Development, Characterization, and Immunomodulatory Evaluation of Carvacrol-loaded Nanoemulsion

Carvacrol (CV) is an essential oil with numerous therapeutic properties, including immunomodulatory activity. However, this effect has not been studied in nanoemulsion systems. The objective of this study was to develop an innovative carvacrol-loaded nanoemulsion (CVNE) for immunomodulatory action. The developed CVNE comprised of 5% w/w oily phase (medium chain triglycerides + CV), 2% w/w surfactants (Tween 80®/Span 80®), and 93% w/w water, and was produced by ultrasonication. Dynamic light scattering over 90 days was used to characterize CVNE. Cytotoxic activity and quantification of cytokines were evaluated in peripheral blood mononuclear cell (PBMC) culture supernatants. CVNE achieved a drug loading of 4.29 mg/mL, droplet size of 165.70 ± 0.46 nm, polydispersity index of 0.14 ± 0.03, zeta potential of −10.25 ± 0.52 mV, and good stability for 90 days. CVNE showed no cytotoxicity at concentrations up to 200 µM in PBMCs. CV diminished the production of IL-2 in the PBMC supernatant. However, CVNE reduced the levels of the pro-inflammatory cytokines IL-2, IL-17, and IFN-γ at 50 µM. In conclusion, a stable CVNE was produced, which improved the CV immunomodulatory activity in PBMCs.

SARS-CoV-2 induces transcription of human endogenous retrovirus RNA followed by type W envelope protein expression in human lymphoid cells.

Patients with COVID-19 may develop abnormal inflammatory response and lymphopenia, followed in some cases by delayed-onset syndromes, often long-lasting after resolution of the initial SARS-CoV-2 infection. As viral infections may activate human endogenous retroviral elements (HERV), we studied the effect of SARS-CoV-2 on HERV-W and HERV-K envelope (ENV) expression, known to be involved in immunological and neurological pathogenesis of human diseases. We demonstrate here that an initial exposure to SARS-CoV-2 virus activates early HERV-W and K transcription in peripheral blood mononuclear cell (PBMC) cultures from healthy donors. Within a week of primary PBMC culture, only HERV-W ENV protein expression was detected in lymphoid cells of some donors, although SARS-CoV-2 infection of PBMC was not observed. HERV activation was reproduced with UV-inactivated virus and with a recombinant spike protein. Interestingly, exposure to SARS-CoV-2 protein induced a significant production of interleukin 6 in PBMC, independently from detectable HERV expression. Altogether, these results show that SARS-CoV-2 viral protein could induce HERV-W ENV expression in lymphocytes from some individuals, underlying the importance to further address the implicated molecular pathways, to understand patients‘ genetic susceptibility associated to the activation of HERV-W and its possible relevance for targeting therapeutic intervention in COVID-19 associated syndromes.

Increased IL-2 and Reduced TGF-β Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis

Granulocyte macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine produced by immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. We investigated the expression and regulation of GM-CSF in different immune cells in MS. We also investigated the differentiation and frequency of GM-CSF-producing Th cells that do not co-express interferon (IFN)-γ or interleukin-17 (IL-17) (Th-GM cells) in MS. We found a significant increase in the percentage of GM-CSF-expressing Th cells, Th1 cells, Th-GM cells, cytotoxic T (Tc) cells, monocytes, natural killer (NK) cells, and B cells in PBMC from MS patients stimulated with T cell stimuli. Stimulated PBMC culture supernatants from MS patients contained significantly higher levels of IL-2, IL-12, IL-1β, and GM-CSF and significantly lower levels of transforming growth factor (TGF-)β. Blocking IL-2 reduced the frequency of Th-GM cells in PBMC from MS patients. The frequency of Th-GM cells differentiated in vitro from naïve CD4+ T cells was significantly higher in MS patients and was further increased in MS with IL-2 stimulation. These findings suggest that all main immune cell subsets produce more GM-CSF in MS after in vitro stimulation, which is associated with defective TGF-β and increased IL-2 and IL-12 production. Th-GM cells are increased in MS. GM-CSF may be a potential therapeutic target in MS.

Altered Indoleamine 2,3-Dioxygenase Production and Its Association to Inflammatory Cytokines in Peripheral Blood Mononuclear Cells Culture of Type 2 Diabetes Mellitus

