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Supplementary information for the article: Đurašinović, T.; Lopandić, Z.; Protić-Rosić, I.; Ravnsborg, T.; Blagojević, G.; Burazer, L.; Jensen, O. N.; Gavrović-Jankulović, M. Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals. International Journal of Molecular Sciences 2024, 25 (11), 5800. https://doi.org/10.3390/ijms25115800.
No full textSupplementary Table S2: A)List of T-cell epitopes in kiwifruit SAHH. B)List of T cell epitopes in latex SAHH.C)List of T-cell epitopes in ryegrass SAH
The beneficial effects of N-methyl-D-aspartate receptor antagonism on experimental autoimmune encephalomyelitis in aged rats
No full textPurpose: Life expectancy is only slightly affected by multiple sclerosis (MS), and it is a disease that often lasts for several
decades. The proportion of patients with late-onset MS has increased significantly and the mean age at diagnosis has
shifted towards older age. Older age at disease onset is associated with a shorter time to disability. Immune cells, such as
macrophages, microglia and lymphocytes, express N-methyl-D-aspartate receptors (NMDARs). Our aim was to elucidate
the effects of ageing on the role of NMDARs in the pathogenesis of experimental autoimmune encephalomyelitis (EAE).
Methods: Young adult (3-month-old) and aged (24-month-old) female Dark Agouti rats were used in this study.
Memantine, a non-competitive NMDAR antagonist, was administered by oral gavage for 7 consecutive days from the
first day post-immunization (dpi) in the first set of experiments or from the 7th dpi in the second set of experiments.
Mononuclear cells were isolated from the spinal cord or draining lymph nodes and analysed by flow cytometry. Spinal
cord tissue was collected for RT-qPCR.
Results: Administration of an NMDAR antagonist during the induction phase of EAE diminished the proportion of Th1
and Th17 cells and increased the proportion of IL-10+
regulatory T cells in the lymph nodes draining the site of
immunization in aged rats. Memantine increased the proportion of CD163+
and IL-10+
cells within CD11b+
cells in the
draining lymph nodes of aged rats, whereas these effects were absent in young rats. Treatment with memantine from the
7
th dpi resulted in a shift of microglia towards the anti-inflammatory M2 phenotype, characterized by an increase in the
expression of CD163 and a decrease in the expression of MHCII molecules, with an increase in the proportion of IL10+
microglia and the expression level of arginase 1 in the spinal cord of aged rats at the peak of the disease.
Conclusion: NMDARs contribute significantly to the pathogenesis of EAE in aged rats in both the induction and effector
phases. Our results suggest that targeting NMDARs in elderly MS patients may help to tailor treatment to the elderly MS
population
Micronutrient supplementation supports immune response to seasonal influenza vaccine in mice
No full textPurpose: Although nutritional gaps are prevalent in several micronutrients reported to support immune function, the
significance of their deficits/supplementation for efficacy of vaccines, particularly those with low efficacy, as it is seasonal
influenza vaccine, has not been fully investigated, yet. The present study examined influence of supplementation
combining vitamins C and D, oligoelements zinc, selenium and manganese, and N-acetyl-cysteine on germinal centre
(GC) and serum IgG responses to seasonal quadrivalent influenza vaccine (QIV) in mice.
Methods: Study encompas female BALB/c mice that were given QIV in two doses (28 days
apart). The supplementation started five days before the first injection. Phenotypic and
functional characteristics of cells from secondary lymphoid organs (SLOs) (draining lymph
nodes and spleens) were analyzed by flow cytometry, whereas serum IgG response to QIV and
levels of cytokines from SLO cell cultures were determined by ELISA. Redox status
parameters were evaluated in spleens by spectrophotometric methods.
Results: The supplementation increased the magnitude of serum IgG response 28 days post the first QIV dose, through
stimulation of GC reaction in SLOs, as indicated by increase in the frequency of GC B cells and follicular CD4+ T helper
(Th) cells, and Th cell IL-21 production. Additionally, 14 days following the second QIV dose the supplementation
supported more favorable (viz. more effective in the context of virus infection clearance) IgG2a response through favoring
Th1 response. This could be ascribed not only to its indirect action related to antioxidant properties (confirmed by redox
status analyses), but also to direct action on Th cell differentiation, as indicating by the ratio in production levels of Th1
(INF-γ) /Th2 (IL-4) signature cytokine ratio upon QIV restimulation in SLO cell cultures.
Conclusion: Thus, the study forms solid base for further studies aimed at repurposing use this safe and inexpensive
micronutrient preparation as an adjuvant for virus influenza vaccine and possible some other virus vaccine
Evaluation of Staphylococcal superantigen-like 7 from Staphylococcus aureus for human serum IgA purification
No full textImmunoglobulin A (IgA) is a crucial antibody class at mucosal surfaces. In serum it remains underexplored. This study focuses on developing an in house method for the purification of serum IgA
using the Staphylococcal superantigen-like 7 (SSL7) protein derived from Staphylococcus aureus.
