Persistence and viable but non-culturable state induced by streptomycin in Erwinia amylovora

Persister cell and viable but non-culturable (VBNC) state of bacteria are survival strategies against antibiotics and various environmental stresses, respectively, but they tend to be ignored in agriculture fields, even though bacteria can regain their abilities to survive and produce disease once those stresses disappear. This study was carried out to determine whether persister cell and VBNC state in Erwinia amylovora are present after exposures to streptomycin, the length of their persistence, and the steps needed to decrease the inoculum. Persister cells were observed using biphasic killed growth curve for 4–8 h when the late stationary phase cells of E. amylovora were cultured in liquid medium containing streptomycin. This state was maintained for up to 12 h based on the colony forming units (CFUs) of the colonies that grew on the mannitol glutamate yeast extract (MGY) medium after streptomycin was removed. The CFUs on the MGY medium were lower than the total count determined using the LIVE/DEAD Kit, suggesting that persister cells and VBNC state might co-exist for up to 12 h after exposure to streptomycin. However, after 12 h, E. amylovora cells did not continue to grow on the medium for 9 days, suggesting that they entered a VBNC state at that time and remained in a persistent state. In addition, based on the Redox Sensor Green staining method, the presence of both states was confirmed for up to 12 h, and only then did the VBNC state became apparent. Furthermore, persister cells were observed for up to 24 h, and damaged cells reduced when E. amylovora cells were culture in distilled water with streptomycin, indicating that the uptake of lower nutrients in E. amylovora led to prolonged persister cells and VBNC state, which are more likely to survive after streptomycin treatments. The addition of sucrose and oxytetracycline to distilled water containing streptomycin reduced persister cells than other sources did. Thus, to inhibit the spread of fire blight, management techniques must consider the hazards of using streptomycin treatments that induce dormancy, such as persister cells and VBNC state, beyond the development of resistant strain

Characterization of the Lytic Bacteriophage phiEaP-8 Effective against Both Erwinia amylovora and Erwinia pyrifoliae Causing Severe Diseases in Apple and Pear

Bacteriophages, bacteria-infecting viruses, have been recently reconsidered as a biological control tool for preventing bacterial pathogens. Erwinia amylovora and E. pyrifoliae cause fire blight and black shoot blight disease in apple and pear, respectively. In this study, the bacteriophage phiEaP-8 was isolated from apple orchard soil and could efficiently and specifically kill both E amylovora and E. pyrifoliae. This bacteriophage belongs to the Podoviridae family. Whole genome analysis revealed that phiEaP-8 carries a 75,929 bp genomic DNA with 78 coding sequences and 5 tRNA genes. Genome comparison showed that phiEaP-8 has only 85% identity to known bacteriophages at the DNA level. PhiEaP-8 retained lytic activity up to 50 degrees C, within a pH range from 5 to 10, and under 365 nm UV light. Based on these characteristics, the bacteriophage phiEaP-8 is novel and carries potential to control both E. amylovora and E. pyrifoliae in apple and pear

Associations of Anti-Aquaporin 5 Autoantibodies with Serologic and Histopathological Features of Sjogren's Syndrome

Biomarkers to stratify the complex and heterogeneous phenotypes of Sjogren's syndrome (SS) are needed. We aimed to validate the prevalence of anti-aquaporin 5 (AQP5) IgG in a non-Korean cohort and to optimize the method to screen the anti-AQP5 IgG. The study cohort included 111 primary SS and 43 non-SS Sjogren's International Collaborative Clinical Alliance (SICCA) controls that were obtained from the Sjogren's International Collaborative Clinical Alliance registry, in addition to 35 systemic lupus erythematosus (SLE) and 35 rheumatoid arthritis (RA) phenotypes. Anti-AQP5 IgG was screened by cell-based immunofluorescence cytochemistry (CB-IFC) assay in the absence or presence of epitope peptides, as well as by ELISA using the epitope peptides as coated antigens. Anti-AQP5 IgG specific to an E1 epitope was best at discriminating between SS and non-SS, and the two different methods (CB-IFC and ELISA) presented comparable performance in diagnostic accuracy (0.690 vs. 0.707). Notably, the SLE and RA groups had substantially lower levels of anti-AQP5 IgG than the SS group. In addition, the presence of anti-AQP5_E1 IgG was associated with serologic and histopathological features of SS. In conclusion, a similar prevalence of anti-AQP5 IgG was confirmed in a non-Korean cohort. Screening anti-AQP5 autoantibodies may help to form subgroups of SS for targeted therapy.Y

