Whole Blood Profiling of T-cell-Derived microRNA Allows the Development of Prognostic models in Inflammatory Bowel Disease.

BACKGROUND: MicroRNAs [miRNAs] are cell-specific small non-coding RNAs that can regulate gene expression and have been implicated in inflammatory bowel disease [IBD] pathogenesis. Here we define the cell-specific miRNA profiles and investigate its biomarker potential in IBD. METHODS: In a two-stage prospective multi-centre case control study, next generation sequencing was performed on a discovery cohort of immunomagnetically separated leukocytes from 32 patients (nine Crohn's disease [CD], 14 ulcerative colitis [UC], eight healthy controls) and differentially expressed signals were validated in whole blood in 294 patients [97 UC, 98 CD, 98 non-IBD, 1 IBDU] using quantitative PCR. Correlations were analysed with phenotype, including need for early treatment escalation as a marker of progressive disease using Cox proportional hazards. RESULTS: In stage 1, each leukocyte subset [CD4+ and CD8+ T-cells and CD14+ monocytes] was analysed in IBD and controls. Three specific miRNAs differentiated IBD from controls in CD4+ T-cells, including miR-1307-3p [p = 0.01], miR-3615 [p = 0.02] and miR-4792 [p = 0.01]. In the extension cohort, in stage 2, miR-1307-3p was able to predict disease progression in IBD (hazard ratio [HR] 1.98, interquartile range [IQR]: 1.20-3.27; logrank p = 1.80 × 10-3), in particular CD [HR 2.81; IQR: 1.11-3.53, p = 6.50 × 10-4]. Using blood-based multimarker miRNA models, the estimated chance of escalation in CD was 83% if two or more criteria were met and 90% for UC if three or more criteria are met. INTERPRETATION: We have identified and validated unique CD4+ T-cell miRNAs that are differentially regulated in IBD. These miRNAs may be able to predict treatment escalation and have the potential for clinical translation; further prospective evaluation is now indicated.

Progressive multifocal leukoencephalopathy with gastrointestinal disease in a pediatric kidney transplant recipient.

PML is a demyelinating disease of the central nervous system caused by infection with JCV. Several cases of PML in bone marrow and solid organ transplant recipients have been reported in recent years. JCV has been isolated from the gastrointestinal mucosa of immunocompromised patients, but there are no published reports of PML associated with symptomatic gastrointestinal involvement in kidney transplant recipients. We report a case of a nine-yr-old girl with a kidney transplant who developed a severe gastrointestinal illness causing pseudo-obstruction in association with PML. JCV was suspected as the causative agent in this patient by the detection of high JCV titer through PCR analysis of the cerebrospinal fluid and blood and positive staining for simian virus 40 in the colon. JCV intestinal infection should be considered in kidney transplant recipients presenting with intestinal pseudo-obstruction.

Immunosuppression by placental indoleamine 2,3-dioxygenase: a role for mesenchymal stem cells.

BACKGROUND/OBJECTIVES: Mesenchymal stem cells (MSC) can be isolated from human placenta and have the potential to contribute to the immunosuppressive properties of placental tissue. The objectives of this study were to investigate the phenotype and differentiation characteristics of MSC derived from human placenta and evaluate the role of the tryptophan degrading enzyme, indoleamine 2,3 dioxygenase (IDO), in mediating their immunosuppressive affect. METHODS: MSC obtained from placental tissue (pMSC) were characterised using flow cytometry and tested for multipotency by determining differentiation into all mesenchymal lineages. The immunosuppressive properties of pMSC were tested in allogeneic mixed lymphocyte reactions and IDO expression and activity were measured by semi-quantitative real-time PCR and HPLC respectively. RESULTS: Multipotent stem cells were isolated from placenta and displayed chondrogenic, osteogenic and limited adipogenic differentiation. Cell surface antigen expression of pMSC was similar to bone marrow MSC (bMSC) with lack of the haematopoietic and common leukocyte markers (CD34, CD45), and expression of adhesion (CD29, CD166, CD44) and stem cell (CD 90, CD105, CD73) markers. Placental MSC were suppressive of allogeneic T-cell proliferation, an effect which was intensified following IDO induction by IFN-gamma. Replenishment of tryptophan or treatment with the IDO-blocker, 1-methyl-tryptophan (1-MT), attenuated the immunosuppressive action of pMSC. CONCLUSIONS: These results suggest that placental tissue contains MSC, which are phenotypically and functionally similar to bMSC, and that IDO is a key mediator of their immunosuppressive effect. Further investigation is needed to determine if pMSC function effects pregnancy outcome.

