Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are elevated in congestive heart failure and are known to induce the production of reactive oxygen species as well as to deteriorate respiratory muscle function.Given the antioxidative effects of exercise training, the aim of the present study was to investigate if exercise training is capable of preventing a TNF-α induced loss of diaphragmatic force in mice and, if so, to elucidate the potential underlying mechanisms.Prior to intraperitoneal injection of TNF-α or saline, C57Bl6 mice were assigned to four weeks of exercise training or sedentary behavior. Diaphragmatic force and power generation were determined in vitro. Expression/activity of radical scavenger enzymes, enzymes producing reactive oxygen species and marker of oxidative stress were measured in the diaphragm.In sedentary animals, TNF-α reduced specific force development by 42% concomitant with a 2.6-fold increase in the amount of carbonylated α-actin and creatine kinase. Furthermore, TNF-α led to an increased NAD(P)H oxidase activity in both sedentary and exercised mice whereas xanthine oxidase activity and intramitochondrial ROS production was only enhanced in sedentary animals by TNF-α. Exercise training prevented the TNF-α induced force reduction and led to an enhanced mRNA expression and activity of glutathione peroxidase. Carbonylation of proteins, in particular of α-actin and creatine kinase, was diminished by exercise training.TNF-α reduces the force development in the diaphragm of mice. This effect is almost abolished by exercise training. This may be a result of reduced carbonylation of proteins due to the antioxidative properties of exercise training. Impaired skeletal muscle function is frequently seen in a large variety of systemic disorders including congestive heart failure (CHF) and sepsis One important mechanism that is common in secondary myopathies is the prevalence of elevated inflammatory cytokines. Increased cytokine levels have been detected in the serum and/or in skeletal muscle tissues both in CHF and sepsis Reactive Oxygen Species (ROS), e.g. hydrogen peroxide, are known to stimulate ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle It is well accepted that exercise training exerts antioxidative effects to the skeletal muscle in CHF, in particular due to an augmentation in the activity of radical scavenger enzymes The aim of the present study, therefore, was to test the hypothesis that an exercise training program is capable of preventing the TNF-α induced loss of muscle contractile force in the diaphragm of mice. In addition, potential molecular mechanisms responsible for the TNF-α induced force reduction and protective effects of exercise training were also analyzed.The local Council for Animal Research (Regierungspräsidium Leipzig/Saxonia/Germany) approved all experimental protocols (TVV 33/09). We performed a power analysis based on our results in soleus muscle prior to the study Mice assigned to the exercise group were accustomed to run on a motorized rodent treadmill with a shock-plate incentive. The slope of the treadmill was kept constant at 5°. Mice were trained at a speed of 15 m/min for 60 min/day with 2-minute rest intervals every 15 minutes for five days a week, as described previously For functional assessment, a diaphragm bundle from the right hemidiaphragm was incubated in an oxygenated physiological buffer. The isolated muscle bundle was fixed with a silk suture at the central tendon and the proximal junction to the ribs, and mounted in an organ bath for measurement of muscle contractile function  as described previously The frozen biopsy samples were homogenized in RIPA buffer containing proteinase inhibitors  and protein content was determined . Enzymatic activities of NAD(P)H oxidase Calpain enzymatic activity was measured as recently described Total RNA was isolated from diaphragm muscle tissue using RNeasy , and reverse transcribed into cDNA using random hexamers and Sensiscript reverse transcriptase . An aliquot of the cDNA was used for quantitative RT-PCR applying the LightCycler™ (Roche Diagnostics Inc). The expression of specific genes was normalized to the expression of Hypoxanthin-Phosphoribosyl-Transferase mRNA (HPRT). For a detailed description of the used primers and conditions, please refer to Frozen tissue samples were homogenized in RIPA buffer containing a mixture of protease inhibitor  and protein expression was quantified by Western blot using specific antibodies to MuRF1  and MafBx . After incubation with a horseradish peroxidase-conjugated secondary antibody, specific bands were visualized by enzymatic chemiluminescence  and densitometry was quantified using a 1D scan software package . Loading differences were controlled by re-probing the blot with an antibody against GAPDH .Oxidized proteins were labeled with Alexa 488 Fluorescent Hydroxylamine and separated on a 2D-gel electrophoresis The peptidase activities of the proteasome in the cytosolic fraction were determined as recently described All data are expressed as mean value ± standard error of the mean. Intergroup comparisons were performed with two-way ANOVA followed by a post test according to Tukey using GraphPad Prism v.2.01, GraphPad Software, San Diego, CA. A probability value of <0.05 was considered statistically significant.Animals of the four groups did not differ with regard to body mass, diaphragm strip lengths, diaphragm strip mass and diaphragm cross sectional area .As shown in Exercise training itself had no influence on the force development since there was no difference between exercised and sedentary sham treated mice.2) and shortening velocity (length/s) and no significant differences in shortening velocity were detectable (data not shown), power followed the same pattern as the above described force development: Since power is calculated as the product of force H oxidase was measured. After TNF-α administration, there was a significantly higher activity of NAD(P)H oxidase in both sedentary and exercised animals .Another important source of ROS, in particular the superoxide anion, is xanthine oxidase. As depicted in As an indirect marker of intramitochondrial ROS, the ratio of aconitase to fumarase activity was reduced in sedentary animals treated with TNF-α compared to shams indicating an increased intramitochondrial ROS concentration. No significant decrease in the ratio of aconitase to fumarase activity was detectable in exercised animals after TNF-α administration.Carbonylated proteins are an established marker of oxidative stress Furthermore, there was a 2.6-fold increase of carbonylated CK in sedentary animals after TNF-α treatment, whereas only a 1.3 fold increase was evident in exercised animals. Comparing sedentary animals treated with TNF-α with exercised animals treated with TNF-α again, the amount of carbonylated CK was significantly lower in the exercise group .Carbonylation of α-actin  as well Given that the muscle specific E3-ligase MurF1 plays a central role in TNF-α mediated loss of force in soleus muscle As shown in The assessment of proteasome activity revealed a significantly higher activity under the influence of TNF-α in sedentary C57Bl6 mice, which was not detectable in exercised mice with respect to trypsin-like activity  and peptFurthermore, calpain activity was not significantly different between the four groups in this experimental setting .Three key findings emerge from this study:A single intraperitoneal injection of TNF-α induced a ∼42% decrease in specific force development in the diaphragm of sedentary mice after 24 hours. This loss of force was almost entirely prevented by intensive exercise training over four weeks prior to TNF-α administration.TNF-α administration resulted in increased oxidative stress indicated by carbonylation of proteins, in particular of α-actin and creatine kinase. Exercise training was able to attenuate the TNF-α induced carbonylation of α-actin and CK. This may be due to an improvement in antioxidative capacity by the exercise program.TNF-α treatment increased proteasome activity by ∼40% in the sedentary group. This increase was impeded by exercise training.It is well accepted that TNF-α administration leads to impaired force development in the diaphragm of several animal models such as mice However, exercise training also exerts antioxidative effects, in particular due to an augmentation in activity of radical scavenger enzymes NAD(P)H oxidase, one major source of ROS in skeletal muscle With regard to aconitase, ROS-mediated inactivation of the enzyme can be overcome, possibly by an exercise induced dephosphorylation of the enzyme As mentioned above, ROS are supposed to play a central role in transmitting the force-impairment effect of TNF-α in the diaphragm For this reason, we searched for carbonylated proteins using 2D-gel electrophoresis. Due to the fast onset of diaphragm dysfunction, we assumed myofibrillar proteins and proteins involved in energy production as possible targets of carbonylation.We detected a 2.6-fold increase in carbonylated α-actin and carbonylated CK in sedentary, TNF-α treated mice. This is in line with the current literature where α-actin and CK are described as the major proteins affected by carbonylation in many scenarios The carbonylation of α-actin also has deleterious effects and destroys the organization of the thin filament. A study of Dalle-Donne and colleagues revealed that exposing actin to hypochlorous acid causes a rapid increase in carbonylation, a result of oxidation of methionines, tryptophans and lysines In contrast to the peripheral soleus muscle Interestingly, proteasome activity, in particular trypsin-like and petidylglutamyl-peptide hydrolyzing activity, was enhanced in sedentary animals by TNF-α which could be prevented by exercise training. Carbonylation of α-actin and CK was significantly correlated to the before mentioned proteasome activities. One possible explanation for this finding may be an ubiquitin-independent proteolytic function of the proteasome which is described for oxidized proteins E. coli lipopolysaccharide despite a clear inhibition of proteasome based proteolysis Furthermore, a study by Supinski and colleagues provides strong evidence that the proteasome system itself is not directly involved in the acute force loss in the diaphragm and seems to be a bystander during this early phase of damage Since it was our aim to investigate the impact of one single inflammatory cytokine (TNF-α) on diaphragmatic function and to elucidate potential biochemical pathways, we used the artificial model of intraperitoneal injection of TNF-α. Therefore, the results of this study cannot be translated into certain diseases like CHF or sepsis. Further studies in animal models of CHF or sepsis are necessary to investigate the proposed mechanisms under the circumstances of a specific disease. In addition, we only investigated one specific exposure time of TNF-α (after 24 hours). For this reason, other mechanisms, e.g. enhanced proteolysis, may be relevant after long term exposure.In conclusion, the results of this study suggest that the administration of TNF-α led to a significant loss of diaphragmatic force and power. This was probably due to a ROS mediated increase in carbonylation of α-actin and CK, two important components of force generation. Exercise training, however, resulted in an elevation of the radical scavenger enzyme GPX as well as a prevention of TNF-α induced increase in xanthine oxidase activity and mitochondrial ROS production, thereby lowering the ROS mediated carbonylation of α-actin and CK to collectively preserve diaphragmatic force .Figure S1(TIF)Click here for additional data file.Manuscript S1(DOC)Click here for additional data file.


