Regulatory T cell abundance and activation status before and after priming with HIVIS-DNA and boosting with MVA-HIV/rgp140/GLA-AF may impact the magnitude of the vaccine-induced immune responses

Background

Little is known about regulatory CD4 T cells (Tregs) in the context of HIV vaccines. Tregs can be differentiated into resting (FoxP3⁺CD45RA⁺ – rTregs), activated (FoxP3^HighCD45RA^? – aTregs) and memory (FoxP3^LowCD45RA^? – mTregs). Tregs, as CD4 T cells, are also frequent targets for HIV infection. We studied how the abundance and phenotypes of Tregs in terms of activation status and expression of HIV-1 binding molecules would have changed during vaccination in healthy volunteers participating in a phase IIa HIV vaccine clinical trial. Subjects were primed three times with HIVIS-DNA and boosted twice with MVA-CMDR-HIV alone (n?=?12) or MVA-CMDR combined with protein CN54rgp140 (n?=?13). The proportions of β7 integrin in all CD4 T cells and in the Tregs subset decreased moderately after the final vaccination (p?=?0.001 and p?=?0.033, respectively) and the rTregs proportion within the total Tregs were also decreased after the final vaccination (p?=?0.038). All these proportions returned to normal values within the three months after the final vaccination. The magnitude of HIV-Envelope-specific IFNγ?+?T cells after vaccination (r?=?0.66; p?=?0.021) correlated directly with the proportion of Tregs, and correlated inversely correlated with ratios of Th17/Tregs (r?=??0.75; p?=?0.0057) and Th17/mTregs (r?=??0.78; p?=?0.0065). Higher titers of IgG gp140 antibodies were observed in subjects with higher mTregs proportions (r?=?0.52; p?=?0.022). Interestingly, pre-vaccination levels of mTregs correlated with vaccine-induced Env-binding antibodies (r?=?0.57; p?=?0.01) and presence of neutralizing antibodies (r?=?0.61; p?=?0.01), while the pre-vaccination Th17/mTregs ratio correlated inversely with the magnitude of cellular IFN-γ ELISpot responses (r?=??0.9; p?=?0.002). Taken together, these results suggest that pre- and post-vaccination Tregs, their activation status, the Th17/Tregs ratio and other host factors affecting Treg abundance, have an impact on the magnitude of HIV vaccine-induced immune responses. Moreover, the DNA-HIVIS/MVA-HIV regimen, alone or in combination with CN54rgp140 induced moderate and temporary alterations of the Tregs activation status. We also show a decrease in expression of the HIV-1 ligand β7 integrin on Tregs and all CD4 T cells.     

Association between CD226 polymorphism and soluble levels in rheumatoid arthritis: Relationship with clinical activity

Objective: To study the relation between CD226 rs763361 gene polymorphism and CD226 serum level and to evaluate their role in susceptibility and disease activity of RA in a cohort of Egyptian individuals.

Methods: The serum level of CD226 was measured using a suitable ELISA kit and the CD226 rs763361 gene polymorphism was typed by PCR-RFLP for 112 RA patients and 100 healthy controls.

Results: Significant association with RA was found with CD226 T allele (OR (95%CI) = 1.6 (1.04–2.4), P = 0.032), and higher CD226 serum level (P = 0.001). Higher CD226 levels were associated with higher ESR values (P = 0.035), positive CRP (0.048), increased number of tender joints (P = 0.045), and higher DAS score (P = 0.035). Serum CD226 is an independent risk factor for the prediction of RA (P = 0.001). No correlations were found between the serum level of CD226 and different CD226 genotypes and also between them and RA activity grades.

Conclusion: The CD226 T allele may be susceptibility risk factors for the development of RA and the higher serum level of CD226 may be involved in the pathogenesis of RA in Egyptian patients. The serum level of CD226 and not CD226 genotypes could be considered as an independent risk factor for the prediction of RA within healthy individuals and also for RA disease activity.     

