Reduced risk of HIV-1 infection correlated with antibody responses to the envelope variable 1 and 2 regions in the RV144 vaccine trial. To understand the relationship between antibody responses, V2 sequence, and structure, plasma samples (n = 16) from an early acute HIV-1 infection cohort from Thailand infected with CRF01_AE strain were analyzed for binding to V2 peptides by surface plasmon resonance. Five participants with a range of V2 binding responses at week 24 post-infection were further analyzed against a set of four overlapping V2 peptides that were designed based on envelope single-genome amplification. Antibody responses that were relatively consistent over the four segments of the V2 region or a focused response to the C-strand (residues 165–186) of the V2 region were observed. Viral escape in the V2 region resulted in significantly reduced antibody binding. Structural modeling indicated that the C-strand and the sites of viral variation were highly accessible in the open conformation of the HIV-1 Env trimer. V2 residues, 165–186 are preferentially targeted during acute infection. Residues 169–184 were also preferentially targeted by the protective immune response in the RV144 trial, thus emphasizing the importance of these residues for vaccine design.  The RV144 HIV-1 vaccine trial in Thailand demonstrated 31.2% efficacy after 3.5 years . 10 helix [The Env V1V2-region has extensive loop length, glycosylation variation, and major sequence differences both within and between clades ,13,14,1510 helix . These a10 helix  in in vi10 helix . Antibodenv sequences from five participants and their relationship to induced V2-specific immune responses during the course of HIV-1 infection. To further deconvolute the V2 antibody responses and their relation to HIV-1 V2 structure, we selected five participants with low or high V2 antibody binding response levels. We divided the V2 region into four designated segments, incorporating viral mutation information based on plasma viral env sequence analysis, and assessed the binding to 16-20 mer V2 peptides with 8–10 overlapping amino acid residues. In addition, we assessed the surface-accessibility of the V2 segments in the context of multiple structurally defined conformations of V2 and HIV-1 Env. Overall, these results reveal the specificity of the humoral response to the HIV-1 V2 region in an early infection cohort and the generation of V2-escape mutants, which strongly suggest that the same region is vulnerable during infection and after vaccination. The limited env sequence variation during the acute phase of the infection coupled with identification of V2 regions that are targeted early during infection could provide information relevant for vaccine design. A vaccine that targets specific vulnerable areas of the V2 region in the Env might prevent acquisition of the virus and thus generate protective immune responses.In the present study, we examined a recent prospective natural history study RV217, the “Early Capture HIV Cohort” (ECHO), conducted in Thailand and East Africa  to assesRV217 is a prospective natural-history observational study conducted at four clinical research sites in Uganda, Kenya, Tanzania, and Thailand (1 site/country). The protocol was approved by the local ethics review boards and the Walter Reed Army Institute of Research  . All indParticipants who tested positive for HIV-1 RNA during the surveillance phase of the protocol and with confirmed HIV-1 infection were enrolled in the long-term follow-up phase. The current analysis focuses on a subset of 5 out of 16 participants from Thailand. None of the participants included here initiated antiretroviral treatment within the timeframe of the current analysis.g at 4 °C for 20 min. The supernatant was stored at −20 °C in small aliquots. Plasma samples were analyzed for the presence of V2-specific antibodies by SPR using a Biacore 4000 system  as previously described with some modifications [Plasma samples from 16 RV217 participants from Bangkok, Thailand, collected pre- and post-infection were analyzed for the presence of binding antibodies by surface plasmon resonance. Plasma samples were heated at 56 °C for 45 min followed by centrifugation at 16,000× ications ,38,39,40Viral RNA was extracted from plasma using the QIAamp Viral RNA Mini Kit . Complementary DNA (cDNA) was synthesized using the SuperScript III RT kit  following the manufacturer’s instructions. cDNA was amplified as a full genome or 2 half genomes overlapping by 1.5 kb as previously described  using sihttps://www.hiv.lanl.gov/content/sequence/VIRALIGN/viralign.html). The aligned sequences were further visualized and refined manually on Geneious Pro 5.6.7,  year 2013 (https://support.geneious.com/hc/en-us/articles/227534768). Amino acid sequences of gp160 were extracted into C1, C2, C3, C4, C5, V1, V2, V1V2, V3, V4, V5, gp120, gp140, and membrane-proximal external region (MPER) using python scripts. The genetic distances within subjects were calculated based on the GTR substitution model using Molecular Evolutionary Genetics Analysis (MEGA) 5.0. The V2 region sequence from the five RV217 participants was sub-divided into four segments and peptides of 16 or 20 amino acid residues in length with overlapping 8-10 residues were synthesized. The V2 peptides for each participant was selected based on the CRF01_AE T/F virus and representative CRF01_AE sequences retrieved from each of the 3 or more visits following first positive test for HIV-1 RNA. The sequences have been submitted to GenBank (Accession numbers MK656525-MK656857).HIV-1 sequences were first aligned with the HXBc2 sequence using HIVAlign via a Hidden Markov Model (2) were determined using PyMol. The clade G strain X1193.c1 HIV-1 SOSIP structure (PDB code: 5FYJ) was used to illustrate the HIV-1 glycan shield in the “closed” conformation. The clade B strain B41 HIV-1 SOSIP structure (PDB code: 5VN8) was used to model the HIV-1 Env in the “open” form. Not all glycans were visible in the experimentally determined “open” structure, so Man-5 ((Man)5 (GlcNAc)2) glycans were modelled at each of the N-linked glycosylation sequons for all three Env protomers using COOT. The atoms of the glycans were modelled with two radii (1.4 and 5.0 Å) to illustrate (i) a single location of the glycans, and (ii) the area covered by the mobile nature of the glycans.All figures were generated using PyMol. The V2 region of the HIV-1 Env is shown in two conformations, sheet conformation (PDB ID: 5FYJ) and helical conformation (PDB ID: 6FY1). Structure-based alignment and calculation of the surface area differences  using a spearman non-parametric correlation test assuming a two-tailed distribution.env sequencing data showed variations in the sequences. The V2 sequences from each of the 5 subjects one-week post-infection showed 64–85% identity with the 92TH023 peptide sequence used to measure V2 binding antibody responses  were examined. Plasma samples (1:100 dilution) from 16 participants from the study located in Thailand, who became infected with CRF01_AE were analyzed  by SPR for the presence of V2-specific antibodies using N-linked biotinylated full-length cyclic V2 peptide based on the sequence of CRF01_AE strain 92TH023 a. Maximuesponses a. Seven esponses c. The thesponses c for furenv sequences at week 21 that appear to be very closely related to the minor T/F)  analysis and sequence alignment for the V2 region of T/F viruses were conducted at each of three or four time points . Sequence reads were aligned for the five selected participants, 40007 , 40123 , 40511F, 40265  and 4051nor T/F) . Particinor T/F)  showed anor T/F) . In contnor T/F)  did not nor T/F) . Particinor T/F) , showed nor T/F) . In all A ribbon representation of the HIV-1 V2 region (residues 157–196) based on the fully glycosylated HIV-1 Clade G X1193.c1 SOSIP.664 Env trimer structure (PDB ID: 5FYJ) is shown in The various biotinylated linear peptides for the five participants were synthesized and used in a SPR-Biacore assay to measure the antibody binding profile of the plasma samples with the aim of determining the influence of V2 sequence changes on antibody responses, identification of antibody escape, and mapping the targeted response within the V2 region . All of In participant 40123, a V2 segment 2 variant Leu184Ile (peptide 2b) did not affect the antibody response compared to peptide 2a b. HoweveIn the case of participant 40265, the antibodies were directed mainly to segment 2 of the V2 region c. Singleenv sequences prior to and following super-infection. Peptide 2a’, derived from the super-infection V2 segment 2 sequence, showed robust binding responses from time of initial infection indicating that the V2 response in participant 40512 had heterologous specificity early in infection  time point . Based oTo determine the accessibility of the V2 region within the trimer in both closed a PDB IDb forms a, the V2env sequence in the first four weeks of infection. However, by the six-month time point, there existed variable but significant sequence diversity resulting in changes in the V2 region amino acid sequence. In participant 40265, targeted deep sequencing analysis detected a minor variant first at day 7 post-infection which remained detectable at low levels (1.1–4.5%) through 3 months post-infection. However, the minor variant contributed only marginally (1.1%) to changes in the Env region even after 6 months post-infection [env and the V2 region following the super-infection event.Prospective HIV-1 infection cohorts, such as RV217, provide an opportunity to assess humoral response in the setting of limited viral diversity during the early stages of HIV-1 infection. It also allows examination of how viral variants can escape the early antibody immune pressure. The continuous and early sampling schedule in the RV217 study  allowed nfection . It is lnfection , as oppoGiven the variation in V2 immune responses, we wanted to understand in detail and to de-convolute the V2 binding response. We divided the V2 region into four segments, and measured binding to a set of peptides with T/F sequence and other variants identified by the SGA sequencing. In general, there were two patterns of immune targeting, (i) a uniform response across the four segments of the V2 region, and (ii) a response targeted to the strand C segment (residues 165–186) of the V2 region. The peak of the immune response to the strand C segment varied between the participants, and while this study only analyzed five individuals, and a limited number of antigens based on the SGA sampling performed only from the plasma compartment, intriguingly, there was a significant correlation between the response to the strand C segment and response to the heterologous 92TH023 V2 region, indicating a relationship between heterologous V2 binding and autologous V2 segment 2 binding. There is no obvious sequence determinant that explains this relationship. In general, the immune response to the strand C region was not dramatically affected by sequence variation, with the exception of participant 40007, where a single V2 mutation caused escape variants that exhibited significantly reduced binding of the peptide. It is intriguing that mutations in the V2 region were observed only around the 6-month time point, despite mutations in other regions of the envelope that were observed earlier in infection. We hypothesize that the increase in V2-specific antibodies by week 24 post-infection probably caused the virus to mutate to escape the immune pressure from the antibodies. It was not possible to follow the responses at later time points as this participant began ART treatment.The V2 region is a significant target for the immune response and known determinant of neutralizing and non-neutralizing protective responses. Given recent structural information, we assessed V2 structural forms and viral sequence variation, in conjunction with our binding data to try to understand the binding responses. HIV-1 Env has significant plasticity, and structural characterization of the V2 region has shown that this region can exist either as a β-strand or a helical form and is targeted by antibodies in either conformation. There has been much supposition over the transition dynamics of the V2 region from β-strand to helical forms with residue 173 hypothesized to play a significant role in this transition. The recent structure of a V2 scaffold with antibody CAP228-16H indicates that the V2 region can form β-strand or helix structures despite similar or identical sequences . ComputaThe observations presented here indicate that residues 165–186 in the HIV-1 gp120 Env V2 are a key region targeted during acute infection, resulting in heterologous V2 recognition, and match the responses seen in the RV144 vaccine trial. Sieve mutations in the V2 region in the RV144 vaccine trial resulted in escape mutants. Viral mutation and variation in this region resulted in reduced humoral response in this acute HIV-1 infection study cohort. This is clearly seen in at least one participant, 40007 where a single amino acid mutation resulted in a significant conformational change and a loss of antibody binding. Despite this portion of the V2 region (residues 165–186) being consistently targeted, it also has significant structural plasticity, which may lead to immune evasion. Structural modeling indicated that this region is significantly occluded in the closed native, prefusion trimer, while accessible in the CD4-liganded open Env conformation. Since the V2 region is targeted in protective vaccine-elicited responses and early in natural HIV-1 infection where there is limited genetic variability, this region should be carefully considered for future vaccine design and subsequent immune responses.


