Familial haplotyping and embryo analysis for Preimplantation genetic diagnosis (PGD) using DNA microarrays: a proof of principle study.

PURPOSE: Development of PGD assays for molecular disorders is based on analysis of a familial mutation together with linked polymorphic STR markers; a process which is lengthy and requires the identification of multiple informative markers prior to PGD analysis. On the other hand, whole genome amplification (WGA), in conjunction with microarray platforms, allows the use of a universal assay for the analysis of a very large number of SNP markers at once. The aim of this study was to test high throughput pre-PGD familial haplotyping for in-case blastomere analysis in order to eliminate time-consuming pre-case preparations for each family. METHODS: A PGD cycle was performed for a couple with paternal Charcot Marie Tooth 1A (CMT1A) using a classic multiplex nested PCR approach. Mutant embryos from the case were blindly reanalyzed, as single or multi-cell biopsies, using a multiple displacement amplification-based WGA protocol and microarray SNP analysis. In parallel, relevant genomic DNA samples from the family were also analyzed by SNP microarray. RESULTS: After applying a 'unique informative allele' selection algorithm to the data, this array-based assay reconfirmed the initial diagnosis in all samples. CONCLUSIONS: We describe a PGD method that is both accurate and feasible during the time-frame required for embryo transfer. This strategy greatly reduces the time for pre-case haplotype preparation.

Characterization of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare (Schultz), and the effect of exposure to methylene blue.

Failure to inflate the swim bladder is regarded a major obstacle in the rearing of many fish species. We present a study of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare. A normal developing primordial swim bladder was first discernable at the end of the first day post-hatch (p.h.) as a cluster of epithelial cells with a central lumen, surrounded by presumably mesenchymal cells. Initial inflation occurred on the fourth day p.h. Prior to inflation the swim bladder epithelium consisted of an outer squamous and inner columnar layer. Cells of the inner layer were filled at their basal region with an amorphous material, which disappeared upon inflation. A pneumatic duct was absent, and larvae presented no need to reach the water surface for inflation, suggesting that angelfish are pure physoclists. A model for the role of the amorphous material in normal initial inflation is proposed. Abnormal swim bladders were apparent from the fourth day p.h., and methylene blue (MB) at a concentration of 5 ppm significantly increased the prevalence of SBN. Histologically, abnormal swim bladders in larvae hatched in 5 ppm MB could not be distinguished from those in fish raised under routine conditions (0.5 ppm MB). We suggest that MB may have a teratogenic effect in angelfish.

Spontaneous rupture of the urinary bladder complicated by extensive fascitis: the importance of a high index of suspicion.

Spontaneous rupture of the urinary bladder is a rare entity. This is, in part, the cause for the low rate of correct preoperative diagnosis, the high rate of delayed diagnosis, morbidity and mortality. We report a case of spontaneous rupture of the urinary bladder in an adult patient, complicated by extensive fascitis of the abdominal wall. The stormy course and long hospitalization in the case described are characteristic of patients with spontaneous bladder rupture and warrant a high index of suspicion in order to achieve an early diagnosis.

[Bilateral hydronephrosis and erythrocytosis].

A 36-year-old woman who developed extreme erythrocytosis following partial correction of severe bilateral hydronephrosis by bilateral ureterolithotomy is presented. Hydronephrosis appears to stimulate renal synthesis and release of erythropoietin. The mechanism could involve increase in medullary hypoxia by increased pressure of the renal pelvis on the medullary vasculature. This might be especially marked when the renal blood supply is compromised by concomitant atherosclerosis, as in this case.

Use of the discriminant index in dynamic treatment to reduce recurrence of calcium oxalate kidney stones.

Treatment with phosphates, thiazides and allopurinol was undertaken in 54 idiopathic calcium oxalate stone formers, 38 of whom were recurrent stone formers. The patients were followed up for 1 1/2 to 4 years (mean 2.6). During the same period at the pre-treatment stage the patients formed 80 stones, but during therapy only one stone was formed. A dynamic scheme of therapy was used. Each patient was tested before the start of drug treatment by the discriminant index (DI) method, which measures the overall inhibitory potential to calcium oxalate crystallisation. About 10 days after the start of treatment the DI was tested again. If the response was positive, therapy was continued; if not, the patient was given another drug. Adjustments were made as required. The stopping of stone formation correlated well with the DI prediction but less well with the hypocalciuric effect of the drugs.