Isoprenylated proteins.

Isoprenoids are synthesized in all living organisms and are incorporated into diverse classes of end-products that participate in a multitude of cellular processes relating to cell growth, differentiation, cytoskeletal function and vesicle trafficking. In humans, the non-sterol isoprenoids, farnesyl pyrophosphate and geranylgeranyl-pyrophosphate, are synthesized via the mevalonate pathway and are covalently added to members of the small G protein superfamily. Isoprenylated proteins have key roles in membrane attachment and protein functionality, have been shown to have a central role in some cancers and are likely also to be involved in the pathogenesis and progression of atherosclerosis and Alzheimer disease. This review details current knowledge on the biosynthesis of isoprenoids, their incorporation into proteins by the process known as prenylation and the complex regulatory network that controls these proteins. An improved understanding of these processes is likely to lead to the development of novel therapies that will have important implications for human health and disease.

Renal transplantation in a 14-year-old girl with vitamin B12-responsive cblA-type methylmalonic acidaemia.

Renal tubular dysfunction and chronic renal failure are well recognised complications of methylmalonic acidaemia (MMA) and can occur even in the context of optimal medical metabolic management. Organ transplantation, such as renal and combined liver and renal transplants, have been utilised in the past for children whose disease cannot be managed by conservative medical practices and those with end stage renal disease. Our patient was diagnosed with B(12)-responsive MMA (subsequently proven to be cblA-type MMA) in the postoperative period following renal transplantation for idiopathic chronic renal failure. She remains well, with excellent graft function and metabolic control 4 years after transplantation. This patient highlights the importance of testing for the inborn errors of metabolism in patients presenting with recurrent acidosis and progressive renal impairment.

Differentiated chondrocytes for cartilage tissue engineering.

Trauma to the articular cartilage surface of the joint represents a challenging clinical problem due to the very limited ability of this tissue to self-repair. Moreover, repair techniques such as microfracture, which introduce cells into the joint, have unpredictable clinical outcomes as they produce a fibrocartilage tissue that degenerates with time. Alternative treatments include tissue reconstruction with autograft and allograft tissue. However, these procedures are restricted by the availability of suitable donor tissue. These limitations have been the driving force behind the emerging field of articular cartilage tissue engineering. This paper will highlight and contrast the key challenges associated with the tissue engineering of this neo-tissue using differentiated adult cells. The various components of the tissue engineering process will be described including the choice of donor cell/tissue type and the selection of scaffolds that guide the formation of tissue. The ability of the tissue engineered implants to stimulate the repair of defects in vivo will also be discussed. Tissue engineering approaches may, in the future, provide an ideal alternative to the current surgical treatments for cartilage repair.

Factors influencing growth and final height after renal transplantation.

Growth retardation occurs commonly in children and adolescents with chronic renal insufficiency. While some children exhibit catch-up growth following renal transplantation, for many children growth remains sub-optimal. The aim of the current study was to review the factors influencing growth and final height following renal transplantation. Data from all children who had a renal transplant performed between 1985 and 1998 at the Royal Melbourne and Royal Children's Hospitals, Melbourne (n = 85), were examined retrospectively. Two children who died in the first year post-transplant and one patient lost to follow-up within 6 months of their transplant were excluded. Children with multiple grafts had only growth following their most recent graft analyzed. The mean height standard deviation score (Ht-SDS) at the time of transplantation was -2.11 (range: -5.05 to 0.27), improving to -1.50 (range: -3.67 to 1.27) at 7 yr post-transplant. On univariate analysis, the dose of cyclosporin at 6 months and at 1 and 3 yr, and the graft function at 1 yr, had a significant positive correlation with the change in Ht-SDS (DeltaHt-SDS) at each of those time-points post-transplant. At all time-points there was a strong correlation between pretransplant height and subsequent growth. A sub-group of children who were 16 yr of age or older at December 1999, and who were considered to have reached their final height, were examined to determine predictors of final height. Multiple regression analysis of clinical and laboratory parameters from the sub-group of patients > or = 16 yr of age showed that height at the time of transplant, age at the time of transplant, and final glomerular filtration rate, were significant independent predictors of growth (r2 = 0.82, p = 0.01). In addition, the immunosuppressive regimen at 1, 3, and 5 yr post-transplant had a significant effect on growth. This study confirms the importance of each of these factors for post-transplant growth.

