Immunomodulatory potential of rapamycin-loaded mesoporous silica nanoparticles: pore size-dependent drug loading, release, and in vitro cellular responses.

Rapamycin is a potent immunosuppressive drug that has been recently proposed for a wide range of applications beyond its current clinical use. For some of these proposed applications, encapsulation in nanoparticles is key to ensure therapeutic efficacy and safety. In this work, we evaluate the effect of pore size on mesoporous silica nanoparticles (MSN) as rapamycin nanocarriers. The successful preparation of MSN with 4 different pore sizes was confirmed by dynamic light scattering, zeta potential, transmission electron microscopy and N2 adsorption. In these materials, rapamycin loading was pore size-dependent, with smaller pore MSN exhibiting greater loading capacity. Release studies showed sustained drug release from all MSN types, with larger pore MSN presenting faster release kinetics. In vitro experiments using the murine dendritic cell (DC) line model DC2.4 showed that pore size influenced the biological performance of MSN. MSN with smaller pore sizes presented larger nanoparticle uptake by DC2.4 cells, but were also associated with slightly larger cytotoxicity. Further evaluation of DC2.4 cells incubated with rapamycin-loaded MSN also demonstrated a significant effect of MSN pore size on their immunological response. Notably, the combination of rapamycin-loaded MSN with an inflammatory stimulus (lipopolysaccharide, LPS) led to changes in the expression of DC activation markers (CD40 and CD83) and in the production of the proinflammatory cytokine TNF-α compared to LPS-treated DC without nanoparticles. Smaller-pored MSN induced more substantial reductions in CD40 expression while eliciting increased CD83 expression, indicating potential immunomodulatory effects. These findings highlight the critical role of MSN pore size in modulating rapamycin loading, release kinetics, cellular uptake, and subsequent immunomodulatory responses.

Influence of Pore Size in Protein G'-Grafted Mesoporous Silica Nanoparticles as a Serum Pretreatment System for In Vitro Allergy Diagnosis.

Particles with the capacity to bind to immunoglobulin G (IgG) can be used for the purification of IgG or to process clinical samples for diagnostic purposes. For in vitro allergy diagnosis, the high IgG levels in serum can interfere with the detection of allergen-specific IgE, the main diagnostic biomarker. Although commercially available, current materials present a low IgG capture capacity at large IgG concentrations or require complex protocols, preventing their use in the clinic. In this work, mesoporous silica nanoparticles are prepared with different pore sizes, to which IgG-binding protein G' is grafted. It is found that for one particular optimal pore size, the IgG capture capacity of the material is greatly enhanced. The capacity of this material to efficiently capture human IgG in a selective way (compared to IgE) is demonstrated in both solutions of known IgG concentrations as well as in complex samples, like serum, from healthy controls and allergic patients using a simple and fast incubation protocol. Interestingly, IgG removal using the best-performing material enhances in vitro IgE detection in sera from patients allergic to amoxicillin. These results highlight the great translation potential of this strategy to the clinic in the context of in vitro allergy diagnosis.

Insulin-regulated serine and lipid metabolism drive peripheral neuropathy.

Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves1,2. Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia3-7, aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes8-10. Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome10-14, but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy15,16. Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.

The role of non-invasive prenatal testing (NIPT) for fetal blood group typing in Australia.

Maternal alloimmunisation against red blood cell antigens can cause haemolytic disease of the fetus and newborn (HDFN). Although most frequently caused by anti-D, since the implementation of rhesus D (RhD) immunoglobulin prophylaxis, other alloantibodies have become more prevalent in HDFN. Recent advances in non-invasive prenatal testing (NIPT) have allowed early prediction of HDFN risk in alloimmunised pregnancies and allow clinicians to focus health resources on those pregnancies that require intervention. This article aims to provide updates on the current status of NIPT in Australia as both a diagnostic and screening tool in pregnancy.

New strategy for liquefying corn stover pellets.

The movement of solid material into and between unit operations within a biorefinery is a bottleneck in reaching design capacity, with formation of biomass slurries needed to introduce feedstock. Corn stover slurries have been achieved from dilute acid, pretreated materials resulting in slurry concentrations of up to about 150 g/L, above which flowability is compromised. We report a new strategy to liquefy corn stover at higher solids concentration (300 g/L) by initially cooking it with the enzyme mimetic maleic acid at 40 mM and 150 °C. This is followed by 6 h of enzymatic modification at 1 FPU (2.2 mg protein)/g solids, resulting in a yield stress of 171 Pa after 6 h and 58 Pa in 48 h compared to 6806 Pa for untreated stover. Mimetic treatment of corn stover pellets minimizes the inhibitory effect of xylo-oligomers on hydrolytic enzymes. This strategy allows for the delivery of solid lignocellulosic slurry into a pretreatment reactor by pumping, improving operability of a biorefinery.

Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice.

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

New prognosis score including absolute lymphocyte/monocyte ratio, red blood cell distribution width and beta-2 microglobulin in patients with diffuse large B-cell lymphoma treated with R-CHOP: Spanish Lymphoma Group Experience (GELTAMO).

