Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.

MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes.

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor- and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)-mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells.

The regulation of interferon type I pathway-related genes RSAD2 and ETV7 specifically indicates antibody-mediated rejection after kidney transplantation.

CONTEXT: Antibody-mediated rejection (ABMR) after kidney transplantation (KTx) remains the crucial obstacle to successful long-term graft function. The identification of gene signatures involved in ABMR could grant the basis for better prevention and treatment strategies. OBJECTIVE: The identification of gene signatures in whole blood cells specific for ABMR after KTx. MATERIALS AND METHODS: Total RNA from blood cells of 16 kidney-transplanted patients with ABMR, stable graft function (SGF), and with T-cell-mediated rejection (TCMR) was isolated. Gene expression was determined by high-throughput sequencing followed by validation and analyses of differentially expressed candidates on mRNA level and on protein level in a large patient cohort (n = 185) in patients with SGF, urinary tract infection (UTI), borderline rejection (BL), TCMR, ABMR, and interstitial fibrosis and tubular atrophy. RESULTS: From the 570 genes detected, 111 discriminated ABMR from SGF and TCMR. A distinct enrichment of interferon (IFN) type I and type II signature gene set was observed. The expression of candidate genes IFIT1, ETV7, and RSAD2 distinguished ABMR patients from patients with SGF and also TCMR, whereas ETV7 and RSAD2 differentiated ABMR also from BL. CONCLUSION: The IFN-inducible genes ETV7 and RSAD2 represent specific biomarkers for ABMR episodes after KTx.

Evaluation of the Intestinal Colonizing Potential and Immunomodulating Capacity of Lactobacilli Microspheres.

Lactobacilli species get degraded by acidic conditions in the stomach. Thus, the objective of this study was to (1) formulate and characterize gastro-resistant Lactobacilli microspheres and (2) evaluate the ability of Lactobacilli microspheres to colonize the intestine and their capacity to have an immunomodulating effect in vivo. The product yield and the encapsulation efficiency were 45% and 100%, respectively. The average microsphere particle size was 5 µm. Lactobacilli microspheres were most stable at 4°C and showed a better suspendibility in distilled water. Without encapsulation, the viability of bacteria decreased within 30 min. In the case of Lactobacilli microspheres, no Lactobacilli were released in the first 3 h, and highest release was observed at 4 h, thus, suggesting the significance of encapsulation of Lactobacilli. Lactobacilli microspheres maintained intestinal colonization only during the dosing period, and the serum IgG, serum IgA, fecal, intestinal, nasal IgA, and the serum interleukin-1ß levels were higher in the Lactobacilli microsphere group compared with the blank microsphere and the lactobacilli solution group, suggesting that the Lactobacilli microspheres were more gastro-resistant and, hence, showed positive effects compared with the Lactobacilli solution. However, the Lactobacilli microspheres did not have a significant effect on the tumor necrosis factor-α levels.

Solid Matrix Based Lipidic Nanoparticles in Oral Cancer Chemotherapy: Applications and Pharmacokinetics.

Chemotherapeutic delivery by oral route in cancer patients has the potential to create "hospitalization free chemotherapy" which is a vision of oncologists, formulation scientists and patients. Such a therapeutic approach will improve patients' compliance, ease the burden of the patients' caregivers and significantly reduce the cost of treatment. In current clinical practice, chemotherapy carried out by intravenous injection or infusion leads to undesired side-effects such as plasma concentrations crossing the maximum safe concentration, rapid body clearance and lower bioavailability. Despite the presence of challenges such as poor aqueous solubility and stability of drugs and the presence of biological barriers like multidrug efflux transporter in the GI tract, oral cancer chemotherapy has the potential to surmount those obstacles. Lipid nanoparticles (LNPs) such as solid lipid nanoparticle, nanostructured lipid carriers, nano lipid-drug conjugates, mixed micelles, liposomes and nanoemulsions have shown some promising results for use in oral anticancer drug delivery through nanotechnological approach. LNPs demonstrate enhanced oral bioavailability owing to their ability to inhibit first pass metabolism via lymphatic absorption by chylomicron-linked and/or M-cell uptake. LNPs reduce the inter- and intrasubject pharmacokinetics variability of administrated drugs. Moreover, certain classes of phospholipids and surfactants used in the formulations of LNPs can suppress the P-glycoprotein efflux system. Here, we shall be discussing the biopharmaceutical challenges in oral cancer chemotherapy and how the LNPs may provide solutions to such challenges. The effect of GI tract environment on LNPs and pharmacokinetics shall also be discussed.

