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The current system to assess transplant center performances needs refinement to ensure that reduced access to transplantation is not an unintended consequence of quality measurement. See the article from Kasiske et al on page 3371.

The waiting list for kidney transplantation continued to grow between 1999 and 2008, from 41?177 to 76?089 candidates. However, active candidates represented the minority of this increase (36?951-50?624, a 37 change), while inactive candidates increased over 500 (4226-25?465). There were 5966 living donor (LD) and 10?551 deceased donor (DD) kidney transplants performed in 2008. The total number of pancreas transplants peaked at 1484 in 2004 and has declined to 1273. Although the number of LD transplants increased by 26 from 1999 to 2008, the total number peaked in 2004 at 6647 before declining 10 by 2008. The rate of LD transplantation continues to vary significantly as a function of demographic and geographic factors, including waiting time for DD transplant. Posttransplant survival remains excellent, and there appears to be greater use of induction agents and reduced use of corticosteroids in LD recipients. Significant changes occurred in the pediatric population, with a dramatic reduction in the use of LD organs after passage of the Share 35 rule. Many strategies have been adopted to reverse the decline in LD transplant rates for all age groups, including expansion of kidney paired donation, adoption of laparoscopic donor nephrectomy and use of incompatible LD.

A biotinylated derivative of alpha-bungarotoxin and tetramethylrhodamine-labeled avidin were employed to fluorescence label the acetylcholine receptors (AcChoR) on the surface of rat myotubes in primary culture. Because of the multivalency of both the biotinylated bungarotoxin and the avidin, this treatment extensivey crosslinks the AcChoR. AcChoR crosslinking immobilizes more than 90% of the normally laterally mobile AcChoR as verified by the fluorescence photobleaching recovery technique; it also redistributes the AcChoR into visible micropatches. Biotinylated alpha-bungarotoxin/avidin-induced AcChoR crosslinking greatly accelerates the rate of internalization of surface AcChoR; this rapid internalization affects both the normally immobile AcChoR in areas of diffuse distribution and the normally immobile AcChoR in preexisting patches. The peculiar pattern of fluorescent avidin binding to AcChoR patches previously bound with biotinylated bungarotoxin suggests that almost all AcChoR patches are in very close contact (< 70 A) with the glass substrate. AcChoR immobilization leads to a partial immobilization of concanavalin A receptors in the myotube membrane.

In vitro tests for predicting the response of tumors to chemotherapeutic agents might be improved if they were modified to take into account tumor-cell heterogeneity. We have studied the heterogeneity of cellular growth rate and drug response in mouse fibroblast NIH 3T3 cells and in NIH 3T3 cells transformed with the human HRAS gene (homologue of the Harvey sarcoma virus oncogene v-Ha-ras) from the EJ human bladder carcinoma cell line. Growth-rate heterogeneity was detected as a broad distribution of numbers of cells per colony. In spite of this heterogeneity, secondary colonies have numbers of cells per colony that resemble that of the primary colony from which they were derived. The variance between unrelated secondary colonies is increased by HRASEJ. Colony-size measurements are reliable because primary colonies divided in half formed two groups of secondary colonies (on two separate plates) that had indistinguishable mean colony sizes. Based on these observations, a divided-colony procedure was devised to detect the drug response of heterogeneous cell populations. Primary colonies are divided into two groups of cells, one of which is treated with a drug and the other is left untreated as a control. The size distribution of treated secondary colonies is then compared to that of the untreated control and to that of the primary colony from which it was derived. The divided-colony procedure is proposed as a modification of the human-tumor-cloning system to increase the sensitivity and reliability of in vitro procedures used to determine the drug response of heterogeneous tumor-cell populations.

We have made direct, quantitative measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture. AChR were fluorescently marked with tetramethylrhodamine-labeled alpha-bungarotoxin and AChR lateral motion was measured by the fluoresence photobleaching recovery technique. We found two coexisting distinct classes of AChR: (i) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (ii) immobile, dense, highly granular AChR in patches of 10-60 mum size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addition, evidence of turnover of AChR labeled with tetramethylrhodamine-alpha-bungarotoxin is seen in the gradual internalization of surface fluorescence within 36 hr after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.

