BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.MethodsCD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.ResultsThe engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.ConclusionCNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

With an overall 5 year survival rate as low as 15% for non-small cell lung cancer (NSCLC), even with surgical intervention and the use of newer molecules in adjuvant chemotherapy, there is an urgent need for new biological targets and associated novel anti-cancer agents. The present study was undertaken to evaluate the potential of the Na(+)/K(+)-ATPase alpha1 subunit as a novel target in NSCLC and revealed that alpha1 expression is markedly higher in a significant proportion of NSCLC clinical samples compared to normal lung tissue. Furthermore, reduction in alpha1 expression in A549 NSCLC cells by anti-alpha1 siRNA resulted in markedly impaired proliferation and migration of these cancer cells. Finally, of three cardenolides investigated, UNBS1450, which is known to bind to Na(+)/K(+)-ATPase and displays potent anti-tumour activity in vivo in experimental models of human NSCLCs, is the most potent inhibitor of Na(+)/K(+)-ATPase isozymes (alpha1beta1, alpha2beta1 and alpha3beta1), most strikingly of alpha1beta1. This was reflected in the compound's more potent anti-proliferative activity in all NSCLC cell lines evaluated (A549, Cal-12T, NCI-H727 and A427); the first three of which over-express alpha1. The marked impairment in A549 NSCLC cell proliferation and migration, and resulting similar morphology following anti-alpha1 siRNA or UNBS1450 treatment, was associated with features of abnormal cytokinesis, mediated in the case of UNBS1450 by disorganization of the actin cytoskeleton. Collectively these data strongly suggest that targeting the Na(+)/K(+)-ATPase alpha1 using specific cardenolides could represent a novel means to combat certain NSCLCs.

Prostasomes are prostate-derived organelles that occur freely in human seminal plasma. They promote forward motility of spermatozoa probably by closely interacting with them in an unknown manner. We have studied the interaction between human prostasomes and spermatozoa by applying them as two separate samples in free-zone electrophoresis. During the run these samples approached each other and finally fused into one single peak that was not further dissociated. Both the spermatozoa and prostasomes displayed a net-negative surface charge, the latter being less negative. This discrepancy in charge was even more pronounced by pretreatment of prostasomes with neuraminidase, which, however, did not affect the interaction. This implies a strong interaction of a probable hydrophobic character between cells and organelles. The presence of prostasomes and spermatozoa in the fused, single peak was confirmed by transmission electron microscopy. Evidence for interaction was apparent in transmission electron microscopy after embedding in a hydrophilic, but not in a hydrophobic, resin. This observation supports the view that the bonds between prostasomes and spermatozoa are of hydrophobic character. This type of interaction enables the prostasomes to act in close vicinity to spermatozoa and may create the prerequisites for a proper microenvironment of the spermatozoa favoring their forward motility.

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