Previous findings suggest our clinical isolate of S. aureus binds human IgA, potentially due to the
surface presence of SSL7. To utilise this fact for IgA purification the SSL7 gene was PCR amplified and
cloned into pQE-Ek expression vector. The sequence identity was confirmed by Sanger sequencing.
Computated protein parameters were Mw 25170,3 Da and pI 8.74. High levels of soluble His-tagged
SSL7 protein were expressed and purified using metal-affinity chromatography from the bacterial
cell lysate. Purified 25 kDa protein was immobilized on a commercial Sepharose matrix. Application
of this matrix demonstrated specific binding, evidenced by protein bands corresponding to immunoglobulin heavy and light chains, alongside a minor fraction (9,7%) of non-corresponding proteins. Western blot analysis confirmed preference for IgA binding over the binding of IgG and IgM.
This study demonstrates the potential of using SSL7 from S. aureus as an affinity ligand for the selective purification of IgA from human serum, but to obtain IgA of higher purity this step should be
followed by protein G affinity purification. Given the known affinity of SSL7 for both human IgA and
complement component C5, future modifications will include site-specific mutagenesis to abolis
NMDA Receptor Antagonist Memantine Ameliorates Experimental Autoimmune Encephalomyelitis in Aged Rats
No full textAging is closely related to the main aspects of multiple sclerosis (MS). The average age of the MS population is increasing and the number of elderly MS patients is expected to increase. In addition to neurons, N-methyl-D-aspartate receptors (NMDARs) are also expressed on non-neuronal cells, such as immune cells. The aim of this study was to investigate the role of NMDARs in experimental autoimmune encephalomyelitis (EAE) in young and aged rats. Memantine, a non-competitive NMDAR antagonist, was administered to young and aged Dark Agouti rats from day 7 after immunization. Antagonizing NMDARs had a more favourable effect on clinical disease, reactivation, and apoptosis of CD4+ T cells in the target organ of aged EAE rats. The expression of the fractalkine receptor CX3CR1 was increased in memantine-treated rats, but to a greater extent in aged rats. Additionally, memantine increased Nrf2 and Nrf2-regulated enzymes’ mRNA expression in brain tissue. The concentrations of superoxide anion radicals, malondialdehyde, and advanced oxidation protein products in brain tissue were consistent with previous results. Overall, our results suggest that NMDARs play a more important role in the pathogenesis of EAE in aged than in young rats
Electrospun Gelatin Scaffolds with Incorporated Antibiotics for Skin Wound Healing
No full textRecent advances in regenerative medicine provide encouraging strategies to produce artificial skin substitutes. Gelatin scaffolds are successfully used as wound-dressing materials due to their superior properties, such as biocompatibility and the ability to mimic the extracellular matrix of the surrounding environment. In this study, five gelatin combination solutions were prepared and successfully electrospun using an electrospinning technique. After careful screening, the optimal concentration of the most promising combination was selected for further investigation. The obtained scaffolds were crosslinked with 25% glutaraldehyde vapor and characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. The incorporation of antibiotic agents such as ciprofloxacin hydrochloride and gentamicin sulfate into gelatin membranes improved the already existing antibacterial properties of antibiotic-free gelatin scaffolds against Pseudomonas aeruginosa and Staphylococcus aureus. Also, the outcomes from the in vivo model study revealed that skin regeneration was significantly accelerated with gelatin/ciprofloxacin scaffold treatment. Moreover, the gelatin nanofibers were found to strongly promote the neoangiogenic process in the in vivo chick embryo chorioallantoic membrane assay. Finally, the combination of gelatin’s extracellular matrix and antibacterial agents in the scaffold suggests its potential for effective wound-healing treatments, emphasizing the importance of gelatin scaffolds in tissue engineering
Teucrium montanum extract ameliorates collagen-induced arthritis in rats
No full textPurpose: Traditional herbal plants attract atention as potential therapeutics in the treatment of arthritis. Teucrium
montanum L. (TM) is a widely distributed plant in almost all Mediterranean countries. The therapeutic potential of TM
extract is insufficiently examined, but its composition implies potential anti-inflammatory effect. We tested amelorating
effects of TM extract on rheumatoid arthritis (RA), in rat collagen-induced arthritis (CIA) model.
Methods: Phytochemical analysis of TM methanol extract was performed using LC-DAD-ESI-MS. Female Dark Agouti
rats were immunized with bovine type II collagen (CII) in incomplete Freund`s adjuvant to induce CIA. CIA rats were
treated with TM extract daily per os (100 or 200 mg/kg) from day when the first signs of disease started. Clinical
evaluation of CIA was performed by scoring and histological analysis. Phenotypic and functional characteristics of
splenocytes and draining lymph nodes (dLNs) cells were analyzed by flow cytometry. Cytokine levels in supernatants
from hind paw tissue culture, and IgG CII-specific antibodies level in blood were determined by ELISA.
