Cell and platelet composition assays by flow cytometry: basis for new platelet-rich fibrin methodologies.

Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are selective blood fractions obtained by cen¬trifugation. They act locally on inflammation and immunity as adjuvant homeostatic modulators during tissue regeneration. In recent years, many methods for achieving these blood concentrates have emerged, whose parameters of time and force of centrifugation presented themselves as critical, conflicting, and poorly understood points. Thus, the present study aimed to evaluate the effect of different centrifugal experimental parameters on the concentration of cells and platelets in samples of anticoagulated blood. Blood samples were centrifuged by forces of 200, 400 and 800 x g for 5, 10 and 15 minutes of centrifugation times to obtain three fractions: a) platelet-poor plasma (PPP), b) leukocyte-rich plasma (L- PRP) and c) red blood cell sed¬iment (RBC). The leukocyte and platelet content of each centrifuged fraction was measured by automated flow cytometry associated with the peroxidase reaction for differential leukocyte count. The application of 200 x g generated a more significant dispersive content of leukocytes and platelets in the supernatant fraction of PPP when compared to the other two strength ranges. However, it presented the highest concentration of platelets in the sediment (P <0.05 ANOVA), representing a loss of total mass during processing. The 400 and 800 x g forces showed leukocytes and platelets condensed in the L-PRP fraction and lower levels in the sedi¬ment, demonstrating the greater effectiveness of buoyancy in the resuspension of these sedimented elements. Our experimental data showed that the concentration and organization of leukocytes and platelets in the centrifuged blood matrices are very sensitive to variations in g force and centrifugation time, thus generating products with different biological composition and characteristics, and with specific potential therapeutic effects. The present study did not focus on comparing authoring methods, but on presenting the impact of methodological variations on the biological nature of centrifuged blood matrices. Further in vivo studies are needed to assess the specific clinical effect of each methodological change.

Combined Endovascular and Microsurgical Hybrid Management of Cerebral Aneurysms: The Preliminary Fujita Experience.

INTRODUCTION: A subgroup of complex aneurysms demands multimodal treatment by microscopic and endovascular means. Partial obliteration or remnant postprocedure demands to go further to the other modality. MATERIALS AND METHODS: All patients between July 2016 and January 2017 who had to undergo multimodality measures for complete obliteration of the aneurysms were included in the study. The patients who had either undergone clipping or coiling for their aneurysms, but with incomplete obliteration of the aneurysm, were also included in the study. RESULTS: Between July 2016 and January 2017, a total of three patients had to undergo coiling after clipping of the aneurysm for complete obliteration. Two patients had to go for clipping following coiling. All five patients had complete obliteration of the aneurysm sac. CONCLUSION: Both microscopic and endovascular means are complementary measures instead of competing procedures. It is important to realize the technical difficulties when surgical therapy follows initial endovascular treatment. Similarly, coiling a previously clipped aneurysm can be difficult, if the clip obscures normal working projections.

Staged Deep Brain Stimulation of Ventral Intermediate Nucleus of the Thalamus for Suppression of Essential Tremors.

Deep brain stimulation (DBS) of the ventral intermediate nucleus (VIM) of the thalamus is a powerful surgical option in the treatment of essential tremors (ETs). However, its therapeutic efficacy depends on the tremor distribution. DBS is highly efficient in the relief of distal appendicular tremor but not other types of tremor. We report a case of staged DBS of ventral intermediate nucleus (VIM) of thalamus for the suppression of ETs.

The role of frozen section biopsy for parotid gland tumour with benign fine-needle aspiration cytology.

OBJECTIVES: This study focused on parotid gland tumours diagnosed as benign by fine-needle aspiration cytology and investigated the necessity of frozen section biopsy. METHODS: There were 104 cases of parotid gland tumour where fine-needle aspiration cytology was benign and frozen section biopsy was subsequently performed, between April 2006 and June 2016. In this retrospective study, the results of frozen section biopsy were analysed and compared with the final histological diagnosis. RESULTS: Among the 104 cases diagnosed as benign by fine-needle aspiration cytology, 102 cases and 2 cases were diagnosed as benign and malignant, respectively, by frozen section biopsy. The final histological diagnoses showed that 98 cases were benign and 6 cases were malignant. The sensitivity and specificity values of frozen section biopsy in detecting malignant tumours were 33 per cent and 100 per cent, respectively. CONCLUSION: The necessity of frozen section biopsy in cases with benign fine-needle aspiration cytology may be low in parotid gland surgery.

