A therapeutic approach to treat cardiovascular dysfunction of diabetes

Diabetes greatly increases risk of cardiovascular dysfunction and interruptions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) have been shown to reduce the risk by alteration in extracellular matrix. We hypothesized that minocycline induced MMP-2 and MMP-9 inhibition can be enhanced by aspirin (through its COX and tPA inhibitory action) and this combination can reduce cardiovascular dysfunction of diabetes. Four weeks after diabetes induction (streptozotocin, 55 mg/kg, i.p.), rats were treated with minocycline (50 mg/kg, p.o.), aspirin (50 mg/kg, p.o.), or minocycline (50 mg/kg, p.o.) plus aspirin (50 mg/kg, p.o.) for a period of next four weeks. At the end of eighth week arterial pressure, heart rate and left ventricular pressure were recorded. Contractile response to phenylephrine (10^?5 M) and relaxation responses to acetylcholine (10^?9–10^?4 M) were obtained from aortic rings of diabetic rats. Gel zymography was performed to evaluate MMP-2 and MMP-9 levels. Heart rate, mean arterial pressure, dp/dt_max and dp/dt_min were found significantly decreased in STZ diabetic rats when compared with normoglycemic group. Treatment with combination of minocycline and aspirin significantly ameliorate these compared to vehicle treated diabetic group. Endothelium-dependent relaxation responses induced by acetylcholine were decreased in diabetic rats and significantly higher in combination treated group. Collagen, MMP-2 and MMP-9 levels were significantly decreased in combined treated group when compared with diabetic control. Present study revealed that aspirin potentate minocycline induced MMP-2 and MMP-9 inhibition to ameliorate cardiovascular dysfunction of diabetes and this combination can be an approach for the treatment.     

Increased VEGFR2 and MMP9 protein levels are associated with epithelial dysplasia grading

The present study aimed to compare levels of VEGFR2 and MMP-9 among control, epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC) groups. We analyzed 48 patients with oral leukoplakia (OL), 20 patients with OSCC and 21 patients without OL and OSCC. Immunohistochemistry of VEGFR2 and MMP9 were performed and compared among groups. Analysis of tissue immunolocalization of VEGFR2 and MMP-9 assumed non-parametrical distribution and comparison between groups was performed using the Mann–Whitney and Kruskal–Wallis statistical tests. VEGFR2 and MMP9 immunoexpression appeared to correlate with the degree of dysplasia and was observed to increase in lesions with more severe dysplasia as compared to those with lower degrees of dysplasia. Immunoreactivity of MMP-9 was lower in the OL samples compared to the OSCC samples (p = 0.004). We observed no difference in VEGFR2 protein levels between OL and OSCC samples. A positive correlation was found between VEGFR2 and MMP-9 in OL samples (r = +0.452, p = 0.001), however, no correlation was found in OSCC samples (r = −0.042, p = 0.861). In conclusion, the results of the current study suggest that expression of MMP9 and VEGFR2 is associated with ED grading and MMP9 levels are increased in OSCC.     

High expression of OX40 (CD134) and 4-1BB (CD137) molecules on CD4+CD25high cells in children with type 1 diabetes

