[Multidisciplinary regenerative treatment and mechanisms for rescuing a severe calciphylaxis patient with human amnion-derived mesenchymal stem cells].

Calciphylaxis is a rare disease with severe pain and high-mortality due to cutaneous ischemic necrosis and infection that currently lacks proved effective therapies. The occurrence of calciphylaxis in end stage kidney disease (ESKD) patients is known as calcific uremic arteriolopathy (CUA), which is characterized histologically by dermal microvessel calcification, intimal fibroplasia and microthrombosis. Here we innovatively treated a severe CUA patient with human amnion-derived mesenchymal stem cells (hAMSCs). A 34-year-old uremic woman was presented with progressive, painful malodorous ulcers in buttocks and mummified lower limbs. Skin pathological features supported the diagnosis of calciphylaxis. The patient was refractory to conventional multidisciplinary symptomatic therapies. With the approval of our hospital ethics committee, she was treated with hAMSCs including intravenous and local intramuscular injection, and external application of hAMSC culture supernatant to the wound area. During 15-month follow-up, the patient had regeneration of skin and soft tissues, with improved blood biochemical, inflammatory, mineral and bone metabolic indices and immunoregulation effects. After 15-month hAMSC treatment, the score of pain visual analog scale (VAS) decreased from 10 to 0, Bates-Jensen wound assessment tool (BWAT) score decreased from 65 to 13, and wound-quality of life (Wound-QoL) questionnaire score decreased from 68 to 0. We propose that hAMSC treatment is promising for CUA patients. The therapy is potentially involved in the multiple beneficial effects of inhibiting vascular calcification, stimulating angiogenesis and myogenesis, modulating adverse inflammatory and immunologic responses, promoting re-epithelialization and restoring skin integrity.

[Analysis of the diagnostic performance of MRI Liver Imaging Reporting and Data System version 2018 for intrahepatic parenchymal substantial lesions ≤3.0 cm].

Objective: To investigate the diagnostic performance of MRI Liver Imaging Reporting and Data System version 2018 in high-risk hepatocellular carcinoma (HCC) patients with intrahepatic parenchymal substantial lesions ≤3.0 cm. Methods: A retrospective analysis was conducted in hospitals between September 2014 to April 2020. 131 pathologically confirmed non-HCC cases with lesions ≤3.0 cm in diameter were randomly matched with 131 cases with lesions ≤3.0 cm in diameter and divided into benign (56 cases), other hepatic malignant tumor (OM, 75 cases), and HCC group (131 cases) in a 1:1 ratio. MRI features of the lesions were analyzed and classified according to LI-RADS v2018 criteria (tie-break rule was applied to lesions with both HCC and LR-M features). Taking the pathological results as the gold standard, the sensitivity and specificity of the LI-RADS v2018 classification criteria and the more stringent LR-5 criteria (with three main signs of HCC at the same time) were calculated for HCC, OM or benign lesions diagnosis. Mann -Whitney U test was used to compare the classification results. Results: The number of cases classified as LR-M, LR-1, LR-2, LR-3, LR-4, and LR-5 in HCC group after applying the tie-break rule were 14, 0, 0, 12, 28, and 77, respectively. There were 40, 0, 0, 4, 17, 14 and 8, 5, 1, 26, 13, 3 cases in benign and OM group, respectively. There were 41 (41/77), 4 (4/14) and 1 (1/3) lesion case in the HCC, OM and benign group, respectively, that met the more stringent LR-5 criteria. The sensitivity of LR-4 combined with LR-5 (LR-4/5) criteria, LR-5 criteria and more stringent LR-5 criteria for HCC diagnosis were 80.2% (105/131), 58.8% (77/131) and 31.3% (41/131), respectively, and the specificity were 64.1% (84/131), 87.0% (114/131) and 96.2% (126/131), respectively. The sensitivity and specificity of LR-M were 53.3% (40/75) and 88.2% (165/187), respectively. The sensitivity and specificity using LR-1 combined with LR-2 (LR-1/2) criteria for the diagnosis of benign liver lesions were 10.7% (6/56) and 100% (206/206), respectively. Conclusions: LR-1/2, LR-5, and LR-M criteria have high diagnostic specificity for intrahepatic lesions with a diameter of ≤3.0 cm. Lesions classified as LR-3 are more likely to be benign. The specificity of LR-4/5 criteria is low, while the more stringent LR-5 criteria has a high specificity for HCC diagnosis.

