Bone metastasis occurs when tumour cells dissociate from primary tumours, enter the circulation , and colonize sites in bone . The bone marrow seems to be a particularly dormancy-inducing environment for DTCs, yet the mechanisms of dormancy initiation, reactivation, and interaction within the bone marrow have to be elucidated. Intriguingly, some evidence has suggested that dormancy is a reversible state that is switched ‘on’ or ‘off’ depending on the presence of various bone marrow resident cells, particularly osteoclasts and osteoblasts. It has become clear that these two cells contribute to regulating dormant tumour cells in bone both directly (interaction) and indirectly (secreted factors). The involved mechanisms include TGFβ signalling, the Wnt signalling axis, the Notch2 pathway, etc. There is no detailed review that specifically focuses on ascertaining the dynamic interactions between tumour cell dormancy and bone remodelling. In addition, we highlighted the roles of inflammatory cytokines during this ‘cell-to-cell’ communication. We also discussed the potential clinical relevance of remodelling the bone marrow niche in controlling dormant tumour cells. Understanding the unique role of osteoclasts and osteoblasts in regulating tumour dormancy in bone marrow will provide new insight into preventing and treating tumour bone metastasis. Tumour metastasis remains an extraordinarily complicated and poorly understood process. During tumour metastasis, cancer cells first undergo epithelial-to-mesenchymal transition (EMT) to dissociate from the primary tumour and enter the microvasculature of the blood and lymphatic systems . Once en0 phase, and able to re-enter the cell cycle and re-proliferate [To survive and grow, cancer cells must navigate themselves to adapt to these features in the bone microenvironment, thus it is sensible for DTCs to enter the dormant state. Tumour dormancy is generally divided into two categories, tumour cell dormancy and tumour mass dormancy. Tumour cell dormancy  refers to cancer cells, including CTCs and DTCs, that are transiently suspended in Gliferate . While tliferate . Dormantliferate . Moreoveliferate . Indeed,liferate –14. Becaliferate , 15. Aftliferate . NotablyAccumulating evidence has revealed the close involvement of bone remodelling during the progression of bone metastasis. Bone remodelling involves a complex set of interactions that result in an overall maintenance of bone mass or an anabolic or catabolic bone state . This dyIntriguingly, evidence has illustrated the unusual role of osteoblasts and osteoclasts in controlling the switch between dormancy versus proliferation in DTCs during bone metastasis . Take mySeveral critical mechanisms involved in osteoclasts/osteoblasts and tumour cell dormancy must be elucidated. a. How do tumour cells initiate and maintain a dormant state via osteoclasts/osteoblasts in the bone? b. What factors released during bone remodelling trigger the reactivation of dormant tumour cells? c. Do dormant tumour cells influence the effects of the bone marrow niche on regulating tumour dormancy? In this review, we clarified and summarized the unique role of osteoclasts and osteoblasts in regulating tumour dormancy in bone marrow, highlighted the link of dormant tumour cells in remodelling the bone marrow niche, and discussed promising therapeutic approaches.Recent findings revealed the role of osteoblasts in promoting tumour cell dormancy through direct interactions with cancer cells Table  Fig. 1)1). By usin vivo study showed that withdrawal of BMP7 significantly abrogated the suppressive effect of osteoblasts and induced metastatic growth of stem-like prostate cancer cells in the bone [Apart from direct interaction between osteoblasts and cancer cells, it has also been observed that factors such as type-I collagen, osteopontin, and Wnt5a produced by osteoblasts or the osteoblastic niche could support tumour cell dormancy. Lawson and colleagues tracked individual myeloma cells by intravital imaging in a green fluorescent protein (GFP) transgenic mouse model. They found that dormant myeloma cells localized directly adjacent to endosteal bone surfaces occupied by type-I collagen-expressing osteoblast cells, while proliferating myeloma cells were preferentially found at locations distant from the bone surface . Anotherthe bone . Wnt sigthe bone . Ren et the bone .in vitro and in vivo, indicating that osteoblast differentiation may affect tumour dormancy [1-cell cycle arrest [Interestingly, the bone-derived transforming growth factor (TGF) β1 and TGFβ2 have been demonstrated to exert opposite functions on the behaviour of tumour cell dormancy in the bone marrow. TGFβ1 facilitates rapid tumour proliferation , while Tdormancy . Mechanie arrest . Similare arrest , might de arrest .Currently, emerging evidence has proven that osteoblast-mediated bone formation and the related osteoblastic niche play a supportive role in dormancy induction and maintenance. This is contrary to previous beliefs that osteoblasts could encourage tumour growth by providing essential growth factors , 38. To https://smart.servier.com/).Osteoblasts can either interact directly with dormant tumour cells or indirectly through secretion of various factors, wherein both ways control the rate of cell proliferation and induce dormancy of the tumour cells near the bone surface. Osteoblast-secreted factors include BMP7, TGFβ2, BMP1, DKK3, vasorin, neogenin, LIF, CXCL12, Wnt5a, Type-I collagen, OPN, and Gas6. In turn, dormant tumour cells preferentially adhere to osteoblasts, thus facilitating bone formation to induce and maintain themselves in a dormant state. In this case, dormant tumour cells upregulate their expression of several signalling receptors, such as Axl, TBK1, and N-cadherin, thereby allowing adhesive attraction of dormant tumour cells to osteoblasts. BMP7, bone morphogenetic protein 7; BMP1, bone morphogenetic protein 1; TGF, transforming growth factor; DKK3, dickkopf-related protein 3; ROR2, receptor tyrosine kinase-like orphan receptor 2; NDRG1, N-myc downstream-regulated gene 1; MAPK, mitogen-activated protein kinases; LIF, leukaemia inhibitory factor; LIFR, LIF receptor; CXCL12, C-X-C motif chemokine ligand 12; CXCR4: C-X-C motif chemokine receptor 4; OPN, osteopontin; Gas6, growth-arrest specific 6; TBK1, TANK binding kinase 1; mTOR, mammalian/mechanistic target of rapamycin. Graphics were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license 2). In exin vivo . Moreovein vivo . Lawson in vivo . These rin vivo [Bone resorption is also mediated by hormones, especially oestrogen and androgen, which might subsequently impact dormant tumour reactivation in bone Table . Bone loin vivo . Additioin vivo . In men,in vivo . In factin vivo –51. The https://smart.servier.com/).Bone metastasis tumour cells exit from dormancy can be influenced by factors produced during osteoclast-mediated bone resorption, including TGFβ1 and MMP-9. To reactivate themselves from dormancy, dormant tumour cells are inclined to promote osteoclastogenesis by upregulating the expression of several receptors, such as VCAM-1 and PTHrP. Hormone-related therapies  could facilitate osteoclastogenesis and increase bone resorption, subsequently releasing dormant tumour cells to reproliferate and consequently inducing overt metastasis. Therapeutic agents that suppress bone resorption could inhibit bone metastasis, such as EGFR tyrosine kinase inhibitor, Cathepsin K inhibitor, Cathepsin B inhibitor. TGF, transforming growth factor; MMP-9, matrix metalloproteinase-9; VCAM-1, vascular cell-adhesion molecule 1; PTHrP, parathyroid hormone-related protein; IL-1β, interleukin-1β; IL-1R, interleukin-1 receptor; PGE2, prostaglandin E2; RANK, receptor activator of nuclear factor-κB; RANKL, receptor activator of nuclear factor-κB ligand; sRANKL, a soluble form of the RANKL; TNF-α, tumour necrosis factor-α; OPG, osteopontin; EGFR, epidermal growth factor receptor. Graphics were partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license  that could determine the conditions of tumour dormancy.Leukaemia inhibitory factor (LIF), an inflammatory cytokine of the IL-6 family, is produced by osteoblasts. LIF promoted breast cancer cell dormancy in the bone by binding to the LIF receptor (LIFR), which induced the expression of crucial dormancy-related genes in breast tumour cells, including thrombospondin-1 (TSP1), tropomyosin-1 (TPM1), TGFβ2, prolyl 4 hydroxylase α-1 (P4HA1), miRNA-190 (miR-190) and SELENBP1. Knockdown of LIFR resulted in lower p53 protein levels and greater c-MYC and pSRC (Y527) protein levels, suggesting that LIFR was crucial for MCF7 cells to remain dormant . CXCL12,In contrast to the dormancy-inducing effect of LIF, CXCL12 and Gas6, the inflammatory cytokine IL-1β promoted tumour proliferation and subsequently triggered overt metastasis of breast tumour cells. Direct interaction between breast tumour cells and osteoblasts promoted IL-1β release from both cell types, which enhanced the progression of EMT, invasion, migration, angiogenesis, and bone colonization . MoreoveOverall, inflammatory cytokines produced mainly by osteoblasts during bone remodelling play a dual role in tumour cell dormancy. This may partially explain why osteoblasts could both induce tumour cell dormancy and promote tumour cell growth in the bone marrow. Inflammatory factors produced during the interactions between dormant tumour cells and osteoblasts may serve as novel biomarkers, which can be utilized to predict the state of tumour cell dormancy and the potential risk of tumour relapse.R2=0.3588, P<0.0001) [There is compelling clinical evidence that tumour cell dormancy exists and that the bone microenvironment may initiate tumour cell dormancy and trigger DTC proliferation. Increasing the understanding that osteoblasts and osteoclasts control the state of dormancy provides essential clinical implications. For instance, identifying dormancy-related factors produced by osteoblasts and osteoclasts provides opportunities to predict and prevent dormant tumours from developing overt bone metastasis. Bone-disseminated prostate tumour cells with lower expression of TGFβRIII caused by differentiated osteoblasts might be less able to enter dormancy. Further prognostic analysis confirmed that lower levels of TGFβRIII expression are associated with metastasis and poor clinical performance outcomes in prostate cancer patients . ROR2 co<0.0001) .Another important implication may come in the context of treatment. Because dormant tumour cell-targeting agents are not yet clinically available, treatments that regulate bone biological processes, especially anti-resorptive therapies, hold promise for controlling tumour dormancy Fig. . In the Undoubtedly, cellular dormancy is a highly complex phenomenon involved during bone metastasis. The interactions between osteoclast/osteoblast-mediated bone remodelling and dormant tumour cells are likely among the most critical rationales of metastatic tumour outgrowth in bone. A majority of strategies focus on keeping dormant tumours ‘asleep’ instead of ‘waking them up’ so that tumour outgrowth and metastasis can be potentially prevented. However, we cannot exclude the possibility that creating more bone niches for supporting dormant tumour cell survival will cause long-term deleterious effects. Dormant tumour cells are resistant to most chemotherapies and radiotherapies that target proliferative tumour cells . Activat