The master pro-inflammatory cytokine, tumour necrosis factor (TNF), has been shown to modulate multiple signalling pathways, with wide-ranging downstream effects. TNF plays a vital role in the typical immune response through the regulation of a number of pathways encompassing an immediate inflammatory reaction with significant innate immune involvement as well as cellular activation with subsequent proliferation and programmed cell death or necrosis. As might be expected with such a broad spectrum of cellular effects and complex signalling pathways, TNF has also been implicated in a number of disease states, such as rheumatoid arthritis, ankylosing spondylitis, and Crohn’s disease. Since the time of its discovery over 40 years ago, TNF ligand and its receptors, TNF receptor (TNFR) 1 and 2, have been categorised into two complementary superfamilies, namely TNF (TNFSF) and TNFR (TNFRSF), and 19 ligands and 29 receptors have been identified to date. There have been significant advances in our understanding of TNF signalling pathways in the last decade, and this short review aims to elucidate some of the most recent advances involving TNF signalling in health and disease. Initially discovered as a serum factor which induced cell death in tumour cells1 and thought at that time to be a promising target as a cancer treatment, TNF was later realised to be a potential target for the treatment of inflammatory diseases, such as rheumatoid arthritis (RA)3, Crohn’s disease (CD)4, ankylosing spondylitis (AS)5, and psoriasis6. However, localised administration of TNF is used as an effective therapy via isolated limb perfusion in patients with melanoma with multiple in-transit metastases7.Since the identification of tumour necrosis factor (TNF) in 19758, albeit in smaller quantities.TNF is produced primarily by monocytes/macrophages, but a number of other cell types, such as T and B lymphocytes, mast cells, natural killer cells, neutrophils, fibroblasts, and osteoclasts, can also secrete TNF9. mTNF and sTNF both perform cellular functions mediated by either of its two receptors: TNFR1, expressed across all human tissues, and TNFR2, expressed primarily in immune cells, neurons, and endothelial cells11. mTNF functions as a ligand transmitting cell-to-cell interactions and, when bound to TNFR2 12, is able to induce a more potent response than sTNF; interestingly, mTNF has also been shown to function as a receptor by initiating a cell signalling cascade through outside-to-inside signalling13. TNFR1 and 2, though similar in their extracellular structures at the mTNF- and sTNF-binding sites, have distinct intracellular structures which bind to a number of adaptor proteins14. TNFR1’s cytoplasmic tail contains a death domain (DD), thereby allowing it to recruit the TNFR1-associated DD (TRADD)15; TNFR2, on the other hand, does not have an intracellular DD and recruits the TNFR-associated factor (TRAF) 1 and 2 proteins instead  and the induction of a cell survival response, TNFR1 is also capable of inducing a cell death response depending on prevailing physiological circumstances; however, the regulation of the two TNFRs is dependent on the cellular environment and is not fully understood  that is expressed on the cell surface, where it either continues to reside or is actively cleaved by TNF-converting enzyme to produce a 17 kDa 157-amino-acid soluble TNF (sTNF) form; sTNF is subsequently released and becomes detectable in the blood plasmainstead 16. Whererstood 17. The 22. TNF-mediated cell death is highly dependent on various cell death check points, which regulate the pro-survival pathway; when these check points are disrupted, TNF-mediated signalling occurs through the formation of complexes IIa, IIb, and IIc23. The ubiquitination status of RIPK1 is a critical determinant of whether a particular signalling pathway promotes cell survival, through complex I24, apoptosis (controlled cell death), via complex IIb26, or necrosis (uncontrolled cell death), through complex IIc28.Upon binding of TNF to TNFR1, TNFR1 undergoes a conformational change in its DD, whereby both TRADD and receptor-interacting serine/threonine protein kinase 1 (RIPK1) are recruited, leading to the formation of complex I and the initiation of cell survival via activation of the NF-κB pathway 20–22. 29. Ub retains seven lysine sites  at the N-terminal methionine (Met-1) and the C-terminal glycine site. The main sites of interest are K48 and K63, as they are essential for the activation of the NF-κB pathway30.TNF signalling is tightly regulated by post-translational ubiquitination, an essential mechanism for the regulation of many biological processes. Ubiquitin (Ub) chains are assembled in response to activation of the TNF receptors and then attached to target substrates to modulate protein function. The type of Ub chains is determined by their specific linkage type, which are assembled to generate distinct intracellular signals. Deubiquitinase (DUB) enzymes reverse the process of ubiquitination by hydrolysing Ub moieties from the modified protein substrates15, RIPK121, TRAF2 or 531, cellular inhibitor of apoptosis protein (cIAP) 1 or 232, and linear Ub chain assembly complex (LUBAC) (33. LUBAC is composed of three proteins: heme-oxidised iron regulatory protein (IRP) 2 Ub ligase 1 (HOIL-1), shank-associated RH domain-interacting protein (SHARPIN), and HOIL-1 interacting protein (HOIP). cIAP and LUBAC add K63-linked and Met1-linked polyUb chains, respectively, to RIPK1, stabilising the Ub structure and amplifying its signal35. This leads to the recruitment of transforming growth factor-beta (TGF-β)-activated kinase (TAK) 1 complex, consisting of TAK-binding protein (TAB) 2 and 3, and inhibitor of κB (IκB) kinase (IKK) complex, composed of the NF-κB essential modulator , IKKα, and IKKβ24. The TAK1 complex phosphorylates mitogen-activated protein kinase (MAPK), leading to a signalling cascade whereby c-Jun N-terminal kinase (JNK), p38, and AP1 transcription factors are activated, and the IKKβ activates NF-κB. This ultimately leads to pro-survival signalling, where inflammation and the proliferation of immune cells are induced24. Following the activation of NF-κB, cellular FLICE-like inhibitory protein long (cFLIPL) translocates to complex IIa in order to prevent caspase-8 activation. However, if the late NF-κB-dependent check point is disrupted and cFLIPL levels are consequently reduced, apoptosis is initiated via complex IIa  through auto-activation of pro-caspase-838. In contrast to the late check point, the early check point, which occurs immediately after ligand binding, is initiated by the ubiquitination of RIPK1 by cIAP and LUBAC (41.The activation of complex I relies on the ubiquitination of RIPK1 and consists of TRADD(LUBAC) 33. LUBAd LUBAC 39–41.42 and form complex IIb, whereby TRADD is replaced by RIPK3, upon degradation of cIAP1 and 243. This, in turn, leads to the cleavage of pro-caspase-8 to caspase-8, and activation of the caspase signalling cascade results in apoptosis  and the induction of a regulated form of necrotic cell death, necroptosis  enzyme deubiquitinates RIPK1, thereby allowing it to disassociate from complex Ioptosis . To ensuoptosis 27,28.46, indicating a regulatory role of the TNFR1 signalling pathway47. It is hoped that, through greater understanding of TNF signalling in health and disease, improved therapies can be developed to inhibit this potent cytokine in a more target-specific manner.TNFR2 interacts directly with TRAF1 or 2 to recruit cIAP1 or 2 to promote cell survival signalling through the formation of complex I and the induction of NF-κB, MAPK, and Akt, promoting cell proliferation and tissue regeneration. The binding of TRAF2 to TNFR2 is considerably weaker than TRAF2’s binding to TRADD49 and its inhibition of the interaction between LUBAC and IKKγ through binding to their linear Ub chains via A20’s ZF7 domain upon TNF stimulation51. A20, as well as the regulatory molecule TAX1BP1, has also been shown to inhibit the E3 ligase activities of TRAF2, TRAF6, and cIAP1 by interfering with E2 Ub enzymes Ubc13 and UbcH5c, disrupting NF-κB signalling52. In 2015, it was documented, in a mouse embryonic fibroblast (MEF) model, that binding of A20 to linear Met1-linked polyUb chains protected the MEFs from CYLD-mediated degradation. This was shown to be dependent on A20’s ZF7 domain but was independent of its DUB activity and protected cells from TNF-induced RIPK1-dependent apoptosis (RDA)53. Another DUB, ovarian tumour (OTU) DUB with linear linkage specificity (OTULIN), acts via its specific cleavage of Met1-linked polyUb chains after it binds to the HOIP component of the LUBAC54. CYLD is another negative regulator of the TNF signalling pathway, removing Ub chains from several proteins such as TRAF2, TRAF6, IKKγ, and RIPK1 to regulate the NF-κB and JNK pathways55.Throughout the TNF signalling pathway, there are a number of proteins that are essential for negative regulation of the pathway. A20 contains both DUB and E3 ligase domains and plays a vital regulatory role at multiple steps of the TNF signalling pathway, such as its removal of K48- and K63-linked Ub chains from RIP1 through its zinc finger (ZF) 4 domain56, termed iuRIPK1, which acts as an intermediate between complex I and cytosolic complex IIb formation and caspase activation. Using a systematic screen for RDA, the investigators found that iuRIPK1 is regulated by Parkinson’s disease-associated leucine-rich repeat kinase 2 (LRRK2), E3 Ub ligase, c-Cbl, and ALS-associated NEK1, suggesting a mechanistic link between RDA and neurodegenerative conditions56. It is well documented that, in addition to its Ub status, kinase activity of RIPK1 is essential for RDA and necroptosis, both being induced by TNF59. In 2018, Menget al. reported that C-terminal DD dimerisation of RIPK1, via K584, serves as an amplification mechanism to promote RIPK1 kinase activity and that increased expression, under pathological conditions, may promote its dimerisation and activation60. In light of these discoveries, the question arises as to whether detergent-insoluble lipid rafts are providing a platform for the assembly of cell surface signalling complexes, under certain pathological conditions, and thus might act as a critical determinant in driving RIPK1-mediated cell death61. Recent research has also found that RIPK1 prevents skin inflammation, via the inhibition of RIPK3-MLKL-dependent necroptosis, mediated by the cytoplasmic DNA sensor protein, Z-DNA-binding protein (ZBP1)63, and that deficiency of RIPK3, as well as an inactive form of RIPK3, reduces necroptosis and thereby the severity of inflammation in mouse models of tissue injury64.The ubiquitination status of RIPK1 is a critical determinant of whether a cell undergoes RDA; a recent study describes a “detergent insoluble, highly ubiquitinated and activated RIPK1 pool”65. A recently published report by Garcia-Carbonellet al.65 reported increased levels ofA20 gene expression in patients with irritable bowel disease (IBD), and intestinal epithelial cells have increased susceptibility to TNF-induced cell death. Similarly, stabilising the function ofA20, in the form of homodimers, facilitated ripoptosome complex (consisting of RIP1 and cIAP1 and 2) assembly by binding linear Ub chains via ZF7, protecting RIPK1 from deubiquitination, resulting in enhanced caspase-8 recruitment and activation65. A20 has also been shown to protect cells from necroptosis through the deubiquitination of RIPK3 in both T cells and fibroblasts66.Controversy hangs over the role of A20 DUB, which has been shown in separate studies to either protect or promote TNF-mediated RDA67. The loss of IAP induces not only the formation of complex IIb and stimulates RDA but also caspase-8-dependent interleukin-1 beta (IL-1β) maturation and NLRP3 inflammasome signalling in macrophages, in a TLR4/TRIF-dependent manner which is independent of TNFR169. Blocking caspase-8 activity, followed by interferon gamma (IFNγ) priming, rendered neutrophils sensitive to TNFR1-dependent necroptosis via the RIPK3-MLKL pathway, leading to NLRP3 activation70. The finding that cell death pathways are distinctly regulated in neutrophils, as compared with other myeloid cells, signifies the importance of cell type on immune signalling pathways in orchestrating a co-ordinated inflammatory response.Alongside their functional roles as E3 Ub ligases, the cIAPs are critical regulators of pro-inflammatory signalling pathwaysIn vitro anti-TNF blockage, used in T-cell monocyte co-cultures of patients with the autoimmune disorder thrombocytopenia, produced a robust proliferation of the immunomodulatory regulatory T (Treg) cells71; interestingly, this Treg cell expansion was dependent on TNFR2 and not TNFR1. Blockage of TNFR2 resulted in a robust expansion of Treg cells, whereas neutralisation of TNFR1 had no effect on this Treg cell expansion71. Therefore, TNFR2 might be considered a potential novel therapeutic target for immunomodulation, not only in thrombocytopenia but also in other unrelated immune disorders associated with decreased levels of Treg cells, such as RA, AS, systemic lupus erythematosus (SLE), IBD, and psoriasis72. For a recent review of anti-TNFR2 therapy, see Zouet al.73.TNF is known to have widespread and profound effects on both the activation and the proliferation of different subsets of immune cells in several disease states.