Wnt signalling is prevented by the proteosomal degradation of β-catenin, which occurs in a destruction complex containing adenomatous polyposis coli (APC), APC-like (APCL), Axin and Axin2. Truncating mutations of the APC gene result in the constitutive stabilisation of β-catenin and the initiation of colon cancer, although tumour cells tolerate the expression of wild-type APCL. Using the colocalisation of overexpressed Axin, APC and APCL constructs as a readout of interaction, we found that Axin interacted with the second twenty amino acid repeat (20R2) of APC and APCL. This interaction involved a domain adjacent to the C-terminal DIX domain of Axin. We identified serine residues within the 20R2 of APCL that were involved in Axin colocalisation, the phosphorylation of truncated APCL and the down-regulation of β-catenin. Our results indicated that Axin, but not Axin2, displaced APC, but not APCL, from the cytoskeleton and stimulated its incorporation into bright cytoplasmic dots that others have recognised as β-catenin destruction complexes. The SAMP repeats in APC interact with the N-terminal RGS domain of Axin. Our data showed that a short domain containing the first SAMP repeat in truncated APC was required to stimulate Axin oligomerisation. This was independent of Axin colocalisation with 20R2. Our data also suggested that the RGS domain exerted an internal inhibitory constraint on Axin oligomerisation. Considering our data and those from others, we discuss a working model whereby β-catenin phosphorylation involves Axin and the 20R2 of APC or APCL and further processing of phospho-β-catenin occurs upon the oligomerisation of Axin that is induced by binding the SAMP repeats in APC. Homeostasis of the colonic epithelium requires the proliferation of the stem cells located at the bottom of the crypts, the subsequent expansion of the daughter cell population, the differentiation and migration of these cells toward the surface and ultimately apoptosis and release into the lumen The mechanistic steps that involve APC are not completely understood. APC is a large protein of 2843 amino acids fig. 1 . We concluded that Axin colocalisation with truncated APCL required the 20R2.In contrast to myc-Axin, N-terminal flag-tagged Axin (flag-Axin) was expressed as cytoplasmic dots in about half of the transfected cells , which exhibited a diffuse cytoplasmic distribution in DLD1 cells upon ectopic expression fig. 3B.We also performed the converse experiment, using yAPCL1179 truncated shortly after the 20R2 and deletion mutants of myc-Axin, to identify a putative second APCL-binding site in Axin that was independent of the RGS domain fig. 4. fig. S3). We identified eight amino acids common to all inspected 20R2 sequences. These eight residues were mutated in the context of yAPCL1257 . The expression of yAPCL(1091–1257) dissolved the flag-Axin dots, whereas the expression of yAPCL(1091–1257)-2μ did not affect these dots. Together, we concluded that the conserved residues within the 20R2 were required for truncated APCL to colocalise with Axin, that the N-terminal residues of APCL were not required and that colocalisation with the 20R2 inhibited Axin oligomerisation.To provide additional evidence that Axin binds to the 20R2 of APCL, we compared the 20R2 sequences of APC and APCL from different species  abolished β-catenin down-regulation and led to the colocalisation of APCL with β-catenin fig. 5C.The immunoprecipitation of yAPCL(1091–1257) followed by western blotting revealed a pattern of three bands fig. 6A.fig. S5). The intracellular localisation of all the constructs was similar  in transiently transfected SW480 cells indicated that each Ser/Thr residue was required to prevent the accumulation of β-catenin and to inhibit its transcriptional activity. Remarkably, the T1155A mutation almost abolished these activities of yAPCL1257. These results qualitatively correlated with the ability of these ectopically expressed proteins to colocalise with Axin , as the mutation of each Ser/Thr residue resulted in a partial loss of colocalisation with flag-Axin. One notable quantitative discrepancy occurred with yAPCL1257-L1168V-T1155A, which colocalised with flag-Axin in a greater proportion of cells than expected based on the β-catenin staining and reporter assays. We hypothesised that T1155 might be involved in other functions in addition to contributing to Axin colocalisation. Together, we concluded that the down-regulation of β-catenin mediated by truncated APCL as well as the Axin/APCL colocalisation depended on the presence of the conserved T1155 residue. The dependence on the conserved Ser residues in the 20R2 suggests a phosphorylation-dependent regulation of truncated APCL impacting on β-catenin activity and Axin colocalisation.We next analysed the functional consequences of individually mutating each of the four conserved Ser/Thr residues in the context of yAPCL1257-L1168V ( similar fig. 6F.fig. S8). This construct did not restore flag-Axin colocalisation (fig. S8A) or β-catenin down-regulation (fig. S8B). Interestingly, the lack of Axin colocalisation with the first SAMP repeat corresponds with previous results showing that both APCL SAMP repeats are required to interact with Axin APCL contains two identifiable SAMP repeats that may contribute to β-catenin degradation and compensate for the deleterious effect of mutating the 20R2. Therefore, we extended the yAPCL1257-2μ construct up to position 1357, shortly after the first SAMP repeat (fig. S9). In contrast to yAPC1641, yAPCL1641-2μ did not down-regulate β-catenin (fig. S9A) and did not affect β-catenin transcriptional activity when expressed at low levels (fig. S9B). Interestingly, yAPC1641-2μ excluded β-catenin from the nucleus (fig. S9A). This correlated with the observation that high levels of yAPC1641-2μ inhibited β-catenin transcriptional activity (fig. S9B). This might be explained by either sequestration APC has three SAMP repeats fig. 1 tTo understand the role of the first SAMP repeat in APC, we performed colocalisation experiments in DLD1 cells using the yAPC1641 and myc-Axin constructs fig. 9. , S10). Inactivating the 20R2 did not affect the efficiency of dot formation with Axin. We concluded that APC-stimulated Axin oligomerisation was independent of the 20R2.To investigate the influence of the 20R2 on dot formation, we repeated the colocalisation experiments and compared yAPC1641 with yAPC1641-2μ fig. 9 a, S10) and flag-Axin  revealed that 20R7 through 20R12 share the four residues that we identified as being important for Axin binding and β-catenin degradation. Moreover, the 20R1 of APC is very similar to the 20R2, suggesting that most of the specificity determinants for Axin binding are provided by sequences outside the original short boundaries of the 20R2. Indeed, a comparison after extending in the N- and C-terminal directions revealed that the extended regions were more conserved in the 20R2s of APC and APCL than in any other homologous 20R pair. The stronger selective pressure on the 20R2 is likely related to the conservation of the elements that create a specific Axin-binding site.Our results indicate that Axin does not colocalise with either the first or third 20R of APCL and that neither APC nor APCL contain another 20R that could functionally complement 20R2 inactivation. Comparing all the 20R sequences from APC and APCL . On the other hand, using an antibody against APC, it has been observed that Wnt induces the partial dissociation of APC and Axin The SAMP repeats are not necessary for targeting β-catenin for degradation when truncated APC retaining the CiD is ectopically expressed Our data provide a hint at the solution for another conflicting situation in colon cancer cells. β-catenin is stabilised as a consequence of mutations that truncate APC before the first SAMP repeat, despite the expression of APCL As a conclusion, the data presented here and elsewhere converge on a working model that attributes β-catenin phosphorylation to Axin bound to the 20R2 of either APC or APCL and the degradation of phosphorylated β-catenin to the oligomerisation of Axin bound to the SAMP repeats of APC.Human embryonic kidney cells expressing SV40 large T antigen (HEK293T) and SW480 and DLD1 colorectal cancer cell lines were obtained from American Type Culture Collection (ATCC) and were maintained in DMEM  supplemented with 10% foetal calf serum , 1% penicillin and 1% streptomycin (PAA Laboratories). The APC mutations in the cell lines are described in The following primary antibodies were used in this study: anti-GFP , anti-β-actin , sheep anti-TGN46 , mouse-anti-GM130 , rabbit anti-Giantin , rabbit anti-flag and anti-β-catenin H102  and anti-myc . Secondary antibodies coupled to horseradish peroxidase or Cy3 were from Dianova, Hamburg, Germany.The plasmids expressing the YFP-APC and YFP-APCL fusion proteins were constructed using standard molecular biology methods. The plasmid sequences are available upon request. pcDNAflag Plasmids were transfected into cells overnight using 5 μl of polyethylenimine (1 mg/ml) per μg of DNA. For DLD1 and SW480 cells, 2 μg of total plasmid DNA was transiently transfected into 250,000 cells in 35 mm dishes. For HEK293T cells, 10 μg of total plasmid DNA was transiently transfected into 5,000,000 cells in 85 mm dishes.The TOPglow reporter consists of a tandem repeat of four TCF/LEF1 binding sites inserted in front of a TATA box Triton-X100 extracts were prepared in a 20 mM Tris-HCl, pH 7.4, buffer containing 150 mM NaCl, 5 mM EDTA, 1% Triton-X100, 1 mM DTT and 1 mM PMSF.Immunoprecipitations were performed using Triton-X100 extracts containing 50 mM NaF. The immunoprecipitates were washed in a 50 mM Tris-HCl, pH 8, buffer containing 150 mM NaCl, 5 mM EDTA and 1% Triton-X100.The blots were developed using Western Lightning™ chemiluminescence reagents , and the signals were detected using a LAS-3000-Fuji camera .Immunofluorescence was performed as previously described Figure S1The intracellular localisation of Axin is either diffuse or dotty. DLD1 and SW480 cells were transiently transfected on day 1 with N-terminal tagged expression constructs . The cells were fixed on day 3 and were stained with an anti-flag or an anti-myc antibody. Bar, 10 μM. A, Representative images of Axin intracellular localisation. The imaging parameters were identical for each type of tag. B, Quantification of the proportion of cells exhibiting a diffuse versus a dotty pattern. The percentages indicate the number of cells with dots (n = 100), and the data are presented as the mean ± standard deviation of three independent experiments.