PurposeDespite the rapidly rising incidence of diabetes in children, with the highest rise in children < 5 years of age, data on mechanisms that trigger severe beta-cells damage are limited. The aim of the study was to assess the frequency of OX40 (CD134) or 4-1BB (CD137) positive cells in the peripheral blood of children with newly diagnosed type 1 diabetes (T1D) in comparison to healthy controls.Material/methodsThe study included 33 children (mean age 7.3 ± 5.4 years) with newly diagnosed T1D and 39 age-matched healthy controls. Separate analysis was performed in children < 5 years. Flow cytometric analysis was performed using the following markers: CD4, CD25, CD137, and CD134. Fasting C-peptide level was assessed as well.ResultsThe frequency of CD4⁺CD25^highOX40⁺ was higher in T1D children than in controls (median value 3.58% vs. 1.1%, respectively; p = 0.003). Moreover, T1D children had higher frequency of CD4⁺CD25^high4-1BB⁺ cells than healthy subjects (median value 5.76% vs. 3.74%, respectively; p = 0.037). A significant correlation was noted between the age of diabetic children and the C-peptide level (r = 0.54, 95% CI [0.19–0.77], p = 0.004). In comparison with age-matched controls, children < 5 years had higher frequency of CD4⁺CD25^highOX40⁺ (p = 0.004) and CD4⁺CD25^high4-1BB⁺ cells (p = 0.079).ConclusionsOur study showed higher frequency of both OX40 and 4-1BB positive cells in T1D children in comparison to controls. It seems that observed differences might be more pronounced in children < 5 years of age than in older subjects. Further clinical studies are needed to determine the age-related differences in the immune system, in the pathogenesis of T1D.     

The effect of the nonionic block copolymer pluronic P85 on gene expression in mouse muscle and antigen-presenting cells

DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO₂₆–PO₄₀–EO₂₆. Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DCs) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c⁺ (DC), and local expansion of CD11c⁺, CD14⁺ (macrophages) and CD49b⁺ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c + CD86⁺, CD11c + CD80⁺, and CD11c + CD40⁺). We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs.     

An updated meta-analysis of transforming growth factor-β1 gene: Three well-characterized polymorphisms with asthma

The association between TGF-β1 polymorphisms and asthma risk has been widely reported, but results were controversial. We performed this meta-analysis based on the Preferred Reporting Items for Systematic Reviews and meta-analyses statement (PRISMA). Electronic database of Pub Med, Web of Science, CBM, and CNKI were searched for eligible articles published up to September, 2013. The effect summary odds ratio (OR) and 95% confidence intervals were obtained. Finally, a total of 20 articles were identified, 17 studies with 3694 cases and 5613 controls for C-509T polymorphism, 7 studies with 1109 cases and 1098 controls for T869C polymorphism and 5 studies with 849 cases and 829 controls for G915C polymorphism. For C-509T, significant associations with asthma were found in Asians (TT + TC vs. CC: P = 0.004, OR = 1.43, 95%CI = 1.12–1.81, P_{heterogeneity} = 0.001) and in Caucasians (P = 0.05, OR = 1.16, 95%CI = 1.00–1.34, P_{heterogeneity} = 0.36). With respect to T869C, a small significant association was observed in overall analysis of allele contrasts(C vs. T: OR = 1.14, 95%CI: 1.01–1.29, P = 0.03) and homozygote comparison (CC vs. TT: OR = 1.29, 95%CI: 1.00–1.65, P = 0.05), but no significant risks were found among Caucasian population and Asian population. For G915C polymorphism, no significant association with asthma risk was demonstrated in overall analysis and subgroup analyses according to ethnicity for all genetic models. This meta-analysis suggested that TGF-β1 C-509T and T869C polymorphisms may be risk factors for asthma.     

Gene expression profile of mouse fibroblasts exposed to a biodegradable iron alloy for stents

Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their superior ductility compared to their counterparts – magnesium alloys. Since the predicted degradation rate of pure iron is considered slow, manganese (35% w/w), an alloying element for iron, was explored to counteract this problem through the powder metallurgy process (Fe–35 Mn). However, manganese presents a high cytotoxic potential; thus its effect on cells must first be established. Here, we established the gene expression profile of mouse 3T3 fibroblasts exposed to Fe–35 Mn degradation products in order to better understand cell response to potentially cytotoxic degradable metallic material (DMM). Mouse 3T3 cells were exposed to degradation products eluting through tissue culture insert filter (3 μm pore size) containing cytostatic amounts of 3.25 mg ml^?1 of Fe–35 Mn powder, 0.25 mg ml^?1 of pure Mn powder or 5 mg ml^?1 of pure iron powder for 24 h. We then conducted a gene expression profiling study from these cells. Exposure of 3T3 cells to Fe–35 Mn was associated with the up-regulation of 75 genes and down-regulation of 59 genes, while 126 were up-regulated and 76 down-regulated genes in the presence of manganese. No genes were found regulated for the iron powder. When comparing the GEP of 3T3 fibroblasts in the presence of Fe–35 Mn and Mn, 68 up-regulated and 54 down-regulated genes were common. These results were confirmed by quantitative RT-PCR for a subset of these genes. This GEP study could provide clues about the mechanism behind degradation products effects on cells of the Fe–35 Mn alloy and may help in the appraisal of its potential for DMM applications.     

