Assessment of the immunomodulatory properties of the probiotic strain Lactobacillus paracasei K5 in vitro and in vivo

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1α (IL-1α), ΙL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character

TLR-6 SNP P249S is associated with healthy aging in nonsmoking Eastern European Caucasians - A cohort study

Background To investigate mechanisms that determine healthy aging is of major interest in the modern world marked by longer life expectancies. In addition to lifestyle and environmental factors genetic factors also play an important role in aging phenotypes. The aged immune system is characterized by a chronic micro-inflammation, known as inflamm-aging, that is suspected to trigger the onset of age-related diseases such as cardiovascular disease, Alzheimer’s disease, cancer, and Diabetes Mellitus Type 2 (DMT2). We have recently shown that a Toll-like receptor 6 variant (P249S) is associated with susceptibility to cardiovascular disease and speculated that this variant may also be associated with healthy aging in general by decreasing the process of inflamm- aging. Results Analyzing the PolSenior cohort we show here that nonsmoking S allele carriers are significantly protected from age-related diseases (P = 0.008, OR: 0.654). This association depends not only on the association with cardiovascular diseases (P = 0.018, OR: 0.483) for homozygous S allele carriers, but is also driven by a protection from Diabetes Mellitus type 2 (P = 0.010, OR: 0.486) for S allele carriers. In addition we detect a trend but no significant association of this allele with inflamm-aging in terms of baseline IL-6 levels. Conclusion We confirm our previous finding of the TLR-6 249S variant to be protective regarding cardiovascular diseases. Furthermore, we present first evidence of TLR-6 249S being involved in DMT2 susceptibility and may be in general associated with healthy aging possibly by reducing the process of inflamm-aging

Oligodeoxynucleotides ODN 2006 and M362 Exert Potent Adjuvant Effect through TLR-9/-6 Synergy to Exaggerate Mammaglobin-A Peptide Specific Cytotoxic CD8+T Lymphocyte Responses against Breast Cancer Cells

Mammaglobin-A (MamA) is overexpressed in 40–80% of all human breast cancers. Recent phase I clinical trials of the MamA DNA vaccine showed encouraging safety outcomes. However, this vaccine elicited only a modest increase in MamA specific CD8+T lymphocyte (CTL) activation. As vaccine adjuvants play a critical role in enhancing the immunotherapeutic efficiency of vaccines, we tested the potential role of three synthetic CpG oligodeoxynucleotides (ODN2216—class A ODN, ODN2006—class B ODN, and ODN M362—class C ODN) to further enhance MamA specific CTL responses. Towards this, naïve CD8+T cells were obtained from healthy HLA-A2+ human donors. The HLA-A2 specific immunodominant epitope of MamA, MamA2.1 (LIYDSSLCDL), was utilized to activate naïve CD8+T cells. The THP-1 (HLA-A2+) cells were used as antigen presenting cells to stimulate naïve CD8+T cells along with (or without) co-treatment of various ODNs mentioned above. Activation of naïve CD8+T cells with the MamA2.1 peptide along with ODNs demonstrated enhanced MamA specific CTL mediated cytotoxicity on AU565 (HLA-A+/MamA+) breast cancer cells following co-treatment with ODN2006 and M362 compared to ODN2216 or MamA2.1 peptide alone. However, no significant cytotoxicity was noted upon treatment of MamA2.1 activated CTLs on MCF7 (HLA-A+/MamA−) cells, suggesting that the activation of CTLs is specific to the MamA antigen. Functional characterization studies demonstrated specific IL-12 mediated cross-talk between TLR-6 and -9 in THP-1 cells following stimulation with ODN2006 and M362, which was critical for the final cytotoxic activation of CD8+T lymphocytes. Based on these data, we conclude that ODN2006 and ODN M362 exerted a strong adjuvant effect through induction of the initial innate immune response through TLR9 upregulation followed by enhanced MamA specific CTL dependent adaptive immune responses. Our current data provide evidence for the application of Class-B/-C-CpG-ODNs as potential vaccine adjuvants towards enhancing the success of MamA based breast cancer vaccination

TlR expression profile of human gingival margin-derived stem progenitor cells

Background: Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-likereceptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. Material and Methods: Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs’ incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated. Results: G-MSCs showed all stem/progenitor cells’ characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test). Conclusions: The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions

Роль полиморфизма генов некоторых молекул иммунного ответа в развитии острого вирус-индуцированного бронхиолита

