Immunophenotypic characterization of reactive and neoplastic plasmacytoid dendritic cells permits establishment of a ten-color flow cytometric panel for initial workup and residual disease evaluation of blastic plasmacytoid dendritic cell neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematopoietic neoplasm whose immunophenotype remains incompletely characterized, particularly with regards to its distinction from reactive plasmacytoid dendritic cells (PDC). This limitation complicates detection of low-level involvement by BPDCN as well as minimal residual disease (MRD) assessment following therapy. We conducted the current study to characterize the immunophenotype of BPDCN in a cohort of 39 patients, and compared it to that of reactive PDC. We found that, in addition to CD56 expression (97%), BPDCN showed a number of aberrancies, including decreased/negative CD38 (82%), positive CD7 (64%), negative CD2 (81%), negative CD303 (56%), increased HLA-DR (69%) and decreased CD123 (78%) expression. Although BPDCN cells were characterized by CD56 expression, reactive PDC consistently included a CD56⁺ subset, ranging from 1.3%-20% (median 4.5%) of all PDC, challenging the detection of MRD. These CD56⁺ reactive PDC were, however, consistently positive for CD2 and CD303, brightly positive for CD38, and negative for CD7, distinctively different from BPDCN. Based on these findings, we set up a ten-color flow cytometry assay for BPDCN and validated it to a sensitivity of 0.01%. This panel was prospectively tested in 19 bone marrow samples from seven patients with BPDCN, and it effectively distinguished BPDCN cells from background reactive PDC in all cases. In summary, by understanding the immunophenotype of reactive and neoplastic PDC, BPDCN can be effectively detected by flow cytometry to a very low level using a panel of markers in addition to CD56. Such an assay could be used for initial bone marrow workup as well as MRD detection after therapy.     

The translational importance of establishing biomarkers of human spinal cord injury

The evaluation of such novel therapies for acute spinal cord injury in clinical trials is extremely challenging. Our current dependence upon the clinical assessment of neurologic impairment renders many acute SCI patients ineligible for trials because they are not examinable. Furthermore, the difficulty in predicting neu-rologic recovery based on the early clinical assessment forces investigators to recruit large cohorts to have sufficient power. Biomarkers that objectively classify injury severity and better predict neurologic outcome would be valuable tools for translational research. As such, the objective of the present review was to de-scribe some of the translational challenges in acute spinal cord injury research and examine the potential utility of neurochemical biomarkers found within cerebrospinal fluid and blood. We focus on published efforts to establish biological markers for accurately classifying injury severity and precisely predict neuro-logical outcome.     

Routine sequencing in CLL has prognostic implications and provides new insight into pathogenesis and targeted treatments

Chronic lymphocytic leukaemia (CLL) is a genetically heterogeneous disease characterised by genomic alterations and gene mutations that may portend worse survival or resistance to treatments. A total of 680 blood or bone marrow samples underwent targeted sequencing of 29 genes previously identified as being mutated in CLL, which were correlated to known prognostic clinical characteristics. Overall, 400 (59%) patients were treatment-naïve (TN) and 280 (41%) were relapsed/refractory (R/R). Most patients (70%) had ≥1 mutation, with TP53 (22%), SF3B1 (18%), NOTCH1 (13%) and ATM (13%) being the most commonly mutated genes. A higher proportion of R/R patients had mutations in SF3B1 (P = 0·01) and TP53 (P < 0·001). Patients with mutated IGHV CLL more often had mutations in KLHL6 (P = 0·001) and MYD88 (P < 0·001). Pairwise associations showed mutational co-occurrences in the TN group including SF3B1/ATM [false discovery rate (FDR) < 0·05] and NOTCH1/POT1 (FDR < 0·01). Recurrent mutations resulting in premature truncation prior to the ubiquitination domains of NOTCH1 in its PEST domain and BIRC3 in its RING domain can produce proteins that constitutively activate CLL. Frequent missense mutations, such as K700E in SF3B1 and E571K in XPO1, have unknown function but are most likely to be activating mutations. Future directions include using these mutations to identify pathways for therapeutic targeting and rational drug design.     

