Decreased rate of blood donors with high ABO antibody titers in Japan and the underlying factors: Comparisons between 2010 and 2021.

BACKGROUND AND OBJECTIVES: Previously (2007), it was reported that ABO antibody titers in Japanese blood donors had decreased significantly compared to 20 years before. Here we evaluated whether further decrease of antibody titers had occurred in recent years, and the potential factors associated with changes in antibody titers. MATERIALS AND METHODS: Serum/plasma from random blood donors in 2010 and 2021 (2010: 3369, 2021: 5796 donors) was classified into low, middle, and high ABO antibody titers according to the reactivity of diluted serum/plasma (2.5-fold and 20-fold) by an automated microplate system. The rates of low/high titer in the two periods were compared. Logistic regression and age-gender-BMI subgroup analyses were conducted to identify the factors that contributed to changes in antibody titers. RESULTS: Compared to 2010, the rate of donors with high ABO antibody titers was　decreased in 2021 for both anti-A and anti-B (anti-A, 2010: 23.8%, 2021: 19.3%; anti-B, 2010: 23.8%, 2021: 16.4%). In logistic regression analysis, age was found to significantly affect both anti-A and anti-B antibody titers (anti-A, adjusted odds ratio 0.36, 95% CI 0.31-0.41; anti-B, 0.42, 0.37-0.47), and BMI (0.82, 0.73-0.92) and other time-related factors (0.79, 0.71-0.88) significantly affect anti-B antibody titers. Subgroup analysis revealed decreased rate of high anti-B titers in the higher age group in 2021. CONCLUSION: The rate of high ABO antibody titers, especially high anti-B titers, was significantly decreased in 2021, and our results suggested an association with aging and obesity of blood donors as well as other time-related factors.

DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

Nuclear factor-κB (NF-κB) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-κB, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-κB (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-κB binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-κB binding site of the competitor. Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (-)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.

Down-regulation of calcium/calmodulin-dependent protein kinase kinase 2 by androgen deprivation induces castration-resistant prostate cancer.

BACKGROUND: Conversion into androgen-hypersensitive state and adaptation to the low concentration of androgen during ADT cause relapse of prostate cancer (PCa). It is important to identify differentially expressed genes between PCa and normal prostate tissues and to reveal the function of these genes that are involved in progression of PCa. METHODS: We performed cDNA microarray analysis to identify differentially expressed genes, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2). Immunohistochemical analysis was conducted to investigate the relationship between the CAMKK2 expression level and prognosis. The function of CAMKK2 was assessed by generating CAMKK2 overexpressed LNCaP cells and by knockdown of CAMKK2. RESULTS: We identified CAMKK2 overexpressed six times in PCa more than normal prostate by cDNA microarray analysis. Immunohistochemical analysis of CAMKK2 protein showed that CAMKK2 protein was expressed more in PCa than normal tissue. However, the expression in the high-grade PCa diminished. Moreover, the narrowness of CAMKK2-positive area before ADT was a poor prognostic factor. Androgen-deprivation treatment from the medium in which LNCaP cells were cultured in the presence of 10 nM DHT repressed CAMKK2 expression. CAMKK2 overexpressed LNCaP cells (LNCaP/GFP-CAMKK2) attenuated androgen-sensitivity. Tumorigenesis of LNCaP/GFP-CAMKK2 cells in male SCID mice was decreased compared with control cells irrespective of castration. Finally, knockdown of CAMKK2 mRNA in LNCaP cells induced androgen-hypersensitivity and stimulated LNCaP cell proliferation. CONCLUSIONS: Induction of androgen-hypersensitivity after ADT may be involved in down-regulation of CAMKK2. This result may provide new therapeutic approach to keep androgen-sensitivity of PCa after ADT.

Rapid detection of epidermal growth factor receptor mutations in lung cancer by the SMart-Amplification Process.

PURPOSE: A positive response to gefitinib in non-small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use. EXPERIMENTAL DESIGN: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied it for analyzing EGFR gene mutations in clinical samples. By using SMAP, we can detect mutations within 30 min including sample preparation. To validate the assay system for potential use in clinical diagnostics, we examined 45 NSCLC patients for EGFR mutations using sequencing and SMAP. RESULTS: The outcomes of the SMAP assay perfectly matched the sequencing results, except in one case where SMAP was able to identify a mutation that was not detected by sequencing. We also evaluated the sensitivity and specificity of SMAP in mutation detection for EGFR. In a serial dilution study, SMAP was able to find a mutation in a sample containing only 0.1% of the mutant allele in a mixture of wild-type genomic DNA. We also could show amplification of mutated DNA with only 30 copies per reaction. CONCLUSIONS: The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology.

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.

[A renocolic fistula due to colonic diverticulitis associated with polycystic kidney].

Renocolic fistulae caused by colonic diverticulitis are rare. We present a case of renocolic fistula caused by colonic diverticulitis associated with polycystic kidney. A 51-year-old male with polycystic kidney on hemodialysis presented with a lasting fever and left lower abdominal pain. Under the diagnosis of the infection of a cyst in a polycystic kidney, puncture of the cyst was performed. Nine hundred ml of turbid fluid, of which culture grew Bacteroides Fragilis, was discharged. Inflammation subsided after the puncture, but soon recurred. Moreover, pneumaturia was observed, and fecaloid fluid was drainaged. Barium enema demonstrated a fistula from the diverticulum of the descending colon into the punctured cyst. The patient underwent a nephrectomy combined with hemi-colectomy.