Warm red blood cell autoantibodies and clinical diagnoses in patients with or without autoimmune hemolysis

ObjectivesRed blood cell autoantibodies (RBC autoAbs) of IgG class are found in the majority of patients with warm autoimmune hemolytic anemia (wAIHA) but sometimes also during the pretransfusion testing of patients with different diagnoses but without hemolysis. The aim of the study was to identify the main differences between these two groups of patients according to age, gender, subclass and titer of IgG RBC autoAbs and diagnosis.Material and methodsIn the 9-year retrospective study, data were collected from records of 291 patients with IgG RBC autoAbs detected by gel technique, from which 111 with wAIHA.ResultsMore than 85% of patients in both groups were over 40 years old, with male to female ratio 1:1.9 in wAIHA vs 1:1.3 in patients without hemolysis (P = 0.0916). The main characteristics of patients with wAIHA vs patients without hemolysis were: IgG only 38% vs 70%, IgG + Complement 62% vs 30%, total IgG1 79% vs 55%, IgG1 + IgG3 35% vs 11%, titer of 100 for IgG1 + IgG3 17% vs 3% (P < 0.0001), respectively, while titer of 100 for IgG1 18% vs 9% (P = 0.0241). The underlying diagnosis in wAIHA vs patients without hemolysis: hematologic disorders 41% vs 22% (P = 0.0006), autoimmune disorders 12% vs 13% (P = 0.8033), solid tumors 5% vs 14% (P = 0.0154) and surgery procedures 6% vs 26% (P < 0.0001).ConclusionWe observed more wAIHA patients with high titer of IgG1 and high prevalence of IgG1 + IgG3 and consider that patients without hemolysis having identical results might be interesting to find out how they are protected from damage by RBC autoAbs.     

Peri-implant reactivity and osteoinductive potential of immobilized rhBMP-2 on titanium carriers

Recombinant human BMP-2 (rhBMP-2) was immobilized non-covalently and covalently as a monolayer on plasma vapour deposited (PVD) porous commercially pure titanium surfaces in amounts of 5–8 μg cm^?2, providing a ca. 10-fold increase vs. previously reported values [37]. Dissociation of the immobilized [¹²⁵I]rhBMP-2 from the surface occurred in a two-phase exponential decay: a first rapid phase (ca. 15% of immobilized BMP-2) with a half-life of 1–2 days and a second slow sustained release phase (ca. 85% of immobilized BMP-2) with a half-life of 40–60 days. Dissociation rate constants of sustained release of k_?1 = 1.3–1.9 × 10^?7 s^?1 were determined, allowing an estimation of the binding constants (K_A) for the adsorbed rhBMP-2 monolayer, to be around 10¹² M^?1. The rhBMP-2-coated surfaces showed a high level of biological activity, as demonstrated by in vitro epifluorescence tests for alkaline phosphatase with MC3T3-E1 cells and in vivo experiments. In vivo osteoinductivity of rhBMP-2-coated implants was investigated in a gap-healing model in the trabecular bone of the distal femur condylus of sheep. Healing occurred without inflammation or capsule formation. The calculated concentration of released rhBMP-2 in the 1 mm gap ranged from 20 to 98 nM – well above the half-maximal response concentration (K_0.5) for inducing alkaline phosphatase in MC3T3-E1 cells. After 4, 9 and 12 weeks the bone density (BD) and bone-to-implant contact (BIC) of the explanted implants were assessed histomorphometrically. Implants with immobilized rhBMP-2 displayed a significant (2- to 4-fold) increase in BD and BIC values vs. negative controls after 4–9 weeks. Integration of implants by trabecular bone was achieved after 4 weeks, indicating a mean “gap-filling rate” of ～250 μm week^?1. Integration of implants by cortical bone was observed after 9 weeks. Control implants without rhBMP-2 were not osseointegrated. This study demonstrates the feasibility of enhancing peri-implant osseointegration and gap bridging by immobilized rhBMP-2 on implant surfaces which may serve as a model for future clinical applications.     