Tissue hydraulic permeability of brain tumours can vary considerably depending on the tissue microstructure, compositions in interstitium and tumour cells. Its effects on drug transport and accumulation remain poorly understood.Mathematical modelling is applied to predict the drug delivery outcomes in tumours with different tissue permeability upon convection-enhanced delivery. The modelling is based on a 3-D realistic tumour model that is extracted from patient magnetic resonance images.Modelling results show that infusing drugs into a permeable tumour can facilitate a more favourable hydraulic environment for drug transport. The infused drugs will exhibit a relatively uniform distribution and cover a larger tumour volume for effective cell killing. Cross-comparisons show the delivery outcomes are more sensitive to the changes in tissue hydraulic permeability and blood pressure than the fluid flow from the brain ventricle. Quantitative analyses demonstrate that increasing the fluid gain from both the blood and brain ventricle can further improve the interstitial fluid flow, and thereby enhance the delivery outcomes. Furthermore, similar responses to the changes in tissue hydraulic permeability can be found for different types of drugs.Tissue hydraulic permeability as an intrinsic property can influence drug accumulation and distribution. Results from this study can deepen the understanding of the interplays between drug and tissues that are involved in the drug delivery processes in chemotherapy. Glioblastoma is the most common primary malignant brain tumour that makes up 16% of all primary brain tumours and 54% of all gliomas . It is cBrain interstitial fluid (ISF) flow is an essential physiological process that contributes to the nutrient and oxygen supply to cells and waste clearance, due to the lack of functional lymphatics in the brain . Fluid fDrug delivery is composed of multiple physiological and physicochemical processes that are determined by the interplays between the biological properties of the tumour and the transport properties of the drugs . MathemaIn this study, a transport-based model is applied to examine the effects of hydraulic permeability of tumour tissue on the performance of nanoparticle-mediated CED. The modelling is based on a 3-D realistic brain tumour model that is reconstructed from the patient Magnetic Resonance (MR) images. The model is designed to capture the key processes in the intracerebral drug delivery, including the CSF and ISF flow, fluid gain from the blood, drug transport by convection with the interstitial fluid flow, drug diffusive transport in the extracellular space, drug release from nanoparticles, drug binding with proteins, elimination due to bioreactions and physical degradation. The treatment is evaluated by the effective distribution volume , cell membrane (CM) and intracellular space (ICS). The transport processes of drugs after being infused are schematically illustrated in Fig. Following the principle of conservation of mass, the concentration of nanoparticles in tissue . The mesh independence test is performed, and the final computational mesh consists of 5.1 million tetrahedral elements. The volumes of brain normal tissue and tumour are measured as 1387 and 25 cmSince the time window of drug delivery is usually much smaller as compared to the rate of tumour growth, the brain and drug properties are assumed to be time-independent. Temozolomide is selected as a representative drug for brain tumour treatment. The values of the model parameters are summarised in Tables 2  for generating the numerical solutions. The 2The pressure on the brain surface and ventricle surface are specified as 1447 Pa , respectThe delivery outcomes of nanoparticle-encapsulated drugs under different conditions are evaluated from the perspectives of drug accumulation, drug spatial distribution and potential treatment effectiveness. These are represented by quantitative indexes defined below.Drug accumulation is determined by the convective and diffusive transport, elimination due to blood drainage, drug release for nanoparticles, and degradation and bioreactions for free chemotherapy drugs. The spatial-averaged drug concentration , paclitaxel (PTX), and carmustine (BCNU). In addition, doxorubicin (DOX) also has the ability to kill brain tumour cells . TherefoThe responses of different chemotherapy drugs to hydraulic permeability of tumour tissue are compared in Fig. The hydraulic permeability of tumour tissue is determined by the tissue microstructure and compositions, particularly the fibre network and polysaccharides in the extracellular matrix. Standing for the tissue resistance to the fluid flow, the low tissue hydraulic permeability leads to high intratumoural pressure and slow interstitial fluid flow upon CED infusion. Such a hydraulic environment can consequently inhibit the fluid gain from the blood and generate a more heterogeneous drug distribution. Although this distribution pattern allows achieving more localised drug delivery, the drug penetration into deep tumour tissue is simultaneously reduced. This would further lead to inadequate drug concentration in the tumour region, which is distant from the infusion site, reducing the treatment effectiveness.CED improves drug delivery outcomes by enhancing the interstitial fluid flow in the tumour ECS. As the sources of ISF in the brain, the fluid gain from the blood and CSF flow from the ventricle contribute to a favourable hydraulic environment that improves the delivery outcomes in the tumour. Notably, the understanding of some underlying mechanisms involved in the complex brain ISF flow remains controversial. For instance, water was reported to be able to cross the BBB through different means including diffusion and vesicular transport , 88. ThiCSF heavily participates in the intracerebral ISF flow. Entering the brain parenchyma from the brain surface next to the subarachnoid space, CSF travels in the perivascular spaces alongside the arteries and eventually leaves the brain tissues through perivenous spaces, perineural spaces and lymphatic vessels in the meninges , 93. ThiThe results from this study show that the four chemotherapy drugs present a similar trend when responding to the changes in hydraulic permeability of tumour tissue. On the contrary, the delivery outcomes vary considerably between the drugs, depending on the properties of the drugs. One of the essential factors is drug elimination due to blood drainage. TMZ and BCNU have been applied in the clinical treatments of brain cancer because of their ability to cross the BBB upon routine intravenous administration. However, as drugs are directly infused into the tumour ECS upon CED, this transvascular transport would enhance the drug loss by blood drainage, thereby reducing delivery outcomes. In contrast, plain DOX is rarely used in routine chemotherapy to treat brain tumours, as it is usually believed unable to penetrate BBB . Whereasin vivo experiments demonstrated that the tumour size has a limited impact on the distribution volume of nanoparticles, which is further confirmed by the modelling study. The predictive power of the transport-based model for predicting drug delivery to solid tumours has been validated in several reported studies using in vivo experimental data. The IFV in the tumour was predicted as 0.17 μm/s  The microvasculature distribution can be highly heterogeneous, depending on the tumour type, location and stage. Since there is a lack of tributed , 38. Thitributed . (II) A tributed . Howevertributed . Moreovetributed . Given iThe effects of hydraulic permeability of tumour tissue on convection-enhanced delivery of nanoparticle-encapsulated drugs to brain tumours have been examined using mathematical modelling. Results show that drugs can transport deep into a permeable tumour with a relatively uniform distribution. This is beneficial for achieving more effective cell killing in a larger tumour region, although the spatial-averaged concentration would be slightly low. The delivery outcomes of CED are more sensitive to the changes in tissue hydraulic permeability and blood pressure as compared to the cerebrospinal fluid flow from the ventricle. High blood pressure and permeable ventricle surface allow more fluid to transport from the blood and ventricle to the brain, respectively. These fluid gains can further enhance the intracerebral ISF flow, and thereby improve the delivery outcomes. Moreover, different drugs present similar responses to the changes in hydraulic permeability of tumour tissue. Results obtained from this study can deepen the understanding of the transport mechanisms involved in drug delivery to brain tumours upon CED.