+ T cells towards a regulatory phenotype with high expression of IL-10 and reduced IFNγ production74. Patients with cardiovascular disease often exhibit high levels of blood TNF, which are positively correlated with the formation of foam cells, and there are further repercussions for the development of plaques and blood clots75. These observations were supported by bothin vitro andin vivo experiments that confirmed that high levels of TNF enhanced the expression of adhesion molecules and scavenger receptors on blood monocytes75. TNF has an important role, not only in immune cells but also in the regulation of circadian rhythms by the central nervous system. One study reported that TNF stimulation of the suprachiasmatic nucleus exerted an important influence on the regulation of circadian rhythms, through the activation of TNFR1 after lipopolysaccharide (LPS) inoculation, mainly during the early period of the night, when TNFR1 showed its highest expression76. This regulation has novel implications for several disorders and might explain some of the observed disruption of circadian rhythms during disease78, perhaps due to higher expression of TNF in activated immune cells.Another report showed that inhibition of TNF signalling, by a number of anti-TNF biological treatments, primed naïve CD479 as well as to restrain the development of Treg cells, as A20-deficient mice present with enlarged thymic and peripheral Treg cell compartments80. A20 has also been shown to exert an important defence role against bacterial infections, as it enhances secondary CD8+ T-cell responses but reduces the primary response81. SHARPIN, a component of LUBAC, has a number of modulating effects on T cells; for example, defective SHARPIN results in a significant reduction in the overall population of Treg cells and their ability to function correctly82. Furthermore, deficiency of SHARPIN leads to reduced numbers of CD4+ CD25+ FOXP3+ Treg cells in the blood, spleen, lymph nodes, and thymus83. HOIL-1, which is another component of LUBAC, has been reported to be cleaved by mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1), leading to its becoming a potent inhibitor of LUBAC-induced NF-κB signalling in activated T cells87; other NF-κB regulatory proteins that are cleaved by MALT1 include A2088, RelB89, and CYLD90, not to mention the auto-proteolytic cleavage of MALT191.A20 has been shown to promote cell survival of CD4 T cells by initiation of autophagy via its inhibition of mammalian target of rapamycin (mTOR)92. TNF has a ubiquitous influence on different cells and tissues and has an important role in the tumour microenvironment. A recent publication reported that regulation of the immunomodulatory check point programmed death-ligand 1 (PD-L1) in tumour-associated macrophages and monocytes was strongly increased by TNF in a B16 melanoma mouse model of disease93. By using TNFR–/– mice (strain B6.129 S-Tnfrsf1atm1Imx Tnfrsf1btm1Imx/J), the researchers found a significant decrease in numbers of tumour-associated macrophages and dendritic cells expressing PD-L1 and an associated reduction in the size of the tumours93. In a separate study, TNF was reported to activate the NF-κB signalling pathway and upregulate PD-L1 in human prostate and colon cancer cells, thereby promoting immunosuppression and favouring the tumour microenvironment94. Immunotherapy has proven to be an effective option in the treatment of several cancers with high expression of PD-L1; thus, incorporating anti-TNF biologics into this therapeutic regimen may result in improved outcomes for certain types of cancers.A recent study focusing on innate immune cells showed the importance of TNF activation of these cells in cerebral tuberculosis, although neuron-derived TNF also plays a limited role96. It was later observed that obese rats expressed increased levels of TNF in their adipose tissue and that obese humans also expressed TNF at higher levels in their muscle tissue99. In obese rats, TNF inhibition led to improved metabolism of glucose and improved insulin sensitivity101; however, when TNF was administered, the opposite effects occurred103.The link between metabolism and immunity first became apparent in the 1980s, when macrophage-conditioned media, stimulated with LPS, was found to increase lipoprotein lipase expression and promote resistance to insulin in adipocytes105, thus showing that the metabolic state of immune cells helps regulate various immune responses. Additionally, the product of GAPDH, nicotinamide adenine dinucleotide (NADH), significantly inhibited TNF secretion in macrophages105.The essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), under low glycolysis conditions has been linked to regulation of the inflammatory response, as it binds to TNF mRNA in monocytes and macrophages and post-transcriptionally inhibits its production106. Immature monocyte-derived dendritic cells have a high expression of the extracellular succinate sensor, a G-protein-coupled receptor known as GPR91, and these cells were shown to have enhanced production of TNF due to succinate when stimulated with the TLR7 ligand imiquimod or the TLR3 ligand polyinosinic-polycytidylic acid (poly[I:C])106. Macrophages have also been recognised to upregulate their expression of GPR91 and release succinate into the extracellular milieu under inflammatory signals, which may lead to exacerbations of RA, as rheumatoid joints have high levels of succinate present107.The anti-inflammatory metabolite itaconate was found to inhibit TNF in an LPS-stimulated mouse model as well as in human peripheral blood mononuclear cells; however, no effects were observed in human macrophages108. Another glycolytic enzyme, alpha-enolase (ENO1), has been shown to have inflammatory effects when expressed on the surface of monocytes and macrophages by contributing to the production of inflammatory cytokines, such as TNF, in the synovium of patients with RA and also in the type II collagen model of arthritis109. ENO1 also plays a role in both the induction and the function of Treg cells via the promotion of Foxp3 splicing110.Cis-aconitate decarboxylase (CAD), the enzyme product of immune-responsive gene 1 (IRG1), catalyses the decarboxylation of cis-aconitate to produce itaconate, and this reaction is induced by heme oxygenase 1 (HO-1) activity. Carbon monoxide induction of HO-1 led to reduced TNF levels via upregulated IRG1 expression, demonstrating IRG1’s anti-inflammatory effects111, and another study demonstrated the importance of mitochondrial citrate carrier (CIC) in TNF-triggered inflammation, whereby the CIC is transcriptionally upregulated under TNF stimulation via NF-κB and plays an essential role in regulating the downstream production of nitric oxide and prostaglandins112. In macrophages, citrate accumulation is essential for fatty acid synthesis, in a murine model of type 2 diabetes, which enhances inflammatory signalling through TNF production114.A recent study showed that inhibition of complex II of the mitochondria reduces the production of TNF in macrophagesTNFRSF1A gene, encoding TNFR1, were found to be associated with episodic fevers and profound localised inflammation; consequently, TNFR1-associated periodic syndrome (TRAPS) was defined as one of the disease entities under the collective heading of autoinflammatory diseases115. The term “autoinflammation” was used to refer to the increasing number of clinical disorders characterised by episodes of seemingly unprovoked inflammation in the absence of high autoantibodies titres or antigen-specific T lymphocytes. Anti-TNF therapy was used for the treatment of TRAPS with some initial success; however, a large number of these patients eventually become resistant to this treatment117, and an increasing number of reports subsequently revealed the clinical benefits of anti-IL-1β and/or anti-IL-6 therapy in the forms of canakinumab and tocilizumab, respectively123. The current standard treatment for the more severe cases of TRAPS is IL-1β blockade.Although TNF plays an essential role in health, it also exerts a double-edged sword effect in several disease states. Indeed, in 1999, mutations in the124. The DUB OTULIN is an essential negative regulator of inflammation and prevention of autoimmunity involving the TNF pathway. Genetic alterations of DUBs have been associated with neurodegenerative diseases and cancers125; also, two unrelated monogenic systemic inflammatory diseases have been associated with defective DUBs124. Otulipenia is caused by loss-of-function mutations in the linear (Met-1) OTULIN with resulting dysregulation of the deubiquitination process127. Increased signalling in the canonical NF-κB pathway with overproduction of TNF, IL-1β, IL-6, IL-12, IL-18, and IFNγ in response to LPS stimulation was reported in these patients. Anti-TNF therapy was found to normalise markers of active inflammation, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and control disease activity in otulipenia124.Recently, the scope of autoinflammation has been broadened to encompass some abnormalities of deubiquitination and redox homeostasis under the name of ubiquitination-associated autoinflammatory diseases (UADs). There are a number UADs, but only two are known to directly affect the TNF signalling pathway, namely otulipenia/OTULIN-related autoinflammatory syndrome (ORAS) and A20 haploinsufficiency (HA20)TNFAIP3 gene, which codes for A20, result in the UAD disorder, HA20, so named because of loss of function of one of the copies of theA20 gene, leading to early onset systemic inflammation and a disorder resembling Behçet’s disease128; for a recent review of HA20, see Kadowakiet al.129. Polymorphisms in TNFAIP3 have also been linked to RA, SLE, psoriasis, and CD susceptibility through genome-wide association studies136. A20 function has also been associated with a protective role in RA, as it negatively regulates NLRP3 inflammasome activity137.Loss-of-function mutations in the138. This differential effect in cell death pathway aetiology provides clues to the benefits of various treatment pathways. Interestingly, patients with LUBAC-inactivating germline mutations, such as HOIL-1 and HOIP mutations, also have primary immunodeficiency and autoinflammation141. Expression of TNFR1 in peripheral blood mononuclear cells from patients with RA has been positively correlated with the efficacy of therapies received142; furthermore, the expression of both TNFR1 and 2 is downregulated in B cells of patients with RA compared with healthy controls142.Keratinocyte-specific deletion of the LUBAC components HOIP or HOIL-1 results in a lethal inflammatory skin disease, caused by TNFR1-induced caspase-8-mediated apoptosis, that occurs independently of RIPK1 kinase activityThe identification of TNF and the development of anti-TNF therapies for associated disorders were revolutionary moments in disease research, resulting in vast improvement in the quality of life for patients with these debilitating diseases. With the ever-expanding understanding of TNF and its signalling pathways, it is hoped that the new research focus on combination therapies, and not just anti-TNF, will further improve these patients’ quality of life.


