Effect of various antioxidants on ovarian tissue vitrification success in sheep.

BACKGROUND: Vitrification is a technique of cryopreservation that has been proposed as a promising alternative method for the preservation of oocytes, embryos and gonadal tissue. OBJECTIVE: To determine the effect of different antioxidants on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue. MATERIALS AND METHODS: Four different antioxidants [i.e., resveratrol (20 uM), ZnSO4 (500 uM), curcumin (25 uM) and quercetin (1 uM)] were evaluated after their addition to the vitrification and warming media for their effects on the viability and morphology of retrieved oocytes and the histology of vitrified ovarian tissue. RESULTS: The number of oocytes retrieved from ovarian tissue from the above mentioned antioxidants and vitrified control were 34, 41, 26, 31 and 46 respectively. Among these the number of viable oocytes were found to be 24 (70.6%), 30 (73.1 %), 20 (76.9%), 26 (83.9%) and 33 (71.7%) and the number of oocytes found morphologically normal were 24 (70.6%), 26 (63.4%), 18 (69.2%), 21 (67.7%) and 34 (73.9%) for the above mentioned different antioxidants and vitrified control, respectively. Non-significant (P. > 0.05) differences were found between different treatment groups. Histomorphological evaluation of the ovarian cortical tissue showed that the percentage of intact follicles was significantly (P < 0.05) higher in the fresh control (84.19±3.9) than in other groups. Non-significant differences were found between resveratrol (50.2±5.5), curcumin (48.7±5.7), quercetin (51.6±4.8) and the vitrified control (42.7±6.1) groups; however, the ZnSO4 supplemented group (23.1±8.54) differed significantly (P < 0.05) from other antioxidant groups but was non-significant (P > 0.05) with the vitrified control group (42.7±6.1). CONCLUSION: The addition of antioxidants resveratrol, curcumin and quercetin at these concentrations tended to non-significantly improve the follicular integrity after vitrification. Doi.org/10.54680/fr24410110212.

Recent advancements in vitrification cryodevices for gamete and gonadal tissue.

Cryopreservation of gametes and gonadal tissue is nowadays primarily accomplished through vitrification. Variables such as cooling rate, viscosity and volume of vitrification solution are critical in gamete vitrification. In addition, sample size and stepwise exposure are also crucial for gonadal tissue vitrification. Recently a class of cryodevices has been developed to reduce the volume of vitrification solution so as to achieve higher cooling rates. Vitrification devices are classified as "open" or "closed" depending on whether the medium comes into direct contact with liquid nitrogen during the process. Examples of the open cryodevices for gamete vitrification are Cryotop, Cryolock, open pulled straw (OPS), etc., and closed devices are Vitrisafe, CryoTip, and high security vitrification kit. Similarly, for tissue vitrification open cryodevices used are needles, cryovials and closed devices used are Cryotissue, ovarian tissue cryosystem, etc. Among all the gamete cryodevices, Cryotop is unique and the best-selling micro-volume storage device. Use of this device has resulted in the highest number of babies born after embryo or oocyte vitrification. Another novel device, Kitasato vitrification system, is a vitrification solution absorber, which is similar to Cryotop but differs in one way, as it possesses a porous membrane that absorbs extra solution from the gamete. This review provides an update on the recent use of cryodevices for gamete and gonadal tissue vitrification. doi.org/10.54680/fr22310110112.

Bilateral Tubercular Mastitis - Case Reports.

Breast tuberculosis is a rare form of tuberculosis. Moreover the disease is often overlooked and misdiagnosed as carcinoma or pyogenic abscess. Reports on breast tuberculosis have been few; reported incidence of breast tuberculosis amongst the total number of mammary conditions varies between 0.1 and 4 percent. Bilateral involvement is still more uncommon (3%). Here we report 3 cases of adult female ranging from 27 to 35 years who presented with 1 to 4 months history of firm lumps in both breasts and no axillary lymphadenopathy. Fine needle aspiration cytology (FNAC) of breast lump in all 3 cases were done but in 1 case showed evidence of tubercular mastitis and other 2 cases findings were inconclusive. Subsequent Excisional Biopsy of those 2 cases showed features of tuberculosis. All 3 were prescribed with four drug anti-tubercular treatments continued for 12 months in 2 cases and 9 months in other case depending upon their response. The lumps disappeared and ulcer healed after anti-tubercular treatment.