To analyze data from children who were previously healthy and presented with post-varicella arterial ischemic stroke upon arrival when admitted to the emergency room, with focus on the clinical/laboratory aspects, and neurocognitive performance after four-year follow-up. Seven children presenting with arterial ischemic stroke after varicella were evaluated at pediatric emergency services in the city of São Paulo (SP), Brazil. Ischemic stroke was determined by magnetic resonance imaging/magnetic resonance angiography in a topography compatible with the areas supplied by the middle cerebral or internal carotid arteries. IgG-class antibodies against varicella zoster virus and varicella-zoster virus DNA by polymerase chain reaction in cerebrospinal fluid were tested. Patients with prothrombotic conditions were excluded. The Pediatric Stroke Outcome Measure was applied upon admission and 4-years after the stroke. All patients (age range: 1.3 to 4 years) included presented chickenpox 5.1 (±3.5) months before. All patients had analysis of anti-varicella-zoster-virus-IgG in cerebrospinal fluid, but only three (43%) had a positive result. Of the patients 43% had no vascular lesions identified in magnetic resonance angiography. All patients showed improvement in their sequela scores. After 4 years, five patients displayed good evolution in the Pediatric Stroke Outcome Measure, and only one patient presented with a score of 2 in the sensorimotor and cognition areas. No recurrence of arterial ischemic stroke was observed. We reinforced the non-progressive course of post-varicella arterial ischemic stroke after 4-year follow-up. The presence of varicella-zoster-virus-DNA detected by polymerase chain reaction, and/or intrathecal IgG antibody against varicella zoster virus, and angiopathy location in magnetic resonance angiography were not determining for the diagnosis. Invasive tests, with low sensitivity, should be well considered in the diagnosis of post-varicella arterial ischemic stroke. Transient cerebral arteriopathy affects the middle cerebral artery (MCA) or the internal carotid artery (ICA), manifesting in the basal ganglia of children with symptoms of AIS. Post-varicella AIS, although rare, accounts for approximately 30% of all AIS in children.Arteriopathy caused by viral infections is one of the most important mechanisms of arterial ischemic stroke (AIS) in the pediatric population. Several viruses were previously associated with AIS, such as the varicella-zoster virus (VZV). This is a DNA virus, like the herpes virus, which is able to remain latent in neuronal cells for months after primary infection. Therefore some complications, such as transient cerebral arteriopathy (TCA), may occur up to one year after varicella.,2-6To date, there is conflicting evidence regarding the identification of antibodies against VZV, or VZV DNA in cerebrospinal fluid for diagnosis of post-varicella AIS in children.To analyze data from children who were previously healthy and presented with post-varicella arterial ischemic stroke to emergency room, and focused on the clinical/laboratory aspects and neurocognitive performance after 4-year follow-up.Instituto da Criança, Hospital Israelita Albert Einstein, Hospital Santa Catarina, Hospital Infantil Darcy Vargas and Hospital São Camilo) in the city of São Paulo (SP), Brazil, from December 2013 to December 2017. This study was approved by the local Ethics Committee of Hospital das Clínicas of the Faculdade de Medicina of the Universidade de São Paulo (USP), CAAE: 43629315.2.1001.0068, protocol 1.036.344, and Hospital Israelita Albert Einstein (HIAE), CAAE: 43629315.2.3002.0071, protocol 1.343.515.This study reported a series of seven cases of patients admitted to the emergency room with post-varicella AIS and longitudinal follow-up. We evaluated charts of these patients from five different hospitals /magnetic resonance angiography (MRA) in a topography compatible with the areas of the MCA or ICA, cerebrospinal fluid sample collected and analyzed, and aged under 18 years. A radiologist reviewed each MRI scan to confirm the presence of AIS. Magnetic resonance imaging and MRA were performed within 24 to 72 hours after the initial visit. All participating hospitals had 1.5 Tesla MRI scanners. Individuals who presented with other risk factors for stroke, such as prothrombotic conditions, cardiopathies, trauma or other cerebral arteriopathies were excluded. Laboratory tests were used to rule out prothrombotic conditions, and two-dimensional Doppler echocardiography was performed to rule out cardiopathies or cervical arterial diseases.Results from the ELISA of cerebrospinal fluid to detect immunoglobulin G (IgG) antibodies against VZV, and polymerase chain reaction (VZV-DNA-PCR) to detect VZV virus DNA were collected up to 10 days after the onset of AIS in all patients. Patients were evaluated by physical therapy, speech therapy, and psychology services for specific follow-up, after hospital discharge.Sensorimotor, language and cognitive sequalae were accessed by the Pediatric Stroke Outcome Measure (PSOM) upon admission and 4 years after AIS.Seven children were included in this study; five of them (71.4%) were male. The median age at stroke was 3.8 years (range: 1.3 to 4), and the mean period between the clinical presentation of AIS and the episode of chickenpox was 5.1 (± 3.5) months.All patients were initially evaluated by computed tomography, and no signs of mineralizing angiopathy could be found. Four patients presented with unilateral vascular lesions in the MCA and/or ICA, while the MRI from three patients were normal. In five children, MRA was repeated after two to eight months to exclude progressive arteriopathy.deficit with slowness of function. These patients had a greater degree of sensorimotor sequelae and were supported by neurology/psychology and physical therapy groups. One of these two patients presented with a score of 2 in the sensorimotor and cognition areas after 4 years. No recurrence of AIS was observed during the 4-year follow-up.In this study we found the presence of viral DNA and/or intrathecal IgG antibodies against VZV, and angiopathy location in MRA, were not determining for diagnosis of post-varicella AIS. Our study adds to previous literature by describing sensorimotor, language and cognitive sequelae evaluated by the PSOM, in a 4-year follow-up after AIS. Therefore, the need of invasive and low-sensitivity tests should be well considered in the diagnosis of post-varicella AIS.-6Only five previous studies included at least four children each, with confirmed radiological imaging characteristic of ischemia in the territories of the MCA or ICA after an episode of varicella .(2-6),6 analyzed the cerebrospinal fluid to look for IgG antibodies against VZV; only five of ten children tested had a positive result for IgG antibodies against VZV. In our study, all patients had an analysis of IgG antibodies against VZV in cerebrospinal fluid and only three (43%) of them had a positive result. Some authors have suggested that, in location where there is universal varicella immunization, tests for IgG antibodies against VZV should be more sensitive than commercial VZV ELISA,which could help explain our anti-VZV IgG detection rates slightly lower than found in previous studies (43% versus 50%). However, given the small sample size, this difference could be explained by variance in the prevalence estimate, and further studies with larger samples are required to clarify this issue. Regarding varicella immunization, there is no association between AIS and live attenuated VZV of varicella vaccine, which has been recommended since 1996. Donahue et al. assessed 3.2 million children, and 1.14 million of them received varicella vaccine in the United States. The authors found varicella vaccine presented no association with increased risk of AIS. In our study, only one patient received one dose of VZV vaccine, probably because in Brazil it was included in the public immunization program only in 2013.Of these, only two identified previously healthy children with AIS, who had a recent history of varicella infection prior to the neurological condition. Nine out of 13 individuals tested had positive results for cerebrospinal fluid-VZV-DNA-PCR. Miravet et al. reported results similar to ours, evaluating children with and without comorbidities. These conflicting findings could be the result of the small sample sizes included; nonetheless, the absence of anti-VZV antibodies in the cerebrospinal fluid does not rule out the diagnosis in children who meet the criteria for post-varicella AIS.,6,11In our study, all children tested had negative results for VZV-DNA-PCR. Helmuth et al.The main limitation of the present study was the fact it is a series of cases based on data collection from medical records. However, there are only two reports that included more than seven patients with post-varicella AIS, confirmed by MRI and MRA. Other diagnostic techniques, such as conventional arteriography, might be more accurate, but are probably very invasive for routine practice.Our study reinforces the importance of assessing the approach to diagnose post-varicella AIS, which considers the clinical presentation, history of varicella, neuroimaging findings and, possibly, anti-VZV antibodies in the cerebrospinal fluid. It should also be noted that MRA was unable to rule out the diagnosis, since we observed three patients whose arterial lesions could not be located by MRA, a finding that had been described previously. After 4 years, the sensorimotor sequelae were more often observed than cognitive and language ones, which reflects the fact post-varicella AIS is mainly subcortical. Additionally, none of our patients presented with new ischemic or hemorrhagic episodes, reinforcing its monophasic course.Our study highlights the nonprogressive character of post-varicella arteriopathy by the PSOM, in the 4-year follow-up. It has been used to better determine the degree of neurological impairment following AIS over time, since the brain of children is still developing, unlike of adults. However, it remains unclear whether acyclovir could prevent recurrence in healthy children. Only one patient in this series received corticosteroids. Patients who were not treated with antivirals or corticosteroids showed no higher risk of neurological impairment than those who were; similar results were found by Bartolini et al.Rehabilitation therapy with neuropsychology, physical therapy and speech therapy may improve the outcomes of post-stroke patients, but these therapies have to be more structured in pediatrics. Regarding treatment, all patients received ASA, and two patients in whom intrathecal antibodies were identified received acyclovir. Treatment with acyclovir may be justified by the finding of VZV DNA in the arteries of a child, who died due to post-varicella AIS.In this study, we reinforced the nonprogressive course of post-varicella arterial ischemic stroke after 4 years of follow-up. The presence of viral DNA and/or intrathecal IgG antibodies against varicella-zoster virus, and angiopathy location in magnetic resonance angiography, were not determining for diagnosis of post-varicella arterial ischemic stroke.In addition, the necessity of invasive and low-sensitivity tests should be well considered in the diagnosis of post-varicella arterial ischemic stroke.