(PDF)Click here for additional data file.Figure S2The APCL dots are not Golgi vesicles. SW480 cells were transiently transfected on day 1 with yAPCL1728. The cells were fixed on day 3 and were stained with the indicated antibodies. Bar, 10 μM.(PDF)Click here for additional data file.Figure S3Comparison of the 20R2 sequences in APC and APCL from different species. Residues common to all 20R2 sequences are highlighted in red.(PDF)Click here for additional data file.Figure S4The 20R2 from a short internal APCL fragment is required to inhibit Axin oligomerisation. DLD1 cells were transiently transfected on day 1 with the N-terminal YFP-labelled APCL constructs or N-terminal flag-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody and Hoechst dye. Where applicable, the percentages indicate the proportion of different localisation patterns observed in the transfected cells. The imaging parameters were identical for each type of tag. Bar, 10 μM.(PDF)Click here for additional data file.Figure S5The L1168V mutation in the 20R2 does not affect the ability of truncated APCL to inhibit β-catenin transcriptional activity. SW480 cells were transiently transfected on day 1 with reporter plasmids and 100 ng of an empty vector (flag) or the indicated N-terminal YFP-tagged APCL constructs. TOP/FOP reporter assays were performed on day 3 to measure β-catenin transcriptional activity . The data are presented as the mean ± standard deviation of three independent values from a representative experiment. In a parallel experiment, the cells were transiently transfected with 1 μg of the indicated plasmids on day 1. Cell extracts were prepared on day 3 and were subjected to western blotting using the indicated antibodies.(PDF)Click here for additional data file.Figure S6Representative cells with or without β-catenin down-regulation upon expression of wild-type or mutant yAPCL1257-LV to illustrate the results presented in. SW480 cells were transiently transfected with the indicated APCL constructs, fixed on day 3 and stained with an anti-β-catenin antibody and Hoechst dye. A schematic of the mutants is presented in (PDF)Click here for additional data file.Figure S7Representative cells with or without Axin colocalisation upon co-expression of wild-type or mutant yAPCL1257-LV to illustrate the results presented in. SW480 cells were transiently transfected on day 1 with the indicated yAPCL1257 constructs and N-terminal flag-tagged Axin. A schematic of the yAPCL1257 mutants is presented in (PDF)Click here for additional data file.Figure S8The first SAMP repeat of APCL restores neither Axin colocalisation (A)or β-catenin degradation (B) after mutation of the 20R2.A, DLD1 cells were transiently transfected on day 1 with the indicated N-terminal YFP-labelled APCL constructs or N-terminal flag-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody and Hoechst dye. B, SW480 cells were transiently transfected with the indicated APCL constructs, fixed on day 3 and stained with an anti-β-catenin antibody and Hoechst dye. The imaging parameters were identical for each type of tag. Bar, 10 μM.(PDF)Click here for additional data file.Figure S9The 20R2 of APC truncated after the first SAMP repeat is required to target β-catenin for degradation (A) and to inhibit its transcriptional activity (B).A, SW480 cells were transiently transfected with a control vector (flag) or the indicated APCL constructs, fixed on day 3 and stained with an anti-β-catenin antibody and Hoechst dye. B, SW480 cells were transiently transfected on day 1 with reporter plasmids and 100 ng of an empty vector (flag) or the indicated N-terminal YFP-tagged APC constructs (see (PDF)Click here for additional data file.Figure S10The 20R2 of full-length APC is not necessary to promote Axin co-oligomerisation. DLD1 cells were transiently transfected on day 1 with the indicated N-terminal YFP-tagged APC constructs or N-terminal myc-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-myc antibody and Hoechst dye. yAPC-2μ contains the mutations shown in (PDF)Click here for additional data file.Figure S11Pairwise comparison of the 20Rs of APC and APCL. Red indicates amino acids common to the 20R2s of APC and APCL from various species (fig. S3), red and blue represent the residues common to the 20R2s of human APC and APCL and the bold black letters indicate the amino acids common within each homologous 20R pair. The asterisks indicate residues from the 20R2 of APCL that are important for Axin binding and β-catenin degradation. Bar, 10 μM.(PDF)Click here for additional data file.


Virus-Like Particles (VLPs) represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN1826 or poly(I:C) adjuvants, has been evaluated in an animal model.H2 pattern, VLPs formulated with either adjuvant elicited a TH1-biased IgG subclasses (IgG2a and IgG3), with poly(I:C) more potent than CpG ODN1826.Adjuvanted VLPs elicited a higher titer of total specific IgG compared to VLPs alone. Furthermore, while VLPs alone induced a balanced TThe results confirmed that adjuvants efficiently improve antigen immunogenicity and represent a suitable strategy to skew the adaptive immune response toward the differentiation of the desired T helper subset, also using VLPs as antigen. The development of a safe and effective HIV-1 vaccine, either prophylactic or therapeutic, remains a major concern and a high priority for the scientific community. In this respect, given that strategies based on attenuated or inactivated pathogens are not suitable as HIV vaccines for safety reasons, alternative effective vaccination strategies are developed and evaluated /2)/(IgG1). According to such calculation, an index value < 1 stands for a TH2 polarization; an index value > 1 stands for a TH1 polarization.To determine whether the addition of adjuvants in the immunization protocol induced a TIntergroup comparisons were performed with the unpaired two-sided Student's t-test. All P values were two-tailed and considered significant if less than 0.05.In order to determine the effect of CpG ODN1826 and poly(I:C) adjuvants on the humoral response elicited by baculovirus-expressed HIV-VLPs developed in our laboratory, Balb/c mice were immunized by intradermal route with 20 μg of VLPs alone or formulated with the two adjuvants. Sera collected after the last boosting from all immunization groups were heat-inactivated and evaluated in ELISA against HIV-1 p24 and trimeric gp140. The results showed that both adjuvants were able to enhance the immunogenicity of HIV-VLPs eliciting a final 1:8,100 (CpG ODN1826) and 1:24,300 (poly(I:C)) titer of IgG to both p24 and gp140 -adjuvanted HIV-VLPs showed a more sustained fashion Figure  and 2B. The pattern of IgG subclasses induced by the different immunization protocols was evaluated by using secondary goat anti-mouse antibodies specific for each IgG subclass.H2 IgG1 and TH1 IgG3 subclasses, while no IgG2a or IgG2b were detected .Sera from animals immunized with HIV-VLPs alone showed anti-p24 low titers 1:300) of both T00 of botH2 IgG1 and TH1 IgG3 subclasses, while no IgG2a were detected. However, in contrast to results for anti-p24, low titers of IgG2b anti-gp140 were detected   adjuvant results in a TH1 skewed IgG pattern.Such results indicate that antibody profile induced by HIV-VLPs in mice is TH1:TH2 index was calculated for each immunization groups. Such index takes into consideration both TH1 IgG2a and IgG3 subclasses compared to TH2 IgG1 subclass. The TH1:TH2 index showed that, in sera of animals immunized with HIV-VLPs alone, the antibody response to p24 and gp140 was TH2 polarized . In contrast, in sera of mice immunized with adjuvanted HIV-VLPs, the antibody response to p24 was markedly skewed towards a TH1 profile (TH1: TH2 index > 1), and such effect was more pronounced when HIV-VLPs were adjuvanted in poly(I:C) (TH1: TH2 index 6) (Table H1 profile (TH1: TH2 index 2)  adjuvants.In order to determine the pattern of IgG subclasses induced by HIV-1 antigens packed in particulate structures as Virus Like Particles and to investigate the modulation of such pattern by TResults shown in the present study confirmed the ability of HIV-VLPs to induce strong humoral immune response and both adjuvants were able to enhance the immunogenicity of HIV-VLPs, inducing a 3-9 fold increase in the total IgG titers for both p24 and gp140. The kinetic of induced humoral immune response showed a similar trend in sera from each immunization group, although the poly(I:C) induced a more sustained titer.H2 for both p24 and gp140 and that formulation in both adjuvants results in a TH1 skewed IgG pattern. In particular, in our experimental model, poly(I:C) scores as the most potent in re-directing the TH pattern, in agreement to previous observations [Evaluation of IgG subclasses demonstrated that HIV-VLPs alone induced anti-p24 and anti-gp140 IgG1 and IgG3 subclasses with no IgG2a elicitation. On the contrary, CpG ODN1826 and poly(I:C) showed the same capacity to enhance titers of anti-p24 and anti-gp140 IgG2a and IgG2b subclasses, compared to HIV-VLPs alone. In addition, only poly(I:C) was able to induce an enhancement also in IgG1 and IgG3 subclasses, resulting in the broadest IgG subclass pattern of immune response. Such results indicate that antibody profile induced by HIV-VLP in mice is Trvations ,86.H1:TH2 index which evaluates the ratio between both TH1 associated IgG subclasses (IgG2a and IgG3) and TH2 associated (IgG1) subclass. Indeed, considering that an index value > 1 stands for a TH1 polarization, the index in sera from animals immunized with poly(I:C)-adjuvanted HIV-VLPs scored the highest.Such finding is further confirmed by the TH2 profile) with other subclasses represented at low titer (IgG2b and IgG3) or not present at all (IgG2a). We demonstrated that TH1-inducing adjuvants, and in particular the poly(I:C), were able to broaden the IgG subclasses response and to re-direct the immune response toward a TH1-biased pattern. In particular, HIV-VLPs formulated in poly(I:C) adjuvant elicited the highest titer of IgG3 subclass which has been claimed to mediate the HIV neutralization in sera from HIV-infected individuals [Our results confirm that HIV-VLPs represent an effective antigenic presentation and delivery system to prime the humoral immune response, eliciting mainly IgG1 subclass antibodies  appropriate for individual causative infectious agents .The authors declare that they have no competing interests.MLV performed the studies and contributed to writing the paper; MT contributed to HIV-VLPs preparation; MLT contributed to the data analysis; FMB supervised the whole project; LB was responsible for the overall planning and coordination of the study as well as writing the paper. All authors read and approved the final manuscript.


