National Science Centre

In this application, studies are planned to assess the degree of metabolic differentiation in P. aeruginosa strains isolated from CF patients at different stages of the disease. This will allow the estimation of the relationship between the evolution of strains leading to the acquisition of multi-drug resistance, the mucus phenotype and the ability to create biofilm, and their metabolic properties, in relation to the ability to use specific carbon and nitrogen sources. The phenotypic microarray technique proposed for the research, due to the possibility of global and simultaneous analysis of the metabolism of various substrates, will allow the comparison of metabolic pathways in P. aeruginosa isolated from patients at various stages of CF. Confirmation of the research hypothesis regarding the specific relationships between the development of characteristic phenotypes of P. aeruginosa in patients chronically suffering from CF and the ability to metabolize certain carbon and nitrogen sources will be important not only for understanding the pathogenesis process itself, but may also open the perspective for the development of new drugs to fight these pathogenic microbes. In addition, the possibility of implementing the proposed research activity will have a significant impact on the scientific development of the Lead, and will enable the planning of further research related to the characteristics of these pathogenic strains, for the implementation of which the Lead will try to obtain funds from subsequent grants. The obtained results, enriched with the previously unpublished results of the preliminary research conducted in the Lead's research group, will most likely be published in a reputable scientific journal, which will also be of great importance for the Lead's scientific path. Additionally, the research cooperation established at IBB will provide the Lead with new skills and opportunities that she will be able to use in her further work.

The leaky gut syndrome (LGS) is a combination of many disorders in the human body resulting from disturbance of the
intestinal barrier's selective permeability. Increased permeability enables penetration of undesirable particles, such as
toxins, outside the intestine lumen. These particles can be hazardous to health and may cause symptoms that reduce
the quality of life (e. g. flatulence, diarrhoea or constipation, vomiting, chronic fatigue, and headache). The cause of LGS
is unknown, but a significant impact of poor diet or lifestyle and infections of enteropathogens, especially
enterotoxigenic E. coli (ETEC), is suspected. The existing methods of therapy for the LGS are inefficient and insufficient.
Therefore, LGS is a subject over which numerous studies are conducted, and new approaches are sought.
Tormentillae rhizoma (TR) is a traditionally used pharmacopeial plant material with a wide range of applications mainly
related to gastrointestinal (GI) tract ailments. The herb originates from Tormentillae erecta (TE), from the Rosaceae
family, which is a widespread species in Eurasia. TR is taken orally in a form of decoctions and infusions, but the most
popular preparation is a tincture (Tormentillae tinctura (TT)). Orally, TR is used for the symptomatic treatment of
dysentery, diarrhoea, poisoning, ulcerative colitis, inflammation of GI.
TR has been used for centuries for treatment of GI tract infections, and its effectiveness has been clinically confirmed.
Despite attributed properties and observed bioactivity, the mechanisms responsible for the beneficial effects of TR in GI
tract ailments have not been yet fully determined. What is more, the chemical principles triggering biological effects are
not unambiguously indicated, especially that from a chemical perspective TR is a very interesting plant material
containing a wide variety of specialized metabolites, which can contribute to its therapeutic properties. What is more, as
medicine taken orally and with activity related to GI tract, its interaction with gut microbiota should be prioritized while
designing studies on its biological properties. The questions of Tormentillae tinctura metabolites (TTM) (TT constituents
metabolized by human gut microbiota) as well as its impact on microbiota homeostasis should be addressed. Despite
crucial role of intestinal epithelium and microbes residing in its lumen, there are no reports about the mechanism of
action of TT and interactions between TT and human gut environment.
In recent years, the significant contribution of gut microbiota in human organism homeostasis is observed. At first, the
microbiota is co-responsible for the intestinal barrier's selective permeability and is a source of Pathogen Associated
Molecular Patterns (PAMPs) migrating through the imbalanced epithelium, which then interact with hosts immune
system. Moreover, the bacteria present in the gut can convert orally taken compounds which can then be absorbed into
the bloodstream and interact with the biochemical processes. For this reason, the final properties of orally applied
preparations containing plant materials may differ from the properties of the compounds which are initially present in
them. On the other hand, orally applied therapeutics may alter the composition of human gut microbiota. The positive
outcome of this activity may include increasing the gut microbiota diversity and acting as probiotics, whereas the
negative outcome may be displayed as microbiome imbalance (dysbiosis). The negative impact on microbiota
communities can be observed in applied therapies of LGS, which are currently based on antibiotics and
chemotherapeutics and result in gut microbiota imbalance leading to significant long-term side effects.
