“Old School” Islet Purification Based on the Unit Gravity Sedimentation as a Rescue Technique for Intraportal Islet Transplantation—A Case Report

Here, we present a case that required a supplemental “old school” islet purification for a safe intraportal infusion. Following pancreas procurement from a brain-dead 26-year-old male donor (body mass index: 21.9), 24.6 ml of islet tissue was isolated after continuous density gradient centrifugation. The islet yield was 504,000 islet equivalent (IEQ), distributed among the following three fractions: 64,161 IEQ in 0.6 ml of pellet, 182,058 IEQ in 10 ml, and 258,010 IEQ in 14 ml with 95%, 20%, and 10% purity, respectively. After a 23-h culture, we applied supplemental islet purification, based on the separation of tissue subfractions during unit gravity sedimentation, a technique developed over 60 years ago (“old school”). This method enabled the reduction of the total pellet volume to 11.6 ml, while retaining 374,940 IEQ with a viability of over 90%. The final islet product was prepared in three infusion bags, containing 130,926 IEQ in 2.6 ml of pellet, 108,079 IEQ in 4 ml of pellet, and 135,935 IEQ in 5 ml of pellet with 65%, 40%, and 30% purity, respectively, and with the addition of unfractionated heparin (70 units/kg body weight). Upon the islet infusion from all three bags, portal pressure increased from 7 to 16 mmHg. Antithrombotic prophylaxis with heparin was continued for 48 h after the infusion, with target activated partial thromboplastin time 50–60 s, followed by fractionated heparin subcutaneous injections for 2 weeks. β-Cell graft function assessed on day 75 post-transplantation was good, according to Igls criteria, with complete elimination of severe hypoglycemic episodes and 50% reduction in insulin requirements. Time spent within the target glucose range (70–180 mg/dl) improved from 42% to 98% and HbA1c declined from 8.7% to 6.7%. Supplemental “old school” islet purification allowed for the safe and successful utilization of a robust and high-quality islet preparation, which otherwise would have been discarded.

Decreased expression of fibronectin and the alpha 5 beta 1 integrin during terminal differentiation of human keratinocytes

We have examined the expression of fibronectin and the alpha 5 beta 1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the alpha 5 and beta 1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the alpha 5 and beta 1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the alpha 5 beta 1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.

Expression of three t-complex genes, Tcp-1, D17Leh117c3, and D17Leh66, in purified murine spermatogenic cell populations

SummaryTransmission ratio distortion (TRD) is a property of the complete t-haplotype which results in the preferential transmission of the t-haplotype chromosome from heterozygous t/+ males to the majority of the progeny. Available data suggest that in t/+ males, a dysfunction of the wild-type sperm within the female reproductive tract is responsible for the observed deviation from Mendelian segregation ratios. Genetically, Lyon has shown that multiple loci within the t-complex are required for maximum levels of TRD. These loci include multiple t-complex distorters (Teds) which act upon a single t-complex responder (Ter). Testis-expressed genes have been cloned which map to the same subregions of the t-complex as the Teds and Ter and are thus considered candidate genes for the products of these loci. To begin to understand how the products of these loci biochemically control TRD, the expression of three TRD-candidate genes (Tcp-1, D17Leh117c3, and D17Leh66) has been determined in populations of spermatocytes and differentiated spermatids purified to near homogeneity by unit gravity sedimentation. Fractions covering the entire gradient were analysed resulting in a more accurate picture of the precise timing of expression than previously reported. Transcription of all three genes was up-regulated in pachytene primary spermatocytes and persisted at stable levels through the haploid spermatid stages. Significantly, only levels of mRNA encoded by D17Leh66, the candidate gene for Tcr, increased from early round to elongating-stage spermatids. If this pattern of expression does, in fact, represent Tcr, these data provide the first direct evidence that wild-type and t-haplotype Tcr elements could be differentially expressed in haploid spermatids.

Purification of dispersed rat adrenal zona glomerulosa cells by Percoll density gradient centrifugation and the isolation of a population of cells highly responsive to adrenocorticotrophin

ABSTRACT Percoll density gradient centrifugation is a simple, inexpensive and convenient method to eliminate contaminating zona fasciculata (ZF) cells from unpurified rat adrenal capsular glomerulosa (ZG) cell preparations (with less than 0·1% ZF cells in the final cell preparation). Basal steroid (aldosterone and corticosterone) output by the purified (PG) cells was unchanged. These purified cells, although free from ZF contamination, were more highly responsive than expected to ACTH (3 nmol/l). When PG cells were further separated by Sephadex column filtration, the filtered PG cells exhibited the steroidogenic response of ZG cells purified by unit gravity sedimentation and Sephadex column filtration, i.e. reduced basal steroid output and an ACTH response reduced to that stimulated by K+ (8·4 mmol/l). Although the cells retained in the column resembled the filtered PG cells ultrastructurally, they showed unchanged basal steroid output and a high ACTH response with increased latepathway activity (the conversion of corticosterone to aldosterone). By combining Percoll density gradient centrifugation and Sephadex column filtration we have a method for the isolation and study of both the high-and low-response rat ZG cells which are free from ZF contamination. J. Endocr. (1986) 109, 351–358