CD40L-stimulated B cells for ex-vivo expansion of polyspecific non-human primate regulatory T cells for translational studies.

The therapeutic applications of regulatory T cells (Tregs ) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded Tregs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific Tregs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. Tregs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to Treg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded Tregs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of Treg -specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these Tregs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these Tregs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific Tregs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific Tregs suitable for use in deceased-donor transplants.

Initial results of selective renal parenchymal clamping with an adjustable kidney clamp in nephron-sparing surgery: an easy way to minimise renal ischaemia.

INTRODUCTION: A renal parenchymal clamp has been used at our centre since March 2012. It is used in position over the kidney to achieve optimal vascular control of a tumour while minimising parenchymal ischaemia. This study aimed to report the feasibility, surgical outcome, and oncological control of a kidney clamp in partial nephrectomy. METHODS: This study was conducted at a teaching hospital in Hong Kong. Partial nephrectomies performed from January 2009 to March 2015 were reviewed. The tumour characteristics and surgical outcomes of kidney clamp were studied and compared with traditional hilar clamping. RESULTS: A total of 92 patients were identified during the study period. Kidney clamps were used in 20 patients and hilar clamping in 72, with a mean follow-up of 27 and 37 months, respectively. For patients in whom a kidney clamp was applied, all tumours were exophytic to a different extent and the majority (90%) were located at the polar region. The PADUA (preoperative aspects and dimensions used for an anatomical) classification nephrometry score was also lower than those in whom hilar clamping was used (7.07 vs 8.34; P=0.002). The clamp was used in open, laparoscopic, and robot-assisted surgery. Operating time was shorter (207 ± 72 mins vs 306 ± 80 mins; P<0.001) and estimated blood loss was lower (205 ± 191 mL vs 331 ± 275 mL; P=0.045) with kidney clamp. No acute kidney injury occurred. Postoperative renal function was comparable between the two groups. CONCLUSIONS: Partial nephrectomy using parenchymal clamping is safe and feasible in selected cases. The postoperative renal function and oncological control were satisfactory.

Development of an E-learning System for the Endoscopic Diagnosis of Early Gastric Cancer: An International Multicenter Randomized Controlled Trial.

BACKGROUND: In many countries, gastric cancer is not diagnosed until an advanced stage. An Internet-based e-learning system to improve the ability of endoscopists to diagnose gastric cancer at an early stage was developed and was evaluated for its effectiveness. METHODS: The study was designed as a randomized controlled trial. After receiving a pre-test, participants were randomly allocated to either an e-learning or non-e-learning group. Only those in the e-learning group gained access to the e-learning system. Two months after the pre-test, both groups received a post-test. The primary endpoint was the difference between the two groups regarding the rate of improvement of their test results. FINDINGS: 515 endoscopists from 35 countries were assessed for eligibility, and 332 were enrolled in the study, with 166 allocated to each group. Of these, 151 participants in the e-learning group and 144 in the non-e-learning group were included in the analysis. The mean improvement rate (standard deviation) in the e-learning and non-e-learning groups was 1·24 (0·26) and 1·00 (0·16), respectively (P<0·001). INTERPRETATION: This global study clearly demonstrated the efficacy of an e-learning system to expand knowledge and provide invaluable experience regarding the endoscopic detection of early gastric cancer (R000012039).

Toxicity of repeated intravenous injection of gene therapeutics for X-CGD in mice.

We made gene therapeutics for X-chronic granulomatous disease (CGD) by transducing murine bone marrow-derived stem cells with MT-gp91 retrovirus and evaluated possible toxicity in mice as a prerequisite for human clinical trials. Male C57BL/6 mice were injected intravenously with gene therapeutics for X-CGD twice at an interval of two weeks at 5 x 10(7) cells/kg and sacrificed 2 weeks after the last administration. Significant changes noted in gene therapeutics for X-CGD-treated animals were an increase in white blood cell counts and a slight decrease in albumin/globulin ratio. The red pulp hyperplasia in the spleen accompanied with an increase in organ weight was considered to result from the accumulation of gene therapeutics for X-CGD, bone marrow-derived stem cells, in the spleen. No anti-gp91 antibody was detected in the sera collected from the animals treated with gene therapeutics for X-CGD. No integration of gp91 DNA from retroviral vector was detected in chromosomal DNA of gonads in animals dosed with the test substance, indicating no potential of genomic integration. In conclusion, the repeated dose of gene therapeutics for X-CGD exerted no toxicity. The splenic red pulp hyperplasia and the increase observed in white blood cell counts and in spleen weights were considered as pharmacological changes induced by the treatment.

