Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Evidence that orexins A and B stimulate insulin secretion from rat pancreatic islets via both receptor subtypes

Orexins are recently identified neuropeptides that appear to play a role in the regulation of energy homeostasis and arousal. They bind to and activate two closely related G protein-coupled receptors (OXR1 and OXR2), previously described as orphans. In this study we examined involvement of orexins in regulation of insulin secretion from rat pancreatic islets utilizing an in situ perfused pancreas and isolated pancreatic islet models. By means of RT-PCR we found that both OXR1 and OXR2 are expressed in rat pancreatic islets. Furthermore, the expression levels of OXR1 were higher than OXR2. In both experimental models applied, orexins A and B (1, 10 and 100 nmol/l) concentration dependently stimulated insulin secretion at two different glucose concentrations (6.66 or 26.4 mmol/l), with orexin A being more potent than orexin B. This study demonstrates that orexins A and B modulate insulin secretion in vitro.     

Homozygosity mapping of achromatopsia to chromosome 2 using DNA pooling

Achromatopsia is an autosomal recessive disease of the retina, characterized clinically by an inability to distinguish colors, impaired visual acuity, nystagmus and photophobia. A genome-wide search for linkage was performed using an inbred Jewish kindred from Iran. To facilitate the genome-wide search, we utilized a DNA pooling strategy which takes advantage of the likelihood that the disease in this inbred kindred is inherited by all affected individuals from a common founder. Equal molar amounts of DNA from all affected individuals were pooled and used as the PCR template for short tandem repeat polymorphic markers (STRPs). Pooled DNA from unaffected members of the kindred was used as a control. A reduction in the number of alleles in the affected versus control pool was observed at several loci. Upon genotyping of individual family members, significant linkage was established between the disease phenotype and markers localized on chromosome 2. The highest LOD score observed was 5.4 (theta = 0). When four additional small unrelated families were genotyped, the combined peak LOD score was 8.2. Analysis of recombinant chromosomes revealed that the disease gene lies within a 30 cM interval which spans the centromere. Additional fine-mapping studies identified a region of homozygosity in all affected individuals, narrowing the region to 14 cM. A candidate gene for achromatopsia was excluded from this disease interval by radiation hybrid mapping. Linkage of achromatopsia to chromosome 2 is an essential first step in the identification of the disease-causing gene.      

Calcium/phosphatidylserine/diacylglycerol-dependent protein phosphorylation in the Aplysia nervous system

It has been shown that intracellular injection of protein kinase C (calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), purified from mammalian brain, or application of the tumor-promoting phorbol diester, 12-O-tetradecanoyl-13-phorbol acetate (TPA), leads to an enhancement of calcium currents in the bag cell neurons of Aplysia. We now present evidence of an endogenous enzyme in bag cell neurons which is activated by TPA and which has properties similar to those of mammalian protein kinase C. Calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity was found in both cytosolic and particulate fractions prepared from isolated clusters of bag cell neurons. This endogenous enzyme phosphorylated an 87,000-dalton protein from bovine brain, which appears to be a specific substrate for protein kinase C, as well as several substrates present in cytosolic fractions prepared from isolated bag cell clusters. Similar results were obtained using preparations made from pooled head ganglia from Aplysia. The pharmacological properties of the calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity in the Aplysia nervous system were similar to those of protein kinase C from mammalian tissues. Thus, the same group of endogenous substrate proteins were phosphorylated when diacylglycerol was replaced by TPA in cytosolic fractions prepared from isolated bag cell clusters. Non-tumor-promoting phorbols (4-alpha-phorbol, 4-alpha-phorbol-12,13-didecanoate, and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate) did not stimulate protein phosphorylation in these preparations. Phosphorylation by the Aplysia calcium/phosphatidylserine/diacylglycerol-dependent protein kinase was inhibited by polymixin B sulfate, by calmodulin, and by the "calmodulin antagonists" trifluoperazine, calmidazolium and W7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple cerebral metastases: detectability with Gd-DTPA-enhanced MR imaging

Sixteen patients with suspected cerebral metastases were studied with magnetic resonance (MR) imaging before and after the intravenous administration of 0.1 mmol/kg of gadolinium diethylenetriaminepenta-acetic acid. The images were interpreted blindly by two neuroradiologists; all clinical, radiologic (computed tomographic and MR imaging), and pathologic data were reviewed to arrive at a final "best diagnosis," which was then compared with the prior blinded interpretations. Of seven patients found to have multiple metastases, six (86%) had at least one tumor nodule depicted by postinfusion MR imaging that was missed by one or both observers on review of preinfusion images alone. Lesions missed on preinfusion studies were usually small nodules hidden by or not detected next to regions of high-signal edema thought to be related to the adjacent tumor nodule. The authors believe that contrast enhancement improves detection of metastatic foci with MR imaging and that the findings indicate broader implications for the detection of multiple lesions from other causes.     