The International Prognostic Index (IPI) is the most widely used score for non-Hodgkin lymphoma but lacks the ability to identify a high-risk population in diffuse large B-cell lymphoma (DLBCL). Low absolute lymphocyte count and high monocytes have proved to be unfavourable factors. Red-cell distribution width (RDW) has been associated with inflammation and beta-2 microglobulin (B2M) with tumour load. The retrospective study included 992 patients with DLBCL treated with R-CHOP. In the multivariate analysis, age, Eastern Cooperative Oncology Group performance status (ECOG-PS), stage, bulky mass, B2M, RDW, and lymphocyte/monocyte ratio (LMR) were independently related to progression-free survival (PFS). A new prognosis score was generated with these variables including age categorized into three groups (0, 1, 2 points); ECOG ≥ 3-4 with two; stage III/IV, bulky mass, high B2M, LMR < 2·25 and RDW > 0·96 with one each; for a maximum of 9. This score could improve the discrimination of a very high-risk subgroup with five-year PFS and overall survival (OS) of 19% and 24% versus 45% and 59% of R (revised)-IPI respectively. This score also showed greater predictive ability than IPI. A new score is presented including complete blood cell count variables and B2M, which are readily available in real-life practice without additional tests. Compared to R-IPI, it shows a more precise high-risk assessment and risk discrimination for both PFS and OS.

Author Correction: Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

Protein-based therapeutics can activate the adaptive immune system, leading to the production of neutralizing antibodies and the clearance of the treated cells mediated by cytotoxic T cells. Here, we show that the sequential use of immune-orthogonal orthologues of CRISPR-associated protein 9 (Cas9) and adeno-associated viruses (AAVs) evades adaptive immune responses and enables effective gene editing using repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major histocompatibility complex (MHC) proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors and 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologues-which we validated for three Cas9 orthologues in mice-yet we anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice that were previously immunized against the AAV vector and the Cas9 cargo. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.

In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.

Oligonucleotide conjugated multi-functional adeno-associated viruses.

Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these 'cloaked AAVs' retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications.

Therapeutic genome engineering via CRISPR-Cas systems.

Differences in genomes underlie most organismal diversity, and aberrations in genomes underlie many disease states. With the growing knowledge of the genetic and pathogenic basis of human disease, development of safe and efficient platforms for genome and epigenome engineering will transform our ability to therapeutically target human diseases and also potentially engineer disease resistance. In this regard, the recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) RNA-guided nuclease systems have transformed our ability to target nucleic acids. Here we review therapeutic genome engineering applications with a specific focus on the CRISPR-Cas toolsets. We summarize past and current work, and also outline key challenges and future directions. WIREs Syst Biol Med 2017, 9:e1380. doi: 10.1002/wsbm.1380 For further resources related to this article, please visit the WIREs website.

Validation of the NCCN-IPI for diffuse large B-cell lymphoma (DLBCL): the addition of ß2 -microglobulin yields a more accurate GELTAMO-IPI.

The study included 1848 diffuse large B-cell lymphoma (DLBCL)patients treated with chemotherapy/rituximab. The aims were to validate the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) and explore the effect of adding high Beta-2 microglobulin (ß2M), primary extranodal presentation and intense treatment to the NCCN-IPI variables in order to develop an improved index. Comparing survival curves, NCCN-IPI discriminated better than IPI, separating four risk groups with 5-year overall survival rates of 93%, 83%, 67% and 49%, but failing to identify a true high-risk population. For the second aim the series was split into training and validation cohorts: in the former the multivariate model identified age, lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, Stage III-IV, and ß2M as independently significant, whereas the NCCN-IPI-selected extranodal sites, primary extranodal presentation and intense treatments were not. These results were confirmed in the validation cohort. The Grupo Español de Linfomas/Trasplante de Médula ósea (GELTAMO)-IPI developed here, with 7 points, significantly separated four risk groups (0, 1-3, 4 or ≥5 points) with 11%, 58%, 17% and 14% of patients, and 5-year overall survival rates of 93%, 79%, 66% and 39%, respectively. In the comparison GELTAMO IPI discriminated better than the NCCN-IPI. In conclusion, GELTAMO-IPI is more accurate than the NCCN-IPI and has statistical and practical advantages in that the better discrimination identifies an authentic high-risk group and is not influenced by primary extranodal presentation or treatments of different intensity.

Analysis of the mutational landscape of classic Hodgkin lymphoma identifies disease heterogeneity and potential therapeutic targets.

Defining the mutational landscape of classic Hodgkin lymphoma is still a major research goal. New targeted next-generation sequencing (NGS) techniques may identify pathogenic mechanisms and new therapeutic opportunities related to this disease. We describe the mutational profile of a series of 57 cHL cases, enriched in Hodgkin and Reed-Sternberg (HRS) cells. Overall, the results confirm the presence of strong genomic heterogeneity. However, several variants were consistently detected in genes related to relevant signaling pathways, such as GM-CSF/IL-3, CBP/EP300, JAK/STAT, NF-kappaB, and numerous variants of genes affecting the B-cell receptor (BCR) pathway, such as BTK, CARD11, BCL10, among others. This unexpectedly high prevalence of mutations affecting the BCR pathway suggests some requirement for active BCR signaling for cHL cell viability. Additionally, incubation of a panel of cHL cellular models with selective BTK inhibitors in vitro constrains cell proliferation and causes cell death. Our results indicate new pathogenic mechanisms and therapeutic opportunities in this disease.