Ceftibuten (CTB) is an oral cephalosporin active against ENT approved by the US FDA in 1995. Avibactam (AVI) is a potent inhibitor of extended-spectrum β-lactamases (ESBLs), serine carbapenemases, and AmpC that can be administered orally. We evaluated the n=1,584; 29 centers) and Europe , but included E. coli isolates from Latin America  and Japan (n=100). A proposed CTB-AVI breakpoint (≤ 2 mg/L) and the current CLSI breakpoint for CTB (≤ 8 mg/L) were applied for comparison.3,216 isolates (1/patient) were consecutively collected from patients with cUTI in 72 hospitals from 25 countries in 2021 then susceptibility (S) tested by CLSI broth microdilution. Isolates were mainly from the US , levofloxacin , and trimethoprim-sulfamethoxazole . Only CTB-AVI, CAZ-AVI, MEM, and AMK were active against >90% of ESBL-phenotype isolates; only CTB-AVI, CAZ-AVI, and AMK were active against > 70% of CRE isolates. CRE rates were 1.8%/1.9% in US/EU; CTB-AVI inhibited 79.3%/81.5% of CRE isolates from US/EU at ≤ 8 mg/L (75.9%/77.8% at ≤ 2 mg/L). The second most active oral agent against CRE was TMP-SMX (24.6%S). Only 17 isolates (0.5%) showed CTB-AVI MICs ≥8 mg/L; these were from the US , EU , and LATAM . Among these 17 isolates, 23.5% were MEM-S, 41.2% were TMP-SMX-S, and 82.4% were AMK-S.The most active agents against ENT were CTB-AVI (99.1%/99.6% inhibited at ≤ 2/≤ 8 mg/L), ceftazidime (CAZ)-AVI (99.6%S), amikacin , and meropenem . CTB-AVI was 4-fold more potent than CAZ-AVI based on MICCTB-AVI was highly active against a large collection of contemporary ENT isolated from patients with cUTIs and exhibited a similar spectrum to CAZ-AVI. CTB-AVI may represent a valuable option for oral treatment of cUTI caused by resistant ENT.Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Michael D. Huband, BS, Pfizer: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.


Intraoral tumours associated with mucosa are commonly oral squamous cell carcinomas followed by minor salivary gland carcinomas, the commonest being mucoepidermoid carcinoma, adenoid cystic carcinoma, and polymorphous adenocarcinoma (PAC). PAC is the second most common malignant tumour that is found in the minor salivary glands of the oral cavity and rarely exhibits high-grade transformation (HGT). We report a case of a 50-year-old woman with a tumour on her buccal mucosa for six months. Histopathologically, the tumour showed more than 15 histopathological patterns with areas of HGT. The high-grade transformed areas predominantly showed solid patterns, increased mitosis, necrosis, vascular invasion, and perineural invasion. An immunohistochemical (IHC) panel inclusive of p63, SOX-10, S-100, calponin, vimentin, and Ki-67 was done to evaluate the tumour and grade PAC. The Ki-67 index was around 25%-30%, confirming the diagnosis of PAC-HGT. This might be the first case of primary PAC-HGT seen on the buccal mucosa on initial clinical presentation. Polymorphous adenocarcinoma (PAC) is a salivary gland tumour that has seen an interesting journey in nomenclature and behaviour from its conception as a tumour way back in 1984 until the WHO 2017 classification and categorization of salivary gland tumours. PAC was classically a low-grade carcinoma called polymorphous low-grade adenocarcinoma (PLGA). Innumerable descriptions of this tumour have been woven together with robust data on histopathology and behaviour and its behaviour has become questionable in the past decades. Cases with high-grade transformation (HGT) or dedifferentiation in PAC have been reported. HGT in salivary gland tumours, though rare, does occur. It has been reported in other salivary gland carcinomas like acinic cell carcinoma (AciCC), adenoid cystic carcinoma (AdCC), epithelial-myoepithelial carcinoma (Epi-myoepi CA), secretory carcinoma (SC), and mucoepidermoid carcinoma (MEC) [A 50-year-old Indian woman presented with a small swelling, approximately one cm in diameter on the buccal mucosa in relation to the maxillary left molars. When the patient first presented to the clinic, she stated that the lesion had been noticed around eight months prior and was an asymptomatic swelling. The patient presented again after a fortnight to the clinic with complaints of an increase in the size of the swelling and ulceration, which may be due to a cheek bite.On inspection, a spherical soft tissue mass of approximately one-and-half cm in diameter was seen on the buccal mucosa, covered by normal-looking mucosa except in one area, which showed a glistening surface representing a probable cystic area Figure . On palpA computed tomography scan showed a well-demarcated tumour nodule in the posterior buccal mucosal area without the involvement of the maxillary bone or sinus region Figure . Lymph-nA provisional clinical diagnosis of a reactive lesion or salivary gland pathology was given. A detailed clinical evaluation revealed a small cystic area in the mass; hence, a diagnosis of salivary gland tumour was considered and an incisional biopsy was done.The microscopic examination of the incisional biopsy specimen showed non-keratinizing stratified squamous epithelium exhibiting hyperplasia and underlying sub-epithelial lesional tissue Figure . The lesThe predominant patterns include filigree pattern, ribbon pattern, cribriform, pseudocribriform, ductal, tubular, layered tubular, trabecular, strands, cords, nests, islands, glomeruloid, solid organoid, sheets, papillary-cystic, Indian file pattern, and rosette patterns Figures -3O.The complete tumour mass was made of monotonous cells with poor cytoplasmic borders but with a large clear vesicular nucleus and few nucleoli Figure . MitoticBased on the findings, a diagnosis of PAC, probably high grade, was considered after assessing the presence of numerous mitotic figures.Hence, IHC was done with a panel of markers such as p63 Figure , SOX-10 The above data confirmed the diagnosis of PAC-HGT. The lesion was excised using both an intraoral and extraoral surgical approach and two large lobular tumour masses, approximately 3.0 x 2.5 cm were removed. The cut surface showed a homogenous soft to firm lesional tissue with no evidence of hemorrhage or necrosis Figures -6C.Histopathological examination of the excised specimen confirmed the presence of various histopathological patterns seen on the incisional biopsy. In addition, the tumour showed a predominance of solid areas in large islands and sheets. Hyalinised nodules and dense areas of hyalinization around tumour islands entrapping the tumour cells were evident Figure . Many isAn IHC panel of p63, SOX-10, S-100, calponin, vimentin, and Ki-67 was repeated on the excisional mass. The lesional tissue showed similar findings as mentioned before.The presence of solid areas of tumour cells, comedo necrosis, high mitotic rate, perineural invasion, vascular invasion, and high Ki-67 index prompted us to give a final diagnosis of high-grade PAC.The surgical wound healing was uneventful on a three-week follow-up. Post-surgical radiation therapy was advised and carried out. On a one-year recall, there is no evidence of recurrence or complications.AdCC, salivary duct carcinoma, and cribriform adenocarcinoma (CAMSG) were some of the tumours considered for differential diagnosis.Although a set of common patterns exist and are shared between PAC and AdCC like the tubular, cribriform and solid areas, the presence of various other patterns, like ductal, Indian file, papillary cystic, layered tubular, tubulo-trabecular, and solid sheet patterns, were also noted, giving clues that lead the diagnosis towards PAC. The tumour cells were predominantly monomorphic with a large vesicular nucleus showing nucleoli sticking to the peripheral nuclear wall, unlike the tumour cells of AdCC, which generally show a hyperchromatic, angular nucleus with prominent central nucleoli, hence ruling out the diagnosis of AdCC.A cystic tumour with no lymph-node involvement at initial presentation and the presence of multiple patterns ruled out the diagnosis of CAMSG.Multiple areas of comedo necrosis were noted, hence a differential diagnosis of salivary duct carcinoma was considered. The presence of different patterns of cell arrangement, monomorphic cells with a vesicular nucleus, and the absence of a characteristic Roman bridge pattern emphasized a diagnosis of PAC. PAC-HGT is an appropriate diagnosis considering the amount of solid pattern seen with comedo necrosis along with a high mitotic rate in the tissue, reconfirmed by 25%-30% of Ki-67 activity in the tumour cells. -7.Salivary gland tumours of the oral cavity involving the mucous membrane of different areas of the mouth are relatively rare and complex. They form an immensely diverse category of carcinomas. PAC is the second most common intraoral minor salivary gland carcinoma ,2, whichThe tumour is seen in the minor salivary glands of women more frequently (70% of cases), with a male to female ratio of 1:2 and a mean age of 59 years. The common sites of presentation are the palate (32%), soft palate (17%), lip (13%), and buccal mucosa (10%). Around 46% of the malignant minor salivary gland tumours are PAC ,12. The An extravagant spectrum of morphological patterns are seen in PAC, namely; solid, glandular, tubular, trabecular, layered tubular, fascicular stream, cord-like cribriform, filigree, cribriform, ductal, cystic, papillary cystic, targetoid, glomeruloid, and Indian file patterns, but the tumour cell type is monotonous ,2,4-6. TThe case we present falls under the rarest category of HGT-PAC. The lesion presented as a cystic tumour on the buccal mucosa with HGT on initial and primary presentation. It is probably the first case of de novo HGT-PAC reported on the buccal mucosa so far, the other de novo HGT-PAC being reported in the palate .The present case showed a well-demarcated tumour mass but did present many small invasive patches. On microscopic examination, the surface epithelium was hyperplastic and the tumour cells were close to the surface epithelium as described in the literature . Classic PAC areas with cribriform, pseudocribriform, cords, islands, nests, ribbons, papillary cystic, tubular, layered tubular, trabecular, glomeruloid, single (Indian) file, and sheet patterns were noted. A rare finding of densely hyalinized