Establishment of monoclonal antibodies against cell surface domains of ASCT2/SLC1A5 and their inhibition of glutamine-dependent tumor cell growth.

Human alanine-serine-cysteine transporter 2 (ASCT2; SLC1A5) is a major transporter of the amino acid glutamine that is known to be overexpressed in certain malignant tumors. In this study, we generated specific monoclonal antibodies (MAbs) against ASCT2 by establishing an ASCT2-expressing Chinese hamster ovary cell line that was used to immunize mice and rats. The MAbs KM4008, KM4012, and KM4018 against ASCT2 were isolated through a cell-based screen; these specifically bound to ASCT2-positive cells, as determined by flow cytometry and immunoprecipitation. In addition, the antibodies suppressed glutamine-dependent growth of WiDr colorectal cancer cells. These results provide evidence supporting the use of MAbs against ASCT2 as an effective therapeutic strategy for cancer treatment.

Glycemic control indicator levels at diagnosis of neonatal diabetes mellitus: Comparison with other types of insulin-dependent diabetes mellitus.

BACKGROUND: Neonatal diabetes mellitus (NDM) is a monogenic insulin-dependent diabetes that develops within 6 months of age. The progression of hyperglycemia until diagnosis is unknown. Glycemic control indicators at diagnosis are useful to estimate the extent and duration of hyperglycemia. We recently established that age-adjusted glycated albumin (GA) is a useful indicator of glycemic control, regardless of age. OBJECTIVE: To compare the levels of various glycemic control indicators at diagnosis between NDM and other types of insulin-dependent diabetes mellitus. PATIENTS AND METHODS: We included 8 patients with NDM, 8 with fulminant type 1 diabetes (FT1D), and 24 with acute-onset autoimmune type 1 diabetes (T1AD). Plasma glucose, glycated hemoglobin (HbA1c), GA, and age-adjusted GA (calculated as previously reported) were measured and compared. RESULTS: There were no significant differences in the plasma glucose levels of the group of patients with NDM and those with FT1D or T1AD. HbA1c and GA levels in the NDM group were not significantly different from those in the FT1D group, and both indicators were lower than those in the T1AD group. Age-adjusted GA levels in the NDM group did not differ significantly from those in the T1AD group, but were higher than those in the FT1D group. CONCLUSIONS: These findings suggest that the time-course of plasma glucose elevation in NDM until diagnosis is similar to that in T1AD. In addition, the high age-adjusted GA value at diagnosis of NDM indicates that this test is useful for assessing chronic hyperglycemia in NDM.

Age-Adjusted Glycated Albumin at Diagnosis is more Correlated with the Product of Age and Plasma Glucose than Plasma Glucose Alone in Patients with Neonatal Diabetes Mellitus.

BACKGROUND: We previously showed that glycated albumin (GA) is a useful glycemic control indicator in patients with neonatal diabetes mellitus (NDM), and that age-adjusted GA (Aa-GA) can reflect more accurately glycemic control status. Here, we investigated whether the age at diagnosis influences Aa-GA at diagnosis of NDM. METHODS: Eight patients with NDM whose GA was measured at diagnosis (age at diagnosis: 39 ± 18 days; GA: 31.3 ± 7.6%; Aa-GA: 47.1 ± 10.3%; plasma glucose: 525 ± 194 mg/dl) were included. Aa-GA was calculated as follows: Aa-GA = GA × 14.0/[1.77 × log-age (days) + 6.65]. Correlations of GA or Aa-GA at diagnosis with its logarithmically transformed age in days (log-age), plasma glucose, and their product were investigated. RESULTS: GA at diagnosis was not significantly correlated with log-age or plasma glucose. On the other hand, Aa-GA at diagnosis was significantly positively correlated with plasma glucose (R = 0.75, P = 0.031) and was more strongly positively correlated with the product of plasma glucose and log-age (R = 0.82, P = 0.012) although it was not correlated with log-age. CONCLUSION: Aa-GA at diagnosis is influenced by both age in days and plasma glucose. This finding is likely to show the aspect that age in days is almost equal to diabetes duration because glycemic control indicators including GA reflect the weighted mean of plasma glucose.

Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

A case of pancreatic agenesis and congenital heart defects with a novel GATA6 nonsense mutation: evidence of haploinsufficiency due to nonsense-mediated mRNA decay.

