Evaluation of Modified Formalin-Ether Concentration Method Using Para Tube in Clinical Settings.

Conventional formalin-ether concentration method is a gold standard for the diagnosis of parasite infection. However, it may be time-consuming and laborious. We aimed to reveal the clinical usefulness of a modified formalin-ether concentration method using the Para Tube (KS Corporation, Korea) compared with the conventional method. A total of 117 fresh, unpreserved fecal samples composed to 90 negative controls and 27 positive controls with ova of Diphyllobothrium latum/D. nihonkaiense, ova of Clonorchis sinensis and cysts of Giardia lamblia were used in this study. Both methods showed comparable correct identification rate (87.2% for the Para Tube vs. 86.3% for the conventional method).When five samples were examined at once, the Para Tube method reduced the procedure time compared with the conventional method (19 min 58 sec vs. 23 min 18 sec, P=0.0286). We concluded that the modified formalin-ether concentration method using the Para Tube is a rapid, simple, and reliable fecal concentration method for clinical use.

Strongyloidiasis in a diabetic patient accompanied by gastrointestinal stromal tumor: cause of eosinophilia unresponsive to steroid therapy.

We report here a case of strongyloidiasis in a 72-year-old diabetic patient (woman) accompanied by gastrointestinal stromal tumor receiving imatinib therapy, first diagnosed as hypereosinophilic syndrome and treated with steroids for uncontrolled eosinophilia. She suffered from lower back pain and intermittent abdominal discomfort with nausea and diagnosed with gastrointestinal stromal tumor. After post-operative imatinib treatment eosinophilia persisted, so that steroid therapy was started under an impression of hypereosinophilic syndrome. In spite of 6 months steroid therapy, eosinophilia persisted. Stool examination was performed to rule out intestinal helminth infections. Rhabditoid larvae of Strongyloides stercoralis were detected and the patient was diagnosed as strongyloidiasis. This diagnosis was confirmed again by PCR. The patient was treated with albendazole for 14 days and her abdominal pain and diarrhea improved. This case highlights the need for thorough investigation, including molecular approaches, to test for strongyloidiasis before and during steroid therapies.

Feasible quantitative detection of cytomegalovirus from urine sediment in stem cell transplant patients.

BACKGROUND: Urine is an important source for the detection of infections caused by CMV in stem cell transplant patients. Currently, there is no agreement about the type of urine specimen. In order to investigate which is the better specimen type for quantitative detection of CMV, we compared the results from urine supernatant and sediment from the same patients. METHODS: Seventy urine specimens were collected from patients with hematological disorders or solid tumors. After performing shell vial culture, residual urine specimens were centrifuged. Then, 10 mL of each urine supernatant and sediment were taken and immediately frozen at -70 degrees C. Afterwards, archived urine specimens were thawed at room temperature and CMV-quantitative PCR was performed on both the supernatant and sediment fraction of urine. The results from each patient were reviewed for CMV antigenemia, blood shell vial culture, CMV-IgM or IgG, and clinical symptoms. RESULTS: CMV-qPCR results for the urine sediment fraction revealed a significant difference (p = 0.012) between the active CMV infection group and the latent CMV infection group. In addition, receiver operating characteristic curves for active CMV infection revealed that CMV-qPCR using urine sediment produced more accurate results than urine supernatant. CONCLUSIONS: These findings suggest that the sediment fraction of urine is a more suitable specimen in CMV-qPCR testing.

Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BACKGROUND: The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications. METHODS: Single cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients. RESULTS: Single cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFß super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn't support the prognostic value of the ASCs in AML. CONCLUSIONS: ASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.

Alteration of the SETBP1 gene and splicing pathway genes SF3B1, U2AF1, and SRSF2 in childhood acute myeloid leukemia.

BACKGROUND: Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. METHODS: Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. RESULTS: Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. CONCLUSIONS: Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.

Profiling of biomarkers for the exposure of polycyclic aromatic hydrocarbons: lamin-A/C isoform 3, poly[ADP-ribose] polymerase 1, and mitochondria copy number are identified as universal biomarkers.

This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs.

Serum parathyroid hormone is a new potential risk factor in multiple myeloma.

