Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs.
We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression.
An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele.
Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors. Neurology® 2012;78:690–695
Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology.
RESEARCH DESIGN AND METHODS
We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates.
Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10−8). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10−4), improved β-cell function (P = 1.1 × 10−5), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10−6). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets.
We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis.
Vascular plants appeared ~410 million years ago then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes (1). We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first non-seed vascular plant genome reported. By comparing gene content in evolutionary diverse taxa, we found that the transition from a gametophyte- to sporophyte-dominated life cycle required far fewer new genes than the transition from a non-seed vascular to a flowering plant, while secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in post-transcriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the tasiRNA pathway and extensive RNA editing of organellar genes.
Human peripheral blood monocytes are permissive for the growth of Mycobacterium tuberculosis, but the fate of nonpathogenic Mycobacterium smegmatis in these cells is not known. Since M. smegmatis may be used as a host with which to express and screen for M. tuberculosis genes needed for survival in monocytes, we determined whether human peripheral blood monocytes could restrict the growth of Mycobacterium smegmatis. Adherent human peripheral blood monocytes were permissive for the growth of M. smegmatis, as measured by ex vivo [3H]uracil uptake. However, human peripheral blood monocytes which were cultured nonadherently in Teflon wells were able to restrict the growth of M. smegmatis while remaining permissive for the growth of M. tuberculosis H37Ra. The loss of viability of M. smegmatis in nonadherent cells was correlated with an increase in nonspacious phagocytic vacuoles. The killing of M. smegmatis was not blocked by NG-monomethyl-L-arginine, suggesting that it was not due to the production of reactive nitrogen intermediates. Incubation of the monocytes for 1 to 7 days before infection had no effect on the fate of M. smegmatis, suggesting that adherence versus nonadherence, and not differentiation, was the key determinant for the difference in functional ability. Nonadherent human peripheral blood monocytes may be a more appropriate model than adherent cells for the study of factors employed by bacterial to survive within monocytes and for selection screening of bacterial genes needed for intracellular survival.
We tested whether maternal hypertensive disorders in pregnancy predict age-related change in cognitive ability in the offspring up to old age.
Using mothers' blood pressure and urinary protein measurements from the maternity clinics and birth hospitals, we defined normotensive or hypertensive pregnancies in mothers of 398 men, who participated in the Helsinki Birth Cohort 1934–1944 Study. The men underwent the Finnish Defence Forces basic ability test twice: first during compulsory military service at age 20.1 (SD = 1.4) years and then in a retest at age 68.5 (SD = 2.9) years. The test yields a total score and subscores for tests measuring verbal, arithmetic, and visuospatial reasoning.
Men born after pregnancies complicated by a hypertensive disorder, compared with men born after normotensive pregnancies, scored 4.36 (95% confidence interval, 1.17–7.55) points lower on total cognitive ability at 68.5 years and displayed a greater decline in total cognitive ability (2.88; 95% confidence interval, 0.07–5.06) after 20.1 years. Of the subscores, associations were strongest for arithmetic reasoning.
Maternal hypertensive disorders in pregnancy predict lower cognitive ability and greater cognitive decline up to old age. A propensity to lower cognitive ability and decline up to old age may have prenatal origins.
Substantial amounts of non-endocrine cells are implanted as part of human islet grafts, and possible influence of non-endocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for non-endocrine cells due to lack of available methods. Aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDC) for clinical islet transplantation, and to characterize them regarding phenotype, viability and function.
We assessed 161 human islet preparations using laser scanning cytometer (LSC/iCys) for phenotypic analysis of non-endocrine cells and flow cytometer (FACS) for PDC viability. PDC and β-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce pro-inflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF), relevant to islet graft outcome.
Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDC, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than β-cells (PDC vs. β-cell: 75.5±13.9 and 62.7±18.7 %; p<0.0001). Although β-cells viability was independent from the density, that of PDC was higher as the density from which they were recovered increased. There was no correlation between PDC and β-cells viability (R2=0.0078). PDC sorted from high-density fractions produced significantly higher amount of pro-inflammatory mediators and VEGF, but not TF.
PDC isolated from different fractions had different viability and function. The precise characterization and assessment of these cells in addition to β-cells in human islet cell products may be of assistance in understanding their contribution to islet engraftment and in developing strategies to enhance islet graft function.
Human pancreatic ductal cells; Islet transplantation; Assessment; Cytokines; Laser Scanning Cytometer; VEGF; IL-8; CA19-9
Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.
