MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

A new potential nano-oncological therapy based on polyamino acid nanocapsules.

A critical objective in cancer therapy is to reduce the systemic toxicity through the modification of the biodistribution of anticancer drugs. Herein, we disclose a new biodegradable nanocarrier, polyglutamic acid (PGA) nanocapsules, and present the in vivo pharmacokinetics/toxicity proof-of-concept for the anticancer drug plitidepsin. These novel nanocapsules were prepared using a modified solvent displacement technique where the polyamino acid was electrostatically deposited onto the lipid core. The nanocapsules exhibited an average size of 200 nm, a negative zeta potential and a great capacity for the encapsulation of plitidepsin (encapsulation efficiency above 90%). In addition, the nanocapsules could be freeze-dried and showed an adequate stability profile upon storage. Finally, the in vivo proof-of-concept studies performed in mice indicated that the encapsulation provided the drug with a prolonged blood circulation and a significantly reduced toxicity. In fact, the maximum tolerated dose of the nanoencapsulated drug was more than 3 times that of the reference formulation (Cremophor® EL plitidepsin solution). Overall, beyond the value of this specific formulation, the work reported here represents the evidence of the potential of polyamino acid nanocapsules in nano-oncological therapy.

Gene therapy in HIV-infected cells to decrease viral impact by using an alternative delivery method.

The ability of dendrimer 2G-[Si{O(CH(2))(2)N(Me)(2) (+)(CH(2))(2)NMe(3) (+)(I(-))(2)}](8) (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection.

Reduction of advanced liver fibrosis by short-term targeted delivery of an angiotensin receptor blocker to hepatic stellate cells in rats.

UNLABELLED: There is no effective therapy for advanced liver fibrosis. Angiotensin type 1 (AT1) receptor blockers attenuate liver fibrogenesis, yet their efficacy in reversing advanced fibrosis is unknown. We investigated whether the specific delivery of an AT1 receptor blocker to activated hepatic stellate cells (HSCs) reduces established liver fibrosis. We used a platinum-based linker to develop a conjugate of the AT1 receptor blocker losartan and the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin (losartan-M6PHSA). An average of seven losartan molecules were successfully coupled to M6PHSA. Rats with advanced liver fibrosis due to prolonged bile duct ligation or carbon tetrachloride administration were treated with daily doses of saline, losartan-M6PHSA, M6PHSA or oral losartan during 3 days. Computer-based morphometric quantification of inflammatory cells (CD43), myofibroblasts (smooth muscle alpha-actin [alpha-SMA]) and collagen deposition (Sirius red and hydroxyproline content) were measured. Hepatic expression of procollagen alpha2(I) and genes involved in fibrogenesis was assessed by quantitative polymerase chain reaction. Losartan-M6PHSA accumulated in the fibrotic livers and colocalized with HSCs, as assessed by immunostaining of anti-HSA and anti-alpha-SMA. Losartan-M6PHSA, but not oral losartan, reduced collagen deposition, accumulation of myofibroblasts, inflammation and procollagen alpha2(I) gene expression. Losartan-M6PHSA did not affect metalloproteinase type 2 and 9 activity and did not cause apoptosis of activated HSCs. CONCLUSION: Short-term treatment with HSC-targeted losartan markedly reduces advanced liver fibrosis. This approach may provide a novel means to treat chronic liver diseases.

Socio-economic impact of antiretroviral treatment in HIV patients. An economic review of cost savings after introduction of HAART.

Star celebrities such as Rock Hudson, Freddie Mercury, Magic Johnson, and Isaac Asimov have unfortunately something in common: they were all victims of the HIV global pandemic. Since then HIV infection has become considered a pandemic disease, and it is regarded as a priority in healthcare worldwide. It is ranked as the first cause of death among young people in industrialized countries, and it is recognized as a public healthcare problem due to its human, social, mass media, and economic impact. Incorporation of new and highly active antiretroviral treatment, available since 1996 for HIV/AIDS treatment, has provoked a radical change in the disease pattern, as well as in the impact on patient survival and quality of life. The pharmaceutical industry's contribution, based on the research for more active new drugs, has been pivotal. Mortality rates have decreased significantly in 20 years by 50% and now AIDS is considered a chronic and controlled disease. In this review we have studied the impact of HAART treatment on infected patients, allowing them to maintain their status as active workers and the decreased absenteeism from work derived from this, contributing ultimately to overall social wealth and, thus, to economic growth. Furthermore, an analysis of the impact on healthcare costs, quality of life per year, life per year gained, cost economic savings and cost opportunity among other parameters has shown that society and governments are gaining major benefits from the inclusion of antiretroviral therapies in HIV/AIDS patients.