Vascular Dysfunction in a Transgenic Model of Alzheimer's Disease: Effects of CB1R and CB2R Cannabinoid Agonists.

There is evidence of altered vascular function, including cerebrovascular, in Alzheimer's disease (AD) and transgenic models of the disease. Indeed vasoconstrictor responses are increased, while vasodilation is reduced in both conditions. ß-Amyloid (Aß) appears to be responsible, at least in part, of alterations in vascular function. Cannabinoids, neuroprotective and anti-inflammatory agents, induce vasodilation both in vivo and in vitro. We have demonstrated a beneficial effect of cannabinoids in models of AD by preventing glial activation. In this work we have studied the effects of these compounds on vessel density in amyloid precursor protein (APP) transgenic mice, line 2576, and on altered vascular responses in aortae isolated ring. First we showed increased collagen IV positive vessels in AD brain compared to control subjects, with a similar increase in TgAPP mice, which was normalized by prolonged oral treatment with the CB1/CB2 mixed agonist WIN 55,212-2 (WIN) and the CB2 selective agonist JWH-133 (JWH). In Tg APP mice the vasoconstriction induced by phenylephrine and the thromboxane agonist U46619 was significantly increased, and no change in the vasodilation to acetylcholine (ACh) was observed. Tg APP displayed decreased vasodilation to both cannabinoid agonists, which were able to prevent decreased ACh relaxation in the presence of Aß. In summary, we have confirmed and extended the existence of altered vascular responses in Tg APP mice. Moreover, our results suggest that treatment with cannabinoids may ameliorate the vascular responses in AD-type pathology.

Stimulation of brain glucose uptake by cannabinoid CB2 receptors and its therapeutic potential in Alzheimer's disease.

Cannabinoid CB2 receptors (CB2Rs) are emerging as important therapeutic targets in brain disorders that typically involve neurometabolic alterations. We here addressed the possible role of CB2Rs in the regulation of glucose uptake in the mouse brain. To that aim, we have undertaken 1) measurement of (3)H-deoxyglucose uptake in cultured cortical astrocytes and neurons and in acute hippocampal slices; 2) real-time visualization of fluorescently labeled deoxyglucose uptake in superfused hippocampal slices; and 3) in vivo PET imaging of cerebral (18)F-fluorodeoxyglucose uptake. We now show that both selective (JWH133 and GP1a) as well as non-selective (WIN55212-2) CB2R agonists, but not the CB1R-selective agonist, ACEA, stimulate glucose uptake, in a manner that is sensitive to the CB2R-selective antagonist, AM630. Glucose uptake is stimulated in astrocytes and neurons in culture, in acute hippocampal slices, in different brain areas of young adult male C57Bl/6j and CD-1 mice, as well as in middle-aged C57Bl/6j mice. Among the endocannabinoid metabolizing enzymes, the selective inhibition of COX-2, rather than that of FAAH, MAGL or α,ßDH6/12, also stimulates the uptake of glucose in hippocampal slices of middle-aged mice, an effect that was again prevented by AM630. However, we found the levels of the endocannabinoid, anandamide reduced in the hippocampus of TgAPP-2576 mice (a model of ß-amyloidosis), and likely as a consequence, COX-2 inhibition failed to stimulate glucose uptake in these mice. Together, these results reveal a novel general glucoregulatory role for CB2Rs in the brain, raising therapeutic interest in CB2R agonists as nootropic agents.

CSF1R Protein Expression in Reactive Lymphoid Tissues and Lymphoma: Its Relevance in Classical Hodgkin Lymphoma.

Tumour-associated macrophages (TAMs) have been associated with survival in classic Hodgkin lymphoma (cHL) and other lymphoma types. The maturation and differentiation of tissue macrophages depends upon interactions between colony-stimulating factor 1 receptor (CSF1R) and its ligands. There remains, however, a lack of consistent information on CSF1R expression in TAMs. A new monoclonal antibody, FER216, was generated to investigate CSF1R protein distribution in formalin fixed tissue samples from 24 reactive lymphoid tissues and 187 different lymphoma types. We also analysed the distribution of CSF1R+, CD68+ and CD163+ macrophages by double immunostaining, and studied the relationship between CSF1R expression and survival in an independent series of 249 cHL patients. CSF1R+ TAMs were less frequent in B-cell lymphocytic leukaemia and lymphoblastic B-cell lymphoma than in diffuse large B-cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma and cHL. HRS cells in cHL and, with the exception of three cases of anaplastic large cell lymphoma, the neoplastic cells in NHLs, lacked detectable CSF1R protein. A CSF1R+ enriched microenvironment in cHL was associated with shorter survival in an independent series of 249 cHL patients. CSF1R pathway activation was evident in the cHL and inactivation of this pathway could be a potential therapeutic target in cHL cases.

Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins.

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5-6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.