Elevated ACE expression in tissues (reflected by blood ACE levels) is associated with increased risk of cardiovascular diseases and is also a marker for granulomatous diseases. We developed a new approach for characterization of ACE status in the blood—ACE phenotyping and established normal values of ACE levels 50–150% of control pooled plasma. ACE phenotyping was performed in citrated plasma of 120 patients with known interstitial lung diseases. In the 1st set of 100 patients we found 22 patients with ACE levels > 150%; ACE phenotyping also objectively identified the presence of ACE inhibitors in the plasma of 15 patients. After excluding these patients and patient with ACE mutation that increases ACE shedding, 17 patients were identified as a suspicious for systemic sarcoidosis based on elevation of blood ACE (> 150% of mean). A new parameter that we have established–ACE immunoreactivity (with mAb 9B9)—allowed us to detect 22 patients with decreased values (< 80%) of this parameter, which may indicate the presence of ACE in the blood that originates from macrophages/dendritic cells of granulomas. In the remaining 20 patients, this new parameter (mAbs binding/activity ratio) was calculated using 3 mAbs , and 8 patients were identified as having decreases in this parameter, thus increasing dramatically the sensitivity for detection of patients with systemic sarcoidosis. Whole body PET scan confirmed extrapulmonary granulomas in some patients with lower immunoreactivity towards anti-ACE mAbs. ACE phenotyping has novel potential to noninvasively detect patients with systemic sarcoidosis.The online version contains supplementary material available at 10.1186/s12931-022-02145-z. Sarcoidosis is a systemic inflammatory disease that occurs throughout the world and affects people of all races and ages. Despite years of study and recent advances in diagnostic strategies, the etiology of sarcoidosis and many details of the pathogenesis remain unknown –5.Serum levels of angiotensin I-converting enzyme (ACE or CD143) are elevated in many patients with sarcoidosis and initially were thought to parallel the activity of sarcoid lesions –9. Howev2+ carboxydipeptidase which is vital for the regulation of blood pressure and also associated with the development of vascular pathology. ACE is constitutively expressed on the surface of endothelial cells, absorptive epithelial and neuroepithelial cells, and cells of the immune system . Blood ACE likely originates from endothelial cell ACE, primarily lung capillary endothelium, due to proteolytic cleavage by a still unidentified ACE secretase-see for review 26] and oBased on these data and analysis of the existing literature, we propose that patients with elevated ACE activity (> 150%) may have systemic sarcoidosis.In the current study, we performed ACE phenotyping in citrated plasma from 120 patients recruited from pulmonary clinics  is not present. Because ACEI significantly alter the local conformation of ACE , 42, theFigure The comparison of ACE activity in the plasma Fig. A and theFigure A major limitation in the use of serum ACE measurements as a valid biomarker for sarcoidosis is the observation that ACE levels can be elevated in other disease processes. Our novel ACE fingerprinting approach helps address this problematic issue. For example, referring back to the data in Fig. However, in patients taking ACE inhibitors this parameter could be applied only for two of these mAbs, because the presence of ACE inhibitors significantly increases mAb i1A8 binding to any ACE/ACE inhibitor complex (data not shown). This effect of ACE inhibitors is reflected in patients #106, #110, #112, 117, 120—red boxed in Fig. The power of our ACE fingerprinting approach to identify sarcoidosis “mimics” is further illustrated by other examples. As noted above, high levels of blood ACE can be caused by certain ACE mutations that increase shedding of the enzyme or its secretion from endothelial cells (reviewed in ). While However, there are additional ACE mutations located outside the stalk region that nevertheless serve to increase ACE shedding by other mechanisms, such as changes in the degree of dimerization  or decreTo summarize the potential of this approach for the 20 subjects described in Fig. To confirm that elevated blood ACE levels, or decreased mAb binding/ACE activity ratios for some mAbs 9B9/3A5/i1A8), indicate association with systemic (extrapulmonary) sarcoidosis, we performed whole body PET scanning in selected patients in which ACE phenotyping was performed. In patient #82B (from another cohort of unrelated patents) with elevated total ACE levels (180%), but no reduction in the mAb binding/ACE activity ratios (data not shown), whole body PET scanning demonstrated no abnormal FDG uptake and no signs of lymph node enlargement : 118.7 (98.9–160.4) vs. 97.3 (81.7–117.5), p < 0.001, respectively) , which varies threefold in the tested population: ranged between 50–150% from control pooled plasma, we found that 31 patients could be considered as possible candidates for systemic sarcoidosis based only on ACE levels. However, an application of conformational fingerprinting allowed us to identify another set of 22 patients based on another parameter of ACE phenotype (ratio of ACE immunoreactive protein (with mAb 9B9) to ACE activity.Our approach incorporates multiple parameters to characterize serum ACE in clinically-relevant ways. These parameters include (1) combination of ACE activity determination with two substrates; (2) quantification of ACE immunoreactive ACE protein with a set of mAbs to ACE; and (3) estimation of ACE conformation in each individual.To summarize the potential clinical importance of these parameters, the ACE fingerprinting approach provides a relatively noninvasive way through blood sampling to eliminate sarcoidosis from consideration in selected patients with elevated ACE levels, as well as detect the presence of sarcoidosis in patients in whom low total ACE levels  may result in the appropriate diagnosis not being considered. It also provides information useful for identifying systemic sarcoidosis in some patients, thus preventing them from undergoing more invasive  and/or expensive testing . Because some extrapulmonary manifestations  are associated with higher mortality and/or more severe morbidity than pulmonary sarcoidosis alone, early detection is an important clinical goal. Thus, this comprehensive approach advances precision medicine in sarcoidosis by providing a noninvasive and relatively low cost method for detecting sarcoidosis patients with low total ACE levels, screening for mutations that result in markedly elevated ACE levels in the absence of sarcoidosis, and also identification of patients with systemic sarcoidosis involvement. As a result, the ACE phenotyping method has the potential add significant value very early in the evaluation process for patients with suspected sarcoidosis. If utility and accuracy is confirmed in additional studies, we propose it be added to the algorithm for sarcoidosis evaluation as early as the first clinical visit in which sarcoidosis is suspected.Additional file 1: Fig.S1. Unremarkable whole body PET scan in a patient with elevated blood ACE levels. A whole body PET/CT was performed on a patient (#82B) with elevated blood ACE level (180%). Top panels shows multi-planar PET reformations –sagittal (A), axial (B) and coronal  (C). Bottom panel illustrates fused PET/CT (D) and CT (E) images demonstrating no increased FDG uptake in the areas of mediastinal or hilar lymph nodes. Further examinations confirmed that the patient had hyperthyroidism rather than sarcoidosis.Additional file: 2: Fig.S2. Comparison of ACE parameters in healthy individuals and sarcoidosis patients. Box-and-whisker plot for ACE activity (A). Box-and-whisker plot for mAb 9B9 binding/ACE activity ratio (B). Line inside the box—median; limits of the box—75th and 25th percentiles; whiskers—10th and 90th percentiles; •—outliers. P values are displayed in the figure.