Different susceptibility to 1-methyl-4-phenylpyridium (MPP+)-induced nigro-striatal dopaminergic cell loss between C57BL/6 and BALB/c mice is not related to the difference of monoamine oxidase-B (MAO-B)

Subcutaneous and intraperitoneal administrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induce selective dopaminergic (DA-ergic) neuronal death in many animal species. After passing through the blood–brain barrier (BBB), MPTP is converted to 1-methy-4-phenylpiridinium (MPP⁺) by astrocytic monoamine oxidase-B (MAO-B). MPP⁺ then induces the dopaminergic neuronal death. In mice, marked strain differences in the susceptibility to MPTP-injection have been reported. To clarify which factor(s) cause the strain differences, MPTP or MPP⁺ was intracerebroventricularly (icv) injected into adult C57BL/6 (highly susceptible to MPTP) and BALB/c (resistant to MPTP) mice. The brain tissues including the striatum and substantia nigra pars compacta (SNpc) were examined immunohistochemically using an antibody to tyrosine hydrocyrase (TH). MPP⁺-injected C57BL/6 mice showed a significant decrease in TH-immunopositive areas in the striatum at Day 3 post injection (p < 0.01), and TH-positive cells in the SNpc at Days 1 and 3 (p < 0.01), respectively, compared to saline-injected control mice. In addition, MPP⁺-injected BALB/c mice showed a significant decrease in TH-positive areas in the striatum at Days 1 and 3, and SNpc TH-positive cells in the SNpc at Day 3, respectively (p < 0.05). However, the decrease rates in the BALB/c mice were lower than that in C57BL/6 mice. MPTP-injected C57BL/6 mice, however, showed no lesions in the striatum and SNpc at Days 1 and 7 after icv injection. All the present findings indicate that factors other than MAO-B can influence the strain susceptibility between C57BL/6 and BALB/c mice after the conversion from MPTP to MPP⁺.     

In vitro investigation of individual and combined cytotoxic effects of aflatoxin B1 and other selected mycotoxins on the cell line porcine kidney 15

In the present study, we evaluated the nephrotoxicity of individual mycotoxins and combinations of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and fumonisin B1 (FB1) to livestock using porcine kidney 15 cells (PK-15) as a disease model via biochemical approaches. The toxicity of individual mycotoxins on cell viability and cell membrane damage was determined using the MTT and lactate dehydrogenase (LDH) assays, respectively. Individual cytotoxicity of mycotoxins in increasing order were FB1 < ZEA < AFB1 < DON. The MTT results of central composite design (CCD) showed synergetic effects after co-exposure of AFB1 + ZEA or AFB1 + DON; however, AFB1 and ZEA showed antagonistic effects in the ternary mixtures. AFB1 and DON significantly induced ROS production and apoptosis in a concentration-dependent manner, but ZEA (10–40 μM) had no effect on cell apoptosis and only slightly induced ROS production. ZEA ameliorated the ROS production caused by 1 μM AFB1; however, ZEA and DON displayed synergistic effects in combination with AFB1 at 5 and 10 μM. The existence of 10 μM ZEA attenuated AFB1-induced apoptosis. In conclusion, AFB1 + ZEA or DON showed synergetic effects on cytotoxicity. Low levels of AFB1 were antagonistic to ZEA, but high doses of AFB1 displayed synergistic effects with ZEA or DON on oxidative damage. ZEA also ameliorated AFB1-induced apoptosis. Generally, the combined effects of mycotoxins acted in a concentration-dependent manner.     

