In this report, we describe the immune-enhancing capability of the nanoparticles to act as a vaccine adjuvant. TNF/CHP nanoparticles showed excellent storage stability and enhanced host immune responses to external immunogens. The nanoparticles were effective via the nasal route of administration for inducing systemic IgG1 as well as mucosal IgA. We applied the nanoparticles in a model experimental influenza virus infection to investigate their adjuvant ability. TNF/CHP nanoparticles combined with a conventional split vaccine protected mice via nasal administration against a lethal challenge of A/PR/8/34 (H1N1) influenza virus. Mechanistic studies showed that the nanoparticles enhanced antigen uptake by dendritic cells (DCs) and moderately induced the expression of inflammation-related genes in nasopharynx lymphoid tissue (NALT), leading to the activation of both B and T cells. Preliminary safety study revealed no severe toxicity to TNF/CHP nanoparticles. Slight-to-moderate influences in nasal mucosa were observed only in the repeated administration and they seemed to be reversible. Our data show that TNF/CHP nanoparticles effectively enhance both humoral and cellular immunity and could be a potential adjuvant for vaccines against infectious diseases, especially in the mucosa.We encapsulated tumor necrosis factor- In the last few decades, outbreaks of emerging infectious diseases such as severe acute respiratory syndrome (SARS)  and avias.c.) and intramuscular (i.m.) one, only induce neutralizing IgG antibody in blood and not mucosal IgA antibody, which is effective in preventing infection. In addition, the efficacy of IgG antibody against mutated viruses is very limited because it has highly restricted cross-protective capabilities. Conversely, IgA antibody on mucosa shows wide cross-protection and can block infection [Vaccines are the most effective interventions against infectious diseases. Many vaccines, however, are effective only in preventing onset and aggravation of symptoms and less effective in preventing infection, in particular, respiratory infections such as influenza. Some reasons for this are that the major administration routes of conventional vaccines, including subcutaneous  or i.m. administration. Generally, children and the elderly tend to respond less to vaccinations, which may lower the preventive power of the population [One significant drawback of mucosal vaccines is that they generally do not induce strong enough immune responses. The recent component-split vaccines, while avoiding many negative patient reactions, tend to be less immunogenic by themselves, even in the case of intravenous  is a major proinflammatory cytokine primarily produced by T cells and macrophages [α exerted adjuvant activities against pathogenic infections [α as a vaccine adjuvant, successful results have not been reported, probably because TNF-α causes unfavorable biological reactions when administered systemically and is rapidly degraded at mucosal surfaces when delivered mucosally. To overcome this, some investigators have attempted to generate protease-resistant mutant TNF-α molecules [Tumor necrosis factor-rophages . Originarophages , 12. Intfections , 14. Altolecules , 16.α, drug-delivery systems (DDS), nanoparticles made of biocompatible polymers, have attracted growing interest recently. One such material, cholesteryl pullulan (CHP), is the polysaccharide pullulan chemically modified with cholesteryl groups [In order to establish safer and more effective ways to administer bioactive substances, such as TNF-l groups , 18. It l groups –21. It hl groups , 23. CHPl groups –26.α into CHP resulting in TNF/CHP nanoparticles. Then, we examined the potential of the nanoparticles as a nasal vaccine adjuvant and protection against lethal influenza infection in a mouse model and conducted mechanistic analyses.In this report, we encapsulated human TNF-α was prepared as previously described [α protein was produced by a human B cell lymphoblastoid cell line, BALL-1, stimulated with hemagglutinating virus of Japan (HVJ) and highly purified through a series of chromatography columns, including a specific monoclonal antibody column. The endotoxin level of the purified TNF-α was determined to be less than 300 pg/mg protein.Human TNF-escribed . BrieflyCHP is a partially (1–3% of glucose units) modified polysaccharide, pullulan, with cholesteryl residues (PUREBRIGHT CP-100T). It was purchased from NOF Co. .μg/mL each).Influenza virus A/Puerto Rico/8/34 (H1N1) strain was provided by Dr. Ayato Takada of the Research Center for Zoonosis Control, Hokkaido University, Japan. Influenza virus HA vaccine (IVV) “SEIKEN” was purchased from DENKA SEIKEN Co., Ltd. . It was a split and trivalent vaccine for seasonal influenza, consisting of the inactivated HA antigens A/Brisbane/59/2007 (H1N1), A/Uruguay/716/2007 (H3N2), and B/Brisbane/60/2008 . The size of the particles was determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS . To estimate the amount of TNF-α encapsulated, nanoparticles were treated with 100 mg/mL methyl-β-cyclodextrin  at 37°C for 2 h to disrupt the particle structure and release TNF-α, as described [Two hundred fifty escribed . The amoα was determined by an ELISA system established in-house in Hayashibara Co., Ltd. Briefly, an anti-human TNF-α-specific mouse antibody (MAb-TNF-α-5) was immobilized in 96-well plates . After incubating with the samples, horse radish peroxidase- (HRPO-) labeled anti-human TNF-α-specific mouse antibody (MAb-TNF-α-1-HRPO) was added to the wells. Then, o-phenylenediamine, a substrate for HRPO, was added to detect the immune complex and the absorbance at 490 nm was measured. Purified human TNF-α protein was used as the standard. This ELISA system recognized the active trimeric TNF-α molecule only and the detection limit was 0.2 ng/mL.The amount of human TNF-BALB/c mice  were obtained from Charles River Laboratories Japan Inc.  and used after a week of quarantine and acclimation. This study was approved by the Laboratory Animal Care Committee of Hayashibara Co., Ltd., and all animal experiments and procedures were in accordance with the Guidelines for the Care and Use of Laboratory Animals at Hayashibara Co., Ltd.μL a nasal cavity, 30 μL/mouse) were administered nasally at the indicated times.Mice were anesthetized with sevoflurane and the antigen and/or adjuvant preparations  and recovered by centrifugation. The nasal passage cells were prepared as reported [Blood and nasal wash were obtained from mice. Briefly, the animals were anesthetized with ether and blood was taken from the abdominal aorta with heparinized syringes. The jaw was incised bilaterally and the nasal cavity, in vicinity to root of the tongue, was exposed. The nasal cavity was washed with saline and the wash was recovered. The blood and the nasal wash were centrifuged at 5,000 rpm for 8 min and stored at −80°C until examination. Then, after thorough washing of the nasal cavity with saline, the upper palates were removed and the NALTs were isolated under the microscope. NALT cells were suspended with 100 reported . Briefly1  for IgG1 and HRPO-labeled goat anti-mouse IgA  for IgA were used with o-phenylenediamine. The absorbance was measured at 490 nm.Antibody responses in the plasma and the nasal wash of the immunized animals were determined using ELISA. IVV was immobilized in microplates and the respective antibody in the plasma or the nasal wash was examined. As the indicators, HRPO-labeled rat anti-mouse IgGSpecificity of antibody response was examined by detection of neutralizing activity in plasma against HAs of influenza type A H1N1, type A H3N2, and type B. Plasma from mice immunized with IVV and TNF/CHP nanoparticles were subjected to hemagglutination inhibition (HI) assay performed by FALCO biosystems Ltd. . The results were expressed as geometric mean titer (GMT).Sixty-eight hours after the 3rd nasal immunization, NALT cells were prepared from mice as described. After being blocked with FcR blocker , the cells were stained with fluorescein isothiocyanate- (FITC-) labeled anti-mouse CD11c antibody and FITC-labeled anti-mouse CD80 antibody, or FITC-labeled anti-mouse CD86 antibody (Milteny Biotec), and subjected to flow cytometry (FCM) analysis using a flow cytometer .μg labeled OVA, the NALT cells and the nasal passage cells were prepared from the mice as described above, blocked with FcR blocker , stained with phycoerythrin- (PE-) labeled anti-mouse CD11c antibody , and subjected to FCM analysis. The results were examined with a flow cytometry analysis software, Gatelogic . The antigen uptake was estimated by the florescence intensity of Alexa 647 against intensity of CD11c+ cells, activated dendritic cells (DCs).Ovalbumin  was labeled with Alexa 647 using the Alexa Fluor 647 labeling kit  according to the manufacturer's instructions. The resulting Alexa-labeled OVA was used as an indicator antigen for nasal administration to mice with or without the TNF/CHP nanoparticles as an adjuvant. Six h after administration of 10 6 cells, using the RNeasy Mini Kit  according to the manufacturer's instructions. After confirming the purity and quality of the RNA, cDNA was synthesized using the RT2 First Strand Kit . The expression of various cytokines and factors related to immune responses was examined with the mouse innate and adaptive immune responses PCR array (SA Biosciences Co.) with a Roche LyteCycler 480 (Roche) according to the instructions.Total RNA was prepared from NALT cells pooled from 8 mice, approximately 4 × 10α (IL-1α), IL-1β, IL-2, IL-4, IL-5, IL-6, CXC ligand 2 (CXCL2), IL-10, IL-12β, IL-17α, IL-18, interferon-β (IFN-β), IFN-γ, TNF-α, fibronectin-1, CD14, toll-like receptor 3 (TLR3), proteoglycan-2, lipopolysaccharide (LPS) binding protein, and lactotransferrin  and represented the difference (Δ) of fluorescence intensity between cases with and without IVV. The culture supernatant was recovered at 16 h and the cytokine-producing cells were examined using the ELISpot PLUS mouse IL-4 and the ELISpot Plus mouse IFN-γ  kits. The results represented the difference of the number (Δ) of cytokine-positive cells/106 cells between cases with and without IVV stimulation.The NALT cells pooled from 8 mice were cultured for 3 d in microplates with feeder splenocytes treated with 50 μL per nasal cavity, 30 μL/mouse) once a week for 3 weeks (n = 10). Samples were prepared by mixing 1 : 1 IVV and the nanoparticle preparation (5 μg/mouse) or Cholera Toxin B-subunit (CTB)  before use. Seven d after the final immunization, the mice were challenged with influenza virus  nasally. The dosage of virus corresponded to 10 LD50 for the mice. The virus-challenged mice were monitored daily for body weight and signs of morbidity. Mice that were moribund or that had lost more than 20% of their body weight were considered to have reached an experimental endpoint and were humanely euthanized by anesthetization with pentobarbital.Mice were anesthetized with sevoflurane and were administered nasally the preparation  in a CHP tetrameric complex (ca. 400 kDa).CHP encapsulated active trimer TNF-ectively . Stoichiα, Me-β-CD was used to disrupt the CHP complex to release the TNF-α inside the particles. The results showed that the nanoparticle retained its integrity and kept TNF-α molecules active inside the complex in aqueous solution at room temperature for at least 21 d  was evaluated at 25°C. To estimate the amount of encapsulated TNF-ast 21 d . The intreported   and with free TNF-α failed to induce significant levels of antibodies compared with IVV alone. The TNF/CHP nanoparticles alone did not induce any measurable antibody response against IVV. Also, we