The immune system is fundamental to tissue homeostasis and is the first line of defense following infection, injury or disease. In the damaged heart, large numbers of immune cells are recruited to the site of injury. These cells play an integral part in both repair by scar formation and the initiation of tissue regeneration. They initially assume inflammatory phenotypes, releasing pro-inflammatory cytokines and removing dead and dying tissue, before entering a reparative stage, replacing dead muscle tissue with a non-contractile scar. In this Review, we present an overview of the innate and adaptive immune response to heart injury. We explore the kinetics of immune cell mobilization following cardiac injury and how the different innate and adaptive immune cells interact with one another and with the damaged tissue. We draw on key findings from regenerative models, providing insight into how to support a robust immune response permissible for cardiac regeneration. Finally, we consider how the latest technological developments can offer opportunities for a deeper and unbiased functional understanding of the immune response to heart disease, highlighting the importance of such knowledge as the basis for promoting regeneration following cardiac injury in human patients. Summary: This Review presents the innate and adaptive immune responses to heart injury in regenerative and non-regenerative organisms to uncover an immune response permissible for cardiac regeneration. The damage caused by a heart attack  leads to a permanent loss of cardiac tissue in adult mammals . MI is oAfter MI, dead cardiomyocytes release damage-associated molecular patterns (DAMPs) that serve as danger signals and trigger mobilization of the first innate immune cell responders  Fig. 1BBFig. 1B.During the initial stages post-MI, mast cells are important initiators of the immune response . After cNeutrophils are initially recruited from the circulating blood and bone marrow to the site of injury by DAMPS and inflammatory signals released by resident cardiac cells, including mast cells . NeutropNeutrophils assume an inflammatory state during their peak response at 1-day post-MI, shifting to an anti-inflammatory and reparative phenotype thereafter . Althoug− resident cardiac macrophages are either maintained independently of circulating monocytes, if expressing the phosphatidylserine receptor TIMD4+, or are partially replaced by CCR2− TIMD4− monocytes, whereas CCR2+ resident macrophage subsets are fully replaced by circulating monocytes (− macrophages decrease in number (+ monocytes expressing the surface glycoprotein lymphocyte antigen 6C (Ly6C) are recruited to the site of injury in response to C–C chemokine ligand 2 (CCL2) expression by resident macrophages, cardiomyocytes, mesenchymal stem cells in the bone marrow and peripheral B cells -dependent transcriptional program  cells are innate lymphocytes that infiltrate the heart following MI  and playInnate lymphoid cells have recently been identified as a new immune cell subset within the repertoire of innate immune cells activated following cardiac injury. Specifically, innate lymphoid cells type 2 (ILC2) cells form a cluster that secrete type 2 cytokines, such as IL5 and IL13, and are activated through IL25, IL33 and IL2 . ILC2s aAfter the initial innate immune response, activation of the adaptive immune system takes place in the injured heart.in situ and then migrate to the draining lymph nodes where they help generate a population of effector lymphocytes (+ (cytotoxic) T lymphocytes in draining mediastinal lymph nodes, establishing an important link between the innate and adaptive immune systems (Dendritic cells (DCs) are the major antigen-presenting cells found within the cardiac tissue, some of which localize in cardiac valves . DCs samphocytes B. In the systems .+ T cells are mobilized to the injured heart and aid myocardial wound healing by promoting CCR2+ pro-reparative macrophage activity (+ T cells can be further subdivided into T helper type 1 (Th1), T helper type 2 (Th2) and regulatory T cell (Treg) subsets, according to their phenotype, cytokine production and function. The IFNγ-producing Th1 population plays a detrimental role during MI healing  T cells, unconventional T cells defined by expression of heterodimeric T cell receptors composed of γ and δ chains in situ tδ chains . γδ T ce healing  + T cells specific for cardiac self-antigen αMyHC escape thymic negative selection  is released in the heart. Activated DCs, mainly conventional type 2 DCs (cDC2s), migrate to the lymph node and present αMyHC to T cells . This iselection . Therefoelection , usuallyelection . Howeverd tissue . Interesd tissue . Furtherd tissue  Fig. 2)+ T cellsAlthough hematopoietic immune cells are the bona fide components of the immune system, immune functions are not unique to hematopoietic cells; other cell types display basic mechanisms of immunity . Recentl+ and CD8+ T cells through an IFNγ-dependent pathway + and CD8+ Ly6Clow reparative macrophages can also contribute to angiogenesis + Ly6Clowogenesis . Furtherogenesis . Cardiacogenesis .Astyanax mexicanus surface fish have remarkable regenerative capacity across organ systems, including the heart. Each of these animals presents an opportunity to investigate how evolution has retained natural regeneration in these species and to understand what has potentially been lost or adapted in adult mammals. Effective cardiac regeneration depends not only on the degree of regenerative competence of the injured heart , based on marker gene expression. However, discriminating state and function within bulk RNA-sequencing of mixed immune cell populations is not possible due to their inherent heterogeneity . Recent hiIgf1hi and MHCII+ macrophages. It also proposes that MGL2 and CD81 are two surface markers that may help distinguish resident macrophage populations from monocyte-derived macrophages in the injured heart. One caveat with scRNA-seq approaches to identify immune cell subtypes is the reliance on existing marker sets to adequately cluster cells that are, by definition, biased (dependent upon previous studies and identification of candidate markers) and may not be entirely accurate to attribute function. Employing broad-based initial cell isolation  enables a more unbiased approach, which computational analysis may cluster into cell subtypes more accurately. Ultimately, by linking both the transcriptome and the proteome, new and reliable cell surface markers may be identified that enable purification and localization of rare immune cell subsets by flow cytometry and confocal microscopy.When unbiased scRNA-seq is combined with immunophenotyping of cells using antibody sequencing (CITE-seq) , the resSingle-cell technologies can profile the molecular heterogeneity of the different immune cell types; however, without spatial context, it is unclear how various immune cells coordinate their function within the injured heart. The advent of spatial transcriptomics is revolutionizing the way we understand tissue biology in homeostasis and disease . CurrentA holistic view of the spatial-temporal heterogeneity of the various immune cells responding to heart injury will allow us to link (epi)genomic diversity to changes in cellular function. Options to selectively modulate immune cell activity have increased dramatically over the past few years. Just a few examples include CRISPR-Cas9 genome editing, pharmacological targeting and nanoparticle delivery of therapeutic cargo; including the latest developments on lipid nanoparticles loaded with mRNA technology further developed and validated during the COVID-19 pandemic . All theThe immune response to cardiac injury is multifaceted. A deeper understanding of how and when immune cell types orchestrate inflammatory and reparative pathways following cardiac injury will lead to effective therapeutic strategies that promote tissue regeneration and improve recovery in post-MI patients. Thus far, studies focusing on macrophages have dominated the landscape. This is understandable given their evolving plasticity in terms of diverse subtypes and function, as well as the fact they are the dominant immune cells in terms of sheer numbers that present following MI. That said, other cell types are very important and have profound effects on both repair and regeneration, as described above. Neutrophils fall into this category, as initial responders within the innate immune spectrum. The role of adaptive immunity is only just being explored in terms of effects on acute injury, chronic disease progression and the development of heart failure. As such, DCs, T cells and B cells require further study to provide basic insight into how these various immune cells interact both with one another and with non-immune cardiovascular cells. A deeper understanding of innate and adaptive immune cell interactions, across both acute and chronic stages of injury, may provide potential therapeutic targets to modify outcome post-MI.To date, many of the immune modifying therapies have tested immunosuppressive drugs that bluntly inhibit inflammation . This ap


The IUGR babies with a protective IGF2 genetic profile had significantly younger parents . The IUGR babies had parents with lower birth weights than AGA babies . In univariable regression analysis, the mother’s and father’s birth weight proved to be associated with IUGR. The father’s birth weight proved to be the only factor significantly associated with IUGR, independent of the mother’s birth weight or the presence of a protective IGF2 genetic profile .Polymorphism of insulin-like growth factor 2 (IGF2) is known to play a role in cell development. Only the paternal IGF2 copy is active, while the copy inherited from the mother is inactive. This study aimed to explore whether maternal and paternal factors influence IGF2 polymorphism in newborns with intrauterine growth restriction (IUGR) compared to appropriate for gestational age (AGA). A cross-sectional exploratory study was conducted from June 2014 to November 2015 at the Neonatology, Gynecology 1 Clinic, Cluj-Napoca, Romania. The ApaI IGF2 genotypes and allele frequencies were similar in the IUGR and AGA groups ( Personal experiences determine the individual’s health during existence. Fetal programming occurs during embryonic and fetal development, a critical period in which tissues and organs are created . The criThe etiology of intrauterine growth restriction (IUGR) is multifactorial and it is based on maternal, fetal, placental, and genetic mechanisms. Maternal risk factors for IUGR include smoking during pregnancy, low pre-pregnancy weight, and low weight gain. Risk factors more strongly associated with preterm IUGR include chronic hypertension and advanced maternal age. Furthermore, low pre-pregnancy body mass index has been associated with an increased risk of term IUGR. Any specific ischemic placental condition in a prior pregnancy is also a risk factor for that same ischemic placental disease in a subsequent pregnancy . PaternaThe