Intravenous fluid resuscitation is recommended first-line treatment for sepsis-associated hypotension and/or hypoperfusion. The rationale is to restore circulating volume and optimize cardiac output in the setting of shock. Nonetheless, there is limited high-level evidence to support this practice. Over the past decade emerging evidence of harm associated with large volume fluid resuscitation among patients with septic shock has led to calls for a more conservative approach. Specifically, clinical trials undertaken in Africa have found harm associated with initial fluid resuscitation in the setting of infection and hypoperfusion. While translating these findings to practice in other settings is problematic, there has been a re-appraisal of current practice with some recommending earlier use of vasopressors rather than repeated fluid boluses as an alternative to restore perfusion in septic shock. There is consequently uncertainty and variation in practice. The question of fluids or vasopressors for initial resuscitation in septic shock is the subject of international multicentre clinical trials. Nonetheless, the evidence underpinning guidelines for early hemodynamic resuscitation is of low quality and the recommendations are graded as weak, leading to clinical uncertainty. The question of fluid dose and the relationship with early vasopressor administration as initial resuscitation in patients with sepsis and septic shock is the focus of this review.It is important to appreciate that many of the IV fluids in clinical use today were introduced into practice at a time when clinical trials were not standard and, thus, not subjected to the same rigorous evaluation as current drug therapies. It was not until the twenty-first century that a systematic evaluation of IV fluids in clinical practice began. Landmark clinical trials have investigated the comparative effectiveness and safety of albumin , 2 and sSepsis is a clinical syndrome characterized by life-threatening organ dysfunction caused by a dysregulated host response to infection . Septic via the hypothalamic-pituitary-adrenal axis, inhibition of pro-inflammatory gene transcription, expansion of regulatory and suppressor T cell lines and apoptosis of pro-inflammatory B and T cells. The relative balance of these elements is governed by a range of host and pathogen factors such as age, comorbid illness, genetic susceptibility, microbial load and virulence. It is the dysregulation of these immune responses which lead to the clinical consequences of organ failure and shock.The pathogenesis of septic shock is complex. The host innate immune response identifies molecular signals from invading pathogenic organisms and tissue injury leads to an initial pro-inflammatory response involving activation of leucocytes, complement and the coagulation pathway . At the via attachment to the CD44 receptor on the endothelial cell surface.The vascular endothelium plays an important role as an initiator of the sepsis response at the site of local pathogen invasion or tissue injury and as a propagator of the inflammatory response . EndotheIn health, the endothelial glycocalyx maintains a barrier between the circulation and the vascular endothelium, while its physical and electrical properties are important in maintaining the oncotic gradient, preventing large molecules such as albumin from crossing into the sub-glycocalyx space. Shedding of the glycocalyx, which occurs during inflammation or ischemia, leads to loss of this barrier function. Activated endothelial cells express intercellular and vascular cell adhesion molecules, and selectin molecules. Collectively, these bind to circulating leucocytes and facilitate the conformational changes to allow diapedesis of leucocytes and extravasation of fluid and molecules from the circulation into the tissues. Finally, the procoagulant state of activated endothelial cells leads to recruitment of platelets and activation of coagulation which can lead to microvascular thrombosis and injury .In contrast to other shock states such as acute hemorrhage or low-cardiac output states, tissue hypoperfusion is usually not the primary mechanism for sepsis-associated organ dysfunction. Tissue hypoxia is less common and widespread tissue necrosis is not a typical post-mortem finding among patients dying of sepsis . Rather The classic sepsis phenotype is distributive shock with peripheral vasoplegia and preserved or increased cardiac output. In practice, there may be a combination of peripheral vasodilatation, hypovolemia due to reduced fluid intake and extravasation and depressed myocardial function secondary to systemic inflammation and acidosis . PeripheIt is important to appreciate the importance of alterations within the microcirculation  in the pathogenesis of sepsis and septic shock . In critBedside assessment of the patient with sepsis requires attention to hemodynamic variables such as heart rate and blood pressure but also an appreciation of microcirculatory, cellular and metabolic factors on which tissue perfusion and organ function depend. Factors such as mental status, respiratory rate, skin perfusion and urine output are clinical features. The measurement of lactate is strongly recommended in patients with suspected septic shock  and formThe rationale for administering an IV fluid bolus is to reverse potential hypovolemia and optimize venous return, with the aim of maximizing stroke volume and cardiac output. The assessment of volume status at the bedside in the emergency department is challenging. Non-invasive dynamic techniques such as echocardiography, pulse-pressure variation or bioimpedance cardiac output monitoring in combination with passive leg raising (PLR) or administration of a fluid bolus are increasingly employed . Static 2) target. This resuscitation algorithm was subsequently adopted into international guidelines for the management of patients with septic shock  incresponse –66, esperesponse  and; (3)response . It is iresponse .via increased preload from increasing the volume of the stressed circulation and increasing myocardial contractility, as well as its effect as a peripheral vasoconstrictor  and net fluid balance at 24 h  (P < 0.001). Similarly, Bai et al. found increased mortality in patients with septic shock when norepinephrine was commenced more than 3 h after the onset of shock   . Decreas 0.0007) . Convers 0.0007) . It is ivia a central venous line, generally in an intensive care setting. This poses a practical and logistical barrier to early initiation of vasopressors. The potential risks associated with peripheral administration need to be balanced against harms associated with delay to restoring adequate perfusion. Several observational studies point to the safety of peripheral administration of vasopressors, at least during the first few hours . The restricted fluid strategy was also associated with a shorter time to vasopressor initiation [34 (interquartile range 15–88) min vs. 150 (interquartile range 63–224) min] and an increased proportion of patients receiving a vasopressor within the ED   ; albeit = 0.011) .A large, multicentre, randomized trial of fluid restriction vs. standard fluid therapy in 1,554 patients admitted to ICU with septic shock (within 12 h of onset) has recently been conducted  . Fluid sNotably, patients in CLASSIC required receipt of a vasopressor or inotropic agent to meet study eligibility and the intervention was not confined to the first 6–24 h after presentation; rather, the resuscitation strategy was delivered for the duration of the ICU stay. In contrast, there are 2 large multicentre, randomized trials currently evaluating a restricted fluids strategy compared to usual care wherein the intervention period is limited to the early resuscitation period (24 h) in patients presenting to the ED with sepsis-induced hypotension; CLOVERS (United States NCT03434028) and ARISE-FLUIDS . The planned sample size for CLOVERS was 2,320 patients with the primary outcome being hospital mortality censored at 90-days. Enrolment was recently closed for futility following the second interim analysis. The ARISE FLUIDS is actively recruiting with a planned sample size of 1,000 patients and a primary outcome of days alive out-of- hospital to 90 days. A third, large, randomized trial, EVIS (United Kingdom NCT05179499) in 3,286 patients with septic shock is evaluating whether an early peripheral vasopressor infusion targeted to a mean arterial pressure > 65 mm Hg for 48 h improves survival to 30-days compared to usual care. The results of all three trials is eagerly awaited and will undoubtedly inform the early hemodynamic resuscitation of patients presenting to the ED with septic shock, particularly the initial fluid volume and the timing of vasopressors.The Surviving Sepsis Campaign guidelines recommend at least 30 mL/kg of fluid within 3 h as first-line therapy for sepsis-induced hypoperfusion or septic shock with the subsequent introduction of vasopressors. The evidence underpinning these guidelines is low quality and the recommendations are, accordingly, graded as weak. A growing body of evidence suggests that initial hemodynamic resuscitation with a restrictive fluid approach, combined with earlier vasopressors, may improve clinical outcomes, including mortality. Several large-scale, multicentre trials are currently evaluating such a strategy. High quality evidence from these trials will inform both bedside clinicians and future international guidelines. Until then, the optimal fluid dose and the timing of vasopressors remains uncertain.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. There are multiple new therapies being developed to improve the current regimens involving amphotericin B, flucytosine, and fluconazole. ATI-2307 is a novel arylamidine which inhibits mitochondrial function in yeasts. We tested ATI-2307 in a rabbit model of CM and evaluated its efficacy, pharmacokinetics, and pharmacodynamics (PK-PD).Immune suppressed rabbits were infected with Cryptococcus neoformans. Starting two days after the infection, they were treated with ATI-2307 , amphotericin B (1 mg/kg), fluconazole (80 mg/kg), a combination of ATI-2307 (1 mg/kg) and fluconazole (80 mg/kg), or they were untreated. ATI-2307 was given daily subcutaneously. Fungal burden and drug levels were quantified in the CSF during the infection and in the brain tissue at study termination, day 4, 10, or 14 post infection. Drug pharmacokinetics in the blood were examined on day 8 post infection. Drug pharmacokinetics in the CSF and brain tissue were examined at day 4 post infection.Daily dosing with ATI-2307 reduced the yeast burden in both the cerebrospinal fluid (CSF) and brain tissue compared to controls. Furthermore, when combined with fluconazole, ATI-2307 reduced the CSF fungal burden below the limit of detection. In fact, we observed a large reduction in fungal burden relative to untreated controls with only three doses, which was sustained for 1 week after the cessation of dosing. The kinetics of ATI-2307 indicated that the drug has a long gamma phase in both blood and CSF. In addition, our analysis of the drug levels in the brain tissue indicated that it dramatically accumulated in the meninges, which may then serve as a site of sustained release.Efficacy of ATI-2307 on CSF Fungal Burden and Distribution of ATI-2307 in Brain TissueATI-2307 was dosed daily starting at day 2 post infection through the study endpoint, day 10 or 14 post infection, with the exception of the 2 mg/kg, 3 dose group, which only received doses on days 2, 3, and 4 post infection.Taken together, these data provide evidence for ATI-2307 as a promising therapy for CM and it may require fewer doses than existing treatments and provide additive anticryptococcal activity with azole treatment.Charles D. Giamberardino, Jr., MR, Affimed NV: Stocks/Bonds|Appili Therapeutics: Grant/Research Support|Interventional Analgesix Inc: Grant/Research Support|Minnetronix: Grant/Research Support|Pfizer: Grant/Research Support|Quoin Pharmaceuticals: Stocks/Bonds|Sfunga: Grant/Research Support Jennifer L. Tenor, PhD, Appili: Grant/Research Support|Pfizer: Grant/Research Support Dena Toffaletti, PhD, Appili Therapeutics: Grant/Research Support|Pfizzer: Grant/Research Support Don Cilla, Pharm.D., M.B.A., Appili Therapeutics: Employee|Appili Therapeutics: Stocks/Bonds William Hope, OBE , Antabio: Grant/Research Support|Appili Therapeutics: Advisor/Consultant|BioVersys: Grant/Research Support|Bugworks: Grant/Research Support|Centauri: Advisor/Consultant|F2G: Advisor/Consultant|F2G: Grant/Research Support|NAEJA-RGM Pharmaceuticals Inc: Advisor/Consultant|NAEJA-RGM Pharmaceuticals Inc: Grant/Research Support|National Institute of Health Research (NIHR).: Co-lead for Infectious Diseases|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Phico Therapeutics: Grant/Research Support|Specialist Advisory Committee for GARDP: Advisor/Consultant|Spero Therapeutics: Advisor/Consultant|Spero Therapeutics: Grant/Research Support|The Global Antibiotic Research and Development Partnership (GARDP): Grant/Research Support|VenatoRx: Advisor/Consultant John R. Perfect, MD, Appili Therapeutics: Advisor/Consultant|Appili Therapeutics: Grant/Research Support|Astellas: Advisor/Consultant|Astellas: Grant/Research Support|Matinas: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Scynexis: Advisor/Consultant.


