Insights into the opportunistic fungal pathogen Cryptococcus and neutrophilic inflammation using zebrafish models

The innate immunity provides the first line of defence against infection and inflammation. Zebrafish are a proven model for understanding the in vivo biology of infection and immunity. Here I describe how I have developed and used zebrafish models to understand three different aspects of infection and immunity: 1) The development and use of a zebrafish model of the human fungal infection Cryptococcus neoformans 2) Understanding the virulence of the hypervirulent Cryptococcus gattii and 3) The mechanisms of action of the immunosuppressive drug mycophenolate mofetil (MMF). I have established an innate in vivo model for macrophage response to Cryptococcus by injecting cryptococci into zebrafish embryos. I have developed a high-content imaging method in a zebrafish model of cryptococcosis. This approach enabled me the discovery that while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. I found that phagocytosis is inhibited early in infection and that increases in cryptococcal number are driven by intracellular proliferation. Moreover, macrophages favourably phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. I then used the zebrafish model of cryptococcosis to determine the virulence of C. gattii. I have identified a mutant in the hyper virulent strain R265 that is attenuated in vivo. The attenuation of the mutant, R265 GFP14 was further confirmed in a mouse model of infection. I analysed the interaction of macrophages and R265 GFP cryptococci in zebrafish and found that the transgenic R265 GFP was rapidly cleared. Whole genome sequences revealed that R265 GFP14 has 32 kb deletion in chromosome 1, resulting in the loss of six genes. R265 wild-type and R265 GFP14 were characterised for carbon sources utilisation. Finally, following up on colleagues’ use of my zebrafish model of cryptococcosis, I investigated the action of MMF on neutrophilic inflammation. I showed that MMF treatment resulted in neutrophil cell death by apoptosis in vivo, thereby reducing neutrophilic inflammation. Thus in this thesis, I demonstrate how I combined the study of infection and immunity to better understand diseases that cause the biggest disease burden in humans. I pioneered novel approaches to studying cryptococcosis using an experimental zebrafish model, which demonstrated for the first time how the early interactions with macrophages determined the outcome of infection. I subsequently used my model to study the virulence of an emerging pathogen, Vancouver strain R265 of C. gattii, identifying a genome region that may be important for virulence. Finally, from my cryptococcosis model a new mechanism for the immunosuppressant mycophenolate mofetil was identified in macrophages. Using my expertise in neutrophilic inflammation I was able to show that there was a second mechanism in neutrophils and this may explain the usefulness of this drug in treating chronic inflammation

Association of Interleukin Genes IL10 and IL10RB with Parameters of Overweight in Military Students

To date, nearly 300 single-nucleotide polymorphisms (SNPs) associated with BMI, waist-to-hip ratio, and other adiposity traits have been identified by GWAS. With regards to IL10, at least 49 IL10-associated polymorphisms have been reported. However, little is known regarding the relationship between SNPs of the IL10 gene and the risk of obesity in young men. The aim of the present study was to investigate the relationship between SNPs of the IL10 and IL10RB genes and the risk of obesity in young men

Inhibition of classical and alternative modes of respiration in Candida albicans leads to cell wall remodelling and increased macrophage recognition

The human fungal pathogen Candida albicans requires respiratory function for normal growth, morphogenesis and virulence. Mitochondria therefore represent an enticing target for the development of new antifungal strategies. This possibility is bolstered by the presence of characteristics specific to fungi. However, respiration in C. albicans, as in many fungal organisms, is facilitated by redundant electron transport mechanisms, making direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we make use of chemicals with low toxicity to efficiently inhibit respiration in C. albicans. We find that use of the Nitric Oxide donor, Sodium Nitroprusside (SNP), and the alternative oxidase inhibitor, SHAM, prevents respiration, leads to a loss of viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo. We propose that SNP+SHAM treatment leads to transcriptional changes that drive cell wall re-arrangement but which also prime cells to activate transition to hyphal growth. In line with this we find that pre-treatment of C. albicans with SNP+SHAM leads to an increase in virulence. Our data reveals strong links between respiration, cell wall remodelling and activation of virulence factors. Our findings demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals, which are not damaging to the mammalian host, but that we need to develop a deeper understanding of the roles of mitochondria in cellular signalling if they are to be developed successfully as a target for new antifungals