examined antigen specificity further by hemagglutinin- (HA-) specific hemagglutination inhibition (HI) assay because the IVV we used was consisting of different types of influenza virus. The TNF/CHP nanoparticles with IVV induced significant HI activity against all types of HA used   . The effγ-producing cells and either adjuvant alone (TNF/CHP nanoparticle or CTB) suppressed the IFN-γ production. Instead, IL-4-producing cells were increased by nasal administration of the vaccine and adjuvant combination  combined with or without TNF/CHP nanoparticles (corresponding to 5 μg TNF-α/mouse) once a week for 3 times. Mice were challenged with influenza virus A/PR8 strain  7 d after the final immunization. The mice that received the IVV only all died by 8 d after the challenge, almost the same as without the IVV immunization. The TNF/CHP nanoparticles without the IVV delayed the onset of death a little, but eventually all the animals died. On the contrary, combined administration of the IVV and the TNF/CHP nanoparticles showed a highly protective effect on the mice and 90% of the animals survived. The effect was comparable to that of CTB as an adjuvant. Free TNF also showed a somewhat protective effect. Interestingly, CHP only (without TNF-α) provided a certain level of protection, as we observed up to 50% survival . These data indicate that the nanoparticles induced systemic immunity and long-lived memory. Overall, our data demonstrate that TNF/CHP nanoparticles are effective as a vaccine adjuvant for nasally delivered IVV.To directly address the stimulatory effect of the TNF/CHP nanoparticles on protective immunity as a vaccine adjuvant, we carried out an experimental lethal influenza virus challenge of immunized mice. The mice were nasally immunized with the IVV  and activation markers for B cells (CD80 and CD86), was examined by flow cytometry. The ratio of CD11cectively . Even th+/CD11c+ cells. Immunization of mice with OVA combined with TNF/CHP nanoparticles activated antigen uptake by both NALT and nasal passage DCs, and the nasal passage DCs responded particularly well. TNF/CHP nanoparticles seemed to stimulate DCs most in the nasal mucosal immune tissue  as a model antigen and assessed antigen uptake by DCs in the NALT and nasal passage cells after 6 h of immunization by flow cytometry. The results were analyzed with Gatelogic software and expressed as fluorescence values of Alexa 647e tissue .β, IL-1 family 9, and IL-6, was found to be significantly upregulated (data not shown). For further analyses of the inflammatory signaling molecules induced by TNF/CHP nanoparticles, we focused on the molecules listed in γ, IL-1α, IL-1β, IL-6, CXCL2, IL-12β, CD14, and LPS binding protein were found. The degree of enhancement varied from gene to gene. In particular, the expression of IL-6 and IL-12β was intensely enhanced. For these genes, free TNF resulted in strong and early increase in expression. The enhancement tended to be greater at 2 h than 6 or 26 h  nasally administered the TNF/CHP nanoparticles combined with the IVV either once or four times were subjected to an acute and a repeated toxicity study, respectively. The general symptoms, ophthalmic examinations, body weight, body temperature, hematology, blood biochemistry, urinalysis, autopsy findings, and histochemical analyses with hematoxylin-eosin staining were inspected.No general symptoms and behavioral anomaly in either male or female animals were correlated with the TNF/CHP nanoparticles and the IVV treatment during the study period. In ophthalmic examinations, there were no test material-related ocular findings observed. Body weights and body temperatures in the both sexes during the treatment were not statistically different among the treatment groups (data not shown).+, K+, Cl−, Ca2+, inorganic phosphate, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), amylase (AMY), γ-glutamyl transpeptidase (γ-GT), total cholesterol, triglyceride, HDL-cholesterol, total bilirubin, and glucose were examined. All differences found during the study fell within historical control value ranges and were not considered test material-related (data not shown).Hematology evaluations were also performed during and at the end of study. There were no differences in any of the parameters  that were considered to be due to the administration of the TNF/CHP nanoparticles and the IVV. For blood biochemistry, total protein, albumin, urea nitrogen, creatinine, NaUrobilinogen, bilirubin, ketone body, glucose, protein, pH, specific gravity, nitrite salt, and leucocyte were examined during the study period. There were no TNF/CHP nanoparticles and IVV-related changes observed (data not shown).1 in blood was examined after the administration of TNF/CHP nanoparticles and IVV. No detectable antibody was induced by the single administration. After the second administration, however, the antibody was induced by only the TNF/CHP nanoparticles combined with the IVV and the titer was maintained even after 2 weeks of cessation period. The effectiveness of the TNF/CHP nanoparticles as vaccine adjuvant was confirmed. In this experiment, females tended to respond to the immunization higher  were measured at the end of the study in all of the animals. No organ weight change was noted as test material-related (data not shown).Histopathology of tissues from animals in each group was examined and there were few histopathological findings, and none of them were considered to be TNF/CHP nanoparticles and IVV-related. As the nasal administration, possible harmful effects on central nervous system were concerned. However, no abnormalities in the brain, especially olfactory bulb, were noted in any animals after histological examination.On the other hand, there were some influences observed in nasal mucosal tissue. First, single administration of any specimen showed no effects. In the case of repeated administration, IVV did not affect the mucosal tissues greatly except slight infusion probably caused by repeated stimulation. On the other hand, TNF/CHP nanoparticles combined with IVV induced slight-to-moderate infusion and infiltration of inflammatory cells . The findings, however, diminished in trace proportions after 2 weeks of cessation period. No excessive inflammatory symptoms, such as formation of edema or fibrosis, were noted .In summary, no significant change due to the administration of the TNF/CHP nanoparticles and IVV was observed in the acute toxicity study. In the repeated toxicity study, slight-to-moderate infusion and inflammatory cell infiltration were observed at the nasal mucosa, the administration site. This response, however, seemed reversible. Overall, no immunotoxicity was detected. Although further evaluation is required, our results demonstrated that the toxicity of TNF/CHP nanoparticles is relatively low.α was successfully incorporated into CHP complexes to form stable nanoparticles. The preparation process was efficient and the resulting nanoparticles were relatively uniform  showed a certain level of efficacy in our study. The reason for the discrepancy between their results and ours is not clear. It is unlikely that TNF-α was replaced in vivo by IVV antigens to form IVV/CHP nanoparticles, which would then preferentially deliver vaccine antigens to the lymph node given the short time frame. Further, Oyewumi et al. reported that particle size was critical for adjuvant activity [To directly address the use of TNF/CHP nanoparticles as a vaccine adjuvant on protective immunity, we employed a lethal influenza challenge mouse model. The mice were immunized with influenza virus A/Brisbane/59/2007 (H1N1), A/Uruguay/716/2007 (H3N2), and B/Brisbane/60/2008, followed by challenge with an antigenically distinct influenza virus A/Puerto Rico/8/34 (H1N1). The nasally administered TNF/CHP nanoparticles induced protective immunity in spite of the distinct antigenicities , suggestTNF/CHP nanoparticles enhanced an IgA response not only at the site of application  but also at distant mucosal sites, including vaginal and salivary glands (data not shown). IgA antibody elicited at the mucosa is of vital importance as the natural route of infection for influenza is via the respiratory mucosa. Hence, local mucosal protection against pharyngeal carriage is likely to be decisive for preventing disease . ConventμL/mouse) in this study, a possibility that TNF/CHP nanoparticles affect immune cells in the lower respiratory tract as well as nasal mucosa should be considered. We plan to explore the possibility in future studies.The mucosal surfaces are known to have abundant B cells, T cells, and plasma (or DC) cells. After the repeated immunization of animals, activation of DC and B cells seemed enhanced in NALT tissue by TNF/CHP nanoparticles, despite being in small extents . Uptake α, IL-1β, IL-6, CXCL2, IL-12β, CD14, and LPS binding protein was found in TNF/CHP nanoparticle-administered mice. Free, exogenous TNF elicited strong and early increases in the expression of inflammatory signaling molecules. In comparison, the pattern was similar but the extent was less with TNF/CHP nanoparticles . Past reports suggested that nasal administration of CTB reached the CNS and accumulated in olfactory tissues. It caused Bell's Palsy in clinical studies, probably due to IL-12 production, and the use of CTB in humans was prohibited [β might be a beneficial safety feature of TNF/CHP nanoparticles.Vaccine adjuvants trigger the innate immune system to enhance humoral and cellular responses to the coadministered vaccine antigens. To understand the mechanisms by which TNF/CHP nanoparticles activate innate immunity, we conducted gene expression profiling in NALT cells. The expression of genes related to inflammation and immunity was found to be upregulated (data not shown). Based on these results, we focused on the inflammatory signaling molecules and performed quantitative PCR analyses. Enhanced expression of IL-1articles . The nanohibited , 39. In 1 and IgA or the expression of inflammation-related genes, it showed a certain level of protection in a lethal influenza virus challenge , another type of saccharide-based material that can form nanoparticles, exhibited adjuvant activity and elicited a strong protective effect against influenza virus in mice and cynomolgus macaques [β-CD at more than 0.5% in vitro, probably because of β-CD's ability to extract cholesterol out of cell membranes [Very recently, Onishi et al. reported that hydroxypropyl-macaques . They suembranes . TNF/CHPPreliminary safety studies revealed no severe toxic findings resulting from the exposure to TNF/CHP nanoparticles. In the repeated toxicity study, slight-to-moderate infusion and infiltration of inflammatory cells were observed at nasal mucosal tissue, the administration site. This response would likely be reversible over time. And enhancing effect of TNF/CHP nanoparticles in the case of repeated administration on induction of antigen-specific antibody in blood was confirmed again . Even thThe results of this study demonstrate that TNF/CHP nanoparticles are effective as a vaccine adjuvant when administered via the nasal mucosal route. Moreover, the ability of TNF/CHP nanoparticles to stimulate comparatively balanced systemic as well as mucosal immune responses makes them a potentially promising vaccine adjuvant for inducing immunity against infectious pathogens. In the short term, TNF/CHP nanoparticles may provide a useful way for developing new nasal influenza vaccines. Further, we propose that combining TNF/CHP nanoparticles with next-generation vaccine platforms that do not rely on the cold chain will offer valuable alternatives for vaccination in a variety of settings.