In this review, we outline the current knowledge on VZV interaction with host signalling pathways, the viral association with proteins associated with epidermal terminal differentiation, and how these interconnect with the VZV life cycle to facilitate viral replication and shedding.The replication of varicella-zoster virus (VZV) in skin is critical to its pathogenesis and spread. Primary infection causes chickenpox, which is characterised by centrally distributed skin blistering lesions that are rich in infectious virus. Cell-free virus in the cutaneous blistering lesions not only spreads to cause further cases, but infects sensory nerve endings, leading to the establishment of lifelong latency in sensory and autonomic ganglia. The reactivation of virus to cause herpes zoster is again characterised by localised painful skin blistering rash containing infectious virus. The development of  Varicella-zoster virus (VZV) is a member of the human alphaherpesvirus family. Primary VZV infection causes chickenpox (varicella) in susceptible individuals, which typically manifests in immunocompetent individuals as a skin blistering rash preceded by flu-like symptoms and is usually self-limiting. In immunocompromised individuals, pregnant women as well as healthy adults, VZV infection may cause serious illness and even be lethal, often as a consequence of bacterial superinfections. A vaccine for VZV exists, which is based on the live attenuated parental VZV Oka strain ,2. BecauVZV is transmitted between hosts by contact with the cutaneous blistering lesions containing the highly infectious cell-free virus or through inhalation of the aerosolized virions released from the skin lesions and to some extent the respiratory tract. During primary infection, VZV initially infects cells of the mucosa lining the upper respiratory tract where it is initially detected by dendritic cells (DCs) and transported to lymphoid tissues, primarily the tonsils, where it infects skin homing markers expressing T-lymphocytes that then deliver it to the skin ,4. The vin vivo VZV infection model that closely mimics VZV infection of human skin in vivo was developed by the Arvin group and is based on the infection with VZV of xenografts of human fetal skin implanted into SCID-hu mice [in vitro representation of VZV infection of the epidermis [in vitro model of VZV infection in human epidermis [The tropism of VZV for the skin is well recognised, however our understanding of the molecular mechanisms of VZV replication in the epidermal tissue of the skin are still incomplete. VZV being a human-restricted virus with limited infectivity for other species, has limited the use of animal models for the study of viral natural history and pathogenicity. Most research into VZV cellular interactions has been in human embryonic lung fibroblasts (HELF), human lung MRC-5 fibroblasts and the human melanoma MeWo cell line. While these cell types are relatively easy to infect with VZV, none of them is representative of the human epidermis, which is the actual site of much VZV replication and shedding. An -hu mice ,6,7. VZVpidermis ,11,12,13pidermis ,9.This review will explore what is understood to date about the mechanisms of VZV replication in the epidermis of the skin, as discovered in the very few existing human skin models of VZV infection.+ T cells expressing activation and skin-homing markers, such as CC-chemokine receptor 4 (CCR4) and cutaneous leukocyte antigen (CLA), but VZV also infects and activates naïve T cells [in vitro that natural killer (NK) cells can also be infected by VZV and consequently increase the expression of skin-homing proteins [in vivo and whether they contribute to the virus dissemination to T cells has not yet been elucidated. The incubation period during VZV infection is about 10–21 days, which is the time between VZV infection and the manifestation of symptoms, including the skin vesicular rash. Studies in SCID-hu mice models indicate that T cells reach the skin within days after infection, but it appears that the virus is able to evade initial antiviral responses through its interaction with interferon (IFN)-mediated innate immunity and apoptotic pathways  , followein vivo  and IE63 (encoded by ORF63 and ORF70), although for IE63 the relevance in VZV infection in vivo is dependent on the phosphorylation status [in vivo encompass the glycoprotein gE (encoded by ORF68), which is essential for VZV replication and cell–cell spread in skin [Early work using skin grafted into SCID-hu mice ,6,7 showiewed in ). Exampliewed in ,45,46. Fn status . The gly in skin ,49, and  in skin , as well in skin ,53,54,55 in skin ,53. The  in skin ). The cy in skin  and the  in skin . It is i in skin .Like other skin epitheliotropic viruses, e.g., cutaneous human papilloma viruses (HPVs) ,59, VZV in vitro models of VZV infection of human epidermis, it was demonstrated that VZV induces the degradation of the suprabasal epidermal differentiation marker K10, as well as the downregulation of a number of corneodesmosomal proteins [In proteins ,9. As K1proteins ,61,62, iproteins .VZV keratinocytes infection is also characterised by upregulation of a number of proteases, most notably kallikreins . KallikrThe formation of syncytia structures is well documented in MeWo cells , and mulK10 not only serves a structural function in VZV infection of the epidermis. In line with increasing studies reporting involvement of keratins in gene expression and signalling pathways ,68, K10 KRT4/13  and KRTegulated . Recent egulated , togetheegulated , seems tMacroautophagy, hereafter called “autophagy”, is a process involving the degradation and recycling of proteins and cellular components, which is activated in response to cellular stress, e.g., following microbes’ infection, to guarantee cell homeostasis. The autophagy events are mediated by a number of proteins called autophagy-related genes (ATGs), as well as not ATG proteins  and invoIn VZV-infected and differentiated keratinocytes and skin explants, NR4A1 has been found to be crucial in the induction of autophagy mechanisms, as demonstrated by the decreased expression of key autophagy markers, such as LC3, when NR4A1 was knocked down . NotablyThe analyses of VZV-infected fibroblasts and MeWo cells, as well as keratinocytes and skin explants, indicate that autophagy has a proviral effect during VZV skin infection and suggest that the virus exploits autophagy events for completion of its life cycle ,79,80. IThe major innate immunity cells involved in the reaction to VZV infection, including DCs, Langerhans and NK cells, have been recently reviewed in . Here, wB adjuvant has recently been shown to be extremely effective in preventing HZ and its commonest sequela, post herpetic neuralgia [The varicella vaccine was generated by Takahashi and colleagues in 1974  by seriaeuralgia .Moffat et al.  demonstrUnderstanding the molecular mechanisms causing attenuation in skin of the existing VZV vaccine will shed light on pivotal events of VZV skin replication that could be targeted by new antiviral drugs, as well as pave the way for the generation of novel vaccines that could be directed to a wider number of recipients, including immunocompromised children.Work on models that reproduce the architecture of human skin or at very least recapitulate keratinocyte differentiation has provided important insights into how VZV interacts with host epidermal pathways. In particular, the pivotal role of the specialised skin keratinocyte cell in the VZV replication cycle is likely to shed light on VZV tropism and pathogenesis. These mechanisms are just beginning to be unveiled and this work is also complicated by VZV infection being highly interconnected with the host epidermal biology, which itself is characterised by molecular pathways that have often been only partially elucidated. An example is epidermal autophagy, whose pathways have only quite recently begun to be uncovered ,78. An iFinally, a better understanding of VZV pathogenesis in the skin could help to develop new vaccines that are not based on the live attenuated virus. The existing vaccine vOka, being attenuated for growth in skin, is actually itself an important tool for the study of VZV skin infection and the comparative analysis with the parental strain pOka can further the understanding of the molecular mechanisms of VZV replication in skin. Moreover, a better knowledge of VZV skin pathogenesis will probably prove to be very valuable to further understand the pathogenesis of other skin epitheliotropic viruses that may share common mechanisms of replication in skin.


