A highly efficient and safe gene delivery platform based on polyelectrolyte core-shell nanoparticles for hard-to-transfect clinically relevant cell types.

While DNA and messenger RNA (mRNA) based therapies are currently changing the biomedical field, the delivery of genetic materials remains the key problem preventing the wide introduction of these methods into clinical practice. Therefore, the creation of new methods for intracellular gene delivery, particularly to hard-to-transfect, clinically relevant cell populations is a pressing issue. Here, we report on the design of a novel approach to format 50-150 nm calcium carbonate particles in the vaterite state and using them as a template for polymeric core-shell nanoparticles. We apply such core-shell nanoparticles as safe and efficient carriers for mRNA and pDNA. We prove that such nanocarriers are actively internalized by up to 99% of primary T-lymphocytes and exert minimal toxicity with the viability of >90%. We demonstrate that these nanocarriers mediate more efficient transfection compared with the standard electroporation method (90% vs. 51% for mRNA and 62% vs. 39% for plasmid DNA) in primary human T-lymphocytes as a model of the hard to transfect type that is widely used in gene and cell therapy approaches. Importantly, these polymeric nanocarriers can be used in serum containing basic culture medium without special conditions and equipment, thus having potential for being introduced in clinical development. As a result, we have provided proof-of-principle that our nanosized containers represent a promising universal non-viral platform for efficient and safe gene delivery.

Mucormycosis in haematological patients: case report and results of prospective study in Saint Petersburg, Russia.

We prospectively observed 36 haematological patients with mucormycosis from nine hospitals of St. Petersburg during 2004-2013. The most frequent underlying diseases were acute leukaemia (64%), and main risk factors were prolonged neutropenia (92%) and lymphocytopenia (86%). In 50% of the patients, mucormycosis was diagnosed 1-65 days after invasive aspergillosis. Main clinical form of mucormycosis was pulmonary (64%), while two or more organ involvement was noted in 50% of the cases. The most frequent aetiological agents of mucormycosis were Rhizopus spp. (48%). Twelve-week survival rate was 50%. Combination therapy (echinocandins + amphotericin B forms) and recovery from the underlying disease significantly improved the survival rate.

Effect of FCGR2A and FCGR3A variants on CLL outcome.

Polymorphisms of activating Fc-γ receptors (FCGRs) on natural killer cells and macrophages result in variable affinity for immunoglobulin G1 monoclonal antibodies and subsequently modulate antibody-dependent cellular cytotoxicity (ADCC) activity. Whether single-nucleotide polymorphisms of FCGRs correlate with survival of chronic lymphocytic leukemia (CLL) patients treated with a monoclonal antibody containing regimen is unclear. We assessed the FCGR3A and FCGR2A genotype of patients enrolled in the REACH trial, where patients received fludarabine and cyclophosphamide (FC) or rituximab plus FC (R-FC). FCGR3A and FCGR2A polymorphisms did not demonstrate prognostic significance in the FC arm (P = .42 and P = .64, respectively) or R-FC arm (P = .41 and P = .88, respectively) with respect to progression free survival. Patients with intermediate affinity genotypes (FV and HR) benefited significantly from addition of rituximab (hazard ratio = 0.55 [0.37-0.8 CI]; P = .0017 and hazard ratio = 0.63 [0.44-0.9 CI]; P = .011, respectively). Similar benefit was suggested for patients with high- affinity VV and HH (hazard ratio = 0.86 [0.4-1.84 CI]; P = .7 and hazard ratio = 0.7 [0.41-1.18 CI]; P = .18, respectively) and low-affinity FF and RR (hazard ratio = 0.85 [0.56-1.29 CI]; P = .44 and hazard ratio = 0.82 [0.47-1.42 CI]; P = .48, respectively). Overall, our results suggest that FCGR2A and FCGR3A polymorphisms do not significantly influence the outcomes of relapsed or refractory CLL patients treated with FC or the monoclonal antibody regimen R-FC.

Rituximab plus fludarabine and cyclophosphamide prolongs progression-free survival compared with fludarabine and cyclophosphamide alone in previously treated chronic lymphocytic leukemia.