Recently, GATA6 heterozygous loss-of-function mutations were reported to cause pancreatic agenesis and congenital heart defects (PACHD [OMIM:600001]). However, the molecular mechanisms resulting from premature termination codons have not been examined in this disorder. The objective of this study was to perform a genetic analysis of a patient with PACHD. A female patient presented with ventricular septal defect, patent ductus arteriosus, and congenital diaphragmatic hernia at birth. Permanent neonatal diabetes mellitus and pancreatic exocrine deficiency due to pancreatic agenesis was diagnosed at 1 month of age. PCR-direct sequencing of GATA6 revealed that the patient is heterozygous for a novel de novo nonsense mutation of c.1477C>T, p. Arg493X in exon 5. RT-PCR direct sequencing of the RT-PCR products of total RNA from peripheral blood of the patient for the region encompassing exons 4-6 revealed only the wild-type allele. This finding provides the evidence for the occurrence of nonsense-mediated mRNA decay (NMD) in the p.Arg493X mutation. Quantitative RT-PCR analysis revealed that the expression of GATA6 transcript in the patient was less than half compared with normal control samples. This is the first evidence that GATA6 haploinsufficiency is caused by NMD in vivo, and we conclude that GATA6 haploinsufficiency causes not only PACHD but may affect other organs derived from the endoderm. Further screenings of GATA6 mutations in patients with various forms of diabetes and/or congenital heart disease with other visceral malformation may reveal the impact of GATA6 mutations on diabetes and congenital malformation.

Glycemic control and motor development in a patient with intermediate DEND.

The most common cause of neonatal diabetes, KCNJ11 gene mutation, can manifest as a neurological disorder. The most severe form consists of a constellation of developmental delay, epilepsy, and neonatal diabetes (DEND). Intermediate DEND (iDEND) refers to a milder presentation without epilepsy. We present a child with iDEND, for whom insulin injections were replaced with glibenclamide therapy at 17 months of age because of poor glycemic control and delayed motor development. Three months after initiation of glibenclamide, HbA1c decreased from 10.2% to 5.6%. Continuous glucose monitoring indicated that blood glucose fluctuations were suppressed while on glibenclamide. Furthermore, after initiating glibenclamide therapy, the developmental quotient (DQ) for motor ability markedly improved from 60 to 91, whereas the DQ for language and adoptive ability remained as they had been before the sulfonylurea treatment. Sulfonylurea treatment improved glycemic control and motor development in the present patient.

Evaluation of glycated hemoglobin and fetal hemoglobin-adjusted HbA1c measurements in infants.

BACKGROUND: As the presence of fetal hemoglobin (HbF) affects the accuracy of hemoglobin A1c (HbA1c) analysis methods, HbA1c measurement may not be a good indicator for patients with neonatal diabetes mellitus, whereas glycated albumin (GA) may be a good indicator. OBJECTIVE: To investigate whether total glycated hemoglobin (GHb) or HbF-adjusted HbA1c (adj-HbA1c) can act as a glycemic control marker in infants. SUBJECTS AND METHODS: Plasma glucose (PG), GA, HbF, GHb measured using the affinity method, and HbA1c measured using the latex-immunoturbidimetry (LA) or the high-performance liquid chromatography (HPLC) methods were determined in 26 full-term newborn infants aged 4-234 d. Adj-HbA1c was calculated as HbA1c/(total Hb - HbF). RESULTS: GHb, adj-HbA1c measured using the LA and the HPLC methods were 4.8 ± 0.5%, 4.5 ± 0.5%, and 4.7 ± 0.6%, respectively. GA was most positively correlated with PG (r = 0.696, p < 0.0001). GHb was positively correlated with both PG (r = 0.479, p = 0.013) and GA (r = 0.727, p < 0.0001). Adj-HbA1c measured using the LA method was positively correlated with GA (r = 0.465, p = 0.017), but not PG (r = 0.304, p = 0.132). Adj-HbA1c measured using the HPLC method was correlated with neither PG (r = -0.077, p = 0.710) nor GA (r = 0.360, p = 0.071). CONCLUSIONS: GHb measured using the affinity method may be a useful glycemic control marker in infants. Although adj-HbA1c measured using the LA method was correlated with GA, it may not be a practical measure because it was not correlated with PG and determining HbF levels using HPLC method can be troublesome. Adj-HbA1c measured using the HPLC method should not be used as a glycemic marker in infants.