Over 45 years ago chronic granulomatous disease (CGD) was ascribed to a failure of neutrophils to mount a respiratory burst, and it is now known to result from primary genetic deficiencies in the phagocyte NADPH oxidase complex. Recent work suggests that reactive oxygen species produced by NADPH oxidases have other important functions as diverse as maturing hormones and promoting protein kinase signal transduction. Candida albicans is an opportunistic pathogen that preys on immunocompromised patients to cause lethal candidemia. We used the transparent zebrafish larva to describe a novel function of both phagocyte oxidase and dual-specific NADPH oxidase in directing phagocyte recruitment to C. albicans infection foci. We demonstrate that NADPH oxidase-dependent attraction of neutrophils and macrophages is instrumental in effective containment of yeast within phagocytes, which prevents the yeast-to-hyphal morphogenetic switch and limits mortality. Remarkably, when the fungal morphogenetic switch is prevented by mutation, NADPH oxidase activity is no longer required for effective fungal containment. Our study suggests that defects in CGD may extend beyond reduced microbial killing by superoxide to include impairment of chemotaxis, and provide a basis for exploring this alternative function in mammals.
Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creERT2 mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5 days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5+ stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5+ stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5+ stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.
Lgr5; dextran sodium sulfate; fish oil; Wnt signaling; colon
Many patients with schizophrenia show a limited symptomatic response to treatment with dopaminergic antipsychotics. This may reflect the additional involvement of non-dopaminergic neurochemical dysfunction in the pathophysiology of the disorder. We tested the hypothesis that brain glutamate levels would differ between patients with first-episode psychosis who were symptomatic compared with those with minimal symptoms following antipsychotic treatment. Proton magnetic resonance spectroscopy (1H-MRS) spectra were acquired at 3 Tesla in the anterior cingulate cortex and left thalamus in 15 patients with first-episode psychosis in symptomatic remission, and 17 patients with first-episode psychosis who were still symptomatic following at least one course of antipsychotic treatment. Metabolite levels were estimated in ratio to creatine (Cr) using LCModel. Levels of glutamate/Cr in the anterior cingulate cortex were significantly higher in patients who were still symptomatic than in those in remission (T(30)=3.02; P=0.005). Across the entire sample, higher levels of glutamate/Cr in the anterior cingulate cortex were associated with a greater severity of negative symptoms (r=0.42; P=0.017) and a lower level of global functioning (r=−0.47; P=0.007). These findings suggest that clinical status following antipsychotic treatment in schizophrenia is linked to glutamate dysfunction. Treatment with compounds acting on the glutamatergic system might therefore be beneficial in patients who respond poorly to dopaminergic antipsychotics.
psychosis; magnetic resonance spectroscopy; glutamate; treatment response; anterior cingulate cortex; thalamus; glutamate, schizophrenia/antipsychotics, imaging, clinical or preclinical, biological psychiatry, psychosis, anterior cingulate cortex, thalamus, magnetic resonance spectroscopy, treatment response
The PNA-FISH Yeast Traffic Light assay was performed on 54 clinical isolates of yeasts inoculated into blood culture bottles. The assay showed high sensitivity (Candida albicans/C. parapsilosis, 100%; C. glabrata/C. krusei, 92.3%; C. tropicalis, 100%) and specificity (C. albicans/C. parapsilosis, 100%; C. glabrata/C. krusei, 94.8%; C. tropicalis, 100%). Case note review estimated a change in therapy in 29% of cases had the PNA-FISH result been available to the clinician.
Our prior research focused on parental treatment acceptability (TA) and
preferences (TP) for preventive dental treatments for young Hispanic children.
We adapted the interview for administration to parents of young African American
In a sample of African American parents, determine parental TA and TP
for 5 dental treatments to prevent early childhood caries.
Interviewed 48 parents/caregivers of African American children
attending Head Start, assessing TA and TP for 3 treatments for children:
toothbrushing with fluoride toothpaste (TB), fluoride varnish (FV), and
xylitol in food (XF); and 2 treatments for mothers: xylitol gum (XG) and
chlorhexidine (CHX) rinse. The interview included verbal information,
illustrated treatment cards, photos/video clips, and samples. Parents
provided TA of each treatment (1–5 scale), TP between each of 10
pairs of the 5 treatments, and open-ended reasons for their preferences. TP
were summed (0–4) to create overall preference.
All treatments were acceptable (means 4.4–4.9). TB was more
acceptable than FV and XF (p<0.05). Summed TP revealed a strong
preference for TB (mean 3.1) above other treatments (all p<0.01).
Primary reasons for preferring TB were: promotes healthy habits;
child-focused; and effectiveness.
All treatments were acceptable, however, parents/guardians strongly
preferred TB. Parents’ emphasis on healthy habits and child-focused
treatment supports efforts for oral health education programs in early
childhood settings. Some parents expressed concerns about FV, XF, and CHX.