The aim of research: To investigate the genetic polymorphism of immune response molecules (TNFα-308G> A (rs1800629), IL4-589C>T (rs2243250), IL10-592C> A (rs1800872), IL10-819C> T (rs1800871), IL10-1082G>A (rs1800896), IL-17A-197G> A (rs2275913), IL- 17F-161His> Arg (rs763780), TLR-2-753Arg>Gln (rs5743708), TLR-6-249Ser>Pro (rs5743810) and assess their prognostic value in the development of acute virus-induced bronchiolitis.Materials and methods. The study included children of the first year of life, whose average age was 4.2 ± 3.7 months. The main group consisted of 106 patients with moderate and severe acute viral bronchiolitis, more often associated with respiratory syncytial virus (56.6%). The control group consisted of 100 healthy children of the same age who had no signs of acute respiratory infection at the time of examination and did not receive passive immunoprophylaxis of respiratory syncytial infection. Genotyping was performed using the polymerase chain reaction method. The analysis of the results included the compliance with the Hardy-Weinberg law, the χ 2 test, the relative chance, and its 95% confidence interval. To assess the distribution of the claimed gene polymorphisms and their alleles, we used the general (χ2 test, df =2) and multiplicative (χ2 test, df =1) inheritance models.Results. It was revealed that the risk of developing acute viral bronchiolitis is increased compared to the healthy population in carriers of the following genotypes: CC, ST gene IL10-819C> T (rs1800871), GG, AA gene IL-17A-197G> A (rs2275913), HisHis gene IL-17F-161His> Arg (rs763780), SerSer, SerPro gene TLR-6-249Ser> Pro (rs5743810), GG gene TNF-α-308G>A (rs1800629). The TT genotype of the IL10-819C>T (rs1800871) gene is associated with a high risk of developing bacterial complications (pneumonia) in viral bronchiolitis. Carriers of genotypes AA, CC of the IL10-592C> A (rs1800872) gene have an increased likelihood of a severe course of viral bronchiolitis.Conclusion. Genetic analysis of gene polymorphism IL10-592C> A (rs1800872), IL10-819C> T (rs1800871), IL-17A-197G> A (rs2275913), IL-17F-161His> Arg (rs763780), TLR-6-249Ser> Pro (rs5743810), TNF-α-308 G>A (rs1800629) can be used as a personalized developmental criterion acute virus-induced bronchiolitis in children, determining the severity of its course and the likelihood of complications.Цель: исследовать генетический полиморфизм молекул иммунного ответа (TNFα-308G>A (rs1800629), IL4-589C>T (rs2243250), IL10-592C>A (rs1800872), IL10-819C>T (rs1800871), IL10-1082G>A (rs1800896), IL-17A-197G>A (rs2275913), IL-17F-161His>Arg (rs763780), TLR2-753 Arg>Gln (rs5743708), TLR-6-249 Ser>Pro (rs5743810) и оценить их прогностическое значение в развитии острого вирус-индуцированного бронхиолита.Материалы и методы. В исследование включены дети первого года жизни, средний возраст которых составил 4,2±3,7 месяца. Основная группа – 106 пациентов с острым вирусным бронхиолитом средней и тяжелой степени тяжести, чаще ассоциированным с респираторно-синцитиальным вирусом (56,6%). Группа контроля – 100 здоровых детей аналогичного возраста, не имевших признаков острой респираторной инфекции на момент обследования и не получавших пассивную иммунопрофилактику респираторно-синцитиальной инфекции. Генотипирование проведено методом полимеразной цепной реакции. Анализ результатов включал соответствие закону Харди – Вайнберга, χ2-тест, относительный шанс и его 95% доверительный интервал. Для оценки распределения заявленных полиморфизмов генов и их аллелей использовали общую (χ2-тест, df=2) и мультипликативную (χ2-тест, df=1) модели наследования.Результаты. Выявлено, что риск развития острого вирусного бронхиолита повышен по сравнению со здоровой популяцией у носителей следующих генотипов: СС, СТ гена IL10-819C>T (rs1800871), GG, АА гена IL-17A-197G>A (rs2275913), HisHis гена IL-17F-161His>Arg (rs763780), SerSer, SerPro гена TLR-6-249Ser>Pro (rs5743810), GG гена TNF-α-308G>A (rs1800629). Генотип ТТ гена IL10-819C>T (rs1800871) ассоциирован с высоким риском развития бактериальных осложнений (пневмонии) при вирусном бронхиолите. Носители генотипов АА, СС гена IL10-592C>A (rs1800872) имеют повышенную вероятность тяжелого течения вирусного бронхиолита.Заключение. Генетический анализ полиморфизма генов IL10-592C>A (rs1800872), IL10-819C>T (rs1800871), IL-17A-197G>A (rs2275913), IL-17F-161His>Arg (rs763780), TLR-6-249Ser>Pro (rs5743810), TNF-α-308G>A (rs1800629) может использоваться в качестве персонифицированного критерия развития острого вирус-индуцированного бронхиолита у детей, определения тяжести его течения и вероятности формирования осложнений