[Etiology and clinical analysis of central nervous system infection caused by Coxsackievirus B5 in severe hand, foot and mouth disease in Qingdao City, 2013-2014].

Objective: To illuminate the gene characteristics and clinical characterization of Coxsackievirus B5 (CV-B5) strains isolated from patients with sevre hand, foot and mouth disease (HFMD) in Qingdao city. Methods: A total of 1 844 patients of HFMD were consecutively admitted to Qingdao Women and Children's Hospital from 2013 to 2014. Information of the study population described above was collected retrospectively. The samples were collected from at least 1 site (throat swab, cerebrospinal fluid), which viral nucleic acid extracted and the entire VP1 gene sequences of CV-B5 isolates were amplified and sequenced, then the homology and phylogeny analysis were conducted by MEGA7.0. The prototype Faulkner strain and other VP1 amino acid sequences were derived from the GenBank database. Results: A total of 8 CV-B5 positive cases were obtained, including 4 males and 4 females; 6 severe hospitalized cases and 2 outpatients. The age of 6 hospitalized patients ranged from 3 to 48 months, with a median of 26 months. For the six inpatients, fever, convulsions vomiting, diarrhea and rash were the main clinical manifestation, and all combined with viral encephalitis. Compared with the prototype strain Faulkner, in the VP1 region,the nucleotide and the amino acid homologies was 77.3%-78.8% and 95.5%-97.0% respectively. Five out of the six severe cases with substitution of serine (S) to asparagine (N) at amino acid site 95 in the VP1 region. The sequences of 8 CV-B5 strains were classified into genogroup D. Conclusion: Hand, foot and mouth disease associated with CV-B5 virus infection can result in nervous system involvement and the main complication was viral encephalitis. The CV-B5 strains associated with severe hand, foot and mouth disease had high nucleotide homology and present a certain regional aggregation.

[Combined multiple artery-first approach to pancreatoduodenectomy].

Objective: To investigate the clinical application of combined multiple artery-first approach to pancreatoduodenectomy. Methods: The clinical data of 53 patients who were diagnosed with peripancreatic head tumor at Department of Biliary-Pancreatic Surgery of Second Affiliated Hospital of Harbin Medical University between June 2013 and June 2015 was retrospectively analyzed.Pancreatic enhanced CT scan, magnetic resonance cholangiopancreatography, ultrasonography and tumor marker detection were applied for all the patients preoperatively.The 53 patients were operated by combined multiple artery-first approach(superior+ posterior approach, superior+ inferior approach, posterior+ inferior approach, superior+ posterior+ inferior approach) according to individualized therapeutic concept.And 42 patients underwent pancreatoduodenectomy, 9 patients underwent palliative operation and 2 patients just received exploratory operation. Results: Forty-two peripancreatic head tumor patients underwent pancreatoduodenectomy by applying combined multiple artery-first approach.The median operation time and intraoperative blood loss were (5.4±3.1)hours and (366±297)ml and the harvested lymph node and duration of hospital stay were 19±5 and (14.0±5.6)days.Nine patients underwent "total mesopancreas excision" and the rate of postoperative pancreatic fistula and R0 resection were 38.1% and 88.1%. Anomalous origin hepatic right artery was detected in one patients during the operation and no death occurred within 30 days postoperatively. Conclusion: According to the tumor location and patient's condition, individualistically applying combined multiple artery-first approach can reduce intraoperative blood loss, terminate unnecessary surgery, detect anomalous origin artery, make the tumor resection more radical and pancreatoduodenectomy more safety.

The predictive value of semaphorins 3 expression in biopsies for biochemical recurrence of patients with low- and intermediate-risk prostate cancer.