human species inherits a copy of most genes from the mother and a copy from the father. Both copies are usually active or “activated” at the cellular level . HoweverIn mice, interruption of the paternal IGF2 allele causes a severe delay in prenatal growth  and postThe ApaI polymorphism of insulin-like growth factor 2 (IGF2) had been reported to be associated with body mass index, fat mass, grip strength  and obesOur study aimed to explore whether maternal and paternal factors influence IGF2 polymorphism in intrauterine growth restriction (IUGR) newborns compared to appropriate for gestational age (AGA) newborns.A cross-sectional exploratory study was conducted to evaluate several parental  factors’ on the ApaI IGF2 polymorphism in newborns with IUGR and AGA. Maternal and paternal anthropometric data, pre-pregnancy and mother’s pregnancy-related pathology, mother weight gain during pregnancy, consumption of toxins , number of pregnancies, and birth weight of parents were collected. Parents’ body mass index was calculated, and subjects were classified as overweight or obese by body mass index (BMI) .Newborns with IUGR diagnosed by fetal ultrasound evaluation and subsequently hospitalized in the Department of Neonatology Gynecology 1 Clinic , level 3 maternity, for 1.5 years (June 2014–November 2015) were included in the study. The AGA group was newborns of the same gestational age as the IUGR group. Newborns with any chromosomopathy or malformation were excluded. p-value > 0.10 [p-value = 0.122), mothers’ pre-pregnancy BMI , ethnicity , and mother’s smoking status  [p-value = 0.049) [The main characteristics of the evaluated groups were previously reported in the scientific literature . Sixty-oe > 0.10 ). The IUe > 0.4) . The new= 0.049) .p-values less than 0.05 were considered statistically significant. Statistical analysis was made with IBM SPSS Statistics for Windows software  at a significance level of 5%, so the Measured data were summarized as mean ± standard deviation whenever data proved to follow the theoretical normal distribution (Shapiro–Wilk test); otherwise, median and IQR (interquartile range defined as (Q1–Q3), where Q1 is the first quartile and Q3 is the third quartile) were reported. The chi-squared or Fisher exact test, or Z-test for the proportion were used to compare IUGR with AGA whenever qualitative variables were tested. The Student t–test for normally distributed quantitative data or otherwise the Mann–Whitney test was used for comparisons between IUGR and AGA groups. p-values ≤ 0.10 in an univariable exploratory regression analysis were considered as potential factors for IUGR and entered into multivariable logistic regression analysis. The ApaI IGF2 genotype (protective vs. non-protective) was included in the multivariable logistic regression analysis as a possible confounder regardless of the univariable logistic regression result. The effect of factors on IUGR was expressed as an odd ratio, inclusive of the associated 95% confidence bounds and significance.All parents and babies . Two mothers in the IUGR group had blood hypertension, and one in the AGA group had diabetes. Fifteen mothers in the IUGR group (37.5%) and two in the AGA group (9.5%) had other diseases . As a result, the mothers with an IUGR baby reported a specific disease significantly more frequently than the mothers of an AGA baby .No mother declared alcohol consumption either in the IUGR or in AGA group. Most mothers declared no medical history . One father of a child with IUGR reported hypertension, one reported diabetes, and another hepatitis B.p-value = 0.2170). In the IUGR group, the parents (mother and father) of children with a protective genetic profile (AA genotype) were significantly younger as compared to the parents of children with non-protective profiles (AG or GG)  and five with AGA (23.8%), had an IGF2 protective genetic profile (AA genotype). No significant association was identified between non-protective genetic profile and IUGR/AGA (Fisher exact test: G or GG) .The univariable and multivariable logistic regression analysis showed that non-protective IGF2 genetic profile (AG or GG) is not significantly associated with IUGR. The fathers’ birth weight is associated with IUGR independent of the IGF2 genetic profile .Our results showed that children with intrauterine growth restriction had parents (mother and father) with lower birth weight than children appropriate for gestational age. Still, only the father’s birth weight remained significantly associated with IUGR in multivariable regression analysis, independent of the ApaI IGF2 genotype.p-value > 0.5). Alcohol and drug abuse compromise maternal nutritional status. Thus, the supply of essential nutrients is not available for the fetus and can lead to fetal abnormalities like IUGR or Fetal Alcohol Spectrum Disorder (FASD) . In our p-value < 0.05). Maternal pathology and preeclampsia can induce disorders in the development of the placenta, and thus there are changes in placental membrane transporters and in the expression of these transporters. A systematic shift in nutrient transporter expression was detected, a factor that could lead to IUGR [Another critical factor in developing IUGR is the maternal pathology during pregnancy, with a particular contribution of preeclampsia (PE). A subset of women develop early-onset PE and are diagnosed with IUGR in special early preeclampsia , suggest to IUGR .Maternal age and ethnicity, and socio-economic and marital status can be associated with child IUGR, and these factors may explain how maternal cultural background and environment influence small for gestational age patterns in a specific population . In our Analyzing the influence of genetic factors, it is known that several imprinted genes are associated with body weight dysregulation. In humans, genetic disorders affecting imprinted genes lead to obesity . ObesityOur results reported no significant association between IGF2 polymorphism and IUGR, similar to results reported by de Mascena Diniz Maia et al. . We founOur study demonstrated lower parental birth weight among IUGR babies than AGA babies . In univThe main limitation of our study is given by the nature of the design, namely, an exploratory study, and thus with a small sample size. The association of maternal and paternal factors with the genetic polymorphism ApaI IGF2 could be valid, but the reported results require validation in larger samples. The relationship between parental birth weight and IGF2 polymorphism in newborns with IUGR and AGA also needs to be validated in larger samples. Thus, our results indicate what we observed in the investigated cohort and generalizing from them is inappropriate. Our results showed that children with intrauterine growth restriction (IUGR) had parents (mother and father) with lower birth weight than children appropriate for gestational age (AGA). Moreover, AGA  babies had an IGF2 (insulin-like growth factor 2) protective genetic profile (AA genotype) more frequently than IUGR (intrauterine growth restriction) babies, although the threshold of significance was not reached. Babies with IGF2 protective genetic profiles in the IUGR group had younger parents than those without IGF2 protective genetic profiles. Still, this result could be observed by chance since the protective profile was observed only in five babies and thus needs validation with larger samples. Only the father’s birth weight remained significantly associated with IUGR in the multivariable logistic regression analysis, but the upper bound of the 95% confidence interval indicates a low relevance. Our results indicate a possible independent paternal genetic role in the development of IUGR.


The aim of this review is to investigate the literature pertaining to the potential risks of low-dose ionizing radiation to Lynch syndrome patients by use of computed tomography (CT), either diagnostic CT colonography (CTC), standard staging CT or CT surveillance. Furthermore, this review explores the potential risks of using radiotherapy for treatment of rectal cancer in these patients. No data or longitudinal observational studies of the impact of radiation exposure on humans with Lynch syndrome were identified. Limited experimental studies utilizing cell lines and primary cells exposed to both low and high radiation doses have been carried out to help determine radio-sensitivity associated with DNA mismatch repair gene deficiency, the defining feature of Lynch syndrome. On balance, these studies suggest that mismatch repair deficient cells may be relatively radio-resistant (particularly for low dose rate exposures) with higher mutation rates, albeit no firm conclusions can be drawn. Mouse model studies, though, showed an increased risk of developing colorectal tumors in mismatch repair deficient mice exposed to radiation doses around 2 Gy. With appropriate ethical approval, further studies investigating radiation risks associated with CT imaging and radiotherapy relevant doses using cells/tissues derived from confirmed Lynch patients or genetically modified animal models are urgently required for future clinical guidance. Lynch syndrome (LS), previously referred to as hereditary non-polyposis colorectal cancer (HNPCC), is the most common cause of hereditary colorectal cancer  . LS patiMLH1, MSH2, MSH6 and PMS2  in synchronized G2 cells. It was found that G2 sensitivity in LS cells was not higher than that observed in control cells even though lymphoblasts from patients heterozygous for MLH1 showed a higher yield of chromatid-type exchanges. The lack of G2 chromosomal sensitivity to IR was also observed in the lymphocytes of LS patients shown in a human study below. It is possible that cells with MMR genes in heterozygous state can still perform sufficient repair function.Chromosomal radio-sensitivity, manifested as an increased yield of chromatid aberrations when cells are exposed to IR during Gsitivity . Howeversitivity  used lym2 /M arrest after IR with no effect on cell survival. Increased sensitivity of d-MMR cells to a number of DNA damaging agents, including high dose-rate IR, was related to inefficient early G2/M checkpoint and decreased DSB repair. In addition to the far different experimental settings between research laboratories, most of these studies used inaccurate outdated methods, such as the cell survival assay; and therefore, more accurate, state-of-the-art cellular and cytogenetic approaches are strongly proposed for updated knowledge in this area.The roles that MMR proteins play in DDR in response to IR remain controversial and some of the conflicting results on the association of MMR proficiency with the radio-sensitivity of cells have been demonstrated and discussed by Martin et al. . BrieflyIt was found from the animal studies that at radiotherapy relevant high doses, IR can induce gastrointestinal or colorectal tumors in d-MMR mice as well as high levels of various types of mutation.Mlh1-knockout mice spontaneously developed gastrointestinal tumors (GIT) and thymic lymphomas by 48 