The incidence of heart failure with preserved ejection fraction (HFpEF) is increasing and its challenging diagnosis and management combines clinical, imagistic and biological data. Natriuretic peptides (NPs) are hormones secreted in response to myocardial stretch that, by increasing cyclic guanosine monophosphate (cGMP), counteract myocardial fibrosis and hypertrophy, increase natriuresis and determine vasodilatation. While their role in HFpEF is controversial, most authors focused on b-type natriuretic peptides (BNPs) and agreed that patients may show lower levels. In this setting, newer molecules with an increased specificity, such as middle-region pro-atrial natriuretic peptide (MR-proANP), emerged as promising markers. Augmenting NP levels, either by NP analogs or breakdown inhibition, could offer a new therapeutic target in HFpEF  by increasing the deficient cGMP levels found in patients. Importantly, these peptides also retain their prognostic value. This narrative review focuses on NPs’ physiology, diagnosis, therapeutic and prognostic implication in HFpEF. The incidence of heart failure (HF) is increasing. If for HF with reduced ejection fraction (HFrEF) there are well-established methods of diagnosis and treatment, this is far from true in HF with preserved ejection fraction (HFpEF) patients. This increasing incidence justifies the need for proper diagnostic, therapeutic and prognostic tools. In the era of cardiac biomarkers, natriuretic peptides (NPs) have a well-established role in HF pathophysiology and patient management. However, both the lack of consensus regarding NPs in HFpEF and the heterogeneous population makes diagnosis and management difficult and unstandardized, leading to a decrease in quality of life and increase in mortality and hospitalization.Heart failure represents a clinical syndrome affecting 2%–3% of the general population . Its preOne of the classifications of HF is made by determining the left ventricular ejection fraction (LVEF). As such, HF can be either with preserved ejection fraction, HFpEF (LVEF > 50%), or reduced ejection fraction, HFrEF (LVEF < 40%), and patients with an LVEF between 40% and 50% are labeled as HF with mid-range EF-HFmrEF . The EurBiomarkers reflect myocardial damage and stress; systemic inflammation and fibrosis and their routine measurements mirror myocardial structural disease . NatriurEssential hypertension and myocardial ischemia are frequent causes of HFpEF and one third of these patients have concomitant atrial fibrillation (AF) . The preThere are three endogenous NPs secreted as pre-prohormones: atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). Subsequently, there are three natriuretic peptide receptors (NPR): natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C or clearance receptor) ,14,15,16Biologically active ANP is a 28 amino acid (aa) peptide. Initially secreted as pre-proANP (151 amino acids), this molecule is cleaved into proANP (126 amino-acids), which is deposited in granules inside atrial myocardium. A transmembrane protease cleaves the secreting proANP into its biologically active short-lived form (ANP- 28 amino acids) and its inactive form, NT-proANP (98 amino-acids) ,11. The The majority of ANP is secreted in the atria in response to myocardial stretch, atrial concentrations being 1000 higher as compared to ventricular ANP levels . VentricThere are three forms of circulating ANP: αANP, βANP and pro-ANP . Found oANP has numerous biological effects, including blood pressure lowering effects and renin-angiotensin-aldosterone system (RAAS) inhibition ,18,19,20Recent studies focusing on AF patients found that NT-proANP levels correlate with AF type and LA dimensions . SeewostThe gene coding ANP contains three exons: the first exon codes the 5′ region (not translated), a signal peptide formed of 25aa (16aa of proANP). The second exon is responsible for coding proANP while the third exon has a role in coding terminal 3′ tyrosine .The biologically active form of human BNP is BNP32 , widely Several studies identified higher BNP concentrations at the level of anterior interventricular vein and coronary sinus, which supports the fact that BNP is mainly secreted by ventricular myocardium . The difImportantly, it seems that HF patients have decreased amounts of BNP32; the latter is cleaved to either BNP3-32 or BNP8-32 by dipeptidyl peptidase IV (DPP IV). The two forms are less biologically active than BNP32, probably due to faster degradation and may account for the presumed resistance to NPs in HF patients ,16.O-glycosylation of proBNP inside the Golgi apparatus is multi-sited (approximately seven sites) and its degree may vary among patients. Heavily glycosylated pro-BNP molecules are recognized as a cause of decreased conversion to its biologically active form, with a subsequent increase in pro-BNP/total BNP ratio. Interestingly, in acute HF the glycosylation level of proBNP decreases as there is a tendency towards forming more mature BNP. In acute HF, the increased proBNP production is accompanied by decreased glycosylation and increased furin activity, leading to elevated BNP and NT-proBNP levels.Both ANP and BNP can undergo post-translational changes, such as phosphorylation and glycosylation. The physiological impact of a phosphorylated proANP form is still uncertain . As one 53 is cleaved in return to CNP22. Both forms have the same functions; however, CNP53 is found in the myocardium while CNP22 is found in plasma and brain [C-type natriuretic peptide is secreted in the myocardium, endothelium, chondrocytes, brain and blood cells . C-type nd brain . CNP alsNPR-A and NPR-B determine NP functions. ANP and BNP activate NPR-A while CNP activates NPR-B ,14,15,16NPR-C is found in the brain, atrium, lungs and aorta. These receptors have an extracellular region for the ligand attachment and a intracellular region with a cGMP dependent proteinkinase ,22.sGC  and particulate guanylyl cyclase (pGC), found in the cellular membrane and activated via NPR. As such, this activation leads to an increase in cGMP, which in turn increases protein-kinase G (PKG) levels [As a second messenger, cGMP is formed from two possible precursors, either soluble guanylyl cyclase- ) levels ,24,25,26NPs have a wide range of biological effects, including endocrine and paracrine. They promote diuresis and natriuresis, vasodilation and inhibit sympathetic nervous system and renin-angiotensin-angiotensinogen. In advanced HF there is a resistance to the effects of NPs, together with either an increased turnover, increased biologically inactive NP secretion or decreased NPR-A activation due to the dephosphorylation of secondary receptors ,2,3,4,5.If ANP levels are influenced by atrial pressures, BNP concentrations are determined by ventricular stretch in response to underlying pressure and/or volume overload. ANP’s short half-life (derived from its higher affinity for NPR-C) precludes its utilization in routine practice while BNP’s stability makes it a desirable biomarker in HF diagnosis and prognosis .A novel NP, middle-range proANP (MR-proANP) has emerged as having a greater stability and both diagnostic and prognostic values, especially in HFpEF patients. It derives from an intermediate region of NT-proANP and exhibits increased stability. Moreover, it correlates with increased LA dimensions and it seems that its diagnostic  and progThe degradation of NPs can be either receptor- mediated (NPR-C) or enzyme-mediated ,7,8,9,10Diagnosing HFpEF remains difficult due to a lack of consensus, patients’ heterogeneity and multiple concurrent pathologies that may mimic not only HF symptoms, but also lead to either increased (AF) or decreased (obesity) NP levels . HFpEF dThe longer plasma half-life of BNP and NT-proBNP  as compared to ANP (2 min), makes the two the preferred NPs for guiding HF diagnosis and, probably, therapy. Moreover, despite having a longer half-life, NT-proANP failed to emerge as a diagnostic marker due to the increased number of cleaved fragments that limit its detection. European Society of Cardiology considers a BNP level of > 35 pg/mL and/or NT-proBNP > 125 pg/mL suggestive of chronic heart failure, with higher values being recommended in acute settings- over 100 pg/mL and > 300 pg/mL, respectively ,33,34,35Several studies agree that both BNP and NT-proBNP retain their diagnostic performance  in acute heart failure, irrespective of LVEF . HoweverRecent studies pointed out the need of different thresholds in HFpEF in regard to sinus rhythm patients. HFpEF and AF coexist in 30% of patients  while AF2 for men and 95 g/m2 for females), E/e’ >13 and mean septal and lateral wall e’ of <9 cm/s [The use of different diagnostic methods in adjunction with increased NPs level is recommended by ESC guidelines. Structural and functional alterations as determined by transthoracic echocardiography include a left atrial indexed volume (LAVI) of >34 mL, increased LV mass index  [Recently, a new NP has emerged as a potential HFpEF diagnostic tool. Cui et al. have revealed increased MR-proANP levels in HFpEF patients, with a significantly higher AUC when compared to NT-proBNP  . Moreove <0.001) . The aut <0.001) .Not only have NPs proven their diagnostic utility, several studies have questioned their ability to identify patients at risk for HF development. The STOP-HF trial referred patients with BNP levels of > 50 pg/mL to further echocardiographic investigations, leading to a decrease in LV dysfunction .Given its difficulties, the diagnosis of HFpEF in its characteristically heterogeneous population usually requires more than one biomarker. As the separation of HF in reduced and preserved EF occurred rather recently, more studies focused on HFrEF. Although both European and American guidelines have yet to consider different thresholds for the two classes of HF, increasing evidence links their distinct pathophysiologies to unique therapeutic strategies. Moreover, taking into consideration the increasing HFpEF incidence and altered prognosis, proper identification of these patients become vital.NPs have been shown to inhibit RAAS, suppressing angiotensin II mediated vasoconstriction, sodium reabsorption  and aldosterone, endothelin and renin secretion ,9. TheirThe rationale behind using NPs as a therapeutic target in HF therapy ,60,61,62The use of recombinant NPs in acute HF is recommended by ESC , but witA different NP used in HF treatment is carperitide, a recombinant form of ANP. Although recommended in Japan for acute HF therapy, it has failed to enter either ESC or American guidelines, especially because of limited studies and decreased half-life ,51. DespAttempts have been made to produce variants by the means of native NP gene alteration, resulting in NPR-A binding peptides resistant to degradation with subsequent increased half-life ,5. SeverAnother possible therapeutic strategy is inhibiting the breakdown of NPs, thus promoting their biological effects ,79,80,81There are several drugs that inhibit NEP . Pure NEA different strategy that has been attempted targets dual endothelin and NP breakdown inhibition. It seems that endothelin-1 (ET-1) and angiotensin II share common pathophysiological mechanisms. Although ET-1 impacts HF progression through vasoconstrictor and fibrotic effects, antagonizing its receptors have failed to show prognostic effects in HF patients . HoweverSacubitril/valsartan (formerly known as LCZ 696) combines a NEP inhibitor (sacubiril) with an angiotensin receptor blocker  in a 1:1 ratio, being the first in its class (ARNI). Its benefits come from combining neprilysin inhibition, which limits NPs breakdown and leads to a subsequent rise in both their levels, and cGMPs with RAAS inhibition given by valsartan ,74. AmonSeveral studies support the sacubitril/valsartan treatment class IB indication in HFrEF patients ,75,76,88Regarding cGMP and sacubitril/valsartan therapy, there is extensive focus on its beneficial effects on cGMP levels and, subsequently, PKG. cGMP deficiency is of paramount importance in HFpEF, as it lowers PKG levels and thus promotes myocardial remodeling (both hypertrophy and impaired relaxation through increased cardiomyocyte resting tension) . ThroughThe PARAMOUNT trial assessed the benefits and safety profile of sacubitril/valsartan treatment in HFpEF patients . NT-proBRecently, the baseline characteristics of patients enrolled in PARAGON-HF trial, which assessed the impact of sacubitril/valsartan therapy in HFpEF on mortality, have been published . 