We hypothesized that serum PTH might be associated with various clinicopathological parameters in multiple myeloma (MM). So we investigated the implications of serum PTH in MM patients and the relationship with other risk factors of MM. A total of 115 patients who were newly diagnosed with MM were enrolled. Serum PTH level was 24.7 ± 34.9 (ranged 0.0-284.1) pg/mL. Serum levels of IgG, IgM, FLC-lambda, albumin, and LDH were in positive correlation with serum PTH. Compared to non-high PTH (<68.3 pg/mL) group, the hazard ratio (HR) for overall survival was higher for group with high PTH level (≥ 68.3 pg/mL) (HR, 1.710). Furthermore, the patient group with high PTH level showed inferior progression-free survival than non-high PTH group (P = 0.056). Interestingly, subgroup analysis showed that serum PTH level at diagnosis was associated with risk factors and clinical outcome in MM patients, especially in complete remission group, transplantation cases, ISS stage II cases, and cases without chromosome abnormality. In conclusion, this study showed that blood PTH level in MM at diagnosis was associated with risk factors and clinical outcome in MM patients.

Circulating mucosal-associated invariant T cell levels and their cytokine levels in healthy adults.

Mucosal-associated invariant T (MAIT) cells have been reported to play an antimicrobial role in infectious diseases. However, little is known about age- and gender-related changes in circulating MAIT cell level and function in healthy population. The purposes of this study were to examine the level and cytokine production of circulating MAIT cells and their subsets in healthy adults and to investigate potential relationships between clinical parameters and MAIT cell levels or their subset levels. One hundred thirty-three healthy subjects were enrolled in this study. MAIT cells, their subset, and cytokine levels were measured by flow cytometry. Circulating MAIT cell levels were found to vary widely (0.19% to 21.7%) in the study subjects and to be significantly lower in elderly subjects (age, 61-92 years) than in young subjects (age, 21-40 years) (p<0.0005). No significant difference was found in the circulating MAIT cell levels between male and female subjects. A linear regression analysis revealed that circulating MAIT cell levels declined annually by 3.2% among men and 1.8% among women, respectively. Notably, the proportion of CD4+ MAIT cells increased with age, whereas that of CD8+ MAIT cells decreased with age. In addition, the production of interleukin (IL)-4 by MAIT cells was found to be significantly increased in elderly subjects and the ratio of interferon (IFN)-γ/IL-4 was lower as compared with young subjects, showing a Th1 to Th2 shift in cytokine profile in elderly subjects. Our data suggest that aging is associated with a reduction in circulating MAIT cells, accompanied with alterations in subset composition and cytokine profile.

Overnight storage of blood in ACD tubes at 4{degrees}C increases NK cell fraction in peripheral blood mononuclear cells.

A considerable variabilility in the effects of sample handling on NK cytotoxicity has been observed. Using flow cytometry, NK cytotoxicity assays and lymphocyte subset analysis of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMCs) isolated from whole blood stored under various conditions were performed. The NK cytotoxicity of samples in heparin tubes stored overnight at 4 and 22°C, as well as at 22°C in acid citrate dextrose (ACD) tubes, was lower than that of a fresh sample. However, the NK cytotoxicity of samples in an ACD tube stored at 4°C was similar to that of a fresh sample. Based on lymphocyte subset analysis, samples in an ACD tube stored at 4°C showed a lower percentage of CD3+ T cells and a higher percentage of CD16/56+ NK cells compared to samples stored under other conditions. The NK cytotoxicity of fresh samples and samples in ACD tubes stored in a Styrofoam cooler box did not differ significantly; however, the differences were inconsistent. Overnight storage of peripheral blood in ACD tubes at 4°C is optimum for retention of NK cytotoxicity, the level of which is similar to that of fresh blood. This may be associated with an increased NK-cell fraction in Ficoll-Hypaque-separated PBMCs after overnight storage.

Over-expression of Her-2 in colorectal cancer tissue, but not in serum, constitutes an independent worse prognostic factor.

BACKGROUND: Currently, conflicting information exists regarding Her-2 over-expression and its clinicopathological implications in colorectal cancer (CRC). This study was undertaken to determine Her-2 over-expression in both serum and tumor tissue of CRC patients, and to assess its clinicopathological and targeted therapeutic implications. METHODS: Ninety five CRC patients and sixty healthy controls were prospectively enrolled. Her-2 expression status in serum and CRC tissue were examined by chemiluminescent immunoassay and immunohistochemical staining, respectively. The results were confirmed using fluorescent in situ hybridization. Clinicopathological parameters were analyzed according to Her-2 expression status. RESULTS: Serum Her-2 levels were found to be increased in CRC patients as compared to those of healthy controls. However, serum Her-2 levels were not found to be significantly associated with prognostic parameters. Her-2 expression analysis of CRC tissues revealed Her-2 over-expression in 23 patients (25%), i.e., 13 patients (14%) showed moderate over-expression and 10 patients (11%) showed strong over-expression. The overall survival of patients negative for Her-2 expression was significantly better than that of patients positive for Her-2 expression (P=0.018). The disease-free survival of patients with Her-2 over-expression was significantly shorter than that of patients with no Her-2 expression (P=0.021). CONCLUSIONS: Her-2 over-expression in CRC tissue, but not in serum, acts as a significant independent worse prognostic factor. Assessment of Her-2 expression status may be valuable for the targeted therapeutic management of CRC.

