Cryopreservation of mobilized blood stem cells at a higher cell concentration without the use of a programmed freezer

Cryopreservation of peripheral blood stem cells (PBSC) mobilized by chemotherapy combined with or without granulocyte colony-stimulating factor (G-CSF) is an essential part of procedure for anti-cancer strategies. We evaluated whether a higher cell concentration (2×10⁸/ml) without the use of a programmed freezer was acceptable for the storage of mobilized PBSC in an autologous setting. Mobilized PBSC were enriched to mononuclear cells (MNC) by Percoll separation and then frozen at cell concentrations of 2–5×10⁷/ml (group I, n=20) or 2×10⁸/ml (group II, n=44) without the use of a programmed freezer using 5% DMSO, 6% hydroxy ethyl starch, and 4% autologous serum or human albumin. CD34+ cells purified by ISOLEX300 were frozen at 2×10⁷/ml (group III, n=22) using the same method. The median recovery rates of CD34+ cells and CFU-GM were, respectively, n.d. (not determined) and 88% in group I, 103 and 64% in group II, and 98 and 53% in group III. There was a statistical significance between the recovery rate of CFU-GM in group III and that in group I (p=0.02). The median percentage of cell viability after thawing in each group was 89, 87, and 75%, respectively. The median numbers of days after PBSCT to achieve a WBC of >1.0×10⁹/l, an absolute neutrophil count of >0.5×10⁹/l, and a platelet count of >50×10⁹/l were, respectively, 11, 11 and 15 in group I; 12, 12 and 16 in group II; and 12, 12 and 27 in group III. These results suggest that enriched MNC from mobilized PBSC could be frozen at a higher cell concentration (2×10⁸/ml) without the use of a programmed freezer, leading to reduction of the toxicities associated with infusion of thawed cells and of costly space required for cell storage.     

Nationwide survey of middle ear cholesteatoma surgery cases in Japan: Results from the Japan Otological society registry using the JOS staging and classification system

ObjectiveThis study was aimed to determine the characteristics of middle ear cholesteatoma and to investigate short-term outcomes regarding the rates of residual and recurrent cholesteatoma and the postoperative hearing results in Japan, via a nationwide survey using staging and classification criteria for middle ear cholesteatoma, as proposed by the Japan Otological Society (JOS).MethodsThe first-round survey was conducted in 2016. The target was patients with middle ear cholesteatoma who were surgically treated in Japan between January and December 2015. Medical information on the patients was anonymized. The questionnaire entries were age, sex, cholesteatoma classification and stage, preoperative hearing level, mastoid development, status of the stapes, and surgical method. There were a total of 1,787 registered patients from 74 facilities from all over Japan. The second survey was conducted in January 2018 and received 1,456 responses from 49 facilities in Japan. Of the 1,456 cases, 1,060 were conducted in the postoperative hearing survey and 1,084 in the residual recurrence survey.ResultsThe most common cholesteatoma type was pars flaccida cholesteatoma (63.3%), followed by pars tensa cholesteatoma (13.0%), congenital cholesteatoma (12.9%), and cholesteatoma secondary to chronic tensa perforation (5.6%). Cholesteatoma of uncertain origin accounted for 5.0% (90 cases). Stage II was predominant in pars flaccida and pars tensa cholesteatoma, which frequently involves the mastoid, whereas about half of cases of cholesteatoma secondary to chronic tensa perforation and congenital cholesteatoma were classified as stage I. One hundred fifty-two of 1,084 cases (14.0%) had recurrent cholesteatoma, residual cholesteatoma, or both following first surgeries. The postoperative rates of hearing success rate was 63.3%.ConclusionWe were able to clarify not only the current epidemiological status of middle ear cholesteatoma but also the current trends of cholesteatoma surgery in Japan. The development of a staging system by the JOS Committee serving an epidemiological database for international or time-dependent comparison. It is possible to use this staging system with reasonable reliability.     