How I do it: underwater endoscopic ear surgery for plugging in superior canal dehiscence syndrome.

BACKGROUND: Underwater endoscopic ear surgery does not require suction and so protects the inner ear from unexpected aeration that may damage its function in the treatment of labyrinthine fistula. A method of underwater endoscopic ear surgery is proposed for the treatment of superior canal dehiscence. METHODS: Underwater endoscopic ear surgery was performed for plugging of the superior semicircular canal through the transmastoid approach. Saline solution was infused into the mastoid cavity through an Endo-Scrub Lens Cleaning Sheath. The tip of the inserted endoscope was filled completely with saline water. RESULTS: Using this underwater endoscopic view, the canal was clearly dissected to expose the semicircular canal membranous labyrinth and dehiscence area. No particular complication occurred during the surgical procedure. CONCLUSION: The underwater endoscopic ear surgery technique for plugging in superior canal dehiscence secures an excellent visual field and protects the inner ear from unexpected aeration.

Phase II/III study of second-line chemotherapy comparing irinotecan-alone with S-1 plus irinotecan in advanced gastric cancer refractory to first-line treatment with S-1 (JACCRO GC-05).

BACKGROUND: In Japan, S-1 plus cisplatin has been used as first-line therapy for advanced gastric cancer (AGC). Patients with no response to first-line treatment with S-1 often receive a taxane-alone or irinotecan-alone as second-line treatment. However, second-line treatment with S-1 plus irinotecan is widely used in patients with AGC resistant to first-line S-1-based chemotherapy. The goal of this trial was to determine whether the consecutive use of S-1 plus irinotecan improves survival when compared with irinotecan-alone as second-line treatment for AGC. PATIENTS AND METHODS: Patients who had disease progression during first-line S-1-based chemotherapy were randomly assigned to receive S-1 plus irinotecan or irinotecan-alone. The S-1 plus irinotecan group received oral S-1 (40-60 mg/m(2)) on days 1-14 and intravenous irinotecan (150 mg/m(2)) on day 1 of a 21-day cycle. The irinotecan-alone group received the same dose of irinotecan intravenously on day 1 of a 14-day cycle. The primary end point was overall survival (OS). RESULTS: From February 2008 to May 2011, a total of 304 patients were enrolled. The median OS was 8.8 months in the S-1 plus irinotecan group and 9.5 months in the irinotecan-alone group. This difference was not significant (hazard ratio for death, 0.99; 95% confidence interval 0.78-1.25; P = 0.92). Grade 3 or higher toxicities were more common in the S-1 plus irinotecan group than in the irinotecan-alone group. CONCLUSION: The consecutive use of S-1 plus irinotecan is not recommended as second-line treatment in patients who are refractory to S-1-based first-line chemotherapy. ClinicalTrials.gov ID: NCT00639327.

16th IHIW: international histocompatibility working group in hematopoietic cell transplantation.

The International Histocompatibility Working Group is a collaborative international effort to understand the HLA and non-HLA genetics of the transplantation barrier. The Working Group is comprised of experts in the fields of histocompatibility and immunogenetics, hematopoietic cell transplantation and outcomes research. Data for 25 855 unrelated donor transplants were submitted in support of research studies for the 16th International Histocompatibility Workshop. Active investigation is in progress in seven key areas: the impact of HLA matching, role of race and ethnicity, identification of permissible HLA mismatches, haplotype-associated determinants, minor histocompatibility antigens, immune response genes and KIR genetics. New hypotheses for the 16th workshop were developed for immunogenetic studies in cord blood and haploidentical-related donor transplantation.

A single nucleotide polymorphism of IL-17 gene in the recipient is associated with acute GVHD after HLA-matched unrelated BMT.