Glycated albumin is lower in infants than in adults and correlated with both age and serum albumin.

BACKGROUND: Glycated albumin (GA) reflects glycemic control in patients with neonatal diabetes mellitus (NDM). However, GA in NDM patients is apparently low in relation to glycemia. OBJECTIVE: To establish the reference intervals for GA in healthy infants. SUBJECTS AND METHODS: Fifty-eight healthy, full-term newborn infants were used to define the GA reference values and to investigate its relationship to plasma glucose (PG) and serum albumin. The infants were categorized into three groups according to age: group A, 5 (4-6) median (range) d: n = 18; group B, 33 (30-38) d: n = 19; and group C, 181 (50-352) d: n = 21. We also studied 212 non-diabetic adults [group D, 53 (28-78) yr old] and the 5 NDM patients previously reported for GA comparisons. RESULTS: In the infants, GA was strongly positively correlated with logarithmic transformation of age [log (age)] (p = 0.831, p < 0.0001). The GA in groups A, B, C, and D were 7.3 ± 1.0%, 8.6 ± 1.1%, 10.9 ± 0.8%, and 14.0 ± 1.1%, respectively. The GA was more strongly positively correlated with serum albumin (r = 0.768, p < 0.0001) than with PG (r = 0.596, p < 0.0001). When GA levels were compared with the age-dependent reference values, GA in the transient NDM patient was normalized although GA in the four permanent NDM patients decreased but remained high after insulin therapy. CONCLUSIONS: This study showed that the reference range for GA in infants is lower than that of adults and increases with age, with which we confirmed that GA in the NDM patients reflected the clinical course. Consequently, GA in NDM patients should be compared with the age-based reference values to assess the accurate glycemic status.

New sandwich-type enzyme-linked immunosorbent assay for human MxA protein in a whole blood using monoclonal antibodies against GTP-binding domain for recognition of viral infection.

OBJECTIVES: To develop a clinically significant and practical enzyme-linked immunosorbent assay (ELISA) for the detection of MxA protein in human whole blood, a biological marker of viral infection. DESIGN AND METHODS: A sandwich ELISA suitable for the measurement of human MxA protein in whole blood was developed using mouse monoclonal antibodies (mAbs) raised against the GTP-binding domain of human MxA protein. Prior to the assay, the whole blood sample was treated with special buffer to extract the MxA protein, improve its stability, and avoid interference from hemoglobin. RESULTS: This ELISA meets all the requirements for use in routine clinical assays, especially in terms of sensitivity (detection limit: 1.3 ng/ml whole blood), accuracy (recovery: 93.0-100.0%), and rapidity (<1.5 h). The present ELISA had a sensitivity of 100% and a specificity of 100% for viral infection when compared to samples from healthy control and 87.1% and 90.9% when compared to samples from the bacterial infection group. CONCLUSION: We have developed a new ELISA for measuring MxA protein in human whole blood using mAbs specific for the GTP-binding domain of MxA. This ELISA has analytical performance enough for routine clinical assay and can be used in detecting the possibility of viral infection.

Bone marrow-derived cells contribute to vascular inflammation but do not differentiate into smooth muscle cell lineages.

BACKGROUND: It has been proposed that bone marrow-derived cells infiltrate the neointima, where they differentiate into smooth muscle (SM) cells; however, technical limitations have hindered clear identification of the lineages of bone marrow-derived "SM cell-like" cells. METHODS AND RESULTS: Using a specific antibody against the definitive SM cell lineage marker SM myosin heavy chain (SM-MHC) and mouse lines in which reporter genes were driven by regulatory programs for either SM-MHC or SM α-actin, we demonstrated that although some bone marrow-derived cells express SM α-actin in the wire injury-induced neointima, those cells did not express SM-MHC, even 30 weeks after injury. Likewise, no SM-MHC(+) bone marrow-derived cells were found in vascular lesions in apolipoprotein E(-/-)mice or in a heart transplantation vasculopathy model. Instead, the majority of bone marrow-derived SM α-actin(+) cells were also CD115(+)CD11b(+)F4/80(+)Ly-6C(+), which is the surface phenotype of inflammatory monocytes. Moreover, adoptively transferred CD11b(+)Ly-6C(+) bone marrow cells expressed SM α-actin in the injured artery. Expression of inflammation-related genes was significantly higher in neointimal subregions rich in bone marrow-derived SM α-actin(+) cells than in other regions. CONCLUSIONS: It appears that bone marrow-derived SM α-actin(+) cells are of monocyte/macrophage lineage and are involved in vascular remodeling. It is very unlikely that these cells acquire the definitive SM cell lineage.