Coffee polyphenols improve peripheral endothelial function after glucose loading in healthy male adults

Brewed coffee is a widely consumed beverage, and many studies have examined its effects on human health. We investigated the vascular effects of coffee polyphenols (CPPs), hypothesizing that a single ingestion of CPP during glucose loading would improve endothelial function. To test this hypothesis, we conducted a randomized acute clinical intervention study with crossover design and measured reactive hyperemia index (RHI) to assess the acute effects of a 75-g glucose load with CPP in healthy, nondiabetic adult men. Blood glucose and insulin levels were elevated after glucose loading with and without CPP, with no significant differences between treatments. The RHI did not significantly decrease after glucose loading without CPP. With CPP, however, RHI significantly (P < .05) increased over baseline after glucose loading. The difference between treatments was statistically significant (P < .05). No significant changes were observed in an oxidative stress marker after glucose loading with or without CPP. These findings suggest that a single ingestion of CPP improves peripheral endothelial function after glucose loading in healthy subjects.     

Aortic squamous metaplasia in a patient with aortoesophageal fistula secondary to thoracic aortic aneurysm: An autopsy case

Aortoesophageal fistula (AEF) is highly lethal. A 74‐year‐old man presented with hematemesis and consciousness loss. He had a long‐term history of hypertension and gout. Computed tomography revealed an aneurysm of the distal descending thoracic aorta, which was treated by insertion of an aortic stent graft. After 24 days of stenting, endoscopic examination revealed an AEF. After 6 months of stenting, he died owing to mediastinitis. On autopsy, macroscopically, we found a 4 × 2.5‐cm, oval, well‐circumscribed AEF. We identified squamous epithelium in the area surrounding the AEF that covered the thoracic aorta inner cavity. Immunohistochemical analysis revealed that the squamous epithelium in the thoracic aorta was positive for p63 and 34βE12. In conclusion, we encountered a long‐term AEF case with aortic squamous metaplasia. To the best of our knowledge, human aortic metaplasia has never been reported. In the present case, aortic squamous metaplasia retained continuity with the esophageal squamous epithelium; therefore, the migration of the squamous epithelium through the AEF may have been induced by aortic erosion.     

Morphological spectrum of renal pathology and its correlation to clinical features in patients with disseminated intravascular coagulation: A study involving a series of 21 autopsy cases

Disseminated intravascular coagulation (DIC), a thrombohemorrhagic disorder, occurs as a secondary complication in many diseases, but the histopathological features of kidneys in DIC have not been extensively characterized thus far. We reviewed 21 autopsy cases of patients with a clinical diagnosis of DIC and studied the repertoire of renal pathology. Eighteen patients had elevated serum creatinine levels and 15 patients had a variable degree of proteinuria. Underlying disorders included malignant neoplasms in 12 patients, and abdominal aortic aneurysm, acute myocardial infarction, and systemic infections in other patients. Coexistent glomerular pathology, such as focal segmental glomerulosclerosis (FSGS) with different morphological variants, and microthrombi formation, was present in many patients. The microthrombi were histologically similar to that seen in thrombotic microangiopathy, but characteristics associated with DIC were detected by special staining. The presence of FSGS correlated with the degree of urinary protein (P = 0.0044), and the presence of acute tubular injury (ATI) and the extent of global glomerulosclerosis both correlated with serum creatinine levels (P = 0.019 and 0.0003, respectively). FSGS was probably due to endothelial cell damage, another potential etiology for FSGS. Global glomerulosclerosis, a result of previous renal injury, can be a determinant of renal function during the acute phase of DIC.     