weeks of age. In their study, 2-week or 10-week old Mlh1+/+, Mlh1+/− and Mlh1−/− mice on a C57BL/6 background were exposed to whole-body X-irradiation at 2 Gy (0.7 Gy/min). It was found that irradiation accelerated GIT development in 10-week old Mlh1−/− mice but had little effect at 2 weeks. In contrast, the vast majority of Mlh1+/- and Mlh1+/+ mice were not susceptible to spontaneous or radiation-induced tumorigenesis until 72 weeks after birth. Thus, a potential elevated risk of secondary cancers should be considered for LS patients after radiotherapy.Tokairin et al.  reportedMlh1 gene plays a key role in the development of CRC [Mlh1−/− and Mlh1/++ mice aged 2 weeks or 7 weeks were given a single whole-body X-irradiation of 2 Gy. At 10 weeks, some were treated with 1% dextran sodium sulphate (DSS) in drinking water for 7 days to induce mild inflammatory colitis. In Mlh1/++ mice, no colon tumors were observed after radiation exposure with or without DSS treatment. DSS treatment alone triggered colon tumor development in Mlh1−/− mice, and exposure to radiation prior to DSS treatment increased the number of tumors in these mice.Inflammatory bowel disease frequently accompanied by silenced t of CRC . In the t of CRC  in 2015,Mlh1/++ and Mlh1/−+ mice harbouring MSI were exposed to 1 or 2.5 Gy of γ-rays and 0.1 or 1 Gy of 56Fe ion particles. It was found that allelic deficiency in Mlh1 significantly increased the risk of hematopoietic malignancy, and the loss of Mlh1 function was associated with high levels of single nucleotide mutations, insertions and deletions in resulting tumors. In contrast, tumorigenesis in Mlh1/++ mice was not significantly increased in both types of radiation used. In addition, a significantly higher mean insertion and deletion size (≥ 5 and ≥ 10 base pairs) in all Mlh1/−+ cohorts compared to the Mlh1/++ cohorts may indicate that Mlh1 not only plays a role in mismatch repair but also in DSB repair.In 2019, Patel et al.  reportedMLH1 and MSH2 genes among the inhabitants and the results showed a significant upregulation of MLH1 in the residents compared to the control group; whilst MSH2 expression showed no significant difference between these two groups. Additionally, the expression of both MLH1 and MSH2 was associated with age and gender as well as the length of residency in the area. The authors suggested the triggering of mismatch repair system by natural radiation which may be associated with hormesis effect and adaptive response.The city of Ramsar, in northern Iran, has the highest level of natural background radiation in the world ; however, research on the inhabitants of this area discovered no significant prevalence of radiation-related diseases or cancer compared to those in normal background areas . One stu2 phase in skin fibroblasts and lymphocytes obtained from both affected and unaffected members of a LS family and found that these cells exhibited indistinguishable responses from normal controls. Again, the skin fibroblasts and lymphocytes are more likely to be heterozygous for the MMR genes, and the expected chromosomal aberrations probably can only be detected in homozygous cells and tumour cells.In 1988, Bender et al.  analysedMSH2 mutation. Extra-colonic cancer risk in MSH2 deficient females and males was 69% and 34%, respectively. No difference in colorectal and extra-colonic cancer risks between MLH1 deficient females and males was identified [Mlh1−/− mice [As with many diseases, factors other than genetic predisposition can also complicate the IR associated radio-sensitivity in LS patients. For example, the CRC risk is reported in one study to be 96% in males and 39% in females with entified . Age may−/− mice . However−/− mice .Low-dose IR is currently used routinely across the world for CT staging and surveillance of Lynch patients with CRC and yet there is no published information, guidance or recommendations available to inform clinicians, radiologists or patients about the relative risks in LS patients compared to sporadic CRC patients. In part this may be due to the contradictory evidence presented in this review which leads to no firm conclusions about the risks of low-dose IR. If IR can be confirmed relatively harmless, then the medical teams and patients can feel reassured to continue utilizing staging/surveillance CT; and the extending of CTC to the role as an adjunct to screening colonoscopy can also be investigated for future practice. However, there is no current data to support an increasing role for CTC in Lynch patients and more compelling evidence of safety would be required before this could be considered. Should low-dose IR be considered harmful then alternative methods for staging and surveillance of Lynch patients with CRC could be recommended, for example whole body MRI has been shown to be as accurate as CT in a large multicentre trial of colorectal cancer staging .Similarly, oncologists routinely offer neoadjuvant radiotherapy preoperatively for Lynch patients with advanced rectal cancer frequently without knowing the MMR status of these patients in advance. Moreover, most oncologists will be unfamiliar with the evidence of risk/benefit when using high radiation dose treatment to the tumor and its surrounding pelvic tissues. Therefore, whether therapeutic doses of radiation can cause MMR mutation and lead to secondary tumorigenesis also require urgent investigation.This review considers the literature associated with the potential radiation risks for LS patients. No direct evidence has been found for low-dose radiation risk of CT scans to high-risk patients with hereditary germline mutations, such as LS patients. Studies using LS associated primary cells or tumor cell lines with defective MMR genes at both low and high doses showed contradictory results in terms of cell radio-sensitivity after radiation exposure. However, there seems to be more evidence supporting relative radio-resistance and higher mutation rate for these cells. Results from animal studies showed elevated radiation risk for d-MMR mice potentially reducing the effectiveness of radiotherapy, worsening tumor prognosis and increasing the risk of new cancers in the surrounding tissues.2 assay at low doses.To date, the radio-sensitivity of primary cells or cell lines is mainly determined using the clonogenic survival assay or the expression of apoptotic markers. However, with ethical approval, it may be feasible and beneficial to study radio-sensitivity by directly analysing the peripheral blood lymphocytes and primary tumor cells from clinically and genetically confirmed LS patients using cytogenetic techniques to detect d-MMR associated chromosome and DNA damage induced by IR. For example, premature chromosome condensation coupled with fluorescence in situ hybridization  and γ-H2It may also be beneficial to use genetically modified animal models or human cell lines with d-MMR and expose them to X-rays mimicking the doses of CT scans and radiotherapy. However, further considerations including the group sizes are needed to identify statistically significant effects following such low dose exposures. Furthermore, whether the presence of germline mutations may increase the risks of radiation toxicity or secondary malignancies is a major concern for clinicians. It would be beneficial to carry out retrospective studies to investigate the risk of somatic and germline MMR mutations and subsequent cancer rates and relate these results to previous CT surveillance and neoadjuvant radiotherapy in a large cohort of CRC patients, taking into consideration the MMR and MSI status, prognosis, gender, and age, etc.Furthermore, quantification for mitochondrial DNA (mtDNA) mutations and deletions could represent a potential biomarker for radiation risks in LS patients. In 2020, Borghini et al.  reportedIn conclusion, there is no current data addressing the risks of using IR for the diagnosis, staging, surveillance and treatment of LS patients and the existing knowledge in this area is outdated. Therefore, further research using the cutting-edge technologies is urgently required to provide the essential information for clinical guidance.


A high degree of deformability of RBCs is required to accommodate the size of micro-vessels with diameters significantly lower than RBCs. The potential therapeutic role of high-density lipoproteins (HDL) in the removal of cholesterol and its activity regarding maintenance of an optimal concentration of RBC membrane cholesterol have not been well investigated. On the contrary, the focus for HDL research has mainly been on the clearance of cholesterol accumulated in atherosclerotic macrophages and plaques. Since all interventions aiming at decreasing cardiovascular diseases by increasing the plasma level of HDL cholesterol have failed so far in large outcome studies, we reviewed the potential role of HDL to remove excess membrane cholesterol from RBC, especially in sickle cell disease (SCD). Indeed, abundant literature supports a consistent decrease in cholesterol transported by all plasma lipoproteins in SCD, in addition to HDL, low- (LDL) and very low-density lipoproteins (VLDL). Unexpectedly, these decreases in plasma were associated with an increase in RBC membrane cholesterol. The concentration and activity of the main enzyme involved in the removal of cholesterol and generation of large HDL particles—lecithin cholesterol ester transferase (LCAT)—are also significantly decreased in SCD. These observations might partially explain the decrease in RBC deformability, diminished gas exchange and tendency of RBCs to aggregate in SCD. We showed that incubation of RBC from SCD patients with human HDL or the HDL-mimetic peptide Fx5A improves the impaired RBC deformability and decreases intracellular reactive oxygen species levels. We propose that the main physiological role of HDL is to regulate the cholesterol/phospholipid ratio (C/PL), which is fundamental to the transport of oxygen and its delivery to peripheral tissues.Cell membrane lipid composition, especially cholesterol, affects many functions of embedded enzymes, transporters and receptors in red blood cells (RBC). High membrane cholesterol content affects the RBCs’ main vital function, O It has been proposed  that dur2 appears to be regulated by membrane cholesterol content and membrane protein gas channels over a wide range spanning 2 to 3 orders of magnitude. Subczynski -cholesterol and then incubated them for 24 h with increasing concentration (0.1 to 100 μg protein/mL) of human HDL or Fx5A. A dose-dependent efflux of labeled [14C]-cholesterol was measured after incubation with both HDL and Fx5A (data not shown) confirming that both peptides do remove cholesterol from RBC.ApoA1, the major protein of HDL, contains a tandem array of amphipathic helices with varying lipid affinity. Fx-5A is produced by combining sphingomyelin with the 5A peptide, which is a bihelical ApoA1-mimetic peptide in which five hydrophobic amino acids are substituted for alanine in the second helix . Fx5A cag at 20 °C). RBCs were washed