Aim: To analyze indoleamine 2,3-dioxygenase (IDO) production in the cell culture supernatant of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from type 2 DM (T2DM) patients and investigate IDO’s association to pro- and anti-inflammatory cytokines. Subjects and methods: PBMC samples were collected from 21 T2DM patients and 17 normoglycemic participants, then stimulated with PHA for 3 days. Cytokine and IDO concentrations were measured in the PBMC culture supernatants. In vitro production of TNF-α, IL-6, interferon-γ, and IL-10 were measured using multiplex immunoassay. IDO concentration was assessed using ELISA. To assess how PHA stimulation altered IDO production and to minimize the unstimulated baseline effect of T2DM, we subtracted the PHA-stimulated IDO concentration from the unstimulated one. IBM SPSS version 23 was used for statistical analysis. Results: The IDO concentrations in the PBMC culture supernatants were significantly higher in T2DM patients regardless of whether they were unstimulated ( P < .001) or PHA-stimulated ( P = .012). Reduced IDO production was observed in 52.8% of T2DM patients and was associated with older age and lower interferon-γ levels. Conversely, 42.8% of T2DM patients showed increased IDO concentrations, which were correlated with the IL-6/IL-10 ratio ( r = 0.683, P = .021) and interferon-γ/IL-10 ratio ( r = 0.517, P = .077). Conclusion: The interferon-γ level was reduced in the PBMC culture supernatant of T2DM patients with reduced IDO production. Reduced IDO production in T2DM patients following PHA stimulation was associated with older age and, notably, higher baseline IDO concentrations. Since IDO is primarily produced by dendritic cells, reduced IDO production after PHA stimulation may indicate dendritic cell dysfunction.

IFN-Gamma Secretion and IL-10 after Stimulation of ESAT-6-CFP-10 (EC610) Fusion Antigen in Patients with Active Tuberculosis and Latent Tuberculosis

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis which is still a health problem in the world including in Indonesia. TB infection arises due to interference with the regulation of the body's defense system. Cellular immune system plays a role in fighting TB infection, namely the role of T lymphocytes that differentiate into Th1 cells secrete pro-inflammatory cytokines IFN-? and Th2 cells that secrete IL-10 anti-inflammatory cytokines. The EC610 fusion antigen is specific and has a strong antigenicity to T cell stimulation. The aim of the study was to determine differences in the mean levels of IFN-? and IL-10 after EC610 antigen stimulation in patients with active TB and latent TB. The design of this study was a quasi-in vitro experiment with PBMC culture stimulated by EC610 antigens in the active TB and latent TB groups. The research and examination were carried out at the South Sumatra Province SSR and the Health Research and Development Laboratory Center, Central Jakarta. There were 21 subjects with active TB and 28 subjects with latent TB who met the inclusion criteria. Measurement of IFN-levels? and IL-10 was performed using ELISA-Reader. The results showed the levels of IFN-? and IL-10 after EC610 antigen stimulation were higher in active TB than latent TB. High levels of IFN-? active TB patients showed protective immune responses against M.tb germs while high levels of IL-10 showed their role as anti-inflammatory. There is no significant difference in the average level of IFN-? in patients with active TB and latent TB (p = 0.769) and there was a significant difference in the level of IL-10 levels after EC610 antigen stimulation was higher in active TB than latent TB (p = 0,000).

Immunogenicity Test of ESAT-6/CFP-10 Mycobacterium tuberculosis (Indonesian Strain) Recombinant Protein Fusion: TNF-α, IL-17 and CD4+ T Cells Expression in PBMC Culture

Background: Bascillus Calmette Guѐrin vaccination has not provided protection against TB in adults. ESAT-6/CFP-10 Mtb recombinant protein fusion as a vaccine candidate can stimulate the body's immune response. Interleukin-17, TNF-α and CD4+ play a major role against TB. This study aims to determine that the recombinant protein fusion ESAT-6/CFP-10 Mtb can stimulate TNF-α, IL-17 and CD4+ T cells expression in PBMC culture. Methods: Design of this study is experimental study. Number of research sample per group of 8 subjects. The subjects: healthy, TB contact and TB patients were taken their peripheral blood sample and treated with ESAT-6/CFP-10 Mtb recombinant protein fusion. TNF-α CD4+ T cells were measured by ELISA. Flow cytometry is used to measure IL-17 and CD4+ Tcells. As standard protocol research on tuberculosis vaccine, each subject also treated without antigen and with PPD. Results: There was no significant increase in the administration of ESAT-6/CFP-10 Mtb fusion compared without antigen on TNF-α expression of CD4+ (P=0.202), expression of IL-17 CD4+ (P=0.994) and percentage of CD4+ T cells (P=0.183). ESAT-6/CFP-10 Mtb Fusion was able to stimulate expression of TNF-α CD4+ in healthy subjects (29.91±1.23pg/ml), TB contact (32.21±4.02pg/ml) and TB patients (33.35±8.41 pg/ml). Expression IL-17 CD4+ in healthy subjects (33.24 ± 39.01%), TB contact (23.88 ± 21%) and TB patients (51.93 ± 36%). CD4+ T cell expression in healthy subjects 30.64 ± 7.63%, TB contact 24.58 ± 5.24% and TB patients (40.73±2.63%). Conclusions: ESAT-6/CFP-10 Mtb recombinant proteins fusion may stimulate the production of TNF-α, IL-17 CD4+ and CD4+ T cells in all subject. Expression of TNF-α, IL-17 CD4+ and CD4+ T cells in the healthy group, indicated that the ESAT-6/CFP-10 recombinant protein fusion has the potential as vaccine candidate. (J Respir Indo. 2019; 39(3):160-8)

Immunogenicity Test of ESAT-6/CFP-10 Mycobacterium Tuberculosis (Indonesian Strain) Recombinant Protein Fusion: IFN-γ and CD8+ T Cells Expression in PBMC Culture