Results may be useful in planning prevention programs for young children in
African American communities.
oral health; acceptability of healthcare; pediatric dentistry; preventive dental treatments; African American
Improved islet isolation has still been important to obtain adequate islet numbers for islet transplantation. Although Ficol-based density gradient is widely used for purification in most islet processing centers, OptiPrep-based density gradient is recently used in the limited centers and their clinical outcomes are excellent. Cytokine/chemokine production from islet preparations for transplantation widely varies. Some cytokines/chemokines have been reported to cause apoptosis in human islet preparations after isolation. Reducing cytokine/chemokine production is a key to improve islet numbers after isolation and islet transplantation outcome. The aim of current study is to investigate the variability of pro-inflammatory cytokine/chemokine production from islet preparations purified by different density gradient. After digestion of human pancreata, pre-purification digests were devided into two groups and purified using semi-automated cell processor with Ficoll-based and OptiPrep-based density gradient. Islet preparations were cultured for 2 days and assessed regarding islet cell viability (FDA/PI), fractional β-cell viability and β-cell content. Cytokine/chemokine production from islet preparations was also examined. After purification, purity, post-purification IEQ and islet recovery rate were comparable between two groups. Although FDA/PI and fractional β-cell viability showed no significant differences, β-cell survival during culture significantly increased in OptiPrep-based density gradient group when compared to Ficoll-based density gradient group. TNF-α, IL-1β, IFN-γ, IL-6 and MIP-1β production from OptiPrep-based density gradient group significantly decreased.
OptiPrep-based density gradient can reduce cytokine-production when compared to Ficoll-based density gradient, resulting in improvement of quantity of β-cell mass. Cytokine profiling could spot new light on assessment of islet preparations before transplantation.
Recent studies demonstrated that prolactin has beneficial effects on β-cells for islet transplantation. We examined the effect of human recombinant prolactin (rhPRL) supplementation to the culture media to determine its potential use in the context of clinical islet transplantation.
Materials and Methods
Each human islet isolated from 14 deceased multi-organ donors was cultured in Miami modified media-1 supplemented with or without rhPRL (500μg/L) for 48 hours. β-cell survival and proliferation (BrdU and Ki-67) were determined by laser-scanning cytometry. The cytoprotective effects of rhPRL against noxious stimuli were assessed by flow cytometry (tetramethylrhodamine ethyl ester). Cytokine/chemokine and tissue factor (TF) production were measured in vitro and islet potency was assessed in vivo into diabetic immunodeficient mice.
β-cell survival during culture was 37% higher in the rhPRL group than in control (p=0.029). rhPRL protected β-cells in vitro from cytokines, Nitric Oxide donor and H2O2. The exposure to rhPRL did not affect human beta-cell proliferation with our protocol. rhPRL treatment did not alter cytokine/chemokine and TF production in vitro nor affected human islet functionality in vivo: recipient mice achieved normoglycemia with a comparable tempo, while loss of graft function was observed in 2/7 mice in the control group and in none of the rhPRL group (p=n.s.).
rhPRL supplementation to islet culture media improved human β-cell-specific survival without altering islet quality. Addition of rhPRL to cultured islets may grant a more viable β-cell mass in culture. The development of β-cell cytoprotective strategies will be of assistance in improving islet transplantation outcomes.
islet; transplantation; prolactin; cytokine; chemokine
N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.
The selection of enzyme blend is critical for the success of human islet isolations. Liberase HI collagenase (Roche) has been introduced in the 1990’s and widely used for clinical islet transplantation. More recently, a blend collagenase NB1 has been rendered available. The aim of this study was to evaluate the isolation outcomes and islet quality comparing human islet cells processed using NB1 and Liberase HI.
A total of 90 isolations processed using NB1 (n=40) or Liberase HI (n=50) was retrospectively analyzed. Islet yield, function in vitro and in vivo, cellular (including β-cell specific) viability and content, as well as isolation related factors were compared.
No significant differences in donor related factors were found between the groups. There were also no significant differences in islet yields (NB1 vs. Liberase; 263,389±21,550 vs. 324,256±27,192 IEQ; P = n.s., respectively). The pancreata processed with NB1 showed a significantly longer digestion time (18.6±0.7 vs. 14.5±0.5 min, P <0.01), lower β-cell viability (54.3±3.4 vs. 72.0±2.1%, P < 0.01), β-cell mass (93,671±11,150 vs. 148,961± 12,812βIEQ, P<0.01) and viable β-cell mass (47,317±6,486 vs. 106,631±10,228 VβIEQ, P < 0.01) than Liberase HI. In addition, islets obtained with Liberase showed significantly better graft function in in vivo assessment of islet potency.