IL-17 has an important role in the host defense against extracellular pathogens and the pathophysiology of autoimmune diseases. This study retrospectively examined the impact of a single-nucleotide polymorphism (rs2275913, G197A) in the IL-17 gene of a total 510 recipients with hematologic malignancies and their unrelated donors on the clinical outcomes in HLA-matched myeloablative (discovery study) and nonmyeloablative (validation study) BMT through the Japan Marrow Donor Program (JMDP). In the discovery study, the presence of a 197A genotype in the recipient resulted in a higher incidence of grades II-IV acute GVHD (hazard ratio (HR), 1.87; 95% confidence interval (CI), 1.23-2.85; P=0.004). The donor IL-17A genotype did not significantly influence the transplant outcomes. The validation study showed a trend toward an association of the recipient 197A genotype with an increased risk of grades III-IV acute GVHD (HR, 5.84; 95% CI, 0.75-45.72; P=0.09), as well as a significantly increased risk for chronic GVHD (HR, 3.86; 95% CI, 1.29-11.59; P=0.02). These results suggest an association of the 197A genotype in the recipient side with the development of acute GVHD.

A single-nucleotide polymorphism of the Fcγ receptor type IIIA gene in the recipient predicts transplant outcomes after HLA fully matched unrelated BMT for myeloid malignancies.

Fcγ receptor type IIIA (FCGR3A) has a functional single-nucleotide polymorphism (rs396991), at which a G-to T-point mutation results in an amino acid substitution at position 158 (valine to phenylalanine; V158F). This study examined the effect of the FCGR3A polymorphism in donors and recipients on the clinical outcomes in unrelated HLA fully matched myeloablative BMT. The FCGR3A-V158F genotype was retrospectively analyzed in a total of 99 recipients with myeloid malignancies, and their unrelated donors. The presence of the 158V genotype in recipients showed a statistically better OS (adjusted hazard ratio (HR) 0.49; 95% confidence interval (CI) 0.26-0.93; P=0.03) and TRM (HR 0.30; 95% CI 0.14-0.67; P=0.003) without significant influence on the relapse rate. The recipient 158V genotype was also associated with a significantly reduced risk of chronic GVHD (HR 0.45; 95% CI 0.20-0.99; P=0.049) and a trend toward a reduced risk of grade II-IV acute GVHD (HR 0.55; 95% CI 0.27-1.10; P=0.09), leading to a significantly reduced GVHD-related mortality (HR 0.22; 95% CI 0.06-0.77; P=0.02). The donor FCGR3A polymorphism did not have any effect on the transplant outcomes. These results suggest an association between the recipient FCGR3A genotype and the clinical outcomes after BMT.

Association between dietary calcium and vitamin D intake and cervical carcinogenesis among Japanese women.

BACKGROUND/OBJECTIVES: To examine the association between dietary calcium and vitamin D intake and cervical neoplasia risk, we conducted a case-control study. SUBJECTS/METHODS: We selected 405 incident cervical neoplasias (333 invasive carcinomas and 72 cervical intraepithelial neoplasias grade III (CIN3)) and 2025 age-matched non-cancer controls. Dietary information was collected using a semiquantitative food-frequency questionnaire (FFQ). The effect on cervical neoplasia risk was evaluated using conditional logistic regression models. RESULTS: The inverse association between invasive carcinoma and milk, yogurt and fish was observed. On the other hand, the marginally significant inverse association between CIN3 and tofu and green leafy vegetables was observed. Compared with the lowest quartile (Q1) of calcium intake, adjusted odds ratios (ORs) for each of the three upper quartiles (Q2, Q3 and Q4) on invasive carcinoma risk were 0.86 (95% confidence interval (CI) 0.63-1.17), 0.50 (95% CI 0.34-0.73) and 0.68 (95% CI 0.48-0.97), respectively (P for trend=0.004). However, no association between calcium and cancer risk was evident among CIN3 cases (P for trend=0.528). Vitamin D intake showed a similar inverse association (Q2: OR 1.03, 95% CI 0.74-1.44; Q3: OR 0.80, 95% CI 0.56-1.15; and Q4: OR 0.64, 95% CI 0.43-0.94; P for trend=0.013). Similar to calcium, no association between vitamin D intake among CIN3 was evident (P for trend=0.109). An inverse association with calcium was evident in women whose vitamin D intake was low. However, this combined effect was not significant (invasive carcinoma: interaction P=0.819; and CIN3: interaction P=0.101). CONCLUSION: We found an inverse association between dietary calcium and vitamin D intake and cervical neoplasia risk among a group of Japanese women.

