICA 草の根事業)を編成し、ネパールデタール村でのフィールドワークを行った。住民208名(年齢5～82歳、男女比82:126)の健康調査を施行し、身体計測とBIA法を用いた体構成成分測定、および血液検査(TP、Albumin、GPT、BUN、Ca)を行った。また住民の内科診療を行い、下痢などの消化管疾患の有無を調査した。さらに、一般家庭における日々の食事内容をデジタルカメラに撮影し、摂取熱量、摂取栄養素を計測すると同時に、村の採水所30ヵ所での飲水を含めた生活用水の細菌学的検査を行った。【結果】身体計測上、BMI<18.5の比率が50%を超え、12歳以下の子供では80%近くに及んだ。体構成成分測定では、特に子供で体内脂肪、筋肉量の減少と、体内水分量の増加が見られた。また一見肥満体系であっても、栄養障害による浮腫や、筋肉量の減少による下腹部の突出(クワシオルコル)を示す症例も、12歳以下の子供で見られた。血液検査は、血清総タンパク、アルブミンの低下以外は、ほぼ正常値内であった。食事は、ネパール特有のダル・バート・タルカリという炭水化物が中心の料理で、摂取栄養素の80%を占めた。1日2食が標準である現地人の平均摂取熱量は800～1000kcal程度と推計された。飲料水の細菌学的検査では、大腸菌、及び大腸菌群が80～100%の頻度で検出された。【結論】未だカースト制度の現存するネパールの山間地方における栄養状態と衛生環境は劣悪であったが、これは過去に先進国が経た道であり、自らの事として手を差しのべる必要があると考えられた。The research team jointed with Kobe Tokiwa University, Kobe University, and Nepal Medical College has been organized to implement the fieldwork at Dithal Village in Nepalese mountainous area for JICA Kusanone Project. In the fieldwork, the nutritional and health state of 208 village inhabitants (5 to 82 years old : 82 of male and 126 of female) were investigated, and their daily dietary foods were analyzed intake of calories and nutrients. The state of hygiene was also studied including of checking the contamination of drinking water microbiologically. The results showed that the high presence of over 50% of thinness, which is defined as less than 18.5 of BMI, was estimated in all age-groups, however among the age group under 12 years, the rate was much higher up to 75~80%. In body component analysis, the decreased fat and muscle volume and increased body water volume were manifested in children. Some of the children showed the distended abdomen, which is the typical sign of Kwashiorkor type malnutrition. The average calorie intake of daily diet was estimated about 800～1000kcal, and the content of it was 80% of carbohydrate, 10% each of fat and protein. The contaminations of Coliform bacilli and E. coli could be detected 80 to 100% of drinking water at 30 water places. The internal medical check revealed the prevalence of water-borne digestive diseases with abdominal pain, vomiting, and diarrhea. The deteriorated nutritional and public health state in Nepalese mountainous area could be disclosed in this study, but these are the similar paths on which the advanced countries have gone through in their past. We are entrusted to give our hands to them

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

publication en ligne. Article dans revue scientifique avec comité de lecture. nationale.National audienceThe human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

<div><p>Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-<i>O</i>-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. <i>In vitro</i> experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides <i>in vivo</i> in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.</p> </div