Analyses of the facilitatory effect of orexin on eating and masticatory muscle activity in rats

The orexins (orexin-A and orexin-B) are neuropeptides that are secreted from neurons in the lateral hypothalamus and that participate in the regulation of feeding behavior. It remains to be determined, however, how the orexins exert their effects on feeding behavior, including masticatory movements. To this end, we analyzed food intake behavior and masticatory muscle activity using video analysis and electromyography (EMG) recording methods. The results showed that the cumulative food intake over 4 h was larger in rats intraventricularly injected with either orexin-A or orexin-B than in saline-injected control rats. The latency to eating and the feeding time for a fixed amount of pellets were shortened by injections of orexins in a dose-dependent manner, with a more potent effect by orexin-A than orexin-B. The shorter feeding time corresponded to a decreased number of chewing cycles. EMG recordings from both the digastric and masseter muscles showed two distinct patterns of bursts corresponding to the gnawing and chewing phases. After the injection of orexin-A, the magnitude of the bursts became larger in both phases in the masseter muscle, the burst duration became longer in the chewing phase in the masseter muscle, and the interburst interval became shortened in the gnawing phase in both muscles. Consequently, the burst frequency in the chewing phase was increased in the digastric muscle and, conversely, reduced in the masseter muscle. These results suggest that the orexin-A-induced facilitatory feeding behavior is characterized by a dynamic jaw-opener activity that opens the mouth rapidly and a powerful jaw-closer activity for crushing the increased amount of food taken into the mouth. The possible involvement of orexin-A in binge eating disorder is discussed.     

Preparation of optimized Naproxen nano liposomes using response surface methodology

The aim of this study was to prepare and optimize a liposomal delivery system for Naproxen (NPX), a practically water insoluble drug. NPX liposomes, which were consisted of cholesterol (CH) and phosphatidylcholine (PC) in different molar ratios, were prepared by modified ethanol injection method and optimized employing response surface methodology (RSM). Proportion of PC:CH and total lipid:drug ratio were selected as the independent variables while the particle size (PS), encapsulation efficiency percent (EE%) and drug release of the liposomes over 24 h (D_24h) were considered as dependent variables. The effects of PC:CH and total lipid:drug ratios were studied and optimized to obtain the liposomal vesicles with desired quality. Graphical response surface and contour plots were also employed to understand the interaction of different variables. The optimum points for the variables were obtained from the optimization plot. The mean PS, EE% and D_24h of NPX liposomes were about 178.11 nm, 53.14 and 46.62 % respectively. The results indicated that PC:CH and total lipids:drug ratios were the major contributing variables for EE% and D_24h. However, only PC:CH ratio was the main contributing variable for PS. The optimum formulation of NPX liposomes, in which PC:CH ratio and lipids:drug ratio were 3.81 and 2.98 respectively, had high EE% (58 %) and D_24h (>50 %) as well as appropriate PS (<162.4 nm). Ethanol injection method besides RSM, are simple, rapid and beneficial approaches for liposome preparation and optimization.     

Characterization of lipid parameters in diabetic and non-diabetic atherosclerotic patients

Background & Objective The relationship between lipid profile perturbation and diabetes associated complications has long been an area of interest. Dyslipidemia is a potent predictor of cardiovascular morbidity and mortality in diabetic patients. The aim of present study was to investigate relationship between aging and lipid profiles in diabetic and non-diabetic atherosclerotic patients. Methods Five hundred and seventy six individuals (45–75 year age) participated in this study. Among these, 192 were having history of diabetes mellitus and atherosclerosis. Individuals are categorized on the base of health (normal, non-diabetic atherosclerosis, diabetic atherosclerosis) and age (45–55 years, 56–65 years, and 66–75 years). All the participants were subjected to the procedures like a detailed history, biochemical analysis for fasting blood sugar, hemoglobin A1c, total cholesterol (TC), triglycerides (TG), low-density lipoprotein-(LDL), very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL). All these parameters were compared between diabetic and non-diabetic atherosclerotic patients of all three age groups. TC/HDL and LDL/HDL were also calculated. Results Diabetic atherosclerotic individuals (both males and females) had high level of TC, TG, LDL, VLDL and low level of HDL in comparison to non-diabetic atherosclerotic and normal control individuals. Among all three age groups, lipoprotein abnormality was observed to be more frequent in females than males. There was a significant increase in TC/HDL and LDL/HDL ratio in diabetic atherosclerotic subjects compared to age and sex matched non-diabetic atherosclerotic and normal control. Conclusions Degree of dyslipidemia increases with increase in age in both genders. Female are more prone to diabetic dyslipidemia and hence have more risk of developing atherosclerosis with increasing age.