A mutation of the ß-domain in POU1F1 causes pituitary deficiency due to dominant PIT-1ß expression.

BACKGROUND: POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1ß, which contains an insertion of 26-amino acids (ß-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1ß is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with ß-domain mutations have been reported. RESULTS: Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1ß (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1ß transcript without other transcripts. The lymphocyte PIT-1ß mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1ß-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells. CONCLUSIONS: We describe, for the first time, that the PIT-1ß mutation can cause CPHD through a novel genetic mechanism, such as PIT-1ß overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1ß mutations.

Establishment of a novel monoclonal antibody against LGR5.

LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.

The role of N-terminal pro-B-type natriuretic peptide in the diagnosis of congestive heart failure in children. - Correlation with the heart failure score and comparison with B-type natriuretic peptide -.

BACKGROUND: Both B-type natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) are useful biomarkers for the assessment of congestive heart failure (CHF) in adults. The purpose of this study was to determine whether BNP and NT-proBNP levels could be used to stratify the severity of CHF in children. METHODS AND RESULTS: The study comprised 181 children with CHF and 232 healthy children aged from 4 months to 14 years who were categorized into CHF grades I, II, III and IV according to the modified Ross scoring system. The plasma BNP and serum NT-proBNP levels were significantly correlated with increasing CHF grades. The NT-proBNP levels were significantly different among the 4 CHF grades. However, only 2 significant differences were observed in the BNP levels between each CHF grade. NT-proBNP testing with cut-off points of >438 pg/ml (> or =grade II), >1,678 pg/ml (> or =grade III) and >7,734 pg/ml (grade IV) in the patients below 3 years of age, and >295 pg/ml (> or =grade II), >1,545 pg/ml (> or =grade III) and >3,617 pg/ml (grade IV) in those above 3 years of age was determined to be highly sensitive and specific by receiver operating characteristic analysis. CONCLUSIONS: The blood levels of BNP and NT-proBNP therefore reflect the severity of CHF in children. In particular, NT-proBNP is a useful biomarker for evaluating CHF in children.

Increase in doses of levothyroxine at the age of 3 years and above is useful for distinguishing transient and permanent congenital hypothyroidism.

There are no recommended diagnostic criteria for transient congenital hypothyroidism (CH) during early childhood. In this study, we aimed to identify the factors that distinguish permanent (P)- and transient (T)-CH. We retrospectively analyzed the clinical, biochemical, and imaging data of 42 children with a definitive diagnosis of P- or T-CH by re-evaluation tests at our institution from November 1986 to October 2019. Patients who continued levothyroxine (L-T4) treatment after the re-evaluation tests were classified as group P (n = 19), while patients who were diagnosed with T-CH and discontinued L-T4 treatment were classified as group T (n = 23). Initial testing performed during infancy showed that the mean serum TSH and free T4 (FT4) levels did not differ significantly between groups P and T. None of the patients in group T required an increased dosage of L-T4 at the age of 3 yr and above while 85% of the patients in group P required increased dosages of L-T4. Hence, T-CH was suspected in patients who did not require an increase in L-T4 dosage at the age of 3 yr and above.

A 34-year-old Japanese patient exhibiting NBAS deficiency with a novel mutation and extended phenotypic variation.

Biallelic neuroblastoma amplified sequence (NBAS) gene mutations have recently been identified to cause a reduction in its protein expression and a broad phenotypic spectrum, from isolated short stature, optic nerve atrophy, and Pelger-Huët anomaly (SOPH) syndrome or infantile liver failure syndrome 2 to a combined, multi-systemic disease including skeletal dysplasia and immunological and neurological abnormalities. Herein, we report a 34-year-old patient with a range of phenotypes for NBAS deficiency due to compound heterozygous variants; one is a SOPH-specific variant, p.Arg1914His, and the other is a novel splice site variant, c.6433-2A>G. The patient experienced recurrent acute liver failure until early childhood. Hypogammaglobulinemia, a decrease in natural killer cells, and optic nerve atrophy were evident from infancy to childhood. In adulthood, the patient exhibited novel phenotypic features such as hepatic cirrhosis complicated by portal hypertension and autoimmune hemolytic anemia. The patient also suffered from childhood-onset insulin-requiring diabetes with progressive beta cell dysfunction. The patient had severe short stature and exhibited dysmorphic features compatible with SOPH, intellectual disability, and epilepsy. NBAS protein expression in the patient's fibroblasts was severely low. RNA expression analysis for the c.6433-2A>G variant showed that this variant activated two cryptic splice sites in intron 49 and exon 50, for which the predicted consequences at the protein level were an in-frame deletion/insertion, p.(Ile2199_Asn2202delins16), and a premature termination codon, p.(Ile2199Tyrfs*17), respectively. These findings indicate that NBAS deficiency is a multi-systemic progressive disease. The results of this study extend the spectrum of clinical and genetic findings related to NBAS deficiency.

