Attempts to improve human ovarian transplantation outcomes of needle-immersed vitrification and slow-freezing by host and graft treatments

Purpose

To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the “improvement protocol” of host melatonin treatment and graft incubation with biological glue?+?vitamin E?+?vascular endothelial growth factor-A.

Methods

Human ovarian tissue was preserved by needle-immersed vitrification or slow-freezing and transplanted into immunodeficient mice, either untreated (groups A and C, respectively) or treated with the improvement protocol (groups B and D, respectively). Grafted and ungrafted slices were evaluated by follicle counts, apoptosis assay and immunohistochemistry for Ki67 and platelet endothelial cell adhesion molecule (PECAM).

Results

Follicle number in the recovered grafts was limited. The number of atretic follicles was significantly higher after vitrification with/without the improvement protocol and slow-freezing than that after slow-freezing?+?the improvement protocol. Stroma cell apoptosis was the lowest in the group D. PECAM staining showed a peripheral and diffuse pattern in the group D (mostly normal follicular morphology) and a diffuse pattern in all other groups (few follicles, mostly atretic), with significantly higher diffuse levels in the vitrification groups. Ki67 staining was identified in all normal follicles. Follicles did not survive transplantation in the vitrification groups.

Conclusions

Ovarian sample preparation with slow-freezing?+?the improvement protocol appears to yield better implantation outcomes than needle-immersed vitrification with/without the improvement protocol. The real quality of frozen tissue can be assessed only after grafting and not after thawing/warming.

     

Management of extreme thrombocytosis in myeloproliferative neoplasms: an international physician survey

Extreme thrombocytosis (ExT) has been associated with an increased bleeding risk in myeloproliferative neoplasm (MPN) patients and is included in the high risk category in treatment guidelines. Treatment of patients with ExT has not been studied in prospective trials. To study physicians’ approaches to ExT, we distributed a web based questionnaire with clinical case scenarios to 202 members of MPN working groups. Cases included low thrombotic risk essential thrombocythemia (ET) with either JAK2V617F or CALR mutation, polycythemia vera with ExT either with or without leukocytosis, an ET patient needing urgent orthopedic surgery, and a poorly controlled ET patient with acute cerebral venous sinus thrombosis. Responses were received from 90 physicians (45 %) and were variable in most case scenarios. Country of practice had the most significant influence on physician response. The USA and Israel physicians responded similarly in most cases and differently to the Europe physicians. Treatment of asymptomatic JAK2V617F positive ET and target platelet count on cytoreduction were significantly influenced by physician years of experience. Responses were not influenced by the volume of MPN practice or by whether MPN was considered a major interest by the physician. Our results show a lack of consensus on how to manage MPN patients with ExT. Randomized controlled trials properly designed to address these questions are needed.     

Ageratum houstonianum toxicosis in zebu cattle

Ageratum houstonianum (Ageratum, flossflower, blue billygoat weed) is an annual plant that tends to become a pest in gardens and pastures. Clinical signs for A. houstonianum toxicosis in cattle are characterized by either an acute hemorrhagic course or sub-acute photodynamic dermatitis. The toxicosis has often been associated with Holstein-Friesian or crossbreed Holstein cattle less resistant to tropical climate conditions. During a recent especially dry spring about 40 adult Zebu cattle were found dead, while another 40/800 animals were sacrificed. The animals had been relocated to the problem area about 4 mo before, where due to the prolonged drought, A. houstonianum was almost exclusively the only pasture available. The intoxicated cattle did not show the characteristic toxic dermatitis reported for A. houstonianum acute toxicosis; but post-mortem examination revealed bloody serous fluid in coccyx-femoral joints and hemorrhages in the large muscle tissues, while liver, kidney and heart also had hemorrhages. To confirm the toxic plant as cause of the toxicosis, phytochemical Qualitative screening and a novel thin-layer chromatographic characterization of plant extracts were done. The chromatographic profiles of coumarin compounds, alkaloids and triterpens in ruminal and intestinal contents were similar to those obtained from A. houstonianum plants from the same area, confirming ingestion of A. houstonianum as cause of the toxicosis. The coincidence of adverse nutritional conditions together with the cattle's ignorance of the grazing area predisposed the plant toxicosis.     

