Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.     

Body mass index correlates with waist circumference in school aged Italian children

This study demonstrates the existence of a linear correlation between Body Mass Index (BMI) and waist circumference in Italian school aged children and suggests an indirect method (from weight and height) to estimate waist circumference, whose increase may be indicative for the diagnosis of the metabolic syndrome.     

Cyclodextrin Protects Podocytes in Diabetic Kidney Disease

Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD⁺) when compared with diabetic patients with normoalbuminuria (DKD⁻) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.Diabetic kidney disease (DKD) is responsible for nearly half of the incidents of end-stage kidney disease in the U.S. (1), yet our current understanding of the pathophysiological processes responsible for DKD has led to limited improvements in patient outcomes. Multifactorial intervention reduces the rate of progression of DKD but does not prevent end-stage kidney disease in type 1 (T1D) or type 2 diabetes (T2D) (2,3). A key factor for this translation gap is the current lack of adequate mechanistic insight into DKD in humans.The kidney glomerulus is a highly specialized structure that ensures the selective ultrafiltration of plasma so that essential proteins are retained in the blood (4). Podocytes are glomerular epithelial cells that contribute to the glomerular filtration barrier through a tight regulation of actin cytoskeleton remodeling (4). Currently, the diagnosis of DKD relies on the detection of microalbuminuria (5). However, a growing body of evidence suggests that key histological lesions precede the development of albuminuria (6,7); among them, decreased podocyte number (podocytopenia) has been described as an independent predictor of DKD progression (8–12). Although we have previously shown that podocyte insulin resistance and susceptibility to apoptosis is already present at the time of onset of microalbuminuria in experimental models of DKD, the cause of podocyte injury in early DKD remains unknown (13).We used a previously established cell-based assay in which differentiated human podocytes are exposed to 4% patient sera for 24 h (14) to identify new pathways and targets in DKD. Podocytes exposed to the sera of patients with DKD showed increased cholesterol accumulation in association with downregulation of ATP-binding cassette transporter 1 (ABCA1) expression that was independent of circulating cholesterol.ABCA1 is a major regulator of cellular cholesterol homeostasis by mediating efflux to lipid-poor apolipoprotein acceptors in the bloodstream (15). ABCA1 genetic variants are strongly associated with the risk of coronary artery disease (16). Furthermore, the capacity of patient sera to induce ABCA1-mediated cholesterol efflux in macrophages is impaired in patients with T2D and incipient or overt nephropathy (17). Excessive cholesterol accumulation has been described in glomeruli of rodent models of T1D and T2D (18–20) and may contribute to DKD development and progression. Finally, inactivating mutations of ABCA1 result in Tangier disease, which causes premature atherosclerosis and proteinuria (21).Although interventions that increase ABCA1 expression (such as liver X receptor agonists) may be beneficial in DKD, they have a relatively high incidence of adverse events (22) as well as intrinsic lipogenic effects (23). We used β-cyclodextrins, cyclic oligosaccharides consisting of seven β(1-4)-glucopyranose rings, to remove cholesterol from differentiated human podocytes in vitro and from diabetic animals in vivo. The exact mechanism by which cyclodextrins (CDs) remove cholesterol from cells is not completely understood, but the formation of cholesterol/CD inclusion complexes at the membrane surface plays an important role in this process (24).We hypothesized that 2-hydroxypropyl-β-cyclodextrin, which was recently approved by the U.S. Food and Drug Administration (FDA) for the cure of Niemann-Pick disorder (25,26), would be an effective way to sequester cholesterol and to protect podocytes from cholesterol-dependent damage in DKD in vivo and in vitro.     