M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.Of those individuals who are infected with  Although a third of the global population has been exposed to tuberculosis the majority harbours a latent form of infection in vitro models and animal studies that simulate M. tuberculosis reactivation M. tuberculosisHost immune factors that allow for mycobacteria to remain in a persistent state of latency have not been clearly defined although considerable insight has been gained through the application of M. tuberculosis infection could be controlled by Tm-TNF but that soluble TNF was required to sustain host immune protection M. tuberculosis was associated with lack of proper bactericidal granuloma formation in latently infected complete TNF−/− mice treated with isoniazid and rifampicin TNF is a multifunctional cytokine, initially synthesized as a 26 Kda non-glycosylated type II trans membrane protein (Tm-TNF) which upon cleavage by the metalloprotease TNF-converting enzyme (TACE) forms a soluble 17 KDa protein. Both molecular forms of TNF are biologically functional as homotrimeric proteins that bind and mediate signaling through either TNFRp55  or TNFRp75  with Tm-TNF binding strongly TNFRp75 With the current development of new TNF inhibitor biologics which specifically inhibit solTNF and spare Tm-TNF in the treatment of chronic inflammatory disorders −/− mice C57Bl/6 wild type (WT) control mice, TNFM. tuberculosis H37Rv was grown in Middlesbrook 7H9 broth  supplemented with 10% Middlebrook OADC enrichment medium , 0.5% glycerol and 0.05% Tween 80 at 37°C until log phase. Prior to usage, mycobacterial aliquots were passed 30× through a 29.5 G needle to minimize bacterial clumping. Pulmonary infection with 100–200 cfu live M. tuberculosis H37Rv bacteria was performed using a Glas-Col Inhalation Exposure System, Model A4224. Inoculum size was confirmed 24 h post-infection by determining the bacterial burden in the lungs of infected mice.M. tuberculosis reactivation model  agar plates containing 10% OADC  and incubated at 37°C for 19–21 days. Colonies on plates were enumerated and bacterial burdens determined.Bacterial burdens in the lungs, livers and spleens of infected mice were determined at specific time points after infection with Mice were euthanized by carbon dioxide inhalation at specific time points. Organs were weighed and fixed in 10% formalin and paraffin-embedded. Two to 3 µm sections were stained with haematoxylin and eosin (H&E) and a modified Ziehl-Nielson (ZN) method as described Whole lungs were removed from infected mice at specific time points and were homogenized in 1 ml 0.04% Tween 80 saline containing protease inhibitor (Sigma) and the supernatants were collected after low-speed centrifugation, aliquoted and frozen at −80°C.Supernatants from organ homogenates or from cultured cells were harvested and assayed for cytokine concentration using commercially available ELISA reagents for IFNγ, IL-10, and IL-12p70 , according to the manufacturer's instructions.The data are expressed as the mean ± SEM. Statistical analysis was performed by ANOVA. For mortality studies, analysis was performed using the log-rank test. For all tests, a p-value of <0.05 was considered significant.et al., 1966, it was observed that immunizing mice with M. tuberculosis then re-infecting them preceding treatment with anti-tuberculous drugs resulted in mice reactivating with lower M. tuberculosis CFU numbers compared to control groups M. tuberculosis. The model used entailed aerosol inhalation infection of WT mice, TNF−/− mice and Tm-TNF mice with 100 viable M. tuberculosis H37Rv bacilli. The infection was allowed to progress for 21 days before commencement of treatment with 25 mg/Kg INH-RIF in drinking water for 6 weeks to reduce bacilli numbers to at least less than 100 CFUs in the lungs after which treatment was withdrawn and tuberculosis reactivation was monitored. Body weights were recorded throughout the infection period and body weight loss was interpreted as severe disease due to reappearing tuberculosis and correlated with susceptibility to infection. Control groups of WT, Tm-TNF and TNF−/− mice that did not receive chemotherapeutic treatment were confirmed to have phenotypes as previously described In a study performed by McCune −/− mice which displayed significantly lower body weights between 69 and 165 days post-infection  compared to WT mice (−/− mice in a model using a 3 fold lower infection dose and chemotherapy was initiated earlier −/− mice was confirmed with a further significant increase (p<0.001) in lung weights noted at 113 days post-infection, 50 days after cessation of therapy. In contrast, lung inflammation in Tm-TNF mice was similar to WT mice during early infection with similar lung weights at 21, 63 and 133 days post-infection (p<0.01) in lung weights was evident in Tm-TNF mice at day 322 post-infection compared to WT mice  fluid was determined after infection. The cellularity in the BAL fluid was significantly higher in TNFnfection  confirminfection . TogetheM. tuberculosis infection whereby upon treatment with TNF-neutralizing antibody, mice displayed severe histopathology marked with excessive inflammation and loss of structured granulomas −/− mice and Tm-TNF mice at the indicated time points post-infection and pulmonary pathology analyzed. Untreated WT mice  via the enzymatic action of macrophage iNOS M. tuberculosis resulted in reactivation of tuberculosis disease with increased organ bacillary burdens and extensive granulomatous response −/− mice and Tm-TNF mice after completion of chemotherapy. iNOS expression patterns were largely within the confinement of granuloma lesions in WT mice (−/− mice where the pattern was dispersed and associated with the diffused granuloma lesions (+ cells) and lymphocyte (CD3+ cells) recruitment to investigate whether a correlation existed between macrophage recruitment and iNOS induction, and also to determine Tm-TNF effects on the structural relationship between macrophages and lymphocytes with respect to granuloma formation during reactivation. It is clear that macrophage distribution correlated strongly with iNOS induction where, in WT mice it was localized on the periphery of established granulomas (We therefore determined whether effector macrophage anti-mycobacterial function was intact in Tm-TNF mice undergoing  WT mice  in contr lesions  analyzed lesions . However lesions , increas lesions  with a ranulomas  but randanulomas  at 322 danulomas  but was anulomas . TherefoM. tuberculosis infection + T cells and subsequent bacilli control during chronic M. tuberculosis infection suggesting that interruption of IL-12 signal transduction contribute to development of reactivation of tuberculosis M. tuberculosis reactivation at this time point. The concentration of anti-inflammatory IL-10 was comparable 133 days post-infection but increased in WT mice by day 322 post-infection and remained significantly lower in Tm-TNF mice (M. tuberculosis chronic immunity and this phenotype is associated with lack of control of bacilli burden in the lung and lethality of these mice.Next we assessed the effect of Tm-TNF mediated immune responses in infected mice subsequent to INH-RIF treatment with particular reference to the quantification of IFNγ and IL-12 levels because of their reported functions in generating and maintaining protective immunity against TNF mice . These dM. tuberculosisEstimates indicate that a third of the global population is latently infected with the tuberculosis pathogen, M. tuberculosis or M. bovis BCG infection −/− mice. This observation confirms findings previously reported by Botha and Ryffel, 2003 M. tuberculosis infection and that solTNF might be required at later stages for sustained protection.We and others investigated the contribution made by the two molecular forms of TNF, solTNF and Tm-TNF, in the induction of protective immunity or immunopathology during et al, 2010 found that neutralization of TNF in cynomolgus macaques resulted in dissemination of disease without compromising the structural integrity of established granulomas M. tuberculosis infection, but referred to the diminished bactericidal capacity of such granulomatous structures et al, 2006 who found typical granulomas in biopsy specimens from patients on anti-TNF therapy who developed tuberculosis et al, 1998 reported adequate protection in ICAM-1 deficient mice despite lacking structured granulomas during M. tuberculosis infection M. tuberculosis. Here, we show that during tuberculosis reactivation, TNF−/− mice formed larger lesions with inflammation occupying larger areas of the lung with some evidence of necrosis compared to WT mice, an observation that corroborates previous findings by Mohan et al., 2001 and Botha and Ryffel, 2003 where a lack of proper defined granulomas in reactivating TNF−/− mice were described The formation of granulomas in response to mycobacterial challenge leads to killing or alternatively, isolation and confinement of bacilli to local sites of infection. They are dynamic structures that promote cellular interaction to enhance bactericidal efficacy, and the initial establishment and continued maintenance of its structural integrity in murine models is critically dependent on TNF. Nevertheless, the relationship between TNF and granuloma structure as an indicator of protection mediated against infectious mycobacteria remains controversial. More recently, Lin in situ within granulomas to understand the lack of sustained Tm-TNF mediated protection during M. tuberculosis reactivation. It is clear that Tm-TNF induced activation of macrophages in a focused manner where iNOS expression was localized to structured granulomas unlike in the complete absence of TNF. The sustainability of this initial focused macrophage response was however compromised during latter stages where iNOS synthesis was notably randomized. The failure to sustain focused bactericidal activity within granulomas was accompanied by a reduction in overall cytokine profile that included IFNγ, IL-12p70 and IL-10. Clinically, similar findings were noted in reactivating tuberculosis patients on anti-TNF therapy who presented with a decreased T cell activation profile and reduction in IFNγ and IL-10 synthesis M. tuberculosis specific phagosome maturation, thereby promoting bacterial survival and replication during long term reactivation. The cytokine profile in reactivating Tm-TNF mice reported here contrasts to that observed in de novo infected Tm-TNF mice de novo M. tuberculosis infection innate cellular responses drive initial protective immunity whereas under conditions of reactivation protective function may primarily be driven by adaptive memory immunity established during infection prior to pathogen resolution after chemotherapeutic treatment.We further assessed iNOS induction as a parameter of macrophage activation M. tuberculosis infection requires both Tm-TNF and solTNF during tuberculosis reactivation. Although Tm-TNF protects mice against acute M. tuberculosis infection, long term protection requires solTNF partly to down regulate inflammatory responses in chronic infection and to sustain immune pressure during recrudescence of M. tuberculosis infection.In conclusion data presented here, illustrates that TNF mediated immunity against