The class-3 semaphorins (Sema3A-F, Sema3s) are initially identified to play an important role in axonal guidance and cell migration. Our previous studies showed that Sema3s are also involved in the lymph node metastasis of prostate cancer, and are likely to modulate the behavior of prostate cancer with a pro-tumoral or an anti-tumoral effect, depending on their subtypes. However, no study has critically investigated the value of Sema3s expression in preoperative biopsy samples for the prediction of biochemical recurrence (BCR) after radical prostatectomy. In this study, we evaluated Sema3s expression by immunohistochemistry on 198 prostate biopsies with low- and intermediate-risk localized prostate cancer. The median follow-up was 42 months (range, 6-60) for all patients. Our results showed that Sema3A (OR: 0.19, P<0.001), Sema3B (OR: 0.38, P=0.003), Sema3E (OR: 0.39, P=0.007), and Sema3C (OR: 2.31, P=0.014) staining were independent predictors of BCR on multivariable analysis. Sema3A, 3B, 3C and 3E expression demonstrated potential values in predicting BCR upon survival analysis (P=0.001, P=0.003, P=0.029, P=0.037, respectively, Log-rank test). Our findings suggested that Sema3A, 3B, 3C, and 3E immunostaining in prostate biopsies, as supplements to clinicopathological parameters, could be used for predicting BCR in low- and intermediate-risk prostate cancer patients after radical prostatectomy. Specially, concurrent Sema3C-positive and Sema3A-negative, 3B-negative, 3E-negative staining is associated with an adverse prognosis. Further prospective studies in larger patient populations are needed to validate the current observations.

Expression profiling identifies new function of collapsin response mediator protein 4 as a metastasis-suppressor in prostate cancer.

Metastasis is the chief cause of mortality from cancer, but the mechanisms leading to metastasis are poorly understood. We used a proteomics approach to screen for metastasis-associated proteins and found that collapsin response mediator protein-4 (CRMP4) expression was inversely associated with the lymph node metastasis of prostate cancer (PCa). Subsequent in vitro and in vivo studies revealed that overexpression of CRMP4 not only suppressed the invasion ability of PCa cells, but also strongly inhibited tumor metastasis in an animal model. Furthermore, methylation of a CpG island within the promoter region of the CRMP4 gene is responsible for downregulation of CRMP4 expression. Thus, in this study, we show new function of CRMP4 as a metastasis-suppressor in PCa. The findings provide new mechanistic insights into metastasis and therapeutic potential for this most common male cancer.

Induced liver allograft immunological tolerance in rats by intramuscular injection of recombinant adeno-associated virus-human cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (rAAV-hCTLA4Ig).

Induction of liver allograft immunological tolerance was performed in rats by intramuscular injection of recombinant adeno-associated virus-human cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (rAAV-hCTLA4Ig). Dark Agouti and Lewis rats were liver allograft donors and recipients, respectively, in four groups: (A) syngeneic control, (B) blank control, (C) rAAV-enhanced green fluorescent protein negative control, (D) rAAV-hCTLA4Ig. Gene transfers occurred 6 weeks before transplantation. Group D had a significantly longer liver graft survival time (> 100 days) than groups B (11.9 +/- 1.3 days) and C (11.6 +/- 1.1 days). Groups B and C showed severe rejection responses and large amounts of CD4(+) and CD8(+) T-lymphocyte infiltration, while only a mild response and few T-lymphocytes were observed in group D. There were no significant differences in interleukin-2 and interferon-gamma levels in liver grafts between groups D and C, but there were significant decreases in granzyme B and lymphotoxin beta levels in group D compared with group C. It is concluded that immunological tolerance to liver allograft could be achieved by gene transfer of rAAV-hCTLA4Ig through intramuscular injection.

Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.

Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.

[Culture of human nasal respiratory epithelial cells in serum free medium supplemented with hormones and growth factors and measurement of human nasal ciliary motility using videomicroscopy].