Transmission of resistant HIV type 1 variants and epidemiological chains in Italian newly diagnosed individuals

Transmission of HIV-1 and drug resistance continue to occur at a considerable level in Italy, influenced mainly by changes in modality of infection. However, the long period of infectivity makes difficult the interpretation of epidemiological networks, based on epidemiological data only. We studied 510 naive HIV-1-infected individuals, of whom 400 (78.4%) were newly diagnosed patients with an unknown duration of infection (NDs), with the aim of identifying sexual epidemiological networks and transmitted drug resistance (TDR) over a 7-year period. Clusters were identified by Bayesian methods for 412 patients with B subtype; 145 individuals (35.2%) clustered in 34 distinct clades. Within epidemiological networks males were 93.1% (n=135); the same proportion of patients has been infected by the sexual route; 62.1% (n=90) were men having sex with men (MSM) of whom 67.8% (n=61) were NDs. Among heterosexuals (n=44), males were predominant (79.5%, n=35) and 77.3% (n=34) were NDs. TDR in clusters was 11.7 % (n=17), of whom 76.5% (n=13) was found in MSM. TDR was predominantly associated with NRTI resistance in individuals with chronic infection (n=11). A high prevalence of epidemiological networks has been found in the metropolitan area of Milan, indicating a high frequency of transmission events. The cluster analysis of networks suggested that the source of new infections was mainly represented by males and MSM who have long lasting HIV-1 infection. Notably, the prevalence of resistance-conferring mutations was higher in chronically infected patients, carrying mainly resistance to thymidine analogs, the backbone of first antiretroviral (ARV) generation. Intervention strategies of public health are needed to limit HIV-1 transmission and the associated TDR.     

Combination of low doses of Enzastaurin and Lenalidomide has synergistic activity in B-non-Hodgkin lymphoma cell lines

Less toxic and more active treatments are needed for indolent lymphomas as there is no curative treatment, and patients eventually die due to complications related to their disease. The purpose of the present study was to assess the antitumour activity of the combination of low doses of Enzastaurin and Lenalidomide (Revlimid) on B-lymphoma cell lines. The combination of Enzastaurin and Lenalidomide, at doses as low as 1 μM, showed strong synergism against indolent lymphomas by reducing cell growth, producing an increase in G0-G1 phase followed by significant decrease in S phase, increasing apoptosis, and inhibiting PI3K/AKT, PKC and MAPK/ERK pathways. These preclinical findings, together with promising results obtained with Lenalidomide for the treatment of non-Hodgkin lymphoma, suggest that further evaluation of the combination of Enzastaurin and Lenalidomide for the treatment of indolent lymphomas is warranted. These compounds, with a favourable toxicity profile, are not classic chemotherapeutic agents, causing severe side effects, and could be considered an example of a new innovative attempt of an anti-cancer 'soft treatment'.     

Influence of upper airway size on volume exhaled under negative pressure during evaluation of upper airway collapsibility

Study objectives  

It has been shown that volume exhaled in the first 0.5 s after application at the mouth of 5 cmH₂O negative pressure (V,NEP_0.5) during wakefulness strongly reflects critical pressure (Pcrit) during sleep but only in males with neck circumference (NC) >37 cm. The aim of this study was to establish the relationship between upper airway (UA) size and V,NEP_0.5, to obtain V,NEP_0.5 values as percent predicted and then correlate them with Pcrit obtained in the same subjects.     

Evaluation of corticospinal excitability in cervical myelopathy, before and after surgery, with transcranial magnetic stimulation: a pilot study

Purpose

A pilot study to examine the impact of cervical myelopathy on corticospinal excitability, using transcranial magnetic stimulation, and to investigate whether motor evoked potential (MEP) and silent period (SP) recruitment curve (RC) parameters can detect changes in corticospinal function pre- and post-surgery.

Methods

We studied six cervical myelopathy patients undergoing surgery and six healthy controls. Clinical and functional scores and neurophysiological parameters were examined prior to and 3 months following the surgery.

Results

MEP latencies for abductor pollicis brevis (APB) and tibialis anterior (TA) muscles and central motor conduction time were prolonged pre- and post-surgery; SP durations were differentially altered. There were significant differences in parameters of RCs for (1) MEP area in APB (max values, S50) and TA (slope) between controls and patients pre- and post-surgery and (2) SP duration in APB (max values) between patients pre-surgery and controls.

Conclusions

The findings of this pilot study suggest an uncoupling of excitatory and inhibitory pathways, which persists at 3 months following cord decompression. RCs for MEP and SP at 3 months provide more information on the functional status of the cord and prompts for a longer term follow-up.