Jon Hildahl

Nanoparticles as Drug Delivery System against Tuberculosis in Zebrafish Embryos: Direct Visualization and Treatment

Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.

Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish

Gonadotropin releasing hormone (GnRH) is a key regulator of sexual development and reproduction in vertebrates. Fish have either two or three pre-pro-GnRH genes, encoding structurally distinct peptides. We identified three pre-pro-GnRH genes in Atlantic cod (Gadus morhua, gmGnRH) using RT-PCR, RACE-PCR and BAC DNA library clone sequencing based on synteny searching. Gene identity was confirmed by sequence alignment and subsequent phylogenetic analysis. The expression of these genes was measured by quantitative PCR in the brain and pituitary of female cod throughout their reproductive cycle and in peripheral tissues. All three gmGnRH genes have highly conserved deduced decapeptide sequences, but sequence and phylogenetic data for gmGnRH1 suggest that this is a pseudogene. gmGnRH1 shares low identity with all fish GnRH variants and grouped with the GnRH3 clade. Although gmGnRH1 is a putative pseudogene, it is transcribed in multiple tissues but at low levels in the brain, indicating the loss of conserved hypophysiotrophic function. Phylogenetic analysis reveals that gmGnRH2 and gmGnRH3 variants are located in variant-specific clades. Both gmGnRH2 and gmGnRH3 transcripts are most abundant in the brain, with lower expression in pituitaries and ovaries. Brain gmGnRH3 gene expression increases in spawning fish and is expressed in the pituitary during puberty. Brain gmGnRH2 transcripts are highly expressed relative to gmGnRH3 before and during spawning. Sequence and expression data suggest that gmGnRH1 is a pseudogene and that gmGnRH3 is likely the hypophysiotrophic form of GnRH in Atlantic cod.

Zebrafish as a model system for characterization of nanoparticles against cancer

Therapeutic nanoparticles (NPs) have great potential to deliver drugs against human diseases. Encapsulation of drugs in NPs protects them from being metabolized, while they are delivered specifically to a target site, thereby reducing toxicity and other side-effects. However, non-specific tissue accumulation of NPs, for example in macrophages, especially in the spleen and liver is a general problem with many NPs being developed for cancer therapy. To address the problem of non-specific tissue accumulation of NPs we describe the development of the zebrafish embryo as a transparent vertebrate system for characterization of NPs against cancer. We show that injection of human cancer cells results in tumor-like structures, and that subsequently injected fluorescent NPs, either made of polystyrene or liposomes can be imaged in real-time. NP biodistribution and general in vivo properties can be easily monitored in embryos having selective fluorescent labeling of specific tissues. We demonstrate in vitro, by using optical tweezer micromanipulation, microscopy and flow cytometry that polyethylene glycol (PEG) coating of NPs decreases the level of adhesion of NPs to macrophages, and also to cancer cells. In vivo in zebrafish embryos, PEG coating resulted in longer NP circulation times, decreased macrophage uptake, and reduced adhesion to the endothelium. Importantly, liposomes were observed to accumulate passively and selectively in tumor-like structures comprised of human cancer cells. These results show that zebrafish embryo is a powerful system for microscopy-based screening of NPs on the route to preclinical testing.

Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: tissue-distribution and changes in transcript abundance during artificially induced sexual development.

Gonadotropin-releasing hormone receptor (GnRH-R) activation stimulates synthesis and release of gonadotropins in the vertebrate pituitary and also mediates other processes both in the brain and in peripheral tissues. To better understand the differential function of multiple GnRH-R paralogs, three GnRH-R genes (gnrhr1a, 1b, and 2) were isolated and characterized in the European eel. All three gnrhr genes were expressed in the brain and pituitary of pre-pubertal eels, and also in several peripheral tissues, notably gills and kidneys. During hormonally induced sexual maturation, pituitary expression of gnrhr1a (female) and gnrhr2 (male and female) was up-regulated in parallel with gonad development. In the brain, a clear regulation during maturation was seen only for gnrhr2 in the midbrain, with highest levels recorded during early vitellogenesis. These data suggest that GnRH-R2 is the likely hypophysiotropic GnRH-R in male eel, while both GnRH-R1a and GnRH-R2 seems to play this role in female eels.

Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish

Abstract Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation.

Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out. An experimental group of silver-stage eel females was maintained at 18 °C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12 weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development. These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.

Differential regulation of GnRH ligand and receptor genes in the brain and pituitary of Atlantic cod exposed to different photoperiod

The onset of puberty and reproduction are tightly controlled by extrinsic and intrinsic inputs combined with genetically determined biological blueprints. Environmental inputs are then mediated by the brain-pituitary-gonad endocrine axis resulting in a unified output. In fish, one of the primary factors controlling the timing of sexual maturation is light, although how these signals are mediated in the brain and pituitary is not well understood. We therefore aimed to elucidate the molecular basis of the control of reproduction during the first spawning season in two year old female Atlantic cod. To this end, we measured GnRH and GnRH-R variant gene expression in brains and pituitaries collected from cod kept under four different photoperiod regimes: natural light (NL), continuous light (LL) and combined treatment of NL-LL and LL-NL. LL inhibited sexual development and spawning and LL-NL delayed sexual development and spawning. LL inhibited the spawning-related increase in brain GnRH3 and pituitary GnRH-R2a gene expression found under NL conditions, and the expression of these genes were delayed in concert with spawning of LL-NL cod. This study indicates that regulation of brain GnRH3 and pituitary GnRH-R2a genes likely mediates photoperiod induced changes in cod gonadal maturation.

Developmental tracing of luteinizing hormone β-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.

Background: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH‐producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue‐specific gene expression was examined. Results: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH‐producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb‐GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb‐GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. Conclusions: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function. Developmental Dynamics 241:1665–1677, 2012.

Four gonadotropin releasing hormone receptor genes in Atlantic cod are differentially expressed in the brain and pituitary during puberty

Gonadotropin releasing hormones (GnRH) are an important part of the brain-pituitary-gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.

A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples

Amicon(®) Ultra centrifugal filters were critically evaluated for various sample preparations, namely (a) proteome fractionation, (b) sample cleanup prior to liquid chromatography mass spectrometry (LC-MS) measurement of small molecules in cell lysate, and (c) separating drug-loaded nanoparticles and released drugs for accurate release profiling in biological samples. (a) Filters of supposedly differing molar mass (MM) selectivity (10, 30, 50 and 100K) were combined to attempt fractionation of samples of various complexity and concentration. However, the products had surprisingly similar MM retentate/filtrate profiles, and the filters were unsuited for proteome fractionation. (b) Centrifugal filtration was the only clean-up procedure in a FDA-guideline validated LC-MS method for determining anti-tuberculosis agents rifampicin and thioridazine in macrophage cell lysate. An additional organic solvent washing step (drug/protein-binding disruption) was required for satisfactory recovery. (c) The centrifugation filters are well suited for separating drugs and nanoparticles in simple aqueous solutions, but significantly less so for biological samples, as common drug-protein binding disruptors can dissolve NPs or be incompatible with LC-MS instrumentation.