Background: BCG vaccination is one way to control tuberculosis (TB) but still poor in efficacy thus new vaccine development is needed. Immunogenicity test is needed in developing new vaccine. The aim of this study was to understand whether the recombinant protein fusion of ESAT-6/CFP-10 Mycobacterium tuberculosis (M. tuberculosis) can stimulate cellular immune response, especially IFN-γ and CD8 + T cell expression in PBMC cultures. Methods: This study was an experimental laboratory research conducted on PBMC cultures of 3 groups of subjects (TB patients, latent TB patients and healthy subjects) at RSUD Dr. Saiful Anwar in April-July 2017. The sample of each groups was 8 subjects. Each groups induced by recombinant protein fusion ESAT-6/CFP-10 M. tuberculosis as a standard protocol and to establish the immunogenicity status. CD8+ T cells IFN-γ expressed by C8+ were measured by flowcytometry. Result: Recombinant protein fusion ESAT-6/CFP-10 can stimulate CD8+ T cells and IFN-γ expressed by CD8+ T cells in all group. The highest stimulation of CD8+ percentage was found in healthy subject (37.533 ± 7.264) and IFN-γ expressed by CD8+ T cells was found in healthy subject (7.908 ± 4.457); There are increase significantly CD8+ T cells (p=0.001) and IFN-γ expressed by CD8+ T cells (p=0.217) not significantly in healthy subject compared in PPD and without antigen. Conclusion: Recombinant protein fusion ESAT-6/CFP-10 M. tuberculosis can stimulate CD8+ T cells and IFN-γ expressed by CD8+ T cells in healthy subject. Recombinant protein fusion ESAT-6/CFP-10 M. tuberculosis potential as a new vaccine candidate. (J Respir Indo. 2018;38: 210-8)

Modular synthesis and modification of novel bifunctional dendrons

The modular synthesis of two generations of highly branched bifunctional dendrons is reported. The first generation dendron–antibody conjugate is shown to selectively detect CD4+ T cells in the PBMC culture.

Expression and Significance of MMPs in Synovial Fluid, Serum and PBMC Culture Supernatant Stimulated by LPS in Osteoarthritis Patients With or Without Diabetes

Objective Patients with Type 2 Diabetes mellitus (T2DM) are prone to osteoarthritis (OA). Matrix metalloproteinases (MMPs), an essential modulator in cartilage matrix homeostasis, increase in T2DM and OA. We aimed to ascertain the expression difference of MMPs and function in mononuclear cells after stimulating by lipopolysaccharide (LPS) in OA patients with or without diabetes. Methods 30 knee OA patients without T2DM (OA group), 20 knee OA patients with T2DM (DM-OA group) and 5 healthy volunteers recruited as control were enrolled from January 2016 to January 2017. The expression levels of MMPs in both serum and synovial fluid were initially detected in three groups by enzyme-linked immunosorbent assay (ELISA). After stimulation of peripheral blood mononuclear cell (PBMC) with LPS, the release of MMPs were determined and evaluated. Results The expression of MMP-1, -7, -8, -9, -10 and -12 in synovial fluid in DM-OA group were significantly higher than in OA group and healthy control. The expression of MMP-1 and -7 in serum were highest in DM-OA group. LPS significantly promotes the production of MMP-1, -8, -9 and -10 in PBMC of each group after 4 h stimulation. It is worth to note that the LPS-stimulated MMP-8 and -9 elevations were more prominent in DM-OA group compared with their counterparts. Conclusion High levels of MMP-1, -7, -8, -9, -10, and -12 in the synovial fluid might be one of important reasons that diabetes patients are more frequently suffered from OA. Inflammation-induced malfunction of mononuclear cells would stimulate MMP-8 and -9 secretion to various extents.

Molecular characterization of duck (Anas platyrynhos) Toll-like receptors, mRNA expressions profile in day-old duckling’s tissues and cytokine response to in vitro TLR agonsists stimulation

TLR repertoire of duck, profiling of their mRNA expression in a range of duckling tissues and cytokine gene expressions upon TLR agonists stimulation in in vitro assay have been investigated. All ten TLR genes orthologous to chicken TLR repertoire were found in duck. Duck TLR genes showed 77-83% similarity at amino acid level to their chicken counterparts. All ten TLRs-TLR1LA, 1LB, 2A, 2B, 3, 4, 5, 7, 15 and 21 mRNA expressions were significantly higher in bursa than other tissues studied, whereas in muscle all TLRs mRNA expressions were significantly lower except for TLR15 (P>0.01). TLR7 gene expression was significantly higher in spleen, bursa and also in lung tissues (P>0.01). The cytokine gene expression levels in duck PBMCs upon LPS and poly I:C stimulation have been quantified. IL-1g gene expression level in LPS stimulated duck PBMC culture was significantly higher at both 12 h and 24 h time intervals (P>0.05). However, there were no significant changes in IFN-ã gene expression levels in poly I:C stimulated duck PBMC culture at both the intervals. TLR gene expression in young ducklings together with cytokine response upon LPS stimulation demonstrates the innate preparedness of younger birds to encounter pathogens and their functional ability to respond to their ligands.