OBJECTIVE: To establish a culture model of human nasal respiratory epithelial cells and a method of measuring human nasal ciliary motility. METHOD: The human nasal respiratory epithelial cells were detached with collagenase and cultured in serum free medium supplemented with hormones and growth factors, the ciliary beat frequency was measured by videomicroscopy. RESULT: After inoculation, cells cultured with this method adhered in 24 hours, confluented in 6-8 days and lived for 16 days. During that time ciliary beating was active, both acidic and neutral mucoitin granules were rich in goblet cells and all chromosome of 23 pairs were normal, the ciliary beat frequency in 29 subjects' nasal mucosa was (411 +/- 24) beats/min (mean +/- s). CONCLUSION: A culture model of human nasal respiratory epithelial cells in serum free medium supplemented with hormones and growth factors and a method of measuring human nasal ciliary motility was successfully established.

[Assay of Epstein-Barr virus in nasopharyngeal tissues and serum of patients with nasopharyngeal carcinoma].

OBJECTIVE: Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). Determination of EBV-VCA-IgA, EBV-EA-IgA in serum and assay of EBV DNA (PCR) in biopsy tissue has been used for diagnosis of NPC. This paper evaluated the value of the three determinations for diagnosis of NPC. METHOD: 146 patients were investigated. For each patient determination of EBV-VCA-IgA, EBV-EA-IgA in serum and EBV-DNA (PCR) in biopsy tissue were performed with double-blind studies, of 146 patients, 76 were NPC and 70 non nasopharyngeal carcinoma as control, identified with histopathological examination. The differences of the determinations between NPC and control were compared statistically with chi 2-test. RESULT: Of 76 NPC, the positive rates of EBV-DNA (PCR), EBV-VCA-IgA and EBV-EA-IgA were 90.8%, 75.0% and 26.3% respectivelly. For 70 control cases, the positive rates 2.9%, 38.6% and 2.9% respectivelly (P < 0.005). CONCLUSION: Our results suggested that assay of EBV-DNA (PCR) has better sensitivity and specificity than EBV-VCA-IgA and EBV-EA-IgA for diagnosis of NPC.

[Monitoring calcium in outer hair cells with confocal microscopy and fluorescence ratios of fluo-3 and fura-red].

Calcium distribution and mobilization during mechanical stimulation in outer hair cells of the guinea pig were monitored using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Spatial calcium gradients were revealed among various subcellular areas. The ratios of the fluorescence intensity of fluo-3 and fura-red were 1.71 +/- 0.85, 1.61 +/- 0.75, 1.47 +/- 0.65 and 1.39 +/- 0.66 for the cytoplasm, the cytoplasmic membrane, the cuticular plate and the nucleus respectively, indicating that free calcium ion concentrations are the highest in the cytoplasm and the lowest in the nucleus. While the calcium concentration remained relatively constant under resting conditions, it increased during mechanical stimulation. The results show that confocal ratio imaging of fluo-3 and fura-red enables us to determine more accurately the subcellular calcium distribution and that the calcium ions make a contribution to the mechanic-electrical transduction in hair cells.

Two types of calcium channels in bullfrog saccular hair cells.

Ca2+ channels were studied in cell-attached recordings from the basolateral membrane of the bullfrog saccular hair cells with the EPC-9 patch-clamp system. Pipettes contained 110 mM Ba2+ and the membrane potential was zeroed with isotonic potassium aspartate. Data acquisition and analysis were performed using E9SCREEN and M2LAB software. L-type channel was distinguished by a single-channel conductance of 26 pS, activation range between -10 and +40 mV and intense activity even at a holding potential of -40 mV. The L-type channel showed characteristic bursts of brief openings (mode 1) interrupted occasionally by longer openings (mode 2). Bay K 8644 promoted the mode 2 activity and nifedipine inhibited L-type channel activity. Another type of calcium channels, 20 pS channel, was detected by -50 to +10 mV depolarizing steps from a holding potential of -40 or -80 mV. This channel was insensitive to dihydropyridines and resembled the N-type channel.

Techniques for isolating hair cells from guinea pig cochlea.