Multivariate linear regression corrected for possible confounders confirmed a significant role of perceived social support in predicting the number of CD8+CD57+ lymphocyte and TNF-α levels. This study supports the association between perceived social support and immune function. In particular, poor social support may be related to a state of chronic inflammation sustained by CD8+CD57+ lymphocyte expansion and increased TNF-α levels.Social support has been supposed to have a positive impact on the function of the immune system. However, the relationship between perceived social support and immune function has not yet been fully investigated. In this cross-sectional study, we investigated the link between perceived social support and lymphocyte subpopulations and cytokines. 232 healthy subjects provided a blood sample and completed the Multidimensional Scale of Perceived Social Support (MSPSS) questionnaire. Lymphocyte immunophenotypes and cytokines were determined. Significantly increased CD8+CD57+ lymphocytes and TNF- Social support from various sources, such as family, friends, organizations, and coworkers, has been linked to many benefits for both physical and mental health –3. PercePsychological stress and negative emotions that could be a consequence of low social support can also have an impact on the immune system –14.Furthermore, alterations in immune activity may be related to increased morbidity and mortality in populations exposed to adverse psychosocial factors such as poor social networks, low socioeconomic status, and portrayed psychological distress –17.Little is currently known about the direct effects of perceived social support on lymphocyte subpopulations and cytokines.In this cross-sectional study, we investigated the link between perceived social support and immune parameters, including lymphocyte immunophenotypes and cytokines, in a group of healthy subjects.Participants in the study were nurses from the local National Health Service Hospital Unit, Ancona, Italy. The study was carried out in accordance with the Declaration of Helsinki's ethical standards. Being part of standard occupational health surveillance it needed no formal approval by the local ethics committee, which was nevertheless consulted and which granted an informal authorization. Inclusion criteria were age <60 years, no infectious disorders or chronic medical diseases, no history of major psychiatric disorders, and no current or recent treatment with drugs affecting the immune system . 232 nurses, meeting the selection criteria, agreed to participate in the study and gave their written informed consent. They were asked to refrain from exercising, smoking, eating, drinking alcohol, or taking medications for at least 12 h before sampling.A blood sample was collected between 08:30 and 09:30 am. Blood was analyzed for leukocyte count, immunophenotype, and cytokines. After blood collection, the nurses completed a self-administered standardized questionnaire on lifestyle and habits. Perceived social support was evaluated by the validated 12-item Multidimensional Scale of Perceived Social Support questionnaire (MSPSS). It is scored on a 7-point scale, where higher scores indicate greater perceived availability of support from family, friends, and other sources .Immunophenotype analysis was performed on fresh whole blood within 2 h of collection using a direct immunofluorescence cytofluorimetric assay. Flow cytometric acquisition was performed on FACScalibur using MultiSet software . At least 10,000 events for each sample were acquired. The proportions of cells expressing CD19+ (B cells), CD3+CD4+ (T helper lymphocytes), CD8+ (cytotoxic/suppressor T lymphocytes), CD8+CD57+ (T lymphocytes with cytotoxic activity), and CD3−CD16+CD56+ (NK cells) were calculated. Absolute values were obtained based on lymphocyte counts provided by an automated Haematology Analyzer .β, IL-6, INF-γ, and TNF-α) were analyzed using a multiplex sandwich ELISA  according to the manufacturer's instructions. Each sample was tested in duplicate. Results are expressed as pg/mL.Proinflammatory cytokines .The values of continuous variables were expressed as median and 25th–75th percentile while ordinal variables were expressed as percentages. The participants' MSPSS score was classified into two categories using the median value as the cutoff. The Mann-Whitney2) and a higher prevalence of alcohol drinkers (40.4% versus 25.0%) compared to the high MSPSS score group. The lymphocyte subpopulation and cytokine values are reported in 3) and TNF-α levels (median 22.6 versus 12.8 pg/mL) were found in the low MSPSS score group compared to the high MSPSS score group.The sociodemographic characteristics of the 232 participants stratified into 2 groups by MSPSS score are reported in P < 0.05) and positively correlated with CD8+ lymphocytes, CD3−CD56+CD16+ lymphocytes, and TNF-α (P < 0.05)  . TNF-α wβ = − 0.17, P < 0.05) and TNF-α  (Multivariate linear regression showed a significant role of MSPSS score in predicting the number of CD8+CD57+ lymphocytes ( < 0.05) .Several studies have pointed out that a poor perceived social support may not only affect emotional well-being but also result in disease development , 20. Chrα levels was found in the low MSPSS score group compared to the high MSPSS score group. TNF-α is a key cytokine, which plays a critical role in tumor immunity as well as in immunity against infection. It is produced mainly by activated macrophages, although it can be produced by many other cell types, such as lymphocytes, NK cells, and neurons [α is in the regulation of immune cells. The deregulation of TNF-α production has been implicated in infectious disease [α is deleterious to the host with regard to coping with autoimmune or chronic inflammatory diseases [An increase in TNF- neurons . The pri disease –29 and i disease –32, Alzh disease , major d disease , and inf disease . TNF-α idiseases –38.α [α levels without providing evidence of a direct relationship between these two immune parameters. Since CD8+CD57+ lymphocytes and/or TNF-α may sustain a state of chronic inflammation [Although it has been reported that CD8+CD57+ lymphocytes may produce proinflammatory cytokines, such as TNF-α –42, our ammation , 37, poo


Heart failure with preserved left ventricular ejection fraction (HFPEF) affects about half of all patients diagnosed with heart failure. The pathophysiological aspect of this complex disease state has been extensively explored, yet it is still not fully understood. Since the sympathetic nervous system is related to the development of systolic HF, we hypothesized that an increased sympathetic nerve activation (SNA) is also related to the development of HFPEF. This review summarizes the available literature regarding the relation between HFPEF and SNA.Electronic databases and reference lists through April 2014 were searched resulting in 7722 unique articles. Three authors independently evaluated citation titles and abstracts, resulting in 77 articles reporting about the role of the sympathetic nervous system and HFPEF. Of these 77 articles, 15 were included for critical appraisal: 6 animal and 9 human studies. Based on the critical appraisal, we selected 9 articles  for further analysis. In all the animal studies, isoproterenol was administered to mimic an increased sympathetic activity. In human studies, different modalities for assessment of sympathetic activity were used. The studies selected for further evaluation reported a clear relation between HFPEF and SNA.Current literature confirms a relation between increased SNA and HFPEF. However, current literature is not able to distinguish whether enhanced SNA results in HFPEF, or HFPEF results in enhanced SNA. The most likely setting is a vicious circle in which HFPEF and SNA sustain each other. Heart failure with preserved left ventricular ejection fraction (HFPEF) affects about half of all patients with a clinical presentation of heart failure (HF).,2 There Irrespective of the definition, we still have much to learn about HFPEF. This is all the more important since no successful treatment is available yet.,4 A numbEven though HFPEF has been the focus of various mechanistic studies, the exact pathophysiology is still unknown. It is geHowever, while the role of the increased sympathetic nerve activity (SNA) in the development and progression of HF with reduced ejection fraction (HFREF) is well established,20, to oThe activity of the SNS can be measured in different ways. Examples are measurement of plasma or urinary norepinephrine (NE) level, assessment of local NE spillover, muscle sympathetic nerve activity (MSNA), iodine 123-metaiodobenzylguanidine (MIBG), or heart rate variability (HRV).This systematic review was conducted and reported in accordance with the “preferred reporting items for systematic reviews and meta-analyses” (PRISMA) statement. We conduAll full-text articles were reviewed by 3 authors  using pre-defined inclusion/exclusion criteria . ArticleCritical appraisal was independently performed using pre-set criteria. These criteria are outlined in th, 2014. A flowchart of the search is depicted in The search was conducted on October 31st, 2013 and identified 7722 unique articles. The search was updated on April 10The animal studies that met the criteria for critical appraisal investigated whether a change in activity of the sympathetic nervous system might induce diastolic dysfunction. All animal studies used a model of isoproterenol (ISO) to mimic an increased SNA. Isoproterenol is a non-selective β-adrenergic agonist structurally similar to epinephrine. The assumed increase in SNA was not measured in any of the animal studies.Three animal studies scored high (4 points or more) in the critical appraisal and were therefore further evaluated.,27,31 ReIn brief, the three selected animal studies demonstrated that administration of a β-adrenergic agonist established diastolic dysfunction in an experimental setting: the first study is the study from Grimm et al. Authors observed that dosages up to 150 mg/kg ISO led to diastolic dysfunction in mice as evaluated by echocardiography. Dosages A major drawback of these three studies is that the percentage of animals in which ISO administration did or did not lead to DD or HFPEF (‘responder rate’) was not clearly reported. Impairment of DD was already observed after a dosage of 2.4 mg/kg/day ISO for 7 days. A single dosage of 150 mg/kg ISO also induced DD. Higher dosages led to systolic heart failure.No studies could be found that prospectively studied a human cohort whether an increased SNA leads to HFPEF during follow-up. In most human studies evaluated by the critical appraisal, patients were included that already had DD based on echocardiographic data. Therefore, the natural progression to HFPEF could not be studied.All studies were hindered by a small sample size of patients (max. 34 patients). Another limitation is that not all studies reported the diastolic parameters: Sugiura and Arora et al. included HF-patients with a good ventricular function LVEF>45%) irrespective of diastolic parameters.,38 Echoc% irrespeIn brief; the 6 human studies selected for further evaluation confirmed a relationship between SNA and HFPEF. Arora et al. observed that patients with HFPEF exhibit a reduction in HRV compared to control subjects. PatientsTo our knowledge, this is the first systematic review investigating the relationship between sympathetic nerve activity and HFPEF in combination with DD. Based on the animal studies we concluded that administration of ISO and therefore increased SNA is related to HFPEF in an experimental setting. Based on the human studies, we concluded that an increased SNA—irrespective of the method of assessment—is indeed related to diastolic dysfunction and/or true HFPEF. However, the available literature about this topic is very scarce, let alone that results could be pooled. Moreover, administration of ISO in animals is not exactly the same as the increase in SNA in humans. However, no animal model to induce HFPEF has been accepted so far and we consider ISO administration the best available at present.As mentioned above, different definitions are used for HFPEF.,5 In theIn the human studies, different ways of assessing sympathetic activity were used. The most straightforward method to measure SNS is measurement of plasma or urinary (NE) level. Plasma NE concentrations however are a resultant of removal rates and not selectively release rates. Also, thThe study of Grassi et al. showed conflicting results; no difference in plasma NE-concentration was observed whereas MSNA values were altered in the patients with DD. In our aFrom a critical point of view, some experts argued that the increased SNA in patients with DD can be attributed to the higher BP since that is often present in patients with DD. To respoSince β-adrenergic stimulation often causes both HFPEF and HFREF, SNS-induced HFPEF could be a precursor state of HFREF or both diseases could be entities resulting from increased SNA. The studREF an ischemic model can be the precursor of increased SNA.