Local inhibition of liver fibrosis by specific delivery of a platelet-derived growth factor kinase inhibitor to hepatic stellate cells.

Liver fibrosis is characterized by excessive proliferation and activation of hepatic stellate cells (HSC), a process in which platelet-derived growth factor (PDGF) plays an important role. Inhibition of liver fibrosis via specific delivery of a PDGF kinase inhibitor to HSC might therefore be an attractive strategy. The HSC-selective carrier mannose-6-phosphate modified human serum albumin (M6PHSA) was equipped with a tyrosine kinase inhibitor, 4-chloro-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (PAP19) (an imatinib derivative), by means of the platinum-based universal linkage system (ULS). The antifibrotic activity of PAP19-M6PHSA was evaluated in culture-activated rat HSC and precision-cut liver slices from fibrotic rats. After 24-h incubation, both free inhibitor PAP19 and PAP19-M6PHSA showed potent activity, as determined by quantitative reverse transcription-polymerase chain reaction analysis of alpha-smooth muscle actin (alphaSMA) and procollagen 1a1. Next, we examined the organ distribution and antifibrotic activity of PAP19-M6PHSA in bile duct-ligated (BDL) rats. Male Wistar rats at day 10 after BDL were administered a single dose of PAP19-M6PHSA and sacrificed at 2 h, 1 day, or 2 days afterward. The accumulation of PAP19-M6PHSA in the liver was quantified by high-performance liquid chromatography analysis (30% of the injected dose at 2 h) and detected in the liver by staining of the carrier. Liver drug levels were sustained at 24 and 48 h after the single dose. Furthermore, PAP19-M6PHSA reduced collagen deposition (Sirius red staining) and alphaSMA staining of activated HSC at these time points in comparison with saline-treated rats. We therefore conclude that delivery of a PDGF-kinase inhibitor to HSC is a promising technology to attenuate liver fibrogenesis.

Delivery of the p38 MAPkinase inhibitor SB202190 to angiogenic endothelial cells: development of novel RGD-equipped and PEGylated drug-albumin conjugates using platinum(II)-based drug linker technology.

Endothelial cells play an important role in inflammatory disorders, as they control the recruitment of leukocytes into inflamed tissue and the formation of new blood vessels. Activation of p38MAP kinase results in the production of proinflammatory cytokines and the expression of adhesion molecules. P38MAP kinase inhibitors are therefore considered important candidates for the treatment of inflammatory disorders. In the present study, we propose a novel strategy to counteract these processes by delivery of the p38MAP kinase inhibitor SB202190 into angiogenic endothelial cells. A drug-targeting conjugate was developed by conjugation of SB202190 to human serum albumin (HSA) using a novel platinum-based linker. Specificity for angiogenic endothelial cells was introduced by conjugation of cyclic RGD-peptides via bifunctional polyethylene glycol linkers. The final products contained an average of nine SB202190 and six RGDPEG groups per albumin. The platinum-based linker displayed high stability in buffers and culture medium, but released SB202190 slowly upon competition with sulfur-containing ligands like glutathione. RGDPEG-SB-HSA bound to alpha(v3)-integrin expressing endothelial cells (human umbilical cord vein endothelial cells) with low nanomolar affinity and was subsequently internalized. When HUVEC were treated with TNF to induce inflammatory events, pretreatment with RGDPEG-SB-HSA partially inhibited proinflammatory gene expression (IL-8, E-selectin; 30% inhibition) and secretion of cytokines (IL-8, 34% inhibition). We conclude that the developed RGDPEG-SB-HSA conjugates provide a novel means to counteract inflammation disorders such as rheumatoid arthritis.

Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology.

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX-M6PHSA at a sustained slow rate. PTX-M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX-M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX-M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.