in phosphate-buffered saline (PBS) and resuspended in PBS at a final hematocrit of 20%. Then, they were incubated at 37 °C for 40 min with 100 μg/mL of human HDL isolated from human plasma by ultracentrifugation [RBCs were incubated with HDL to measure its effect on deformability. Blood obtained from 7 healthy controls (HbAA) and 12 HbSS patients was collected in EDTA tubes and immediately centrifuged  in citrate tubes. RBCs were processed as above and incubated at 37 °C for 40 min with 100 μg/mL of Fx5A or with PBS (control condition). RBCs were washed and incubated for 20 min with 10 uM of DCFDA. Then, the samples were analyzed by flow cytometry, as described above.t-test for paired samples or one-way ANOVA for repeated measures completed by Tukey’s post-hoc tests, when appropriate. Comparisons between HbAA and HbSS subjects were performed using Student’s t-test. A p-value < 0.05 was set for statistical significance.Statistical analysis: data are presented as means +/− SEM. The effects of incubation on RBC deformability and ROS levels were analyzed using Student’s As expected, RBCs from HbSS patients were less deformable than those of HbAA patients at both shear stress levels A–D. RBC p < 0.05) in RBCs from HbSS vs. HbAA subjects. RBC incubations with HDL did not affect ROS levels in HbAA  and 30 Pa (p < 0.01) at both tested doses (100 μg/mL and 200 μg/mL) (The ApoA1 mimetic Fx5A significantly increased HbSS RBC deformability at 3 (0 μg/mL) A,B. A cl0 μg/mL) .In conclusion, both HDL and the ApoA1 mimetic peptide Fx5A can modulate the deformability and level of ROS in RBCs from SCD (SS) patients in vitro. It is noticeable that L4F, another ApoA1 mimetic peptide, has been shown to be beneficial in a mouse model of SCD .Additional studies aiming to quantify the concentration dependence of cholesterol in the membranes of RBCs of SCD (HbSS) patients following incubations with HDL and to determine the concentrations of HDL and Fx5A necessary to normalize RBC membrane cholesterol and RBC deformability are recommended.SCD appears to be characterized by low plasma cholesterol both in HDL and LDL/VLDL lipoprotein fractions with a simultaneous increase in RBC membrane cholesterol, suggesting an inefficient clearance of excess membrane cholesterol. The changes in the RBC membrane very likely affect RBC deformability and ROS levels. Our data suggest that HDL or HDL mimetics may be beneficial to patients suffering from SCD and its microvascular complications.The role of HDL and therapeutic interventions aiming at raising plasma HDL mainly addressed cardiovascular diseases associated with high plasma cholesterol and atherosclerotic plaque regression. Similarly, the majority of atherothrombosis studies are focused on leukocytes. The demonstration that RBCs can enter the arterial wall during the earliest stages of atheroma and are phagocytosed by smooth muscle cells—a pathogenic mechanism enhanced by hypercholesterolemia, endothelial injury and RBC senescence ,63—turneThis review highlights the relationship between HDL formation through LCAT activity and clearance via the SRB1 receptor and RBC physiology and pathology. The potential link between HDL and RBC membrane cholesterol and RBC functions may be exacerbated in SCD and may have implications for more frequent diseases such as T2D and ACS.Low HDL levels, high RBC membrane cholesterol, high blood viscosity  and low Li et al.  reviewedp < 0.001). In a small group of diabetics with lipid levels normalized with hypolipidemic treatments [Tziakas’s team is one of the most active research groups investigating cardiovascular diseases and RBC membrane cholesterol. They showed for the first time that RBC membrane cholesterol is significantly higher in patients with acute coronary syndrome (ACS) compared with chronic stable angina patients ,76. Theieatments  and in aeatments , an incr2. Thus, we believe, as proposed by Pavlaki et al. [As one would expect, patients with both SCD (or the sickle cell trait) and diabetes have a higher incidence of diabetes-related complications, including retinopathy, nephropathy and hypertension ,81. Oxidi et al. , that fu


To assess the accuracy of preoperative sonographic staging for prediction of limited axillary disease  and to identify factors associated with high prediction–pathology concordance in patients with early-stage breast cancer meeting the Z0011 criteria.Patients treated between January 2015 and January 2020 were included in this retrospective, multicentric analysis of prospectively acquired service databases. The accuracy of LAD prediction was assessed separately for patients with one and two suspicious lymph nodes on preoperative sonography. Test validity outcomes for LAD prediction were calculated for both groups, and a multivariate model was used to identify factors associated with high accuracy of LAD prediction.p ≤ 0.01].Of 2059 enrolled patients, 1513 underwent sentinel node biopsy, 436 primary and 110 secondary axillary dissection. For LAD prediction in patients with one suspicious lymph node on preoperative ultrasound, sensitivity was 92% (95% CI 87–95%), negative predictive value (NPV) was 92% (95% CI 87–95%), and the false-negative rate (FNR) was 8% (95% CI 5–13%). For patients with two preoperatively suspicious nodes, the sensitivity, NPV, and FNR were 89% (95% CI 84–93%), 73% (62–83%), and 11% (95% CI 7–16%), respectively. On multivariate analysis, the number of suspicious lymph nodes was associated inversely with correct LAD prediction ([OR 0.01 (95% CI 0.01–0.93), Sonographic axillary staging in patients with one metastatic lymph node predicted by preoperative ultrasound showed high accuracy and a false-negative rate comparable to sentinel node biopsy for prediction of limited axillary disease. As the mere preoperative identification of axillary disease was no longer sufficient to triage patients with early-stage breast cancer to appropriate axillary surgical treatment, a clinical need for preoperative quantification of the extent of axillary disease arose. Consecutively, preoperative breast imaging studies focused on the distinction of patients with limited axillary disease  who would not undergo ALND after a positive sentinel due to the lack of therapeutic implication from patients with extensive axillary disease  who would benefit from ALND. These studies revealed a correlation between the number of abnormal lymph nodes identified by preoperative axillary ultrasound and the number of metastatic nodes on final pathology.9 Given these findings, the recent NCCN Clinical Practice Guidelines in Oncology (NCCN guidelines) advise consideration of SNB for patients meeting the Z0011 criteria in whom two or fewer suspicious lymph nodes are found on preoperative imaging, even when one node shows biopsy-proven positivity.10 If limited histopathological axillary disease could be identified safely by preoperative ultrasound and core needle biopsy, SNB may constitute overtreatment and omission of SNB in this setting due to a lack of consequence in selected patient populations might be an approach for further scientific evaluation. The purposes of this study were to assess the accuracy of preoperative sonographic axillary staging for prediction of LAD  in patients with early-stage breast cancer meeting the Z0011 criteria, who underwent ALND, and to identify factors associated with high concordance between sonographic prediction and histopathology.The approach to axillary surgery for patients with breast cancer has evolved tremendously over recent decades, leading to significant changes in the role of preoperative axillary imaging.The study was approved by the institutional review committee and met the guidelines of their responsible governmental agency. The requirement for participants’ written informed consent was waived. All patients treated for primary breast cancer between January 2015 and January 2020 at one of the two study sites were identified retrospectively from prospectively maintained breast service databases. All patients who underwent preoperative ultrasound including axillary staging and surgery at one of the participating sites were eligible for inclusion in this study. Patients who completed neoadjuvant chemotherapy before axillary surgery were excluded. Other exclusion criteria were pathological T3/T4 or metastatic disease and incomplete clinical data. Breast cancer subtypes were defined by immunohistochemistry (IHC) as luminal A , luminal B , HER2/neu positive (positive/negative expression of estrogen or progesterone receptor and HER2/neu overexpression (defined as 3+ expression or 2+ expression by IHC and a ratio of ≥ 2.0 by fluorescence in situ hybridization)), and triple negative (absence of estrogen and progesterone receptor expression and lack of HER-2/neu overexpression (defined as 1+ expression or 2+ expression by IHC and a ratio of < 2.0 by fluorescence in situ hybridization)). Data used in this study, including treatment and outcome data, were extracted from the patients’ medical records.13 In cases of sonographic suspicion of axillary nodal involvement , axillary biopsy (core needle biopsy) of the most suspicious node was performed.15 The number of axillary nodes suspected to be metastatic on ultrasound examination was recorded. Patients with suspicious lymph nodes other than axillary lymph nodes were excluded from the analysis.Four breast imaging professionals with 5–25 years of experience in breast imaging performed the preoperative ultrasound examinations independently using Voluson E8/10  and Hitachi Hi Vision Ascendus  devices equipped with 5–12 MHz and 13–3 MHz linear-array transducers. These examinations were performed a median of 13 (range 8–27) days before surgery. According to institutional standard, sonographic evaluation of the axilla was performed routinely in all breast cancer patients as part of the diagnostic workup and included standardized assessment of level I, II, III, internal mammary, supra-, and infraclavicular lymph nodes.Preoperative axillary staging was defined as positive when preoperative ultrasound showed suspicion of axillary nodal involvement and this suspicion was confirmed by percutaneous biopsy. Negative preoperative axillary staging was defined as either absence of nodal involvement on preoperative ultrasound or suspicion of axillary nodal involvement on preoperative ultrasound but negative axillary core needle biopsy.15Patients with negative preoperative axillary staging proceeded to sentinel node biopsy. Patients with positive preoperative axillary staging proceeded to ALND. Secondary ALND was also performed in all patients with positive SNB findings. LAD was defined as presence of one or two metastatic axillary lymph nodes. EAD was defined as presence of three or more metastatic axillary lymph nodes. Pathological staging was performed according to the 8th edition of the American Joint Committee on Cancer’s cancer staging manual.Preoperative sonographic axillary findings were compared with final pathology for prediction of absence of nodal involvement (N0) in