[As Rosendorff previously discussed, it is not yet clear whether DD potentiates the sympathetic activation or whether the increased SNA causes DD. Based onased SNA. However,ased SNA.With the current available evidence, we cannot simply state that one causes the other. Paulus et al. has clearly set out that different comorbidities contribute to a systemic inflammatory state, which induces oxidative stress in the coronary microvascular endothelium.  The presUntil now, no treatment has yet convincingly shown to improve clinical status, morbidity and mortality in HFPEF.,64 It isEchocardiography is a reliable tool to objectively assess diastolic function. The effects on echocardiography should be taken into account when conducting a therapeutic study in a HFPEF population. Echocardiographic parameters have been used in some studies investigating aldosterone antagonists, beta-blockade, exercise training, and RAS-inhibition. Although some studies reported an improvement in clinical state, no clear effects on echocardiographic parameters were observed.,69,70 BaFirst of all, the available evidence is limited and heterogeneous in design. This heterogeneity may have influenced our results. We tried to uniform the different studies by using the critical appraisal.Although our search strategy was extensive and also focused on studies that showed HFPEF and SNA to be unrelated, it is possible that our search resulted in a relative over-representation of positive studies. By checking references of selected articles we tried to obtain studies that reported negative findings about the suggested relation. This search did not result in any relevant full text articles.In the human studies, we could not report about medical history, medication use and other determinants for heart failure because these determinants were not clearly reported. It should, however, be taken into account that most patients studied were treated with drugs that affect sympathetic activity. Since most antihypertensive drugs (indirectly) lower SNA, the curNot all human studies included patients with HFPEF; half of the human papers studied patients with DD instead of HFPEF. However, DD is an important, if not the main, precursor of HFPEF. Moreover, Sugiura et al. showed a higher cardiac sympathetic activity in HFPEF patients with a higher NYHA class. Though, A possible confounding factor in this review is that HFPEF-patients are more often older. Age itself is related to sympathetic activity and may be a confounder in the available literature. SelectedBased on our results, we conclude that current literature confirms a relation between increased SNA and HFPEF. However, current literature is not able to distinguish whether enhanced SNA results in HFPEF, or HFPEF results in enhanced SNA. The most likely setting is a vicious circle in which HFPEF and SNA sustain each other. Disruption of this vicious circle may be an attractive treatment option.S1 Checklist(DOC)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Immunization information systems (IIS) are confidential, computerized, population-based systems that collect and consolidate vaccination data from vaccination providers that can be used in designing and sustaining effective immunization strategies ,2. To moOf the 56 immunization program grantees  to increase to 95% the proportion of children aged <6 years whose immunization records are in fully operational, population-based IIS  participated in an IIS in 2012. This child participation measure is used to track a ased IIS . Child pased IIS . Nationaased IIS . Two IISFunctional standards for IIS were developed in 2001 and revised in 2007. The standards have been approved by NVAC ,4 for asChild participation in IIS increased steadily from 2006 to 2012, reaching 86%; adult participation, however, only reached 25% in 2012. Eight of 12 IIS functional standards were met by ≥90% of grantees in 2012. Increases in grantees meeting minimum functional standards for IIS data quality and interoperability, including the presence of core data element fields, timeliness for vaccination records, and HL7 messaging functionality, also have been demonstrated from 2011 to 2012, although challenges remain for IIS to reach their full potential in these areas, and for improving the timeliness of birth records in IIS.Historically, the primary focus of IIS and immunization programs has been pediatric populations. This focus was warranted because of the increasing complexity of the routine pediatric immunization schedule, mobility of children among different providers resulting in vaccination record scattering  that makCurrently, 53 of 56 immunization program grantees have IIS with lifespan systems, yet adult participation in IIS remains low. Challenges to increase adult participation in IIS include 1) identifying and enrolling the diverse providers that serve adults, 2) a lack of adult immunization reporting mandates in many grantees’ jurisdictions, and 3) competing priorities for state and local immunization programs. To support increased adult provider participation in IIS, CDC is supporting several new initiatives, including partnering with the Veterans Administration, the Indian Health Service, and federal occupational health clinics; providing supplemental funding to IIS Sentinel Sites to support adult provider enrollment and completeness of adult data in IIS as part of pandemic preparedness; and collaborating with the American Immunization Registry Association to better understand barriers and opportunities for pharmacy reporting to IIS. CDC also has initiated the Clinical Decision Support for Immunization (CDSi) project for the adult vaccine schedule, which will provide a single, authoritative, software-independent foundation for development and maintenance of evaluation and forecast systems .** By caWhat is already known on this topic?In 2011, 84% of U.S. children aged <6 years (19.2 million) participated in immunization information systems (IIS).What is added by this report?In 2012, 86% of U.S. children aged <6 years participated in IIS. Adult participation (25%) in IIS lags behind. Eight of 12 minimum functional standards for IIS published by the National Vaccine Advisory Committee have been met by ≥90% grantees, but gaps still exist in meeting Health Level 7 (HL7) interoperability and some data quality standards.What are the implications for public health practice?To realize the full benefits of IIS, progress is needed to reach lifespan participation in IIS, advanced bidirectional HL7 messaging between IIS and electronic health records, and improved data quality in IIS. Initiatives designed to increase adult participation in IIS, and promote HL7 messaging and electronic health records use among providers, are expected to support progress in these areas.In addition to capturing the complete population of children and adults within each IIS jurisdiction, IIS must maintain and enhance system functionality to ensure that data quality is high, protect the confidentiality of data, and serve multiple stakeholders. Although IIS have made great strides in implementing functional standards, progress can still be made in areas such as timeliness of record submission, completeness of core data elements, and HL7 functionality. Several ongoing and new initiatives are expected to support these functional standards, including the use of IIS to support vaccine ordering and inventory management, the use of two-dimensional barcodes to record vaccination information, and interoperability between IIS and electronic health records . ImplemeThe findings in this report are subject to at least two limitations. First, although CDC provides guidance to grantees to validate IISAR responses, data are self-reported and self-validated, which might result in overestimation or underestimation of participation rates. Second, because two of the 56 grantees did not report data during the period studied, the percentage of grantees meeting each of the functional standards might be higher or lower than calculated.New functional standards for IIS for 2013–2017 have been developed by CDC through a consensus process involving input from IIS managers and technical experts nationwide . Those s


Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.The online version of this article (doi:10.1186/s13287-015-0080-7) contains supplementary material, which is available to authorized users. In vitro, tumor cell products that are present in tumor-derived conditioned medium (CM) convert MSCs to CAFs which become fully and potently functional in promoting malignancy in vivo [The development and progression of breast tumors are multifactorial processes that are influenced by the tumor microenvironment (TME). Recent studies demonstrated that breast tumors are populated by myofibroblasts that express pro-cancerous functions -4, known in vivo -14.via angiogenesis and matrix-remodeling activities [The activities of CAFs and MSCs do not take place in the void, but rather are integrated in their intimate TME. In many cancers, the TME is dominated by inflammatory elements, including inflammatory leukocytes and inflammatory soluble factors that generally promote disease progression -18. The tivities -37. Overtivities ,38-40.We recently reported that TNF-α and IL-1β were minimally expressed by normal breast epithelial cells, but were highly expressed in tumor cells of biopsies from most breast cancer patients . In suchRecent studies indicate that CAFs and MSCs promote malignancy through the expression of inflammatory chemokines ,54-65. IOur overall goal in this study was to investigate aspects of inflammation–stroma interactions in breast cancer. To this end, we determined the influence of breast tumor-derived factors and of inflammatory cytokines on the inflammatory phenotype of CAFs and MSCs, manifested by the release of the pro-cancerous chemokines CCL2, CXCL8 and CCL5 by these cells. The novel findings obtained in this study show intensive inflammation–stroma interactions that act specifically through the designated pathway of nuclear factor κB (NF-κB) activation in stromal cells. Through these interactions, the inflammatory microenvironment shapes the tumor-promoting phenotype of MSCs and CAFs and may thus enhance tumor progression.l-glutamine .Human breast tumor MDA-MB-231 cells were obtained from ATCC  and MCF-7 cells were kindly provided by Prof. Kaye, Weizmann Institute of Science, Rehovot, Israel. The MCF-7 cells