Vomocytosis of live pathogens from macrophages is regulated by the atypical MAP kinase ERK5

Vomocytosis, or non-lytic extrusion, is a poorly understood process through which macrophages release live pathogens that they have failed to kill back into the extracellular environment. Vomocytosis is conserved across vertebrates and occurs with a diverse range of pathogens, but to date the host signaling events that underpin expulsion remain entirely unknown. Here we use a targeted inhibitor screen to identify the MAP-kinase ERK5 as a critical suppressor of vomocytosis. Pharmacological inhibition or genetic manipulation of ERK5 activity significantly raises vomocytosis rates in human macrophages whilst stimulation of the ERK5 signaling pathway inhibits vomocytosis. Lastly, using a zebrafish model of cryptococcal disease, we show that reducing ERK5 activity in vivo stimulates vomocytosis and results in reduced dissemination of infection. ERK5 therefore represents the first host regulator of vomocytosis to be identified and a potential target for the future development of vomocytosis-modulating therapies

Selected aspects of the treatment of epilepsy in the elderly.

Padaczka jest jedną z poważniejszych chorób neurologicznych, o charakterze społecznym. Na świecie na padaczkę choruje prawie 50 mln osób. W Polsce liczba chorych wynosi ok. 400 tys. Częstość występowania padaczki jest ściśle skorelowana z wiekiem. Największa zachorowalność przypada na dwa okresy życia człowieka. Pierwszą grupę stanowią małe dzieci, a drugą, osoby starsze po 60 roku życia. Właśnie na tę dekadę życia przypada ponad 50% rozpoznań nowych przypadków padaczki. Należy spodziewać się, że w najbliższych latach, w związku z wydłużaniem się długości życia populacji europejskiej, oraz wzrostem zapadalności na choroby naczyniowe i zwyrodnieniowe mózgu, osób z padaczką, zwłaszcza po 60 roku życia, będzie przybywać. Celem niniejszej pracy było omówienie wybranych aspektów leczenia padaczki u osób starszych. Analizowano problemy psychologiczno – społeczne i farmakologiczne u starszych osób z padaczką, w oparciu o największą bibliograficzną bazę danych na świecie, z zakresu medycyny oraz nauk pokrewnych (PubMed MEDLINE) oraz o zasoby biblioteki Instytutu Farmakologii PAN w Krakowie, oraz biblioteki Collegium Medicum UJ. Większość dostępnych danych wskazuje, że padaczka u osób w zaawansowanym wieku, stanowi istotny problem kliniczny. Trudności pojawiają się już na etapie diagnostyki napadów padaczkowych. Podstawę odpowiednio postawionej diagnozy, stanowi zebranie rzetelnego wywiadu od pacjenta, co niestety nie jest łatwe w przypadku osób starszych. Dzieje się tak, głównie ze względu na pojawiające się problemy z pamięcią, związane z postępującą demencją. Kolejnym ważnym problemem w diagnostyce padaczki w podeszłym wieku, jest współwystępowanie u tych osób, wielu epizodów mogących imitować napad padaczkowy. Stąd też w diagnostyce napadów u osób w starszym wieku, oprócz podstawowego badania – elektroencefalografii (EEG), pomocne są metody neuroobrazowania takie jak tomografia komputerowa, czy rezonans magnetyczny. Rozpoznanie padaczki w zaawansowanym wieku, wymaga wdrożenia odpowiedniego leczenia. W większości przypadków, skuteczne okazuje się leczenie farmakologiczne, przy pomocy odpowiednich leków przeciwpadaczkowych. Farmakoterapia musi uwzględniać zmiany