Successful third kidney transplantation with intensive immunosuppression in a highly sensitized recipient.

HLA sensitization associated with previous kidney transplantation is a major drawback to retransplantation. Recently we successfully performed a third graft using intensive immunosuppression for a highly sensitized recipient. The patient was a 31-year-old man who had previously undergone a living donor graft from his father at our institute in 1999. His kidney graft function had deteriorated due to chronic allograft nephropathy, returning to hemodialysis therapy in 2005. He received a second graft from a deceased donor in another country on August 14, 2006. It rejected on postoperative day 3 possibly due to acute accelerated rejection. He was offered a third kidney from his brother. Panel-reactive antibody (PRA) tested before the third procedure revealed positive class I (88%) and class II (96%) PRAs. Mycophenolate mofetil (MMF) was started 3 weeks before the third transplantation, and preoperative plasmapheresis performed thrice. He underwent the living donor graft on March 9, 2007. Immunosuppression consisted of tacrolimus, MMF, methylprednisolone, and basiliximab. Immediately afterward there was a sudden decrease in allograft blood flow and urine output, implying hyperacute rejection. Following treatment with plasmapheresis and a single dose of rituximab (200 mg), the kidney allograft function recovered, although the PRA at 3 weeks was still positive. Six months posttransplantation, he is well with a creatinine of 0.9 mg/dL. Our protocol may reduce the risk for graft loss in a highly sensitized transplant recipient.

Extended middle cranial fossa approach for vestibular schwannoma: technical note and surgical results of 896 operations.

From 1976 to 2006, 896 vestibular schwannomas were operated on using an extended middle cranial fossa approach. With this approach, the operative field can be extended according to tumor size and the facial and cochlear nerves can be preserved more easily with cooperation between the neurosurgeon and ENT surgeon. The mortality rate among 896 vestibular schwannoma patients was 0.3%. In the 760 initially operated vestibular schwannomas with total removal of the tumor, facial nerves were anatomically preserved in 715 or 94.1% of the cases. In 61.0% of 270 cases in which hearing preservation was attempted, hearing was preserved, and in 46.7% of those 270 cases useful hearing was preserved postoperatively. However, in the last 10 years the useful hearing preservation rate of the 140 attempted cases was 53.6%. Most of the complications of this approach were cerebrospinal fluid leakage; by using fat tissue, fibrin glue and spinal drainage from 1992 to 2005, cerebrospinal fluid leakage occurred in 59 or 10.6% of 569 cases, with 13 or 2.3% being repaired surgically. Moreover, in the last 10 years, the surgical results have improved along with improved surgical experience, improved instruments and better monitoring methods.

Four subtypes of petroclival meningiomas: differences in symptoms and operative findings using the anterior transpetrosal approach.

BACKGROUND: Petroclival meningiomas are vaguely defined as tumours arising from the antero-medial zone to the internal auditory meatus. This report subclassifies petroclival meningiomas based on their origin determined by using radiological and intra-operative findings. METHOD: Ninety-one patients with petroclival meningioma underwent surgery via the anterior transpetrosal approach. The Meckel's cave was routinely opened. Tumour origin was classified into four subtypes according to the main attachment and trigeminal nerve deviation into, upper clivus (UC), cavernous sinus (CS), tentorium (TE), and petrous apex (PA). Their characteristic clinical symptoms and anatomical features were investigated. FINDINGS: The characteristic symptom was ataxia in the UC type (37.5%), abducens nerve palsy in the CS type (64.3%) and trigeminal neuropathy, mainly neuralgia in the PA type (80.0%) with a higher statistical difference from other subtypes. The rate of tumour invasion into Meckel's cave reached 70.3% in average, with the lowest rate in the PA type (25.0%). The rate of middle fossa extension was the highest in the TE type (59.5%). The middle fossa approach was considered to be ideal for UC and TE types because of easier access to the Meckel's cave. Radical dissection without complications was difficult in the CS type. Both the anterior transpetrosal approach and the lateral suboccipital approach could be indicated in the PA type due to the rare invasion of Meckel's cave and middle fossa, and frequent extension into the internal auditory meatus. CONCLUSIONS: This classification is useful to predict the relation between the tumour and the cranial nerves based on symptoms and images. The anterior transpetrosal approach could be used for all four subtypes and with an absolute indication in the UC and TE types showing middle fossa extension.