Emerging advances in cancer nanotheranostics with graphene nanocomposites: opportunities and challenges.

As an inorganic nanomaterial, graphene nanocomposites have gained much attention in cancer nanotechnology compared with the other inorganic nanomaterial in recent times. Although a relatively new drug carrier, it has been extensively explored as a potential chemotherapeutic carrier and theranostic because of its numerous physicochemical properties, including, capability of multiple pay load, functionalization for drug targeting and photothermal effect. Despite potential benefit, its translation from bench to bed-side in cancer therapy is challenged due to its toxicity concern. Here, we discussed the present progress and future possibilities of graphene nanocomposites as a cancer theranostic. Moreover, the paper also exemplifies the effects of graphene/graphene oxide on tissues and organ functions in order to understand the extent and mechanism of toxicity.

The effect of antisense to NF-κB in an albumin microsphere formulation on the progression of left-ventricular remodeling associated with chronic volume overload in rats.

BACKGROUND: Increased NF-κB levels play a crucial role in the pathophysiology of heart failure and are known to cause ventricular remodeling. Antisense therapy can be used for blocking the expression of NF-κB and subsequently avoiding heart failure. However, as with most biotechnology products, molecular instability and overall cost are often the major issues and concerns limiting the advancement of most antisense drugs to the market. Therefore, a cost-efficient biodegradable sustained release particle drug delivery system to transport and target NF-kB antisense to its intended site of action would be ideal. PURPOSE: To evaluate the in vivo performance of a sustained release spray-dried albumin microsphere formulation for effective delivery and treatment of left ventricular remodeling with antisense to NF-κB. METHODS: Albumin-based microspheres encapsulating antisense to NF-kB were prepared by spray drying and studied in a rat model to treat congestive heart failure. RESULTS: The NF-κB activation and TNF-α release seen in treated animals were significantly lower than control animals. Ventricular remodeling was controlled in animals with antisense-treated AV fistulas as ΔV0-25 and ΔV0 were significantly lower compared to animals with untreated AV fistulas. CONCLUSION: This treatment was successful in curbing ventricular remodeling by suppressing NF-κB activation.

In vitro and ex vivo characterization of lectin-labeled Mycobacterium tuberculosis antigen-containing microspheres for enhanced oral delivery.

PURPOSE: Oral immunization for mucosal protection against Mycobacterium tuberculosis would be the best option for effective tuberculosis (TB) control. However, this route of vaccine delivery is limited due to the short residence time of the delivery system at the site of absorption. Cytoadhension has made it possible to optimize the targeted delivery of oral vaccine to lymphoid tissues. The purpose of this project was to evaluate the ability of human M-cell specific lectin-labeled microparticles to target the human M-cells of the Peyer's patches. METHOD: Albumin microspheres containing Mycobacterium tuberculosis cell lysate antigens were coupled with Wheat germ agglutinin and Aleuria aurantia lectins and their ability to bind to M cell models as well as their preferential distribution in the Peyer's patches were investigated. RESULTS: The study demonstrated an enhanced delivery of targeted polystyrene and BSA/Lysate microspheres to M cells. It was demonstrated that alpha-l-fucose sugar residue might be the target of these lectins. CONCLUSION: It can be concluded from the study that the lectin-coupled microspheres had better affinity for M-cells and showed preferential binding to the Peyer's patches. This means that the coupling enhanced the targeted delivery of the antigens to the M cells.

