Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

Common genetic variants do not associate with CAD in familial hypercholesterolemia

In recent years, multiple loci dispersed on the genome have been shown to be associated with coronary artery disease (CAD). We investigated whether these common genetic variants also hold value for CAD prediction in a large cohort of patients with familial hypercholesterolemia (FH). We genotyped a total of 41 single-nucleotide polymorphisms (SNPs) in 1701 FH patients, of whom 482 patients (28.3%) had at least one coronary event during an average follow up of 66 years. The association of each SNP with event-free survival time was calculated with a Cox proportional hazard model. In the cardiovascular disease risk factor adjusted analysis, the most significant SNP was rs1122608:G>T in the SMARCA4 gene near the LDL-receptor (LDLR) gene, with a hazard ratio for CAD risk of 0.74 (95% CI 0.49–0.99; P-value 0.021). However, none of the SNPs reached the Bonferroni threshold. Of all the known CAD loci analyzed, the SMARCA4 locus near the LDLR had the strongest negative association with CAD in this high-risk FH cohort. The effect is contrary to what was expected. None of the other loci showed association with CAD.     

Clinical approaches to non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD)ranges from simple steatosis to nonalcoholic steatohepatitis(NASH),leading to fibrosis and potentially cirrhosis,and it is one of the most common causes of liver disease worldwide.NAFLD is associated with other medical conditions such as metabolic syndrome,obesity,cardiovascular disease and diabetes.NASH can only be diagnosed through liver biopsy,but noninvasive techniques have been developed to identify patients who are most likely to have NASH or fibrosis,reducing the need for liver biopsy and risk to patients.Disease progression varies between individuals and is linked to a number of risk factors.Mechanisms involved in the pathogenesis are associated with diet and lifestyle,influx of free fatty acids to the liver from adipose tissue due to insulin resistance,hepatic oxidative stress,cytokines production,reduced very low-density lipoprotein secretion and intestinal microbiome.Weight loss through improved diet and increased physical activity has been the cornerstone therapy of NAFLD.Recent therapies such as pioglitazone and vitamin E have been shown to be beneficial.Omega 3 polyunsaturated fatty acids and statins may offer additional benefits.Bariatric surgery should be considered in morbidly obese patients.More research is needed to assess the impact of these treatments on a long-term basis.The objective of this article is to briefly review the diagnosis,management and treatment of this disease in order to aid clinicians in managing these patients.     

Comparing human and mouse salivary glands: A practice guide for salivary researchers

Mice are a widely utilized in vivo model for translational salivary gland research but must be used with caution. Specifically, mouse salivary glands are similar in many ways to human salivary glands (i.e., in terms of their anatomy, histology, and physiology) and are both readily available and relatively easy and affordable to maintain. However, there are some significant differences between the two organisms, and by extension, the salivary glands derived from them must be taken into account for translational studies. The current review details pertinent similarities and differences between human and mouse salivary glands and offers practical guidelines for using both for research purposes.     

Cytokine activation during attacks of the hyperimmunoglobulinemia D and periodic fever syndrome

The hyperimmunoglobulinemia D and periodic fever (hyper-IgD) syndrome is typified by recurrent febrile attacks with abdominal distress, joint involvement (arthralgias/arthritis), headache, skin lesions, and an elevated serum IgD level (> 100 U/mL). This familial disorder has been diagnosed in 59 patients, mainly from Europe. The pathogenesis of this febrile disorder is unknown, but attacks are joined by an acute-phase response. Because this response is considered to be mediated by cytokines, we measured the acute-phase proteins C-reactive protein (CRP) and soluble type-II phospholipase A2 (PLA2) together with circulating concentrations and ex vivo production of the proinflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, and tumor necrosis factor alpha (TNF alpha) and the inhibitory compounds IL-1 receptor antagonist (IL-1ra), IL-10, and the soluble TNF receptors p55 (sTNFr p55) and p75 (sTNFr p75) in 22 patients with the hyper-IgD syndrome during attacks and remission. Serum CRP and PLA2 concentrations were elevated during attacks (mean, 213 mg/L and 1,452 ng/mL, respectively) and decreased between attacks. Plasma concentrations of IL-1 alpha, IL-1 beta, or IL-10 were not increased during attacks. TNF alpha concentrations were slightly, but significantly, higher with attacks (104 v 117 pg/mL). Circulating IL-6 values increased with attacks (19.7 v 147.9 pg/mL) and correlated with CRP and PLA2 values during the febrile attacks. The values of the antiinflammatory compounds IL-1ra, sTNFr p55, and sTNFr p75 were significantly higher with attacks than between attacks, and there was a significant positive correlation between each. The ex-vivo production of TNF alpha, IL-1 beta, and IL-1ra was significantly higher with attacks, suggesting that the monocytes/macrophages were already primed in vivo to produce increased amounts of these cytokines. These findings point to an activation of the cytokine network, and this suggests that these inflammatory mediators may contribute to the symptoms of the hyper-IgD syndrome.     

T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28

To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross- linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI- MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T- cell deficiency in patients with Hodgkin's disease.