Relationship between cell proliferation, cell-cycle phase, and retroviral vector production in FLYRD18 human packaging cells.

The relatively low concentrations of retroviral vectors produced by most packaging cells requires the optimization and intensification of their production to make a commercially viable product for gene therapy. While a number of reports exist concerning target cell-cycle effects on retroviral vector infection efficiency, no studies have been reported on the effects of packaging cell cycle on vector production. We have studied the effect of proliferation of the human packaging cell line, FLYRD18, on vector production. In addition, the titer levels of vector produced by cells in each phase of the cell cycle were compared. Numerous studies suggested progression of the cells through the cell cycle to be essential for vector production. However, vector release was found not to be predominant in any particular phase of the cell cycle. These findings indicate that packaging cell proliferation is important for optimal virus production and that arrest of the cells in any particular phase of the cell cycle affords no benefits in retroviral vector production. In contrast to previous reports (using other cell lines), we observed no temporary inhibition of cell cycle progression after detachment of cells from their substratum and that virus production occurred immediately after re-plating of the cells. The findings in this report are important for determining the optimal culture conditions for vector production by packaging cells in vitro.

Clinical spectrum of Denys-Drash and Frasier syndrome.

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are two related conditions caused by mutations of the Wilms tumor gene, WT1. Both syndromes are characterized by male pseudohermaphroditism, a progressive glomerulopathy, and the development of genitourinary tumors. DDS and FS have previously been distinguished by differences in nephropathy, with DDS patients demonstrating diffuse mesangial sclerosis (DMS) in contrast to focal and segmental glomerulosclerosis (FSGS) in FS patients. The clinicopathological features and genotype analysis of two patients with WT1 mutations are presented in this report. Genotype analysis of the first patient revealed a previously undescribed mutation in exon 8 of the WT1 gene. The second patient presented with a rapidly progressive nephropathy characterized histologically by DMS, but was found to have the genetic mutation seen in FS patients. A summary of all reported patients with the characteristic mutation associated with FS demonstrates the clinical overlap of this syndrome with DDS. This suggests that both these conditions should be considered as part of the spectrum of disease due to WT1 gene mutations rather than as separate diseases. Clinical classification remains important for prognosis, as the underlying renal disease appears to predict the progression of nephropathy independently of the genetic abnormality.

Children's illness drawings and asthma symptom awareness.

This study examines the relationship between children's abilities to perceive their symptoms of asthma via several previously researched subjective and objective procedures compared with their performance on a standardized children's drawing task and scale criteria. Results indicated that girls verbalized significantly more emotions about their drawings and were better able to detect airflow changes in their small airways than boys. The Gabriels Asthma Perception Drawing Scales (GAPDS) is a promising clinical tool for assessing children's perceptions and emotions about asthma via nonverbal methods. Varying methods of measuring asthma symptom awareness are not highly correlated; thus, more than one methodology is appropriate for use with children.

Effects of culture parameters on the production of retroviral vectors by a human packaging cell line.

The use of retroviral vectors for human gene therapy requires the production of large quantities of high titer vector stocks. Maintaining high titers during the prolonged culture of packaging cells will require that critical parameters be controlled. The aim of this study was to determine which culture parameters critically affect the production/decay of retroviral vectors produced by the human packaging cell line FLYRD18/LNC-hB7. The stability of retroviral vectors released by this cell line was found to be temperature dependent (half-life of 6.9, 11.0, and 64.3 h when incubated at 37, 32, and 0 degrees C, respectively). Titers increased up to 10-fold when the packaging cells were cultured at 32 degrees C, compared to 37 degrees C, despite a decrease in cell yield (cell-specific titers were 20-fold higher). Virus titers were also over 10-fold higher when the packaging cells were cultured in a reduced serum concentration (1%) compared to 5%. Retrovirus production at a range of pH levels revealed a significant decrease in virus titer at pH levels below 6.8 and above 7.2, optimum titers being achieved in cultures at pH 7.2. Dissolved oxygen levels in the range 20-80% did not significantly affect titers under the conditions tested. Finally, a packed bed system containing the packaging cells immobilized on porous microcarriers was shown to sustain the production of active retroviral vectors for over 1 month, in relatively large volumes.

Evaluation and long-term outcome of pediatric renovascular hypertension.