Therapeutic antitumor efficacy of monoclonal antibody against Claudin-4 for pancreatic and ovarian cancers.

Claudin-4 (CLDN4) is a tetraspanin transmembrane protein of tight junction structure and is highly expressed in pancreatic and ovarian cancers. In this study, we aimed to generate an anti-Claudin-4 monoclonal antibody (mAb) and evaluate its antitumor efficacy in vitro and in vivo. To isolate specific mAb, we generated CLDN3, 4, 5, 6, and 9, expressing Chinese hamster ovary (CHO) cells, and then used them as positive and negative targets through cell-based screening. As a result, we succeeded in isolating KM3900 (IgG2a), which specifically bound to CLDN4, from BXSB mice immunized with pancreatic cancer cells. Immunoprecipitation and flow cytometry analysis revealed that KM3900 recognized the conformational structure and bound to extracellular loop 2 of CLDN4. Furthermore, binding of KM3900 was detected on CLDN4-expressing pancreatic and ovarian cancer cells, but not on negative cells. Next, we made the mouse-human chimeric IgG1 (KM3934) and evaluated its antitumor efficacy. KM3934 induced dose-dependent antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro, and significantly inhibited tumor growth in MCAS or CFPAC-1 xenograft SCID mice in vivo (P < 0.05). These results suggest that mAb therapy against CLDN4 is promising for pancreatic and ovarian cancers.

Establishment of humanized anti-interleukin-5 receptor alpha chain monoclonal antibodies having a potent neutralizing activity.

Human interleukin-5 is the key cytokine involved in regulating the production and function of human eosinophils. IL-5 binds to its specific receptor composed of two heterogeneous alpha and beta polypeptide chains (hIL-5Ralpha and betac) that are expressed on the cell surface. The hIL-5Ralpha specifically binds IL-5 without involvement of the betac. It has been suggested that neutralizing antibodies to hIL-5Ralpha could serve as a therapeutic agent in eosinophil-associated diseases. We describe here the creation and biologic activities of a mouse monoclonal antibody against hIL-5Ralpha that blocks the following IL-5 dependent activities (a) binding of the IL-5 ligand to its receptor, (b) IL-5 dependent growth of hIL-5R expressing cells, and (c) IL-5-induced adhesion of human eosinophils. We also describe the process for humanization of the mouse Mab towards development of a therapeutic MAb. The humanized version of the monoclonal antibody also displayed potent neutralizing activity against IL-5 dependent activities.

Impaired negative feedback suppression of bile acid synthesis in mice lacking betaKlotho.

We have generated a line of mutant mouse that lacks betaKlotho, a protein that structurally resembles Klotho. The synthesis and excretion of bile acids were found to be dramatically elevated in these mutants, and the expression of 2 key bile acid synthase genes, cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1), was strongly upregulated. Nuclear receptor pathways and the enterohepatic circulation, which regulates bile acid synthesis, seemed to be largely intact; however, bile acid-dependent induction of the small heterodimer partner (SHP) NR0B2, a common negative regulator of Cyp7a1 and Cyp8b1, was significantly attenuated. The expression of Cyp7a1 and Cyp8b1 is known to be repressed by dietary bile acids via both SHP-dependent and -independent regulations. Interestingly, the suppression of Cyp7a1 expression by dietary bile acids was impaired, whereas that of Cyp8b1 expression was not substantially altered in betaklotho mice. Therefore, betaKlotho may stand as a novel contributor to Cyp7a1-selective regulation. Additionally, betaKlotho-knockout mice exhibit resistance to gallstone formation, which suggests the potential future clinical relevance of the betaKlotho system.