Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR.

AIMS--To evaluate the ability of four rapid DNA extraction methods to provide DNA for the polymerase chain reaction (PCR) from routinely fixed, paraffin wax embedded archival tissues. METHODS--Eighteen blocks of various tissues, 18 blocks of cervical cancer specimens, and nine blocks of B cell lymphomas were investigated. Both normal and biopsy specimen sized tissues were studied. DNA was extracted using four methods: boiling for 20 minutes in distilled water; boiling for 20 minutes in 5% Chelex-100 resin solution; 3-hour proteinase K digestion; and 3-hour proteinase K digestion, followed by boiling in 5% Chelex-100. Different exons of the p53 gene, human papillomavirus type 16 (HPV 16) sequence, and immunoglobulin heavy chain (IgH) gene rearrangement were amplified from the extracts. RESULTS--The Chelex boiling, proteinase K digestion, and proteinase K digestion-Chelex boiling methods produced DNA suitable for amplification in all of the 45 samples. Boiling in water yielded insufficient template for the PCR in three of the 45 cases (7%), and in six of 42 positive cases (14%) much fainter bands were observed, mostly when the processed material was either biopsy specimen sized or a B cell lymphoma sample. Fragments of the p53 gene were successfully amplified up to 408 base pairs in water boiled extracts, up to 647 in Chelex boiled preparates, and up to 984 in proteinase K digested and proteinase K digested-Chelex boiled samples, although with decreased sensitivity in the last case. All of the templates were reusable after 3 months of storage at -20 degrees C. CONCLUSIONS--Chelex boiling, proteinase K digestion, and proteinase K digestion followed by Chelex boiling produce suitable templates for the PCR from a large variety of paraffin wax embedded tissues. As the simple 20 minute boiling method in 5% Chelex-100 solution requires minimal manipulation and time, it could be useful, especially in the routine processing of large amounts of material.     

The effect of prednisolone on substance P-induced vascular permeability in mice

The effect of prednisolone on the substance P (SP)-induced vascular permeability increase in male ddY, WBB6 F₁–+/+ (control) and WBB6 F₁-W/W^v (no mast cell in skin or internal organs) mice was investigated. 1) SP (1–10 000 pg/site) increased vascular permeability in ddY, WBB6 F₁–+/+ and WBB6 F₁-W/W^v mice ears. 2) SP (100 pg/site)-induced vascular permeability was inhibited by prednisolone (10 mg/kg) administered intraperitoneally 3 to 12 hours prior to the elicitation of the reaction in ddY mice. When dexamethasone at a dose of 1 mg/kg was administered intraperitoneally 2 to 24 hours prior to the elicitation of the reaction, significant inhibition was observed. When prednisolone was administered intraperitoneally 8 hours prior to the elicitation of the reaction, the SP-induced capillary permeability increase in both ddY and WBB6 F₁-W/W^v mice was clearly inhibited by the drug at doses of 5 and 10 mg/kg. 3) Diphenhydramine (1 and 10 mg/kg) inhibited SP-induced vascular reaction in ddY mice but not in WBB6 F₁-W/W^v mice. 4) Atropine (10 mg/kg) inhibited SP-induced vascular reaction in both ddY and WBB6 F₁-W/W^v mice. But acetylcholine did not cause an increase of vascular permeability in ddY and WBB6 F₁-W/W^v mice ears. 5) Prednisolone (5 mg/kg) inhibited histamine- and serotonin-induced vascular permeability in ddY and WBB6 F₁-W/W^v mice ears. 6) Prednisolone (5 and 10 mg/kg) inhibited the SP-induced histamine release from ddY mice peritoneal mast cells. These results suggest that the vascular effect of SP is mediated by both mast cell dependent (release of histamine from mast cells) and mast cell independent mechanisms. Prednisolone inhibits the SP-induced vascular permeability mediated by both mechanisms in mice.     

Genotype,serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus

Hepatitis B virus (HBV) genotypes have distinct geographical distribution. HBV sequences among hepatitis B carriers in Malawi have not been evaluated thus far. HBsAg serotype and genotype of HBV was determined in 20 serum samples from Malawian chronic HBV carriers, and two complete genomes and 13 entire pre-S2/S genes were sequenced directly. Genotype A HBV isolates were found in all of the samples, and serotype with adw2 and ayw2 were detected in three and 17 samples, respectively. In phylogenetic analyses, two complete genomes were classified into a subgroup A' that was described previously in South African isolates of the virus, and were separated from HBV isolates in Western countries with nucleotide differences ranging from 4.1-6.2%. The separation of subgroup A' was also evident in the tree topology of the entire pre-S1/S2, X and precore/core region, but not evident in the small-S region. The nucleotide divergences in subgroup A' were higher than those among genotype A without subgroup A' in the complete genomes as well as each of four open reading frames. All of the 13 pre-S2/S sequences were classified into the subgroup A', and clustered with known HBV isolates with ayw2 in carriers from South Africa and Zimbabwe. Three amino acids in the pre-S2/S gene were characteristic of subgroup A' with ayw2. In conclusion, unique HBV isolates of subgroup A' with ayw2 are prevalent in Malawi, and subgroup A' with a relatively higher nucleotide diversity may be a HBV isolate characteristic of the indigenous population of some African countries.     

