Early barriers to neonatal porcine islet engraftment in a dual transplant model.

Porcine islet xenografts have the potential to provide an inexhaustible source of islets for ß cell replacement. Proof-of-concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood-mediated inflammatory reaction and a thorough understanding of early xeno-specific immune responses. A paucity of data exist comparing xeno-specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1 hour, 24 hours, and 7 days. Within the first 24 hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])-positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24 hours compared with 1 hour in both NPI (wild-type) and AIs. At 7 days, IgM and macrophages were highly specific for NPIs (α1,3-galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.

Multi-omics integration identifies key upstream regulators of pathomechanisms in hypertrophic cardiomyopathy due to truncating MYBPC3 mutations.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways and key regulators causing the disease. Therefore, we employed a multi-omics approach to study the pathomechanisms underlying HCM comparing patient hearts harboring MYBPC3 mutations to control hearts. RESULTS: Using H3K27ac ChIP-seq and RNA-seq we obtained 9310 differentially acetylated regions and 2033 differentially expressed genes, respectively, between 13 HCM and 10 control hearts. We obtained 441 differentially expressed proteins between 11 HCM and 8 control hearts using proteomics. By integrating multi-omics datasets, we identified a set of DNA regions and genes that differentiate HCM from control hearts and 53 protein-coding genes as the major contributors. This comprehensive analysis consistently points toward altered extracellular matrix formation, muscle contraction, and metabolism. Therefore, we studied enriched transcription factor (TF) binding motifs and identified 9 motif-encoded TFs, including KLF15, ETV4, AR, CLOCK, ETS2, GATA5, MEIS1, RXRA, and ZFX. Selected candidates were examined in stem cell-derived cardiomyocytes with and without mutated MYBPC3. Furthermore, we observed an abundance of acetylation signals and transcripts derived from cardiomyocytes compared to non-myocyte populations. CONCLUSIONS: By integrating histone acetylome, transcriptome, and proteome profiles, we identified major effector genes and protein networks that drive the pathological changes in HCM with mutated MYBPC3. Our work identifies 38 highly affected protein-coding genes as potential plasma HCM biomarkers and 9 TFs as potential upstream regulators of these pathomechanisms that may serve as possible therapeutic targets.

The metabolism and binding properties of 3H-aldosterone in plasma and its sex dependence in adrenalectomized rats.

The rates of clearance of plasma 3-H radioactivitivity following intravenous injection of 3-H aldosterone was demonstrated to be sex-dependent in adrenalectomized rats. The perchantage plasma radioactivity which is CH-2CL-2extractable is greater in female than in male rats from 5 min to 90 min postinjection; however the quantities of CH2-CL2-extractable label are not significantly different until 60 min postinjection. The quantities of nonextractable, water-soluble metabolites of adosterone (NEPD), which are markedly greater in the plasma of males, reach peak levels 30 min after injections of aldosterone, during the latent period of the hormone.N females, these polar metabolites (NEPD)are rapidly cleared from the blood. The quantities of 3-H-radioactivity associated with the plasma binding proteins are similar in both males and females. The unbound levels of aldosterone and its metabolities are significantly greater in the plasma of males. These findings indicate that the sex hormones may influence not only the metabolism of aldosterone in rats, but also the plasma levels of unmetabolized aldosterone and its metabolites.

The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia--caspase-3 as a case study.

Quantitative reverse transcription and polymerisation chain reaction (RT-PCR) using Taqman¿trade mark omitted¿ fluorogenic probes has been used to measure changes in gene expression in the cerebral cortex of rats in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia. The mRNA levels of three housekeeping genes have been analysed in this model to determine which gene showed least change following experimental insult. In the lesioned cortex, beta-actin mRNA increased at 24 h, while the levels of cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not change. We have also used this methodology to examine modulations in the level of caspase-3 mRNA during focal ischemia in the rat. Caspase-3 mRNA showed a 41% increase at 6 h post-MCAO, which was specific to the lesioned cortex. This change became more pronounced with time, showing an increase of 220% at 24 h. This methodology enables changes in mRNA expression to be analysed more sensitively and quantitatively than other available techniques and highlights the need for careful choice of control or housekeeping genes used for RNA comparisons.

Caspase mRNA expression in a rat model of focal cerebral ischemia.

Proteins of the caspase family are involved in the signalling pathway that ultimately leads to programmed cell death (apoptosis), which has been reported to occur in some experimental models of stroke. In a previous paper we used quantitative reverse transcription and polymerase chain reaction (RT-PCR) to characterise changes in the mRNA expression of one member of this family, caspase-3, in a rat model of permanent focal ischemia. Here we have used this technique to study the expression of a further three caspases which are involved in different aspects of caspase signalling. Caspase-8, involved in Fas-mediated apoptosis, was upregulated in the cortex of ischemic rats. Caspase-11, which leads to the synthesis of the functional form of the cytokine interleukin-1 beta, also showed increased expression, but with a different temporal profile from caspase-8. In contrast, caspase-9, which forms part of the pathway signalling through the mitochondria, showed a decrease in expression. The expression of a further four caspases (1, 2, 6 and 7) has also been characterised in a simpler experiment. These caspases all showed distinctive patterns of expression following the induction of ischemia. These data lead us to conclude that caspase expression as a whole is under very strict transcriptional control in this model. Certain elements of caspase signalling, such as the Fas-induced pathway and the events upstream of IL-1 beta processing, are upregulated, while others are not. This may be due to some form of genetic program activated in response to ischemia in the brain and may highlight which biological pathways are modulated.