Immunological evaluation of OMV(PagL) + Bap(1-487aa) and AbOmpA(8-346aa) + Bap(1-487aa) as vaccine candidates against Acinetobacter baumannii sepsis infection

Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients with sepsis form. The surface exposed virulence proteins and serum resistance factors helping to dissemination of this bacterium to bloodstream are the most promising vaccine candidates against this microorganism. In this project we immunologically evaluated OMV_(PagL) + Bap_(1-487aa) and AbOmpA _(8-346aa) + Bap_(1-487aa) as combination forms as well as Bap_(1-487aa), AbOmpA_(8–346aa) and OMV_(PagL) singly, with addition of alum adjuvant as vaccine candidates. The titers of total IgG, IgG1 and IgG2c as well as concentration of IL-4 and IFN-γ and survival rates were measured in a C57BL/6 murine model with disseminated sepsis. The ratio of IgG1/IgG2c and profile of IL-4/IFN-γ in OMV _(PagL) + Bap _(1–487aa) formulation shows the humoral and cellular immune responses have been induced robustly and have created a full protection against A. baumannii ATCC 19606 and MDR AB-44 strains. We found that the two combination vaccine candidates were protective and induced both Th1 and Th2 responses.     

Mobilization of hematopoietic stem cells in patients undergoing HSCT as a treatment of early diabetes type 1

BackgroundThe immunoablation with autologous hematopoietic stem cell transplantation is a new experimental treatment of early diabetes type 1. The treatment is based on destruction of immune system with cytotoxic drugs which leads to halt of immune reaction directed against beta cells of pancreas. During that treatment young patients with diabetes type 1 who are otherwise healthy undergo mobilization with cyclophosphamide (CY) and G-CSF. They are naïve to cytotoxic drugs and mobilization is their first contact with chemotherapy. We analyzed the efficiency of mobilization with cyclophosphamide and G-CSF in this population.MethodsWe analyzed the medical records of 25 patients with diabetes who underwent mobilization with cyclophosphamide and G-CSF.ResultsThe median white blood cell count on the first day of apheresis was 14.6 × 10³/μL (range 1.5–33.3) in CY + G-CSF mobilized patients. Median absolute CD 34+ cell count in peripheral blood on the first apheresis day was 0.095 127 × 10³/μL (range 0.026–0.477). The median total number of collected CD34+ cells during one or two (if needed) aphereses was 466 × 10⁶ (range 204–816) or 7.24 × 10⁶ CD34+ cells per kg of patient body weight (range 3.03–13.1). There were no poor mobilizers who were unable to collect sufficient cell numbers.ConclusionThe mobilization of hematopoietic stem cells with CY + G-CSF in patients with early diabetes type 1 is efficient and the underlying diabetes does not impair the efficiency of hematopoietic stem cell collection.     

Induced elastin regeneration by chronically activated smooth muscle cells for targeted aneurysm repair

