No abstract

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level.

CD8+ T cells are key players during infection with the malaria parasite Plasmodium berghei ANKA (PbA). While they cannot provide protection against blood-stage parasites, they can cause immunopathology, thus leading to the severe manifestation of cerebral malaria. Hence, the tight control of CD8+ T cell function is key in order to prevent fatal outcomes. One major mechanism to control CD8+ T cell activation, proliferation and effector function is the integration of co-inhibitory and co-stimulatory signals. In this study, we show that one such pathway, the HVEM-CD160 axis, significantly impacts CD8+ T cell regulation and thereby the incidence of cerebral malaria. Here, we show that the co-stimulatory molecule HVEM is indeed required to maintain CD8+ T effector populations during infection. Additionally, by generating a CD160−/− mouse line, we observe that the HVEM ligand CD160 counterbalances stimulatory signals in highly activated and cytotoxic CD8+ T effector cells, thereby restricting immunopathology. Importantly, CD160 is also induced on cytotoxic CD8+ T cells during acute Plasmodium falciparum malaria in humans. In conclusion, CD160 is specifically expressed on highly activated CD8+ T effector cells that are harmful during the blood-stage of malaria.

Despite being one of the most fundamental biological processes, the process of speciation remains poorly understood in many groups of organisms. Mouse lemurs are a species-rich genus of small primates endemic to Madagascar, whose diversity has only recently been uncovered using genetic data and is primarily found among morphologically cryptic, allopatric populations. To assess to what extent described species represent reproductively isolated entities, studies are needed in areas where mouse lemur taxa come into contact. Hybridization has previously been reported in a contact zone between two closely related mouse lemur species (Microcebus murinus and M. griseorufus) based on microsatellite data. Here, we revisit this system using RADseq data for populations in, near, and far from the contact zone, including many of the individuals that had previously been identified as hybrids. Surprisingly, we find no evidence for admixed nuclear ancestry in any of the individuals. Re-analyses of microsatellite data and simulations suggest that previously inferred hybrids were false positives and that the program NewHybrids can be particularly sensitive to erroneously inferring hybrid ancestry. Using coalescent-bases analyses, we also show an overall lack of recent gene flow between the two species, and low levels of ancestral gene flow. Combined with evidence for local syntopic occurrence, these data indicate that M. murinus and M. griseorufus are reproductively isolated. Finally, we estimate that they diverged less than a million years ago, suggesting that completion of speciation is relatively rapid in mouse lemurs. Future work should focus on the underpinnings of reproductive isolation in this cryptic primate radiation, which are mostly unknown. Our study also provides a cautionary tale for the inference of hybridization with microsatellite data.

Microsatellites have been a workhorse of evolutionary genetic studies for decades and are still commonly in use for estimating signatures of genetic diversity at the population and species level across a multitude of taxa. Yet, the very high mutation rate of these loci is a double-edged sword, conferring great sensitivity at shallow levels of analysis (e.g. paternity analysis) but yielding considerable uncertainty for deeper evolutionary comparisons. For the present study, we used reduced representation genome-wide data (restriction site-associated DNA sequencing (RADseq)) to test for patterns of interspecific hybridization previously characterized using microsatellite data in a contact zone between two closely related mouse lemur species in Madagascar ( Microcebus murinus and Microcebus griseorufus ). We revisit this system by examining populations in, near, and far from the contact zone, including many of the same individuals that had previously been identified as hybrids with microsatellite data. Surprisingly, we find no evidence for admixed nuclear ancestry. Instead, re-analyses of microsatellite data and simulations suggest that previously inferred hybrids were false positives and that the program N ew H ybrids can be particularly sensitive to erroneously inferring hybrid ancestry. Combined with results from coalescent-based analyses and evidence for local syntopic co-occurrence, we conclude that the two mouse lemur species are in fact completely reproductively isolated, thus providing a new understanding of the evolutionary rate whereby reproductive isolation can be achieved in a primate.