The positions of some fiducial markers appeared not stable on the 4DCT throughout the respiratory phases. Markers’ center of mass tends to be a more reliable measurement. Distance errors of tumor location based on registration versus markers center of mass were less than 2mm. There were no statistically significant differences between the reference and the sequential registration, i.e., consistency and errors were comparable to resolution errors. We demonstrated that intensity-based DIR is accurate up to resolution level for locating the tumor in the liver during breathing motion.We investigate the accuracy of intensity-based deformable image registration (DIR) for tumor localization in liver stereotactic body radiotherapy (SBRT). We included 4DCT scans to capture the breathing motion of eight patients receiving SBRT for liver metastases within a retrospective clinical study. Each patient had three fiducial markers implanted. The liver and the tumor were delineated in the mid-ventilation phase, and their positions in the other phases were estimated with deformable image registration. We tested referenced and sequential registrations strategies. The fiducial markers were the gold standard to evaluate registration accuracy. The registration errors related to measured versus estimated fiducial markers showed a mean value less than 1.6 Stereotactic body radiotherapy (SBRT) is one of the treatment modalities used for managing tumors in the liver . The SBRThe feasibility of using deformable image registration (DIR) to estimate breathing motion and tumor positions in the liver has been demonstrated , aiding Liver tumors are often not visible without admission of intravenous contrast on either CT or cone-beam CT (CBCT) , 19. TheRegarding the DIR algorithms on the liver, the finite element model-based DIR proposed by Brock et al.  is the oIn this paper, we aim to demonstrate that the use of intensity-based DIR is accurate in estimating the position of liver tumors in low contrast images. We applied DIR to respiratory correlated 4DCT and used implanted fiducial markers as a surrogate of tumor locations.This study was based on data from eight patients treated with SBRT for metastases in the liver at Rigshospitalet  between March 2018 and September 2019. The patients were selected from a group of patients, participating in a prospective clinical protocol investigating liver SBRT in breath-hold . All patients signed an informed consent before the enrollment in the study. This consent included use of their imaging data for research purposes. See details on patient selection in mm. The image resolution in each slice was 512 × 512 pixels and a pixel size of 0.98 × 0.98 mm.Respiratory correlated 4DCT with intravenous contrast injection was performed for all patients on a SOMATOM Definition AS scanner . 4DCT image data were phase-sorted into ten phase bins throughout a respiratory cycle based on an external respiratory signal monitored with Real-Time Position Management . The slice separation in each phase of the 4DCT was 2In addition, MR scans in deep inspiration breath-hold without visual guidance were performed for all patients to achieve better visualization of the tumor. The MR images  were acquired in a 1.5T SIEMENS MAGNETOM Avanto scanner .The mid-ventilation phase of the 4DCT was determined based on fiducial marker (tumor) motion for each patient and used for treatment planning . FurtherThree fiducial markers were implanted percutaneously near the tumor in each patient using ultrasound guidance. The fiducial markers were placed within one week prior to the imaging for treatment planning. Goldlock fiducial markers  were implanted in the first five patients and Gold Anchor fiducial markers  were implanted in the latter three patients.mm). The uncertainty of the fiducial markers centroids were expected to be greater in the SI plane due to the 2 mm slice separation.The centroid position of each fiducial marker was calculated automatically in each phase and chosen as its ground truth locations. The automatic procedure consisted of three steps. First, a threshold of 500 Hounsfield units was applied to the images. Second, the fiducial markers were delineated in the axial plane in all 4DCT phases. Third, the delineations were used to create a volume of the fiducial marker and then the centroid is computed. The accuracy of this method was compared to the manual method for locating the center of the fiducial markers . The comThe fiducial markers center of mass (COM) are calculated for each patient as the average position of the three fiducial markers. From here, we will refer to this measurement simply as markers COM, while ‘fiducial markers’ refer to the  positions of the markers centroids.The publicly available Symmetric Image Normalization algorithm (SyN)  was usedIn this work, the DIR was computed in two ways, depicted in The liver and GTV delineations were propagated to the other phases of the 4DCT with the DIR algorithm using only the sequential approach. The appropriated transformation was chosen as the lowest number of composition of deformations from the mid-ventilation phase to the other phases. After DIR, a volume was created in each phase for each patient to compute the center of mass (COM) of the tumor and the liver. From here, we will refer to this positions as liver COM and GTV COM. This methodology is referred as segmentation based on DIR or contour propagration . The accThe DIR algorithm is voxel intensity-based. In this scenario, the fiducial markers became a problematic surrogate for registration, since they create high intensity values around their location . In ordeThe image inpainting was performed with the algorithm proposed by Telea et al. . For extThe fiducial markers were used as the direct measurement to evaluate the accuracy of image registration. Two distance metrics were used: the first one for DIR evaluation and the second one to verify the relative distance variations of markers, tumor and liver. xf, j, where the subscripts f and j refer to the particular fiducial marker and the image phase respectively. The transformation found by registration φj was applied to the fiducial markers in the reference phase xf,ref, producing the estimated fiducial markers The first metric was the registration error calculated as the euclidean distance between the ground truth positions of the fiducial markers and the estimated positions of the fiducial markers. The ground truth coordinate was a point The registration errors are calculated for the reference and sequential registration algorithms, as well as for images registered with and without inpainting. The statistical tool used to compared the data is the Kolmogorov-Smirnov test .The second metric was the relative distance variation. In this case, the distance between two points was compared to check if the distance was the same across the phases. The metric was calculated between the fiducial markers, between the GTV COM and the fiducial markers, between the GTV COM and the markers COM, and between the GTV COM and the liver COM. As an example, the relative distance of the fiducial marker 1 and fiducial marker 2 is presented here, as:μ = 7.9 mm with a standard deviation of σ = 2.9 mm. The fiducial markers distances to the GTV COM ranged from 17 to 55 mm, with mean value μ = 32 mm and standard deviation of σ = 10 mm.The sequential registration was used to estimate volume variations in the liver and the GTV calculated on every phase. The baseline volume measured in mid-ventilation phase is shown in We tested the registration algorithm with end expiration and end inspiration phases  where the maximum displacement occurs. The algorithm was parametrized to achieve convergence in this scenario. For further details see mm for the referenced registration and 1.6mm for the sequential registration, shown in mm and 1.4mm respectively. An increased mean and standard deviation was noted for the images further away of the reference phase for both reference and sequential registration. There were no statistically significant differences per phase related to the registration method. See more details in We tested the registration errors  in all pmm and 3.7mm for the referenced registration and the sequential registration respectively. More details are presented in mm for each patient.The maximum registration errors were 5.3The inpainted images were tested only for the referenced registration case with the registration error metric and we found no statistically significant differences compared to the original images, see As we observed with the registration errors that the average maximum obtained was equivalent or lower than the resolution in the axial slices. Since the reference and the sequential registration did not produce statistically significant differences this indicates a consistency in the registration method to accurately align the images.mm. This error is justified in For patient 8 a high registration error of marker 1 in phase 90% is related to the position of the marker close to the edge of the liver. For this case, image qualitative verification is a valid tool of assessment, as suggested in the AAPM TG-132 report on image registration .mm. Assuming that the real position of the GTV was followed by the marker COM the accuracy of registration was as good as the resolution of the image. GTV COM to liver COM distance errors were lower than 1.2mm. Both COMs were computed from registration and they followed the same motion pattern.mm. This suggested that intensity-based DIR is accurate for liver radiotherapy .We found no statistically significant differences between registering the images sequentially or to a reference. These results are in accordance to the results found by Boldea et al. for lungs . In contIt could be argued that the high intensity artifacts created by the fiducial markers influenced the convergence and accuracy of the registration algorithm. In order to investigate this hypothesis, we performed a test with the same algorithm on the same images where inpainting was applied to remove the fiducial markers. Only the reference registration is tested. No statistically significant difference was found from the results obtained using the original images (without inpainting). This also suggest that the DIR is driven by the whole abdomen (liver) intensities values.We expose the other capabilities of DIR applied to the liver. These are the estimation of liver and tumor volumes in the breathing cycle. The liver tissue is not highly compressible, but some deformation is expected . Anothermm for each patient. Some of the maximum values in registration errors could be seen as outliers, and may be due to uncertainties in the measurement of individual fiducial markers.We always evaluated the registration accuracy with features or points, i.e. fiducial markers. Other metrics such as image similarity or volume overlapping are less recommended in DIR . The regAnalysis of the individual results of each patient in mm and 55mm. We used two types of fiducials in our study. We observed that the generated imaging artifacts are similar in intensity and volume. Therefore we consider for our case that the COM estimation is equivalent and fiducial type independent.Compared to some of the previous studies related to fiducial markers in liver radiotherapy , 43–45, One of the limitations of our study is the small number of patients. A larger number of patients would allow pinpointing of situations with lower accuracy. Furthermore, including more patients would strengthen the results, however for a proof of principle, the included number seems sufficient, since a wide range of tumour positions within the liver and respiratory motion amplitudes was represented in our patient material. Another drawback prior to a potential clinical implementation is current DIR computational time. This may be overcome in a near future with SyN algorithm implementations on GPU  or emergmm. Such accuracy also seems adequate for radiotherapy.We have demonstrated that intensity-based DIR is accurate for liver radiotherapy. Furthermore, we found no statistically significant differences between the images sequentially or referenced DIR methods. We propose a GTV localization strategy on 4DCT using DIR, that is consistent with GTV localization based on fiducial markers’ COM with less variability than what is seen between individual markers. The DIR algorithm using the liver COM as a reference to locate the GTV, resulted in our small patient cohort in a maximum error of 1.2S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(PDF)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file.S7 Appendix(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Many factors can affect the recurrence of endometriosis after surgery, however, whether endometriotic cyst fluid contributes to endometriosis recurrence after surgical excision of ovarian endometriomas remains unclear. The objective of this study was to determine the presence of endometrial cells in ovarian endometriosis cyst fluid and the