Cardiac energetics analysis after aortic valve replacement with 16-mm ATS mechanical valve

The 16-mm ATS mechanical valve is one of the smallest prosthetic valves used for aortic valve replacement (AVR) in patients with a very small aortic annulus, and its clinical outcomes are reportedly satisfactory. Here, we analyzed the left ventricular (LV) performance after AVR with the 16-mm ATS mechanical valve, based on the concept of cardiac energetics analysis. Eleven patients who underwent AVR with the 16-mm ATS mechanical valve were enrolled in this study. All underwent echocardiographic examination at three time points: before AVR, approximately 1 month after AVR, and approximately 1 year after AVR. LV contractility (end-systolic elastance [Ees]), afterload (effective arterial elastance [Ea]), and efficiency (ventriculoarterial coupling [Ea/Ees] and the stroke work to pressure–volume area ratio [SW/PVA]) were noninvasively measured by echocardiographic data and blood pressure measurement. Ees transiently decreased after AVR and then recovered to the pre-AVR level at the one-year follow-up. Ea significantly decreased in a stepwise manner. Consequently, Ea/Ees and SW/PVA were also significantly improved at the one-year follow-up compared with those before AVR. The midterm LV performance after AVR with the 16-mm ATS mechanical valve was satisfactory. AVR with the 16-mm ATS mechanical valve is validated as an effective treatment for patients with a very small aortic annulus. The cardiac energetics variables, coupling with the conventional hemodynamic variables, can contribute to a better understanding of the patients’ clinical conditions, and those may serve as promising indices of the cardiac function.     

Continuous fever-range heat stress induces thermotolerance in odontoblast-leneage cells

Objective

Heat shock during restorative procedures can trigger damage to the pulpodentin complex. While severe heat shock has toxic effects, fever-range heat stress exerts beneficial effects on several cells and tissues. In this study, we examined whether continuous fever-range heat stress (CFHS) has beneficial effects on thermotolerance in the rat clonal dental pulp cell line with odontoblastic properties, KN-3.

Methods

KN-3 cells were cultured at 41 °C for various periods, and the expression level of several proteins was assessed by Western blot analysis. After pre-heat-treatment at 41 °C for various periods, KN-3 cells were exposed to lethal severe heat shock (LSHS) at 49 °C for 10 min, and cell viability was examined using the MTS assay. Additionally, the expression level of odontoblast differentiation makers in surviving cells was examined by Western blot analysis.

Results

CFHS increased the expression levels of several heat shock proteins (HSPs) in KN-3 cells, and induced transient cell cycle arrest. KN-3 cells, not pre-heated or exposed to CFHS for 1 or 3 h, died after exposure to LSHS. In contrast, KN-3 cells exposed to CFHS for 12 h were transiently lower on day 1, but increased on day 3 after LSHS. The surviving cells expressed odontoblast differentiation markers, dentine sialoprotein and dentine matrix protein-1. These results suggest that CFHS for 12 h improves tolerance to LSHS by inducing HSPs expression and cell cycle arrest in KN-3 cells.

Conclusions

The appropriate pretreatment with continuous fever-range heat stress can provide protection against lethal heat shock in KN-3 cells.     

Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides

Most virulence (vir) genes of Agrobacterium tumefaciens that are required for the formation of crown gall tumors are expressed in response to such plant signal molecules as acetosyringone and lignin precursors. The phenolic signals are transduced through a receptor VirA protein in the inner membrane of the bacterial cell. The expression of these genes triggers the transfer of a specific DNA segment, called transferred DNA (T-DNA), from the Ti plasmid to plant cells, and its integration into their nuclear DNA. We show here that a group of aldoses (L-arabinose, D-xylose, D-lyxose, D-glucose, D-mannose, D-idose, D-galactose, and D-talose) can markedly enhance acetosyringone-dependent expression of vir genes when the concentration of acetosyringone is limited (10 microM) but does not enhance the expression of noninducible genes. Likewise, a 2-deoxy-D-glucose, a nonmetabolized sugar, is also effective. When a deletion was introduced into the virA gene in the region encoding the periplasmic portion of the VirA protein, enhancement by glucose disappeared, but vir expression was induced by acetosyringone in this mutant. These results suggest that these sugars directly enhance a signaling process initiated by phenolic inducers that results in an increase in expression of the vir genes.