4822 paβ concentration as NEP is responsible with enzymatic clearance of amyloid, thus augmenting the risk of developing Alzheimer’s. An ongoing trial will assess the impact of the novel drug as compared to valsartan treated HFpEF patients on cognitive impairment, with results being scheduled for 2022 [An interesting possible side-effect of sacubitril/valsartan therapy may be an increase in amyloid-for 2022 .Apart from novel therapies, several studies investigated the benefits of mineralocorticoid receptors antagonists (MRAs) therapy in HFpEF ,90,91,92Patients with HFpEF present increased fibrosis biomarkers, as shown by Zile et al.  and OikoIt appears that HFpEF with lower matrix metalloproteinase 9 levels benefit most from eplerenone treatment . Chen etIt is agreed upon that although NP values tend to be lower in HFpEF patients with no consensus regarding diagnostic thresholds, they retain their prognostic utility irrespective of LVEF ,87.In a different study, increased BNP levels were associated with increased mortality, irrespective of LVEF . Also, iThe NPs concentrations retain their prognostic utility albeit their lower baseline levels in HFpEF patients . SeveralIt appears that different NPs concentrations threshold might be necessary for prognosis in HFpEF patients as compared to HFrEF ,105,106.Anand et al. associated a baseline NT-proBNP of 339 pg/mL with a four year mortality of 21.1%, which translates into a 5% annual mortality for an NT-proBNP level between 300–500 pg/mL .As NT-proBNP is also influenced by AF , increasA recent study launched the possibility of increased NT-proBNP playing a causative role in AF . This idAnother emerging prognostic biomarker is MR-proANP . It seemThere are controversies regarding the prognostic value of NPs in sacubitril/valsartan treated patients. As the formerly known LCZ696 interacts with NPs levels, there still are questions regarding their accuracy in determining HF patients’ prognosis. Some studies agree that both BNP and NT-proBNP retain their prognostic utility in these patients, emphasizing the fact that during therapy initiation, NT-proBNP is the preferred biomarker  . HoweverAnother issue regarding NPs prognostic value in HFpEF is whether these patients would benefit from NPs guided therapy. So far, studies are controversial ,126,127,Recent studies have focused on MR-proANP as a possible biomarker to guide HF therapy. So far, it seems that MR-proANP levels might be able to predict incident HF, as it shown in a study conducted by Sabatine et al. . InteresRegardless of the biomarker used, NP guided medical therapy remains controversial, especially in HFpEF patients. In these patients, more studies are required to assess the benefits of optimizing medical therapy after serial NP measurements. The most studied NPs are BNP and its inactive form, NT-proBNP, especially due to their proven diagnostic capacities. The novel MR-proANP, with its increased half time as compared to ANP, shows promise both in prognosis and guiding medical therapy, but more studies are required to confirm its safety profile and utility.Several on-going studies on NPs in HFpEF have been announced. The results of PARAGON-HF trial will clear the impact of sacubitril/valsartan therapy in HFpEF , while aMoreover, in the context of the pro-inflammatory state of HFpEF, there is increasing body of evidence linking non-NPs biomarkers to HFpEF as a better diagnostic tool . EmerginThe number of HFpEF patients is increasing; moreover, these patients show similar prognosis with HFrEF patients.The role of NPs in HF is both diagnostic and prognostic. Guidelines regard a BNP > 35 pg/mL or an NT-proBNP > 125 pg/mL as being diagnostic in non-acute setting, while emphasizing the fact that although several factors alter NP concentrations, their diagnostic utility rather lies in their negative predictive ability. For decompensated HF, either a BNP > 100 pg/mL, an NT-proBNP > 300 pg/mL or a MR-proANP of > 120 pmol/L is diagnostic. Authors agree that lower levels may be considered for HFpEF, without any thresholds being agreed upon. The absence of a consensus regarding diagnostic thresholds for NPs in HFpEF has led to inhomogeneous inclusion criteria in large clinical trials, thus affecting the results.The role of NPs extends beyond diagnosis, as they can be regarded as a therapeutic target per se through nesiritide and the novel ARNI, sacubitril/valsartan. Augmenting the concentrations of NPs leads to lower blood pressure levels as well as an improvement in symptoms and quality of life. A degree of reverse remodelling with a subsequent decrease in myocardial fibrosis and LA dimensions could be determined.Higher NPs levels are associated with poorer prognosis, especially in acute settings, irrespective of LVEF. Moreover, BNP and MR-proANP predict not only incident HF, but also AF. As AF is one of the most frequent causes of HFpEF, this becomes of the utmost importance. For AF patients it must be remembered that NP levels tend to be higher, irrespective of the presence of HF. Also, for the same BNP concentration, they show a better prognostic as compared with their sinus-rhythm counterparts.Finally, guiding medical therapy by serial NP measurements is controversial, especially in HFpEF. Authors recommend in the absence of consensus that no change in patient management should be taken if the NP levels remain constantly elevated throughout treatment.The need for further studies assessing diagnostic NP thresholds and prognostic values in HFpEF patients is enormous. Furthermore, given the novel therapies that interact with NPs, such as sacubitril/valsartan, these levels might be altered.


Tumor necrosis factor-α (TNF-α) antagonists, most of which are monoclonal antibodies, became a widespread treatment for autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel diseases, psoriasis, psoriatic arthritis, hidradenitis suppurativa and uveitis. Their use is based on the blockage of TNF-α, which plays an important role in granulomas formation, development of phagosomes, activation and differentiation of macrophages, immune response against viral pathogens. The multiple adverse effects of TNF-α inhibition have been identified, including a two-to four-fold increased risk of active tuberculosis and other granulomatous conditions and an increased occurrence of some other serious bacterial, fungal and certain viral infections.A 34-year-old male patient with disseminated varicella and pneumonitis was admitted to our hospital. The diagnosis of varicella was established serologically by enzyme immunoassay (EIA) and by polymerase chain reaction confirmation of the virus in vesicular fluid. The patient has been receiving adalimumab and methotrexate for the last 3 years due to ankylosing spondylitis and was seropositive to varicella zoster virus prior to the introduction of TNF-α antagonists. Acyclovir was administered for 10 days with the resolution of clinical illness and radiological signs of pneumonitis.Due to the use of biological agents, particularly TNF-α inhibitors, as a well-established therapy for some autoimmune diseases, new potential adverse events can be expected in the future and we wanted to point out one of them. To our knowledge this is the first case of recurrent disseminated varicella in a patient taking TNF-α antagonists. Tumor necrosis factor-α (TNF-α) antagonists, most of which are monoclonal antibodies , became a widespread treatment for autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel diseases, psoriasis, psoriatic arthritis, hidradenitis suppurativa and uveitis. Their use is based on the blockage of TNF-α, which plays an important role in granulomas formation, development of phagosomes, activation and differentiation of macrophages, immune response against viral pathogens –3. AdaliHowever, multiple adverse effects of TNF-α inhibition have been identified, including a two-to four-fold increased risk of active tuberculosis and other granulomatous conditions and an increased occurrence of some other serious bacterial, fungal and certain viral infections –6.We report a case of a 34-year-old patient with a medical history of fever, malaise, cough, and generalized vesicular rash that started 1 day before admission. 14 days prior to disease onset, the patient’s son developed chickenpox. The patient had a history of ankylosing spondylitis and has been treated with adalimumab 40 mg subcutaneously biweekly in addition to methotrexate10 mg per week for the last 2 years. He had had chickenpox at the age of 5, and positive IgG antibodies  to varicella-zoster virus (VZV) using EIA in 2014, prior to the initiation of adalimumab treatment.On admission to the hospital, physical examination revealed a subfebrile (37.3 °C) patient with papular and vesicular rash over the entire body Fig. . LaboratPatients receiving TNF-α antagonists therapy remain at a selectively increased risk for more severe primary varicella infections compared with the general population, with the estimated incidence rate of hospitalization due to chickenpox of 26 cases per 100,000 (95% CI 10–69) compared with the expected rate of 1.9 (95% CI 1.8–2.0) in the general population . ContradSeveral large population-based studies have been conducted, with those performed in the United States not finding an increased risk with TNF-α inhibitors, contrary to those in Europe generally showing an increased risk , 8, 9. TDue to the use of biological agents, particularly TNF-α inhibitors, as a well-established therapy for some autoimmune diseases, new potential adverse events can be expected in the future and we wanted to point out one of them. To the best of our knowledge, this is the first case of confirmed recurrent varicella with disseminated disease and pneumonia in a patient receiving adalimumab since this drug has been approved for use in 2002 in the United States and 2003 in the European Union , 23.