Influenza virus-like particle (VLP) vaccines are one of the most promising approaches to respond to the constant threat of the emergence of pandemic strains, as they possess the potential for higher production capabilities compared to traditional vaccines made in egg-based technology. VLPs are particles produced in cell culture utilizing recombinant protein technology composed of viral antigens that are able to elicit an immune response but lack viral genetic material. Thus far, influenza VLPs have been produced in mammalian, insect and plant based platforms , with prVLPs from both systems were characterized using currently available influenza quantification techniques such as Single Radial Immunodiffusion (SRID) assay, Hemagglutination (HA) assay, Negative Staining Electron Microscopy (NSEM) and western blot.It was found that VLPs from the HEK 293SF system were present in the culture supernatant in a heterogeneous mixture in terms of particle shape and size. Particles were spherical and also pleomorphic in shape and ranged from sizes of 100-400 nm. Sucrose cushion concentrated samples contained broken particles and a lot of debris. Additionally, it was found that VLPs were associated with the cell pellet after harvest in relatively the same amount as released into the supernatant in the form of unreleased VLPs from NSEM and HA assay analysis. This is possibly due to the sticky nature of the HA protein or from cell clumping during production that worked to trap the VLPs, preventing release into the supernatant. Sf9 cells produced more uniformly shaped VLPs that were spherical in shape, around 100 nm in size and were found to be mainly in the supernatant, not associated with the cell pellet. Sucrose cushion concentrated VLPs contained noticeably less debris than VLPs produced from HEK 293SF cells. It was found that VLP production in Sf9 cells produced 1.5 logs more VLPs than in HEK 293SF cells and had 30× higher HA activity. However, Sf9 VLP samples contained 20× more baculovirus than VLPs, which can contribute to HA activity in both the HA and SRID assays which has to be acknowledged during process development stages. This is the first time to our knowledge that specific production values for influenza VLPs in terms of total particles/ml have been reported.From this study, the insect-cell baculovirus system produced a more homogeneous population of VLPs compared to its counterpart in HEK 293SF cells. However, this study also highlights the major problem of baculovirus contamination in the Sf9 system, which requires removal for final vaccine formulations and to help ease the optimization of process production conditions.


The role of cytokines in the immunopathogenesis of oral lichen planus (OLP) has received much attention. The aim of this study was to evaluate the serum levels of TNF-α and TGF-β in patients with OLP in an Iranian population. Thirty-two patients with OLP and 32 age-matched healthy volunteers as a control group were included in this study. Serum tests including TNF-α and TGF-β was performed in both groups. Data analysis was performed using descriptive statistics and Mann–Whitney U test using SPSS software version 16.0. The mean of TNF-α in study and control groups were 157 ± 115 pg/ml and 14 ± 10 pg/ml, respectively. The difference between the two means was statistically significant (P < 0.001). Moreover, the mean of TGF-β in study and control groups were 155 ± 26 pg/ml and 175 ± 57 pg/ml, respectively. The difference between the two means was statistically significant (P = 0.03). According to the results of the present study, there was a significant decrease in the serum levels of TGF-β and a significant increase in the serum levels of TNF-α in patients with oral lichen planus. The increase in TNF-α serum levels in patients with OLP explains the inflammatory process in the course of the disease. In recent years, there has been increasing interest in the role of various cytokines in the immunopathogenesis of lichen planus, which include interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β).5 Another study showed that TGF-β suppresses the immune response to auto-antigens, and the severity of inflammation from lichen planus increases in areas with reduced TGF-β activity.6In a study on the salivary levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in a group of patients with oral lichen planus, the results indicated a significant increase in the levels of both cytokines.7 Another important cytokine is TNF-α, which has a role in the host immune response to infection, angiogenesis induction, tissue repair and regeneration, regulation of proliferation and tissue differentiation.8TGF-β has various functions, including growth inhibition, induction of apoptosis, effect on extracellular matrix structure and immunosuppression. It has also been demonstrated that TGF-β can increase the release of vascular endothelial growth factor (VEGF) in addition to having a significant anti-inflammatory effect.8 serum levels of TNF-α are significantly higher in patients with oral lichen planus compared to those in healthy controls. The serum levels of TGF-β were also significantly higher in patients with the erosive form compared to those with the reticular form. However, no statistically significant differences were found in TGF-β levels between patients with oral lichen planus and healthy controls. The results of another study indicated an increase in the salivary levels of TNF-α in patients with oral lichen planus; in addition, a significant relation was noted between TNF-α levels and different forms of oral lichen planus.3 TNF-α levels were higher in atrophic and erosive forms compared to the reticular form. Also, an immune response has been observed against TNF-α in 17 out of 22 patients with oral lichen planus.9 However, in the tissue samples of healthy controls a negative result was observed for TNF-α. Therefore, it was reported that TNF-α might have a role in the pathogenesis of oral lichen planus.9According to a previous study,Despite already published literature, there is still a limited number of studies that have evaluated the role of TNF-α and TNF-β in recent years and the experimental data are rather controversial with no general agreement. The aim of this study was to evaluate the serum levels of these two cytokines in patients with oral lichen planus and compare them with those in a group of healthy controls.The subjects consisted of patients, aged 18-60 years, with oral lichen planus, who were selected based on clinical diagnosis  or clinical-pathologic diagnosis from the patients referring to the Department of Oral Medicine, Faculty of Dentistry, Tabriz University of Medical Sciences.Thirty-two subjects were selected by simple sampling method during a nine-month period. The controls consisted of 32 healthy individual, without lichen planus, who lived in the same geographic location and had presented to the same center.After taking an accurate medical history and completing a preliminary checklist and taking biopsies (if necessary), TNF-α, and TGF-β serum level tests were administered. ELISA kit  was used to this end. Briefly, the specific human anti-TNF-α and anti-TGF-β antibodies were placed in the wells of each row of plates. The samples, including standard samples of the kit with known concentrations of TNF-α and TGF-β, the control samples and the sera of the subjects were introduced into these wells. During the initial incubation, TNF-α and TGF-β bind to the fixed antibody after irrigation, the specific biotin-bound antibodies for TNF-α and TGF-β are incorporated. During the second incubation phase, this antibody binds to TNF-α and TGF-β, which have been fixed during the initial incubation. After three irrigation procedures, streptovidin-bound peroxidase was added, which bound to the biotin-bound antigenic agent. After the third incubation procedure and irrigation to remove unbound enzymes, a chromogen substrate is incorporated, which reacts with the bound enzyme and produces color. The intensity of the light produced is directly proportional to TNF-α and TGF-β concentrations in the initial sample.In the control subjects, CBC, FBC, HDL, LDL, TG and cholesterol tests, which are all considered screening tests, were ordered along with TNF-α and TGF-β serum level tests to encourage cooperation and to contribute to the evaluation of the health status of the subjects.All subjects gave written informed consent to participate in the study. The study protocol and