Lithium-mediated phosphorylation of glycogen synthase kinase-3β involves PI3 kinase-dependent activation of protein kinase C-α

Lithium, a known mood-stabilizer frequently used in treatment of bipolar disorders, is an effective glycogen synthase kinase-3beta (GSK-3beta) inhibitor. This led to the idea that GSK-3beta is an in vivo target directly inhibited by lithium. As lithium is a weak in vitro inhibitor of GSK-3beta (IC50=2 mM), however, we speculated that it inhibits GSK-3beta via an indirect, yet unknown, mechanism. The present studies show that lithium increased the phosphorylation of a key inhibitory site of GSK-3beta, serine-9 (Ser-9), in HEK293 cells and in PC12 cells. This phosphorylation was significantly reduced by protein kinase C (PKC) inhibitors GF109203X and Ro31-8425, as well as GO6976, an effective inhibitor toward conventional PKC isoforms (cPKC). Consistent with these results, lithium increased PKC-alpha activity approximately twofold in both cell lines. Because PI3 kinase is a potential upstream regulator of cPKC, its inhibition by wortmannin or LY294002 also abolished the lithium-induced serine phosphorylation of GSK-3beta in HEK293 and PC12 cells. Moreover, lithium did not activate PKB, and in addition, its activity was not dependent on the presence of medium inositol nor did it affect the autophosphorylation activity of GSK-3beta. Finally, intracerebroventricular injection of lithium increased GSK-3beta Ser-9 phosphorylation and enhanced PKC-alpha activity 1.8-fold in mouse hippocampus, confirming this lithium response in vivo. Our studies propose a new mechanism by which lithium indirectly inhibits GSK-3beta via phosphatidylinositol 3 kinase- dependent activation of PKC-alpha.     

Effect of D-003, a mixture of very high molecular weight aliphatic acids, on prednisolone-induced osteoporosis in Sprague-Dawley rats

BACKGROUND: Drugs inhibiting cholesterol biosynthesis may affect bone metabolism through inhibition of the mevalonate pathway resulting in the inhibition of protein prenylation required for osteoclast activity. D-003 is a mixture of high molecular weight aliphatic primary acids purified from sugar-cane (Saccharum officinarum) wax, with cholesterol-lowering effects demonstrated in experimental and clinical studies. D-003 inhibits cholesterol biosynthesis through indirect regulation of HMG-CoA reductase activity. A previous study demonstrated that D-003 prevented bone loss and bone resorption on ovariectomy-induced osteoporosis in rats. Corticosteroid-induced osteoporosis is the result of changes affecting calcium homeostasis, but the hallmark of corticosteroid-induced bone loss is the direct effects on bone cells, such as inhibition of osteoblastogenesis, promotion of apoptosis of osteoblasts and osteocytes, and decrease in bone formation. OBJECTIVE: To determine whether D-003 could prevent the bone loss induced with prednisolone in Sprague-Dawley rats. METHODS: Rats were randomly distributed in five groups (ten rats per group): a sham-operated control and four groups orally treated with prednisolone 6 mg/kg for 80 days; a positive control orally treated with vehicle; and three groups orally treated with D-003 at 5, 25 and 200 mg/kg, respectively. Rats were killed, bones removed and histological variables of bone resorption and formation studied for histomorphometry. RESULTS: Compared with the sham group, prednisolone significantly (p < 0.01) reduced trabecular bone volume (TBV), while D-003 significantly (p < 0.001) and dose-dependently prevented the prednisolone-induced reduction of TBV. Treatment with prednisolone lowered (p < 0.001) trabecular thickness (TbTh) and number (TbN), while increasing (p < 0.001) the gap between trabeculae. D-003 (5, 25 and 200 mg/kg/day) significantly (p < 0.001) and dose-dependently prevented the reduction of TbTh and TbN and the increase of trabecular gap induced with prednisolone. Treatment with prednisolone increased both the surface and number of osteoclasts compared with sham (p < 0.001). D-003 (5-200 mg/day), however, prevented this effect (p < 0.001 for all comparisons). D-003 also prevented (p < 0.001) the reduction of osteoblast surface (ObS/BS) induced by prednisolone. Osteonecrotic areas were observed in all positive controls, but in none of the sham animals. Positive controls showed hypertrophy of bone marrow adipocytes and lipid-laden pluripotential stromal cells in bones. A significant and dose-dependent reduction of the frequency of animals showing prednisolone-induced osteo-necrosis was observed across the doses of D-003 (5, 25 and 200 mg/kg) investigated here. CONCLUSIONS: D-003 (5, 25 and 200 mg/kg) prevented trabecular bone loss and femoral neck osteonecrosis induced with prednisolone in Sprague Dawley rats, also increasing osteoblast surface and reducing bone resorption parameters. These results suggest that D-003 could be useful for managing corticosteroid-induced osteoporosis.     