nodules with numerous small islands of tumour cells undergoing degeneration was seen. Hyalinization can be seen in PAC or AdCC . In the The greater part of the tumour mass consisted of HGT areas (around 50%-60%). The HGT areas presented predominantly as large solid islands with comedo necrosis and cells consisting of large basophilic appearing but vesiculated nuclei in many areas. An increase in normal mitoses of around 5-7 per 10 HPF; perineural and vascular invasion at many sites involving small and large nerve trunks and vessels, respectively was noticed. These features were similar to the cases described by Kikuchi et al. and Simpson et al. ,11.Generally, the basaloid salivary tumours include either AdCC, AciCC, basal cell adenoma, or basal cell adenocarcinoma. The present case also appeared as a basophilic tumour as many cells showed an increased nuclear-cytoplasmic ratio with prominent vesicular nuclei. This feature rightly correlates and PACs can be seen as blue or basaloid tumours as described by Sheetala et al. ,8. Some The current case did not show any regional lymph node metastasis or distant metastasis on a whole-body scan. Neither was there a history of previous carcinoma or radiation exposure. In cases of PAC, regional and distant metastasis is quite an isolated phenomenon, but the papillary variants of PAC and CAMSGs have a higher preponderance to show metastasis ,6,11.An immunohistochemical panel was done to confirm the final diagnosis and HGT. A Ki-67 activity of 25%-30% was seen in the tumour mass spread across every pattern. Calponin was negative in all tumour cells but was taken up by a few connective tissue components. The nominal staining for calponin ruled out myoepithelial cell participation. The p63 was taken up mainly in diffuse patchy areas in the cells of tumour islands. S-100 was strongly and diffusely positive in low-grade areas and almost negative in solid areas showing high-grade differentiation. Vimentin staining was predominantly seen in connective tissue cells. SOX-10 was strong, nuclear positive in large numbers in the tumour cells.In IHC, the vital marker for a diagnosis of HGT is Ki-67 index. It will be raised on a comparative correlation with conventional PAC areas. Similarly, in our case, the conventional areas showed nearly 5% activity but was markedly raised to 25%-30% in HGT areas. Furthermore, minimal expression of myoepithelial cells was demonstrated with the use of calponin. According to Kikuchi et al. and Simpson et al., numerous other IHC markers can have overlapping expressions; like S-100, CK, vimentin, cyclin D1, p53, p63, oestrogen receptor, androgen receptor, and SOX-10 in cases of PAC, AdCC, and a few other salivary gland tumours . RecentlA classic monomorphic cell type with a vesicular nucleus arranged histologically in numerous patterns along with characteristic IHC expression and valuation using multiple markers helped us to conclude the diagnosis of PAC-HGT as against conventional PAC, AdCC, CAMSG, or other salivary carcinomas.Here, we report a rare case of PAC-HGT in the oral cavity, which is probably the first case of PAC-HGT on the buccal mucosa on initial presentation. The tumour presented with a minimum of 15 well-defined histopathological patterns of PAC along with classic HGT areas.It is very important to have complete clarity on histopathology along with some valuable IHC markers to differentiate between PAC and AdCC, also to differentiate between high-grade PAC and de-differentiated AdCC.Although extremely rare, the aggressive behaviour of PAC with HGT does exist and needs to be considered as it alters the prognosis and long-term survival of the patient. Such cases add value in understanding, utilizing experience, and IHC for differentiation of PAC in aggressive and high-grade forms. Missing out on this will alter the prognosis and morbidity rates. It is also vital to differentiate between HGT salivary gland tumours as they dictate altered treatment protocols.


CIC-rearranged sarcoma. The efficacy was tested in seven patient-derived xenograft models, which were generated by the engraftment of tumour fragments from patients directly into nude mice. The treatment lasted 22 days, and the efficacy of the drug was assessed and compared to the doxorubicin and vehicle groups by volumetric analysis, histopathology and immunohistochemistry. We observed tumour volume control in all the tested histological subtypes. Additionally, in three sarcoma subtypes, extensive central necrosis, associated with significant tumour regression, was seen. This histological response is explained by the drug’s vascular-disruptive properties, reflected by a decreased total vascular area in the xenografts. Our results demonstrate the in vivo efficacy of plocabulin in the preclinical models of soft tissue sarcoma and corroborate the findings of our previous study, which demonstrated similar vascular-disruptive effects in gastrointestinal stromal tumours—another subtype of soft tissue sarcoma. Our data provide a convincing rationale for further clinical exploration of plocabulin in soft tissue sarcomas.A clinically relevant subset of patients with soft tissue sarcoma presents with either locally advanced or upfront metastatic disease, or will develop distant metastases over time, despite successful treatment of their primary tumour. The currently available systemic agents to treat such advanced cases only provide modest disease control and are not active in all histological subtypes. Thus, there is an unmet need for novel and more efficacious agents to improve the outcome of this rare disease. In the current preclinical in vivo study, we evaluated plocabulin, a novel tubulin inhibitor, in five distinct histological subtypes of soft tissue sarcoma: dedifferentiated liposarcoma, leiomyosarcoma, undifferentiated sarcoma, intimal sarcoma and  Soft tissue sarcomas (STS) are a highly heterogeneous group of solid tumours of mesenchymal origin and represent around 1% of all adult malignancies . The treThe first line of therapy for advanced disease has been doxorubicin for over 40 years, despite providing a modest response rate of around 15%. Multiple clinical trials have since been carried out to evaluate either doxorubicin combined with other cytotoxic agents or regimens with other chemotherapeutic compounds to improve clinical outcomes, though none of them were able to show a reproducible benefit in overall survival ,8,9,10. Lithoplocamia lithistoides  in n cells” . They ren cells” . It is hn cells” . Interesn cells” . Future DDLPS and -STS22_2FLMS received doxorubicin after a donation of tumour tissue for the establishment of the PDX models. Both patients progressed under the systemic therapy [Despite being the current first-line standard of care in settings of advanced STS, we could not show any meaningful impact on the tumour volume of doxorubicin in the seven different sarcoma models in the current study. This finding is concordant with the very modest efficacy of doxorubicin observed in clinical settings . Interes therapy , which iIn the current study, we observed a high rate of death in the groups treated with doxorubicin, which was administered i.p. A total of 23 mice were lost out of a total of 68 mice in this group (death rate of 34%). Gilliams et al. have previously demonstrated that doxorubicin i.p. causes oxidative stress and lipid oxidation in the peritoneal cavity, resulting in phrenic weakness and drug-induced peritonitis. Conversely, these adverse effects were not seen if the drug was administered i.v. . AdditioA facet that could be considered is the replacement of the human tumour stroma with a murine one after successive passaging. Repeated implantation through passaging and engraftment replaces the native human tumour stroma with a murine one  and, thuA limitation of the current study is the collection of the tumours only at the very end of the experiment—the setup we had successfully used in our previous in vivo studies when studying STS in the PDX models ,37,38. Tnu/nu mice were purchased from Janvier Labs. The animals were housed in groups of 4 in individually ventilated cages. Autoclaved wood chips  were used as bedding material, and cotton cylinders  were provided as nesting material. A commercial diet in pellet form  and autoclaved tap water were given ad libitum. The mice were housed under 12-h light/dark cycles with a room temperature of 22 °C (±2 °C).Six-week-old female, athymic Rj:NMRI-Foxn1DDLPS and -STS124DDLPS), leiomyosarcoma , undifferentiated soft tissue sarcoma , intimal sarcoma  and CIC-rearranged sarcoma . These xenografts were established, as described previously, by the bilateral implantation of tumour fragments from donor patients directly into the subcutaneous tissue of nude mice [DDLPS, -STS124DDLPS, -STS22FLMS, -STS111LMS, -STS84XUSTS, -STS122FIS and -STS134CIC were used, respectively. The collection of human tumour tissue and its usage for xenografting was approved by the Medical Ethics Committee of the University Hospitals Leuven . All donor patients gave written informed consent for the use of their tissue for this purpose. The Animal Ethics Committee of KU Leuven approved the creation of the PDX models and the in vivo experiments , which were performed according to the local guidelines and Belgian regulations.The in vivo efficacy of plocabulin was evaluated using 7 STS PDX models, characterised previously . These rude mice . Once thPlocabulin and its non-active vehicle were obtained as lyophilized vials from PharmaMar S.A. The working solutions were prepared under sterile conditions, according to the manufacturer’s instructions. Doxorubicin hydrochloride was purchased from Sigma-Aldrich (D1515). The working solutions were prepared with sterile water for injection to reach a concentration of 0.5 mg/mL. The aliquots were kept at −80 °C, and individual vials were thawed for each dosing. All solutions were brought to room temperature before administration.3. On the final day of drug administration, all mice were euthanised by cervical dislocation after an intraperitoneal injection of pentobarbital sodium . The tumours were then collected, partly snap-frozen in liquid nitrogen and partly fixed in 10% neutral buffered formalin for further analyses.For