Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction.

OBJECTIVE: To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products. METHODS: We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method. RESULTS: The detection sensitivity of 16S rDNA PCR in spiked whole blood was 10¹ to 10² colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested. CONCLUSIONS: Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine.

Alterations in HbA1c resulting from the donation of autologous blood for elective surgery in patients with diabetes mellitus.

BACKGROUND: The aim of this study was to confirm the change in haemoglobin A1c consequent to pre-operative donation of autologous blood for elective surgery in patients with diabetes mellitus. MATERIAL AND METHODS: For enrolment in this prospective study, patients had to be scheduled for multiple autologous blood donations at different times and have a haemoglobin A1c level more than 5.8% at the first donation. The values of four factors, haemoglobin, haemoglobin A1c, glycated albumin, and glycated albumin/haemoglobin A1c ratio were determined. Changes in the values of these four factors between before and after the blood donations were calculated. RESULTS: In all 24 patients studied, haemoglobin and haemoglobin A1c decreased as a result of the autologous blood donations. The group with a reduced glycated albumin/haemoglobin A1c ratio had short intervals between blood donations. Correlations were observed between donation interval and change in haemoglobin A1c (r=-0.63, P=0.003), and between donation interval and change in the glycated albumin/haemoglobin A1c ratio (r=0.489, P=0.045). DISCUSSION: Haemoglobin A1c levels are likely to be underestimated after autologous blood donation by patients with diabetes mellitus, so glycated albumin may be a better indicator of these patients' glycaemic control.

SS-A/Ro52 promotes apoptosis by regulating Bcl-2 production.

SS-A/Ro52 (Ro52), an autoantigen in systemic autoimmune diseases such as systemic lupus erythematosus and Sjögren's syndrome, has E3 ligase activity to ubiquitinate proteins that protect against viral infection. To investigate Ro52's role during stress, we transiently knocked it down in HeLa cells by siRo52 transfection. We found that Ro52(low) HeLa cells were significantly more resistant to apoptosis than wild-type HeLa cells when stimulated by H(2)O(2)- or diamide-induced oxidative stress, IFN-α, IFN-γ and anti-Fas antibody, etoposide, or γ-irradiation. Furthermore, Ro52-mediated apoptosis was not influenced by p53 protein level in HeLa cells. Depleting Ro52 in HeLa cells caused Bcl-2, but not other Bcl-2 family molecules, to be upregulated. Taken together, our data showed that Ro52 is a universal proapoptotic molecule, and that its proapoptotic effect does not depend on p53, but is exerted through negative regulation of the anti-apoptotic protein Bcl-2. These findings shed light on a new physiological role for Ro52 that is important to intracellular immunity.

[A case of Salmonella-infected thoracoabdominal aortic aneurysm making final diagnosis difficult].

We report a case of thoracoabdominal aortic aneurysm (TAAA) due to Salmonella Enteritidis making final diagnosis difficult. A 63-year-old man with a history of diabetes mellitus, hypertension, and cerebral infarction was seen elsewhere for a 40 degrees C fever, vomiting, and shaking on day 1 after onset. He was diagnosed with Salmonella bacteremia and hospitalized by us for intensive care. Computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound imaging did not, however, show critical findings of aneurysm, endocarditis, or osteomyelitis, and laboratory testing suggest significant inflammatory symptoms. He did not respond to antibiotics, but had an intermittent low fever during the first hospitalization. On day 48 after onset during the second hospitalization, abdominal CT showed an aneurysm -3 cm in diameter in the thoracoabdominal aorta above the renal artery- small enough to have been missed in earlier diagnosis. Surgery and TAAA graft replacement were done on day 64. Bacterial culture of the graft showed no Salmonella growth due to long-term in vivo antibiotic exposure. He recovered without significant complications, with oral ciprofloxacin antibiotic therapy continued to the present. This case indicates the importance of an early diagnosis through continuous blood culture and imaging for Salmonella sp blood stream infection.

[Comparison of detection sensitivity in rapid-diagnosis influenza virus kits].

Rapid-diagnosis kits able to detect influenza A and B virus by immunochromatography developed by different manufacturers, while useful in early diagnosis, may vary widely in detection sensitivity. We compared sensitivity results for eight virus-detection kits in current use--Quick Chaser FluA, B (Mizuho Medy), Espline Influenza A & B-N (Fujirebio), Capilia Flu A + B (Nippon Beckton Dickinson & Alfesa Pharma), Poctem Influenza A/B (Otsuka Pharma & Sysmex), BD Flu Examan (Nippon Beckton Dickinson), Quick Ex-Flu "Seiken" (Denka Seiken), Quick Vue Rapid SP Influ (DP Pharma Biomedical), and Rapid Testa FLU stick (Daiichi Pure Chemicals)--against influenza virus stocks, contained five vaccination strains (one A/H1N1, two A/H3N2, and two B) and six clinical strains (two A/H1N1, two A/H3N2, and two B). Minimum detection concentrations giving immunologically positive signals in serial dilution and RNA copies in positive dilution in real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were assayed for all kits and virus stock combinations. RNA log10 copy numbers/mL in dilutions within detection limits yielded 5.68-7.02, 6.37-7,17, and 6.5-8.13 for A/H1N1, A/H3N2, and B. Statistically significant differences in sensitivity were observed between some kit combinations. Detection sensitivity tended to be relatively higher for influenza A than B virus. This is assumed due to different principles in kit methods, such as monoclonal antibodies, specimen-extraction conditions, and other unknown factors.

DNA-PK: the major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway.

The effect of wortmannin posttreatment was studied in cells derived from different species (hamster, mouse, chicken, and human) with normal and defective DNA-dependent protein kinase (DNA-PK) activity, cells with and without the ataxia telangiectasia (ATM) gene, and cells lacking other regulatory proteins involved in the DNA double-strand break (DSB) repair pathways. Clonogenic assays were used to obtain all results. Wortmannin radiosensitization was observed in Chinese hamster cells (V79-B310H , CHO-K1), mouse mammary carcinoma cells (SR-1), transformed human fibroblast (N2KYSV), chicken B lymphocyte wild-type cells (DT40), and chicken Rad54 knockout cells (Rad54-/-). However, mouse mammary carcinoma cells (SX9) with defects in the DNA-PK and chicken DNA-PK catalytic subunit (DNA-PKcs) knockout cells (DNA-PKcs-/-/-) failed to exhibit wortmannin radiosensitization. On the other hand, SCID mouse cells (SC3VA2) exposed to wortmannin exhibited significant increases in radiosensitivity, possibly because of some residual function of DNA-PKcs. Moreover, the transformed human cells derived from AT patients (AT2KYSV) and chicken ATM knockout cells (ATM-/-) showed pronounced wortmannin radiosensitization. These studies demonstrate confirm that the mechanism underlying wortmannin radiosensitization is the inhibition of DNA-PK, but not of ATM, thereby resulting in the inhibition of DSB repair via nonhomologous endjoining (NHEJ).