procedures were approved by the Research Ethics Committee of Tabriz University of Medical Sciences.Patients with the following conditions were excluded from the study:Presence of any stimulus leading to lichenoid reactions, including use of any medications such as anticholinerigic agents, saliva-reducing agents, antihistamines, antihypertensives and adrenergic Beta-blockers during the past 6 months.Presence of any congenital or acquired immune system disorders, such as AIDS, or a history of chemotherapy, injection addictives, and individuals undergoing dialysis.Presence of signs and symptoms of systemic infections, allergies, smoking and use of medications for the above-mentioned conditions.Data were analyzed with descriptive statistical methods , and Mann–Whitney U test using SPSS software version 16.0. Statistical significance was defined at P < 0.05.Fisher’s exact test did not reveal any significant differences between the study and control groups in relation to gender (P = 0.45).Means of TNF-α serum levels were 157.41 ± 115.30 pg and 14.44 ± 10.16 pg in the case and control groups, respectively. Mann–Whitney U test revealed statistically significant differences in TNF-α serum levels between the two groups . This finding emphasizes the role of this cytokine in the occurrence of autoimmune diseases including lichen planus. The results of a large number of studies have also confirmed the role of this key cytokine in inducing OLP.10 In addition, TNF-α induces apoptosis in a number of cells. Furthermore, some studies have shown that this cytokine induces the necrosis of epithelial cells, reducing the thickness of the epithelium.11 Therefore, TNF-α might be considered a factor involved in the incidence of ulcerations in and atrophy of the epithelium in OLP.TNF-α is involved in the secretion and initiation of the activity of matrix metalloproteinases of T-cells, which destroy the basal layer membrane proteins in conjunction with kimase and tripase secreted by mast cells.12 found significantly high levels of nuclear factor-kappa B dependent cytokines  in tissue transudate of OLP patients. Pezelj-Ribaric et al3 also showed increased salivary levels of TNF-α in OLP. The observation of significantly higher TNF-α serum levels in OLP patients assessed in the present study is in line with these findings.Rhodus et al13 TGF-β inhibits proliferation and differentiation of T-cells, activation of macrophages, and secretion of pro-inflammatory cytokines.10As mentioned earlier, TGF-β levels in the present study were significantly lower in OLP patients compared to healthy individuals (P < 0.037). T-cells secreting TGF-β have been identified and named Th-3 cells as a distinct population of antigen-specific CD4 T-cells. There is evidence that these cells inhibit the response of immune response to auto-antigens and prevent autoimmune reactions.15TGF-β induces the synthesis of extracellular matrix proteins such as collagen and cellular receptors for matrix proteins such as integrins. These functions result in tissue regeneration after controlling local inflammatory and immunologic reactions.16A decrease in TGF-β levels makes the individual susceptible to autoimmune lymphocytic inflammation while its administration in autoimmune disorders, in which T-cells have a prominent role, might have a therapeutic effect.8 reported significantly higher serum levels of TNF-α in patients with OLP compared to healthy controls and higher serum levels of TGF-β in the erosive form of the disease compared to the reticular form. However, the overall serum levels of TGF-β in OLP patients were not significantly different from those in the controls,8 which is not consistent with the results of the present study. Considering this discrepancy, it should be noted that a decrease in TGF-β levels is consistent with the pathogenesis of the disorder. The different results of the latter study which do not coincide with the course of the disorder might be attributed to the fact that the etiology and pathogenesis of the erosive form of lichen planus may be different from other forms of the disorder. It is also possible that the TGF-β secreted is non-functional or it does not have sufficient number of specific receptors; however, further studies are necessary in this regard.16The decrease in TGF-β levels in the present study might be attributed to depletion of TGF-β-secreting T-cells (Th-3) or regulatory T-cells, inhibition of TGF-β secretion, secretion of non-functional TGF-β, inadequate or incomplete expression of TGF-β receptors, and inadequate intracellular signals from TGF-β receptors. Zhou et alFinally, it should be pointed out that OLP is a multifactorial disorder with an unknown etiology and several risk factors have been suggested as its etiologic agents and TGF-β is only one of them. On the other hand, TGF-β has been known to have several roles in the etiology of OLP and it is possible that a change in its serum level cannot be considered as the sole etiologic factor. It is suggested that the counts and function of regulatory T-cells be evaluated in oral lichen planus patients.TNF-α and TGF-β serum levels be compared in patients with the atrophic, erosive and reticular forms of the disorder.The relationship between serum levels of TNF-α and TGF-β and the clinical severity of the disorder and the response to treatment be evaluated.Significant changes were evident in the serum levels of TNF-α and TGF-β in patients with oral lichen planus. The increase in TNF-α serum levels in patients with OLP may, in part, explain the inflammatory process in the course of the disease. The results of the present study indicate a decrease in the factors controlling the autoimmune reactions, i.e. the activity of regulatory T-cells, which are the principle cells to secrete TGF-β. Therefore, it can be concluded that a decrease in the activity of regulatory T-cells might have a role in the pathogenesis of OLP.


Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs).VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes  was greatly reduced.Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entireHPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres.D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics. Most vaal warts -7. HPV val warts . L1 alonal warts ,10 or real warts ,12. The The spontaneous organization inside yeast cells of pentameric L1 capsomeres into periodically packed quasi-symmetric VLPs is controlled by thermodynamic constraints via the combination of many intra- and intercapsomeric forces. However, heterogeneity due to assembly polymorphism is common for VLPs lacking genetic material -15, in a® (licensed for human use in 2006). A panel of non-overlapping mAbs  being just the purified VLPs (without D/R). High consistency was demonstrated for the VLP D/R process at production scale when multiple lots of post-D/R VLPs were analyzed. Figure Interestingly, the D/R process, similar to viral uncoating and virion assembly inside the cell, could be performed with recombinant L1 reliably with high yield and consistency. Neutralizing epitopes on L1 VLPs were improved as desired, whereas a decrease of binding was observed for mAbs targeting linear epitopes as a result of D/R. A set of epitope-specific antigenicity data from IC50 assays with four mAbs is presented in Table The impact on the antigenicity of VLPs probed by different mAbs is shown in Figure It was particularly striking that the epitope for H5 was no longer detectable upon D/R treatment Figure . In the To complement the IC50 ELISA analyses of antibody reactivity against HPV VLPs, we directly measured the antibody binding affinities for pre- and post-D/R VLPs SPR Figure  by analyThe structure and morphology of various types of HPV VLPs before and after D/R have been compared directly by negatively stained transmission EM and atomic force microscopy (AFM) in previous studies ,16,37. TA significant fraction of the particles in the cryoEM images have a diameter and capsomere arrangement that is consistent with the atomic force microscopy (AFM) data Figure . MoreoveTo understand why the epitopes of certain antibodies become more exposed upon D/R treatment of VLPs while other epitopes become less exposed, we generated an atomic model of the entire HPV16 (T = 7)VLP Figure , 4. ResiH16.J4 is a weakly neutralizing antibody originally identified by the immunoreactivity of the sera from HPV infected patients with cervical cancer ,30. LikeIn contrast to H16.H5, H16.O7 and H16.J4, antibodies H16.V5, H16.E70 and H263.A2 are neutralizing and their binding to VLPs was enhanced upon D/R treatment Figures , 8C. Of Our observations on H16.H5 and other linear targeting mAbs are consistent with the loss in binding when (pre-D/R) baculovirus derived HPV16 L1VLP-coated plates are compared to HPV16 L1 + L2 pseudovirion-coated plates . AnotherBiophysical data from AFM, cryoEM, dynamic light scattering and sedimentation experiments show a significant improvement in the morphology, homogeneity and thermal stability of the HPV16 VLPs upon D/R treatment ,37. AntiAmong an array of immunochemical assays for HPV VLP epitope characterizations, including competition ELISAs (IC50), sandwich ELISA, equilibrium dissociation constant determination, relative antigenicity and pair-wise epitope mapping, the epitope specific relative IC50, or rIC50, assay is the most sensitive assay with straightforward data interpretation due to the use of a single mAb in the assay. A ~3-fold enhancement in IC50 was observed for H16.V5 after D/R treatment of VLPs. In the relative antigenicity assay by SPR, the enhancement was ~2-fold. In addition, in a sandwich ELISA in which VLPs were captured with H16.J4 and detected with H16.V5, a moderate 30%-50% increase was observed ,20. ThisThe events that are likely to occur during D/R treatment on purified VLPs are: (1) breakdown of non-specific aggregates including disulfide-bonded