Heart failure with preserved ejection fraction (HFpEF) is an increasingly studied entity accounting for 50% of all diagnosed heart failure and that has claimed its own dignity being markedly different from heart failure with reduced EF in terms of etiology and natural history . Recentl The burden of heart failure nowadays is constantly increasing and even if the most presentation is still characterized by the reduction in ejection fraction, the phenotype of heart failure with Preserved Ejection Fraction (HFpEF) is becoming constantly more common, mainly because of the improvement in diagnostic tools, the higher clinical awareness, and the steady increase in life expectancy. Accordingly, HFpEF now accounts for about 2.5 million cases in the United States, which means almost half of all the recorded heart failure diagnosis. Although the prevalence of this condition is growing worldwide, its complex pathophysiology is yet to be fully elucidated. For many years, it was simply considered a mere problem of myocardial stiffness, mostly due to chronic hypertension and increased circulatory demand, with elevated left ventricular filling pressures eventually resulting in concentric hypertrophy and consequent diastolic dysfunction . NonetheHFpEF nowadays accounts for approximately half of the heart failure (HF) cases with incidence trends showing that it might become the most prevalent form of HF. In the general population, incidence of HFpEF shows a marked imbalance in sex prevalence , with elWithin the considerably wide spectrum of HF presentations, HFpEF undoubtedly covers the slice where the diagnosis is most challenging. Recognizing HFpEF may be complex since clinical manifestations are not specific, requiring a high degree of clinical suspicion. Indeed, the strong multimorbidity background in HFpEF often imposes diagnostic dilemmas because HF-like symptoms might be attributed to non-cardiac concomitant causes and diseases. Moreover, primary HFpEF must be distinguished from diastolic dysfunction not directly arising from myocardial disease but secondary to bystander cardiac conditions  .It has lately been recognized that patients with obesity, decreased exercise capacity, increased cardiac filling pressures, and normal left ventricular ejection fraction (LVEF) do not suffer from a homogeneous disorder but might present with at least three phenotypes of heart failure that should be clinically differentiated since they might have different response to available therapeutic armamentarium .According to 2016 ESC guidelines on HF diagnosis and treatment, diagnosis can be made in the presence of symptoms and/or signs of heart failure, a LVEF ≥50%, increased levels of natriuretic peptides and either the presence of structural heart disease and/or diastolic dysfunction.According to the ESC guidelines, the two recommended biomarkers are N-terminal pro B-type natriuretic peptide  and brain natriuretic peptide  .NT-proBNP strongly pairs the increase in LV diastolic filling pressure , has a vNonetheless, none of the cut-off values considered can discriminate between the two types of HF (HFrEF vs. HFpEF). Recently, galectin-3 and soluble ST-2 are showing promising results mostly in terms of outcome prediction in HFpEF with the first one having received a class IIB recommendation for risk stratification in 2013 ACC/AHA guidelines. Additionally, several circulating molecules  are currently being investigated in order to evaluate the possible clinical and prognostic implication. So far, contradictory results have been produced possibly due to the different contexts and the heterogeneous HFpEF pathophysiology .2 or a left ventricular mass index (LVMI) ≥115 g/m2 for males and ≥ 95 g/m2 for females. Functional echocardiographic abnormalities are mostly focused on the signs of diastolic dysfunction such as the ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e′) being equal or higher than 13 and/or mean septal and lateral wall mitral annular early diastolic velocity (e′) being <9 cm/s. Other indirect functional abnormalities that can aid in the echocardiographic diagnosis of HFpEF are abnormalities in the global longitudinal strain (GLS) or elevated tricuspid regurgitation velocities (TRV), whereas speckle tracking echocardiography has been shown to provide early signs of LV diastolic dysfunction in a large animal model of metabolic dysfunction  >34 ml/mfunction . In the function . Moreovetiffness .Since the original description of HFpEF, the relation between the clinical syndrome and the presence of comorbities has been described: epidemiological studies and prospective clinical trials showed consistently that hypertension, especially in the elderly, and irrespective of the administration of blood pressure medications  was assoCertainly, the high workload weighting on the heart of hypertensive patients triggers a structural remodeling which can be an important determinant of diastolic failure, but the long-lived “mostly mechanic” paradigm of increased afterload as the major, if not the only, mechanism underlying the pathophysiology of this condition has been recently challenged. In recent studies, hypertension-related diastolic dysfunction was already contextualized in a more “systemic” view, with the evidence of paracrine and autocrine signaling of the renin-angiotensin-aldosterone system (RAAS), involved both in inappropriate blood pressure elevation and reactive ventricular hypertrophy, myocardial fibrosis, vascular inflammation, and dysfunction . In 2013This new paradigm, having the microvascular dysfunction at the center of the disease evolution, has gained support over years. Recently, Graziani and Crea proposed an innovative theory that identifies microvascular dysfunction as the “common soil” for the occurrence of both microvascular angina and HFpEF   rather than elevated arterial pressure, suggesting that comorbidities could somehow blunt heart function in a more complex way than previously thought  pathway comprising cyclic guanosine monophosphate (cGMP) and activated protein kinase G (PKG) . DiabetiThis proinflammatory milieu implies elevated levels of IL-6, TNF-α, sST-2, pentraxin-3 and changes in endothelial adhesion molecular phenotype with the increased expression of vascular cell adhesion protein 1 (VCAM-1) and E-selectin which together promote monocyte migration . EventuaParvovirus B19 infection of these cells and impaired diastolic function with poor response to acetylcholine tests has been observed  comprises structural and functional abnormalities affecting heart microcirculation in different clinical conditions, not only as a mere epiphenomenon, but also as a pathogenetic factor and prognostic marker. The first acknowledgment of microvascular role in heart disease came from the observation that almost 50% of patients with stable angina and 10% of patients presenting with myocardial infarction do not have obstructive epicardial CAD: in both cases, CMD could be the hidden culprit causing symptoms. Camici and Crea have classified CMD on the basis of clinical context in which it occurs, obtaining four main types: (1) CMD in the absence of obstructive CAD and myocardial disease; (2) CMD in the presence of myocardial disease; (3) CMD in the presence of obstructive CAD; (4) iatrogenic CMD .per se to drive HFpEF evolution  frame count and myocardial blush grade in HFpEF patients without overt CAD . FurtherTherefore, physiology behind the increase in IMR and reduction in CFR probably rests on both the rarefaction in capillary concentration and the reduced bioavailability of NO .The obvious consequence of microvascular dysregulation is the presence of areas of local chronic ischemia. Accordingly, several studies proposed latent ischemia as the center of a vicious circle in which reduction in oxygen delivery impairs myocyte relaxation, whereas diastolic dysfunction promotes intramyocardial tension, increasing oxygen consumption demands . AdditioN2BA to the stiffer isoform N2B  stiffness in HFpEF occurs primarily due to the changes in sarcomeric titin. In this context, an isoform shift, from the more compliant isoform form N2B , has beeform N2B , leads tform N2B .+/Ca2+ exchange activity resulting in excessive cytosolic calcium load during the phase of diastole , induced by TGF-β  pathway ignaling . EndoMT ignaling  and the ignaling : when Enignaling . Interesignaling . It has ignaling .An important role in inflammation and microvascular dysfunction may be related to the accumulation and inflammation of epicardial adipose tissue that ultimately leads to fibrosis . In chroThe latest frontier in the pathophysiology of HFpEF involves the role of miRNA in inflammation and endothelial dysfunction: miRNA 146 and 155 seem to be relevant for the inflammatory cytokine production and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation. miRNA 126 is instead implied in the loss of endothelial homeostasis and vascular integrity, that leads to “leaky vessels,” correlated to VCAM expression and increased monocyte recruitment .It has been proposed that increased insulin signaling might exert adverse effects on cardiac remodeling, vasculature, kidneys, and adipose tissue, thus predisposing an individual with diabetes to HF and HFpEF in particular . ClinicaObstructive sleep apnea (OSA) is known to be marked by sleep disturbance, chronic intermittent hypoxia, hemodynamic changes, and sympathetic activation and this might contribute to development or sustaining of the HF . Accordi2max , have been consistently shown to be less effective in HFpEF. Cleland et al. suggested that beta-blockers conferred similar beneficial effects in patients with HFrEF and those with midrange LVEF (HFmrEF) phenotype, while these effects were absent in patients with preserved LVEF implicating that different targets might exist in patients with preserved vs. impaired systolic function (HFmrEF being a milder form of systolic dysfunction) . With miGiven the breakdown in NO metabolism is likely to be the cause of endothelial dysfunction in HFpEF, it is reasonable to propose that organic NO donors could be a potentially useful therapeutic tools . UnfortuRedfield and colleagues showed in the NEAT-HFpEF trial that isosorbide mononitrate (ISMN) did not improve but, rather, tended to reduce the total physical activity level with no improvement in submaximal exercise capacity. This evidence conflicts with the microvascular hypothesis of HFpEF, but it must be noticed that in this cohort of patient, there was a high incidence of hypotension, a common adverse reaction to this drug class, which could have limited effort tolerance in enrolled patients which are extremely sensitive to alterations in their hemodynamic state .On the other hand, the inorganic form of NO donor, nitrite, seems to have some benefits, improving arterial stiffness, diastolic LV pressures, and pulmonary artery pressure during exercise . Trials Therefore, although inorganic NO donors seem like an attractive therapeutic option in tackling LV stiffness in HFpEF, major limitations in their use currently exist, such as tachyphylaxis, impaired bioactivation of nitrates group, and the important preload reduction secondary to the peripheral vasodilation exerted by these agents.risk-reward ratio of previously described drugs, research focused on direct stimulation of NO-activated guanylyl cyclase (GC) by the experimental drug BAY41-8543, with preliminary data on animal model observing the beneficial role in terms of morbidity and mortality in HFpEF; interestingly, these advantages were independent of hypertrophy reversal , enzymes responsible for cyclic GMP (cGMP) degradation. Ideally, suppressing PDEs activity would thus increase cGMP levels independently from NO stimulation, thereby leading to improved microvascular regulation. Clinical hopes