Role of Endoscopic Ultrasound Guided Fine Needle Aspiration in the Diagnosis of Pancreatic Lesions.

To evaluate the diagnostic utility of endoscopic ultrasound guided fine needle aspiration (EUS-FNA) of pancreatic lesions, EUS-FNA was carried out on a total of 28 cases at the Department of Surgery, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh from June 2015 to August 2016. Clinical impression was compared with the final cytological diagnosis and the percentage of non diagnostic smears was calculated. The lesions were categorized according to cytological report. Out of 28 cases, 3(10.71%) cases were normal, 6 cases (21.43%) were reported as inconclusive while a definite diagnosis was given in 19 cases (67.85%). The mean patient age was 47.82 years. There were 16(57.1%) males and 12(42.9%) females. The most common site biopsied was pancreatic head in 21cases (75%) followed by tail in 5 cases (17.9%) & body in 2(7.1%) cases. The average number of passes made was two. Endoscopic Ultrasound Guided Fine-Needle Aspiration in the diagnosis of pancreatic lesion is a useful procedure.

Penetrating keratoplasty: outcomes from a corneal unit compared to national data.

AIMS: To determine long term graft survival rates and visual results for different indications for penetrating keratoplasty from a single institution over 10 years and compare these to national outcome data. METHODS: Retrospective chart analysis. 784 records were available for review of 1096 consecutive penetrating keratoplasty procedures performed between 1990 and 1999 (72%). Outcomes of graft survival, visual acuity, and astigmatism were analysed and compared to national outcome data supplied by the UK Transplant Service. RESULTS: At 5 year follow up, overall graft survival was 66%. This was subdivided into 98% for keratoconus, 86% for viral keratitis, 85% for Fuchs' dystrophy, 84% for pseudophakic bullous keratopathy, 55% for regrafts, and 57% for other diagnoses. There was a significantly higher graft survival rate for all diagnostic subgroups except Fuchs' dystrophy at 3 years of follow up compared to the national average. Best corrected visual acuity at 5 years was 6/18 or better in 53% of cases. The mean keratometric astigmatism was 3.4 dioptres. CONCLUSION: Penetrating keratoplasty is a safe and effective treatment for selected corneal disorders. Penetrating keratoplasty for viral keratitis may achieve good results with long term antiviral treatment. Patients may achieve better outcomes if their surgery is performed at specialist centres.

In vivo cell cycle characteristics of pediatric leukemia patients.

Following i.v. bromodeoxyuridine infusion, a double-label technique using in vitro tritiated thymidine was used to determine the labeling index (LI), duration of S-phase (Ts), and cell cycle time (Tc) in pediatric leukemia patients. Eleven patients with acute lymphoblastic leukemia (ALL) and six patients with acute nonlymphoblastic leukemia (ANLL) were studied. Results of cell cycle kinetic studies are given for each group. Although median values for AML and ALL patients are similar to values reported in previous studies, there is a wide range of values among individual patients. The variation among the kinetic properties of blast cells in these patients reflects the heterogeneity of the acute leukemias of childhood. Further studies will be done to determine if these parameters correlate with outcome of therapy for pediatric leukemia patients.

In vivo determination of cell cycle kinetics of non-Hodgkin's lymphomas using iododeoxyuridine and bromodeoxyuridine.

Using sequential infusions of two S-phase-specific drugs, iododeoxyuridine and bromodeoxyuridine, we have developed an in vivo method for determining the labeling index (LI), the S-phase duration (Ts), and total cell cycle times (Tc) of non-Hodgkin's lymphomas. In nine non-Hodgkin's lymphomas studied, the LI ranged from 1.5% in a follicular small cleaved-cell lymphoma to 29.6% in a diffuse large-cell lymphoma. The Ts ranged from 16 hr in a large-cell lymphoma (immunoblastic type) to 117 hr in a follicular small cleaved-cell lymphoma. The Tc varied from 69 hr in a large-cell lymphoma (immunoblastic type) to over 1000 hr in all low-grade lymphomas studied. Immunohistochemical methods using anti-BrdU antibodies were used to detect cell incorporation of the two S-phase-specific drugs. In this manner, cell cycle times could be calculated while the architecture of the tumor specimen was preserved. Difficulties in using this methodology, specifically in the calculation of the growth fraction and total cell cycle times, are pointed out. This in vivo method does, however, allow for Ts calculations independent of growth fraction considerations. Correlations of cell cycle data with various biological and clinical factors await further patient follow-up.

Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.

In situ cell cycle kinetics in bone marrow biopsies following sequential infusions of IUdR/BrdU in patients with hematopoietic malignancies.

Examination of the proliferative characteristics of myeloblasts was undertaken in situ in bone marrow (BM) biopsies of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) following sequential infusions of iodo- (IUdR) and bromodeoxyuridine (BrdU). The ability to identify S-phase cells which have incorporated both or either one of the labels in vivo by using two monoclonal antibodies in vitro permitted the measurement of labeling index (LI) and durations of S-phase (Ts) and the total cell cycle (Tc) both from the BM aspirates and biopsies. While the LI is 2-3 times higher in biopsies, Ts and Tc are fairly comparable in the two samples in 8/10 cases (p = 0.02 and 0.003 respectively). Advantages associated with the determination of cell cycle parameters in BM biopsies have been discussed at length.

Cell cycle and clinical characteristics of patients with acute myeloid leukemia and myelodysplasia whose biopsies are reactive with anti-factor VIII antibody. A Leukemia Intergroup Study.

Presence of megakaryocytic cells in patients with myeloid disorders were investigated by staining plastic embedded biopsy sections with an anti-Factor VIII antibody (AFA). Two hundred and fifty cases were studied, 207 of whom had acute myeloid leukemia (AML) while 43 had myelodysplastic syndromes (MDS). Abnormal clusters of AFA positive cells indicating multilineage disease were identified in 17% with primary AML (30/175), 38% with secondary AML (12/32) and 42% cases of MDS (18/43). Biological characteristics of these 60 AFA positive cases were investigated. No unique differences in cell cycle characteristics following bromodeoxyuridine (BrdU) were identified. We confirm several recent reports that the incidence of multilineage involvement in AML is substantial.

Detection of single-stranded DNA damage using monoclonal anti-thymidine antibody.

A method to detect single-stranded DNA damage from individual cells has been developed using a monoclonal anti-thymidine antibody (MoAb20B7). Initially, HL-60 cells were incubated with daunomycin at different concentrations, and processed by MoAb20B7. While 73.5% of the cells incubated with 5 micrograms/ml of daunomycin for 24 h reacted positively with MoAb20B7, 83.5% cells at 10 micrograms/ml daunomycin dose were positive. Next, this method was combined with unscheduled DNA synthesis to simultaneously measure repair and damage from individual cells. Finally, patients with acute myeloid leukemias were studied before and 24 h after therapy with a daunomycin containing regimen. In vivo damage could be determined in a prompt fashion.

Cell-cycle characteristics - alterable determinants of remission duration in a study of 179 standard risk newly diagnosed patients with acute myeloid-leukemia.

Prognostic factors were related to remission duration among 179 standard risk newly diagnosed acute myeloid leukemia (AML) patients who received identical induction and consolidation therapies. Following a bromodeoxyuridine infusion, labeling indices of bone marrow aspirate/biopsy, durations of S-phase and cell cycle (Tc) were determined. Patients with slowly cycling myeloblasts had longer remissions (Log rank p=0.03) than those with rapidly cycling myeloblasts. Multivariate analysis demonstrated that both WBC and Tc contributed to remission duration (p=0.01 and 0.005 respectively). Patients with slowly proliferating leukemias have longer remissions probably due to slower regrowth of leukemia between chemotherapy courses.

Echo-planar functional MR imaging of epilepsy with concurrent EEG monitoring.