Telocytes in human fetal skeletal muscle interstitium during early myogenesis

A new peculiar stromal cell type called telocyte (TC)/CD34-positive stromal cell (i.e. cell with distinctive prolongations named telopodes) has recently been described in various tissues and organs, including the adult skeletal muscle interstitium of mammals. By forming a resident stromal three-dimensional network, TCs have been suggested to participate in different physiological processes within the skeletal muscle tissue, including homeostasis maintenance, intercellular signaling, tissue regeneration/repair and angiogenesis. Since a continuous interplay between the stromal compartment and skeletal muscle fibers seems to take place from organogenesis to aging, the present study was undertaken to investigate for the first time the presence of TCs in the human skeletal muscle during early myogenesis. In particular, we describe the morphological distribution of TCs in human fetal lower limb skeletal muscle during early stages of myogenesis (9–12 weeks of gestation). TCs were studied on tissue sections subjected to immunoperoxidase-based immunohistochemistry for CD34. Double immunofluorescence was further performed to unequivocally differentiate TCs (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). Our findings provide evidence that stromal cells with typical morphological features and immunophenotype of TCs are present in the human skeletal muscle during early myogenesis, revealing differences in either CD34 immunopositivity or TC numbers among different gestation ages. Specifically, few TCs weakly positive for CD34 were found between 9 and 9.5 weeks. From 10 to 11.5 weeks, TCs were more numerous and strongly reactive and their telopodes formed a reticular network in close relationship with blood vessels and primary and secondary myotubes undergoing separation. On the contrary, a strong reduction in the number and immunopositivity of TCs was observed in fetal muscle sections from 12 weeks of gestation, where mature myotubes were evident. The muscle stroma showed parallel changes in amount, density and organization from 9 to 12 weeks. Moreover, blood vessels appeared particularly numerous between 10 and 11.5 weeks. Taken together, our findings suggest that TCs might play a fundamental role in the early myogenetic period, possibly guiding tissue organization and compartmentalization, as well as angiogenesis and maturation of myotubes.     

Oxidized-low density lipoprotein in gingival crevicular fluid of patients with chronic periodontitis: a possible link to atherogenesis

Objective. To investigate a possible link between periodontitis and atherogenesis by examining the levels of anti-oxidized low density lipoprotien (ox LDL) and low density lipoprotien (LDL) in gingival crevicular fluid (GCF) and serum of healthy subjects and chronic periodontitis patients. Methods. Sixty male subjects (35–55 years) were grouped into 30 healthy individuals and 30 subjects with chronic periodontitis. Serum and GCF samples were obtained from each subject and were assessed for anti-ox LDL and LDL levels. Results. A significant difference (p < 0.001) was found between the anti-ox-LDL levels in GCF of healthy vs chronic periodontitis groups. Also the ratio of GCF anti-ox LDL to GCF LDL was significantly higher (p < 0.001) in chronic periodontitis patients as compared to the healthy group. Conclusions. A significant rise in ox LDL level in otherwise systemically healthy chronic periodontitis patients may put these subjects at an increased risk of developing atherosclerosis.     

Assessment of Interleukin‐6 Receptor Inhibition Therapy on Periodontal Condition in Patients With Rheumatoid Arthritis and Chronic Periodontitis

Background: Overproduction of interleukin (IL)‐6 may play a pathologic role in rheumatoid arthritis (RA) and chronic periodontitis (CP). The present study assesses IL‐6 receptor (IL‐6R) inhibition therapy on the periodontal condition of patients with RA and CP. Methods: The study participants were 28 patients with RA and CP during treatment with IL‐6R inhibitor, and 27 patients with RA and CP during treatment without IL‐6R inhibitor. Periodontal and rheumatologic parameters and serum levels of cytokine and inflammatory markers and immunoglobulin G against periodontopathic bacteria were examined after medication with IL‐6R inhibitor for 20.3 months on average (T1) and again 8 weeks later (T2). Results: No differences were observed between the groups in any parameter values at T1, except for serum IL‐6 levels. The anti–IL‐6R group showed a significantly greater decrease in gingival index, bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL), and serum levels of IL‐6 and matrix metalloproteinase (MMP)‐3 from T1 to T2 than the control group (P <0.05). A significant correlation was found between changes in serum anticyclic citrullinated peptide levels and those in PD and CAL in the anti–IL‐6R group (P <0.05), whereas both groups exhibited a significant association between changes in serum MMP‐3 levels and those in BOP (P <0.05). Conclusion: Changes in periodontal and serum parameter values were different between the patients with RA and CP during treatment with and without IL‐6R inhibitor.     