Ex vivo expansion of natural killer cells using cryopreserved irradiated feeder cells.

Currently, feeder cells are γ-irradiated immediately before use for the ex vivo expansion of natural killer (NK) cells from human peripheral blood. Storing irradiated feeder cells by cryopreserving them in multiple vials would be more convenient than irradiating cells each time they are needed. We compared NK cell expansion using cryopreserved-irradiated feeder cells (cryopreserved group) and freshly-irradiated feeder cells (fresh group). To expand NK cells, peripheral blood mononuclear cells were isolated and co-cultured with-100 Gy-irradiated K562 leukemia cells that had been modified to express 4-1BB ligand and membrane-bound (mb) interleukin (IL)-15 (K562-mb15-41BBL cells) for three weeks in the presence of IL-2 and IL-15. Fresh and cryopreserved K562-mb15-41BBL feeder cells expressed similar levels of 4-1BB ligand, whereas membrane-bound IL-15 expression was lower in the cryopreserved cells than in the fresh cells. The NK cell expansion rate did not differ between the two groups (980-fold vs. 1058-fold, respectively), although the mean NK cell purity was higher in the fresh-group than in the cryopreserved-group at day 14 (94.1% vs. 92.5%, respectively) and day 21 (97.1% vs. 95.4%, respectively). The NK cells from the two feeder cell groups did not differ in cytotoxicity against various malignant cell lines at effector-to-target ratios of 4:1, 2:1, and 1:1, or in the expression pattern of NK cell receptors [cluster of differentiation (CD)-16, natural killer group-2, member D (NKG2D), CD69, NKp30, NKp44, NKp46, and CD158b] and level of interferon-γ secretion. Our results demonstrate that cryopreserved irradiated feeder cells can be used for the ex vivo expansion of human NK cells and provide a convenient improvement on current methods.

Diagnostic and prognostic value of mitochondrial DNA minisatellites after stem cell transplantation.

Mitochondrial DNA has been used to investigate phylogenetic relationships and pathophysiologic roles in aging, degenerative diseases, and cancer. We investigated the prognostic usefulness of mitochondrial DNA minisatellite (mtMS) markers compared with nuclear short tandem repeat markers by evaluating the laboratory performance and clinical value of these markers in a large sample of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) with various simulated conditions in vitro and in serial follow-up samples. We examined the value of mtMS markers as a prognostic indicator in 100 patients with various hematologic disorders undergoing allo-HSCT, including 35 patients with longitudinal follow-up for 55 months. The mtMS markers showed high sensitivity and accuracy for the quantitative detection of chimerism compared with nuclear short tandem repeat markers, particularly in unrelated transplantation and under inappropriate sampling conditions. Longitudinal follow-up after allo-HSCT disclosed that chimerism precisely reflected the status of engraftment or relapse during the clinicopathological course. Moreover, changes in mtMS markers in recipients before allo-HSCT were associated with clinical outcomes. Our data indicate that mtMS markers have multiple functions in monitoring mixed chimerism and predicting prognosis after allo-HSCT.

Allelic expression imbalance of JAK2 V617F mutation in BCR-ABL negative myeloproliferative neoplasms.

The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs.

Sex-specific differences in the association between ABO genotype and gastric cancer risk in a Korean population.

BACKGROUND: Although previous studies have demonstrated an association between ABO blood group and the risk of gastric cancer (GC), only one study has identified these associations using the ABO genotype; however, that study did not evaluate sex differences in this association. The aim of the present study was to investigate whether there are sex-specific differences in the ABO genotype-associated risk of GC. In addition, we explored the association of the ABO genotype and the clinicopathologic characteristics of GC in a Korean population. METHODS: We conducted a large-scale case-control study of 3245 GC patients (2204 males, 1041 females) and 1700 controls (821 males, 879 females). The ABO genotype was determined by multicolor real-time polymerase chain reaction (PCR) using displacing probes. RESULTS: As compared with genotype OO, genotypes AA and AO in females, but not in males, were associated with a significantly increased risk of GC (odds ratio [OR] 1.56 and 95 % confidence interval [CI] 1.08-2.26 for AA; OR 1.57 and 95 % CI 1.21-2.03 for AO). In a subgroup analysis, blood group A had a significantly increased risk of diffuse-type GC (OR 2.00, 95 % CI 1.43-2.78), but not of intestinal-type (OR 1.31, 95 % CI 0.96-1.79) or mixed-type GC (OR 1.43, 95 % CI 0.92-2.24). CONCLUSION: The ABO genotypes AA and AO were significantly associated with GC only in females and only for diffuse-type GC. These data suggest that the association between ABO blood group and GC risk may differ according to sex and histological type.