BACKGROUND AND PURPOSE: The role of functional MR (fMR) imaging in the evaluation of patients with epilepsy has not been systematically studied. Our purpose was to identify the fMR correlates of interictal epileptiform discharges. METHODS: Twenty patients with epilepsy and frequent interictal discharges were studied with concurrent EEG monitoring on a 1.5-T echo-planar magnet to acquire blood-oxygenation-level-dependent (BOLD) images in the baseline (OFF) and immediate post-discharge (ON) states. Analysis was performed using subtraction of average ON and OFF data (method I); cross-correlation analysis between the ON and OFF states (method II); and individual spike analysis (ISA), with which signal intensity in the individual ON states was statistically analyzed using a weighted comparison with the mean and variance of the OFF states (method III). Agreement of fMR activation with EEG localization was determined. RESULTS: Eighteen of 20 patients had interictal discharges during the monitoring period. Method I yielded visually detectable sites of BOLD signal differences in only one patient. Method II resulted in two patients with sites of BOLD activation. Method III, ISA, resulted in regions of increased BOLD signal corresponding to the EEG focus in nine of 10 patients. CONCLUSION: fMR studies can often reveal sites of increased BOLD signal that correspond to sites of interictal EEG discharge activity. Because of variable intensity changes associated with discharge activity, ISA resulted in increased sensitivity.

Penetrating keratoplasty: indications over a 10 year period.

AIMS: To determine the indications for penetrating keratoplasty (PK) at the Corneoplastic Unit and Eye Bank, UK, a tertiary referral centre, over a 10 year period. METHODS: Records of all patients who underwent PK at our institution between 1990 and 1999 were reviewed retrospectively. Of the 1096 procedures performed in this period, 784 records were available for evaluation (72%). RESULTS: Regrafting was the most common indication, accounting for 40.9% of all cases. Keratoconus was the second most common indication (15%), followed by Fuchs' endothelial dystrophy (9.3%), pseudophakic bullous keratopathy (7.6%), and viral keratitis (5.9%), which included both herpes simplex and herpes zoster and showed a statistically significant decreasing trend using regression analysis (p<0.005). Among the regraft subgroup, viral keratitis accounted for 21.2% as the underlying primary diagnosis. The most common cause for graft failure in the regraft subgroup was endothelial failure (41.8%). CONCLUSION: Regrafting is the leading indication for PK; viral disease-although declining-is the leading primary diagnosis.

A novel triple label method validates the double label technique of defining cell cycle kinetics in HL-60 cells.

HL-60 cells were sequentially labeled with the thymidine analogues iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU). The labeling index (LI), the duration of S-phase (Ts) and the total cell cycle time (Tc) were measured immediately. It was therefore possible to predict the next time when the single versus double labeled cells would re-enter the S-phase. In our study, the Tc was calculated to be 20 hours. The third label, tritiated thymidine (3HTdR), was introduced at the predicted time of 20 hours to confirm the validity of the previously calculated Tc. The actual percentage of cells which were labeled by (3HTdR) was very similar to the predicted value. We conclude, therefore, that the calculated cell cycle time correlated well with the actual cell cycle time, at least in a controlled in vitro culture system. This novel triple label method validates our double-label technique developed for cell cycle measurements.

Comparison of two double labeling techniques to measure cell cycle kinetics in myeloid leukemias.

Five patients with acute myeloid leukemia (AML) received a one hour infusion of iododeoxyuridine (IUdR) 100 mg/M2 to label S-phase cells in vivo. The aspirate was labeled in vitro either with tritiated thymidine (3HTdr) or bromodeoxyuridine (BrdU) to measure the duration of S-phase (Ts). The mean Ts using 3HTdr (Ts1) was 15.9h (13.1-19.8h) and using BrdU (Ts2) was 17.1h (14.5-20.6h). Total cell cycle time (Tc) ranged between 44.7h to 158.8h using Ts1 and 54.0h to 170.5h using Ts2. Based on this close approximation between the results, we confirm the reliability of the newly developed method that relies purely on immunohistochemical reaction.

Cell cycle kinetic studies in human cancers. Development of three DNA-specific labels in three decades.