The main aim of the submitted project is to justify the use of TT in the treatment of the leaky gut syndrome.
Hypotheses:
H1. TT contains condensed and hydrolyzable tannins contributing to its therapeutic properties.
H2. Chemical constituents of TT are metabolized by human gut microbiota and beneficially influence its diversity ex vivo.
H3. TT and TTM inhibit the growth of enterotoxigenic E. coli (ETEC) and Clostridium sp.
H4. TT and TTM influence the Caco-2 cells monolayers integrity and permeability parameters.
The studies to confirm the stated hypotheses will be carried out in cooperation between Microbiota Lab (Centre for
Preclinical Studies, Medical University of Warsaw and Institute of Animal Nutrition (Freie Universität Berlin).

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes severe pandemic COVID-19. When activation of
the innate immune system fails to eliminate pathogen or to induce adequate adaptive response, persistent
inflammation, manifesting by cytokine storm, ARDS and organ failure may develop. The Spike glycoprotein (S-gp) is a
glycosylated trimeric protein protruding from the viral surface, highly homologous with that of SARS-CoV-1. The
complement system is a major branch of immune response, cross-talking with various mechanisms of immunity, both
innate and adaptive. We hypothesize that SARS-CoV-2 S-gp exposed N-glycans may be recognized by complement lectin
pathway (LP)-activating collectins and/or ficolins – pattern recognition molecules with affinity to carbohydrates. We
based our hypothesis on published data, concerning SARS-CoV-1 and SARS-CoV-2/COVID-19: 1. SARS-CoV-1 S-gp is as
target for mannose-binding lectin (MBL) and surfactant protein D (SP-D) 2. Glycosylation site of S-gp at Asn343 is
essential for MBL binding 3. Data from murine SARS-CoV-1 model showed a crucial role of the complement in the
development of cytokine shock and ARDS 4. Complement activation contributes to the COVID-19 and higher MBL serum
levels are associated with severe adverse effects in patients 5. Inhibition of complement (also with LP-inhibitors gives
promising effects of treatment 6. High expression of MBL and deposition of MASP-2 in lung tissues from COVID-19
patients.
Interaction of complement with S-gp may be essential for clearance of infection but on the other hand, it may hinder
the access of neutralizing antibodies to the viral epitopes. Furthermore, complement over-activation may lead to lifethreatening
events. To determine the role of S-gp glycosylation pattern in the interaction with these factors, we intend
to test the binding of recombinant and natural (complexed with MASP serine proteases, able to activate complement)
lectins MBL, collectin liver-1 (CL-L1), collectin kidney-1 (CL-K1) and ficolins: ficolin-1, -2, -3. to recombinant SARS-CoV-2
S-gp and its glycosylation variants, as well as investigate whether lectin-glycoprotein interaction leads to complement
activation. Furthermore, complement non-activating collectin SP-D will be included. We plan to express five
glycoproteins: 1. wild-type SARS-CoV-2 S-gp; 2. S-gp only with high-mannose N-glycans; In addition, to examine the role
of particular N-glycans, we will express 3 variants substitution at N-glycosylation sites: Asn343; Asn234; Asn801 as well
as 3. wild-type SARS-CoV-1 S-gp (control). These products will be used for analysis of glycopeptides and glycans derived
from the S-gp and its glycosylation variants, and for evaluation of the role of N-glycans in SARS-CoV-2 recognition by
selected lectins and SARS-CoV-2 specific antibodies.
Although recombinant antigens are widely used in vaccines/diagnostics, to confirm the functionality of our preparations,
we will test their reactivity with commercially available anti-S-gp antibodies as well as with antibodies present in sera
from patients diagnosed with paediatric inflammatory multisystem syndrome – temporally associated with SARS-CoV-2
(PIMS-TS). The usage of such sera will allow also determination of S-gp-specific antibody profile in patients and to
investigate an interplay between complement and antibodies. We intend to answer the following questions: 1. Which
PRM, specific for the lectin pathway (or SP-D) bind SARS-CoV-2 S-gp glycans? 2. What is the molecular basis of such
interaction? 3. Does it lead to complement activation? 4. Does it inhibit specific/cross-reacting antibody binding? 5. Do
antibodies raised in response to SARS-CoV-2 infection modify interaction of lectins with S-gp? 6. Are investigated factors
involved in pathogenesis of PIMS-TS?