The utilization of collagenase NB1 in human islet isolation was associated with significantly lower β-cell viability, mass and islet potency in vivo in our series when compared to Liberase HI even though there was no significant difference in islet yields between the groups. Evaluation of viable β-cell mass contained in human islet preparations will be useful for selecting enzyme blends.
islet; transplantation; isolation; collagenase; cell viability; potency; nude mice
Several reports suggest that islets isolated from younger donor pancreata are of better quality for clinical islet transplantation. The relative inefficiency of the continuous gradient purification process (CGP) is one of the major obstacles to the utilization of these younger donor pancreata. This study demonstrates the benefits of utilizing an additional purification step, rescue gradient purification (RGP), to recover trapped islets and examines the possible superiority of these rescued islets.
Seventy-three human islet isolations purified by RGP following CGP were divided into 2 groups based on age, and the isolation results were retrospectively analyzed (Group I; age ≤40, Group II; age >40). The quality of islets from both CGP and RGP were assessed by β-cell fractional viability (βFV) and ADP/ATP ratio.
Significant increases in the % islet recovery from RGP and the % trapped islets in Group I compared to Group II were observed. Donor age correlated negatively to the % islets recovered from RGP (R=0.440) and to the % of trapped islets (R=0.511). RGP islets had higher βFV and better ADP/ATP ratio compared to CGP islets.
In conclusion, RGP improved the efficiency in the purification of trapped islets, which often come from younger donor pancreata. The better quality of β-cells in RGP islets encourages us to perform RGP, considering the higher quality as well as the quantity of remaining islets.
Islet transplantation; purification; viability; trapped islet; young donor
Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose induced insulin secretion similar to exendin-4. It has been reported that systemic administration of PACAP maintained beta-cell mass, delayed the onset of hyperglycemia and protected beta cells from glucose toxicity. Moreover, PACAP increases glucose-stimulated insulin release in vitro and in vivo. In this study, we investigated the possibility of PACAP use in human islet transplantation.
Human islets were cultured in the presence or absence of PACAP (10−12M) for 48 hours. Beta-cell viability by FACS, cellular composition analysis by iCys/LSC and glucose stimulated insulin secretion were assessed. In vivo, islets were transplanted beneath the kidney capsule of Streptozotocin induced diabetic immunodeficient mice. Intravenous glucose tolerance test (IVGTT) was also performed in presence or absence with PACAP (Peptide International; 1.3 nmol/kg).
There were significant improvements in terms of beta cell viability and cellular composition between islets cultured with or without PACAP (respectively; P<0.05). Moreover, glucose stimulated insulin secretion significantly improved in islets cultured with PACAP, compared to controls, respectively; P<0.05). Treatment of recipient mice with PACAP resulted in beneficial effects on insulin secretion (PACAP vs. control, l3.2 vs. 1.9 mU/l), in IVGTT. However, no significant difference in glucose level between the two groups was observed.
Our study indicated that PACAP significantly improved beta cell viability and survival during culture, and increased insulin secretion in vitro and in vivo. However, blood glucose levels in vivo after an IVGTT did not significantly improve probably due to increased glucagon secretion from alpha cells. PACAP supplementation to culture medium could be of assistance in improving clinical islet transplantation outcomes.
Islet isolation and purification using a continuous density gradient may reduce the volume of tissue necessary for implantation into patients, therefore minimizing the risks associated with intraportal infusion in islet transplantation. On the other hand, the purification procedure might result in a decreased number of islets recovered due to various stresses such as exposure to cytokine/chemokine. While a Ficoll-based density gradient has been widely used in purification for clinical trials, purification with Iodixanol (OptiPrep) has been recently reported in islet transplant series with successful clinical outcomes. The aim of the current study was to compare the effects of the purification method using OptiPrep-based and Ficoll-based density gradients.
Human islet isolations were performed using a modified automated method. After the digestion phase, pre-purification digests were divided into two groups and purified using a semiautomated cell processor with either a continuous Ficoll or OptiPrep-based density gradient. The quantity, purity, viability and cellular composition of islet preparations from each group were assessed. Cytokine/chemokine and tissue factor production from islet preparations after 48h culture were also measured.
Although islet purity, post-purification IEQ, islet recovery rate, FDA/PI and fractional β-cell viability were comparable, β-cell mass after 48h culture significantly improved in OptiPrep group when compared to the Ficoll group. The production of cytokine/chemokine including IL-1β, TNF-α, IFN-γ, IL-6, IL-8, MIP-1β, MCP-1 and RANTES but not tissue factor from the OptiPrep group was significantly lower during 48h culture after isolation. Each preparation contained the similar number of ductal cells and macrophages. Endotoxin level in both gradient medium was also comparable.
The purification method using OptiPrep gradient media significantly reduced cytokine/chemokine production but not tissue factor from human islet preparations and improved β-cell survival during pre-transplant culture. Our results suggest that the purification method using OptiPrep gradient media may be of assistance in increasing successful islet transplantation.
islet; transplantation; OptiPrep; Ficoll; cytokine; purification