Elastin breakdown in vascular aneurysms is mediated by cytokines such as tumor necrosis factor α (TNF-α, which induces vascular smooth muscle cell (SMC) activation and regulates their deposition of matrix. We previously demonstrated that exogenous supplementation with TGF-β1 (1 ng ml^?1) and hyaluronan oligomers (0.786 kDa, 0.2 μg ml^?1) cues the upregulation of elastin matrix synthesis by healthy cultured SMCs. Here, we determine whether these cues likewise enhance elastin matrix synthesis and assembly by TNF-α-stimulated SMCs, while restoring their healthy phenotype. Adult rat aortic SMCs were treated with TNF-α alone or together with TGF-β1/hyaluronan oligomeric cues and the release of inflammatory markers were monitored during over a 21 day culture. Biochemical analysis was used to quantify cell proliferation, matrix protein synthesis and cross-linking efficiency, while immunofluorescence and electron microscopy were used to analyze the elastin matrix quality. It was observed that SMC activation with TNF-α (10 ng ml^?1) induced matrix calcification and promoted production of elastolytic MMP-2 and MMP-9. However, these effects were attenuated by the addition of TGF-β1 and HA oligomer cues to TNF-α-stimulated cultures, which also enhanced tropoelastin and collagen production, improved elastin matrix yield and cross-linking, promoted elastin fiber formation and suppressed elastase activity, although the release of MMP-2 and MMP-9 was not affected. Overall, the results suggest that TGF-β1 and HA oligomers are potentially useful in suppressing SMC activation and inducing regenerative elastin repair within aneurysms.     

Association of IL-1β +3953 C and HLA-DRB1*15 with Coronary Artery and Rheumatic Heart Diseases in South India

Among the various candidate genes predisposing for cardiovascular diseases, HLA-DRB1* and IL-1β +3953C/T alleles have been implicated repeatedly. To test these in South India, we carried out a case control study of 323 Coronary Artery Disease (CAD) patients, 56 Rheumatic Heart Disease (RHD) patients and 254 endemic controls. The polymorphisms were studied by PCR – SSP and ARMS-PCR methods and results analyzed for various clinical and demographic parameters. In CAD, HLA-DRB1*14 allele showed significant predisposition (OR: 2.19; 95% CI: 1.04–4.58; p value = 0.023), particularly in male patients (OR: 4.07; 95% CI: 1.20–13.81; p value = 0.01) and further in males with Triple Vessel Disease (OR: 5.49; 95% CI: 1.45–20.60; p value = 0.007). On the other hand, HLA-DRB1*15 predisposed for RHD (OR: 3.56; 95% CI: 1.87–6.78; p value = 0.001) in both the genders. Population stratification showed this higher risk association in Vanniyar caste (OR: 5.00; 95% CI: 1.27–19.59; p value = 0.022). Among the IL1-β +3953C/T polymorphism, the ancestral allele ‘C’ showed a significant high risk association with CAD (OR: 1.83; 95% CI: 1.24–2.70; p value = 0.001), particularly in Mudaliar (OR: 6.07; 95% CI: 1.77–20.74; p value = 0.003; AF = 0.7) and Vanniyar castes (OR: 3.67; 95% CI: 0.92–14.57; p value = 0.05; AF = 0.660). Two different cardiac ailments studied, RHD & CAD thus showed varied associations in this South Indian cohorts. RHD having an infectious aetiology shared a HLA-DRB1*15 high risk association, while HLA-DRB1*14 and IL-1β +3953C predisposed for CAD, an inflammatory disorder, reiterating the diverse genetic predisposition of the two cardiac ailments studied.     

Stronger Toll-like receptor 1/2, 4, and 7/8 but less 9 responses in peripheral blood mononuclear cells in non-infectious exacerbated asthmatic children