Results: TM extract contained phenylethanoid glycosides, mainly verbascoside, phenolic acids, flavonoid and iridoid
glycosides. Applied TM extract doses significantly reduced arthritic score and severity of ankle joint inflammation in CIA
rats. This was accompanied by the more pro-oxidant profile in serum, and shifted pro-inflammatory (TNF-α and IL-6)
and anti-inflammatory (IL-10) cytokines balance toward later in paws of treated rats. This was in conjunction with less
inflammatory phenotype of dLN and spleen CD11b+ cells (monocytes/macrophages) judged by their lower expression of
CD86, MHCII, and TLR-4, and lower production of TNF-α and IL-1β. The frequency of TCR+ cells, and IL-17- and
IFN-γ- producing CD4+ cells were lower in dLNs and spleen, while frequency of regulatory CD4+CD25+FoxP3+ cells
was higher. The lower frequency of CD45RA (B cells) in dLNs and spleen was accompanied with lower levels of serum
anti-CII antibodies in treated rats.
Conclusion: These findings suggest, for the first time, that TM extract effectively ameliorated clinical presentation of RA
in model of rat CIA, and that this therapeutic effect may be associated with its immunoregulatory/anti-inflammatory
action
Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination
No full textTranscription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.Supplementary information: [https://intor.torlakinstitut.com/handle/123456789/866
Description of a new potential aggregation factor from the Streptococcus thermophilus genome
No full textAutoaggregation, the ability to self-aggregate, is widespread among both Gram-positive and Gram-negative bacteria. The functional role of aggregation is not fully understood, but it is believed to be involved in the adaptation of bacteria to environmental conditions (PMID: 31294207). One interesting class of compounds responsible for the aggregation of lactic acid bacteria is aggregation factors—surface high-molecular-weight proteins rich in threonine and lysine (PMID: 30027759). Recently, our research group discovered a new strain of Streptococcus thermophilus in the mountainous regions of Serbia, exhibiting an aggregation phenotype. Aggregation phenotype was confirmed visually and using microscopy. Complete genome of Agg+ strain was sequenced using NGS and a gene encoding a potential aggregation factor, which was named aggS was identified. The predicted threonine (12.5%) and lysine (10.5%) rich protein contains 2367 amino acids, with an average molecular weight of 255986.63 Da. AggS also contains two cysteine residues, whereas previously well-described aggregation factors of this type did not contain any cysteine residues. The predicted protein includes an N-terminal YSIRK-like signal sequence and an LPXTG cell wall anchor domain. It has 6 Mucin binding domain repeats alternating with 6 Mub B2-like domain repeats. Additionally, we found a region resembling an ice-binding domain. Given that these bacteria endure prolonged periods of low temperatures, it can be speculated that this surface membrane protein also helps the bacteria withstand freezing. The fact that the alignment using BLASTp revealed AggS to be most closely related to an uncharacterised protein from the genome of Lactococcus garvieae, along with the discovery of a transposase gene sequence upstream of the gene, suggests that the aggregation factor was likely acquired through horizontal gene transfer. We plan to clone it into a shuttle vector and investigate the aggregation phenotype using a heterologous expression system in Lactococcus lactis, as well as explore its other functions
Pertussis vaccine overview
No full textPertussis or whooping cough is a highly contagious, vaccine-preventable, respiratory disease caused by Bordetella pertussis, and transmitted through the respiratory tract. According to the reports of the World Health Organization and Centers for Disease Control and Prevention, the incidence of pertussis shows periodical variations in certain regions of the world. As humans are the sole reservoir of this bacteria complete vaccination against pertussis and high vaccination coverage is of utmost importance for reducing the incidence and severity of the disease. Two types of pertussis vaccine are available: wholecell (wP) and acellular pertussis vaccine (aP). wP contains whole nonviable bacteria, while aP usually contains two or more protein components. These protein components include inactivated pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae. The acellular vaccine was developed in response to reports of adverse reactions upon administering the whole-cell vaccine in certain countries. Both vaccines are usually formulated with diphtheria and tetanus toxoids, and more recently a trend of combining more antigenic sources such as Haemophilus influenzae type b, hepatitis B, and inactivated poliovirus vaccine has been accepted in many countries, including Serbia. The wP vaccine stimulates a strong immune response more similar to infection, while the response to aP vaccine differs in this respect. Due to the difference in the types of immune response predominating with different types of pertussis vaccines, there are differences in the duration of protection, and it has been reported that wP induces more durable protection. For countries that have adopted aP increased monitoring is advised as well as the inclusion of booster doses. The special focus is on the vaccination of pregnant women to protect the newborns. Incited by the recent surge in pertussis cases in Serbia here we provide a comprehensive literature overview of pertussis vaccines, covering their historical development, current status, challenges, and potential future directions