Emphysematous renal and perirenal infection in nondiabetic patient.

Emphysematous renal infection is a severe, rare complication of urinary tract infection associated with gas production, which frequently causes renal destruction and has a high mortality rate. It is termed emphysematous pyelitis when gas is confined to the collecting system, or emphysematous pyelonephritis if it also involves the parenchyma with or without the perirenal space. Only 7 cases of perirenal emphysema have been described previously; all were in diabetics. Here we present a rare case of gas-producing renal infection in the collecting system and perirenal tissues, in a nondiabetic patient, and discuss the management of the various types of emphysematous renal infections.

Evaluation of the relative inhibitory potential of fractionated urinary macromolecules.

UNLABELLED: Ultrafiltration membranes of 10,000 d, 1,000 d and 500 d were used to remove urinary macromolecules from the urine of normal subjects and from the urine of stone forming patients. The filtrated urines were examined for their residual inhibitory potential for calcium-oxalate precipitation, by the discrimination method of Sarig et al. (D.I. test). The results of testing the filtrate were complementary to the information gained by analyses of retentates obtained in successive ultrafiltration. The method has an inherent advantage because the manipulation of solids retained on membranes may inadvertantly modify their inhibitory potential. At least two distinct groups of inhibitors were found in 20 normal urines. The first group has MW above 10,000 d while the second group of inhibitors has MW in the range of 500-1,000 d. The mean of the D.I. values increased dramatically from the normal range (less than 0.6) to the stone former range (greater than 1.1) (p less than 0.001) after the 500 d filtration. Some of the normal urines, even after the 500 d filtration, still had a degree of inhibitory potential. This inhibitory potential may be related to the inorganic compounds which were found in the urines. The inhibitory activity of macromolecules with MW above 10,000 d and below 500 delta was negligible in 7 stone formers (SF) urines. The relative contribution of 500-1,000 d macromolecules is the highest both in SF and normal urines. CONCLUSIONS: 1) inhibitors in human urine are of wide range in MW; 2) stone formers and normals differ in the level of inhibitor activity at all MW ranges; especially in above 10,000 d and below inhibitors.

Change in inhibitory potential in urine of hyperuricosuric calcium oxalate stone formers effected by allopurinol and orthophosphates.

Allopurinol and orthophosphates were used in the treatment of 25 hyperuricosuric calcium oxalate stone formers. Their urines were tested by the Discriminating Index method, which measures the potential of a urine to retard calcium oxalate precipitation in vitro. The tests were performed before any treatment was begun and about 10 days after the commencement of drug intake. An insignificant effect by allopurinol and a markedly significant effect of orthophosphates on Discriminating Index were found. In a few of the patients, allopurinol seemed to cause a decrease in the urine's potential to retard calcium oxalate precipitation. Therefore the effect of allopurinol in 11 hyperuricosuric patients with no history of calcium oxalate stone formation was tested. In 5 of the 11 patients, the urine's potential to retard calcium oxalate precipitation decreased to levels similar to those of calcium oxalate stone formers. The results of this study suggest that hyperuricosuric calcium oxalate stone formers suffer from the same yet-undefined etiology of stone formation as other calcium oxalate stone formers. The results also question the role of uric acid in calcium oxalate stone formation and the efficacy of allopurinol in preventing this disease. It is suggested that allopurinol may even be related to stone formation in some patients.

Repeated subselective renal embolization following pyelolithotomy: angiographic salvage of a kidney.

A bleeding intrarenal aneurysm secondary to pyelolithotomy was treated successfully by super-selective catheterization of the feeding arteries and embolization using stainless steel Gianturco coils. Three therapeutic procedures were performed because of recurrent hemorrhage. The advantages of this mode of treatment are that it is well tolerated, relatively safe and allows maximal preservation of functioning renal tissue.

Determination of GOT activity on nucleation and crystal growth of calcium oxalate.

Crystal Size Distribution (CSD) and the yield of Calcium Oxalate Crystals in solutions with an admixture of 5 normal and 3 stone forming urines, were determined. A positive correlation was found between the median size, the number of particles and the overall inhibitory potentials of the urines toward calcium oxalate precipitation in vitro as reflected by Discriminating Index (DI) measurements. Incubation of two samples of stone formers' (SF) urines with glutamic-oxalacetic-transaminase (GOT) caused a reduction of aspartic acid concentration, an increase in glutamic acid concentration and a parallel decrease in the DI values. After 90 min of SF urine incubation with GOT the DI in three samples was improved and both the median size and number of particles reduced, by 28% and 45% respectively. These results could indicate that GOT activity changes the inhibitory power of the SF urine by transforming aspartic acid into glutamic acid, having thus most probably a part in the inhibition of CaOx stone formation.