Toll-like receptors (TLR) initiate innate and often affect adaptive immune response. This study aimed to determine if TLR response and T regulatory cell (Treg) function in peripheral blood mononuclear cells (PBMC) correlate with clinical severity in non-infectious asthma. TLR1–9 expression and representative response cytokine TNF-α, IL-6, and IFN-β secretions were analyzed after stimulation by TLR1–9 ligands from 17 non-infectious asthmatic children. TNF-α production was higher in TLR1/2 (median 385.4 vs. 250.3 pg/ml in 1 μg/ml Pam3CSK4, p = 0.0078), TLR4 (2392.4 vs. 1355.9 in 1 μg/ml LPS; p = 0.0005), and TLR7/8 (10,776.2 vs. 4237.0 pg/ml in 1 μg/ml R848, p = 0.0079) of patients in exacerbation than those in convalescence and healthy controls despite equal TLR expression. TNF-α production stimulated by TLR9 agonist was significantly lower in exacerbation (17.7 vs. 34.9 pg/ml in 1 μg/ml ODN2216, p = 0.0175), while IL-6 production had similar patterns but was significantly lower in TLR3 signaling (119.7 vs. 245.0 pg/ml in 0.1 μg/ml poly(I:C), p = 0.0033). IFN-β production by TLR3 agonist also decreased in exacerbation but not statistically significant. Six older children showed decreased FOXP3 percentage in CD4 + CD25^high and decreased suppression capability in exacerbation but restored in stabilization (82.8% vs. 90.0%, p = 0.0061 and 60.9% vs. 81.7%, p = 0.0071; respectively). In conclusion, normalizing imbalanced TLR signaling and enhancing Treg cell capability may guide possible therapeutic strategies for non-infectious asthma in exacerbation.     

The stimulation of CD8+ T cells by dendritic cells pulsed with polyketal microparticles containing ion-paired protein antigen and poly(inosinic acid)–poly(cytidylic acid)

New adjuvants and delivery strategies are needed to optimize the ability of protein-based vaccines to elicit CD8⁺ T cell responses. We have developed a model vaccine formulation containing ovalbumin (OVA) and the double-stranded RNA analog poly(inosinic acid)–poly(cytidylic acid) (poly(I:C)), a TLR3 agonist. OVA and poly(I:C) were each ion-paired to cetyltrimethylammonium bromide (CTAB) to produce hydrophobic complexes, which were co-encapsulated in pH-sensitive polyketal (PK3) microparticles (1–3 μm) using a single emulsion method. Loading levels ranged from 13.6 to 18.8 μg/mg OVA and 4.8 to 10.3 μg/mg poly(I:C). Murine splenic dendritic cells (DCs) pulsed with PK3-OVA–poly(I:C) microparticles, at antigen doses of 0.01 and 0.1 μg/mL, induced a higher percentage of IFNγ-producing CD8⁺ T cells than DCs treated with PK3-OVA particles or soluble OVA/poly(I:C). A higher antigen dose (1 μg/mL) was less effective, which can be attributed to CTAB toxicity. At the lowest antigen dose (0.01 μg/mL), PK3-OVA–poly(I:C) microparticles also enhanced TNF-α and IL-2 production in CD8⁺ T cells. These data demonstrate the potential of polyketal microparticles in formulating effective CD8⁺ T cell-inducing vaccines comprising protein antigens and dsRNA adjuvants.     

Characterizations of CD4-1, CD4-2 and CD8β T cell subpopulations in peripheral blood leucocytes,spleen and head kidney of Japanese flounder (Paralichthys olivaceus)

In the previous study, antibodies against CD3 molecule have been produced and were used in labeling T cells in Japanese flounder (Paralichthys olivaceus). In this paper, CD4⁺ and CD8⁺ lymphocytes subpopulations in peripheral blood leucocytes (PBL), spleen and head kidney of flounder were investigated. The flounder CD4-1, CD4-2 and CD8β recombinant proteins and their antibodies (Abs) were produced, then the cross-reactivity of the Abs to CD4-1, CD4-2 and CD8β was detected by Western blotting, respectively, and the reactions of Abs to PBL were analyzed by immunofluorescence staining (IFS) and flow cytometry (FCM). CD4-1⁺/CD3⁺, CD4-2⁺/CD3⁺, and CD8β⁺/CD3⁺ lymphocytes in PBL, spleen and head kidney were observed by double IFS, then their proportions were analyzed using two-color FCM, respectively. Further, CD4-1/CD8β, CD4-2/CD8β, or CD4-1/CD4-2 lymphocytes were analyzed using double-IFS and two-color FCM. Finally, CD4-1⁺, CD4-2⁺, and CD8β⁺ lymphocytes in spleen and head kidney were observed by immunohistochemistry. The results showed that the Abs were specific for CD4-1, CD4-2 and CD8β molecules, respectively. The proportions of CD4-1⁺/CD3⁺, CD4-2⁺/CD3⁺, and CD8β⁺/CD3⁺ lymphocytes were 6.7 ± 2.0%, 8.6 ± 2.8%, 2.1 ± 1.3% in PBL; 13.6 ± 3.6%, 15.6 ± 5.2%, 2.8 ± 1.4% in spleen; 20.0 ± 4.6%, 20.5 ± 4.6%, 3.2 ± 1.5% in head kidney, respectively. Most CD4⁺ and CD8⁺ cell subpopulations belonged to CD3⁺ cells; there were no cross-reactivity between CD4⁺ and CD8⁺ cells. CD4-1⁺/CD4-2⁻, CD4-1⁻/CD4-2⁺, and CD4-1⁺/CD4-2⁺ cells presented different proportions in PBL, spleen and head kidney, among them, CD4-1⁺/CD4-2⁺ cell is the majority of CD4T cell subpopulation.     