were authenticated on the basis of published characteristics of MCF-7 cells ,77 by vel-glutamine, minimum essential medium nonessential amino acids, antibiotics and 60 mM β-mercaptoethanol . Cells propagated in these conditions had typical fibroblastic phenotype: their identity was verified by the absence of the epithelial marker cytokeratin and the presence of the typical mesenchymal markers vimentin, α-smooth muscle actin (αSMA), fibroblast-specific protein 1 (FSP1) and fibroblast-activation protein alpha (FAPα)  in DMEM, filtered  and plated in DMEM supplemented with 20% FBS, 1 mM sodium pyruvate, 2 mM Two human patient-derived CAF isolates were generated and characterized for CAF phenotype as described above. CAFs #1 cells were derived from a lung metastasis of breast cancer and were immortalized by human telomerase reverse transcriptase (hTERT), as described previously . CAFs #2in vitro, to determine multipotency (data not shown). Second, at the advanced stages of the study, fully characterized MSCs were purchased from Lonza. As indicated in Lonza’s datasheet, the cells had typical MSC characteristics. They expressed CD44, CD29, CD105, and CD166 and did not express CD45, CD14 and CD34. Also, the cells can differentiate to adipogenic, chondrogenic and osteogenic lineages when cultured in the recommended differentiation medium. The cells were thawed into enriched mesencult medium (Stemcell Technologies Inc.) and then were subcultured every 5 to 7 days in enriched DMEM medium. In all cases, cell growth was continued in DMEM medium for a limited number of passages .Two sources of human BM-derived MSCs of healthy individuals were used in the study. First, in the earlier stages of the study, human MSCs were isolated from unprocessed BM of healthy individuals obtained from Lonza  and were cultured in mesencult medium . Following establishment in culture, the isolated MSCs were cultured in α-minimum essential medium containing 10% FBS and penicillin-streptomycin (as in ). In linin vivo in past experiments [MDA-MB-231 and MCF-7 breast tumor cells were grown in enriched DMEM culture medium (as above). Tumor CM from these cancer cells was collected after 18 to 24 hours, centrifuged at 1,200 rpm for 5 minutes and supernatants were passed through sterile filters with 0.45 μm polyvinylidene difluoride membrane . MSCs were exposed to such fresh Tumor CM twice a week for a ~30-day time period, following the protocol described in  that haseriments . In paraCAFs obtained by stimulating MSCs for ~30 days and their control MSCs were trypsinized, centrifuged and counted. The cells were plated at equivalent density and, following an additional 24 hours of incubation, supernatants from the differentiated CAFs and from control MSCs were frozen in −20°C. In parallel, an aliquot of the original Tumor CM was frozen at −20°C. All supernatants were then thawed at the same time  for determination of CCL2, CXCL8 and CCL5 levels in these supernatants by enzyme-linked immunosorbent assay (ELISA).In other assays, this same procedure was utilized in the presence of short stimulation by TNF-α , carried out during the last 24 hours of culture. TNF-α working concentrations were chosen based on criteria described next in Stimulation of MSCs by TNF-α or IL-1β. As control for TNF-α, the cells were incubated with the vehicle of the cytokine ). CCL2, CXCL8 and CCL5 levels in cell supernatants were determined by ELISA.in vitro in other studies  or IL-1β (500 pg/ml) (PeproTech). Cytokine working concentrations were chosen based on calibrations performed in other cell systems in our laboratory and are in the accepted range used ications -81). CheIn specific cases, neutralizing antibodies or small interfering RNAs (siRNAs) were used. For experiments with antibodies to TNF-α receptors, MSCs were plated in enriched DMEM medium for 24 hours. The cells were then washed and incubated in DMEM medium supplemented with 1% FBS, in the presence of neutralizing antibodies for tumor necrosis factor receptors TNF-RI and TNF-RII  or isotype-matched nonrelevant antibody control  for 1 hour. Antibody concentrations were chosen based on preliminary analyses, in which several concentrations of neutralizing antibodies were tested (data not shown). Following this incubation period, TNF-α (50 ng/ml) or its vehicle (as above) was added to cells. After 24 hours, cell supernatants were collected for ELISA assays.For experiments with siRNAs to c-Jun or p65, MSCs were plated in enriched DMEM medium for 24 hours. The cells were then transiently transfected with siRNA to c-Jun , siRNA to p65  or control siRNA (same concentrations) by Lipofectamine RNAiMAX™ transfection reagent  according to the manufacturer’s instructions. The cells were washed and incubated in DMEM medium overnight, and then stimulated with TNF-α  for 24 to 48 hours. Cell supernatants were collected for ELISA assays.MDA-MB-231 cells were transiently transfected with pcDNA3.1 vector or with the same vector coding for human TNF-α, by ICAFectin™ 441 DNA transfection reagent  according to the manufacturer’s instructions. After 24 hours, Tumor CM was harvested and TNF-α expression was determined by ELISA, as described below. In parallel, on the day of MDA-MB-231 cell transfection, MSCs were plated in enriched DMEM medium. The MSCs were then incubated for 24 hours with Tumor CM from MDA-MB-231 cells transfected by control vector or by TNF-α-expressing vector, or with enriched DMEM medium as control. Part of the above MDA-MB-231 Tumor CM was frozen in −20°C, as was also the case for CM of MSCs alone. All CM was then thawed at the same time . As appropriate, CCL2, CXCL8, CCL5 and TNF-α levels in these supernatants were determined by ELISA.CAFs #1 and CAFs #2 isolates were cultured for 24 hours in CAF growth medium (as above). The cells were then washed twice in LPM starvation medium , and incubated for 48 hours in LPM starvation medium in the presence of TNF-α (50 ng/ml), IL-1β (500 pg/ml) or cytokine vehicle (as above). CCL5 levels in the supernatants of the cells were determined by ELISA.Human BM-derived MSCs that were grown in the absence of or in the presence of MDA-MB-231-derived or MCF-7-derived Tumor CM for ~30 days (as above) were cultured for 24 hours on sterilized cover slips at 37°C. The cells were washed, fixed in 8% paraformaldehyde, permeabilized with 0.2% triton, blocked with 2% BSA in PBS, and then incubated with antibodies against αSMA  and vimentin . Negative controls included samples in which the primary antibodies were replaced by isotype-matched nonrelevant antibody controls (data not shown). The cells were then stained with the secondary antibodies, as appropriate. In parallel, cells were counterstained with the nuclear dye 4′,6-diamidino-2-phenylindole. Cell-coated cover slips were removed from the wells, embedded in mounting gel and imaged by confocal microscopy .To determine the expression of human TNF receptors, total RNA was isolated by the EZ-RNA kit  from patient CAFs #1 and CAFs #2 isolates, from MSCs and from HL-60 cells that served as positive control. First-strand complementary DNA was generated using the M-MLV reverse transcriptase . The expression of TNF-RI (TNFRSF1A) and TNF-RII (TNFRSF1B) was determined using the following primers: TNF-RI, forward 5′-GCACTGCCGCTGCCACACT-3′ and reverse 5′-AAGGCGATCTCGCAGGACG-3′, expected PCR product size 1,480 base pairs; and TNF-RII, forward 5′-ATGGCGCCCGTCGCCGTCT-3′ and reverse 5′-CCTGGTTAACTGGGCTTCATC-3′, expected PCR product size 1,390 base pairs. PCR amplification of TNF-RI was performed over 40 cycles . PCR amplification of TNF-RII was performed over 40 cycles . The sequence of the resulting PCR products was verified as TNF-RI and TNR-II. No-template controls were negative.2SO4, and was measured at 450 nm.ELISA assays were performed in the linear range of absorbance using standard curves generated with recombinant proteins. The ELISA for CCL2 involved mouse monoclonal antibodies (mAbs) against human CCL2  for coating, and biotinylated rabbit anti human CCL2 antibodies  for detection. The ELISA for CXCL8 involved mouse mAbs against human CXCL8  or rabbit polyclonal antibodies against human CXCL8  for coating, and biotinylated goat anti human CXCL8 antibodies  or biotinylated rabbit anti human CXCL8 antibodies  for detection. The ELISA for CCL5 involved mouse mAbs against human CCL5  for coating, and biotinylated goat anti human CCL5 antibodies  for detection. The ELISA for TNF-α involved mouse mAbs against human TNF-α  for coating, and biotinylated rabbit anti human TNF-α antibodies  for detection. Following the addition of streptavidin–horseradish peroxidase, the substrate TMB/E  was added. The reaction was stopped by addition of 0.18 M HThe expression of CCL2 by CAFs in biopsies of patients diagnosed with invasive ductal carcinoma (IDC) was determined as described previously . BrieflyTo determine the activation of activator protein 1 (AP-1) and NF-κB, TNF-α stimulation was carried out at time points that were determined by preliminary kinetics analyses . In all of the experiments described below, control cells were incubated with the relevant vehicles of the reagents. The following primary antibodies were used: phosphorylated p65 , total p65 , phosphorylated c-Jun , total c-Jun  and IκBα . To determine the characteristics of patient CAFs and of Tumor CM-derived CAFs, the following primary antibodies were used: pan-cytokeratin , αSMA , vimentin , FSP1  and FAPα . Loading controls were determined using antibodies against glyceraldehyde 3-phosphate dehydrogenase , β-tubulin  and Erk , as indicated in the figures. After washing, the membranes were incubated with the appropriate secondary antibodies. In all cases, conventional western blot procedures were taken and membranes were subjected to enhanced chemiluminescent (ECL) solution and X-ray film.Monocytic THP-1 cells were grown in suspension in enriched RPMI medium and their migration in response to CM of MSCs, stimulated or not stimulated by TNF-α, was determined. To this end, MSCs were stimulated by TNF-α (50 ng/ml) for 24 hours, while control cells were exposed to the vehicle of the cytokine (0.1% BSA) in DMEM starvation medium. Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 ; and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody . Recombinant human CCL2 (rhCCL2) was used at 100 ng/ml as positive control in the migration assays. rhCCL2 was either untreated or treated by neutralizing antibodies against CCL2 (as above). All antibody treatments were performed for 30 minutes at 37°C. The migration of human THP-1 monocytic cells in response to these cell supernatants was assessed by a 48-well modified Boyden chamber through a polycarbonate polyvinylidene difluoride (PVDF) filter . After 2 hours, the filters were fixed by methanol, and stained with a Diff-Quik kit . The cells were counted in five high-power fields (HPF) by light microscopy in triplicate.t test was used to calculate P values for ELISAs and chemotaxis assays. P <0.05 was considered significant. Adjustment for multiplicity of comparisons was done using the Benjamini–Hochberg procedure. Using this procedure, all of the significant results that were presented in the manuscript remained statistically significant after correcting for their multiplicity. The ELISA results presented in the paper show a representative experiment out of n ≥ 3 independent experiments that have shown similar trends. An exception is shown in Figure Student’s in vivo in promoting breast cancer [Published reports indicate that prolonged stimulation of BM-derived human MSCs with breast tumor-derived factors (Tumor CM) has led to conversion of MSCs into functional CAFs that promoted tumor growth -14. We bt cancer , by stimt cancer ,13,82,83The generation of such CAFs by Tumor CM has been only partly accompanied by enhanced inflammatory profile of the resulting CAFs. Tumor CM of MDA-MB-231 cells has increased the release of CCL2 and CXCL8 but not of CCL5 . Of the two cytokines, TNF-α is of particular interest because of its divergent and at times opposing roles in the TME, and also because it induces the release of inflammatory chemokines by breast tumor cells and normal tissue cells . To determine the effects of TNF-α stimulation on Tumor CM-generated CAFs, MSCs that were exposed to Tumor CM for ~30 days were stimulated by TNF-α during the last 24 hours of culture (MSCs + MDA CM + TNF-α or MSCs + MCF-7 CM + TNF-α) followed by determination of CCL2, CXCL8 and CCL5 expression in cell supernatants. In parallel, chemokine expression was determined in MSCs that were exposed to Tumor CM alone for the same prolonged time period (MSCs + MDA CM or MSCs + MCF-7 CM) and in MSCs that were grown in culture for ~30 days without Tumor CM and were then stimulated by TNF-α during the last 24 hours (MSCs + TNF-α). The results shown in Figure These findings suggest that, following their recruitment to the tumor site, MSCs are exposed to tumor constituents that promote their differentiation to CAFs and to some extent can also elevate their ability to release pro-malignancy chemokines. However, since the TME is enriched with TNF-α released by the tumor cells, this inflammatory cytokine turns into a most powerful inducer of chemokine release by CAFs. TNF-α thus enriches the TME with high levels of inflammatory/tumor-promoting chemokines presenting many deleterious pro-cancerous effects.To examine the potential clinical relevance of these observations we tested the content of inflammatory chemokines in CAFs obtained directly from a lung metastasis of a breast cancer patient (CAFs #1) and from a different patient’s primary breast tumor (CAFs #2). Two such CAF isolates showed production of CCL2 and of CXCL8 Figure A1,B1.FigTo better understand the regulation of chemokine release by patient CAFs, we asked whether CCL5 – which was not released constitutively by these cells – would be upregulated by inflammatory cytokines such as TNF-α and IL-1β that are known to prevail at the TME of breast tumors. Indeed, in line with the expression of both TNF-RI and TNF-RII by the two patient CAF isolates  patients (Figure The AP-1 and NF-κB transcription factors are known to induce the expression of inflammatory chemokines in the immune context; however, their extent of involvement in inducing such chemokines depends on the cell type and stimulus. From the therapeutic perspective it is crucial to know whether both or only one of these transcription factors are mediating the response of MSCs to inflammatory stimuli that give rise to increased production of pro-malignancy chemokines. We began this part of the study by determining the involvement of AP-1 in induction of CCL2 and CXCL8, the two inflammatory chemokines that we found to be most relevant to the clinical setting, as shown by their expression by patient CAF isolates #1 and #2 Figure . While ADifferent results were obtained for NF-κB, showing that this transcription factor is cardinal in CCL2 and CXCL8 upregulation by TNF-α in MSCs. First, we found that TNF-α stimulation induced significant reduction in the expression levels of IκBα, the negative regulator of the NF-κB pathway, in MSCs Figure A. SimilaIn contrast to the results obtained with TNF-α stimulation, a different pattern was obtained with regards to the impact of Tumor CM on the cells: while factors released by tumor cells resulted in MSC conversion to CAFs (Figure S2 in Additional file To summarize, the results of this part of the study clearly indicate an NF-κB-based pathway that regulates the expression of inflammatory and pro-malignancy chemokines such as CCL2 and CXCL8 in MSCs and in CAFs in response to TNF-α. NF-κB, but not AP-1, was involved in TNF-α-induced inflammatory patterns of CAFs and MSCs, despite the fact that the promoter domains of both these chemokines contain binding sites for c-Jun. This dichotomy between the NF-κB and AP-1 transcription factors in controlling CCL2 and CXCL8 production in MSCs indicates that the inflammatory profile of MSCs is tightly regulated. The preference of one transcription factor over another needs to be considered when potential therapeutic measures are directed to intracellular components involved in inflammation-induced tumor progression, such as NF-κB and AP-1.Thus far, we have demonstrated that the inflammatory cytokines TNF-α and IL-1β induced inflammatory traits in CAFs and MSCs; furthermore, we have delineated the molecular mechanisms involved in these processes. To follow on these observations, we asked whether the inflammatory traits induced by TNF-α in MSCs have functional relevance to tumor-promoting events taking place at the TME.in vitro migration of monocytic cells and found that it was induced by supernatants of TNF-α-stimulated MSCs (Figure CCL2 induces the recruitment to tumors of myeloid subpopulations that exert prominent pro-cancerous effects, including TAMs and MDSCs ,67,71-75s Figure , which hs Figure A. Furthes Figure . These rFurthermore, our findings suggesting that MSC-derived CCL2 promotes the recruitment of TAMs and MDSCs to breast tumors emphasize the important roles of inflammation–stroma interactions. These findings gain high relevance with the fact that CCL2 is indeed expressed in breast cancer cells, as we have shown in our study of biopsies of invasive ductal carcinoma patients Figure .Much prominence has been given to inflammation-driven activities that promote tumor progression by acting on cancer cells, infiltrating leukocytes and adjacent stromal cells. In the current study we provide new findings on the networks that control the inflammatory phenotype of CAFs and MSCs, demonstrating key roles for the inflammatory cytokines TNF-α and IL-1β in upregulating the release of inflammatory, tumor-promoting chemokines by these two cell types.TNF-α and IL-1β are highly relevant to the inflammatory setup of breast tumors -49. TheyIn this study we have also revealed the important role of NF-κB, but not of AP-1, in mediating the activities of TNF-α on MSCs and CAFs. These findings support other reports ,92,93, iCurrently, the inhibition of NF-κB in cancer has not entered the stage of clinical trials, and may be hampered by the fact that this pathway regulates a number of essential cellular processes and immune activities. Such hurdles can be overcome by directing the NF-κB-inhibiting modalities to specific target cells, in which this transcription factor is the key inducer of undesired inflammatory, tumor-promoting effects. In this respect, the results of our study imply that MSCs and CAFs could serve as appropriate targets for NF-κB inhibition, through which downregulation of the inflammatory TME could be achieved.


Furthermore, the use of Au nanoparticles also generated enhanced immunogenicity of CpG and VLPs on both humoral and cellular immune pathways, as followed from increased expressions of total HBc-specific antibody titer, CD4+ T cells, CD8+ T cells, cytokine interleukin-4, and interferon-gamma. These findings demonstrated that CpG-Au@HBc VLP nanocomposite could induce robust cellular and humoral immune response, which could be a potential vaccine for future prophylactic and therapeutic application.Different types of vaccines have been developed to elicit active immunization to treat various diseases, while suffer from limitation of efficacy. Herein, a novel immunostimulatory nanocomposite (CpG-Au@HBc VLP) was rationally designed by self-assembling engineered virus-like particles encapsulating CpG-gold nanoparticle conjugates through electrostatic interactions. The monodispersed and uniformly sized CpG-Au@HBc VLP showed increased CD4 Escherichia coli (E. coli)) [Vaccines aim at generating the body’s protective and therapeutic immune responses against many diseases, such as infectious diseases and cancers . Convent. coli)) , 11. HBc. coli)) . Moreove. coli)) . Thus, t. coli)) , 14. HowAdjuvant is responsible for stronger efficacy of vaccine and subsequently significant immune responses. Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) have been utilized as potent adjuvants with negligible toxicity, due to their wide stimulation to macrophage cells, dendritic cells, natural killer (NK) cells, and B cells . CpG ODNIn the present work, we developed CpG-Au@HBc VLPs as an immunostimulatory nanocomposite in which the CpG ODNs adjuvant was conjugated with Au NPs and co-encapsulated by genetically engineered HBc VLPs. The designed nanocomposite showed the ability to induce robust specific T cell response and enhanced Th1-type immune response than HBc VLPs co-immune with conventional adjuvant. It was also revealed that Au NPs could enhance the immune stimulation of CpG ODNs on both humoral and cellular immune response. As a result, the CpG-Au@HBc VLPs might be a potential vaccine for future prophylactic and therapeutic application.4·3H2O), sodium citrate, sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic (Na2HPO4), potassium phosphate monobasic (KH2PO4), 10 % sodium azide (NaN3), Tris(hydroxymethyl)aminomethane (Tris-HCl), isopropyl-β-d-thiogalactoside (IPTG), Triton, ammonium sulfate ((NH4)2SO4), sodium dodecyl sulfate (SDS), glycerol, glycine, and Tween-20, were purchased from Sinopharm Chemical Reagent Co., Ltd . Bovine serum albumin (BSA), Freund’s adjuvant complete and Freund’s adjuvant incomplete were purchased from Sigma-Aldrich . Horseradish peroxidase (HRP) conjugated anti-mouse antibody, 3, 3′ 5, 5′-tetramethylbenzidine (TMB), PE/Cy5 anti-mouse CD8a, and FITC-CD4 anti-mouse CD4 were purchased from Ebioscience Biotechnology (USA). High affinity purification purified CpG ODNs (5′-TCCATGACGTTCCTGACGTT-SH-3′) were synthesized by Sangon Biotech Co., Ltd . Unless otherwise noted, all chemicals were analytical grade and used without further purification. Milli-Q water (less than 18.2 MΩ cm) was used throughout the whole experiment.Chemicals including chloroauric acid trihydrate (HAuClE. coli BL21 (DE3) strain containing the expression plasmid was cultured and induced to express HBc VLPs. After the expression, HBc VLPs were separated and purified successively (detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis) by salting out with saturated ammonium sulfate, DEAE ion-exchange chromatography, and Sepharose CL 4B molecular exclusion chromatography. The purified HBc VLPs were ultrafiltered to reach a final concentration of 2 mg/mL in PBS and stored at −20 °C.The coding sequence of the full-length HBc antigen (HBcAg) was amplified from the plasmid pET-43.1a. Amino acid sequence of the full-length HBcAg was the same as the published report . The exp4 aqueous solution  was added to 100 mL ultrapure water and heated to boiling, then trisodium citrate solution  was added quickly under vigorous stirring. After reaction for 20 min, the Au solution was cooled down to room temperature. The as-synthesized Au NPs were kept at 4 °C.Citrate-stabilized gold nanoparticles with small size were prepared using a modified seed-mediated method reported by Frens et al. . BrieflyThe assembly of Au NPs with CpG ODNs was conducted by using a salt-aging approach as follows. Firstly, sulfhydryl-modified CpG ODNs  and Au NPs solution  were mixed at 16 °C for 12 h. The solution was adjusted to 0.1 % SDS and 10 mM of PBS buffer (pH 7.4). Then the concentration of NaCl in the solution was slowly increased to 0.1 M in a 6-h time window by stepwise addition of 1 M NaCl aqueous solution. The mixture solution was shaken for 12 h at room temperature to make sure the Au NPs were fully covered by thiolated CpG ODNs. CpG-Au NP conjugates were then centrifuged  