PURPOSE: Rituximab, a monoclonal antibody that targets the CD20 cell surface antigen, has clinical activity in patients with non-Hodgkin's lymphoma and other B-lymphocyte disorders when administered alone or in combination with chemotherapy. Promising results have previously been reported in nonrandomized studies in patients with chronic lymphocytic leukemia (CLL). This trial was designed to compare chemoimmunotherapy with chemotherapy alone in patients with previously treated CLL. PATIENTS AND METHODS: This international, multicenter, randomized trial compared six cycles of rituximab plus fludarabine and cyclophosphamide (R-FC) with six cycles of fludarabine and cyclophosphamide alone (FC) in patients with previously treated CLL. A total of 552 patients with Binet stage A (1%), B (59%), or C (31%) disease entered the study and were randomly assigned to receive R-FC (n = 276) or FC (n = 276). RESULTS: After a median follow-up time of 25 months, rituximab significantly improved progression-free survival in patients with previously treated CLL (hazard ratio = 0.65; P < .001; median, 30.6 months for R-FC v 20.6 months for FC). Event-free survival, response rate, complete response rate, duration of response, and time to new CLL treatment or death were also significantly improved. Although the rates of adverse events, grade 3 or 4 events, and serious adverse events were slightly higher in the R-FC arm, R-FC was generally well tolerated, with no new safety findings and no detrimental effect on quality of life. CONCLUSION: R-FC significantly improved the outcome of patients with previously treated CLL.

High-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation as a treatment option in multiple sclerosis.

High-dose immunosuppressive therapy (HDIT) with autologous hematopoietic stem cell transplantation (auto-HSCT) is a new and promising approach to the treatment of multiple sclerosis (MS) patients because currently there are no effective treatment methods for this disease. In this article, we present results of a prospective clinical study of efficacy of HDIT + auto-HSCT in MS patients. The following treatment strategies were employed in the study: "early," "conventional," and "salvage/late" transplantation. Fifty patients with various types of MS were included in this study. No toxic deaths were reported among 50 MS patients; transplantation procedure was well-tolerated by the patients. The efficacy analysis was performed in 45 patients. Twenty-eight patients achieved an objective improvement of neurological symptoms, defined as at least 0.5-point decrease in the Expanded Disability Status Scale (EDSS) score as compared to the baseline and confirmed during 6 months, and 17 patients had disease stabilization (steady EDSS level as compared to the baseline and confirmed during 6 months). The progression-free survival at 6 years after HDIT + auto-HSCT was 72%. Magnetic resonance imaging data were available in 37 patients before transplantation showing disease activity in 43.3%. No active, new, or enlarging lesions were registered in patients without disease progression. In conclusion, HDIT + auto-HSCT suggests positive results in management of patients with different types of MS. Identification of treatment strategies based on the level of disability, namely "early," "conventional," and "salvage/late" transplantation, appears to be feasible to improve treatment outcomes.

Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection.

Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.

Mosquito densonucleosis viruses cause dramatically different infection phenotypes in the C6/36 Aedes albopictus cell line.

Mosquito densoviruses generally establish persistent infections in mosquito cell lines including the C6/36 Aedes albopictus cell line. In contrast, the closely related Haemagogus equinus densovirus (HeDNV) causes dramatic cytopathic effects in the C6/36 Aedes albopictus cell line. Infection of C6/36 cells by HeDNV causes internucleosomal fragmentation of host chromosomal DNA, changes in cellular morphology (membrane budding, apoptotic bodies), caspase activation and exposure of phosphatidylserine on the cellular membrane. This is accompanied by a higher rate of infection and more vigorous production of virus in these cells. These observations are consistent with the induction of apoptosis during infection. In contrast, expression of AeDNV proteins in C6/36 cells does not cause obvious cytopathic effects although NS1 expression causes accumulation of cells in G2 phase. C6/36 cells persistently infected with AeDNV were not protected from superinfection with HeDNV. Thus, there does not seem to be an antiviral state induced by AeDNV persistent infection.