patients undergoing SNB and for number of metastatic lymph nodes in patients undergoing ALND. Patients with preoperative suspicion of LAD were assessed separately. The accuracy of preoperative sonographic axillary staging for identification of patients with histopathological LAD  was assessed separately in patients with ultrasound-detected LAD undergoing ALND.This group was further divided according to number of predicted metastatic lymph nodes (one or two) on preoperative ultrasound. The first group (group 1) consisted of patients with one suspected metastatic lymph nodes on preoperative ultrasound and ≤ 2 metastatic lymph nodes on final pathology, and the second group (group 2) of patients with sonographic suspicion of two metastatic lymph node and ≤ 2 metastatic lymph nodes in final pathology.P values < 0.05 were considered significant. G.W. and A.K. performed the statistical analysis using SPSS .The accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-negative rate (FNR), and false-positive rate (FPR) were calculated for the sonographic prediction of N0 disease in patients undergoing SNB and for the two groups with LAD  undergoing ALND. A negative test outcome was defined as LAD on final pathology. For assessment of sonographic prediction of N0 disease, the FNR was defined as negative preoperative axillary staging but positive SNB, and FPR as positive preoperative axillary staging but negative findings on ALND. For prediction of LAD, FNR was defined as prediction of LAD on preoperative sonography but EAD on ALND, and FPR as prediction of EAD on preoperative sonography but LAD on ALND. For assessment of age-dependent variations of accuracy of axillary sonographic staging, the performed analyses were additionally conducted for the subgroup of patients ≥ 70 years. A multivariate model was used to identify factors associated with high accuracy of axillary sonographic staging in terms of correct identification of patients with LAD. We used the chi-squared test for univariate analysis and stepwise binary logistic regression for multivariate analysis (MVA). Variables that were significant on univariate analysis were included as covariates in the MVA. n = 81), final pathological staging of T3/T4 or metastatic disease (n = 49), and incompleteness of clinical data (n = 31), leaving 2059 patients for final analysis  were BRCA1/2 positive, 31 (1.5%) were negative, and 2018 (98%) were not tested. The results showed that 1165 (57%) patients were diagnosed with T1 and 894 (43%) with T2 breast cancer.Of 2220 patients, 161 were excluded due to performance of axillary surgery after completion of neoadjuvant chemotherapy (n = 1550 (75%)] had invasive carcinoma of no special type (NST) histology. Based on immunohistochemical analysis, 919 (45%) patients had luminal A, 735 (35%) patients had luminal B, 189 (9%) patients had HER2/neu positive, and 216 (11%) patients had triple-negative breast cancer. Regarding type of breast and axillary surgery, 1433 (70%) patients had breast-conserving therapy and 626 (30%) mastectomy, 1513 (74%) underwent SNB, 436 (21%) ALND, and 110 (5%) ALND after positive SNB. The median number of lymph nodes removed was 2 (range 1–11) [SNB] and 10 (range 1–40) [ALND], and the median number of metastatic lymph nodes removed was 0 (range 0–7) [SNB] and 0 (0–35) [ALND].On preoperative axillary sonography, 1623 (79%) patients showed no suspicious axillary nodes and 436 (21%) patients showed suspicious axillary nodes on ultrasound, which were proven to be metastatic by core needle biopsy. Of these 436 patients, 178 (41%) appeared to have EAD (three or more lymph nodes) and 258 (59%) appeared to have LAD (one or two lymph nodes) on preoperative ultrasound. On final pathology, 1513 (74%) patients who underwent SNB had no axillary metastatic lymph node (N0) and 546 (26%) patients who underwent ALND had positive axillary nodes (N1–3). Most patients , sensitivity [90% (95% CI 82–95%)], specificity [80% (95% CI 59–93%)], and NPV [67% (95% CI 57–83%)] decreased while the FNR [10% (95% CI 5–18%)] and FPR increased [20% (95% CI 7–41%)] , p ≤ 0.01], G3 , lymphovascular invasion , and the number of suspicious lymph nodes on preoperative sonography . On multivariate analysis, including these parameters, the number of suspicious lymph nodes on preoperative ultrasound was the only parameter that correlated inversely with correct prediction of LAD . Similar results were obtained for the subgroup of patients ≥ 70 years , 11 However, the FNR in the present study might have greater clinical implications since it means that these patients had EAD, whereas the FNR in SNB patients only means these patients have additional nodal disease. The actual percentage of false-negative SNB patients with EAD is not known, but in the original Z0011 population only 27.3% patients who underwent ALND after a positive SNB had additional metastasis in lymph nodes removed by ALND, and 21% of these had EAD.5In this retrospective analysis of a prospectively maintained service database, we found high rates for preoperative identification of limited axillary disease (LAD) defined as one or two metastatic nodes on final pathology, in patients with early-stage breast cancer in whom one metastatic lymph node was predicted on preoperative ultrasound and who underwent ANLD. With an FNR of 8% for this prediction, these findings are comparable to those of SNB (5–9%).16 Although the authors did not provide statistical measures on test validities, they reported a high probability of having two or fewer metastatic lymph nodes on final pathology in patients with one abnormal node on preoperative axillary sonography and concluded that these patients should be offered SNB.16 Large clinical trials have shown that systemic therapy decisions for postmenopausal women with early-stage, hormone receptor-positive breast cancer and LAD should be based on genomic assays, rather than on the number of affected lymph nodes.19 In these patients, the benefit of identifying additional metastatic lymph nodes by SNB after preoperative image-based LAD confirmation seems questionable. Given this lack of additional therapeutic implications and the low FNR rate for patients with one preoperatively suspicious lymph node in this study, SNB omission might be an option worth considering in these patients. However, our results indicate that this approach cannot be recommended for patients with up to two suspicious lymph nodes on preoperative sonography. Although 89% of patients with histopathologically confirmed LAD were correctly identified via ultrasound, 11% of patients with one or two suspicious nodes on preoperative sonography were diagnosed false negative, and axillary surgery omission would have constituted undertreatment. Similarly, accuracy of LAD prediction in patients fulfilling the Z0011 criteria seemed to be dependent on the number of suspicious nodes identified by preoperative sonography in several smaller studies.16 In our analysis, the number of preoperatively suspicious lymph nodes was the only factor associated with correct prediction of LAD. Our findings add to accumulating evidence demonstrating an association between an increasing number of suspicious lymph nodes on preoperative ultrasound and a higher number of metastatic nodes on final histology.24 Thus, and following widespread implementation of the Z0011 approach into clinical practice, many imaging studies have focused on preoperative extensive axillary disease and reported PPVs of up to 92.9%, supporting the high accuracy of prediction of heavy nodal burden, and imply that these patients can safely proceed to axillary dissection without SNB.26Two smaller studies have demonstrated the feasibility of preoperative LAD prediction.A collective in whom the benefit of SNB has been the focus of current clinical research are patients with clinical node-negative disease on preoperative imaging. As these patients represent more than 70% of primary breast cancer patients, omission of SNB in these patients would be a significant contribution to deescalation of axillary surgery.27In this study, 20% of preoperative sonographic N0 diagnoses were false negative. Similar results were reported in a metaanalysis assessing preoperative sonographic staging of the axilla including ultrasound-guided biopsy of suspicious nodes in 9212 patients with primary breast cancer. The authors found a sensitivity of 50% and an FNR of 25% regarding prediction of nodal involvement, concluding that preoperative axillary staging could not replace SNB in these patients.30 A National Cancer Database study compared patients aged ≥ 70 years, with clinical negative axilla, where 99,764 patients underwent axillary surgery and 31,531 did not. Beside an improved overall survival in the group of patients who were treated with axillary surgery, the authors found 14% of the clinical node-negative patients to be node positive on final pathology, which is well in line with our false-negative rate of 16%.31 As these sonographically N0 patients would not receive postoperative axillary radiation, SNB omission might lead to undertreatment in these cases. Several ongoing studies are exploring this issue and will improve the selection of elderly patients in whom omission of axillary surgery can be safely performed and help to identify clinical features that refine a low-risk collective in this setting.33The use of SNB in older patients has been questioned lately, and current recommendations advise that SNB in patients ≥ 70 years with early-stage, hormone receptor-positive, HER2-negative breast cancer and no palpable axillary lymph node treated with endocrine therapy can be considered individually with regard to its impact on radiation recommendations and systemic therapy decisions.5The generalizability of our findings is limited, given the variability in ultrasound image acquisition and interpretation, although the inclusion of data from two centers increases their reliability. The transfer of the findings to other populations has to be done cautiously as the collective assessed mostly comprised screening patients, with low rates of axillary nodal burden and moderate numbers of involved lymph nodes. However, the therapeutic consequences identified in the Z0011 trial are transferable to our sample, which was similar to the Z0011 population (medians of two and one metastatic lymph nodes).In patients with early-stage breast cancer fulfilling the Z0011 criteria with one abnormal axillary lymph node identified by preoperative sonography who underwent ALND, the prediction of limited axillary disease on final pathology was highly accurate, with a false-negative rate comparable to that of sentinel node biopsy. These findings imply that omission of sentinel node biopsy might be an option that can be considered in these patients. With the ability to correctly distinguish limited from extensive nodal disease, axillary imaging represents a key clinical decision-making tool for management of the axilla and can be used to further individualize and deescalate surgical staging.