potential differences between these cells and those in the cyst wall.n = 39) and drainage fluid (n = 14) were collected from patients with ovarian endometriomas undergoing laparoscopic surgery. Drainage fluid from 14 patients without endometriosis was used as a control. The presence of endometrial cells in cyst fluid and drainage fluid was determined by cell culture in vitro and immunostaining. In addition, cyst fluid endometrial fragments and viscosity were analysed by transcriptome sequencing analysis and apparent diffusion coefficients, respectively. An animal model was used to confirm the ability of endometrial cells in cyst fluid to form new lesions.Samples of cyst fluid (P < 0.05). In addition, living endometrial cells in the cyst fluid were able to adhere and alive in the animal model.We found endometrium-like tissues in 71.8% (28/39) of cyst fluid and 71.4% (10/14) of drainage fluid samples by histopathological examination, and the presence of endometrioid tissue in cyst fluid was related to the viscosity of the cyst fluid. The living endometrial cells in cyst fluid and drainage fluid were confirmed by cell culture in vitro and immunostaining. Moreover, the adhesion ability of endometrial fragments in cyst fluid was significantly higher than that of ectopic tissues in the cyst wall (The existence of living endometrial cells with high adhesion ability in ovarian endometriotic cyst fluid may contribute to the recurrence of endometriosis after surgical excision of endometriomas due to cyst fluid outflow during the surgical procedure.The online version contains supplementary material available at 10.1186/s13048-022-01018-9. Endometriosis is a chronic systemic disease characterized by the implantation and growth of endometrial-like tissue outside the uterus and affects 6-10% of reproductive-age women . The maiAll types of endometriosis have a high rate of recurrence after conservative surgery . For ovaTherefore, in this study, we aimed to determine the presence of endometrial cells in ovarian endometriotic cyst fluid and postoperative peritoneal drainage fluid and the potential differences in endometrial tissue between cyst fluid and the cyst wall . We found living endometrial cells in ovarian endometriotic cyst fluid and postoperative peritoneal drainage fluid. Moreover, the function of living endometrial cells in the cyst fluid was proved by animal experiments. Our results indicate that endometrial cells in cyst fluid caused by cyst fluid outflow during the surgical procedure may contribute to the postoperative recurrence of endometriosis.− 3 mm2/s) was inversely proportional to the viscosity value defined by us. The viscosity cyst fluid had a smaller ADC value  patients  in the Cyst, Ec and Eu groups, including transition, A-G, C-T, transversion, A-C, A-T, C-G and G-T SNPs. According to the violin plot, there was a slight increase in the number of mutations in the Ec and Cyst groups compared to the Eu group, whereas there was no significant difference in the variance and frequency distribution in the three groups Fig. A. We fouWe detected ICAM2 and Claudin5 expression in the endometrial fragments in cyst fluid and ectopic lesions by IHC. Both the ICAM2 and Claudin5 levels were higher in the endometrial fragments in cyst fluid than in the corresponding ectopic tissue Fig. A, B. Basn = 16) mice at 2 weeks, and 9  mice at 3 weeks . For incision aspiration, a small breach of the cyst wall was made with scissors, and the cyst fluid was collected with an aspirator connected to a 50 mL sterile syringe. Puncture aspiration was performed using a Transfix needle connected to a 50 mL sterile syringe. Approximately 5-100 mL of the endometriotic cyst fluid was aspirated into the syringe. Cyst fluid was obtained from both sides of the cysts in the patients with bilateral ovarian endometriomas. The cyst fluid was transported to the laboratory within 30 minutes on ice.The drainage fluid was obtained from the pelvic drainage bag. Twenty-four hours after surgery, the drainage fluid (5-20 mL) was collected using a 50 mL sterile syringe from the bag and immediately transported to the laboratory in a cold chain.The endometriotic cyst wall and eutopic endometrium were collected during the surgery and immediately transported to the laboratory in a cold chain. After being washed with cold sterile saline three times, the ectopic endometrial tissues from the cyst wall and the eutopic endometrium from the corresponding patients were immersed in liquid nitrogen for RNA extraction. The other ectopic tissues on the cyst wall were washed and immersed in 4% neutral buffered formalin for the histological experiment.All the endometriomas were removed during the surgery. In addition, none of the patients had received sex-hormone therapy within the 6 months before surgery. Detailed information on the patients enrolled in the study and the samples used in each experiment is shown in Additional file The endometriotic cyst fluid was filtered through a 100 μm filter membrane, and the viscosity of the cyst fluid was classified into three types according to the difficulty of the cyst fluid passing through the filter membrane: type 1, the cyst fluid was not viscous and could directly pass through the 100 μm filter membrane, marked as “-1”; type 2, the cyst fluid had some viscosity and thereby needed to be repeatedly blown through the pipette to pass through the filter membrane, marked as “0”; type 3, the cyst fluid was very viscous and could not pass through the filter membrane even after repeated blowing, marked as “1”. It was necessary to dilute the type 3 cyst fluid more than twice until it could pass through the sieve.Thirty-two endometriosis patients underwent MRI examination before surgery. Apparent diffusion coefficient (ADC) values were obtained from 18 patients. Fourteen patients underwent MRI in the outpatient department, and the MRI image was unavailable for analysing when they underwent the surgery. MRI scans were performed on a 1.5 Tesla MR system  with a phased-array abdominal coil. The patients were in a supine position and breathed freely during acquisition. The scanning range was from the inferior pubic symphysis to the renal hilum and extended beyond the dome of the cyst in patients with large masses. Diffusion-weighted imaging (DWI) was performed in axial planes at b values of 0, 800 s/mm2 (TR/TE 4600 ms/72 ms). The scanning parameters were as follows: 5 mm slice thickness, 1.2 mm gap, 256× 256 matrix, 296 mm field of view and four excitations.2 was placed at targeted areas with the possibly lowest ADC values in the cyst components of ovarian endometrial cysts, by referring to conventional MR imaging. At least three measurements were obtained and averaged.ADC values were measured on ADC maps. A circular region of interest (ROI) of at least 1 cmAll cyst fluid or drainage fluid samples were filtered with 100 μm apertures sieves under sterile circumstances. To separate the debris and the endometrial fragments, the remnants in the 100 μm aperture sieves were washed with 1x phosphate-buffered saline (PBS) several times. Fresh, endometrial fragments were collected and washed with 1x PBS. Three endometrial fragments from cyst fluid were immersed in liquid nitrogen for RNA extraction. Ten endometrial fragments from the cyst fluid were immersed in 4% neutral buffered formalin for the histological experiment. Fifteen endometrial fragments from the cyst fluid were used for in vitro cell culture. All the endometrial fragments acquired from drainage fluid were immersed in 4% neutral buffered formalin for the histological experiment.Fragments were collected under sterile conditions and digested with type I collagenase  in a shaker at 37 °C. After 45 minutes, digestion was stopped with Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12)  containing 12% foetal bovine serum. A 100 μm nylon cell strainer was used to remove the debris. Stromal cells and epithelial cells were not separated. The filtered liquid was centrifuged and resuspended in DMEM/F12 containing 12% foetal bovine serum. The cells were cultured in a humidified incubator at 37 °C with 5% CO2.Total RNA of the endometrial fragments in the cyst fluid (Cyst), ectopic tissues on the cyst wall (Ec) and eutopic endometrium (Eu) was extracted using TRIzol reagent (Invitrogen). Cleaved RNA fragments were reverse-transcribed to create a cDNA library by the protocol for the mRNA Seq sample preparation kit (Illumina), with a resulting average insert size for paired-end libraries of 200 bp (± 50 bp). Samples were subjected to paired-end sequencing on an Illumina HiSeq 4000 (LC Sciences) by Lc-Bio Technologies  Co., Ltd.2 (fold change) > 2 or log2 (fold change) > − 2 and with statistical significance (P < 0.05). The SNP sites in coding regions were analysed based on the transcriptome level. According to the Hisat2 alignment results of each sample and the reference genome, mpileup processing was performed with SAMtools software, and the possible SNP and INDEL information of each sample was then annotated with ANOVA. Bioinformatic analysis was performed using OmicStudio tools at https://www.omicstudio.cn/tool/. We used the gene differential gene expression in the cyst, ectopic and eutopic groups as the input, and the volcano plot, heatmap, GSEA, KEGG pathway enrichment, and Venn diagram were exported from the websites. Circos graphs of INDELs were generated by Lc-Bio Technologies  Co., Ltd.Mapped reads for each sample were assembled using String Tie. All transcriptomes were merged to reconstruct a comprehensive transcriptome using Perl scripts. Following generation of the final transcriptome, String Tie and edge R were used to estimate the expression levels of all transcripts. String Tie was used to determine the expression level of mRNAs by calculating the fragments per kilobase per million (FPKM). Differentially expressed mRNAs and genes were selected as logThe endometrial fragments from cyst fluid and drainage fluid were embedded in paraffin and cut into 4 μm sections. Haematoxylin and eosin (H&E) staining was utilized to confirm the endometrial structures in the fragments. To determine the epithelial and interstitial structure, the slides were incubated with anti-cytokeratin 19  and anti-CD10  overnight. For the slides of negative control, the primary antibody was replaced by PBS. The corresponding secondary antibodies were incubated for 60 min. Slides were visualized with DAB , counterstained with haematoxylin and observed under a microscope.For immunofluorescence, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 10 min. Unspecific binding was blocked by using 8% bovine albumin in PBS at room temperature for 1 hour. Cells were incubated overnight at 4 °C with anti-vimentin  and anti-cytokeratin 19  antibodies. The next day, the slides were washed 3 times in PBS and incubated with goat anti-mouse antibody  and donkey anti-rabbit antibody  at room temperature for 1 hour. Nuclear DNA was labelled in blue with DAPI . The cells were visualized under a confocal microscope .For the histochemical assay, paraffin-embedded slides of endometrial fragments from cyst fluid and ectopic endometrial tissue from the cyst wall were incubated with anti-ICAM2  and anti-Claudin5  antibodies. A semiquantitative evaluation of the immunostaining intensity was carried out using ImageJ, and the mean optical density (MOD) was used to represent the levels of protein expression. The average MOD of five different fields of a slide was regarded as the expression of the molecule.n = 16) or three (n = 27) weeks. The abdominal of the mouse was carefully probed and lesions were collected for histological examination.This study was carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Six-week-old female BALB/C nude mice were purchased from Shanghai Animal Center, Chinese Academy of Science. The mice were housed in an environment at 21 °C ± 0.5 °C under a 12 h light/dark cycle and with free access to food and water. The endometriotic cyst fluid used in this part was collected from 12 patients . All the results are expressed as the mean ± standard error of the mean (SEM). The normality of the data was tested with the Shapiro-Wilk test. An unpaired Student’s t-test (two-tailed) was used to identify statistically significant differences between the two groups. Pearson correlation was used to analyse the correlation between the viscosity of the cyst fluid and the ADC mean value. A Additional file 1. Clinical characteristics of endometriosis and control patients in this study.Additional file 2. The ADC values.Additional file 3: Fig. S1. Animal model establishment and new lesion formation. A Schematic diagram of the animal experiment. B The white arrow shows endometriotic lesions in the mice after model establishment. C Representative image of an endometriotic lesion with ZsGreen fluorescence, × 200; bar = 50 μm. D The short arrow shows the lesions found in the peritoneal cavity of mice in the experimental group. E Representative image of a new lesion with ZsGreen fluorescence; × 200; bar = 50 μm.