the cohort expansion, a total of 140 mice were implanted bilaterally and subcutaneously with tumour fragments that were generated after passaging under general anaesthesia. Ultimately, 211 tumours were generated, as not all implanted tumour fragments resulted in tumour growth. Each mouse was engrafted with a tumour from a single model. The tumour-bearing mice were randomised into 3 treatment groups per model: (i) the vehicle given intravenously (i.v.), once weekly (QW); (ii) doxorubicin 3 mg/kg given intraperitoneally (i.p.), QW; and (iii) plocabulin 16 mg/kg i.v., QW. Both the vehicle and plocabulin were administered by bolus injection into the lateral tail veins. The tumours were measured three times per week using a digital calliper during the 22-day treatment period. The body weights were measured daily, and the animals’ well-being was checked daily. Mice were sacrificed for ethical reasons: in case of severe body weight loss (>20% of starting body weight) or if the tumour burden exceeded 2000 mmThe tumours were approximated as ellipsoids, and volumes were calculated using the following formula: volume = 4/3 × π × length/2 × width/2 × height/2, with a length defined as the greatest dimension of the tumours and the other two axes perpendicular to the previous ones. To take into account the different starting volumes of the tumours, the volumes were standardised as relative tumour volumes: relative tumour volume = (volume on day x)/(volume on day 1) × 100%. The tumours from the mice sacrificed before the final day of the experiment were excluded from the volumetric analysis.CIC-rearranged sarcoma model. Ki-67 and phospho-histone H3 (pHH3) were used as markers of cell proliferation. Cleaved poly-ADP-ribose-polymerase (cleaved PARP) was used as an immunohistochemical marker of apoptosis. Sections of 4 µm were cut for the haematoxylin-eosin (H&E) and immunohistochemical staining from the formalin-fixed and paraffin-embedded tumours. Tissue sections were deparaffinized and rehydrated in UltraClearTM  and ethanol series, respectively. The endogenous peroxidase activity was either quenched by a 15-min treatment with 0.9% hydrogen peroxidase dissolved in distilled water for CD 31, or a 20-min treatment with 0.09% hydrogen peroxidase in methanol for all the other antigens. The epitopes were unmasked with a heat-induced epitope retrieval using a citrate buffer (pH = 6.0) for Ki-67, MDM2 and pHH3. A Tris-EDTA buffer was used for αSMA, caldesmon, CD31, CD99 and WT1. Reveal Decloaker  was used for cleaved PARP. The following primary antibodies were used for the immunohistochemistry: Ki-67 , MDM2 , pHH3 , αSMA , caldesmon , CD31 , CD99 , WT1  and cleaved PARP . EnVision®+ System-HRP Labelled Polymer Anti-Rabbit  was used as a secondary antibody for Ki-67, pHH3, caldesmon, CD99 and WT1. EnVision®+ System-HRP Labelled Polymer Anti-Mouse  was used as a secondary antibody for MDM2 and αSMA. Biotinylated Goat Anti-Rat IgG Antibody, mouse adsorbed , was used as a secondary antibody for CD31. SignalStain® Boost IHC Detection Reagent  was used as a secondary antibody for cPARP. The antigen-antibody complexes were visualised using diaminobenzidine , and the slides were counterstained with Gill’s haematoxylin .Immunohistochemical staining for the mouse double minute 2 homolog (MDM2) was used to demonstrate the MDM2 overexpression in the DDLPS models as the oncogenic driver and to confirm the stability of the models. Caldesmon and α-smooth muscle actin were used as smooth muscle markers in the LMS models. CD99 and Wilms’ tumour protein (WT1) expression were used to confirm the stability of the 2 area) on H&E. The Ki-67 index was calculated as the average percentage of the Ki-67 positive cells in 5 images captured at 400× magnification. Immunohistochemistry for pHH3 and cleaved PARP were used as additional markers for proliferation and apoptosis, respectively, and were assessed by calculating the average number of positive cells in 10 HPF. CD31 stains were used to assess tumour vasculature. The mean vascular density and the total vascular area were defined as the average number of vessels and the average area of those vessels covered on 5 digital micrographs captured at 200× magnification (0.418 mm2 area), respectively. Individual microvessels were identified as described previously [The mitotic and apoptotic activities of the treated tumours were evaluated by counting the number of mitotic figures and apoptotic cells in 10 high-power fields .The absolute tumour volumes on day 1 vs. day 22 of the treatment were compared using the Wilcoxon matched-pairs signed-rank test to categorise the tumour volume evolution into stabilisation, shrinkage or growth. Tumour shrinkage or growth was defined as a statistically significant difference, whereas stabilisation was defined as the cases where no statistically significant difference could be detected using the aforementioned test. A comparison of the non-parametric variables of the different treatment groups  was done using a Kruskal–Wallis test with Dunn’s multiple comparisons test post hoc. In the case of only two evaluable groups, the Mann–Whitney U test was used instead. Two tumours engrafted onto the same mouse were regarded as not dependent statistical events and were analysed as separate lesions . GraphPaIn conclusion, we demonstrated the efficacy of the novel tubulin inhibitor plocabulin in seven PDX models of other STS, spanning five histological subtypes. We confirmed its efficacy, corroborated the findings of our previous in vivo study, and provided a convincing preclinical rationale for further clinical exploration of the agent.


Epetraborole (EBO), an orally available bacterial leucyl transfer RNA synthetase inhibitor with potent activity against nontuberculous mycobacteria, is under clinical development for treatment of MAC lung disease. A population pharmacokinetic (PK) model describing the disposition of EBO after oral (PO) and intravenous (IV) administration was developed to support EBO PK-PD analyses and dose selection for patients with MAC lung disease.Table 1).Model development was conducted using NONMEM (v.7.4.3). Models were attempted for oral absorption, systemic compartments, and linear vs. non-linear elimination. Model evaluation involved goodness-of-fit plots and prediction-corrected visual predictive plots, which describe the ability of model-based simulations to capture the observed data. Included were data from 5 Phase 1 (3 IV and 2 PO) and 2 Phase 2 studies (IV only) (Table 2). The impact of body weight on PK was included using an allometric scaling approach to accommodate observed lower body weight in MAC lung disease patients. PO dosing was modeled using an absolute bioavailability term (F) and transit compartments with separate absorption rates for fed and fasted administration. Interindividual variability (IIV) in systemic clearance was low (7.9%), but IIV in F (32.7%) contributed to slightly higher variability in PK for PO vs. IV administration. Moderate shrinkage was observed for the IIV in the model parameters. This was considered acceptable given inclusion of patients with limited PK data in the model and the objective to facilitate simulations designed to inform dose selection. The prediction-corrected visual predictive check plots for the data obtained from a recently completed Phase 1b study evaluating 28-day oral dosing regimens (NCT04892641) are provided in Figure 1.The pooled dataset included 2637 EBO PK samples from 138 subjects/patients. A robust fit to observed data across studies was obtained using a three-compartment model with linear elimination (This model is useful for describing expected PK in MAC lung disease patients and was used to conduct simulations for the advancement of the oral EBO 500 mg q24h dosing regimen into clinical studies in patients with MAC lung disease.Harish Ganesan, MS, Adagio Therapeutics, Inc.: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co.Meiji Seika Pharma Co., Ltd: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support M. Courtney Safir, PharmD, Adagio Therapeutics, Inc: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited,: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A.: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Sujata M. Bhavnani, PharmD; MS; FIDSA, Adagio Therapeutics, Inc: Grant/Research Support|Amplyx Pharmaceuticals, Inc: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc: Grant/Research Support|B. Braun Medical Inc: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Bravos Biosciences: Ownership Interest|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Ownership Interest|ICPD Biosciences, LLC.: Ownership Interest|Insmed Inc: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Sujata M. Bhavnani, PharmD; MS; FIDSA, Adagio Therapeutics, Inc: Grant/Research Support|Amplyx Pharmaceuticals, Inc: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc: Grant/Research Support|B. Braun Medical Inc: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Bravos Biosciences: Ownership Interest|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Ownership Interest|ICPD Biosciences, LLC.: Ownership Interest|Insmed Inc: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Sujata M. Bhavnani, PharmD; MS; FIDSA, Adagio Therapeutics, Inc: Grant/Research Support|Amplyx Pharmaceuticals, Inc: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc: Grant/Research Support|B. Braun Medical Inc: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Bravos Biosciences: Ownership Interest|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Ownership Interest|ICPD Biosciences, LLC.: Ownership Interest|Insmed Inc: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Kevin M. Krause, MBA, AN2 Therapeutics: Employee|AN2 Therapeutics: Stocks/Bonds Christopher M. Rubino, PharmD, Adagio Therapeutics: Grant/Research Support|Amplyx Pharmaceuticals, Inc: Grant/Research Support|AN2 Therapeutics: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Bravos Biosciences: Ownership Interest|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|ICPD: Ownership Interest|ICPD Biosciences, LLC.: Ownership Interest|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics,: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support.