and washed with 0.1× PBS buffer for three times to remove the extra CpG ODNs. The final precipitate was dissolved in PBS for further use.To quantify the accurate amount of CpG ODNs bound to each Au NP, a fluorescence-based method was used . BrieflyThe HBc VLPs were firstly dissociated by adding dissociating buffer  with a final concentration of 1 mg/mL and incubated for 3 h at room temperature. Then CpG-Au NP conjugate solution  or free CpG ODNs  were added and incubated for 30 min under slight shaking to form CpG-Au@HBc VLPs or CpG@HBc VLPs, respectively. Finally, samples were dialyzed overnight against reassembling buffer  at 4 °C, and detected by agarose gel electrophoresis.Six-week-old pathogen-free female BALB/c mice were obtained from Shanghai Laboratory Animal Center of Chinese Academy of Sciences (China) and kept at the Animal Center of Xiamen University. All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee. The mice were randomly divided into five groups (six mice per group). Then the mice were injected intraperitoneally with following vaccine formulations (0.2 mL) (a) 50 μg HBc VLPs only, (b) CpG@HBc VLPs: 50 μg HBc VLPs encapsulated 10 μg CpG ODNs, (c) CpG-Au@HBc VLPs: 50 μg HBc VLPs encapsulated CpG-Au NP conjugates containing 10 μg CpG ODNs, (d) CpG-Au + HBc VLPs: 50 μg HBc VLPs mixed with CpG-Au NP conjugates containing 10 μg CpG ODNs, (e) CFA/IFA + HBc VLPs: HBc VLPs mixed with Freund’s adjuvant (positive control): 50 μg HBc VLPs mixed with 50 μg complete Freund’s adjuvant (CFA) for the first injection, and 50 μg HBc VLPs mixed with 50 μg incomplete Freund’s adjuvant (IFA) for the other three injections, and (f) PBS  as blank control. Mice were immunized on days 0, 7, 14, and 21, respectively. After immunization for the 4 weeks, all mice were sacrificed on day 28. The sera and splenocytes of mice were harvested for further analysis.2SO4 was added to have color reaction. Finally, the absorbance of samples at 450 nm was measured with microplate reader.96-well plates were coated with HBc VLPs in coating buffer  overnight at 4 °C. Wells were washed three times with PBS containing 0.05 % Tween-20 then blocked by 1 % BSA at 37 °C for 1 h. After washing with PBS, diluted mouse serum samples collected after each immunization were incubated at 37 °C for 1 h, followed by washing three times, 100 μL of horseradish peroxidase (HRP)-conjugated anti-mouse antibody at different dilutions  were added to wells. The plates were incubated at 37 °C for 1 h and washed again. TMB substrate solution was added to each well and incubated for 15 min at 37 °C. Then H3 and 0.5 % BSA. The expression of CD4+ and CD8+ T cells were determined by flow cytometry analysis.The splenocytes were separated after mice of all five groups were euthanized. Then, splenocytes were stained by fluorescent antibodies  on ice for 30 min, and cells were subsequently washed with ice-cold PBS, containing 0.1 % NaNγ), interleukin-2 (IL-2), and interleukin-4 (IL-4) from mice serum in the presence of the four different vaccine formulations were analyzed with the cytokine enzyme-linked immunosorbent assay (ELISA) kits . All operations were performed following the manufacturer’s instructions.The level of cytokine interferon-gamma (IFN-Transmission electron microscopy (TEM) images were obtained by a JEM-1400  operated at an accelerating voltage of 100 kV. Protein samples for TEM analysis were prepared by spreading a drop of the sample dilute dispersion on carbon film-coated copper grids followed by staining with 1 % uranyl acetate. Dynamic light scattering (DLS) and zeta potential distribution were measured on a Malvern Zetasizer Nano ZS . UV-vis absorption spectra were acquired with a DU 800 UV-vis spectrophotometer . The fluorescence emission spectra were obtained by FluoroMax-4 spectrometer . Flow cytometry analysis was performed on a Coulter Epics XL flow cytometer . Optical absorbance measurements in ELISA assays were performed on Bio-Rad Model 680 microplate reader .t test. p values below 0.05 were considered as significant and indicated with * in the figures.Data for each experiment were presented as mean values ± standard deviation (SD). Comparisons between two groups were performed by two-tailed Student’s E. coli system and purified by salting out, ion-exchange chromatography, and molecular exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result , which were widely applied to induce both humoral and cellular immune response, respectively. Humoral immune responses were documented on days 7, 14, 21, and 28 by evaluating the anti-HBc titer in the sera of mice. As shown in Fig. + and CD8+ T cells in splenocytes, which were known as two prime T cell subsets. The CD4+ T cells are mainly T helper (Th) cells, and their responses can be divided into two types, T helper 1 (Th1) and T helper 2 (Th2), based upon cytokine secretion and effector function [+ T cell subset was significantly higher in mice immunized with HBc than those immunized with PBS (p < 0.05), suggesting that all of the vaccine formulations  could activate CD4+ T cell subsets. The CD4+ T cell in mice immunized with CpG-Au@HBc VLPs (26.3 %) was much higher than those immunized with CpG@HBc VLPs  and CFA/IFA + HBc VLPs , while has no significant difference with CpG-Au + HBc VLPs . The other T cell subset evaluated here was CD8+ T cells. The antigen-specific CD8+ T cells are mainly CTL, which are critical for protecting against intracellular pathogens. Flow cytometry analysis of CD8+ T cells in Fig. + T cells. The production of CD8+ T cell subsets were also significantly higher in mice immunized with HBc than those immunized with PBS (p < 0.05). The production of CD8+ T cells elicited by CpG-Au@HBc VLPs (13.4 %) revealed higher level than those induced by CpG@HBc VLPs  and CFA/IFA + HBc VLPs . Hence, CpG-Au@HBc VLPs had great potential as immunostimulatory vaccine formulation for the generation of specific T cell responses than CpG@HBc VLPs and CFA/IFA + HBc VLPs. Besides, Au NPs made the vaccine formulation much more efficient to induce immune response.T cells play key roles in cellular immune response . The T cfunction . Strong function . It was function . These ep < 0.05), but is considerably less than that triggered by CFA/IFA + HBc VLPs (p < 0.01), and showed no significant difference with CpG-Au + HBc VLPs. This result was consistent with the result of anti-HBc titer in Fig. p < 0.05). However, the secretion of cytokine IL-2 presented no significant difference (p > 0.05) among CpG@HBc VLPs, CpG-Au + HBc VLPs, CpG-Au@HBc VLPs, and CFA/IFA + HBc VLPs. It was thus indicated that strong Th1 cell immune response was also stimulated by CpG-Au@HBc VLPs.It is well known that cytokines IFN-γ can induce Th1 differentiation and IL-4 can induce Th2 differentiation. Another cytokines IL-2 can induce both Th1/2 differentiation . The secIn general, the above results suggested that CpG-Au@HBc VLPs could induce stronger cellular immune response when compared with CpG@HBc VLPs and CFA/IFA + HBc VLPs, respectively. Meanwhile, this nanocomposite could also stimulate greater humoral immune response when contrast with CpG@HBc VLPs. Such robust immune responses caused by low dosage of adjuvant (CpG ODNs) could help to reduce the side effects in mice. Besides, Au NPs of this nanocomposite played an important role in inducing both Th1- and Th2-dominated response.In summary, a highly monodisperse and uniform CpG-Au@HBc VLPs nanocomposite was developed through a dissociation and reassembling process. The obtained adjuvant-containing VLPs exhibited both strong humoral and cellular immune stimulation ability. Noteworthy, Au NPs, which were conjugated with CpG ODNs, could enhance the immunogenicity of the nanocomposite through helping to stimulate both Th1- and Th2-dominated immune response. This new type of immunostimulatory nanocomposites is thus expected to be as a potential vaccine for prophylactic and therapeutic application, even for tumor immunotherapy.E. coli, Escherichia coli; HBc VLPs, hepatitis B core protein virus-like particles; HBcAg, HBc antigen; IFA, incomplete Freund’s adjuvant; IFN-γ, interferon-gamma; IL-2, interleukin-2; IL-4, interleukin-4; NK cells, natural killer cells; Th, T helperAu NPs, gold nanoparticles; Au NRs, gold nanorods; CFA, complete Freund’s adjuvant; CFA/IFA-HBc VLPs, HBc VLPs mixed with conventional Freund’s adjuvants; CpG ODNs, oligodeoxynucleotides-containing un-methylated CpG motifs; CpG@HBc VLPs, HBc VLPs encapsulated CpG ODNs; CpG-Au@HBc VLPs, HBc VLPs encapsulated CpG-Au conjugates; CTL, cytotoxic T lymphocyte;


Globally, falls from height (FFH) are a substantial public health jeopardy and are among the important leading causes of serious and fatal injuries for construction workers. A comprehensive understanding of the causal factors in FFH incidents is urgently required; however, the literature appears to lack a scientific review of FFH. In this study, 297 articles that contribute to the topic of fall incidents were reviewed. Seventy-five (75) articles met the criteria for relevance and were aggregated in a database to support a critical review. A synthesis of macro-variables approach was adopted rather than a structured meta-analysis. Such a method of analysis provides the flexibility to combine previous studies' findings. The most common factors associated with FFH are risky activities, individual characteristics, site conditions, organizational characteristics, agents (scaffolds/ladders) and weather conditions. The outcomes contributed to identifying the most significant research area for safety enhancement by improving engineering facilities, behaviour investigations and FFH prevention methods. The construction industry has grown over the last decades and resulted in improvements in company profits, financial accessibility and increased commodities demand in Australia , as it hSlipping, tripping and falling incidents in construction have received significant attention and have been widely researched due to their severity and frequency . HoweverOf all construction accidents, falls are the leading cause of serious injuries (48%) and fatalities (30%) . In partConstruction fatalities and serious injuries caused by falls have attracted several researchers’ attention. For instance, Hu et al.  conducteBecause there is a lack of studies that address fall factors, this is an appropriate time for a critical scientific review of the existing literature on FFH incidents and to look for further information for future research directions. It would be beneficial to have a comprehensive review of the literature, which could assist researchers and practitioners to grasp the body of knowledge and pave the way for future research to fill an important research gap. The present overview has covered several FFH facets, including data collection types, analysis methods, fall factors and prevention strategies. This review has provided an aggregated database that attempts to fill the knowledge gap.The literature was first searched to retrieve the relevant journal papers and conference proceedings from the accessible databases. As we are in the technological era, online databases were reviewed by utilizing the well-known search engine Google Scholar and the online library of the Queensland University of Technology to access the most common databases, including Science-Direct, Taylor & Francis, J-STOR, the American Society of Civil Engineering, Emerald Insight and many others that have published material on the topic of FFH incidents. As a starting point, a web search was conducted with typical keywords related to the subject, such as “fall(s)/falling from height(s)”,” fall(s)/falling from elevation”, “fall(s) injuries/accidents /incidents” and “construction industry/sector”. The search process