Surgery is a fundamental part of the multimodal therapy concepts in oncological patients, especially in the early stage of pancreas tumour. There are numerous studies on the impact of primary tumour resection on the immune status, but to our knowledge, the impact of tumour-unrelated surgery on the anti-tumour immune response to the primary tumour it is not yet understood. Therefore, we used a murine model combining an orthotopically implanted adenocarcinoma of the pancreas and the model of surgically-induced immune dysfunction to assess the impact of postoperative immunosuppression on the growth of the primary tumour, on mortality and on the most important immune cell compartments in tumour defence. This knowledge might contribute to a basic understanding of the interaction of the primary tumour and the immune system and could guide new approaches to therapeutic strategies.(1) Background: Surgery is the most important element of multimodal treatment concepts in oncological patients, especially in the early stages of pancreatic tumours. While the influence of primary tumour resection on the immune status was analysed in several studies, the impact of tumour-unrelated visceral surgery on the tumour-bearing organism and on the primary tumour itself is not yet fully understood. (2) Methods: We combined a murine model of orthotopically implanted adenocarcinoma of the pancreas with the model of surgically-induced immune dysfunction (SID). Mortality and general condition including body weight were observed over a period of 28 days. Tumour growth was analysed by MRI scans on days 8 and 27 following tumour implantation. On day 28, the immune cell populations in the blood and spleen as well as the serum cytokines were quantified. (3) Results: SID results in a significant deterioration of the general condition and a reduced increase in the body weight of tumour-bearing mice compared to the control groups, while mortality and tumour growth rate were not influenced. The numbers of spleen macrophages and neutrophils were increased in tumour-bearing animals following SID. Furthermore, both macrophage and neutrophil levels were increased in the peripheral blood. (4) Conclusions: The presented results might contribute to the basic understanding of the interaction of tumour and immune system and could contribute to new approaches to immunotherapeutic strategies. Surgical procedures induce a state of immunosuppression for up to several weeks postoperatively. The immune dysfunction affects both the innate and the acquired immune system . The innSurgery is a very important element of multimodal treatment concepts in oncological patients. However, the surgical procedure and the associated stressors have also been shown to increase the susceptibility for the development of cancer metastasis and local recurrence . SeveraMost of the in vivo and in vitro studies have evaluated the effects of SII on cancer recurrence and the development of metastasis in the context of the resection of the primary tumour. Since primary tumours have been shown to supress the development of metastasis , it is Animals. Nine week old male C57BL/6N mice purchased from Charles River Laboratories  were held under specific pathogen-free conditions at least two weeks before inclusion into the experiments in order to adapt to the housing conditions. Mice had free access to food and water. The animal experiments were approved by the veterinary government authority ; experiment numbers: TSD/7221.3-1-038/16 and TSD/7221.3-1-038/16-1). Anaesthesia for tumour implantation, operations, and organ removal was performed by intraperitoneal injection of 100 mg/kg body weight ketamine  and 10 mg/kg body weight xylazine . Anaesthesia for MRI examination was performed by inhalational anaesthesia with 1% isoflurane. After interventions, drinking water was supplemented with 250 mg/100 mL tramadole . At the end of the experiment or when the termination criteria of the stress score were fulfilled, mice were euthanised under intraperitoneal injection narcosis with ketamine and xylazine by cervical dislocation.Cell line. The murine pancreatic adenocarcinoma cell line 6606PDA was a kind gift from Prof. David Tuveson, Cambridge, UK  and was isolated from pancreatic adenocarcinoma cells in transgenic C57BL6/N mice carrying a KrasD12G allele [G allele .Experiment design. Animals were randomly divided into six experimental groups: SID + tumour, laparotomy + tumour, without operation (w/o op) + tumour, SID, laparotomy, and the control. On day 0, 6606-PDA-cells were xenografted in mice by orthotopic injection into the pancreas as described before [5 pancreatic carcinoma cells dissolved in PBS  and Matrigel  were injected into the head of the pancreas. On days 8 and 27, the tumour volume was determined by magnetic resonance imaging using a 7.1 tesla ClinScan device , generating T2-weighted axial and coronal images. Images were analyses with the MIPAV software . On day 11 and on day 21, surgery was performed .Spleen cell isolation. After cervical dislocation, the spleen was removed and stored on ice in PBS with 10% FCS  until further processing. The spleen was dispersed by squeezing the organ through a 70 µm cell strainer  and erythrocytes were eliminated by a red blood cell lysis buffer . Absolute cell numbers were determined using BD™ Trucount tubes .Flow cytometry. Nonspecific binding was blocked with an anti-FcγIII/II antibody . The following antibodies and conjugates were used in the experiments: anti-Ly6G-Alexa647 (clone 1b8) , anti-CD3e-V500 (clone 500A2) , anti-CD19-V450 (clone 1D3) , anti-CD45-allophycocyanin (APC)/Cy7 (clone 30f11) , anti-CD4-Alexa700 (clone RM4-5) , anti-CD8-phycoerytrhin (PE)/Cy7 (clone 53-6.7) , CD11b-brilliant violet (BV)650 (clone M1/70) , and anti-NKp46-PE (clone 29A1.4.) . For dead cell exclusion, the cells were stained with 7-AAD Viability Staining Solution  before analysis. Stained cells were analysed on a BD LSR II Flow Cytometer  and evaluated with FlowJo software . Total spleen immune cells were defined as 7AAD−CD45+ cells, macrophages as 7AAD−CD45+CD11b+Ly6G−, neutrophils as 7AAD−CD45+CD11b+Ly6G+, B-cells as 7AAD−CD45+CD19+, T-cells as 7AAD−CD45+CD3+, and NK-cells as 7AAD−CD45+NKp46+ cells. T-cells were subdivided into T-helper cells (CD4+) and cytotoxic T-cells (CD8+).Cytokine analysis. Cell suspension from the spleen was incubated with RPMI  + 10% FCS + 1% penicillin/streptomycin  for 24 h at 37 °C and 5% CO2. The serum of 100 µL heparinised blood was frozen at −20 °C until further analysis. Serum and supernatants of the spleen were analysed by the Cytometric Bead Array (CBA) using the BD™ CBA Mouse/Rat Soluble Protein Master Buffer Kit and BD™ CBA Mouse Flex Sets  according to the manufacturer’s protocol. The following cytokines were analysed: IL-6, IL-10, TNF-α, INF-γ, IL-4, IL-1β, IL-6, IL-13, MCP-1, and IL-21. Stained beads were analysed on a BD LSR II Flow Cytometer  and evaluated with the FCAP Array™ software .Statistical analysis. Statistical analysis and graphs were performed with the GraphPad Prism software . The groups were tested for Gaussian distribution with the Shapiro–Wilk test. The three tumour-bearing operation groups were compared to its corresponding non-tumour-bearing group by the Student’s t-test when they were normally distributed; otherwise, the Mann–Whitney–U test was used. For multiple comparisons within the tumour-bearing groups (and control) or within the non-tumour-bearing groups, a one-way ANOVA, when normally distributed, or a Kruskal–Wallis test was performed. The survival rate was determined by Kaplan–Meier curve. p values < 0.05 were considered significant (* p < 0.05 and ** p < 0.01). The graphs show the mean and standard error when normally distributed and the median and interquartile ranges otherwise.The general condition of mice included in the experiments was assessed using the stress score modified from Partecke et al. . As expeWhile the body weight of the control animals increased by 10.4% during the observation period, the tumour-bearing animals showed significantly smaller increases in body weight. Among the tumour-bearing mice, those undergoing SID showed a tendency to increase their body weight to the smallest extent among all of the experimental groups observed .In order to assess whether SID resulted in increased growth of the primary tumour, the growth of the orthotopic pancreatic tumour was assessed by MRT scan on day 8 (8 days after tumour implantation and 3 days before SID) and day 27 (after two episodes of SID). Tumour growth was similar in animals with no surgical trauma, minor trauma (laparotomy), and SID .Visceral surgery unrelated to the primary tumour had no impact on the survival of tumour-bearing mice. The survival in tumour-bearing animals was similar in animals subjected to limited  or extensive  surgical trauma or no surgical trauma at all (73%) . Tumour-The total leucocyte counts were increased in tumour-bearing animals subjected to SID even 7 days after the last surgical procedure. In contrast, tumour-bearing mice without intercurrent surgery or with only minor surgical trauma showed no increase in the total leucocyte numbers compared to the healthy controls a. This dThe absolute spleen cell numbers were significantly increased in the tumour-bearing mice subjected to intercurrent SID compared to healthy control animals, while no significant increase could be detected between the later and tumour-bearing animals after intercurrent laparotomy or after no intercurrent surgery a. This oThe comparison of relative cell numbers showed a significant decrease in both the splenic B- and T-cells in tumour-bearing SID animals compared to the healthy controls, but also to tumour-bearing animals without intercurrent surgery d,e. Both+ T-helper cells and CD8+ cytotoxic T-cells in the tumour-bearing SID animals compared to the tumour-bearing animals without operations as well as the tumour-free control animals  are characterized by the capability to exercise a profound modification and inhibition of both the local and systemic immune response . Thus, sIn the present study, SII had no impact on the overall survival. This is in contrast to the results reported by Menges et al. . In thisOur results indicate that SID leads to a decline in the general condition of the tumour-bearing mice, which is supported by the tendency to a reduced weight gain in the period of 28 days following the tumour implantation as well as a significantly increased stress score. It is well-established that surgical stress provokes a physiological response with the release of glucocorticoids and the neurotransmitters epinephrine and norepinephrine in a multifactorial context . The resIn accordance with previous studies, we observed no influence of SID on the tumour growth as analysed by MRI scan on day 8 and day 27 following the tumour implantation . The incThe tumour generated by the 6606PDA cell line is considered to be valuable in characterising several aspects of cancer immunology: inflammatory cells are shown to surround but not to infiltrate the carcinoma . The tumThe immune microenvironment of PDAC has been characterised as immunosuppressive and up to now, immunotherapeutic approaches to this tumour entity have mostly shown unsatisfactory results ,29. ThisA profound knowledge of the impact of visceral surgery, which is part of the multimodal treatments against PDAC, on the immune system of tumour-bearing animals is of importance. In our model, SII was characterised by changes in the immune system, mostly described to hamper the anti-tumour immune response.