Genetic variation was studied in several mouse L cell lines containing tandemly repeated herpes simplex virus thymidine kinase (TK) genes introduced by DNA-mediated gene transfer. Variants were obtained after alternate positive and negative selection for TK expression. Three classes of molecular alteration are described. One class consisted of a concerted wave of hypermethylation affecting many sites in all or nearly all of the TK genes. This resulted in genetically stable TK- variants. Of five TK+ transformants from independent transfer experiments, only one, named HM, showed this class of methylation. Hypermethylation was a reproducible phenomenon in HM, yielding TK- variants after selection with either bromodeoxyuridine or acycloguanosine [Acyclovir or 9-(2-hydroxyethy-oxymethyl)guanine]. A second class of alteration consisted of methylation affecting some, but not all, genes in the cluster. This happened in all TK+ (HAT [hypoxanthine-aminopterin-thymidine]-resistant) cell lines investigated, and this second class of methylation was incapable of generating TK- variants. Neither type of methylation was accompanied by genomic rearrangements. The third class of molecular alteration was found among TK+ (HAT-resistant) back revertants of hypermethylated HM TK- derivatives. It consisted of a 10-fold amplification of the hypermethylated TK genes. Demethylation of hypermethylated HM variants was not observed. Thus, hypermethylation in this system can be compensated for by amplification but cannot be reversed.

We used Southern blot hybridization to titrate and map restriction enzyme cleavage sites of a 6.3-kilobase-pair species of extrachromosomal viral DNA found in derivatives of the 745A line of murine erythroleukemia cells, which vary in their ability to be induced to differentiate by dimethyl sulfoxide (DMSO). Greater than an eightfold variation was observed in the amount of this DNA, with the largest amounts being found in cells that were resistant to the induction of differentiation by DMSO. This increase in the level of extrachromosomal viral DNA was found to be dependent upon the continued presence of DMSO in the culture medium. The increase was shown not to be due to an immediate stimulatory effect of this agent on the synthesis or maintenance of this DNA, since cell lines sensitive to the differentiation-inducing effects of DMSO were shown to undergo a transient reduction in the amount of extrachromosomal viral DNA after the addition of DMSO to the culture medium. In addition to the 6.3-kilobase-pair linear form found in the cytoplasm, in some preparations two hybridizing bands were observed that migrated in agarose gels in the position expected of covalently closed circular species of viral DNA. Restriction enzyme mapping of the cytoplasmic linear form indicated a close relationship of this DNA to two polycythemic strains of spleen focus-forming virus that have been molecularly cloned by other workers. No obvious change in the number or arrangement of chromosomal viral sequences could be detected after treating cells with DMSO. Thus, the exposure of murine erythroleukemia cells to DMSO caused an obvious change in the amount of extrachromosomal spleen focus-forming virus DNA but no obvious change in the integration of the provirus.

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.

The use of Bayesian methodology to evaluate center performance has both potential benefits and drawbacks that the transplant community will need to carefully monitor in the coming years. See viewpoint (page 1271) and article (page 1310) by Salkowski et al.

Analysis of large and small plaque mutants of simian virus 40 using antisera prepared against each has revealed quantitative and possibly qualitative antigenic differences for each plaque type. A sensitive micro radioisotope precipitation test permitted evaluation of immunochemical similarities and differences of capsid antigens by inhibition of precipitation.

A series of polymers of polyglutamic acid have been tested as plasma volume expanders. The results indicate that those polymers prepared from backbones of 40,000 molecular weight or over and side chains of 3,000 molecular weight or over, will have plasma volume retention half-life of 15 hours or longer. Measurements of the oncotic efficiency of these polymers in vivo indicated a blood stream fluid retention of approximately 51 ml./gm. of polymer. Similar measurements of polymer-serum albumin solutions in vitro showed a retention of 52 ml./gm. of polymer. The clinical response to infusion of solutions of these polymers indicated no untoward pharmacological properties. Although the number of trials presented is too small for ultimate conclusion regarding either the physiological or clinical effects of these polymers, they do provide a strong indication of the desirability of further clinical testing of this polymer and a sound basis on which to plan the larger scale preparation of polymer. Both measures are currently under way.

SIGNIFICANT quantities of residual urine are conducive to urinary-tract infection. Retention of this pool of urine also makes treatment of the infection difficult, and predisposes to reinfection. At times, there is a reluctance or a relative contraindication to use of a catheter simply to determine the quantity of residual urine. Phenolsulfonphthalein (PSP) excretion can be used, without catheterization, to screen patients for residual urine and by modifying the method can measure the volume of residual urine.The fractional PSP excretion test is widely used to assess renal function. In this test, urine collections are made 15, 30, and 60 minutes after the dye is given. Since the rate of dye excretion by the kidney steadily decreases, the amount of dye in the urine collections should also decrease. If it does not, it is an indication of clinically significant residual urine.If a known amount of dye is added to an