A Fatal Case of Severe Hemolytic Disease of Newborn Associated with Anti-Jkb

The Kidd blood group is clinically significant since the Jk antibodies can cause acute and delayed transfusion reactions as well as hemolytic disease of newborn (HDN). In general, HDN due to anti-Jkb incompatibility is rare and it usually displays mild clinical symptoms with a favorable prognosis. Yet, we apparently experienced the second case of HDN due to anti-Jkb with severe clinical symptoms and a fatal outcome. A female patient having the AB, Rh(D)-positive boodtype was admitted for jaundice on the fourth day after birth. At the time of admission, the patient was lethargic and exhibited high pitched crying. The laboratory data indicated a hemoglobin value of 11.4 mg/dL, a reticulocyte count of 14.9% and a total bilirubin of 46.1 mg/dL, a direct bilirubin of 1.1 mg/dL and a strong positive result (+++) on the direct Coomb's test. As a result of the identification of irregular antibody from the maternal serum, anti-Jkb was detected, which was also found in the eluate made from infant's blood. Despite the aggressive treatment with exchange transfusion and intensive phototherapy, the patient died of intractable seizure and acute renal failure on the fourth day of admission. Therefore, pediatricians should be aware of the clinical courses of hemolytic jaundice due to anti-Jkb, and they should be ready to treat this disease with active therapeutic interventions

Reliability and Validity of the Korean Cancer Pain Assessment Tool (KCPAT)

The Korean Cancer Pain Assessment Tool (KCPAT), which was developed in 2003, consists of questions concerning the location of pain, the nature of pain, the present pain intensity, the symptoms associated with the pain, and psychosocial/spiritual pain assessments. This study was carried out to evaluate the reliability and validity of the KCPAT. A stratified, proportional-quota, clustered, systematic sampling procedure was used. The study population (903 cancer patients) was 1% of the target population (90,252 cancer patients). A total of 314 (34.8%) questionnaires were collected. The results showed that the average pain score (5 point on Likert scale) according to the cancer type and the at-present average pain score (VAS, 0-10) were correlated (r=0.56, p<0.0001), and showed moderate agreement (kappa=0.364). The mean satisfaction score was 3.8 (1-5). The average time to complete the questionnaire was 8.9 min. In conclusion, the KCPAT is a reliable and valid instrument for assessing cancer pain in Koreans

Expansion of cytotoxic natural killer cells in multiple myeloma patients using K562 cells expressing OX40 ligand and membrane-bound IL-18 and IL-21.

BACKGROUND: Natural killer (NK) cell-based immunotherapy is a promising treatment approach for multiple myeloma (MM), but obtaining a sufficient number of activated NK cells remains challenging. Here, we report an improved method to generate ex vivo expanded NK (eNK) cells from MM patients based on genetic engineering of K562 cells to express OX40 ligand and membrane-bound (mb) IL-18 and IL-21. METHODS: K562-OX40L-mbIL-18/-21 cells were generated by transducing K562-OX40L cells with a lentiviral vector encoding mbIL-18 and mbIL-21, and these were used as feeder cells to expand NK cells from peripheral blood mononuclear cells of healthy donors (HDs) and MM patients in the presence of IL-2/IL-15. Purity, expansion rate, receptor expression, and functions of eNK cells were determined over four weeks of culture. RESULTS: NK cell expansion was enhanced by short exposure of soluble IL-18 and IL-21 with K562-OX40L cells. Co-culture of NK cells with K562-OX40L-mbIL-18/-21 cells resulted in remarkable expansion of NK cells from HDs (9,860-fold) and MM patients (4,929-fold) over the 28-day culture period. Moreover, eNK cells showed increased expression of major activation markers and enhanced cytotoxicity towards target K562, U266, and RPMI8226 cells. CONCLUSIONS: Our data suggest that genetically engineered K562 cells expressing OX40L, mbIL-18, and mbIL-21 improve the expansion of NK cells, increase activation signals, and enhance their cytolytic activity towards MM cells