Compared to simple percutaneous coronary intervention (PCI), complex PCI is associated with higher bleeding and thrombotic risk. No previous study has evaluated the use of protamine after PCI with contemporary technologies. This study aimed to evaluate the safety and efficacy of manual compression with and without protamine after transfemoral complex PCI.We retrospectively analyzed 160 patients  who underwent complex PCI via the femoral artery. The primary outcome was a composite of in-hospital death, myocardial infarction, stent thrombosis, stroke/systemic embolism, bleeding requiring blood transfusion, and vascular access complications.p = 0.006). This was driven mainly by the lower incidences of hematoma in the protamine group . Furthermore, the protamine group had a significantly shorter hospital stay than the non-protamine group . While > 90% of the patients had acute coronary syndrome, there were no incidences of myocardial infarction or stent thrombosis in either group.The primary outcome was significantly lower in the protamine group than in the non-protamine group (4.3% vs. 17.6%; Among patients who underwent complex PCI via transfemoral access, immediate protamine administration was associated with a significantly lower rate of vascular access complications, especially hematoma, and shorter hospital stay than no protamine administration. Complex percutaneous coronary intervention (PCI) is likely to increase the risk of bleeding complications due to the use of larger French sheath systems via a transfemoral approach, dual access, and a large volume of heparin , 2. TherFrom January 2015 to December 2019, 3976 consecutive patients (7007 lesions) underwent PCI at the Pusan National University Yangsan Hospital. The inclusion criteria were (1) femoral artery puncture requiring a > 7-Fr guiding system; (2) complex PCI, which was defined as a procedure with at least one of the following angiographic characteristics: unprotected left main disease, ≥ 3 lesions treated, lesion length ≥ 60 mm, bifurcation treated with two-stent technique, or chronic total occlusion (CTO) lesions as the target lesion; and (3) manual compression, which was not suitable for arterial closure devices such as Perclose Proglide  and Angio-Seal  because of access site locations, dense calcifications, and small size of the femoral artery. The exclusion criteria were as follows: (1) use of oral anticoagulants, (2) presence of an intracoronary thrombus, and (3) hemodynamic instability. In this study, only the first PCI was considered for analysis for patients who underwent multiple PCI. A total of 160 patients who met the inclusion and exclusion criteria were retrospectively analyzed. The study population was divided into patients who received protamine (protamine group) and those who did not receive protamine (non-protamine group). This study was approved by the Pusan National University Yangsan Hospital Institutional Review Board (IRB No. 05-2020-105), and the requirement for written informed consent was waived because of the retrospective study design.The baseline clinical and procedural data were retrospectively collected. The primary outcome was a composite of in-hospital death, myocardial infarction (MI), stent thrombosis, stroke/systemic embolism (SSE), bleeding requiring blood transfusion, and vascular access complications. Vascular access complications were defined as the presence of a major groin hematoma (> 5 cm in diameter), pseudoaneurysm, arteriovenous fistula, or surgical repair. Protamine-related adverse effects included hypotension, bradycardia, anaphylactic reactions, and pulmonary hypertension.Before the procedure, all patients received dual antiplatelet therapy (DAPT), including clopidogrel , ticagrelor , or prasugrel  with acetylsalicylic acid . PCI was performed using standard techniques. Unfractionated heparin was administered as an initial bolus of 70–100 UI/kg, and additional boluses were administered during the procedure to achieve an activated clotting time (ACT) of 250–300 s. The choice of stent type and device was left to the discretion of the interventional cardiologist. Intravascular ultrasound (IVUS)-guided PCI was performed in all patients. A successful angiographic procedure was defined as residual stenosis < 30%, TIMI grade 3 distal flow, and absence of significant dissection. At the end of PCI, the ACT was checked. The use of protamine was maintained at the physician’s discretion. If ACT was < 200 s, no protamine was administered. If ACT was ≥ 200 s, 25–50 mg of protamine  was administered intravenously for 10 min. The protamine injection rate was maintained at ≤10 mg/min to avoid hypotension and pulmonary edema [All continuous variables are expressed as the mean ± standard deviation. Normally and non-normally distributed continuous variables were compared using the Student’s t-test and Mann–Whitney U test, respectively. Categorical variables were expressed as absolute numbers (frequency) and percentages and compared using Pearson’s chi-square test or Fisher’s exact test. Independent predictors of the primary outcome were identified by first including the parameters in a univariate regression analysis and subsequently entering the significant predictors in a stepwise multivariate logistic regression model.p value of ≤ 0.05. Statistical analyses were performed using SPSS version 18.0 for Windows .Statistical significance was defined as a two-tailed A total of 160 patients were enrolled and divided into the protamine group (n = 92) and the non-protamine group (n = 68). The baseline patient characteristics are shown in Table p = 0.072). There were significantly more cases of CTO as a target lesion treated in the protamine group than in the non-protamine group . All patients underwent IVUS-guided PCI using second-generation drug-eluting stents (DES).The angiographic and procedural characteristics of the two groups are shown in Table p = 0.006) (Table p = 0.020). No stent thrombosis or MI was observed in either group. There were no adverse reactions related to protamine, such as hypotension, bradycardia, anaphylactic reaction, bronchospasm, and pulmonary hypertension. In addition, the protamine group had a significantly shorter length of in-hospital stay than the non-protamine group .The primary outcome was significantly lower in the protamine group than in the non-protamine group  was independently associated with the primary endpoint  immediate administration of protamine resulted in significantly lower rates of the composite of in-hospital death, MI, stent thrombosis, stroke/systemic embolism, bleeding requiring blood transfusion, and vascular access complications; (2) use of protamine after PCI was safe with lower rates of hematoma and without increasing any stent thrombosis and MI; and (3) patients with protamine administration had significantly shorter hospital stays than patients without protamine.Although the incidence of bleeding complications after PCI has been decreasing, it remains the main challenge in complex PCI, which requires a femoral approach, larger sheath size, and more prolonged ACT. Stent thrombosis is a rare but serious complication of complex PCI due to procedure-related factors, such as bifurcation, long calcified lesions, and CTO .Protamine has been used clinically for prompt reversal of the anticoagulant effect of heparin. Although protamine has been widely used in cardiovascular surgery  for a loDespite the inclusion of patients who underwent complex PCI with high anatomical risk, the reasons for the absence of stent thrombosis events in the protamine group should be considered. First, at the time of PCI, we only included patients who were already treated with DAPT, including new-generation P2Y12 inhibitors such as ticagrelor or prasugrel. DAPT has been established as a standard-of-care treatment for preventing stent- and non-stent-related ischemic events after PCI with DES –15. StenThis study had several limitations that should be addressed. This was a retrospective, non-randomized, single-center study. However, to the best of our knowledge, this is the first study to evaluate patients receiving heparin reversal with protamine for complex PCI. In addition, the relatively small study population may have affected the outcome. The cumulative incidence of stent thrombosis with DES at one year was very low at less than 1% ; therefoAmong patients undergoing complex PCI via transfemoral access, immediate protamine use with manual compression after PCI resulted in significantly lower rates of the primary endpoint, driven mainly by significantly lower rates of vascular access complications, especially hematoma. In addition, protamine administration after complex PCI significantly shortened the hospital stay. Further prospective studies are needed to validate the safety and efficacy of protamine following complex PCI.