Policosanol prevents bone loss in ovariectomized rats

Osteoporosis is characterized by reduced bone mass, abnormal bone architecture and increased fracture risk. Ovariectomy impairs bone mass and metabolism in rats and ovariectomized rats are considered as a suitable model of postmenopausal osteoporosis. Mevalonate is required for producing lipoids that are important in osteoclast activity and thus drugs affecting mevalonate production can prevent bone loss in rodents. Policosanol is a cholesterol-lowering drug isolated from sugar cane wax that inhibits cholesterol biosynthesis through an indirect regulation of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity. The purpose of this study was to determine whether policosanol could prevent bone loss in the bones of ovariectomized rats by comparing its effects with those induced by estradiol. Sprague Dawley female rats were randomly distributed in four groups: a sham-operated group treated with Tween/H2O vehicle and three groups of ovariectomized rats treated with 17beta-estradiol (30 microg/kg/day) or policosanol (50 and 200 mg/kg/day), respectively, for 3 months. At treatment completion the rats were sacrificed, their bones removed and variables of bone resorption and formation were investigated by histomorphometry. Ovariectomy increased trabecular separation but diminished the number and thickness of trabecules. Estradiol and policosanol prevented these effects compared with ovariectomized controls. Both treatments also prevented an increase in the number of osteoclasts and their surface area induced by ovariectomy. Estradiol, but not policosanol, significantly prevented an increase of osteoblast surface area compared with ovariectomized controls. In conclusion, policosanol prevented bone loss and decreased bone resorption in ovariectomized rats, suggesting that it should be potentially useful in preventing bone loss in postmenopausal women.     

C-reactive protein as a marker for active coronary artery disease in patients with chest pain in the emergency room

BACKGROUND: Markers of inflammation, such as C-reactive protein (CRP), were found to be related to risk for cardiovascular disease (CVD) events in patients with angina pectoris. In addition, recent studies have shown that, in the case of atherosclerosis, increased CRP concentration reflects the inflammatory condition of the vascular wall. HYPOTHESIS: The study was undertaken to determine whether CRP levels in individuals with chest pain attending the emergency room (ER) may be used as a marker of active CVD. METHODS: Serum CRP level was measured in 226 of 326 consecutive patients (128 men, 98 women; mean age 61.3 +/- 5.9 years; range 19-87 years) referred to the ER with chest pain. The decision whether to admit orrelease the subjects was determined without taking the CRP level into account. Follow-up was then performed for 1 year. RESULTS: Eighty-four patients were admitted to the hospital. Of these, 9 with acute coronary syndrome (ACS) had very high levels of CRP (25-40 mg/l), 35 had had an acute coronary event within the preceding 3 months, with levels of CRP 14-20 mg/l. Only eight patients with nonsignificant CVD had elevated CRP levels. Twenty-eight subjects who were released from the ER had elevated CRP levels (7-14 mg/l); 8 of these, in addition to 4 subjects with normal CRP levels, had a late coronary event. CONCLUSION: This study indicates that in patients referred to the ER with chest pain and no other indication for hospitalization, a normal level of CRP suggests safe release. Most hospitalized patients with normal CRP will not have acute coronary syndrome. Patients who will develop early coronary events have very high CRP levels. High serum CRP level, after excluding other inflammatory sources, was proven to be a sensitive diagnostic and prognostic marker for significant coronary disease.