+ and CD4+ T-cell populations had been reduced in tumour-bearing animals after SID, indicating that a reduced T-cell associated anti-tumour immune response induced by trauma due to surgery is not related to the primary tumour. Further characterisation of the tumour infiltrating T-cell populations in this model is required to address the local consequences of this observation.Increased numbers of cytotoxic CD8+ T cells and T-helper 1 T-cells have been shown to mediate the anti-tumour response in murine models of PDAC. Increased numbers of these cell types are correlated with an improved survival in human PDAC patients . In our Tumour-associated macrophages (TAM) and tumour-associated neutrophils (TAN) are essential components of the TME and have a profound impact on the biological behaviour of PDAC . IncreasIL-4 and IL-13 have both been shown to supress the anti-tumoural immune response, and moreover to directly drive the proliferation of KRAS-mutant tumour cells . The levIn this paper, we describe the systemic consequences of surgical procedures on the systemic immune status in mice bearing a pancreatic tumour. However, further research based on the findings in the present manuscript is required to complete the general picture of an immunomodulating influence of non-tumour related abdominal surgery in tumour-bearing mice. First, a histopathological analysis of the primary tumour is required to assess the local consequences of the observed systemic changes of the immune system. In pancreatic cancer, both the tumour cells and the TME can exert local immunosuppressive actions through a wide range of different mechanisms (reviewed in ). TherefOn the basis of the composition of the immune cell composition of the microenvironment, PDCA can be subdivided into distinct subtypes that are each associated with characteristic clinicopathological features and the presence of mutational changes in the tumour cells . The mutIn summary, the results presented herein revealed a profound impact of surgery-associated trauma on the immune system of the tumour-bearing animal. Several of the modified immune parameters observed have been described to be of major importance in the host’s immunological anti-tumour response. Given that anti-tumour immunotherapy is a promising therapeutic modality in many solid tumours but has mostly failed so far in PDAC , surgery


Background: Tumours are among the most lethal diseases that heavily endanger human health globally. Xuefu Zhuyu Decoction (XFZYD) is a prescription used to treat blood-activating stasis. Although XFZYD has been shown to suppress migration and invasion of tumour cells, the active ingredients, potential targets, and underlying mechanism remain largely elusive.Purpose: To identify the prospective ingredients and major targets of XFZYD against tumours, and evaluate the efficacy and potential molecular mechanisms of XFZYD extract on tumour growth and invasion.Methods: We predicted that XFZYD might act on 80 targets through 128 active components using the network pharmacology analysis method. In addition, we prepared an XFZYD aqueous extract and employed the RasV12/lgl−/−-induced Drosophila tumour model to carry out experimental verification.Results: XFZYD did not exhibit any side effects on development, viability, and fertility. Furthermore, XFZYD significantly impeded tumour size and invasion at moderate concentrations and suppressed the increased phosphorylation of JNK but strongly enhanced the expression of Caspase 3 in the RasV12/lgl−/− model. Finally, the mRNA level of the transcription complex AP-1 component c-FOS was remarkably reduced. In contrast, the transcription of three pro-apoptotic genes was significantly increased when XFZYD was used to treat the tumour model.Conclusion: The study findings suggest that XFZYD may promote tumour cell apoptosis by activating caspase signalling to control primary growth and hinder tumour cell invasion by suppressing JNK/AP-1 signalling activity, thus providing a potential therapeutic strategy for XFZYD in the clinical treatment of cancer and other related diseases. Cancers can be fatal diseases with high morbidity and mortality, posing a serious threat to global human health. According to Global Cancer Observatory, an estimated 19.3 million new cancer cases, and approximately 10.0 million cancer deaths occurred worldwide in 2020 . The occSalvia miltiorrhiza and Paeoniae radix extract induce apoptosis in a manner associated with Bcl-2 and the activation of Caspases 3 and 9 in HepG2 cells. Moreover, an extract from Astragalus membranaceus inhibits breast cancer cell growth through the PI3K/Akt/mTOR pathway  plays a role in the treatment of tumours through multiple factors, pathways, and targets . Reporte pathway . Meanwhi pathway .Xuefu Zhuyu decoction (XFZYD) has been widely researched and applied clinically for the treatment of blood stasis syndrome , which h, UAS/GAL4, FLP/FRT, mosaic analysis with a repressible cell marker (MARCM) system  cooperated with lgl4 homozygous mutant (RasV12/lgl−/−) mosaic clone-induced fly invasive tumour model  system , and the in vivo . Therefour model , to explAngelica sinensis (Oliv.) Diels. (Dang Gui), and Rehmannia glutinosa Libosch. (Sheng Dihuang), Prunus persica (L.) Batsch (Tao Ren), Carthamus tinctorius L. (Hong Hua), Citrus aurantium L. (Fuchao Zhiqiao), Paeonia lactiflora Pall. (Chi Shao), Bupleurum chinense DC. (Bei Chaihu), Glycyrrhiza uralensis Fisch. (Gan Cao), Platycodon grandiflorum (Jacq.) A. DC. (Jie Geng), Ligusticum chuanxiong Hort. (Chuan Xiong), and Cyathula officinalis Kuan (Chuan Niuxi) (http://tcmspw.com/tcmsp.php). All chemical composition information of the 11 drugs in XFZYD were collected, and further screening was performed based on two key evaluation indicators: oral bioavailability (OB ≥ 30%) and drug-like parameters (DL ≥ 0.18). In addition, domestic and foreign literature was consulted as a supplement, and the potentially effective active ingredients in XFZYD were obtained.XFZYD is composed of n Niuxi) . These dhttps://www.uniprot.org/) under the ‘Homo sapiens’ condition and was converted into a unified gene name.The targets of XFZYD bioactive components were also searched from the TCMSP databases. The target corresponded with the target protein and gene information correction in the UniProt database (www.genecards.org/) was used to acquire the relevant anti-tumour genes using the term ‘anti-tumour’ to screen for candidate targets. Only “Homo sapiens” proteins were selected, and the screening conditions were set at scores ≥1.81.GeneCards (https://cytoscape.org/) was used to build network relationship diagrams and analyse the results. The node size was determined by the degree value, which varied from high to large. The transparency of the node colour was determined by the degree of the high value to the deep colour.Cytoscape 3.7.0 (http://string-db.org) to demonstrate the interaction between target proteins. The species was set as “Homo sapiens”, and the “Interaction Score” was set to “Medium Confidence-0.400”. CytoHubba  was used to perform the Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis. Statistical significance was set at p < 0.05. The histogram was plotted using http://www.bioinformatics.com.cn, a free online platform for data analysis and visualisation.The DAVID database  to control the quality of XFZYD. The analyses were performed using a Shimadzu LC-20A instrument. The chromatographic column was an Agilent Eclipse XDB-C18 . The mobile phase flow rate was 1 ml/min, and the column temperature was maintained at 35 ± 1°C. Quercetin, kaempferol, isorhamnetin, luteolin, and baicalein were dissolved in methanol as controls to be detected by HPLC. Quercetin, kaempferol, isorhamnetin, and luteolin were eluted using a gradient system consisting of methanol (A) and 0.4% phosphoric acid solution (B) . Baicalein was eluted using a gradient system consisting of methanol (C) and 0.2% phosphoric acid solution (D) .Drosophila stock w1118 (#3605) was obtained from the Bloomington Drosophila Stock Centre (BDSC). To produce fluorescently labelled invasive tumours in the eye discs, the following strains were previously described  and starved for 2 h under adverse food conditions (0.8% agar in PBS). Then the larvae were transferred to fresh dye-added food (0.05% Brilliant Blue) for 20 min. After feeding, larvae were washed with PBS, dried on tissue paper, and homogenized in 100 μL of lysis buffer (PBS + 0.1% Triton X-100). After centrifugation, 2 μL of the supernatant was analysed with a spectrophotometer at 630 nm. A small amount of dye-containing food was weighed, processed as described above, and used as a standard to calculate the amount of ingested food. Triplicate measurements on each group were conducted .V12/lgl−/−-induced tumour invasion model is shown in V12/lgl−/− were kept on the XFZYD-added medium from the egg stage to the third instar larval stage or pupal stage. Female flies with genotype ey-Flp act>y+>GAL4 UAS-GFP were crossed with male flies of strain w1118 on regular food, and the progeny larvae with genotype ey-Flp act>y+>GAL4 UAS-GFP/+ were collected as the control group. Female flies with genotype yw ey-Flp; tub-GAL80 FRT40A; act>y+>GAL4 UAS-GFP were crossed with male flies of strain w; lgl4 FRT40A UAS-RasV12/Cyo; UAS-Puc/TM6B.Tb on regular food, and the progeny larvae with genotype yw ey-Flp/+; tub-GAL80 FRT40A/lgl4 FRT40A UAS-RasV12; act>y+>GAL4 UAS-GFP/UAS-Puc (RasV12/lgl−/−+Puc) were selected as the positive control.The crossing scheme for establishing the Rasw1118 flies were crossed, and eggs were laid in regular food or medium with XFZYD. Thirty healthy L1 larvae were collected 24 h after egg deposition (AED) and reared in 30 animals/tube with a new corresponding medium. The percentage of white pupae was estimated every 4 h to generate a developmental curve, and the percentage of emerging adults was recorded to assess viability  PBST, the tissues were stained with primary antibodies overnight at 4°C and then with secondary antibodies at RT for 4 h. The following antibodies were used: rabbit anti-pJNK , rabbit anti-cleaved Caspase 3 , and goat anti-Rabbit-Cyanine3 (Cy3) . Vectashield mounting medium  with 4′, 6-diamidino-2-phenylindole (DAPI) was used for mounting. The fluorophores DAPI, GFP, and Cy3 were excited and visualised using an inverted fluorescent microscope .rd instar larvae, and qRT-PCR was performed as previously described  was used to isolate total RNA from five 3escribed . Rp49 waPrimers used are provided:rp49 Sense: 5′-TACAGGCCCAAGATCGTGAA-3′For Antisense: 5′-TCTCCTTGCGCTTCTTGGA-3′c-Fos Sense: 5′-CCTTCAAATGGGCAGACAA-3′For Antisense: 5′-TGGGAGCGACGAAACACC-3′rpr Sense: 5′-AAAGTCCGGCAAATATCGCAAG-3′For