Immunogenicity and safety profiles of genetic vaccines against human Her-2/neu in cynomolgus monkeys.

Her-2/neu is a well-characterized tumor-associated antigen, the overexpression of which in human carcinomas correlates with a poor prognosis. Here, we evaluated Her-2/neu-specific humoral and cellular immune responses in immunized monkeys after immunization with nonreplicating adenovirus (AdHM) expressing the extracellular and transmembrane domain of human Her-2/neu (HM) and/or naked DNA vaccine (pHM-hGM-CSF) expressing human granulocyte-macrophage colony-stimulating factor together with HM. Priming of monkeys with AdHM generated Her-2/neu-specific long-lasting antibody production. Furthermore, these Her-2/neu-specific antibodies produced by AdHM immunization, some of which shared epitope specificity with Herceptin, were able to induce antibody-dependent cellular cytotoxicity against Her-2-expressing target cells. Cellular immune responses were elicited in all monkeys immunized with Her-2/neu-expressing vaccine; interferon-gamma was secreted when these splenocytes were restimulated with Her-2/neu-expressing autologous cells, and immunization with AdHM induced Her-2/neu-specific lymphoproliferative responses. Further, immunization with pHM-hGM-CSF before AdHM immunization noticeably enhanced cytotoxic T-lymphocyte activity. In addition, we observed no abnormalities that would indicate that the genetic vaccines had toxic effects in the immunized monkeys. Thus, we can conclude that our genetic vaccines efficiently elicited Her-2/neu-specific humoral and cellular immune responses without causing severe adverse effects in nonhuman primates and that as such they warrant further clinical investigation.

A combination of rough-based feature selection and RBF neural network for classification using gene expression data.

This paper presents a novel rough-based feature selection method for gene expression data analysis. It can find the relevant features without requiring the number of clusters to be known a priori and identify the centers that approximate to the correct ones. In this paper, we attempt to introduce a prediction scheme that combines the rough-based feature selection method with radial basis function neural network. For further consider the effect of different feature selection methods and classifiers on this prediction process, we use the NaIve Bayes and linear support vector machine as classifiers, and compare the performance with other feature selection methods, including information gain and principle component analysis. We demonstrate the performance by several published datasets and the results show that our proposed method can achieve high classification accuracy rate.

Recurrent prostatic stromal tumour of uncertain malignant potential (STUMP) presenting with urinary retention 6 Years after transurethral resection of prostate (TURP).

CLINICAL PRESENTATION: A 56-year-old Chinese male with previously diagnosed prostatic stromal tumour of uncertain malignant potential (STUMP) presented with urinary retention 6 years after transurethral resection of prostate (TURP). TREATMENT AND OUTCOME: Cystoscopy showed a papillary tumour of the prostatic urethra causing near-complete obstruction. Repeat TURP was performed. He has been asymptomatic since. CONCLUSION: There has been fewer than 100 cases of this lesion reported worldwide. Definitive treatment is not well established. Longterm follow-up to monitor progression and possible recurrence is required, and repeat TURP or radical surgery may be necessary.

Viral vector-mediated transduction of a modified thrombospondin-2 cDNA inhibits tumor growth and angiogenesis.

Gene therapy represents a possible alternative to the chronic delivery of recombinant antiangiogenic proteins to cancer patients. We have constructed retroviral and adenoviral vectors that express murine N-terminal fragments of thrombospondin-2 (NfTSP2), a potent endogenous inhibitor of tumor growth and angiogenesis. To test the possibility of anticancer gene therapy using NfTSP2, we tested whether an ex vivo retrovirus-mediated procedure could be used for the treatment of tumors. The treatment of tumor-bearing mice with syngenic immortalized cell lines expressing NfTSP2 led to a tumor volume reduction up to 70% as compared with the controls (P<0.005). In addition, the established tumors were eradicated in 40% of the mice treated with NfTSP2-expressing cells. Furthermore, the intratumoral injection of the NfTSP2-expressing adenoviral vector to the human squamous cell carcinoma in nude mice resulted in a significant reduction of the growth rates and the volumes of the carcinoma (P<0.05). Immunohistochemical staining of the tumors indicated that the total area and the average size of tumor vessels were significantly reduced in the treatment group versus the controls (P<0.05). In conclusion, the present study clearly demonstrates that the viral vector-mediated transfer of the NfTSP2 gene could inhibit the growth of tumors by perturbing tumor-associated angiogenesis.

