Achieved within the project (01.04.2022 – 31.03.2024)

Summary of the project’s theme

According to the hygiene hypothesis, the increasing prevalence and incidence of autoimmune diseases such as multiple sclerosis (MS), particularly in industrialized countries, results from an underdeveloped immune system during the formative years. Although the studies in this aspect have not established a clear connection for the pathogenesis of MS, it prompted the investigations in intestinal microbiota and the fermentation metabolites of the commensal bacteria. The gut bacteria ferment dietary fibers to produce short and medium fatty acids (SCFA/MCFA). SCFA have demonstrated immunomodulatory properties and can transverse the blood-brain barrier and hypothetically exert their effects inside the central nervous system, by modulating inflammation and promoting neuroplasticity and remyelination. In the periphery, SCFA promote the differentiation of regulatory T lymphocytes and secretion of anti-inflammatory cytokines such as interleukin – 10. MCFA, such as caproic acid, have contrasting, pro-inflammatory effects depending on the microbial pattern of the patients. Caproic acid promotes the immune differentiation toward a pro-inflammatory phenotype, Th1 and Th17, with secretion of proinflammatory cytokines. The selective permeability of the blood brain barrier ensures that immune cells have no way of passage into the brain parenchyma. In MS, the blood-brain barrier permeability is altered and inflammatory cells pass through, exerting the inflammatory cascade leading to demyelination, neurodegeneration and axonal loss. Matrix metalloproteinases (MMPs) are a set of enzymes involved in the maintenance and structure of the endothelial cells that compose the blood-brain barrier. MMP9 in particular has been linked with destructive effects on the blood-brain barrier and also the intestinal barrier.

The gut brain axis represents a dynamic communication between the intestinal tract and the central nervous system, functioning as a bidirectional signaling pathway through which the enteric system influences the brain and vice-versa. The central element of this ecosystem is represented by the intestinal microbiota. The direct connection between the intestinal compartment and the brain is partially mediated via the vagus nerve. One of the most important neuromodulators involved in this bi-directional communication is brain-derived neurotrophic factor (BDNF), which modulates the excitability of the vagal neurons. BDNF is also highly influenced by the SCFA, particularly butyric acid. The experimental approach of the intestinal microbiota is challenging due to the multitude of short-term influencing factors such as alcohol consumption, diet, treatment with pre/pro biotics, stress, unhealthy diet, etc.

Therefore, the present project proposed two important directions of research:

1. The analysis of inflammation biomarkers from a quadri dimensional perspective (see schematic below):

   • Intestinal compartment: assessment of peripheral levels of SCFA/MCFA

   • Interaction between the gut and the brain via the vagus nerve: BDNF

   • Pro and anti-inflammatory cytokines

   • Integrity of the blood-brain barrier: MMP

2. Implementing and validating a working method by liquid chromatography-mass spectrometry (LC-MS/MS) to assess SCFA and MCFA from serum samples

Concrete achievements

1. Implementation and validation of a working method for LC-MS/MS which allowed for the analysis of SCFA and MCFA from human serum, namely acetic acid, propionic acid, butyric acid and caproic acid, metabolites of the human microbiome with demonstrated immunomodulatory effects in MS. The method was developed and applied in the Center for Advanced Medical and Pharmaceutical Research, CCAMF (https://eeris.eu/ERIF-2000-000C-0801) from the George Emil Palade University of Medicine, Pharmacy, Science and Technology of Targu Mures.

The development and validation of a selective, sensitive, precise, and accurate LC-MS method within this project enable the conduct of various types of future clinical, preclinical, biomonitoring, or metabolomic studies at reduced costs and with short analysis times, also allowing for efficient planning of such studies.