Non-Invasive Diagnosis for Acute Rejection Using Urinary mRNA Signature Reflecting Allograft Status in Kidney Transplantation

Urine has been regarded as a good resource based on the assumption that urine can directly reflect the state of the allograft or ongoing injury in kidney transplantation. Previous studies, suggesting the usefulness of urinary mRNA as a biomarker of acute rejection, imply that urinary mRNA mirrors the transcriptional activity of the kidneys. We selected 14 data-driven candidate genes through a meta-analysis and measured the candidate genes using quantitative PCR without pre-amplification in the cross-sectional specimens from Korean kidney transplant patients. Expression of 9/14 genes (CXCL9, CD3ϵ, IP-10, LCK, C1QB, PSMB9, Tim-3, Foxp3, and FAM26F) was significantly different between acute rejection and stable graft function with normal pathology and long-term graft survival in 103 training samples. CXCL9 was also distinctly expressed in allografts with acute rejection in in situ hybridization analysis. This result, consistent with the qPCR result, implies that urinary mRNA could reflect the magnitude of allograft injury. We developed an AR prediction model with the urinary mRNAs by a binary logistic regression and the AUC of the model was 0.89 in the training set. The model was validated in 391 independent samples, and the AUC value yielded 0.84 with a fixed manner. In addition, the decision curve analysis indicated a range of reasonable threshold probabilities for biopsy. Therefore, we suggest the urine mRNA signature could be used as a non-invasive monitoring tool of acute rejection for clinical application and could help determine whether to perform a biopsy in a recipient with increased creatinine

Evaluation of the TEST 1 erythrocyte sedimentation rate system and intra- and inter-laboratory quality control using new latex control materials

Background: The erythrocyte sedimentation rate (ESR) test has been considered to be a simple procedure, not requiring quality control (QC). However, QC is essential for accuracy and precision. We evaluated the TEST 1 ESR system and performed QC procedures using newly developed latex control materials in three hospitals. Methods: Using tripotassium ethylenediaminetetraacetic acid blood samples (n=184), we compared TEST 1 ESR values with Westergren ESR data and evaluated intra-assay precision. Three levels of latex control materials were used to assess inter-assay precision. Reference range assessment was done using samples from 220 healthy individuals. Inter-laboratory QC with latex control materials in three hospitals was performed. Results: Correlation between TEST 1 ESR and Westergren ESR results was good (p<0.001). Intra-assay precision [coefficients of variation (CV) 6.6%-21.7%] with patient samples and inter-assay precision (CV 0.0%-6.8%) with latex control materials were satisfactory. The reference ranges of 2-10 mm/h for males and 2-19 mm/h for females were established. Inter-laboratory QC data with latex control materials in three hospitals demonstrated good accuracy and satisfactory precision (CV 0.0%-14.4%). Conclusions: Our results demonstrate that the TEST 1 QC is reliable and the latex control materials are valuable for inter-laboratory proficiency testing. Clin Chem Lab Med 2010; 48: 1043-8.Cha CH, 2009, AM J CLIN PATHOL, V131, P189, DOI 10.1309/AJCPOU1ASTLRANIJ*CLIN LAB STAND I, 2008, C28A3 CLSIPiva E, 2007, CLIN BIOCHEM, V40, P491, DOI 10.1016/j.clinbiochem.2006.12.002Padro-Miquel A, 2007, CLIN CHEM LAB MED, V45, P930, DOI 10.1515/CCLM.2007.150Romero A, 2003, CLIN CHEM LAB MED, V41, P232Plebani M, 2002, AM J CLIN PATHOL, V117, P621LEE BH, 2002, J CLIN PATHOL QUALIT, V24, P167GIAVARINA D, 2002, CLIN LAB, V48, P459Piva E, 2001, CLIN CHEM LAB MED, V39, P451*CLIN LAB STAND I, 2000, H2A4 CLSIPlebani M, 1998, AM J CLIN PATHOL, V110, P334BULL BS, 1993, J CLIN PATHOL, V46, P198BULL BS, 1991, PRACTICAL LAB HEMATOFABRY TL, 1987, BLOOD, V70, P1572WESTERGREN A, 1926, AM REV TUBERC, V14, P94