The use of defined S-gp glycosylation variants may shed more light on complement activation and reveal the underlying
molecular mechanisms. The project should contribute to better understanding of the COVID-19 and PIMS-TS
pathogenesis by exploring early immune response: interaction of N-glycans of S-gp with the complement. The outcome
of the project may significantly influence the development of disease diagnostics (based on patients’ genotype related
to the complement), treatment (supplementation/inhibition therapy regarding the complement system) and prevention
(vaccination strategies based on the identified glycans).

Immunotherapy is currently considered as one of the most exciting areas in cancer therapy, however it still needs
further improvement. The failure to prevent, control and completely eradicate malignancies by infiltrating immune cells
can be exacerbated by the metabolic flexibility of neoplastic cells. All forms of immunotherapy, including immune
checkpoint inhibitors (ICIs), CAR-T and CAR-NK cells are likely to be impacted by the build-up of specific metabolites and
signaling molecules in the tumor microenvironment (TME). Transcriptomics analysis of TCGA data revealed that in many
cancer types high expression of glutaminase (GLS) is usually associated with poor survival rate. GLS is an enzyme that,
together with glutamate dehydrogenase, metabolize glutamine into alpha-ketoglutarate and 2 molecules of ammonia.
Ammonia, a waste byproduct of amino-acid metabolism, tends to accumulate in the TME at supra-physiological
concentrations, 50-100x higher than the level of ammonia in plasma. Therefore, I hypothesized that a TME rich in
ammonia would be more immunosuppressive. My preliminary data support the idea that ammonia is an
immunosuppressive factor since it strongly inhibits the cytotoxic activity of NK cells toward target cancer cells and may
ultimately impact all forms of cancer immunotherapies. The research proposal will tackle the following questions:
1) How could immune effector cells, such as NK cells and cytotoxic T cells (CTLs) sense ammonia?
2) How can we prevent the accumulation of ammonia in TME and recover the cytotoxic function of immune cells?
3) Will the reduction in ammonia production stimulate anti-tumor immunity and lead to tumor growth inhibition?
In order to uncover the ammonia’s mechanism of action, I have already tested if ammonia affects the release of
cytolytic granules by effectors cells. I found that degranulation process is not inhibited by ammonia. Therefore, we plan
to carry out a proteome and secretome analysis of ammonia-treated cytotoxic NK and T cells. We may find changes in
the levels of activating or inhibitory receptors, lytic enzymes or secreted cytokines, since they all are necessary for
efficient killing of target cancer cells. Additionally, ammonia was reported to induce autophagy in cancer cells. Indeed, I
found that ammonia upregulates the level of Sequestosome-1/p62, a cargo receptor for the autophagic degradation of
proteins. Therefore, we plan to confirm the hypothesis that ammonia inhibits cytotoxicity function of NK and CTLs by
inducing autophagy. Recent reports also showed that function of CTLs can be suppressed by increased concentration of
extracellular potassium (K+) ion, due to autophagy. Thus, we plan to measure extracellular K+ concentration,
accompanied with metabolome analysis of ammonia-treated NK and CTLs.
In order to inhibit the build-up of ammonia in TME, we will test several pharmacological metabolism modulators that:
i) inhibit GLS and glutamine transporters SLC1A5 and SLC7A5 in cancer cells, previously screened for high expression of
GLS and ammonia production;
ii) activate the carbamoyl phosphate synthase-1 to trap ammonia in the urea cycle using carglumic acid, an FDAapproved
treatment for hyperammonemia.
The efficacy of these drugs on the production of ammonia and the cytotoxic activity of NK cells and CTLs will be
evaluated. Lastly, we will evaluate in vivo efficacy of the most efficient metabolism modulators that were able to reduce
the ammonia production by cancer cells. Their efficacy in reducing ammonia levels in tumor interstitial fluids, tumor
infiltration by CTL and NK and tumor growth will be tested in the syngeneic mouse model. The selected compound, that
achieved the best control of tumor growth will be next combined with immune checkpoint inhibitors (ICIs), such as anti-
PD1 or anti-CTLA4, to test whether the inhibition of ammonia build-up in tumors enhances the response of the tumors
to immunotherapy.