Varicose ulcer of the upper extremity.

Serious complications of local venous hypertension occur rarely in the upper extremity. These are most frequently the result of a dysfunctional arteriovenous fistula and may produce changes indistinguishable from those of chronic lower-extremity venous insufficiency. A combination of arterial and venous contrast imaging is essential for identifying major venous outflow obstruction and for planning appropriate surgical therapy.

The vanishing elective cholecystectomy. Trends and their consequences.

During the 1980s, there have been ongoing changes in the policies and programs that govern the health care provision system. To assess the effect of these changes on patient care, the charts of patients undergoing cholecystectomy in 1980 (132 patients) and 1986 (108 patients) were reviewed and compared. The patients are increasingly over 60 years of age. The elective cholecystectomy is being replaced by procedures for more advanced disease as referrals of patients to the surgeon are delayed. The morbidity more than doubled. Compared with 1980, the policies and programs in effect during 1986 allowed fewer patients to undergo cholecystectomy (-18%), but required a markedly increased cost for those patients (+22%).

Gastrointestinal lipomas.

We reviewed 15 years of experience with gastrointestinal (GI) lipomas at our institution. A series of eight patients was included, six of whom had lipomas in the colon and two, in the stomach. All eight patients were symptomatic. The diagnosis was not made preoperatively in seven cases, which accounted for surgical intervention in these patients. Colonoscopy was helpful in the diagnosis and treatment of the eighth patient. Advances in the diagnosis and treatment of GI lipomas include the use of computed tomographic scanning and colonoscopy.

Experience with lobular carcinoma of the breast. Emphasis on recent aspects of management.

A total of 105 patients with lobular carcinoma of the breast were seen at two hospitals between 1966 and 1978. Mammography was found to be of limited value in the preoperative assessment of these patients. As reported by others, there was a high incidence of bilateral lobular neoplasia. In the patients with lobular carcinoma in situ for whom follow-up data were available, all were alive and well. In contrast to this, infiltrating lobular carcinoma was found to be as malignant a lesion as any other infiltrating breast cancer regardless of the operative procedure performed. These latter tumors were found to bind substantial levels of estrogen receptor proteins. From the information gained, it is suggested that early hormonal manipulation and/or chemotherapy may be advantageous in the patient with recurrent infiltrating lobular carcinoma in particular.

Ependymal cyst of the quadrigeminal plate: case report.

A patient with an ependymal cyst of the quadrigeminal plate is reported. The patient presented with headache, diplopia, and evidence of increased intracranial pressure. A nonenhancing cyst with hydrocephalus was demonstrated by computed tomographic scan. The cyst was subtotally resected using a transtentorial approach. The causes, clinical presentation, and treatment of quadrigeminal plate ependymal cysts are discussed, together with a review of the literature.

Isolated central nervous system metastasis from transitional cell carcinoma of the bladder: report of a case and review of the literature.

Although systemic metastases from transitional cell carcinoma of the bladder occur frequently, involvement of the central nervous system is uncommon. We describe a patient with an isolated cerebral metastasis who had previously undergone resection of a Grade III, Stage B2 carcinoma of the bladder. We have been able to find only one previous case report of a solitary intracerebral metastasis from transitional cell carcinoma of the bladder without evidence of primary recurrence or additional sites of spread. Central nervous system metastasis from bladder carcinoma must be considered in the differential diagnosis of solitary intracerebral lesions.

Management of solitary nonparasitic liver cyst.

Solitary nonparasitic liver cysts are clinically rare and mostly nonsymptomatic. Occasionally, cases present with symptoms mainly because of the presence of a large cyst. In this report, the clinical features, diagnostic modalities, and current management of such cysts are described.

Paraganglioma of the cauda equina: case report with magnetic resonance imaging description.

The case of a 35-year-old white man with a cauda equina syndrome is presented. Magnetic resonance imaging confirmed the diagnosis, and radiation treatment successfully treated the condition.

The fungal flora of loganberries in relation to storage and spoilage.

Botrytis cinerea, Cladosporium spp., Penicillium spp., Aureobasidium pullulans, Mucor mucedo, Phoma state of Didymella applanata, Cryptococcus laurentii var. laurentii, C. albidus var. albidus, Candida sake and Sporobolomyces roseus were consistently present on freshly harvested loganberries. Botrytis cinerea was the main spoilage organism with Mucor mucedo of minor importance. The importance of benomyl-resistant strains of B. cinerea is discussed.

Tc-99m MDP uptake in osteopoikilosis.

A Tc-99m bone scan of a patient with classic roentgenographic findings of osteopoikilosis revealed multiple foci of increased activity that corresponded to many of the sclerotic foci on the roentgenograms. The authors presume that the abnormal bone scan in this patient reflects active osseous remodeling, similar to what has been observed in bone islands. Previous reports have emphasized the critical role of the radionuclide bone scan for distinguishing osteopoikilosis from osteoblastic bone metastases in patients with a known or suspected primary malignancy. In a young patient, an abnormal bone scan does not exclude the diagnosis of osteopoikilosis if the roentgenographic findings are characteristic of that entity.

The ERA implant-supported overdenture.

Extracoronal Resilient Attachment (ERA) implant-supported overdentures, Class I, Division 1 or Division 3 prostheses, provide an exceptionally stable and retentive prosthetic design for edentulous patients with osseointegrated implants. The ERA System uses either an individual female implant abutment or plastic female patterns that can be incorporated into a bar. The male portion of the attachment is a nylon piece of varying retentive quality. The attachment is resilient, stable, and can be easily serviced.

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands.

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.).