To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR).+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed.Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05).EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na Hamlyn et al.,+-K+-ATPase activity, promote the excretion of sodium from the kidneys and cross-react with digoxin antibody. EO also demonstrated the cardiotonic, diuretic, vasoconstrictive, and other digitalis-like effects. The vascular effect of EO includes inhibition of the Na+-K+-ATPase α2 subunit that increases intracellular Na+, which promotes Ca2+ entry into the intracellular by reducing the activity of the sarcolemmal Na+/Ca2+ exchanger, and thereby increases the vascular smooth muscle tone+ transport across the tubular cell by activating the basolateral Na+-K+-ATPase α1 subunit, ultimately contributing to increase blood pressure (BP)Endogenous ouabain (EO) was first identified in human plasma in 1991+-K+-ATPase in cell membrane, which results in altered gene expression of Na+-K+-ATPase α-subunit and enzyme conformation responsible for its activitiesDisorders of water and sodium metabolism are the pathophysiological basis of essential hypertension,,-Sympathetic nerve fibers innervate all organs that are involved in cardiovascular control, such as the heart, peripheral blood vessels and, perhaps most importantly in the current context, the kidneys. The sophisticated network of efferent sympathetic and afferent sensory nerve fibers residing in the kidney and their signaling pathways provide the basis for the modulating influence of central integrative structures in the brain stem on renal effectors contributing to BP control+-K+-ATPase α1 and β1 subunits in spontaneously hypertensive rats (SHR), and further to explore the role of renal sodium handing treatment by RDN.The clinical implication and perspectives of RDN-based hypertension management strategy have caught the attention of many scientific groups. Animal studies pointed to increase EO content triggering sympathetic nerve activity elevation and evoking hypertensionAll procedures were performed following the Animal Ethics Committee guidelines of Guangxi Medical University and had approval. Ten-week-old male spontaneously hypertensive rats  and age-matched Wistar-Kyoto rats , normotensive controls, were purchased from Vital River Laboratories Co., Ltd. . Specifically, after two weeks of adaptive feeding, the SHR were randomly divided into two groups, the renal denervated group  and sham operation group . Animals were maintained in standard laboratory animal housing conditions.After sterilization, a midline incision was made. Then, bilateral RDN was induced by painting the renal vessels with 10% phenol under 2% pentobarbital sodium anesthesia  in RDNX group. While the sham group and the WKY group underwent the same procedure as the RDNX group, the renal vessels were painted with normal saline. Two weeks after surgery, under ketamine anesthesia , blood was collected via the abdominal aorta, and the kidneys, adrenal glands and hypothalamus were removed and stored at -80 °C until analysis. Furthermore, the effectiveness of RDN had already been testified in a previous examination from our laboratory125I RIA kit. The intra- and inter-assay coefficients of variation, respectively, were less than 10 and 15% for EO.After blood collection from the abdominal aorta, the blood samples were allowed to stand at room temperature for 30 min. Then, the serum was separated at 1,500 rpm for 10 min. Tissue samples were thawed and minced, and 2% homogenate was made by adding appropriate amount of saline on ice and centrifuged at 2,500 rpm for 10 min to extract the supernatant. The concentrations of EO in serum and tissue were determined using the radioimmunoassay method with an +-K+-ATPasewas determined in the supernatant spectrophotometrically with the corresponding kit . Enzyme activity was expressed as micromoles of Pi released per hour per milligram of protein.The kidneys were thawed, weighed, and homogenized in ice-cold saline to 10%, and the homogenates were centrifuged at 2,500 rpm for 10 min at 4 °C. Then, 200 μL of supernatant was extracted and diluted into 2% tissue homogenate by adding 800 μL of saline. The concentration of proteins was measured by Coomassie brilliant blue method. The activity of Na® Premix Ex Taq™ Kit (TaKaRa), under universal cycling conditions . The products were confirmed by appropriate size by agarose gel electrophoresis. The samples were assayed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control to normalize the transcript levels, and data were analyzed according to the comparative threshold cycle method.RNA was extracted from kidneys using RNAsimple Total RNA Kit (Tiangen Biotech). Total RNA was reverse transcribed using a cDNA Synthesis Kit (Takara) to acquire cDNA. The sequences for the specific primers used are shown in 2O2 for 5 min to suppress endogenous peroxidase activity after heating in citrate buffer sodium (pH 6) and blocked with 10% normal goat serum for 10 min to prevent nonspecific binding. Next, the slides were incubated with anti-Na+-K+-ATPase antibody for 3 h at room temperature in a humidified chamber. All sections were incubated with SP-9000 reagent for 20 min at 37 °C. Immunoreactivity was observed with 3,3’diaminobenzidine reagent and applied with Harris hematoxylin to counterstain. All kidney sections were washed, dried, and mounted. Images were acquired using Digital Microscope Imaging System  and expressed by mean density using computer-assisted image analyzer system (IPP 6).In brief, kidney slices (3 μm) were deparaffinized in xylene and dehydrated in a graded series of ethanol after formalin fixation and paraffin embedding. The sections were incubated in 3% HResults are provided as means ± standard error and processed with SPSS 16.0 statistical software . Differences were determined using analysis of variance (ANOVA) when more than two groups were compared followed by the Student-Newman-Keuls-q test or Dunnett-t’s test. Pearson correlation analysis was used to evaluate the relationship between variables. Significance was declared at p < 0. 05.As shown in +-K+-ATPase activity of renal tissue homogenate was significantly increased in the sham group compared with the WKY group . Na+-K+-ATPase activity was slightly higher in RDNX group than in WKY group, but the difference was not significant (p > 0.05). Results of the above experiments indicated that RDN significantly decreased Na+-K+-ATPase activity of renal tissue. Data on norepinephrine (NE) renal tissue content was as previously reported+-K+-ATPase activity of kidney , as shown in +-K+-ATPase activity in kidney may have been upregulated by NE. However, the correlation analysis showed no statistical difference between EO levels and Na+-K+-ATPase activity in the kidney (P = 0.93).As shown in +-K+-ATPaseα1 subunit in the renal tissue . In addition, the mRNA of Na+-K+-ATPase α1 subunit was markedly downregulated under chemical sympathectomy conditions compared to sham group with intact renal innervation (P < 0.05). A similar decrease was detected in the WKY group (P < 0.05). However, there was no significant difference in mRNA of Na+-K+-ATPase β1 subunit between groups (P = 0.63).As shown in +-K+-ATPase staining in the kidneys of the experimental groups was illustrated as shown in +-K+-ATPase proteins in the renal cortex, outer stripe and inner stripe of the outer medulla as compared to sham group , while RDNX group was not significantly different from WKY group (p > 0.05). Sham and WKY groups showed significant differences for the expression of Na+-K+-ATPase proteins in the renal cortex, outer stripe and inner stripe of the outer medulla .With positive expression as cytoplasm into brownish-yellow staining, the mean density of Na+-K+-ATPase activity in SHR. The main findings of our study are as follows: first, EO levels significantly reduced after decreasing renal sympathetic nerve activity in SHR kidneys. Second, renal denervations decreased Na+-K+-ATPase activity of kidney, the expression of Na+-K+-ATPaseα1 subunit mRNA of kidney and the expression of Na+-K+-ATPase proteins in the renal cortex, outer stripe and inner stripe of the outer medulla in SHR compared with the sham group. Third, we observed a positively correlation between NE content and Na+-K+-ATPase activity. However, the correlation analysis showed no statistical difference between Na+-K+-ATPase activity and EO levels in the kidney. EO has high intrinsic affinity and selectivity for the Na+-K+-ATPase by interacting with the cardenolide receptor on the Na+-K+-ATPase+-K+-ATPase in the kidney. Taken together, these new results speculate that sympathetic nerve is involved in the regulation of Na+-K+-ATPase activity through reduction of hypothalamic EO secretion, thus playing an important role in the water and sodium balance.In this study, we explored whether sympathetic nerve involve water and sodium balance by regulating EO secretion and kidney Na,,,et al.EO can regulate the sodium pump on the membrane of central nervous system (CNS), heart, blood vessel wall and renal tubules, and EO functions as regulator of water and sodium metabolism and blood vessel wall tension, which indicates that EO is essential to the pathogenesis of hypertensionet al.et al.EO elevated BP by inhibiting the reuptake of NE from the perivascular adrenergic nerve endings and increasing sympathetic nerve activityet al.et al.EO is closely associated with the metabolism of water and sodium in hypertension. EO-induced modulatory pathway may be the key roles resulting to the long-term elevation of BP in central and peripheralet al.+ diet. In addition, our previous studies showed that RDN improved impaired sodium excretion metabolism, delayed glomerular damage and reduced renal vascular resistance in RDNX group compared with the Sham groupThese findings indicated the mutually independent and compensatory relationship between RAAS and EO in water and sodium metabolism. Leenen et al.The hypothalamus and adrenal glands are the main sites of EO synthesis and secretion+-K+-ATPase pumped sodium ions outside and potassium ions inside living cells, converting chemical energy into work. EO existing in human circulation has high intrinsic affinity and selectivity for the Na+ pump by interacting with the cardenolide receptor on the Na+ pump,,+-K+-ATPase incorporates in the cell membrane, which obtains energy through ATP hydrolysis and transports ions depending on an inverse electrochemical gradient to regulate intracellular sodium-potassium homeostasis, cell membrane excitability and metabolism+-K+-ATPase activity.Na+-H+exchange (NHE) in the kidney+-K+-ATPase and NHE1 activity, as well as NHE1 is required for the ouabain-induced increase in BP+-K+-ATPase and NHE1 activities on renal tubular was involved in endogenous ouabain-like factors stimulated hypertensionet al.+-K+-ATPase in renal tubules. The activity of Na+-K+ -ATPase was upregulated after oral administration of EO inhibitor (PST 2238) at the dose of 10 mg/kg/day for four weeks in rats with hypertension. After five days with EO inhibitor (PST 2238), the expression of Na+ -K+ -ATPase was increased in renal tubular epithelial cells.Our previous study showed that the renal sympathetic nerve may play an important role in the regulation of Na-A series of clinical trials had evidenced efficacy and safety of RDN to treat hypertension+-K+-ATPase play an important role in hypertension and related organ complications. The EO content and Na+-K+-ATPase activity were both reduced after treatment through RDN, indicating that the hypertensive patients may benefit from RDN. However, extensive clinical trials were required to solve existing problems including patient selection criteria and the indicators for evaluating RDN efficacy. So, RDN may be a viable option and a promising approach for hypertension in the future.Several experimental and clinical studies provide insights into the notion that EO and Na