Antisense: 5′-TGTTGTGGCTCTGTGTCCTTGACT-3′hid Sense: 5′-TGCGAAATACACGGGTTCA-3′For Antisense: 5′-CCAATATACACCCAGTCCCG-3′grim Sense: 5′-TCGGAGTTTGGATGCTGGGATC-3′For Antisense: 5′-AGTCACGTCGTCCTCATCGTTGT-3′p < 0.05 and p ≥ 0.05 was considered insignificant.The data reported were verified using at least three independent experiments. Image-Pro Plus 6.0 was used to calculate tumour size in pixels. Results were presented as bar graphs or line charts performed using GraphPad Prism 8.0. For statistical significance verification of the experiment, one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test or chi-squared test was applied. The centre values were taken as the mean, and error bars indicated standard deviation. Statistical significance was set at Angellica sinensis (Oliv) Diels., two from Rehmannia glutinosa Libosch, 17 from Bupleurum chinense, 23 from Prunus persica (L.) Batsch, 22 from Carthamus tinctorius L., five from Citrus aurantium L., 29 from Paeonia lactiflora Pall., 17 from Bupleurum chinense DC., 92 from Glycyrrhiza uralensis Fisch., 7 from Platycodon grandiflorum (Jacq.) A. DC., 7 from Ligusticum chuanxiong Hort, and 4 compounds from Cyathula officinalis Kuan.Based on the TCMSP databases, 186 active ingredients of XFZYD were screened, including two compounds from Based on these active ingredients in XFZYD, 273 active ingredients-related targets were acquired via TCMSP, and 289 anti-tumour-related targets were obtained from GeneCards. UniProtKB was used to obtain the standard names of the predicted target proteins. After merging active ingredient and anti-tumour-related targets, 80 overlapping targets were identified as candidates . A totalTo further elucidate the key active ingredients and targets, we analysed the complex relationship between XFZYD, active ingredients, anti-tumour activity, and gene targets. The drug-ingredient-disease-target network of XFZYD was constructed using Cytoscape. As a result, 220 nodes and 1,572 edges were included in this network. The network showed 11 herbs, 128 active ingredients, and 80 key targets and their interrelationships with XFZYD .A PPI network for the 80 key targets was constructed using Cytoscape. The network consists of 80 nodes and 1,692 edges in total . The topTo investigate the cell functions primarily impacted by the candidate targets, we first performed GO enrichment analysis, which includes biological processes (BP), molecular functions (MF), and cellular components (CC) to elucidate the multiple biological functions of potential targets on the anti-tumour activity at a systemic level . For BP,p < 0.05) were screened -Flp/MARCM system-mediated RasV12/lgl−/− tumour invasion model at the Drosophila 3rd instar larval stage to investigate the anti-tumour effects of XFZYD in vivo. In line with previous studies, ectopic expression of activated Ras (RasV12) in lgl4 homozygous mutant (lgl−/−) mosaic clones triggered noticeable tumour-like overgrowth at the cephalic complexes, which consist of the brain hemispheres (BH) and the eye-antennal discs (EA), labelled with GFP   . Further < 0.05)  reduced  < 0.05) . The tum < 0.05) , which s < 0.05) . The effNext, we measured the level of food intake and observed a normal ingestion rate in both the XFZYD-added and the control groups , ruling V12/lgl−/−-induced tumour growth and invasion, we considered TNF/JNK and caspase signalling as putative central targets. First, these factors, including TNF, MAPK8 , MYC, JUN, and MMP1, ranked in the top 30 of the PPI analysis  (c-FOS transcription was notably impeded by XFZYD at the concentration of 3.13 g/L through a qRT-PCR assay (reaper (rpr), head involution defective (hid), and grim  , and theCR assay . Furtherand grim , which aosophila . Hence, We applied network pharmacology and drug target prediction to further explore the anti-tumour mechanism of XFZYD. Our study showed that XFZYD anti-tumour activity might act on 80 targets through 128 active components. The main active components were β-sitosterol, stigmasterol, quercetin, kaempferol, campesterol, baicalein, luteolin, naringenin, and isorhamnetin. The PPI network showed that CASP3, JUN, TNF, MMPs, and MAPK are the core hubs of XFZYD in tumour treatment. XFZYD has the advantages of multiple active ingredients and targets, which can possess various anti-tumour activities providing new opportunities for discovering targeted small molecules of “yet to be drugged” or “undruggable” proteins.Drosophila melanogaster has been considered a good model system for studying the genetic and molecular mechanisms of tumourigenesis and metastasis  elicits an obvious inhibitory effect on cell invasion (V12/lgl−/−-induced tumour study based on the previous experiment. Collectively, 1.57 g/L, 3.13 g/L, and 6.25 g/L of XFZYD all reduced tumour size. Additionally, 3.13 g/L XFZYD strongly suppressed tumour invasion and rescued the pupariation rate in the RasV12/lgl−/− tumour model.Over the last 20 years, tastasis . Additioinvasion . Thus, wDrosophila food intake (Based on quantifications of daily d intake , it is ed intake . Thus, aDrosophila tumour models caused by different gene mutations. The effect of XFZYD on the invasion percentage and tumour size is not decreased in a dose-dependent manner. Our findings suggest that the dosage of XFZYD should be determined according to the specific situation of the patients receiving the clinical treatment for tumour metastasis.Personalized treatment based on TCM theory plays a key role in cancer treatment and is consistent with the concept of precision medical treatment for cancer . TCM comV12/lgl−/− tumour invasion model. However, the expression of pJNK decreased, while that of cleaved Caspase3 increased following XFZYD treatment. Furthermore, XFZYD decreased the expression of c-FOS and increased the mRNA levels of rpr, hid, and grim in the RasV12/lgl−/− background, indicating that XFZYD may hinder tumour cell invasion by suppressing JNK/AP-1 signalling and promoting tumour cell apoptosis by activating caspase signalling.Our previous study showed that XFZYD inhibits tumour cell migration by regulating MMP1 expression . MoreoveIn summary, our work demonstrates that XFZYD displays anti-tumour activity at moderate concentrations. XFZYD impedes tumour invasion, presumably by inhibiting JNK signalling, and stimulating caspase-mediated apoptosis, thus, highlighting specific key drug targets and crucial strategies for cancer clinical treatment.


VZV responsiveness was defined according to IFN-γ release from VZV-stimulated PBMC. Upon VZV stimulation, we found that allo-HSCT recipients at a median time of 6 [5-8] months post-transplant had lower IFN-γ release  and fewer proliferating T cells  than HI. A subset of allo-HSCT recipients  distinguished themselves from VZV-non-responders  by higher IFN-γ release  and T-cell proliferation , suggesting recovery of VZV-specific CMI. Interestingly, VZV responders had a significant fold increase in ifn-γ gene expression, whereas ifn-γ mRNA was not detected in whole blood of VZV-non-responders (P <.0001). This study is the first to suggest that measurement of ifn-γ gene expression in 24-h-stimulated whole blood could be an accurate test of VZV-specific CMI. The routine use of this immune functional assay to guide antiviral prophylaxis at an individual level remains to be evaluated.Herpes zoster, which is due to the reactivation of Varicella zoster virus (VZV), is a leading cause of morbidity after allogeneic hematopoietic stem cell transplantation . While cell-mediated immunity (CMI) is critical to inhibiting VZV reactivation, CMI is not routinely assessed due to a lack of reliable tests. In this study, we aimed to evaluate VZV-specific CMI among allo-HSCT recipients (n = 60) and healthy individuals  through a panel of three immune functional assays after  Allogeneic hematopoietic stem cell transplantation  recipients are at increased risk of reactivation of latent varicella zoster virus (VZV) infection, with up to half of patients experiencing herpes zoster (HZ) during the first 5 years post-transplant –4. In adCurrently, prevention of VZV disease in seropositive allo-HSCT recipients relies on antiviral prophylaxis with acyclovir or famciclovir recommended for at least 12 months post-transplant or longer in case of protracted immune recovery , 16. Durin vitro stimulation by VZV antigens. These assays can be performed on peripheral blood mononuclear cells (PBMC) or whole-blood samples, and different readouts of VZV-specific cell-mediated immunity can be used, such as evaluation of lymphocyte proliferative response or quantification of interferon (IFN)-γ secretion   which evaluate cellular immune responses to y, IGRA) –23. NeveComité de Protection des Personnes Sud-Est V, Grenoble, France, number 69HCL17_0769) and is registered in ClinicalTrials.gov (NCT03659773).Consecutive, adult allo-HSCT recipients transplanted at the hematology department of the Lyon University Hospital (France) and participating in the prospective, single-center “VaccHemInf” cohort study between May 2018 and August 2020 were enrolled. The VaccHemInf cohort study aims at studying vaccine response and immune reconstitution in HSCT recipients, as described elsewhere . BrieflyEtablissement Français du Sang, EFS), were obtained to serve as controls. According to the EFS-standardized procedures for blood donation and to provisions of the articles R.1243–49 and following ones of the French public health code, a written non-opposition to the use of donated blood for research purposes was obtained from HI. The blood donors’ personal data were anonymized before transfer to the research laboratory. Regulatory authorizations for the handling and conservation of these samples were obtained from the regional review board (Comité de Protection des Personnes Sud-Est II) and the French ministry of research . Blood samples from children (VZV-naïve controls) were provided as part of a study approved by the regional review board , French Data Protection Authority , and French Advisory Committee on Healthcare Research Data Processing . Parents’ written consent was obtained for each participant.Concomitantly, blood samples from healthy individuals (HI), recruited among adult donors to the Lyon (France) blood bank  according to the manufacturers’ instructions. An IgG antibody titer ≥ 150 mIU/ml was considered positive (VZV-specific immunoglobulin (Ig)G was measured in sera by a chemiluminescence immunoassay , or MRC-5 control antigen  at 20 μg/l, or medium alone  for 7 days. A 3-day stimulation with phytohemagglutinin  at 4 µg/l served as mitogen control and medium alone (NUL) as its related negative control condition. Concentrations of stimulants, as well as length of incubation, had been determined by preliminary assays (data not shown).As previously described, fresh PBMC were purified from a heparin-coated blood tube . Fresh PAt each time point, IFN-γ was measured in PBMC supernatants using the ELLA nanofluidic system  according to the manufacturers’ instructions. All values below the lowest limit of quantification (LLOQ = 0.17 pg/ml) provided by the manufacturer were reduced to a value equal to LLOQ/√2 (= 0.12 pg/ml). Patients were classified into “VZV-responders” and “VZV-non-responders” according to the concentration of IFN-γ in supernatants from VZV-stimulated PBMC, with a positivity threshold arbitrarily fixed at twice (4.83 pg/ml) the highest secretion obtained in the control condition (2.415 pg/ml).