were high when it was documented that a PDE-5a inhibitor administered to an animal model of HFpEF improved LV relaxation and reduced diastolic LV stiffness . HoweverThe fortune of PDE inhibitors resurged when PDE-9 inhibition proved to be superior to the inhibition of other, more widely known isoform. Indeed, upregulation of this protein was seen in LV biopsies in HFpEF patients and its blockage in HFpEF animal models regressed hypertrophy, fibrosis, and vascular dysfunction. Likely, the key of the superiority of PDE-9 inhibition compared to PDE-5 inhibition could be in the ability to modulate cGMP signaling independently from NO pathway, whereas PDE-5 inhibition still requires active synthesis of NO . It has Statins have gained the attention of researchers for both their well-known anti-inflammatory action and their pleiotropic effects on endothelial homeostasis that went beyond established cholesterol-lowering effects. Endothelial effects of statins mainly consisted of restoring NO downstream signaling and improving endothelium-dependent relaxation through the amelioration of redox balance. The molecular benefit of statin-treated HFpEF patients has been highlighted by endomyocardial biopsy data showing less myocardial nitrotyrosine presence, higher myocardial PKG activity, less cardiomyocyte hypertrophy, and lower cardiomyocyte resting tension . Recent + current and therefore may minimize intramyocyte Na+ accumulation and the resultant Ca2+ overload in HFpEF  share many similarities with microvascular dysfunction being a fundamental part of both disease evolution . TherefoPhysical activity can mitigate the effects of excessive oxidative stress and reduce the level of total ROS produced, by slowing NADPH oxidase activity . It has 2, again certifying the role of inflammation in clinical manifestations of heart failure . Although a nominally significant reduction in the secondary outcome of hospitalization for heart failure was observed with spironolactone, overall the results of the TOPCAT trial were neutral.In Aldo-DHF trial on spironolactone, it was shown that the improvement in diastolic function was associated with reverse LV remodeling; nevertheless, no effect on maximal exercise tolerance and patient symptoms were observed . The TOPpost hoc analysis, spironolactone seemed to benefit patients in the Americas but not those in Russia or Georgia , treatment with spironolactone was beneficial in patients who were enrolled in the natriuretic peptide stratum but not those in the hospitalization stratum . This di Georgia .p = 0.693) or HF hospitalization  , a national registry of hospitalized HF patients evaluated the clinical effectiveness of aldosterone antagonists in older patients with HFpEF. The main outcomes of the study were a composite endpoint of all-cause mortality or HF hospitalization at 6 years of follow-up (primary outcome) and all-cause mortality, HF hospitalization, and all-cause hospitalization (secondary outcome). The authors were not able to find any association between aldosterone antagonists and all-cause mortality  .The first study to evaluate the effect if eplerenone in patient with HFpEf was the single-center RAAM-PEF study, in which Deswal and colleagues did not show any significant improvement in exercise capacity compared to placebo in the eplerenone group, but a significant reduction in markers of collagen turnover and improvement in diastolic function was detected in the latter . More reIn the recent meta-analysis of 14 available RCTs, mineralocorticoid antagonist (MRA) reduced hospitalizations and improved diastolic function and quality of life, despite the lack of favorable effects on all-cause mortality . In the p = 0.35). Overall rates of death were 52.6 and 52.3 per 1,000 patient-years, respectively . When the authors evaluated the death modality, treatment with irbesartan did not impact on the overall mortality or the distribution of mode-specific mortality rates (In the Irbesartan in patients with heart failure and preserved ejection fraction (I-PRESERVE) trial, patients who were at least 60 years of age and had New York Heart Association class II, III, or IV heart failure and an ejection fraction of at least 45% were randomly assigned to receive 300 mg of irbesartan or placebo per day. The primary composite outcome was death from any cause or hospitalization for a cardiovascular cause . During a mean follow-up of 49.5 months, the primary outcome occurred in 742 patients in the irbesartan group and 763 in the placebo group. Primary event rates in the irbesartan and placebo groups were 100.4 and 105.4 per 1,000 patient-years, respectively  than in the placebo group were admitted to hospital for CHF once  or multiple times. Composite outcomes that included non-fatal myocardial infarction and non-fatal stroke showed similar results to the primary composite . It is worthy of notice that the rates of study-drug discontinuation due to adverse events or laboratory abnormalities were significantly higher in the candesartan group , having the most common cause increase in creatinine level , hypotension , and hyperkalemia . Another potential confounder is the fact that the statistical borderline significance in the reduction in HF hospitalizations was only reached after adjusting for covariates  , but fewer patients in the candesartan group (ctively) .The study populations of I-PRESERVE and CHARM-preserved trial were highly representative of the HFpEF patients having a median EF of 59% and a mean EF of 54% respectively.Therefore, even if there could be a pathophysiological rationale for RAAS targeting in HFpEF, clinical results are inconclusive, with more consistent advantages mainly seen on diastolic dysfunction, LV remodeling, and hospitalization events. Likely, spironolactone is beneficial only in selected subgroups of patients while ineffective in others; this theory was recently backed by another analysis of ALDO-DHF trial that linked specific collagen I phenotype, which exhibit extensive cross-links, with spironolactone resistance .in vivo serelaxin reversed endothelial dysfunction by enhancing NO-mediated relaxation thus improving ventricular hypertrophy and apoptosis  is evaluating the potential role of adenosine receptor stimulation in HFpEF . The expEven though the total amount of natriuretic peptides in these patients is lower than in HFrEF, this inhibitor of the neprilysin system is being investigated in HFpEF in the PARAGON HF trial, after data collected from the exploratory phase II PARAMOUNT study pointed toward a reduction in the total amount of natriuretic peptides with an improvement in NYHA class end renal function. Even if it is not known whether these effects are related to an improvement in microvascular function, the results of these studies are eagerly awaited .p = 0.002) in hospitalization for HF compared with placebo group . Designed for patients with diabetes, reducing the kidney blood glucose resorption, rapidly SGLT2i gained attention among HF specialist since they were able to demonstrate a beneficial prognostic impact in patients affected by HF and diabetes, even independently from the glycemic control. Three SGLT2 inhibitors have been widely studied so far: canagliflozin, dapagliflozin, and empagliflozin. The EMPA-REG OUTCOME trial (EMPAgliflozin Cardiovascular OUTCOME Events in Type 2 Diabetes Mellitus Patients) included 7,020 patients with diabetes and established cardiovascular disease, showed a reduced risk of major CV events, CV mortality, all-cause mortality, and hospitalization for HF in 2TD patients, after a median observation time of 3.1 years when treated with empagliflozin. In particular, a 14% reduction in cardiovascular death, nonfatal myocardial infarction, nonfatal stroke and CV and all-cause mortality, despite a no significant difference about myocardial infarction or stroke. Patients treated with empagliflozin has a relevant reduction  is a large multinational study that analyzing data from six countries reported a lower risk of hospitalization and mortality for HF with several SGLT2i . And eveA novel target of research efforts has focused on this little electron transporter, moving from the hypothesis that supplementation of these molecules could yield to a more efficient ATP synthesis and cardiomyocyte performance, eventually reducing ROS production . Indeed,Cardiosphere-derived cells (CDCs) are a type of cardiac progenitor cells with anti-fibrotic, anti-inflammatory, and angiogenic properties . It is iThe tantalizing research for a prognosis-improving therapy in HFpEF should not neglect the humbler attempt to enhance the quality of life, looking on the bright side of all those clinical trials failing in their primary endpoint but proving to relieve symptoms and reduce physical limitations. Albeit the novel paradigm, which puts the coronary microvascular endothelial inflammation on the culprit mechanistic pedestal and the common pathway in the complex HFpEF pathophysiology, the dismal outcomes revealed by RCTs testing one-therapy strategies prompt to perpetuate the research for pathophysiological subtypes.The advance in mechanistic comprehension should aim to better understand the genetic and environmental etiological factors that might contribute to HFpEF onset and progression. This missing tile of the puzzle may facilitate the primary prevention of incidental HFpEF through accurate identification of vulnerable individuals and HFpEF-prone phenotypes. On the other hand, the discovery of more specific, reliable biomarkers may facilitate both early diagnosis and prognostic assessment.DD, SM, JB, AR, NA, MG, RV, and FC conceived and wrote the manuscript. AL, GN, JB, RM, and SM reviewed the manuscript and made the final figure.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tectona grandis L.F.), the difference of chemical composition between the heartwood and sapwood of teak was investigated by gas chromatography–mass spectrometry (GC-MS) based on the acetone extractive compounds. The results showed that the difference in content of the main components between heartwood and sapwood was not obvious. However, the amount of extractives in heartwood was higher than that in sapwood, especially for phenols, quinones, and ketones. The most obvious different substances in the acetone extractive between heartwood and sapwood were 4-tert-butyl-2-phenyl-phenol,2-methyl-anthraquinone, and 2,3-dimethyl-1,4,4a,9a-tetrahydro-9,10-anthracenedione, which might be the main composition for the chromatic aberration of teak. This paper focuses on a preliminary study and further work such as high-performance liquid chromatography (HPLC) with ultraviolet photometric detector (UV)/mass spectrometry (MS) will be carried out.In order to clarify the chemical color change of teak ( Tectona grandis L.F.), as one of the most famous timber species all over the world, is distributed from 73° E in India to 104°30′ E in Thailand, and from the island of Java at 8°45′ S to the low hills and plains which is below 800 m in northern Myanmar in terms of its origin [Teak  into mixture powder from 6 discs with 40–60 mesh respectively. The moisture of sapwood and heartwood of teak were determined by reference of GB/T 2677.2-1993 “Fibrous raw material–Determination of moisture content”.The relative Chinese standards were found based on the book of Shi and HE ; 5 × 5 ×Determination of α-cellulose and hemicellulose content were measured by reference of GB/T 744-1989 “Pulps–Determination of α-cellulose” and GB/T 2677.10-1995 “Fibrous raw material– Determination of holocellulose”, respectively.The determination of lignin was carried out in accordance with GB/T 2677.8-1994 “Fibrous raw material–Determination of acid-insoluble lignin”.The pH value was measured referring to GB/T 6043-1999 “Method for determination of pH of wood”. 