Finally, the implementation of the project will provide the necessary evidence regarding the role of ammonia in inducing
immunosuppression and resistance to immunotherapeutics. The results will demonstrate its mechanism of action and
provide proof of concept that abrogation of ammonia accumulation in the TME could be a beneficial approach for
effective immunotherapy.

The aim of this study is to check the presence and dynamics of changes in new biomarkers in urine and blood in the early stage of acute kidney injury in hematooncological patients and to develop an artificial intelligence algorithm that allows for faster and more effective detection of this syndrome based on the collected data. One of the most promising biomarkers of kidney damage are circular RNAs, which, due to their structure, are highly stable in the urine and blood of patients, which makes them easier to detect. The research planned in the project will allow to determine whether the selected panel of biomarkers and the designed algorithm are effective in this group of patients. Based on the available literature and scientific research, the project included biomarkers that were found in the urine or blood of patients with kidney damage in the course of cancer.

Primary Sclerosing Cholangitis (PSC) is an increasingly recognized chronic liver disease, currently defined as one of the greatest challenges of modern hepatology. In its course, as a result of not fully understood mechanisms, the bile ducts are damaged. The disease may be accompanied by a number of troublesome ailments, including chronic fatigue, persistent itching of the skin, recurrent episodes of cholangitis threatening sepsis. The disease is associated with a significantly increased risk of an aggressive neoplasm such as bile duct cancer. The current pharmacological treatment options for PSC are significantly limited. The effectiveness of the only drug used, which is ursodeoxycholic acid (UDCA), remains controversial.
S-adenosylmethionine (SAMe) is a precursor of two very important metabolic pathways (transmethylation and transsulfuration) improving the elimination of toxic compounds from the body. The end product of SAMe metabolism is glutathione, one of the key antioxidant compounds. Chronic liver diseases lead to acquired SAMe deficiency, which can significantly aggravate the already impaired liver function. Unfortunately, in the world literature there are no well-planned, randomized trials with the use of SAMe in cholestatic diseases, therefore this preparation is still awaiting its place in the treatment of liver diseases.
Our previous studies have demonstrated the additive beneficial effect of SAMe in combination with UDCA in various experimental cholestasis models. In our recent MAESTRO research, conducted in patients with another cholestatic disease, which is Primary Biliary Cholangitis (PBC), we showed not only an improvement in the biochemical parameters of liver function, but, very importantly, a significant improvement in the quality of patients' lives. Our subsequent research, also within the MAESTRO project, has shown that SAMe can also inhibit the production of autoantibodies that lead to biliary damage.
The project is divided into two parts: clinical and laboratory.
In the clinical part, the effect of SAMe treatment will be assessed in a group of 60 patients with stable PSC. The study will be randomized and will last 6 months. SAMe and placebo will be purchased commercially. Patients will receive either UDCA + SAMe or UDCA + placebo for a period of 6 months. The influence of SAMe on clinical, biochemical and serological parameters as well as the result of the elastographic examination of the liver will be analyzed. The effects of SAMe therapy on quality of life, which is quite often severely impaired in PSC patients, will be analyzed in great detail. This is an area of great importance, but unfortunately neglected for years in the context of autoimmune liver diseases. The quality of life study will be carried out with the use of a series of well-verified questionnaires, assessing, among others, symptoms of chronic fatigue, itching, depression and anxiety. This issue has been the subject of our interest for many years, which is confirmed by numerous publications of our team in recent years. The results of the study will help to define the role of SAMe in the treatment of PSC and therefore could be directly translated into clinical practice.The aim of the laboratory part is to expand our knowledge about the mechanisms of hepatoprotective action of SAMe and it will be a continuation of our previous research, financed, among others, from the NCN MAESTRO grant. Using biochemical, molecular methods and tandem liquid chromatography in mass spectroscopy (LC-MS / MS), we will deepen the scope of our still incomplete knowledge of the antioxidant role of SAMe and its potential protective activities as a sulfone and methyl donor group. Both parts of the project are complementary and should significantly contribute to a better understanding of the protective mechanisms in the liver.