Extracorporeal shockwave lithotripsy and intracorporeal lithotripsy for proximal ureteric calculi--a comparative assessment of efficacy and safety.

INTRODUCTION: Extracorporeal shockwave lithotripsy (ESWL) is the treatment modality of choice of many urologists for proximal ureteric calculi. In this study, we compared the efficacy and safety of ESWL versus ureteroscopy with holmium laser lithotripsy for the treatment of this group of stones. MATERIALS AND METHODS: Between May 1999 and October 2000, 50 patients had ESWL and another 51 patients underwent ureteroscopy with holmium laser lithotripsy for proximal ureteric calculi. The two groups were similar in age, sex ratio and stone size. ESWL was performed with the Dornier Compact lithotriptor whereas holmium laser lithotripsy was performed via retrograde ureteric access with a Wolf 7.5 Fr semirigid ureteroscope. RESULTS: Ureteroscopy with holmium laser lithotripsy was significantly better in terms of the mean procedure time (56 min in ESWL; 25 min in ureteroscopy; P < 0.001) and the 1-month stone free rate (50% in ESWL; 80% in ureteroscopy; P = 0.001). The 3-month stone free rate was also higher for ureteroscopy (78% in ESWL; 90% in ureteroscopy) but this difference was not statistically significant (P = 0.09). Minor complications of steinstrasse (6%) occurred in ESWL and proximal stone migration (8%) occurred during ureteroscopy. CONCLUSION: Ureteroscopy with holmium laser lithotripsy is a viable and safe alternative to ESWL for the management of proximal ureteric calculi.

Electro-gene therapy of collagen-induced arthritis by using an expression plasmid for the soluble p75 tumor necrosis factor receptor-Fc fusion protein.

Tumor necrosis factor (TNF) is a proinflammatory cytokine involved in the pathogenesis of rheumatoid arthritis, and antagonism of TNF may reduce the activity of the disease. Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. In this study, we attempted to treat collagen-induced arthritis (CIA) with anti-TNF gene therapy by transferring the plasmid encoding soluble p75 TNF receptor linked to the Fc portion of human IgG1 (sTNFR:Fc) using in vivo electroporation. DBA/1 mice were immunized with bovine type II collagen and boosted with the same antigen. At 2 days after boosting, the plasmid vector containing cDNA for the sTNFR:Fc was injected into one selected site in the gastrocnemius muscle followed by electroporation. Serum levels of sTNFR:Fc reached 2.3 ng/ml on day 5 when gene expression reached its peak. Macroscopic analysis of paws for redness, swelling and deformities showed that the onset of moderate-to-severe CIA in mice treated with sTNFR:Fc was prevented on a significant level compared with the control mice (P<0.05). The beneficial effect of sTNFR:Fc DNA transfer lasted for at least 18 days following treatment. In addition, both the synovitis and the erosion of cartilage in the knee joints were dramatically reduced in mice treated with sTNFR:Fc (P<0.05). The expression of IL-1beta and IL-12 in the paw was also decreased by sTNFR:Fc treatment (P<0.01) while there was little change in the levels of IL-17 and vWF. These data showed that sTNFR:Fc expression plasmid was effective in the prevention of CIA, and in vivo electroporation-mediated gene transfer may provide a new approach to cytokine therapy in autoimmune arthritis.

Penile colour duplex ultrasonography as a screening tool for venogenic erectile dysfunction.