fizjologiczne związane ze starzeniem się organizmu, gdyż mogą one w znaczący sposób wpływać na wahania stężenia leku przeciwpadaczkowego we krwi. Osoby starsze są grupą szczególnie wrażliwą, również ze względu na większą podatność na działania niepożądane, a także możliwość wystąpienia interakcji leków przeciwpadaczkowych z lekami stosowanymi na inne schorzenia. Z drugiej strony chorzy z padaczką w starszym wieku,spotykają się z przejawami dyskryminacji, napiętnowania i wykluczenia społecznego. Często mają problemy z prawidłowym funkcjonowaniem w codziennym życiu.Epilepsy belongs to the one of the main neurological diseases with great social matter. Globally almost 50 million people suffer from epilepsy. In Poland the amount of patients with this illness is estimated at 400 thousands. The incidence of epilepsy is strongly correlated with age. Probability of occurring the epilepsy falls into two periods of life: the first group are small children and the second group are people over 60 years old. In this decade of life more than 50% of new cases of epilepsy are diagnosed. In the coming years it is expected that the amount of patients suffering from epilepsy will be increased. It is connected with the lengthening of the life of the European population as well as with rise of the frequency of occurrence the incidence of vascular and degenerative diseases of the brain. The aim of this study was to discuss some aspects of the epilepsies treatment in group of elder patients. The psychological, social and pharmacological problems concerning treatment of elder people with epilepsy were analyzed based on the largest bibliographic database in the world in the field of medicine and related sciences (PubMed MEDLINE) and the library resources of the Institute of Pharmacology, Polish Academy of Sciences and the Jagiellonian University Collegium Medicum library. The most of the available data indicates that cure of epilepsy in people in advanced age, is an important clinical problem. The right diagnosis of seizures is the first main difficulty. The basis of appropriate diagnosis is to collect reliable information from the patient, which unfortunately is not easy in case of elderly people. This group of patients suffer also from memory problems and progressive dementia. Another important aspect of correct diagnosis in group of elder patients is occurrence of episodes that may mimic seizure. Thus, apart from elementary medical examination of Electroencephalography (EEG) more tested are necessary. Neuroimaging methods such as computed tomography or magnetic resonance imaging are very helpful. The diagnosis of epilepsy at an advanced age, allows for the implementation of appropriate treatment. For most of the patients the proper pharmacological treatment with anti-epileptic drugs is effective. Pharmacotherapy of epilepsy have to take into account the physiological changes connected with aging of the body because they may have a significant influence on antiepileptic drugs concentration in the blood. Elderpeople are a special group of patients, they are sensitive to side effects of drug activity and also to possibility of occurring interactions with drugs used for other diseases. What more, elder patients with epilepsy meet with discrimination, stigma and social exclusion. They have got problems with the proper functioning in everyday life