resulted in accumulated knowledge through reviewing the collected literature. An additional number of keywords boosted and expanded the search process. They included “fall from roof/scaffolds/ladder/platform”, “fall to lower level/same level/opens”, “falls protection”, “hazardous/risky fall(s)”, “fatal fall(s)”, “and occupational accidents/injuries”.After a comprehensive search on FFH publications, a relevancy selection criterion was adopted to select the closest study/paper to the topic. Papers that met the criteria of homogenous title, bibliographic keywords and abstracts were added to the database to be analysed by macro-variables. The review methodology towards the selected articles has taken two paths. Firstly, examining the title, keywords, and abstracts were checked to determine whether the papers were related or not. 297 publications related to FFH were found covering journal articles, preceding conference and reports. The second path was to check the references of the first path articles to reach the 75 articles to be synthesized in a database. The timeframe of the search for reviewed papers was about twenty years published from 1994 to 2014.The 75 articles, which have been included with references, were comprehensively reviewed and synthesised into different categories to obtain the main variables that served the study aim. The data extracted from each article included the author, journal, title, keywords, study type, fall type, fall causes, fall effects, statistical /methods used, worker’s trade, activities performed, geographical location, building type, final results and conclusions see . Table 1As shown in FFH studies were conducted in different countries, as shown in FFH were analysed in different construction sectors, such as residential, non-residential, light/heavy commercial (including public buildings), and highway/road construction . Table 5The methodological approaches implemented in the 75 articles were based on their research objectives. Data collection methods varied from one study to another, ranging from using archival data , survey questionnaires, interviews, experiments and simulations, as shown in Quantitative analysis approaches were mainly implemented to analyse and evaluate FFH incidents. Over 80% of the studies (65 articles) have taken only a statistical analysis approach. More than two thirds of the studies (76%) relied on archival datasets for the purpose of statistical estimations for evaluating and predicting accidents’ severity and fall patterns. Eleven percent of the studies used questionnaires/surveys and interviews.The studies conducted earlier tend to use autopsy records to analyse demographic data , fall locations and heights, and autopsy findings . On the other side, the majority of the studies conducted their analysis by investigating the interventions of fall factors , weather and workers’ safety behaviours and attitudes)  or speciQuantitative analysis approaches were mainly implemented to analyse and evaluate FFH incidents. Over 80% of the studies (65 articles) have taken only a statistical analysis approach. More than two thirds of the studies (76%) relied on archival datasets for the purpose of statistical estimations for evaluating and predicting accidents’ severity and fall patterns. Eleven percent of the studies used questionnaires/surveys and interviews.The studies conducted earlier tend to use autopsy records to analyse demographic data , fall locations and heights, and autopsy findings . On the other side, the majority of the studies conducted their analysis by investigating the interventions of fall factors , weather and workers’ safety behaviours and attitudes)  or speciFFH is at the forefront of construction industry incidents compared with other industries . Yet invFFH incidents have adverse physical effects. The most common injuries are fractures, trauma, contusions, concussions, bruises and abrasions. They may lead to death. After the physical injury which has had a direct influence on a worker’s body, in some cases, a psychiatric illness may also occur due to the loss of a job opportunity or from having a permanent disability . Table 9FFH are influenced by a number of factors, including risky activities, individual characteristics, site conditions, organisation/management, agent, and weather/environmental conditions. FFH injuries are more likely to occur when the construction crew performs hazardous tasks/activities . The risIndividual variables play a significant role in FFH accidents. The responsibility for falling is partially situated in the individual characteristics of the construction workers, including their demography , knowledge level (education and experience), human behaviours and attitudes, physical characteristics, and health . At the top of the list of characteristics is the workers’ demography. Demographic attributes such as age, gender, weight, marital status, etc., have strong relationships with the workers' health, education and experience and nutrition system. For instance, workers who are suffering from obesity or who are overweight are exposed to fatigue more often, and fatigue is an important leading factor to FFH . Older wIn one hand, platform/surface conditions can impact those who work at elevated heights in several ways. FFH could occur when there are defects in the work surface, such as unprotected walkways, improper guardrails, slippery or sloped surfaces. Unexpected modifications in surface properties can be sufficient for FFH to occur ,49. On tOrganisation’s variables are significantly associated with FFH accidents and it has several components that lead to FFH. Firstly, the Australian construction companies are often small businesses, and 97% of general construction businesses employ 20 workers or less. Given that more workers are increasingly involved in small businesses due to short employment periods and high wages ,28,69. CThe second component is contractors and sub-contractors. A lack of capability or of resources of contractor/sub-contractors also leads to FFH. Although contractors and subcontractors have more direct control in construction sites, they need to implement a complicated system to process the safety regulations on a day-to-day basis . FFHs caThe third component is project management. Project timelines or shift work generate pressure on workers’ attitudes/behaviours to complete on time or ahead of schedule because of the urgency or supervisors’ pressure to accelerate the tasks with incompatible safety standards , which mAgents are one of the influential variables, and were mentioned in more than 25% of the studies e.g., ,18,23). ,23. 18,2The weather and surrounded environment in some cases have contributed to FFH incidents. Regionally, construction workers face heat, cold, rain or windy weather. The trades most exposed to weather conditions are carpenters and roofers, as shown in The significance of studying fall accidents is to prevent and mitigate the seriousness of injuries. Around half of the articles (51%) have given either recommendations or preventive methods to avoid falling incidents. These articles have been divided into passive or proactive methods. 10% of the articles have provided precautionary/proactive measures inside construction sites. The most important protective methods are to have on-site precautionary measures, and education and training are at the forefront of preventing FFH accidents, while the importance of passive methods is sometimes lies on analysing the fall accidents data for future plans.Proactive methods can be the optimal strategy to reduce FFH incidents. Educating and training construction workers can also reduce serious incidents. Furthermore, designing short safety training courses for the workers, seminars and talks focusing on work at height risks, might have positive impacts on workers’ behaviour that reduce FFH incidents ,70. FurtMajority of industries, including the construction industry, are subjected to specific safety standards/guidelines. The importance of such regulations is to enhance the on-site health and safety management to prevent  injuries while working. Regular safety regulations revisions and regular inspections are among the potential remedies that can include any variation in job design, for example, weather or platform conditions . Table 1This paper conducted a comprehensive review of FFH literature. It contributes to summarising the research trend of FFH, highlighting the inadequacies, and providing signposts for future research. The review shows that most of the existing FFH research focused on analysing archival data. Sources of the archival data are mainly accident databases or accident reports. There are limitations of using accident databases or accident reports which are secondary data for analysis.Accident databases may not have been designed in a way to fit the purpose of the FFH research. Categorization of the data in the accident databases may not align with the needs of the researchers conducting FFH research. This data limitation restrains FFH research from providing further in-depth analysis.The problem of solely using accident reports as the data for FFH research is that the research has the problem of hindsight bias . As explThere is no doubt that learning from incident is important. However, most of the extant FFH literature did not embrace learning theories and methods. This limits the potential of the FFH literature to provide theoretically supported recommendations and strategies for organizations to develop the learning capability to prevent FFH accidents. Learning from accident investigation is an emerging topic of safety research. To effectively learn from FFH incident, interdisciplinary learning science theories and research methods should be embraced. For example, learning from incidents involves a number of critical steps, namely investigating and analysing incidents, planning interventions, intervening, and evaluating . These sA vast majority of the existing FFH research studies did not consider the holistic systems approach, that is, how the factors are interdependent with one and other. As evidenced in the review, individual factors were the most frequently mentioned. Interdependencies of the factors are underexplored in the existing literature. Factors leading to FFH accidents at the blunt end are likely to be undermined but the factors at the sharp end tend to be overestimated. In an increasingly complex socio-technical environment, it is unlikely that accidents have a single root cause or follow a sequence event pattern . Rather,To further advance FFH research and make an impact, it is suggested that other sources of data and research methods should be employed. Cognitive mapping for interviews and critical incidents can be useful in extracting the details of how FFH accidents happened without oversimplifying the complexities or losing important details . GettingAlthough design for safety has been one of the common recommendations found in the existing FFH research, this concept has yet to be adopted from the whole lifecycle perspective. Design for safety has huge potential to design out or minimize the FFH risk not only during construction but also long-term repair and maintenance of the building or infrastructure. Future FFH research should put more emphasis on the intervention of technology, such as building information modelling (BIM), augmented virtual reality, wearable monitoring devices for safety and health. Recently, there are studies ,90 whichTo conclude, a critical review has been undertaken to summarise the existing FFH literature, highlight the inadequacies and provide future research directions. The reviewed FFH literature was mainly drawn from peer-reviewed journal articles and conference proceedings ,79,80,83