House keeping genes and gene expression analysis in transplant recipients: a note of caution

BACKGROUND: House keeping genes are often used as a means of standardising results obtained in gene expression investigations. This study was performed to investigate whether beta-actin, beta2-microglobulin (two genes frequently quoted as house keeping genes) and/or transferrin receptor would be suitable house keeping genes for use in gene expression analysis of renal transplant recipients. METHODS: Sequential expression of all three genes was measured in the peripheral blood mononuclear cells of 13 living donors and 45 renal transplant recipients, pre-operatively and then daily for up to 2 weeks. Fifteen of the recipients experienced an episode of biopsy proven acute rejection. Gene expression measurement was performed using quantitative real time 'TaqMan' PCR technology. RESULTS: Gene expression of all three genes was unchanged in the living donor cohort. However, in the transplant recipients there were significant increases in expression following transplantation in the non-rejectors, and preceding the diagnosis of acute rejection. In the latter group, levels returned to pre-transplant values after the commencement of anti-rejection therapy. CONCLUSIONS: Beta-actin, beta2-microglobulin and transferrin receptor gene expression, although not influenced by surgery, is influenced by transplantation, acute rejection and anti-rejection therapy making these genes unsuitable as house keeping genes following renal transplantation. These findings may cast doubt on the results of some studies that used these genes for the purposes of standardisation when looking at cDNA measurement. We suggest that any group wishing to use a house keeping gene ensure that its expression is independent of study parameters prior to the start of the study.     

Amelioration of diabetic nephropathy in SPARC-null mice

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular protein that inhibits mesangial cell proliferation and also affects production of extracellular matrix (ECM) by regulating transforming growth factor-beta1 (TGF-beta1) and type I collagen in mesangial cells. This study is an investigation of the role of SPARC in streptozotocin (STZ)-induced diabetic nephropathy (DN) of 6-mo duration in wild type (WT) and SPARC-null mice. SPARC expression was evaluated by immunohistochemistry (IHC) and by in situ hybridization (ISH). Deposition of type I and IV collagen and laminin was evaluated by IHC, and TGF-beta 1 mRNA was assessed by ISH. Renal function studies revealed no significant difference in BUN between diabetic SPARC-null mice and diabetic WT mice, whereas a significant increase in albumin excretion was detected in diabetic WT relative to diabetic SPARC-null mice. Diabetic WT animals exhibited increased levels of SPARC mRNA and protein in glomerular epithelial cells and in interstitial cells, in comparison with nondiabetic WT mice. Neither SPARC mRNA nor protein was detected in SPARC-null mice. Morphometry revealed a significant increase in the percentage of the glomerular tufts occupied by ECM in diabetic WT compared with nondiabetic WT mice, although there was no difference in the mean glomerular tuft area among groups. In contrast, diabetic SPARC-null mice did not show a significant difference in the percentage of the glomerular tufts occupied by ECM relative to nondiabetic null mice. Tubulointerstitial fibrosis was ameliorated in diabetic SPARC-null mice compared with diabetic WT animals. Further characterization of diabetic SPARC-null mice revealed diminished glomerular deposition of type IV collagen and laminin, and diminished interstitial deposition of type I and type IV collagen correlated with decreases in TGF-beta 1 mRNA compared with WT diabetic mice. These observations suggest that SPARC contributes to glomerulosclerosis and tubulointerstitial damage in response to hyperglycemia through increasing TGF-beta 1 expression in this model of chronic DN.     

No anesthetic or analgesic benefit of neostigmine 1 mg added to intravenous regional anesthesia with lidocaine 0.5% for hand surgery

BACKGROUND AND OBJECTIVES: Neostigmine has shown analgesic benefit when used as an adjunct to epidural or intrathecal anesthesia and analgesia, but evidence of benefit in the peripheral nervous system is controversial. This study aimed to determine if neostigmine 1 mg added to intravenous regional anesthesia (IVRA) provided any advantage in terms of intraoperative anesthesia or postoperative analgesia. METHODS: We recruited 54 patients booked for hand surgery into this randomized, double-blind study. For the IVRA technique, patients were administered 3 mg/kg of 0.5% lidocaine (maximum 45 mL). The treatment group (group N) had 1 mg neostigmine added to lidocaine before dilution. The control group (group C) had no additives to the IVRA solution. At the completion of surgery and after transfer to the recovery room, patients had verbal response pain scores measured at 30 minutes, 1 hour, and 2 hours after cuff deflation. Time to first request for analgesic, side effects, and analgesic consumption at 24 hours were also recorded. RESULTS: Significantly more patients in group N had motor block at 5 and 10 minutes after injection of study solution. There were no other significant differences in sensory block onset, intraoperative anesthesia, postoperative analgesia, or adverse effects between groups. CONCLUSIONS: Neostigmine 1 mg provides no anesthetic or analgesic advantage when added to IVRA for upper limb surgery.     

Unusual radiological presentation of renal cell carcinoma

We present an unusual case of renal cell carcinoma in a 59-year-old Saudi male less than 3 cm in size showing a pelvicalyceal filling defect on excretory urography and retrograde pyelography. Renal stones and blood clots were excluded by ultrasound and computerized tomography scanning. A urothelial tumor was initially diagnosed; finally surgery revealed a papillary renal cell carcinoma.     