Variation of dose distribution of stereotactic radiotherapy for small-volume lung tumors under different respiratory conditions.

PURPOSE: To clarify the effects of respiratory condition on dose calculation for stereotactic radiotherapy of small lung tumors. METHODS AND MATERIALS: Computed tomography (CT) data were obtained for nine tumors (diameter, 2.1-3.6cm; mean, 2.7cm) during the stable state, deep expiration, and deep inspiration breath-hold states. Rotational Irradiation with 3 non-coplanar arcs (Rotational Irradiation) and static irradiation with 18 non-coplanar ports (Static Irradiation) using 6-MV photons were evaluated using Fast Fourier Transform (FFT) convolution and Multigrid (MG) superposition algorithms. Dose-volume histograms (DVHs), mean path-length (PL) and mean effective path-length (EPL) were calculated. RESULTS: Although the PL was larger for the inspiration state than for the stable state and the expiration state, the EPL was 0.4-0.5cm smaller in the inspiration state than in the expiration state (p=0.01 for Rotational Irradiation; p=0.03 for Static Irradiation). The isocenter dose obtained by the FFT convolution algorithm was 7-12% higher than that obtained with the MG superposition algorithm. A leftward shift of the DVH obtained by MG superposition was noted for the inspiration state compared with the expiration state. CONCLUSIONS: The choice of the proper algorithm is important to accounting for changes in respiration state. Differences in isocenter dose were not large among the respiratory states analyzed. EPL was a little shorter for inspiration than for expiration, although there were larger and reverse trends in path length. A leftward shift of the DVH obtained for the inspiration state when MG superposition was used.

p53 target gene AEN is a nuclear exonuclease required for p53-dependent apoptosis.

DNA degradation is one of the biochemical hallmarks detected in apoptotic cells, and several nucleases have been reported to function cooperatively in this process. It has also been suggested that different sets of nucleases are activated by different stimuli, and induce distinct patterns of DNA degradation. Here we report that apoptosis-enhancing nuclease (AEN) is a novel direct target gene of p53. AEN is induced by p53 with various DNA damage, and its expression is regulated by the phosphorylation status of p53. We demonstrate that AEN is a typical exonuclease with conserved exonuclease domains Exo I-III, and it targets both single- and double-stranded DNA and RNA. AEN induces apoptosis by itself, and the conserved domains are essential for both AEN nuclease activity and its apoptosis-inducing ability. AEN possesses nuclear and nucleolar localization signals, and it translocates from the nucleolus to nucleoplasm upon apoptosis induction. We also show the dislocation of nucleophosmin in conjunction with the translocation of AEN to the nucleoplasm, indicating the ability of AEN in nucleolus disruption. In addition, AEN is shown to be required for efficient DNA fragmentation in p53-dependent apoptosis. These results suggest that AEN is an important downstream mediator of p53 in apoptosis induction.

Endodermal cyst of the cranio-cervical junction.

We reviewed 36 patients with endodermal cysts occurring at the craniocervical junction. They were aged between 3 and 66 years. Headache, motor weakness, and neck pain were commonly observed symptoms. Radiographically, T1-weighted magnetic resonance imaging of the tumours demonstrated a hypointense, isointense, or hyperintense signal according to the cystic content. In most cases, the cyst walls did not enhance after gadolinium administration. Histologically, the cysts were found to be lined by a single layer of epithelium. Histochemical and immunohistochemical studies showed that almost all were reactive to periodic acid schiff stain, epithelial membrane antigen, and carcino-embryonic antigen, but negative to glial fibrillary acidic protein. Mainly, the suboccipital approach with or without a laminectomy, or the trans-oral approach were selected for surgical excision of these tumours. In 17 of the 36 patients, total or gross total resections were performed, and subtotal resections were achieved in sixteen. Three patients developed recurrences.