Seventeen children with renovascular hypertension were managed at the Royal Children's Hospital, Melbourne, over the 20-year period from 1975 to 1996. The age at presentation ranged from 10 days to 18 years. All children presented with severe hypertension with mean systolic blood pressure 7 standard deviations above age-matched averages and mean diastolic blood pressure 5.5 standard deviations above age-matched averages. Neurofibromatosis was the most common etiology (58% of patients) and there were no cases of Takayasu's arteritis. Patients underwent a variety of biochemical and imaging investigations but in all cases renal angiography was necessary for definitive diagnosis and for planning therapy. Ten of the 17 patients had surgical procedures performed. Percutaneous transluminal angioplasty was performed in four patients but led to cure in only one patient following thrombosis of the affected artery producing segmental renal infarction. Other vascular reconstructive procedures, including the use of autologous or synthetic bypass grafts and autotransplantation, produced cure of hypertension in 50% of children with improvement in a further 30%. The long-term outlook for children treated with surgical reconstructive procedures was excellent. One patient underwent surgery for avulsion of an arterial graft following a pubertal growth spurt. No other patient originally cured by surgery has required reoperation with no cases of restenosis at a mean follow-up of 11 years 3 months.

Familial occurrence of idiopathic infantile hypercalcemia.

Idiopathic infantile hypercalcemia (IIH) is a rare cause of hypercalcemia in the 1st year of life and was initially considered part of a spectrum encompassing vitamin D intoxication, Williams syndrome, and idiopathic hypercalcemia. Identification of the gene for Williams syndrome now allows a clear separation of IIH from Williams syndrome. The inheritance and pathogenesis of IIH remains largely unknown, with only sporadic cases reported to date. This report describes a family with two siblings with IIH. The pedigree is consistent with autosomal recessive inheritance, but more complex inheritance is suggested by the occurrence of hypercalciuria in a number of family members. Although one affected patient demonstrated elevated 1,25-dihydroxyvitamin D(3) levels, no conclusions regarding the pathogenesis of this condition could be drawn.

Streptococcus pneumoniae-induced haemolytic uraemic syndrome.

Haemolytic uraemic syndrome secondary to infection with neuraminidase producing Streptococcus pneumoniae is well recognised, but was previously considered to be rare. This case report describes the course of a 9-month-old male with pneumococcal pneumonia, T activation and haemolytic uraemic syndrome. The clinical features of three other cases treated in Southeast Queensland in the past 2 years and 12 previously reported cases are summarised. The widespread availability of rapid diagnostic testing for this entity should allow for increased recognition, enabling appropriate use of low plasma volume blood products with improved patient outcome.

Monoclonal antibodies to high-incidence Kell epitopes: characterization and application in automated screening of donor samples.

Monoclonal antibodies, LM313/706 and LM357/828, recognize high-frequency epitopes which are absent on red cells of Ko phenotype. Both antibodies have proved suitable for automated screening of blood donor samples, with 5 ml of each culture supernatant sufficient to screen 3,000 donor samples. In a screen of 45,545 samples, LM313/706 revealed 11 samples (1:3686) of Kp(a+b-) phenotype. Thirty-seven samples of K+k- phenotype were identified in 15,235 samples screened with LM357/828 antibody (0.24%). The epitope recognized by LM313/706 was inactivated by 2 mM DTT suggesting a possible association with the Jsa/Jsb antigen structure. The expression of the k-like epitope detected by LM357/828 antibody was found to be influenced by pH change.

Fc receptors induced by herpes simplex virus. I. Biologic and biochemical properties.

Radioiodinated normal rabbit IgG was used to detect Fc receptors on the surface of HeLa S3-2 cells infected with herpes simplex virus (HSV). Unlike the Fc receptors present on most leukocytes, these virus-induced Fc receptors were found to be sensitivite to trypsin at concentrations of enzyme as low as 0.1 mg/ml. Treatment of infected cells with neuraminidase enhanced the binding of IgG. Yet the HSV-induced Fc receptor(s) is probably a glycoprotein because its synthesis was inhibited by 2-deoxy-D-glucose at concentrations inhibiting glycoproteins but not total proteins. Binding of radioiodinated IgG to Fc receptors on infected HeLa S3-2 cells was unaffected by F(ab')2 fragments prepared from antisera against uninfected HeLa S3-2 cells or against human beta2-microglobulin. By contrast, anti-HSV F(ab')2 or Fab' fragments decreased binding of radioiodinated IgG to HSV-infected cells by 85%, and binding of radioiodinated Fc was also inhibited by anti-HSV Fab'.