Molecular identification of Schizophyllum commune as a cause of allergic fungal sinusitis.

Schizophyllum commune, a basidiomycetous fungus, rarely causes disease in humans. We report a rare case of allergic fungal sinusitis caused by S. commune in a 14-yr-old girl. The patient presented with nasal obstruction and a purulent nasal discharge. Materials obtained during endoscopic surgery of the frontal recess revealed allergic mucin and a few fungal hyphae. A potato dextrose agar (PDA) culture from the allergic mucin yielded a rapidly growing white woolly mold. Although no distinctive features including hyphae bearing spicules or a clamp connection were present, the case isolate disclosed compatible mycological features including growth at 37℃, susceptibility to cycloheximide, and production of a tart and disagreeable smell. S. commune was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA. We believe this is the first report of allergic fungal sinusitis caused by S. commune in Korea. Moreover, this report highlights the value of gene sequencing as an identification tool for non-sporulating isolates of S. commune.

Age- and gender-related differences in circulating natural killer T cells and their subset levels in healthy Korean adults.

Natural killer T (NKT) cells have been reported to play crucial roles in a variety of diseases, including infectious diseases, autoimmunity, and cancers. Previous studies have reported wide age- and/or sex-related variations in circulating NKT cell levels in healthy subjects, but reported results are discrepant. In this study, the authors examined NKT cell levels in the peripheral blood of healthy Korean subjects and investigated potential relationships between clinical parameters and NKT cells and their subset levels. One hundred and thirty-eight age- and sex-matched healthy subjects were enrolled in this study. NKT cell and NKT subset levels were measured by flow cytometry. Circulating NKT cell levels were found to vary widely (0.01-5.15%) in the study subjects and to be lower in men than in women (P<0.05). Notably, gender-related differences in NKT cell levels were more prominent in elderly subjects (P<0.05). Furthermore, alterations in NKT subset compositions were found in elderly men, in whom the proportion of CD4+ NKT cells was elevated and that of double-negative NKT cells was reduced. Our data suggest that circulating NKT cells and NKT subset levels are affected by age and gender in the Korean population.

Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence of dysplasia.

BACKGROUND: Barrett's esophagus (BE) is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA). The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA) mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities) in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT) and tissue (BT) from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53%) exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS) level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE.

Interleukin-21 increases direct cytotoxicity and IFN-γ production of ex vivo expanded NK cells towards breast cancer cells.

BACKGROUND/AIM: Interleukin-21(IL-21) stimulates cytotoxicity and interferon-γ (IFN-γ) production in natural killer (NK) cells. However, little has been reported on the stimulatory effect of IL-21 on ex vivo expanded NK cells. In this study, we examined the cytotoxicity and IFN-γ production of ex vivo expanded, IL-21-stimulated NK cells against trastuzumab-coated breast cancer cells. MATERIALS AND METHODS: To expand NK cells, peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with irradiated K562-mb15-41BBL cells in the presence of IL-2 and IL-15 for 3 weeks. After a 4-day stimulation with IL-21, NK cell cytotoxicity and IFN-γ production were measured. RESULTS: NK cells were expanded up to median of 911-fold and represented approximately 94.93% of expanded cells after 21 days. Cytotoxicity of the expanded NK cells against the MCF-7, SKBR3, and T47D cell lines was significantly increased following 4-day stimulation with IL-21. However, antibody-dependent cellular cytotoxicity mediated by trastruzumab was significantly increased only in the SKBR3 cell line, which highly expresses the HER2/neu antigen. IL-21 pre-treatment also increased IFN-γ production in the expanded NK cells in response to the trastuzumab-coated breast cancer cells. CONCLUSION: IL-21 significantly enhances the cytolytic activity and IFN-γ production of ex vivo expanded NK cells in response to trastuzumab-coated breast cancer cells.