A novel Fc receptor for IgA and IgM is expressed on both hematopoietic and non-hematopoietic tissues

By contrast to well-defined Fc gamma and Fc epsilon receptors, the structural and functional characteristics of Fc mu receptor are unclear. We have recently described a novel mouse Fc receptor, designated Fc alpha/mu receptor, and its human homologue, which bind both IgM and IgA. Here we show that the Fc alpha/mu receptor is expressed on mature, but not immature, B lymphocytes and acquires the ability to bind IgM and IgA antibodies after stimulation of B lymphocytes. Moreover, stimulation with phorbol 12-myristate 13-acetate increased endocytosis of IgM-coated microparticles mediated by the Fc alpha/mu receptor expressed on pro-B cell line Ba/F3 cells. We also show that the Fc alpha/mu receptor is expressed in secondary lymphoid organs, such as lymph node and appendix, kidney and intestine, suggesting an important role of the receptor for immunity in these organs.     

A new-type titanium intervertebral spacer and its insertion device used in posterior lumbar interbody fusion

We have recently developed a new-type trapezoid mesh cage (TPM cage) together with an insertion device, which for use as a new titanium mesh intervertebral spacer in posterior lumbar interbody fusion (PLIF). The TPM cage has sufficient mechanical strength, a large contact area that gives good long-term stability, and preserves the initial disc height to provide good balance. The insertion device for the TPM cage is useful not only for handling the implant but also for controlling the implant insertion direction. The TPM cage and its insertion device are promising for use in PLIF.     

Magnetic Resonance Imaging Assessment of Abductor Muscles Shortly After Curved Periacetabular Osteotomy

BackgroundCurved periacetabular osteotomy (CPO) is performed via an anterior approach without detachment of the hip abductor muscles. This study aimed to evaluate the abductor muscle status shortly after CPO on magnetic resonance imaging (MRI).MethodsWe prospectively evaluated 38 hips in 38 patients 1 week and 3 months after CPO between October 2017 and July 2019. The status of the abductor muscles was assessed on MRI using the following criteria: grade 0, normal; grade I, strain/edema; grade II, partial tear; and grade III, complete tear. We also evaluated associations between muscle status and patients’ characteristics.ResultsOne week after CPO, the gluteus maximus was classified as grade 0 in all patients. The gluteus medius was grade 0 in 84.2% of patients and grade I in 15.8%. The gluteus minimus was grade I in 55.3% of patients and grade II in 44.7%. Three months after CPO, both the gluteus maximus and gluteus medius were grade 0 in all patients, while the gluteus minimus was still grade I in 47.4%. There were no significant differences between patients with a grade 0 and grade I gluteus minimus at 3 months after CPO in patients’ characteristics (age and body mass index) or clinical scores (Harris Hip Score and Japanese Orthopedics Association score).ConclusionBoth the gluteus minimus and medius showed abnormal appearances on MRI 1 week after CPO, whereas only the gluteus minimus showed abnormalities 3 months after CPO. This abductor muscle status did not affect the postoperative Harris Hip Score or Japanese Orthopedics Association score.     

Minimum-Dose,Short-Term Methotrexate With Tacrolimus for Graft-vs-Host Disease Prophylaxis Following Unrelated Cord Blood Transplantation in Adults: A Retrospective Analysis at a Single Institution

BackgroundMethotrexate (MTX) or mycophenolate mofetil with tacrolimus (TAC) is used for graft-vs-host disease (GVHD) prophylaxis in unrelated cord blood transplantation (CBT). However, there is no consensus regimen for GVHD prophylaxis in CBT. We aimed to assess the efficacy and feasibility of minimum-dose, short-term MTX (MS-MTX) for GVHD prophylaxis in CBT.MethodsWe retrospectively evaluated 35 consecutive adult patients who underwent CBT and received MS-MTX (6 mg/m² day 1; 3 mg/m² days 3 and 6, intravenously) with TAC for GVHD prophylaxis in our hospital between 2015 and 2019. Transplantation outcomes with respect to time to hematopoietic recovery, engraftment, incidence and severity of GVHD, adverse events, relapse, nonrelapse mortality (NRM), and overall survival were evaluated.ResultsThe median time to neutrophil, platelet, and reticulocyte recovery was 22, 38, and 32 days, respectively. Cumulative neutrophil engraftment was 91.4%. After a median 3.2-year follow-up, the 2-year overall survival was 64.3%. The 2-year cumulative incidence of relapse and NRM was 20.4% and 14.9%, respectively. The 100-day cumulative incidence of grade II-IV acute GVHD and 2-year cumulative incidence of chronic GVHD were 28.6% and 36.6%, respectively. No grade IV acute GVHD was observed. Sixteen patients experienced oral mucositis and/or pharyngeal pain (46%; grades 1-2, n = 15; grade 3 pharyngeal pain, n = 1). No patients suffered from human herpesvirus 6 encephalitis/myelitis.ConclusionsMS-MTX with TAC is feasible and safe and yields lower rates of severe oropharyngeal mucositis and human herpesvirus 6 encephalitis/myelitis without increasing GVHD, graft failure, relapse, or NRM.