+ proliferating cells (among CD3+ cells) was obtained by flow cytometry analyses performed on a Navios flow cytometer . For each experiment, a minimum of 2.5 × 103 CD3+ cells were recorded. Data were analyzed using Kaluza software . Values equal to “zero” proliferation were set to 0.01% to be plottable on a logarithmic scale.Pellets collected from paired wells were analyzed for T-cell proliferation using the Click-It™ EdU AF488 flow kit  to measure incorporation of 5-ethynyl-2′-deoxyuridine (EdU) according to the previously published protocol . Briefly2. Total RNA was extracted using the RNeasy Blood Mini Kit  according to the manufacturer’s instructions. Briefly, the cells were harvested and lysed in RLT buffer (lysis buffer) supplemented with β-mercaptoethanol and stored at -80°C until further processing. RNA quantity and quality were determined using NanoDrop . For mRNA detection, RNA was retro-transcribed using SuperScript VILO cDNA Synthesis kit (Thermo Fisher) followed by qPCR performed using commercial TaqMan probes for ifn-γ  and normalized using the mean of PPIB and DECR1 housekeeping genes on CFX Connect Real-Time (RT)-PCR Detection System . The relative differential expression of the ifn-γ gene was expressed as a fold change using the 2-ΔΔCt method . ΔΔCT is the difference of ifn-γ gene ΔCT obtained in stimulated conditions (VZV or CTRL) compared to the unstimulated control condition (NUL).Heparinized whole blood (100 µl) was distributed into a 96-well plate. VZV antigen at 20 µg/l (VZV), medium alone , or supernatants from uninfected MRC-5 cells  were added to a single well and incubated for 24 h at 37°C and 5% of COt method . The fir+CCR7+) CD4+ and CD8+ T-cells, central memory (CD45RA-CCR7+) CD4+ and CD8+ T-cells, effector memory (CD45RA-CCR7-) CD4+ and CD8+ T-cells, and differentiated memory (CD45RA+CCR7-) CD4+ and CD8+ T-cells were counted (cells/μL), in addition to routine absolute CD4+ and CD8+ T lymphocyte counts.A large panel of T-cell membrane markers was measured by flow cytometry  on whole blood. Naïve  and R .Qualitative variables were expressed as counts (percentages), and quantitative continuous data were expressed as mean and range or as median and interquartile range [IQR]. Quantitative non-gaussian data were compared using the Kruskal–Wallis test or the Mann–Whitney test, where appropriate. Qualitative data were compared between groups using Fisher’s exact test. The association between quantitative data was quantified by a non-parametric Spearman rank-correlation coefficient . Two-sided tests were performed with statistical significance defined by P = .39) and sex . Allo-HSCT recipients were enrolled at a median [IQR] time of 6 [5-8] months post-transplant. All HI were VZV-seropositive, 98% , and 75% (n = 45/60) of allo-HSCT recipients had positive VZV IgG titers prior to transplantation and at enrollment, respectively. Nineteen (32%) allo-HSCT recipients were on immunosuppressive therapy, and 41 (68%) had received intravenous Ig infusions at a median [IQR] time of 4 [3-6] months before inclusion (P = .16). Pediatric controls (n = 2) were VZV-naïve.HI (n = 17) and allo-HSCT recipients (n = 60) were included. HI and allo-HSCT recipients did not significantly differ in terms of age  (P <.0001 for both) compared to control conditions, indicating that these tests were appropriate to assess lymphocyte functionality in the study population. However, IFN-γ release  and proliferation capacity of T-cells  were significantly lower in allo-HSCT recipients than in HI (+ CD3+ cells) upon PHA stimulation were 52.6–210.3 pg/ml and 38.5%–45.4%, respectively.First, to verify that HI and allo-HSCT recipients were able to mount a detectable immune response upon mitogen stimulation, IFN-γ in supernatants and proliferation of PHA-stimulated PBMC were determined. HI and allo-HSCT recipients developed a significantly higher IFN-γ release and percentage of EdUP <.0001) and a significantly lower percentage of EdU+ proliferating cells (among CD3+ cells)  among allo-HSCT recipients as compared to HI (+ proliferating CD3+ cells: 0%–0%) and had IFN-γ release below LLOQ (0.104–0.131 pg/ml).A 7-day stimulation of PBMC with VZV antigen resulted in a significantly lower IFN-γ release in the supernatant . The IFN-γ release among the VZV-responder group was lower than among HI , but the difference did not reach statistical significance (P = .64). Of note, similar observations were made for the VZV-specific proliferative response, with a significant higher proliferation capacity in the VZV-responder group, as compared to the VZV-non-responder group . Proliferative response among the VZV-responder group was lower than in HI without reaching statistical significance .Results indicated that allo-HSCT recipients had an impaired VZV-specific T-cell function based on lower proliferation and IFN-γ release post-stimulation. When focusing on allo-HSCT recipients’ results, two subsets were observed and classified as VZV responders  or non-responders  based on IFN-γ secretion level above the positivity threshold Table 2.P = .005). Altogether, these results suggest an ongoing recovery of VZV-specific immunity in the VZV-responder group. Only 2/15 (13%) had reported HZ-compatible clinical manifestations at 4 and 5 months post-transplant, respectively, while none in the VZV-non-responder group.Interestingly, 13/15 (87%) of VZV responders had a positive VZV-specific humoral response, as depicted by higher IgG titers , as compared to the VZV-non-responder group, where 29/42 (69%) had a positive titer  had a significant fold increase in ifn-γ gene expression in whole blood , whereas the relative expression of ifn-γ mRNA was not detected for other allo-HSCT recipients   . Overall, results of VZV-specific IFA in VZV-naïve pediatric controls confirmed the specificity of these assays.To investigate the VZV-specific immune response at the transcriptional level, the relative differential expression (fold change) of + T-cell count was lower than normal values (+CCR7+) and central memory (CD45RA-CCR7+) CD4+ T-cells, as well as of naïve (CD45RA+CCR7+) CD8+ T-cells , 0.28 (95% CI [0.02–0.52]) and 0.36 (95% CI [0.10–0.58]), respectively). When comparing VZV responders and non-responders, median naïve and central memory CD4+ and naïve CD8+ T-cell counts were significantly higher in the VZV-responder group . Of note, no associations were observed between VZV responsiveness and clinical determinants  T-cell counts and central memory (CD45RA-CCR7+) CD4+ T-cell counts correlated with the reconstitution of the VZV-specific immune repertoire.This study provides insights into VZV-specific immune reconstitution after allo-HSCT. We found that 15/57 (26%) of allo-HSCT recipients had recovered a VZV-specific IFN-γ response by 6 months post-transplant, in the absence of any HZ-related clinical event for the majority of them. Interestingly, the measurement of in vitro assays showed detectable VZV-specific immune cell responses. Relative to the entire cohort of allo-HSCT recipients, this proportion of subclinical VZV reactivations  is overall consistent with previous studies that reported 26% to 41% of subclinical VZV reactivations among adult bone marrow transplant recipients  or evaluating cellular response to VZV vaccination.Taken together and considering that one quarter of allo-HSCT recipients had detectable VZV-specific cell-mediated immunity by the time of study initiation (6 months post-transplant), measuring the VZV-specific Beyond antiviral prophylaxis, VZV-related disease can be prevented by vaccination. The inactivated RZV has demonstrated a clinical efficacy of 68.2% to prevent HZ after autologous HSCT and has been recently shown in a prospective cohort study to be safe after allo-HSCT, although immunogenicity data in this setting are lacking , 40. Thein vivo . The time of 6-month post-transplant corresponds to a pivotal period, when allo-HSCT recipients are usually off immunosuppressive therapy and when immune reconstitution is well underway from a quantitative point of view. We did not assess in this study kinetics of antiviral immune reconstitution. Due to the high seroprevalence of VZV (>95%) among adults in France, the specificity of VZV IFA was confirmed on pediatric samples , 43. Thein vivo , 45. Pepin vivo , 47; simin vivo .ifn-γ gene expression assay from a whole-blood sample and immunophenotyping of T-cell subsets could help to monitor antiviral immune repertoire reconstitution after allo-HSCT and ultimately enable a more rationale use of antiviral prophylaxis.In conclusion, as the management of immunocompromised individuals evolves to increasingly personalized agendas, measurement of VZV-specific cell-mediated immunity by a fast (24-h) The datasets presented in this article are not readily available because no patient-level data can be made available, in accordance with data protection laws. Upon reasonable request, aggregate data with different levels of resolution may be possible to provide. Requests to access the datasets should be directed to florence.ader@chu-lyon.fr.The studies involving human participants were reviewed and approved by Comité de Protection des Personnes Sud-Est V, Grenoble, France (69HCL17_0769). The patients/participants provided their written informed consent to participate in this study.MB contributed to the conception and design of the study and to the acquisition and interpretation of the data and drafted the manuscript. AC and WM contributed to the acquisition and interpretation of the data and drafted the manuscript. CR-S and EF performed the VZV serology assays and contributed to the revision of the paper for important intellectual content. BR provided pediatric control samples and contributed to the revision of the paper for important intellectual content. FVa, VA, HL-W, HG, and KB-P contributed to the interpretation of the data and to the revision of the paper for important intellectual content. FVe contributed to the acquisition of the data of immune functional assays, to the interpretation of the data, and to the revision of the paper for important intellectual content. ST-A contributed to the conception and design of the study, to the acquisition and interpretation of the data, and to the revision of the paper for important intellectual content. FA conceived and designed the study, participated in the analyses and interpretation of the data, and drafted the manuscript. All authors have read and have approved the final version of the manuscript.This research was funded by an internal grant from the Hospices Civils de Lyon  and by the Région Auvergne-Rhône-Alpes .in vitro diagnostic company.WM has a PhD grant CIFRE 2019  half-funded by Lyon University and half-funded by bioMerieux SA. KB-P is an employee of bioMérieux SA, an The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.