Controversy has epitomized the prognostic and clinical significance accorded to cell cycle kinetic studies in neoplastic diseases. Recent introduction of monoclonal antibodies to thymidine analogues such as iododeoxyuridine and bromodeoxyuridine has unveiled a spectacular new area of research. Large numbers of patients with liquid and solid tumors have been interrogated by introducing iododeoxyuridine and bromodeoxyuridine as DNA-specific probes in vivo. Further in vitro incubation of S-phase cells already double-labeled in vivo with tritiated thymidine has added a whole new dimension to the kinds of questions that can now be addressed with alacrity. Time is rapidly approaching when this information will be available in a prompt enough fashion to be useful for planning therapeutic strategies. Reviewed are the salient features of the rapid progress achieved in the last decade in this exciting discipline.

Site-specific delivery of iloprost during experimental angioplasty suppresses smooth muscle cell proliferation.

PURPOSE: The authors have previously reported that intramural delivery of iloprost during angioplasty suppresses local platelet aggregation at 1 hour in undiseased porcine arteries. In this study, the authors sought to quantify the effect of such treatment on medial vascular smooth muscle cell proliferation, an event implicated in the development of intimal hyperplasia. MATERIALS AND METHODS: Three Yorkshire pigs underwent percutaneous transluminal angioplasty with hydrogel-coated balloons for a total of 10 iloprost-treated (experimental) and 10 saline-treated (control) arterial sites. The balloons were prepared with previously reported techniques and loaded with 2.25 microg of iloprost for the experimental sites. On the eighth day after angioplasty, these sites were harvested and prepared for immunohistochemical staining. Thin (4 microm) sections of the specimens were stained with use of monoclonal antibody to proliferating cell nuclear antigen (PCNA). Appropriate positive and negative controls were used. Approximately 350-500 vascular smooth muscle cells were randomly counted under high power (100x) by an experienced physician who was blinded to the origin of the specimen. A PCNA index (%) was calculated as follows: [(#PCNA [+] cells)/(#PCNA [+] cells + #PCNA [-] cells)]x 100. A paired t test was used for statistical comparison. RESULTS: The PCNA indices for eight (n = 8) paired large vessels (iliac, carotid, subclavian) were 7.98 (+/- 1.8)%, for the iloprost-treated experimental sites, and 14.58 (+/- 3.8)% for the saline-treated control sites. This difference was statistically significant (P = .003). One large vessel pair was not available for analysis. When the pair of renal arteries of animal 3 were included (n = 9), the PCNA indices were 8.32 (+/- 2.3)% for the experimental sites, and 13.79 (+/- 4.2)% for the control sites. The differences were again significant (P = .01). CONCLUSION: Intraarterial site-specific delivery of iloprost during angioplasty with drug-loaded, hydrogel-coated balloons significantly suppresses medial smooth muscle cells in swine at the expected peak period of proliferation of 7 days after angioplasty.

Cell cycle parameters and DNA ploidy in colorectal carcinomas.

Seven patients with colorectal adenocarcinoma and one with squamous cell carcinoma of anorectal region were infused preoperatively with iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU) in sequence (100 mg/m2 x 1 hr each with 1-hr interval in between) to label S-phase cells. The tumor biopsy specimens were embedded in glycol-methacrylate and 2-microns thick sections were treated with two monoclonal antibodies which permitted the identification of cells which incorporated IUdR only, BrdU only, both IUdR and BrdU, or neither IUdR or BrdU. The labeling index of tumors varied from 17.3 to 35.6% (mean = 25.78 +/- 6.162), duration of S-phase ranged from 14.0 to 23.9 hr (mean = 18.73 +/- 3.712), and total cell cycle time ranged from 39.4 to 123.4 hr (mean = 76.78 +/- 24.165). The architecture of the tumor was well preserved and a variable number of DNA synthesizing mononuclear cells were identified within and around the tumor. Image analysis of Feulgen-stained smears of the tumors was done to measure the DNA content of seven tumor samples. Each tumor was found to be hyperdiploid with multiple modal values. The studies described here demonstrate the feasibility of performing cell cycle kinetic measurements on gastrointestinal tumors which have been labeled in vivo. The ability to perform these measurements on tumor biopsies allows the avoidance of artifacts introduced when solid tumors are disaggregated in vitro for study.