Analysis of YKL‐40 Acute‐Phase Protein and Interleukin‐6 Levels in Periodontal Disease

Background: YKL‐40, a new acute‐phase protein, is shown to be elevated in inflammatory diseases, such as rheumatoid arthritis, type 2 diabetes mellitus, and coronary artery diseases. However, there is no data indicating a relationship between YKL‐40 and periodontal disease. Interleukin‐6 (IL‐6) is the major regulator of acute‐phase protein synthesis and one of the most studied inflammatory markers in periodontal disease. The purpose of the present study is to evaluate YKL‐40 and IL‐6 levels in gingival crevicular fluid (GCF) and serum of patients with periodontal disease and healthy individuals. Methods: Periodontally healthy individuals (n = 15), patients with gingivitis (n = 15), and patients with severe chronic periodontitis (CP) (n = 15) without any systemic disease were included in the study. Clinical measurements were recorded; GCF and blood samples were obtained from each participant. GCF and serum YKL‐40 and IL‐6 levels were analyzed by enzyme‐linked immunosorbent assay. Statistical analysis was performed by parametric and non‐parametric tests. Results: Total amounts of YKL‐40 and IL‐6 in GCF as well as serum YKL‐40 and IL‐6 levels were significantly higher in patients with gingivitis and CP compared with healthy controls (P <0.01). YKL‐40 levels in GCF and serum as well as serum IL‐6 levels were significantly higher in patients with CP compared with patients with gingivitis (P <0.01). Conclusions: YKL‐40 levels in GCF as well as serum YKL‐40 and IL‐6 levels increased from gingivitis to periodontitis. Within the limits of the present study, the YKL‐40 molecule might be a potential novel inflammatory marker of periodontal disease.     

Tooth Storage,Dental Pulp Stem Cell Isolation,and Clinical Scale Expansion without Animal Serum

Introduction

Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers.

Methods

In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4°C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT]), cell surface marker expression, and multipotency were compared between DPSCs in FBS and DPSCs in HS.

Results

The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P < .01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P < .01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P < .05) higher for DPSCs in HS (PDT = 1.59 ± 0.46) than that for DPSCs in FBS (PDT = 2.84 ± 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS.

Conclusions

These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties.     

Temporal-controlled Release of Bovine Serum Albumin from Chitosan Nanoparticles: Effect on the Regulation of Alkaline Phosphatase Activity in Stem Cells from Apical Papilla

Introduction

The controlled delivery of bioactive molecules is crucial for the regulation of stem cell differentiation. In this study, we examined the effects of temporal-controlled release of bovine serum albumin (BSA) from chitosan nanoparticles (CSnp) to regulate the alkaline phosphatase activity (ALP) in stem cells from apical papilla (SCAP).

Methods

BSA-loaded CSnp were synthesized by 2 methods to achieve the variant temporal-controlled release: (1) the encapsulation technique (BSA-CSnpI) and (2) the adsorption technique (BSA-CSnpII). After characterization of the size, charge, and release kinetics, SCAP were cultured in the presence of these bioactive molecule–loaded nanoparticles. SCAP viability was analyzed at 1, 7, 14, 21, and 28 days, and ALP activity was analyzed every 7 days until 21 days to determine the effect of these bioactive molecule–releasing nanoparticles on the cytotoxicity and differentiation potential, respectively.

Results

BSA-CSnpI and BSA-CSnpII presented distinct in vitro release profiles of BSA in a time-controlled manner. Cell viability was significantly enhanced over time in the presence of BSA-CSnpI and BSA-CSnpII (P < .01), when compared with BSA nonloaded CSnp. ALP activity was significantly higher (P < .01) in the presence of BSA-CSnpI after 3 weeks than in BSA-CSnpII.

Conclusions

BSA-loaded CSnps were synthesized and characterized in this study. Based on the physical/chemical interaction of BSA with CSnp (encapsulation or surface adsorption), different time-controlled release profiles were observed that influenced the ALP activity of SCAP in vitro. This study highlighted the potential of temporal-controlled bioactive molecule release technology in the differentiation of stem cells in dentin pulp regeneration.     

新型壳聚糖基引导骨再生膜的蛋白缓释性能研究

目的：制备负载牛血清白蛋白的壳聚糖/β-甘油磷酸钠(CS/β-GP)可吸收性膜,探讨其缓释蛋白的性能。方法：利用CS/β-GP体系的温敏相转变特性,同时向其中添加蛋白制成新型生物膜。进行膜厚度及拉伸强度等力学性能测试。利用BCA蛋白浓度试剂盒（加强型）检测不同时点的蛋白浓度,绘制膜的蛋白缓释曲线。结果：利用SPSS16.0统计分析软件进行分析,负载蛋白后复合膜的理化性能均未发生明显变化（P>0.05）。不同浓度的复合膜可缓慢释放蛋白12天以上,并且随着β-GP浓度的升高,蛋白缓释总量增大。结论：新型壳聚糖温敏凝胶膜可以作为蛋白缓释的载体,是在引导骨再生领域具有应用潜力的生物膜材料。