Nucleated red blood cell counts: not associated with brain injury or outcome

The objective was to determine whether an elevated nucleated red blood cell count at birth after perinatal depression is associated with brain injury as measured by (1) proton magnetic resonance spectroscopy and (2) abnormal neurodevelopmental outcome at 30 months of age. The nucleated red blood cell counts from the first 24 hours of life were statistically analyzed in 33 term infants enrolled in a prospective study of the value of magnetic resonance imaging for the determination of neurodevelopmental outcome after perinatal depression. Nucleated red blood cell counts were elevated in 13/33 (39%). Abnormal outcome (19/33, 54%) was associated with Score for Neonatal Acute Physiology-Perinatal Extension (P = 0.04), decreased N-acetylaspartate to choline ratio in the basal ganglia (P = 0.009), and increased lactate to choline ratio in the basal ganglia (P = 0.02), but not with cord pH, Apgar score, or nucleated red blood cell value. In a logistic regression model, increasing nucleated red blood cell counts did not increase the odds of an abnormal outcome at 30 months of age (OR 1.02, P = 0.17). In a population of neonates with perinatal depression, the nucleated red blood cell count at birth does not correlate with magnetic resonance spectroscopy or 30-month neurodevelopmental outcome. The nucleated red blood cell count should not be used as a surrogate marker for subsequent brain injury.     

Word length modulates neural activity in auditory cortex during covert object naming

Several lines of evidence show that posterior portions of left auditory cortex participate in aspects of speech production. A current hypothesis is that these regions play a specific role in processing phonological codes. We used event-related fMRI to test this hypothesis. Subjects covertly named objects that had names varying in length from one to four syllables. Behavioral data, both in previous work and in the present study, show an increase in naming reaction time as words get longer, reflecting the increase in phonological load. If left posterior auditory areas participate in phonological aspects of production, word length should modulate neural activity in this region. Object naming activated the left planum temporale in each subject, confirming previous findings of auditory cortex involvement in speech production. The timecourse of activation in this region showed a length effect, consistent with its hypothesized role in phonological processing. Similar effects were also observed in premotor cortex, but not in occipital-temporal cortex, which presumably supports visual processing of the pictures to be named.     

Lipid rafts mediate the interaction between myelin-associated glycoprotein (MAG) on myelin and MAG-receptors on neurons

The interaction between myelin-associated glycoprotein (MAG), expressed at the periaxonal membrane of myelin, and receptors on neurons initiates a bidirectional signalling system that results in inhibition of neurite outgrowth and maintenance of myelin integrity. We show that this involves a lipid-raft to lipid-raft interaction on opposing cell membranes. MAG is exclusively located in low buoyancy Lubrol WX-insoluble membrane fractions isolated from whole brain, primary oligodendrocytes, or MAG-expressing CHO cells. Localisation within these domains is dependent on cellular cholesterol and occurs following terminal glycosylation in the trans-Golgi network, characteristics of association with lipid rafts. Furthermore, a recombinant form of MAG interacts specifically with lipid-raft fractions from whole brain and cultured cerebellar granule cells, containing functional MAG receptors GT1b and Nogo-66 receptor and molecules required for transduction of signal from MAG into neurons. The localisation of both MAG and MAG receptors within lipid rafts on the surface of opposing cells may create discrete areas of high avidity multivalent interaction, known to be critical for signalling into both cell types. Localisation within lipid rafts may provide a molecular environment that facilitates the interaction between MAG and multiple receptors and also between MAG ligands and molecules involved in signal transduction.     

Structural differentiation of self-reported depression and anxiety in late life

Research has shown impressive support for tripartite models of anxiety and depression that include a common factor of negative affect, and the unique factors positive affect and arousal. It is not clear whether this structure extends into later life. The current study used confirmatory factor analysis to model the structural relationship of anxiety and depression in two samples of older adults: a large probability sample (N = 1429) and a smaller convenience sample (N = 210). Across all analyses, a correlated, two-factor, psychometric model was most parsimonious. The tripartite model could be fit to the data, but added no explanatory power; in some cases a one-factor model also fit. The results suggest that there is a unitary factor of "distress" that incorporates anxiety and depression, but that the structure is not consistent with factor structures found in younger samples. Instead, the broad constructs may be represented in a more complex manner among older adults, and are less easily differentiated.     

Treating hypertension in diabetic nephropathy

OBJECTIVE: Control of hypertension in patients with diabetic nephropathy improves mortality and slows progression to end-stage renal disease. However, blood pressure is difficult to treat; multiple drug combination therapy is required and treatment algorithms to establish this are lacking. We used a stepped-care algorithm, centered on maximum doses of an ACE inhibitor or angiotensin II receptor blocker, to treat hypertension according to American Diabetes Association recommended blood pressure target goals (<130/80 mmHg) in patients with diabetic nephropathy. RESEARCH DESIGN AND METHODS: We treated 49 consecutive patients with diabetes (13 with type 1 and 36 with type 2), diabetic nephropathy, and proteinuria > or =500 mg/24 h with a stepped-care blood pressure treatment algorithm. The level of blood pressure control achieved at most recent follow-up was assessed. RESULTS: Patients were followed for a median of 18 months (range 9-48). Mean blood pressure achieved was 140/75 +/- 23/14 mmHg in patients with type 1 diabetes and 146/76 +/- 22/14 mmHg in patients with type 2 diabetes. Target blood pressure was reached in 16 (33%) patients, 6 of 13 patients with type 1 diabetes and 10 of 36 patients with type 2 diabetes, whereas systolic blood pressure remained above the target level in the remaining patients. There was no difference in baseline blood pressure, proteinuria, or serum creatinine level between patients who were treated to target and those who were not. CONCLUSIONS: Levels of blood pressure control similar to those achieved in clinical trials in diabetic nephropathy were obtained with a stepped-care algorithm. However, in most patients, systolic blood pressure was difficult to control to target despite the use of multiple drug combination therapy.