2. Publications: (1) ISI-Q1 (published – review): Barcutean L, Maier S, Burai-Patrascu M, Farczadi L, Balasa R. The Immunomodulatory Potential of Short-Chain Fatty Acids in Multiple Sclerosis. Int J Mol Sci. 2024; 25(6):3198. https://doi.org/10.3390/ijms25063198. FI: 5.6.; (2) ISI-Q2 (published – original research): Barcutean L, Maier S, Bajko Z, Stoian A, Mosora O, Sarmasan Emanuela, Manescu I-B, Balasa R. Long-Term Disability Outcomes in Relapsing–Remitting Multiple Sclerosis Patients: Impact of Clinical and Demographic Factors on Disease Progression. J Clin Med. 2024; 13:1813. https://doi.org/10.3390/jcm13061813, IF: 3.9.; (3) ISI-Q1 (under review): sent to IJMS, manuscript ID 2956963, title: Peripheral fatty acids dynamics in response to interferon beta treatment in naïve multiple sclerosis patients: insights into inflammation, gut-brain connection and blood-brain permeability.

3. Organisation of a special session dedicated to microbiota within an international conference:

1. • Microbiome alterations and implications in patients with multiple sclerosis – targeting neuroinflammation from the intestinal compartment; International Conference: Toward Precision Medicine in Multiple Sclerosis Management, 7-8 Oct 2022, Targu Mures, Romania

     • Presented papers:

       1. Rodica Balasa: Gut-brain axis: is MS a digestive triggered disease?

       2. Anca Negovan: Environmental risk factors altering the intestinal microbiota

       3. Monica Tarcea, Gliga Florina: Diet and gut microbiota implications in multiple sclerosis

       4. Laura Barcutean, Lenard Farczadi, Rodica Balasa: Potential mechanisms of immune modulation targeting the intestinal microbiota

       5. Adina Hutanu: Inflammatory biomarkers for microbiota dysregulation in multiple sclerosis

       6. Farczadi Lenard, Laura Barcutean, Silvia Imre: The role of liquid chromatography-mass spectrometry in modern medicine – assessing the impact of microbiota by short-chain fatty acids assay

4. Active presentations in national and international conferences in the field of research:

   • Oral presentation in the 10^th World Congress of the International Society of Microbiota: Targeting Microbiota, Towards Clinical Revolution, Venice, 17-19 Oct, Italy with the paper: “The role of microbiome in multiple sclerosis: a multidimensional approach”

   • Oral presentation in the Scientific Session of University Academic Staff with the paper “The role of brain-derived neurotrophic factor in interferon beta-treated multiple sclerosis patients”, 11-15 Dec, Targu Mures, Romania

   • Accepted abstract (poster, will be presented) in the 10^th Congress of the European Academy of Neurology, 29 Jun – 2 July, Helsinki, Finland with the paper: “Brain-derived neurotrophic factor and progression in multiple sclerosis.”

   • Accepted abstract (oral presentation) in the The 22nd Congress of the Romanian Society of Neurology, 5-7 June 2024 with the paper: “Microbiota and Multiple Sclerosis”.

5. Student’s master and bachelor’s degree

   • I coordinated the bachelor’s degree for the student Andrei Manea, Faculty of General Medicine, within George Emil Palade University of Medicine, Pharmacy, Science and Technology, with the title “Implications of nutrition in the evolution of patients with multiple sclerosis”. The thesis was presented in July 2022, final grade 10.

   • I am coordinating a master’s thesis for the student Andreea Kelemen, master student in the program Clinical and Community Nutrition, within the George Emil Palade University of Medicine, Pharmacy, Science and Technology, with the title “Dietary Considerations for Individuals with Multiple Sclerosis”.

6. Student dedicated workshop

   • I coordinated the first workshop for medical students within the XXVII^th International Congress for Students, Young Doctors and Pharmacists, Mariensis, in Targu Mures regarding the implications of gut-brain axis in demyelinating diseases, with a focus on MS, entitled: “Gut-brain axis. The hidden agenda of autoimmune disorders – focus on multiple sclerosis”, which took place on 17-21 may 2023, Targu Mures, Romania

   • The 2^nd workshop entitled “Microbiota in multiple sclerosis: truth or dare?” has been announced and will be held on 23^th of May, in Targu Mures, Romania, within the XXIII^th International Congress for Students, Young Doctors and Pharmacists, Marisiensis.