3 g wood flour sample was extracted by alcohol-benzene, 1% NaOH, cold water, hot water, and the acetone, respectively referring to GB/T 10741-1989 “Pulps–Determination of alcohol-benzene soluble”, GB/T 2677.5-1993 “Fibrous raw material Determination of 1% sodium hydroxide solubility”, GB/T 2677.4-1993 “Fibrous raw material–Determination of water solubility” and GB/T 2677.6-1994 “Fibrous raw material–Determination of solvent extractives”. After the extraction complete, the extractives were transferred to 50 ml volumetric flasks and bring to volume.y = 0.00059 + 0.1149x .The total phenol content was measured with the cold water extractives and hot water extractives by spectrophotometry. The standard solution of gallic acid was detected by wavelengths at 780 nm. Regression analysis of the data to obtain the regression equation is: http://webbook.nist.gov/chemistry/, Maryland, USA) with retention time and indices.The analysis was carried out by Fisher TSQ Quantum GC (gas chromatography) . The chromatographic conditions were Agilent  DB-1MS (60 m × 0.25 mm × 0.25 μm) with column flow rate of 1.0 ml/min. The inlet temperature was 250 °C, and split ratio was set at 15:1. The initial temperature was 40 °C and rose to 280 °C at 10 °C/min, then retained for 15 min. The mass spectrometry conditions were an EI (Electronic ionization) ionization source with electron bombardment energy of 70 eV, ion source temperature of 250 °C, filament current of 100 μA, and transmission line temperature of 250 °C. The compounds were identified by comparing the MS (mass spectrometry) spectra to the National Institute of Standards and Technology (NIST) library  with R2 of 0.9987. It can be seen from As the typical substance of teak, 2-methyl-anthraquinone was suspected to be the reason for the special color and surface properties of teak. According to the external standard method, the area of the standard solution at different concentration was detected by GC–MS. The standard curve drawn according to the test results is shown in The quantitative analysis of the other substance in the acetone extractive detected by GC-MS depended on the relationship of the peak areas between them and the 2-methyl-anthraquinone. The results of GC-MS analysis of acetone extracts in heartwood and sapwood of teak are shown in The acetone extractives in heartwood were higher in composition and content than those in sapwood . A totalThe components in acetone extractives of heartwood and sapwood are accumulated according to the type and shown in Alkanes, ketones, phenols and quinones  in acetoComponents like lappaol, deoxylactam and its isomers, such as squalene, chloranol, palmitic acid, etc. were all detected in teak indigenous to Java Island, Yogyakarta and Panama. Similar substances were found in this research. Due to the different origins, the content of each component was different. The tectol with higher content detected in teak from Indonesia, did not appear in this experimental result; 4-tert-butyl-2-phenylphenol and 2,3-dimethyl-1,4,4a,9a-tetrahydro-9,10-nonanedione, which had a high content in this research, were undetected in teak from Indonesia and Panama. Although ketones appeared less than in other literature, 2,3-dimethyl-1,4,4a,9a-tetrahydro-9,10-nonanedione was similar to lappaol, deoxylapram and its isomers in structure. Extractives of the teak grown in the B region of Panama, increased in the amount from the sapwood to heartwood, which was consistent with the results obtained in this paper that the heartwood extractives were greater than the sapwood ,31. It sAlthough the proportion of extractives in wood is small, they play an important role in the particular characteristics such as the color and smell of the wood, which is highly related to wood discoloration ,33. The The increase of chromophoric group and auxochrome group in the extractives after illumination caused a change in wood color . The yelThe difference in content of the main components between heartwood and sapwood was slight. They both were appeared to be weakly acidic. The moisture of heartwood was slightly lower than that of sapwood. The content in alcohol–benzene and acetone were obviously different between heartwood and sapwood.The acetone extractives in heartwood were higher in composition and content than those in sapwood; 49 components were detected in the acetone extractive of the heartwood, and 26 components were detected in the sapwood. The high content in the teak heartwood was mostly detected in the sapwood.In the acetone extractives, the substances of phenols, quinones and ketones in sapwood were obviously less than those in the heartwood. These substances may be the main components that made the heartwood darker than the sapwood. The most obvious distinct substances in acetone extractives between heartwood and sapwood were 4-tert-butyl-2-phenyl-phenol, 2-methyl-anthraquinone and 2,3-dimethyl-1,4,4a, 9a-tetrahydro-9, 10-anthracenedione#.This paper on the issue was a preliminary study. Since GC–MS was not the most accurate analysis method, a liquid chromatography system like high-performance liquid chromatography (HPLC) with UV–MS will be used to confirm the possible substance for the chromatic aberration in teak from China in a further study.


In the past decade, nanomedicine research has provided us with highly useful agents (nanoparticles) delivering therapeutic drugs to target cancer cells. The present review highlights nanomedicine applications for breast cancer immunotherapy. Recent studies have suggested that tumour necrosis factor (TNF) and its receptor 2 (TNFR2) expressed on breast cancer cells have important functional consequences. This cytokine/receptor interaction is also critical for promoting highly immune-suppressive phenotypes by regulatory T cells (Tregs). This review generally provides a background for nanoparticles as potential drug delivery agents for immunomodulators and further discusses in depth the potential of TNF antagonists delivery to modulate TNF-TNFR2 interactions and inhibit breast cancer progression. Amo. Amo5].  Commiphora myrrha and Boswellia sacra extracts to induce trisodium citrate dihydrate reduction which leads to cytotoxicity in breast cancer and normal cells. The study reported cytotoxicity in breast cancer cells, but no harm in normal breast cells , 5251, 5In recent years, along with the extensive identification of molecular markers on breast cancer, several novel nanomedicine applications have been developed to specifically target these pathways . TargetiTNF is a multifunctional cytokine secreted by various types of cells as well as being responsible for leukocyte recruitment, monocyte chemoattraction, and increased regulation of adhesion molecule expression and may also promote apoptosis . TNF is  TNFR1 gene located on chromosome 12p13.31, consisting of 10 exons, and codes for a 55/60 kDa membrane receptor. TNFR2 (p75) is encoded by TNFR2 gene located on chromosome 1p36.22, consisting of 10 exons, and codes for a 75/80 kDa membrane receptor [κB) pathway [κB pathway plays a key role in the expression of genes that are responsible for encoding antiapoptotic proteins and several proinflammatory cytokines, including TNF, IL-6, and IL-1 [Both sTNF and mTNF are regulated by binding with their two receptors localised at the cellular surface. TNFR1 (p55) is encoded byreceptor . These t. TNFR2 p is encodreceptor , 77. TNFreceptor . Also, T pathway . Activatand IL-1 . FurtherκB pathway [κB pathway by the deactivation of the NFκB-inducing kinase (NIK)-TRAF3-TRAF2 axis, which results in aggregation of NIK in activated T cells [κB signalling pathway in turn regulates expression of some chemokines needed in adaptive immunity and structuration of the secondary lymphoid organ, such as B cell chemoattractant (CXCL13) [−/− mice were able to double the number of normal TNFR2-expressing Tregs [Upon binding of TNF to TNFR1, TNFR1 interacts with receptor-interacting protein 1/2 (RIP1/2) and TNFR1-associated DD protein (TRADD) to build a receptor complex  that ind pathway . On the  pathway . The exp T cells . Noncano(CXCL13) . Althougng Tregs . Tregs, ng Tregs , and expng Tregs . Therefong Tregs –92.κB or AKT serine/threonine kinase 1 signalling pathways, which in turn regulate DNA damage and repair of poly (ADP-ribose) polymerase (PARP) protein [The implication of TNF in almost all steps of tumourigenesis has been reported, both as an angiogenic and antiangiogenic factor, depending on the TNF doses and nature (soluble and membrane-bound) . Since T protein . Moreove protein  as well  protein . TNFR1 s protein . Due to  protein . However protein . All togNumerous studies have explored the association of TNF and its receptors in breast cancer progression as well as the therapeutic possibilities. However, only a few investigated the impacts of TNFR2 expression in breast cancer . In 2008As TNFR2 exists without DD, it can enhance proliferation and activation of Tregs via 3 main pathways, namely, NF-kB, activator protein 1 (AP1), and MAPK pathways , therefoIt has previously been shown that TNF antagonism (anti-TNF) is a successful therapeutic option that has been applied in various inflammatory cases, including inflammatory bowel disease (IBD), spondyloarthritis (SpA), psoriasis, and rheumatoid arthritis (RA) . TNF antAnti-TNF biology functions by mopping up excess soluble TNF and reducing the endocrine activity of these cytokines. They would bind to TNF complexes to block cell-to-cell contact and/or trigger reverse signalling, lastly acting as agonists on Fc receptor (FcR)-expressing cells as they are fused to human IgG1 . HoweverTaken together, these findings suggest that targeting TNF-TNFR2 interaction with pharmacological agents, in an attempt to reduce the number and function of Tregs while enhancing the function and number of Teffs, could provide stronger immune responses against cancer cells and serve as a promising cancer therapeutic approach , 114. OnAs we discussed before, nanomedicine platforms offer a variety of potentially efficient solutions for the development of immunotherapeutic agents that can be exploited for breast cancer treatment . As mentStudies have demonstrated that TNF-TNFR2 axis is implicated in the suppression of immune response and affects tumour progression and invasion by its oncogenic roles, which results in enhanced proliferation, angiogenesis, and migration of breast cancer. This receptor is also responsible for enhancing the proliferation and activation of Tregs and MDSCs, thus promoting tumour immune escape. Hence, neutralisation of TNF as well as TNFR2 by using TNF-antagonist drugs might be an effective therapeutic strategy for breast cancer cells. However, no studies to date have investigated the modulation of TNF antagonists targeting TNF-TNFR2 axis and their immunoregulation on breast cancer cells, while the potential of nanoparticles to mediate these effects in breast cancer is still unknown. The field of nanomedicine has provided the possibility of targeting the TNF-TNFR2 axis to not only deliver therapeutic drugs to targeted sites but also restore the immune response to suppress the cancer cells. Based on accumulating evidence suggesting that tumour progression is governed not only by genetic changes intrinsic to cancer cells but also by environmental factors, future studies might use nanoparticles as a model for inert environmental stimuli. In summary, nanoparticles have the potential to be used as drug delivery vehicle in the future for nanomedicine development in breast cancer therapy. Therefore, future studies should investigate how the presence of nanoparticles with specific characterisation would alter the function of breast cancer cells via TNF-antagonist effects by TNF-TNFR2 signal and their contribution to distinct immunological effects.