An electrochemical study on self-ordered nanoporous and nanotubular oxide on Ti–35Nb–5Ta–7Zr alloy for biomedical applications

Highly ordered nanoporous and nanotubular oxide layers were developed on low-rigidity β Ti–35Nb–5Ta–7Zr alloy by controlled DC anodization in electrolyte containing 1 M H₃PO₄ and 0.5 wt.% NaF at room temperature. The as-formed and crystallized nanotubes were characterized by electron microscopy, energy-dispersive X-ray spectrometry and X-ray diffraction. The electrochemical passivation behavior of the nanoporous and nanotubular oxide surfaces were investigated in Ringer’s solution at 37 ± 1 °C employing a potentiodynamic polarization technique and impedance spectroscopy. The diameters of the as-formed nanotubes were in the range of 30–80 nm. The nanotubular surface exhibited passivation behavior similar to that of the nanoporous surface. However, the corrosion current density was considerably higher for the nanotubular alloy. The surface after nanotube formation seemed to favor an immediate and effective passivation. Electrochemical impedance spectra were simulated by equivalent circuits and the results were discussed with regard to biomedical applications.     

Attenuation of alpha-naphthylisothiocyanate (ANIT)-induced biliary fibrosis by depletion of hepatic macrophages in rats

Biliary fibrosis is a complex process in which macrophages and myofibroblasts may play central roles. We investigated biliary fibrosis lesions induced in the Glisson’s sheath in rats by alpha-naphthylisothiocyanate (ANIT) administration under macrophage depletion. Hepatic macrophages were depleted in F344 rats with liposome-encapsulated clodronate (CLD) (10 mL/kg body weight, i.v) followed by bile duct injury with ANIT (75 mg/kg body weight, i.p) (ANIT + CLD group). Rats received empty-liposomes (Lipo) followed by ANIT, and served as control (ANIT + Lipo group). In both ANIT + Lipo and ANIT + CLD groups, ANIT-induced bile duct injury with inflammatory cell infiltration was seen on days 1–3, and subsequently reparative fibrosis occurred on days 5 and 7. In comparisons between the two groups, macrophages reacting to CD68, CD163, MHC class II and CD204 were less in numbers in ANIT + CLD group; the most sensitive immunophenotype was of CD163-positive. Furthermore, in ANIT + CLD group interstitial mesenchymal cells/myofibroblasts reacting to vimentin, desmin and α-smooth muscle actin were also less in grades and tended to be delayed in appearance. Interestingly, MCP-1, IFN-γ, IL-10, and TGF-β1 mRNAs were significantly increased mainly on day 2 in ANIT + Lipo group, while the levels of these factors were prominently lower in ANIT + CLD group. Collectively, depletion of hepatic macrophages plays roles in attenuating biliary fibrogenesis by production of inflammatory factors. The present results indicated clearly importance of macrophage functions in the pathogenesis of biliary fibrosis.