TNF-alpha binding ability as a sign of peripheral blood neutrophils preactivation in the course of multiple sclerosis

Usually neglected is the role of neutrophils in causing of immunological disturbances in patients with multiple sclerosis (MS). Nevertheless, it has been indicated over the recent years that these cells possess a sufficient potential to affect both immune response and inflammation. This potential may result in MS through the process of priming of these cells by proinflammatory cytokines like TNF. We studied TNF and its soluble receptors sp55 and sp75 serum levels and binding of fluorescein isothiocyanate (FITC)-stained TNF by neutrophils. We studied three different groups of MS patients: 10 patients in relapse of the disease, 13 in its remission, and 11 in its chronic progressive form (CP-MS). The control was provided by 14 neurological patients (OND) with non-inflammatory diseases. The performed studies showed higher TNF sp55 and sp75 soluble TNF receptors serum levels in the patients with relapse, comparing with other MS patients and OND. TNF binding by neutrophils of MS patients during relapse was also higher, than other MS patients and OND. These result suggest the preactivation of neutrophils in the relapse of MS.     

Diagnostic and Perinatal Management of Fetal Extrasystole

Fifty fetuses referred to the Polish Mother's Memorial Hospital for fetal echocardiography between January 1, 1991 and June 1, 1995 were evaluated. The mean fetal gestational age at the time of diagnosis of arrhythmia was 34.1 weeks, and the mean gestational age at the time of delivery was 38.7 weeks. Checkup echocardiographic examinations were performed every 10–14 days, for a mean 2.4 studies per fetus. In most cases (48/50, 96%), premature atrial contractions were present during the first echocardiography examination. The fetal heart study was normal in 30 cases; in 7 (14%) there was tricuspid valve regurgitation, in 7 (14%) an atrial septal aneurysm, in 4 congenital heart defects, in 1 myocardial hypertrophy, and in 1 disproportion in the four-chamber view. Of the 50 fetuses, 43 underwent regular echocardiographic monitoring alone; in 7 cases, based on the presence of additional echocardiographic findings, pharmacotherapy was applied (digoxin, verapamil, or both). Three neonates died after delivery owing to malformations in two cases (one critical aortic stenosis, one spina bifida plus hygroma colli) and due to myocarditis in one case. In six of seven newborns treated in utero, myocarditis was diagnosed after birth (including the one with neonatal demise). Most of the newborns were in good condition after birth, their mean Apgar score being 8.6 and the mean birth weight 3259 g. We concluded that most extrasystoles represent an isolated anomaly, not affecting the fetal condition. Their presence should not influence the obstetric care and may require only echocardiographic monitoring. In most of our cases the premature contractions subsided after birth, although sometimes they preceded fetal supraventricular tachycardia or appeared after congenital myocarditis.     

Survival and developmental disability in infants with birth weights of 501 to 800 grams, born between 1979 and 1994

OBJECTIVE: Because the survival rate has increased for extremely low birth weight neonates, many have raised the concern that the rate of developmental disability among survivors will also increase. To address this concern, we analyzed changes over time in survival and major neurosensory impairment in a sample of extremely low birth weight infants born between July 1, 1979, and June 30, 1994. METHODS: The study sample included 513 infants with birth weights of 501 to 800 g who were cared for in either of the two neonatal intensive care units that serve a 17-county region in northwest North Carolina and who were born to mothers residing in that region. At 1 year of age (corrected for gestation), survivors were examined by a pediatrician and were tested using the Bayley Scales of Infant Development. Major neurosensory impairment was defined as cerebral palsy, a Bayley Mental Developmental Index <68, or blindness. A total of 209/216 (97%) of survivors were examined at 1 year of age. Epoch of birth was defined as follows: epoch 1, July 1, 1979 to June 30, 1984; epoch 2, July 1, 1984 to June 30, 1989; and epoch 3, July 1, 1989 to June 30, 1994. RESULTS: Survival rates for epochs 1, 2, and 3 were, respectively, 24/120 (20%), 63/175 (36%), and 129/218 (59%). In contrast, the proportions with a major neurosensory impairment did not increase over time; rates for successive epochs were 6/24 (25%), 17/61 (28%), and 26/124 (21%). Rates of cerebral palsy were 3/24 (13%), 12/61 (20%), and 9/124 (7%); rates of delayed mental development were 4/24 (17%), 12/61 (20%), and 17/124 (14%); and rates of blindness were 2/24 (8%), 0/62, and 5/124 (4%), respectively. CONCLUSIONS: This analysis suggests that the increasing survival of extremely low birth weight neonates since the late 1970s has not resulted in an increased rate of major developmental problems identifiable at 1 year of age.