Mesenteric desmoplasia in small intestinal neuroendocrine neoplasms (SINENs) is associated with increased morbidity and mortality. In this paper, we discuss the development of desmoplasia in SINENs.The fibrotic reactions associated with these tumours could be limited to the loco-regional environment of the tumour and/or at distant sites. Mesenteric fibrotic mass forms around a local lymph node. Formation of desmoplasia is mediated by interactions between the neoplastic cells and its microenvironment via number of profibrotic mediators and signalling pathways. Profibrotic molecules that are mainly involved in the desmoplastic reaction include serotonin, TGFβ (transforming growth factor β) and CTGF (connective tissue growth factor), although there is some evidence to suggest that there are a number of other molecules involved in this process.Desmoplasia is a result of autocrine and paracrine effects of multiple molecules and signalling pathways. However, more research is needed to understand these mechanisms and to develop targeted therapy to minimise desmoplasia. Desmoplasia could be defined as formation of fibrous connective tissue and forms a key component of solid tumour stroma , 2. NENsA study involving 824 patients with SINENs showed that 88% of all participants had mesenteric lymph node metastases. Among 538 patients with stage IV SINENs in this study had fibrosis associated with mesenteric nodal metastases which were detected among 50% of the participants [Small intestinal NENs (SINENs) are derived from the enterochromaffin cells and may give rise to carcinoid syndrome , 7 and rMesenteric desmoplasia is known to cause intestinal obstruction due to fibrotic adhesions or from mass effect. In addition, superior mesenteric vessels could be effected with arterial involvement causing small bowel ischaemia and superior mesenteric venous occlusion leading to ascites, small bowel venous ischaemia and varices , 10. It Desmoplasia is radiologically diagnosed through its characteristic spoke wheel appearance with linear soft tissue opacities radiating outward from a central mesenteric mass , 9. The This paper elaborates the current evidence on pathogenesis of desmoplasia in SINENs with an emphasis on tumour microenvironment, molecular mediators and downstream intracellular signalling pathways.It is believed that the nidus of mesenteric desmoplasia is a small bowel lymph node. Desmoplasia is the result of complex interactions mediated via profibrotic and anti-fibrotic factors between the SINEN cells and the tumour microenvironment . These mNENs do not only comprise proliferating cancer cells, but also consist of a plethora of heterogeneous non-cancerous cells and a non-cellular matrix which composite the tumour microenvironment •, 12–14.The tumour microenvironment consists of stromal cells, extracellular matrix (ECM), endothelial cells and inflammatory cells •, 12–14.The stromal cells in the tumour microenvironment predominately consist of fibroblasts. However, these fibroblasts are of a modified activated phenotype and are best named as cancer-associated fibroblasts (CAF) ••, 17, aSINENs demonstrate a high rate of α smooth muscle actin expressing fibroblasts than the other NENs ••, 17. AThe ECM consists of a complex network of crosslinked collagens, glycoproteins and water that continuously undergoes remodelling through enzymatic and non-enzymatic post-translational changes , 18. TheThe inflammatory cells in the tumour microenvironment is known to contain a large number of tumour-associated macrophages which effectively supress the immune system and promote tumour growth and fibrosis by secreting profibrotic factors ••, 20. ISINENs originate from the enterochrromafin cells of the diffuse endocrine system and secrete various amines and peptides promoting the tumour growth via autocrine and paracrine effects , 7. SomeSerotonin (5 HT) is an amine derived from the amino acid tryptophan and is produced in SINENs and excreted by the body predominantly in the urine in the form of 5 hydroxyindole acetic acid (5 HIAA) , 6. SeroSvejda and colleagues developed an in vitro co-culture model containing small intestinal neuroendocrine tumour cells (KRJ-I) , which wTransforming growth factor-β (TGF-β) is also a pro-fibrotic agent that is secreted by the SINENs ••, 27. AConnective tissue growth factor (CTGF) is a downstream mediator of TGF-β and serotonin  and it iCTGF may enhance profibrotic actions of TGF-β, FGF and EGF by stimulating fibroblast proliferation, collagen production and differentiation of fibroblasts into α smooth muscle actin producing fibroblasts. The location of these CTGF-secreting neoplastic cells was identified close to the α smooth muscle actin producing fibroblasts which could also suggest a potential role of CTGF to desmoplastic reaction of SINENs , 18. MorPlatelet-derived growth factor (PDGF) is a strong profibrotic agent and exerts its effect via PDGF-α and PDGF-β receptors and also stimulates the proliferation of fibroblasts. In SINENs, PDGFβ receptors are found in 65–85% of the cases and the receptors are probably on α smooth muscle actin producing fibroblasts but not on tumour cells. Evidence suggests that the tumour cells and the macrophages in the tumour microenvironment induce PDGFRβ expression on adjacent fibroblasts and upregulate PDGFRβ activity in a paracrine fashion promoting PDGF-mediated fibrogenesis .Transforming growth factor α (TGF α) is a peptide related to epithelial growth factor (EGF) and is known to express its effects via the EGF receptors. While it is overly expressed by the SINEN cells (> 90%), it also plays a crucial role in fibrosis and induce tumoural angiogenesis and proliferation. TGF-α produced by tumour cells acts on fibroblasts via the EGF receptors located on the fibroblasts ••, 30.Fibroblast growth factor (FGF) type 2/acidic FGF is known to be expressed by the SINEN cells and the adjacent stromal cells indicating its possible role in fibrosis , 31. HowInsulin-like growth factors (IGFs) 1 and 2 are known to increase the proliferation of fibroblasts in chick embryos, mammals and humans . These gSubstance P (SP) is tachykinin that is secreted by SINENs and is a known mediator of carcinoid syndrome . In addiNerve growth factor (NGF) is known to potentiate effects of TGF-β and is considered a profibrotic agent. SINENs are known to express NGF , 40. In Histamine produced by the SINENs is considered as a mediator of carcinoid syndrome  and is aWhile the above details express the potential roles of individual profibrotic growth factors in the development of desmoplasia, there is developing evidence to suggest that complex cross-talk between the above molecules and post-receptor signalling intracellular cascades is involved in the development of SINEN-associated desmoplasia ••, 42. IMoreover, cancer-associated fibrosis is known to be mediated via complex intracellular signalling pathways namely Hedgehog (Hh), Notch and Wnt/β-catenin and it is worth to discuss the evidence for these complex pathways in the context of SINEN-associated desmoplasia ••, 44.Hh cascade is activated by several ligands  which bind to Patch receptors stimulating production of transcription factors (Gli 1–3). Gli 1 transcriptional factor increases the expression of Snail which in turn leads to suppression of E-cadherin promoting epithelial to mesenchymal transition (EMT) and formation of myofibroblasts and subsequent desmoplasia , 16••. FNotch signalling also drives the epithelial to mesenchymal transition increasing the production of fibroblasts/myofibroblast population and thus promoting extensive fibrosis. In addition, activation of this pathway also stimulated TGFβ production which may further stimulate the fibrotic process . A studyβ catenin levels in the cytoplasm are maintained by binding to a destructive protein complex. Upon activation of Wnt signaling pathway, β catenin levels in the cytoplasm rise and then these molecules translocate to the nucleus activating target gene transcription . HoweverDespite our existing knowledge of the pathogenesis of mesenteric fibrosis, there are hardly any medical therapeutic strategies to treat mesenteric desmoplasia to the date . AvailabTherefore, the treatment of mesenteric fibrosis mainly involves surgical resection of the mesenteric fibrotic mass, palliative bypass surgeries in the presence of inoperable tumour and obstruction and stenting .SINEN-associated desmoplasia is associated with increased morbidity and mortality. Desmoplasia is the result of a complex interaction between SINEN cells and the tumour microenvironment mediated via a plethora of molecules potentiating their profibrotic effects independently or via a cascade of downstream signalling pathways. The key molecules known to be involved in the pathogenesis of SINEN-associated desmoplasia are serotonin, TGFβ and CTGF. Yet, to date, there is no effective medical therapy to treat or reverse this phenomenon. Further research into the pathophysiology of this desmoplastic reaction should provide insights into future development of targeted anti fibrotic therapies for SINEN-associated desmoplasia.