Management of ureteropelvic junction obstruction associated with lower polar vessels.

Four cases of hydronephrosis due to ureteropelvic junction (UPJ) obstruction by lower polar vessels are presented. Regular propagation of peristaltic waves across a patent UPJ and rapid emptying of the pelvis were the basic conditions before accepting the lower polar vessels as the primary cause of the hydronephrosis. Transposition or division of the lower polar vessels, accompanied sometimes by a limited lower polar nephrectomy dependent on the resultant ischemia, without any pyeloplasty was the selected procedure in each of the cases. No hypertension and no immediate or delayed postoperative complications occurred in any of our patients. Relief of symptoms, a remarkably improved postoperative pyelogram, and a sterile urine were achieved in all our cases. Detection of every case in which the lower polar vessels play a primary role in the establishment of UPJ obstruction is urged since transposition or division of these vessels in such cases is a superior procedure to pyeloplasty.

Busulfan-induced hemorrhagic cystitis.

We report a case of severe hemorrhagic cystitis induced by prolonged busulfan therapy. The clinical course and pathological results were similar to irradiation and cyclophosphamide cystitis. Only 1 such case has been described previously. Although hemorrhagic cystitis induced by busulfan is a rare complication it should be considered in the differential diagnosis and busulfan should be discontinued immediately.

Follow-up of drug therapy efficacy to prevent recurrence of calcium oxalate kidney stone formation.

Results of a prospective study of response to phosphate treatment after surgery in a group of kidney stone formers, some of them recurrent are presented. The follow-up, of 1 year duration, was carried out by periodic tests of the stone formers' urine using the Discriminating Index (DI) procedure. In a group of 32 stone formers, 30 individuals showed a remarkable change in the DI value; i.e., the inhibiting potential of their urine was similar to that of normals. Two of the patients did not respond well, and their drug treatment was adjusted accordingly. During this year no recurrency was reported.

Adrenergic response of bladder muscle in prostatic obstruction. Its relation to detrusor instability.

In vitro isometric studies of bladder dome muscle taken at operation from 47 patients with benign prostatic obstruction, revealed an alpha-adrenergic response instead of the normal beta response in 23 per cent. The relationship between this finding and the presence of irritative symptoms and an unstable detrusor is examined. This finding may explain the beneficial effect of alpha-blockers on these symptoms in many patients with prostatic obstruction.

A method for discrimination between calcium oxalate kidney stone formers and normals.

Calcium ion concentration versus time was measured in solutions containing admixture of 10 per cent tested urine of normals and stone-formers, during induced calcium oxalate precipitation. A Discriminating Index was formulated by statistical analysis of the data. It was found that stone-formers and normals differ significantly with respect to the measurements and the Discriminating Indices. An equation to evaluate the odds of stone-forming based on results of an individual test has been derived. The Discrimination Index may be recommended as a diagnostic tool.

Retardation of calcium oxalate precipitation by glutamic-oxalacetic-transaminase activity.

It has been found that calcium oxalate stone formers have low UGOT activity compared to healthy individuals (controls). Urine from stone formers with no GOT activity and no effect on calcium oxalate precipitation was incubated with GOT for various periods. Subsequently calcium oxalate precipitation was decreased and found to be considerably retarded i.e., the pathological urine after the incubation acted in a way similar to that of normal urine. The yield of Glutamic-Oxalacetic Transaminase (GOT) activity is glutamic acid. It was shown that glutamic acid has a significant retardation effect on the precipitation of calcium oxalate stone formation. Therefore it may be suggested that GOT activity involved in glutamic acid creation in situ, has a role in kidney stone formation.

Comparative experimental study on the use of the carbon dioxide laser beam in partial nephrectomy.

A report is given on an experimental study in 20 dogs designed to compare the use of the CO2 laser beam as a cutting instrument in resection of the kidney parenchyma, with the simple scalpel and the cutting diathermy current. It was found that the laser can be used with impunity. It shortens the time of operation by facilitating hemostasis, diminishes blood loss, and does not interfere with the final kidney function. The damage to the kidney parenchyma compares favorably with the produced by the other instruments.