Spray-dried doxorubicin-albumin microparticulate systems for treatment of multidrug resistant melanomas.

As multidrug resistance continues to be a problem in cancer treatment, controlled release delivery systems, such as microspheres, may aid to give a slower release of anticancer drugs into drug resistant tumor cells. In this study doxorubicin microspheres microencapsulated in an albumin matrix were prepared via the spray-drying method and characterized for particle size, content analysis, and release studies. They were then evaluated in vitro using drug resistant murine melanoma tumor cells for uptake and efflux studies. Spray-drying produced a dispersed powder with a mean particle size of 4.91 ± 1.2 µm, 60% product yield, and encapsulation efficiency of 85% and a ζ potential range of 37 to -40 mV. Intracellular doxorubicin concentrations were higher in drug resistant tumor cells treated with microspheres as opposed to solution, and efflux of doxorubicin from the tumor cell was inhibited. Greater cytotoxic effects were seen in tumor cells treated with doxorubicin microspheres versus solution up to and after 3 days. In vivo pharmacokinetic studies conducted in male Sprague-Dawley rats, revealed a plasma-level time curve indicative of a two-compartment model, and showed prolonged half-life of doxorubicin, greater area under the plasma concentration time curve, and increased plasma concentrations of doxorubicin in rats at 8 and 24 h after administration of doxorubicin microspheres.

Formulation, characterization and testing of tetracaine hydrochloride-loaded albumin-chitosan microparticles for ocular drug delivery.

Most ocular surgical procedures take approximately 60 min to complete, the anaesthetic property of the safest drug, tetracaine, is initiated in a few minutes and lasts approximately 10-15 min. The purpose of the present study was to develop an ocular tetracaine formulation which can produce an immediate onset of action and/or longer duration of action during the entire surgical procedure. Tetracaine-loaded microparticle formulation was prepared by the method of spray-drying and characterized in terms of size, zeta potential, morphology, thermal stability and release pattern. The study reports a microparticulate ocular formulation with minimum cytotoxicity and optimum cellular uptake. In addition, microencapsulated tetracaine was found to significantly increase the duration of action of the drug up to 4-fold. Taken together, the results presented in this work described albumin-chitosan microparticles to be an effective delivery platform for ocular anaesthetic agents and a potential treatment of various ocular diseases.

The effect of intracellular delivery of catalase and antisense oligonucleotides to NF-kappaB using albumin microcapsules in the endotoxic shock model.

UNLABELLED: Microencapsulated (MC) catalase has been shown to inhibit H(2)O(2) and tumor necrosis factor (TNF) in vitro after endotoxin stimulation. It is the purpose of this study to determine whether MC catalase improves pro-inflammatory cytokine inhibition and mortality in an endotoxic shock model in vivo. We also examined whether MC catalase and antisense oligonucleotides (ASO) to nuclear factor kappaB (NF-kappaB) together improved survival by inhibiting pro-inflammatory cytokines using different mechanisms. METHODS: Albumin microcapsules containing catalase and ASO to NF-kappaB were prepared 2-7 microm in size by using a Büchi spray dryer. Progressively increasing doses of MC catalase, MC ASO to NF-kappaB, and the combination were given to rats before the administration of Escherichia coli endotoxin. Results demonstrated 60% survival in rats given 15 mg/kg MC catalase, 70% survival with 20 mg/kg MC ASO NF-kappaB, and 80% survival with the combination. TNF was inhibited by 53% in the MC catalase group 4 h after endotoxin administration, 43% in the ASO NF-kappaB group, and 78% in the combination group compared to controls. In conclusion, this study demonstrates the effectiveness of MC intracellular delivery of the naturally occurring antioxidant catalase in improving animal survival. The addition of ASO to NF-kappaB improved both cytokine inhibition and animal survival in endotoxic shock.