aggregates ; (2) unmasking of some virion-like epitopes previously hidden due to non-covalent or covalent association with other VLPs or capsomeres through aggregation or non-native disulfide bond formation; (3) promotion of complete assembly of the closed icosahedral VLPs; (4) reduction of non-native disulfide bonds and formation of native and thermodynamically preferred intra- and intercapsomeric disulfide bonds; (5) formation of native electrostatic and hydrophobic interactions upon correct capsomere assembly by formation of intercapsomeric disulfides ,41. MakiMapping of the previously identified antibody epitopes onto the HPV16 VLP atomic model shows that intercapsomere contacts in the VLP can explain the observed changes in antibody reactivity upon D/R and allow more precise and more consistent mapping of the antibody epitopes. Within the H16.H5 epitope (residues 174-185), residues 175, 177 and 178 are occluded by the C-terminal arm of an adjacent capsomere in the HPV16 VLP but not in pentameric capsomeres, suggesting that the observed reduction of H16.H5 and H16.O7 antibody reactivity to VLPs upon D/R is due to the occlusion of one or more of these residues. Cys175 forms an intercapsomeric disulfide with Cys428 in the C-terminal arm of a neighboring capsomere ,43. WhenIn contrast to H16.H5, VLP binding to antibodies H16.V5, H16.E70 and H263.A2 is enhanced upon D/R treatment. The 3.6-Å resolution cryoEM structure of BPV1 and our atomic model of HPV16 do not, however, suggest that any epitopes become exposed upon virus assembly . We conc® have undergone D/R treatment to produce more homogeneous VLPs with improved neutralizing epitopes, structure and stability. The effects on VLPs due to D/R treatment are evident in improving particle uniformity and size distribution from AFM and cryoEM images. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. For VLP-based vaccine design and production, generating the virion-like epitopes consistently, keeping them stable and knowing how to analyze them accurately are of paramount importance. These mAbs targeting different epitopes function as the indicators for the consistency and correctness of VLP assembly. Significant binding to surface or buried linear epitopes  is an indication of poor folding and/or incomplete assembly, as explained by the atomic model in this report. A similar approach, using the immunoreactivity of a mAb recognizing a linear epitope, was employed to probe the "incorrectly" or poorly folded erythropoietin [HPV16 L1 VLPs in Gardasilopoietin ,46. MultFull length HPV16 L1 protein was over expressed in yeast. Purification of the VLPs, and the D/R process at lab scale was previously described ,40 and tMouse mAbs ,18,27 weIn order to prevent cross-linking of the VLPs during cryoEM experiments (with both IgG and Fab), Fab antibody fragments were prepared from the H16.V5 (IgG2b). Reduction of disulfide bonds and specific cleavage at the hinge region were achieved by adding a solution of H16.V5 (1.0 mg/ml) in 20 mM L-cysteine hydrochloride monohydrate to immobilized papain on agarose beads (Pierce), followed by gentle mixing for complete digestion over the course of two days at ambient temperature. Supernatant containing the Fab fragments was purified via gentle mixing with immobilized Protein A (Repligen) for one hour. Fab fragments in solution were concentrated using a 10 kDa filter . The completeness of the cleavage of IgG and purified Fab was assessed with a size-exclusion HPLC. Concentrations of the H16.V5 Fab fragments were determined before and after filtration by UV absorbance measurements at 280 nm using an Agilent 8453 UV-Visible spectrophotometer.50) of antigen in solution with half- maximal binding. Briefly, a fixed amount of HPV 16 VLPs on plate and varying amount of HPV L1 VLPs in solution were allowed to compete for the binding to a constant concentration of anti-HPV 16 mAbs at 10 or 20 ng/mL. This assay was used to quantitatively measure the solution antigenicity of HPV L1 VLPs against a given mAb. Reagent VLPs were passively adsorbed to a high-binding microtiter plate at a fixed concentration . 1.5-fold serial dilutions of a reference lot and the test samples were performed on the VLP coated assay plate using a Beckmann Biomek® 2000 or 3000 Laboratory Automation Workstation. A constant amount of the anti-HPV 16 antibody was then added to the solution HPV 16 VLP dilution series and the plate was incubated for 60 min. After washing away components in solution , the antibody bound to the surface immobilized VLPs was quantitated with an alkaline phosphatase-conjugated rabbit anti-mouse IgG antibody. For detection, 4-methylumbelliferyl phosphate (4-MUP) was used as a substrate. The substrate forms the fluorescent substance 4-methylumbelliferone (4-MU) in the presence of the immobilized alkaline phosphatases. This fluorescence intensity was measured with a fluorescence plate reader after 60-70 min fluorescence development. Percentage of inhibition was calculated based on the wells without any antigen present in solution as a control, obtained on the same plate. Curve-fitting and IC50 calculation were performed with GraFit [Competitive fluorescence ELISA (FL-ELISA) of HPV VLPs was employed to assess the median inhibitory concentrations (ICh GraFit .VLP) to a given amount of captured IgG (RUIgG). Results  from the samples are compared to results from a reference lot tested in adjacent cycles.Epitope specific antigenicity assessments using SPR with a panel of mAbs were carried out using similar procedures as described . BrieflyFor pair-wise epitope mapping, VLP was first captured by H16.J4  onto the sensor chip surface. Then the VLP surface was saturated with mAb1 by injecting mAb1 in high concentration (200 μg/mL), then the remaining VLP surface was probed by the binding of mAb2 to compare the degree of overlapping or the decrease in mAb2 binding to the mAb1-saturated VLP surface as compared to mAb2 binding to the free VLP. Then, in a separate assay cycle, the order of binding (mAb1:mAb2) was reversed, by saturating VLP with mAb2 first, followed by binding of mAb1.-/Å2, and a nominal defocus of ~3 μm. The images of the V5-Fab with VLP were obtained by mixing them at 1:1 ratio based on protein concentration, prior to applying to the grid.Samples were preserved in a thin layer of vitrified ice over C-Flat holey carbon films  supported on 400 mesh copper grids. Samples were not diluted prior to vitrification. Electron microscopy was performed using a Tecnai F20 electron microscope (FEI Co.) operating at 120 keV equipped with a Gatan 4 k × 4 k digital camera. Images were acquired, using the Leginon data collection software , at a no3OAE) [3IYJ) [The atomic homology model of the HPV16 VLP was constructed as follows. The crystal structure of the HPV16 L1 pentamer bound to heparin oligosaccharides (PDB code 3OAE)  was supe) [3IYJ) . A singl) [3IYJ) . The ene) [3IYJ) . The atoAFM: Atomic force microscopy; BPV: Bovine papillomavirus; cryoEM: Electron cryomicroscopy; D/R: Disassembly/reassembly; HPV: Human papillomavirus; IC50: Half-maximal inhibitory concentration; mAb: Monoclonal antibody; SPR: Surface plasmon resonance; VLP: Virus-like particle.The authors declare that they have no competing interests.QZ, KH, VT, Yuan Meng, YW, JA, MK, and MWW disassembled and reassembled the virus-like particles and carried out the ELISA and surface plasmon resonance experiments. DA, DW and MK generated the virus-like particles. Yorgo Modis carried out the molecular modeling studies, wrote the corresponding sections of the manuscript, edited the final manuscript and figures and handled the submission and peer review process. BC and CSP carried out the electron microscopy. QZ, MWW and RDS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Macrophages and their pro-inflammatory cytokines, including TNF, are pivotal mediators of chronic synovitis in Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA). Despite similar levels of synovial macrophage infiltration and clinical responses to TNF-blockade, SpA is characterized by more pronounced infiltration with alternatively activated macrophages and ongoing osteoproliferation. Here, we investigated whether these differences were related to differential expression and/or function of TNF between both diseases.Expression of membrane-bound TNF (mTNF) and soluble TNF (sTNF) was measured in IFN-γ, IL-4 or IL-10 polarized macrophages obtained from healthy donors. Expression of TNF and its receptors was also measured in synovial fluid (SF) and synovial tissue (ST) of actively inflamed knee joints. Mice transgenically overexpressing mTNF were evaluated for spondylitis and arthritis.The expression of mTNF was increased in IL-4 and IL-10 polarized macrophages compared to IFN-γ polarized macrophages. Moreover, secretion of sTNF was impaired in both alternatively polarized macrophages, indicating a shift from sTNF to mTNF. In line with these data, the sTNF SF levels were lower in SpA compared to RA (p=0.01) despite similar TNF mRNA levels in ST. This was not related to altered expression of TNF receptors as both TNF-R1 and TNF-R2 were similarly expressed in ST. mRNA levels of TACE, the enzyme responsible for the cleavage of TNF, were also similar between SpA and RA ST. To investigate the relevance of overexpressed mTNF in SpA pathophysiology, we characterized mTNF transgenic mice. As previously described, these mice develop a moderate arthritis, resulting in deformation of paws and loss of grip. Histological, the joints were characterized by synovitis, lymphoid aggregates and osteoproliferation. Besides arthritis, all mice spontaneously developed spondylitis as evidenced by a crinkled tail, a hunchback and histological pathophysiology.Membrane-bound TNF is relatively overexpressed by alternatively polarized macrophages in SpA synovitis and leads to an axial and peripheral SpA phenotype in transgenic mice.