Polyphenol Supplementation and Antioxidant Status in Athletes: A Narrative Review

Antioxidants in sports exercise training remain a debated research topic. Plant-derived polyphenol supplements are frequently used by athletes to reduce the negative effects of exercise-induced oxidative stress, accelerate the recovery of muscular function, and enhance performance. These processes can be efficiently modulated by antioxidant supplementation. The existing literature has failed to provide unequivocal evidence that dietary polyphenols should be promoted specifically among athletes. This narrative review summarizes the current knowledge regarding polyphenols’ bioavailability, their role in exercise-induced oxidative stress, antioxidant status, and supplementation strategies in athletes. Overall, we draw attention to the paucity of available evidence suggesting that most antioxidant substances are beneficial to athletes. Additional research is necessary to reveal more fully their impact on exercise-induced oxidative stress and athletes’ antioxidant status, as well as optimal dosing methods

Impact of the DRD2 Polymorphisms on the Effectiveness of the Training Program

Dopamine receptor D2 gene (DRD2) polymorphisms have been associated with cognitive abilities, obesity, addictions, and physical-activity-related behaviors, which may underlie differences in the effectiveness of training programs. What is not yet clear is the impact of DRD2 polymorphisms on the effectiveness of exercise programs. Thus, the aim of this study was to investigate the association between the DRD2 polymorphic sites (rs1076560, rs12364283, rs1799732, rs1800497, and rs1800498) and the body’s response to regular physical activity. We studied genotypes and haplotypes distribution in a group of 165 females measured for body mass and body composition measurements, lipid profile, and glucose levels before and after realization of a 12-week training program. When tested individually, statistical analyses revealed one significant genotype by training interaction under the general model (for the basal metabolic rate, BMR, p = 0.033). Carriers of the rs1076560 CC genotype exhibited a decrease in BMR in response to training (p = 0.006). Haplotype analyses also showed that (i) the CACCC and CACTT haplotypes were associated with a post-training decrease in glucose level (β = −4.11, p = 0.032; β = −6.86, p = 0.020, respectively); (ii) the CGCCT with an increase in BMR (β = 0.65, p = 0.003) and fat free mass (FFM, β = 1.20, p = 0.009); (iii) the CA-CT with a decrease in low-density lipoprotein cholesterol (LDL, β = −17.26, p = 0.046). These results provide some evidence that the DRD2 polymorphisms may play a role in post-training changes in lipid and carbohydrate metabolism, and, as a consequence, in the effectiveness of training programs

Analysis of Industrial <i>Bacillus</i> Species as Potential Probiotics for Dietary Supplements

So far, Bacillus species bacteria are being used as bacteria concentrates, supplementing cleaning preparations in order to reduce odor and expel pathogenic bacteria. Here, we discuss the potential of Bacillus species as ‘natural’ probiotics and evaluate their microbiological characteristics. An industrial microbiological concentrate CS-4 of mixed Bacillus species cultures was tested, which may be a promising bacteria source for food probiotic preparation for supplementary diet. In this study, antagonistic activities and probiotic potential of Bacillus species, derived from an industrial microbiological concentrate, were demonstrated. The cell free supernatants (CFS) from Bacillus licheniformis mostly inhibited the growth of foodborne pathogenic bacteria, such as Escherichia coli O157:H7 ATCC 35150, Salmonella Enteritidis KCCM 12021, and Staphylococcus aureus KCCM 11335, while some of Bacillus strains showed synergistic effect with foodborne pathogenic bacteria. Moreover, Bacillus strains identified by the MALDI TOF-MS method were found sensitive to chloramphenicol, kanamycin, and rifampicin. B. licheniformis and B. cereus displayed the least sensitivity to the other tested antibiotics, such as ampicillin, ampicillin and sulfbactam, streptomycin, and oxacillin and bacitracin. Furthermore, some of the bacterial species detected extended their growth range from the mesophilic to moderately thermophilic range, up to 54 °C. Thus, their potential sensitivity to thermophilic TP-84 bacteriophage, infecting thermophilic Bacilli, was tested for the purpose of isolation a new bacterial host for engineered bionanoparticles construction. We reason that the natural environmental microflora of non-pathogenic Bacillus species, especially B. licheniformis, can become a present probiotic remedy for many contemporary issues related to gastrointestinal tract health, especially for individuals under metabolic strain or for the increasingly growing group of lactose-intolerant people

Alternative oxidase induction protects Candida albicans from respiratory stress and promotes hyphal growth

The human fungal pathogen Candida albicans possesses two genes expressing a cyanide-insensitive Alternative Oxidase (Aox) enzymes in addition to classical and parallel electron transfer chains (ETC). In this study, we examine the role of Aox in C. albicans under conditions of respiratory stress, which may be inflicted during its interaction with the human host or co-colonising bacteria. We find that the level of Aox expression is sufficient to modulate resistance to classical ETC inhibition under respiratory stress and are linked to gene expression changes that can promote both survival and pathogenicity. For example we demonstrate that Aox function is important for the regulation of filamentation in C. albicans and observe that cells lacking Aox function lose virulence in a zebrafish infection model. Our investigations also identify that pyocyanin, a phenazine produced by the co-colonising bacterium Pseudomonas aeruginosa, inhibits Aox-based respiration in C. albicans. These results suggest that Aox plays important roles within respiratory stress response pathways which C. albicans may utilise both as a commensal organism and as a pathogen