Isolated live hair cells are important models for studies on the electrophysiology, pathology and pharmacology of the hair cell. Using mechanical isolation method after papain treatment, 70 +/- 27 hair cells, including 0 to 4 inner hair cells, could be obtained from each cochlea of 4 guinea pigs. The criteria for a good viability of isolated cochlear hair cells were: 1) a smooth hair cell membrane; 2) hair cells not swollen; 3) the nucleus in normal position; 4) the cytoplasm in a state of translucence with a halo at the periphery (birefringence); and 5) no Brownian movement of the organelles within the cytoplasm. With short-term culturing at room temperature, approximately 90% of the isolated hair cells retained a good viability at the end of two hours. Subsequently, the hair cells gradually degenerated but still, at the end of five hours, about 40% of them appeared intact. The degeneration patterns have been carefully observed and described.

[Potassium channels in saccular hair cells in the bullfrog].

By means of cell-attached and excised inside-out patch clamp techniques, we studied ion channels of the basolateral membrane of hair cells isolated from the bull-frog's sacculus. Three types of K+ channels were recorded, with conductances of 19-23PS, 46PS and 78PS respectively. The reversal potential was -56 +/- 20mV. The kinetics of the high- and low-conductance type K+ channels was different. The former was identified as KCa and the latter was suspected as KDR or KIR. They have important function in the sensory coding of hair cells.

[Significance of typing in the survival of malignant lymphomas].

The significance of typing and subtyping in the survival rate of 116 patients suffering from pathologically proved malignant lymphoma is presented. Our data show that Hodgkin's disease was least malignant, having the highest survival curve (P less than 0.005). Lymphoblastic lymphoma had the highest degree of malignancy, having the lowest survival curve. Yet, the middle portion of its curve resembled that of the peripheral T cell lymphoma. Although the degree of malignancy and the curve of peripheral T cell lymphoma and B cell lymphoma were similar (0.05 greater than P greater than 0.05), their middle portion were separated, a fact worthy of further study. Typing of malignant lymphomas is of practical importance.

Calcium distribution and mobilization during depolarization in single cochlear hair cells. Imaging microscopy and fura-2.

Intracellular distribution of cytoplasm-free Ca2+ concentrations ((Ca2+)i) and dynamic changes during stimulation of viable hair cells were studied using digital imaging microscopy and the Ca2(+)-sensitive dye fura-2. (Ca2+)i was visualized on pseudo-colour images and three-dimensional computer graphics. In the resting state, the intra-cellular distribution of (Ca2+)i in both the outer and inner hair cells was heterogeneous, and the amount of (Ca2+)i in most of the peripheral cytoplasm just beneath the plasma membrane was greater than that throughout the entire cytoplasm. Cell depolarization, induced by elevated K+, led to an increase in (Ca2+)i in the outer hair cells. The increase in (Ca2+)i was not observed under conditions of depolarization in Ca2(+)-free medium. These observations are interpreted to mean that the increase in (Ca2+)i is induced by depolarization with the result that there is an influx of extracellular Ca2+ into the cytoplasm. When Mn2+ was applied during depolarization, a fluorescence quenching occurred. By such means the site of Ca2+ channels was elucidated.

[Thymus pathological changes in perinatal babies and children].

The most outstanding pathological changes of perinatal babies and children, based on the review of the autopsy files in the 1st Affiliated Hospital of West China University of Medical Sciences, are lymphocytic depletion, and reticuloepithelial cell swelling and/or fusiform malformation in the cortex, while in the medulla apoptosis is more prominent. We suppose that these alterations are due to immaturity of cortical thymocytes, and in the diseased condition, they are easily affected by extraordinary factors, especially the influence of corticosterone inducing acute severe necrosis, so the number of lymphocytes are obviously diminished. But, in the medulla, as intact mature lymphocytes exist, physiological phenomenon such as apoptosis is rather prominent in it. While in the medulla, Hassall's corpuscles have various characteristic changes, such as cornification, calcification, fusion, cystic change and disintegration. Besides, we observed a new alteration in the thymus defined as vacuolization. All the above pathological changes reached the peak in the 28- day group; there after, they might become either worse or better according to the condition of the disease and growth of the body. However, these are still problems to be further studied separately.