P < 0.05).Glycemia in NPDR: 6.6 mmol/L (range: 5.8–6.3); in PDR: 6.7 mmol/L(range: 6.1–7.2); in control: 5.7 mmol/L (range: 4.9–6.1); microalbuminurea inNPDR: 10.6 mg/L (range: 5.6–20); in PDR: 25.2 mg/L(range: 17–40); in control: 5.3 mg/L (range: 2.6–10); Body mass index inNPDR: 26 Kg/m2 (range: 20.3–40);in PDR: 28 Kg/m2 (range 20.3–52);in control: 21 Kg/m2 (range 19–26).The TNF-alpha concentrations in tears increase with the severity of pathologyand were lower in control group than in diabetic subjects. In the end, the level of TNF-alphais highly correlated with severity of diabetic retinopathy and with nephropathy.Tear fluid collection may be a useful noninvasive method for the detection of proliferative diabetic retinopathy.We assess the level of tumour necrosis factor alpha  in tear fluids and other serumparameters associated with diabetes in different degrees of diabetic retinopathy. We have performed a prospective,nonrandomized, observational study. Study population consisted of 16 healthy subjects (controls) and 32 type 2diabetic patients: 16 affected by proliferative diabetic retinopathy (PDR) and 16 with nonproliferative retinopathy. Body mass index, urinary albumin, blood glucose, HbA1c, and tear levelsof TNF-alpha were measured in all subjects. The value of glycaemia, microalbuminurea, and Body mass indexin diabetic retinopathy groups were higher than those in control group ( Diabetes is a pathologic condition strongly associated with both microvascular  and macrovascular  complications. Microvascular complications include retinopathy, nephropathy, and neuropathy; whereas macrovascular complications include cardiovascular diseases, strokes, and insufficiency in blood flow to legs. These complications are triggered by the same culprit: elevated blood glucose levels. In fact, chronic hyperglycaemia plays a major role in the beginning of diabetic vascular complications through many metabolic and structural derangements, that is, the production of advanced glycation end products, abnormal activation of signaling cascades, elevated production of reactive oxygen species, and abnormal stimulation of hemodynamic regulation systems. Thus, metabolic control in diabetes can delay the onset and progression of these complications , 2.Diabetic retinopathy (DR) is a leading cause of blindness and visual disability. DR is a serious consequence of long-standing and poorly controlled diabetes and the most important cause of vision loss in the working-age population in developed countries . One hun TNF-alpha is an inflammatory cytokine, strongly correlated with insulin resistance and chronic inflammation . RecentlThe present study was designed to verify the feasibility of using the tear TNF-alpha assay as a novel biomarker to assess the degree of eye involvement in type 2 diabetic patients with and without proliferative diabetic retinopathy.The study was conducted in accordance with the tenets of the Declaration of Helsinki and the Institutional Review Board approved the study protocol. All participating subjects gave their informed consent after a detailed description of the procedure used and of the aim work. Thirty-two diabetic patients and 16 healthy subjects were recruited for this study. Diagnosis of type 2 diabetes was carried out according to the guidelines of American Diabetes Association . Diabeti Body height and weight and waist circumference were measured to calculate the Body Mass Index (BMI). The following formula was used: weight in kilograms/height in meters squared (kg/m²). The BMI results are classified as: (i) underweight (BMI < 18.5); (ii) healthy weight (BMI between 18.5 and 24.9); (iii) overweight (BMI between 25 and 30); (iv) obese (BMI > 30); (v) morbidly obese (BMI > 40). Blood glucose, HbA1c , blood urea nitrogen (BUN), and protein urine  were performed to all subjects. The occurrence of nephropathy was assessed measuring the glomerular filtration rate (GFR), which was calculated according to the Cockcroft formula . A valueμL/5 mm of phosphate-buffered saline (PBS) for 3 h to elute tear proteins. TNF-alpha was measured using a 96-well plate coated with antibody specific for human TNF-alpha, according to manufacturer's instructions . Nonstimulated tears were collected from each eye from the inferior tear meniscus between the 6 o'clock and lateral canthus positions using a standard clinical Schirmer's strip. Following collection, the strip was immediately transferred into a sterile labelled tube and frozen at −40°C until analysed. The wet portion of each Schirmer's strip was then cut into smaller pieces and soaked in 50 U-test was used to compare the control and DR groups. A P value of  <0.05 was considered statistically significant. Data were recorded in an Excel 2007 spreadsheet and analyzed by SPSS statistical software. For analysis of statistical differences, we used the Wilcoxon signed-rank test to evaluate the intragroup significance between variables. Conversely, the Mann-Whitney P = not significant. The blood glucose difference between diabetic patients with and without proliferative retinopathy was also not significant. The HbA1c percentage was significantly higher in the groups of diabetics than in controls: NPDR: 7.2% (range: 6.5–11.5); PDR: 7.8% (range: 7.2–12.5); controls: 5.6% (range: 5.3–6.1); P < 0.05. However, there were no significant intragroup differences . Diabetic subjects exhibited levels of protein urine significantly higher (P < 0.001) than controls: NPDR: 10.6 mg/L (range: 5.6–20); PDR: 25.2 mg/L (range: 17–40); controls: 5.3 mg/L (range: 2.6–10). The intra-group difference (PDR versus NDPR) was also highly significant (P < 0.001). The values of BMI between diabetic and nondiabetic subjects were significantly different: NPDR: 26 Kg/m2 (range: 20.3–40); PDR: 28 Kg/m2 (range 20.3–52); control: 21 Kg/m2 (range 19–26); P < 0.05. Contrarily, the difference between diabetic patients with and without proliferative retinopathy was not significant. Duration of diabetes was higher in patients with PDR (P < 0.05). Lastly, diabetic patients showed a significant reduction (P < 0.001) of GFR: from 95 mL/min/1.73 m2 (73–91) of controls to 82 mL/min/1.73 m2 (46–89) and 64 mL/min/1.73 m2 (56–78) of NPDR and PDR patients, respectively. The intra-group difference (PDR versus NDPR) was also highly significant (P < 0.001). Tears TNF-alpha levels were lower in control than those in PDR group (P < 0.05) and the TNF-alpha concentration significantly increased along with the severity of pathology: control: 1.9 Kg/mL (range 1.1–6.9); NPDR: 2.8 pg/mL (range: 1.2–5.5); PDR: 13.5 pg/mL (range 9.2–21.7). . . To the best of our knowledge, this is the first report in which TNF-alpha in tears from patients with type 2 diabetes has been measured. The levels of TNF-alpha were associated with the degree of DR, being lower in NPDR and higher in PDR. These findings of ours tally with those reported by previous investigators which showed that proangiogenic cytokines are more highly represented than antiangiogenic cytokines in the tears of type 2 diabetic patients with retinopathy , 36. 2), glomerular filtration rate (GFR), albuminuria, blood urea nitrogen, and levels of TNF-alpha in the tears has been found, further confirming that TNF-alpha tear levels may be considered a good predictor of microvascular complications. TNF-alpha is a central regulator of inflammation, and TNF-alpha antagonists may be effective in treating inflammatory disorders in which TNF-alpha plays an important pathogenetic role. Strong correlations between enhanced inflammatory biomarkers, including TNF-alpha, and the occurrence of diabetic retinopathy have been reported through extensive studies by many researchers [ Several studies have shown the association between TNF-alpha and diabetic vascular damage , 37. Kuoearchers , 40. In earchers . Serum cearchers . TNF-alpearchers . Altoget Early detection and treatment of diabetes can decrease the risk of developing its complications. Randomized trials have established the benefits of interventions to prevent or delay diabetes and reduce diabetes-related complications . Screeni The proposed method does not replace eye exams by an ophthalmologist, but it could be seen as a “filter” to exclude diabetic patients without diabetic retinopathy. Furthermore, increasing rate of patients with diabetes soon will outpace the supply of eye care providers, and then some communities have poor or even no access to ophthalmologic care. It was seen that the tear levels of TNF-alpha increases in various inflammatory pathologies of the ocular surface  –52. Once


Mycobacterium tuberculosis can give protection in animal models when administrated with specific adjuvants. In the present study, we explored the use HBcag-VLPs for delivery of tuberculosis antigens.Growing prevalence of TB and the emergence of XDR-TB have stimulated substantial efforts to develop better vaccines for TB. Recent researches have shown that some of the antigenic proteins and fusion of different proteins produced by E. coli and purified. VLPs formation was confirmed with TEM. BALB/c mice were immunized with VLPs and controls without any adjuvants. Sera were analysed for antibody responses (ELISA). Splenocytes were cultured and restimulated with purified antigens and CF (culture filtrate) of M.tb. The cell proliferation was measured using cell proliferation assay kit and the culture supernatants were analysed for IL-2, IFN-γ and TNF.HBcVLPs bearing ESAT-6 and CFP-10 were constructed using PCR and recombinant DNA methods. Proteins were expressed in M. tuberculosis antigen. Restimulation with mycobacterial CF also produced the same effect.The recombinant VLP induces preferentially a Th1-type immune response against mycobacterial antigen even though Th2 has been reported as the predominant response in BALB/c mice. IFN-γ, IL-2, TNF and proliferation were significantly higher in mice immunised with HBcVLPs-The humoral and cellular responses suggest that the VLP containing fusion constructs generated immune response in a Th1 dependent manner. By virtue of its self-adjuvant nature, HBc VLPs are a better vaccine delivery system for use with newer antigens identified in the course of recent developments in subunit protein vaccine research in tuberculosis.