Clinical characteristics of chronic graft-versus-host disease following umbilical cord blood transplantation for adults.

Chronic GVHD is a significant complication following allogeneic hematopoietic stem cell transplantation; however, the clinical characteristics of chronic GVHD following cord blood transplantation (CBT) in adults have not been well described. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers of the Nagoya Blood and Marrow Transplantation Group. Of 77 patients, 29 survived without graft failure or progression of underlying diseases for at least 100 days after transplantation. The median age of the 29 patients was 42 years (range, 18-67 years). Seven patients developed chronic GVHD (extensive, n=4; limited, n=3) disease. The cumulative incidence of chronic GVHD 1 year after day 100 was 24% (95% confidence interval (CI), 11-41%), and the organs involved were the skin (n=6), oral cavity (n=4), liver (n=1) and gastrointestinal tract (n=1). In three patients, chronic GVHD was resolved with supportive care. The remaining four were successfully treated with additional immunosuppressive therapy. Event-free survival rates of the 29 patients with and without chronic GVHD 3 years after day 100 were 83 (95% CI, 27-97%) and 36% (95% CI, 17-56%), respectively (P=0.047). These results suggest that chronic GVHD following CBT is mild and has a graft-versus-malignancy effect.

Olfactory schwannoma.

We report a case of olfactory schwannoma with calcification. Intraoperative findings indicated that the tumour originated from the olfactory groove. Intraoperative findings of previous studies have not indicated a clear relationship between subfrontal schwannoma and the olfactory nerve, which seems strange, given the association between tumours and cranial nerves at other sites. We suggest this observation has not been reported because the growing olfactory schwannoma changes the local morphology, affecting the appearance of the olfactory nerve.

Effect of HLA-A2 allele disparity on clinical outcome in hematopoietic cell transplantation from unrelated donors.

Population-based differences in clinical outcome after unrelated donor hematopoietic cell transplantation suggest that the significance of human leukocyte antigen (HLA) mismatching may be related to locus-specific and allele-specific differences that distinguish ethnically diverse transplant donors and recipients. We studied the risks associated with HLA-A locus mismatching in two large transplant populations from the International Histocompatibility Working Group in hematopoietic cell transplantation data set to better understand permissible and nonpermissible HLA-A mismatches.

The human cathepsin H gene encodes two novel minor histocompatibility antigen epitopes restricted by HLA-A*3101 and -A*3303.

Minor histocompatibility antigens (mHags) play crucial roles in the induction of graft versus host disease (GVHD) and/or graft versus leukaemia (GVL) effects following human leucocyte antigen (HLA)-identical haematopoietic stem cell transplantation (HSCT). Using HLA-A*3101- and -A*3303-restricted cytotoxic T lymphocyte (CTL) clones generated from different post-HSCT recipients, we identified two novel mHag epitopes encoded by the leader sequence of cathepsin H (CTSH) isoform a. The nonameric sequence ATLPLLCAR was defined as an HLA-A*3101-restricted epitope (CTSH(R)/A31), while a decameric peptide featuring a one N-terminal amino acid extension, WATLPLLCAR, was presented by HLA-A*3303 (CTSH(R)/A33). The immunogenicity of both epitopes was totally dependent on the polymorphic C-terminal arginine residue and substitution with glycine completely abolished binding to the corresponding HLA molecules. Thus, the immunogenicity of this mHag is exerted by differential HLA binding capacity. CTSH is relatively ubiquitously expressed at protein levels, thus it may be involved in GVHD and anti-leukaemic/tumour responses. Interestingly, however, CTL clones predominantly lysed targets of haematopoietic cell origin, which could not be explained in terms of the immunoproteasome system. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that CTSH-encoded mHags could be targets for GVL effects.