Numerous trials using dendritic cell (DC)-based vaccinations for the treatment of cancer are being carried out. However, an improvement of the quality of DC used is highly warranted. We here generated human monocyte-derived dendritic cells using a 3 day protocol and stimulated the cells using a combination of OK432 (Picibanil), TLR7/8 ligand CL097, and reduced amounts of prostaglandin (PG)E. We analyzed phenotype, migratory, and T-cell stimulatory capacity compared to a cytokine cocktail consisting of IL-1β, IL-6, TNF, and PGE. The OK432 cocktail stimulated cells had a similar mature phenotype with upregulated co-stimulatory molecules, HLA-DR and CCR7 as the cytokine cocktail-matured cells and a similar cytokine profile except increased amounts of IL-12p70. Chemotaxis towards CCL19 was reduced compared to the cytokine cocktail, but increased compared to OK432 alone. The T-cell stimulatory capacity was similar to the cytokine cocktail stimulated cells. In conclusion, the OK432 cocktail has the advantage of inducing IL-12p70 production without impairing phenotype or T-cell stimulatory capacity of the cells and might, therefore, be an advantageous alternative to be used in DC-based immunotherapy.

Helminths still infect a quarter of the human population. They manage to establish chronic infections by downmodulating the immune system of their hosts. Consequently, the immune response of helminth-infected individuals to vaccinations may be impaired as well. Here we study the impact of helminth-induced immunomodulation on vaccination efficacy in the mouse system. We have previously shown that an underlying Litomosoides sigmodontis infection reduced the antibody (Ab) response to anti-influenza vaccination in the context of a systemic expansion of type 1 regulatory T cells (Tr1). Most important, vaccine-induced protection from a challenge infection with the 2009 pandemic H1N1 influenza A virus (2009 pH1N1) was impaired in vaccinated, L. sigmodontis-infected mice. Here, we aim at the restoration of vaccination efficacy by drug-induced deworming. Treatment of mice with Flubendazole (FBZ) resulted in elimination of viable L. sigmodontis parasites in the thoracic cavity after two weeks. Simultaneous FBZ-treatment and vaccination did not restore Ab responses or protection in L. sigmodontis-infected mice. Likewise, FBZ-treatment two weeks prior to vaccination did not significantly elevate the influenza-specific Ig response and did not protect mice from a challenge infection with 2009 pH1N1. Analysis of the regulatory T cell compartment revealed that L. sigmodontis-infected and FBZ-treated mice still displayed expanded Tr1 cell populations that may contribute to the sustained suppression of vaccination responses in successfully dewormed mice. To outcompete this sustained immunomodulation in formerly helminth-infected mice, we finally combined the drug-induced deworming with an improved vaccination regimen. Two injections with the non-adjuvanted anti-influenza vaccine Begripal conferred 60% protection while MF59-adjuvanted Fluad conferred 100% protection from a 2009 pH1N1 infection in FBZ-treated, formerly L. sigmodontis-infected mice. Of note, applying this improved prime-boost regimen did not restore protection in untreated L. sigmodontis-infected mice. In summary our findings highlight the risk of failed vaccinations due to helminth infection.

The world health organization estimates that more than a quarter of the human population is infected with parasitic worms that are called helminths. Many helminths suppress the immune system of their hosts to prolong their survival. This helminth-induced immunosuppression “spills over” to unrelated antigens and can suppress the immune response to vaccination against other pathogens. Indeed, several human studies have reported a negative correlation between helminth infections and responses to vaccinations. Using mice that are infected with the parasitic nematode Litomosoides sigmodontis as a model for chronic human filarial infections, we reported previously that concurrent helminth infection impaired the vaccination-induced protection against the human pathogenic 2009 pandemic H1N1 influenza A virus (2009 pH1N1). Vaccinated, helminth-infected mice produced less neutralizing, influenza-specific antibodies than vaccinated naïve control mice. Consequently helminth-infected and vaccinated mice were not protected against a challenge infection with influenza virus but displayed high virus burden in the lung and a transient weight loss. In the current study we tried to improve the vaccination efficacy using vaccines that are licensed for humans. We either introduced a prime-boost vaccination regimen using the non-adjuvanted anti-influenza vaccine Begripal or employed the adjuvanted influenza vaccine Fluad. Although both strategies elevated the production of influenza-specific antibodies and protected mice from the transient weight loss that is caused by an influenza challenge infection, sterile immunity was not achieved. Helminth-infected vaccinated mice still had high virus burden in the lung while non-helminth-infected vaccinated mice rapidly cleared the virus. In summary we demonstrate that basic improvements of influenza vaccination regimen are not sufficient to confer sterile immunity on the background of helminth-induced immunosuppression, despite amelioration of pathology i.e. weight loss. Our findings highlight the risk of failed vaccinations in helminth-endemic areas, especially in light of the ongoing vaccination campaign to control the COVID-19 pandemic.