INTRODUCTION: Vasculogenic impotence is one of the major causes of erectile dysfunction. Cavernosometry and cavernosography is traditionally the gold standard for evaluation of venogenic impotence. However, it is invasive and there are potentially significant complications. Penile colour flow Doppler imaging (PCDI) is non-invasive and can be used to assess venous incompetence. MATERIALS AND METHODS: One hundred and sixty-eight patients were referred for PCDI assessment from March 1998 to February 2001. Forty-three of these also had cavernosogram and cavernosometry done and were included in the study. RESULTS: The sensitivity was 93.9%, the specificity was 90.0%, the accuracy was 93.0% with a negative predictive value of 81.8% and a positive predictive value of 96.9%. Kappa value of 0.81 was obtained, indicating excellent agreement between PCDI and cavernosogram and cavernometry. CONCLUSIONS: Penile colour flow Doppler imaging is accurate in the assessment of venogenic erectile dysfunction. It can replace cavernometry and cavernosogram as a screening tool. Cavernometry and cavernosogram should only be done in cases when PCDI suggests venogenic impotence, and when surgery is contemplated.

Holmium laser lithotripsy in the management of ureteric calculi.

INTRODUCTION: To evaluate the efficacy and safety of the holmium:YAG laser in retrograde ureteroscopic laser lithotripsy of ureteric calculi. METHODS: Three hundred and twenty-five patients (256 males and 69 females) with mean age of 45 years underwent 328 retrograde ureteroscopic laser lithotripsy as the primary therapy for ureteric calculi using semirigid mini-ureteroscopes and the holmium:YAG laser (Coherent, Palo Alto, California, USA). Four consultants and three registrars performed the procedures over a period of 31 months. RESULTS: The mean largest diameter of the calculi was 8.4 mm. The mean hospital stay was 1.8 days and the mean duration for the operation was 25 minutes. One hundred and twenty-two (37%) procedures were done as day surgery cases. Complete stone-free rates after single primary treatment using only the holmium:YAG laser were 75% and 91% at 4 weeks and 12 weeks of follow-up, respectively. Including 22 (7.6%) renal units which required additional procedures, the overall stone-free rate was 97%. Seventeen cases (5.2%) needed additional extracorporeal shock wave lithotripsy (ESWL) and 5 cases (1.5%) needed an additional repeat ureteroscopy and lithotripsy for complete stone clearance. Significant complications include 1 case of ureteric perforation. CONCLUSIONS: The holmium:YAG is a safe and effective intracorporeal lithotripter when deployed in a retrograde manner in conjunction with a mini-ureteroscope.

Bioactivities of methyl allyl disulfide and diallyl trisulfide from essential oil of garlic to two species of stored-product pests, Sitophilus zeamais (Coleoptera: Curculionidae) and Tribolium castaneum (Coleoptera: Tenebrionidae).

Two of the major constituents of the essential oil of garlic, Allium sativum L., methyl allyl disulfide and diallyl trisulfide, were tested against Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) for contact toxicity, fumigant toxicity, and antifeedant activity. The contact and fumigant toxicities of diallyl trisulfide were greater than that of methyl allyl disulfide to the adults of these two species of insects. These two compounds were also more toxic to T. castaneum adults than to S. zeamais adults. Older T. castaneum larvae were more susceptible to the contact toxicity of the two compound, whereas younger larvae were more susceptible to the fumigant toxicity of these compounds. Both compounds reduced egg hatching of T. castaneum and subsequent emergence of progeny. Diallyl trisulfide totally suppressed egg hatching at 0.32 mg/cm2, and larval and adult emergence at 0.08 mg/cm2. Methyl allyl disulfide significantly decreased the growth rate, food consumption, and food utilization of adults of both insect species, with feeding deterrence indices of 44% at 6.08 mg/g food for S. zeamais and 1.52 mg/g food for T. castaneum. However, it did not affect any nutritional indices of T. castaneum larvae. Diallyl trisulfide significantly reduced all of the nutritional indices in all of the insects tested. Feeding deterrence indices of 27 and 51% were obtained in S. zeamais adults and T. castaneum larvae, respectively, at the concentration of 2.98 mg/g food, whereas feeding deterrence of 85% was achieved in T. castaneum adults at a much lower concentration of 0.75 mg/g food. Hence, diallyl trisulfide is a more potent contact toxicant, fumigant and feeding deterrent than methyl allyl disulfide.

Subjective visual experience during phacoemulsification and intraocular lens implantation under topical anesthesia.

OBJECTIVE: The aim of this study was to investigate the subjective visual experience of patients during phacoemulsification and intraocular lens (IOL) implantation under topical anesthesia. DESIGN: Postoperative questionnaire survey. PARTICIPANTS: The study cohort consisted of 52 patients with cataracts. There were 18 male (34.6%) and 34 female (65.4%) patients. Their mean (+/- SD) age was 67.5 (+/-10.8) years. INTERVENTION: The patients underwent routine phacoemulsification and IOL implantation under topical anesthesia. MAIN OUTCOME MEASURES: The patients were interviewed on the same day after their operation regarding their visual experience in the operated eye during surgery. RESULTS: All patients (100%) reported that they could see at least some light during the surgery. Some patients reported they could also see one or more colors (50 patients, 96.2%), movements (32 patients, 61.5%), flashes (24 patients, 46.2%), the surgeon's fingers/hands (13 patients, 25%), instruments (12 patients, 23.1%), and/or the surgeon (4 patients, 7.7%). The colors seen included red (24 patients, 46.2%), yellow (23 patients, 44.2%), blue (12 patients, 23.1%), green (7 patients, 13.5%), and orange (6 patients, 11.5%). Eight patients (15.4%) saw the spectrum of colors similar to that of the rainbow. Twenty-four patients (46.2%) reported that the brightness of light changed during the course of the operation. Eight patients (15.4%) found their visual experience frightening. There was no statistically significant association between those who found the visual experience frightening and the sex or age of the patient, a history of cataract operation in the fellow eye, the type of visual sensation experienced, or the presence of coexisting ocular pathology. CONCLUSION: All patients undergoing phacoemulsification under topical anesthesia experience a variety of visual sensations that may be frightening in a small proportion of patients.

Induction of NG108-15 cells differentiation by human bone marrow stromal cells.

The survival and differentiation of neuronal cells is dependent on factors such as neurotrophins, cytokines and components of extracellular matrix. Bone marrow stromal cells have been shown to support the growth and differentiation of neuroblastoma cells. In an attempt to study the effects of bone marrow stromal cells on neuronal differentiation, we have co-cultured neuroblastoma x glioma hybrid NG108-15 cells with human bone marrow stromal cells. After co-culturing, clones exhibiting morphological differentiated phenotype and high level of neurofilament expression were isolated. Interestingly, these clones maintain their ability to proliferate in contrast to differentiated NG108-15 cells induced by dibutyryladenosine 3',5'-cyclic monophosphate. These results suggested that bone marrow stromal cells can induce partial differentiation of NG108-15 cells.

Structural modeling of differential diagnosis, treatment, and results for allergic rhinitis.

This paper analyzed the relationship among the differential diagnosis, treatment, and results for allergic rhinitis using the covariance structural model. The data were collected from 274 patients with suspected allergic rhinitis who visited the Otorlaryngology Department of the Paik Hospital during 1991-1993. After each patient's characteristics was categorized and combined into several common factors, covariance structure analysis was performed to analyze the structural relationships among the differential diagnosis, treatment, and results of treatment using the significant factors obtained from discriminant analysis. The significant characteristics influenced the diagnosis were the results of skin test from mite/animal, and from mugworts, the results from laboratory tests, rhinorrhea and sneezing, and nasal polyps. The significant characteristics that influenced the method of treatment were: nasal polyps, headache/general symptom, family history/medication, and septal deviation. Headache/general symptom was the only significantly influencing factor for the treatment results.

Reactions of serum creatine kinase in early phase of spherical steel bullet injury.

On the basis of experimental study on 17 dogs, this paper reports on the variation of serum creatine kinase (SCK) with time in the early phase of wounding by spherical steel bullets at three different velocities and its relation with the kinetic energy absorbed by the tissue and with the amount of the excised necrotic tissue surrounding the wound tracks. The results showed that the energy absorbed by the tissue and the amount of the excised necrotic tissue surrounding the wound tracks increased with increasing velocity. Within a period of 6 hours after wounding the SCK activity increased correspondingly.

Differential labeling of the subunits of respiratory complex III with [3H]succinic anhydride, [14C]succinic anhydride, and p-diazobenzene-[35S]sulfonate.

Exposure of antimycin-treated Complex III (ubiquinol-cytochrome c reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex II depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by 14C- and 3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7,8, and 9. Two additional polypeptides of molecular masses 23 and 12kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of 14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in 14C/3H labeling ratios of core proteins I and II, cytochrome c1, and a polypeptide of molecular mass 13kDa identified as an antimycin-binding protein.