The evidence supports the occurrence of environmentally-induced paternal epigenetic inheritance that shapes the offspring phenotype in the absence of direct or indirect paternal care and clearly demonstrates that sperm epigenetics is one of the major actors mediating these paternal effects. However, in most animals, while sperm makes up only a small portion of the seminal fluid, males also have a complex mixture of proteins, peptides, different types of small noncoding RNAs, and cell-free DNA fragments in their ejaculate. These seminal fluid contents (Sfcs) are in close contact with the reproductive cells, tissues, organs, and other molecules of both males and females during reproduction. Moreover, their production and use are adjusted in response to environmental conditions, making them potential markers of environmentally- and developmentally-induced paternal effects on the next generation(s). Although there is some intriguing evidence for Sfc-mediated paternal effects, the underlying molecular mechanisms remain poorly defined. In this review, the current evidence regarding the links between seminal fluid and environmental paternal effects and the potential pathways and mechanisms that seminal fluid may follow in mediating paternal epigenetic inheritance are discussed. Epigenetic mechanisms, including DNA methylation, histone modification and small non-coding RNA (sncRNA) transmission, and their functions in genome regulation and cell-to-cell transmission are not considered revolutionary topics in today’s biology research. Organisms are exposed to different external and internal environmental conditions that alter their phenotype, which is regulated by these epigenetic mechanisms during their development and lifetime. However, the heritability of epigenetically-acquired phenotypes through the transmission of the alternative functional states of these mechanisms to the next generations is a hot topic of interest ,2,3,4,5.Studies on paternal epigenetic inheritance through sperm reveal the significant role of sperm epigenetic status in altering the offspring phenotype in an adaptive or maladaptive manner ,29,30,31transgenerational epigenetic inheritance parents to (grand)offspring and seminal fluid can encompass both. Epi is a Greek prefix often used to refer to something that is “upon, on, over, near, or at” of something else, and is rarely used to refer to something that is “beyond” something else. Indeed, epigenetic mechanisms work tightly with, on, or near genes throughout the genome. Therefore, I have also chosen not to use the term non-genetic or non-DNA inheritance when referring to a parental effect derived from one or more of these defined epigenetic mechanisms, mainly to avoid confusion about the concept of epigenetic inheritance.In this mini-review, I focus on the evidence for seminal fluid-mediated paternal effects on offspring and discuss the proximate pathways and mechanisms related to Sfcs within the concept of sequence ,53), excsequence . Note thTelostylinus angusticollis, Crean et al. (2014) used a so-called telegony approach, in which the female mates consecutively with two conditionally manipulated males, and the effects of the first male’s condition on the phenotype of the second male’s offspring were estimated [The supportive evidence of the link between the environmentally-induced Sfc composition of the father and the phenotype of the offspring comes from a limited number of studies in the field. In their pioneering study in the neriid fly stimated . In theiTribolium castaneum, where the seminal fluid of males exposed to bacterial infection was used when one of the two males mated with the female, it was shown that the step-offspring’s immune resistance is altered by the seminal fluid of the exposed males [Coregonus lavaretus, the presence of foreign seminal fluid in the ejaculate has been shown to the increase swimming performance in the offspring [Using a similar approach in the red flour beetle ed males . In anotffspring . The linffspring ,59,60.Teleogryllus oceanicus. These results undoubtedly indicate a relationship between Sfc composition and the reproductive success of seminal fluid donors. However, caution must be exercised when interpreting these results in the context of paternal inheritance, as the population in question died before the data were collected.Embryo survival as an observed offspring phenotype has been measured in some other studies using similar experimental approach to test for paternal effects through seminal fluid. For example, variance in embryo viability is partially explained by Sfc composition , and semDrosophila melanogaster, it was shown that females that received seminal fluid had daughters with higher fertility than females that did not receive seminal fluid [D. melanogaster, increased fecundity of the step-daughters was found due to the effects of the seminal fluid–maternal interactions [Another line of studies focuses specifically on the effects of the absence of seminal fluid or its producing and storage organs. These studies often result in a lack of fertilization or embryo growth ,66,67,68al fluid . Furtherractions . Overall, the compelling evidence suggests that non-sperm fraction of the seminal fluid, which is altered by the paternal environment, can exclusively affect offspring development and phenotype. In addition, the abovementioned examples come from a variety of taxa including insects, fish, and mammals which suggests that seminal fluid-mediated paternal effects may be widespread among animals. Unlike most other organic molecules, Sfcs are in close contact with various reproductive tissues and germ cells of both sexes, and eventually reach the embryos. Thus, it would not be surprising that Sfcs can have unique pathways that give them the ability to act on the epigenetic regulations of both sexes . In thisSperm epigenetics and its role in the formation of offspring health and phenotype are now well known ,28,29,74First of all, there is ample evidence for a link between seminal fluid and the control of sperm RNA composition. In animals, seminal fluid carries various kinds of extracellular vesicles  containing Sfcs ,91,92,93The environmentally-exposed somatic cells that produce Sfcs can transmit their exposure to the germline through the transmission of extracellular vesicles or directly as vesicle-free molecules that can induce changes in the offspring ,121,122.Locusta migratoria offspring as a function of population density, and it has been suggested that hsp functions mediate paternal epigenetic regulation and maintain transgenerational plasticity [D. melanogaster [Tribolium castaneum [Second, the level of Sfc-producing gene expression and/or the amount of a particular protein/peptide in the seminal fluid may also act as a signal of a male’s status and be associated with the epigenetic status of sperm. For example, the expression level of genes encoding heat shock proteins (hsp) was found to be related to changes in the morphology of asticity ,129. Supnogaster ,131, theastaneum , boar [1astaneum , and humastaneum , this suEpigenetic modifications such as DNA methylation and chromatin modification in spermatozoa often occur prior to ejaculation, where spermatozoa are stored with Sfcs in the storage organs . AlthougOn the other hand, the genes responsible for the production of Sfcs might themselves be subject to specific DNA methylations and/or histone modifications which can be heritable, as many genes are expressed at the appropriate time and levels required in male reproductive organs. For example, DNA methylation profiling of seminal vesicles, where some Sfcs are produced in mice, has shown that some toxic exposure of vesicles in early development affects their level of methylation and subsequent epigenetic reprogramming in adulthood . AnotherAs evidence has pointed out, seminal fluid can mediate sperm epigenetics through various pathways, which ultimately affects the phenotype of the offspring and therefore can be considered in the context of epigenetic inheritance. The novel idea that the paternal environment can influence the offspring through mechanisms involving information transfer between seminal fluid and sperm via Sfcs is a very promising and exciting hypothesis idea. The causes and the mechanisms of when, where and how seminal fluid composition is formed and used in the somatic cells and how it alters the epigenetics of the sperm are yet to be explained in detail. D. melanogaster and, studies found changes in female remating behaviors , ovulation, oogenesis, sperm storage and survival, egg-laying, and a number of other reproductive processes [Changes in the maternal environment can dramatically affect the phenotype of the offspring, as we have learned a lot from the outcome of research on maternal effects ,145,146.rocesses ,156,157.rocesses ,165,166.rocesses ,167,168.rocesses ,167. Amorocesses . In boarrocesses . Femalesrocesses ,171, androcesses ,174,175.D. melanogaster in response to receiving sex peptides in the seminal fluid [Gallus g. gallus influences maternal investment in the eggs and ultimately the body weight of the offspring [Among the most common genes whose expression changes in response to Sfcs are genes related to egg development, immunity, and nutrient sensing in females which suggests that Sfcs may serve as signals to impose paternal effects on offspring by controlling on gene regulation of maternal investment. For example, it has been shown that females can moderate their food intake and investment in eggs in al fluid ,177. Othal fluid . In domeal fluid ,178. Relffspring . D. melanogaster [Overall, it is likely that the effects of Sfcs on female gene regulation altering immune and nutritional responses go far beyond the induction of successful fertilization and can change the offspring phenotype. Therefore, a male’s environment altering Sfc composition may mediate the female’s transcriptional response according to this compositional change based on the information that has come from his environment. Supporting the idea, a study has shown that the variation in transcriptional change in females is explained more by the male’s environmental background than by his genotype in bed bugs . On the nogaster ,131, thenogaster ,181. TheT. oceanicus [Drosophila species [Anastrepha suspensa [D. melanogaster, a few Sfcs of interest were also detected on the surface of eggs [Apart from the Sfc-mediated epigenetic changes in sperm and in female reproductive tissues, seminal fluid may also reach developing or mature oocytes and embryos and affect their epigenetic status, leading changes in the offspring phenotype. We have as of yet limited evidence of the direct fusion of Sfcs to the eggs/embryos. For example, some studies have shown that the radio-isotopically labelled amino acids of seminal fluid proteins from the ejaculate are incorporated into the ovaries and eggs of the cricket ceanicus , several species , and Anasuspensa . In D. m of eggs . It is a of eggs ,186,187. of eggs . HoweverCaenorhabditis elegans, it was shown that an important sperm protein, which is responsible for oocyte maturation in females, is provided by the sperm to the eggs in extracellular vesicles of seminal fluid [C. elegans [On the other hand, there is growing evidence that the sperm do not just pass on DNA to the eggs, but also cytoplasmic mRNA and sncRNAs that can originate from the seminal fluid ,189. Foral fluid . Further elegans . These s elegans ,195,196. elegans . Taken tThe literature on paternal effects on offspring phenotype, health, and fitness is extensive ,51,197, To estimate the paternal effects mediated by Sfc and to define the mechanisms exclusively, experiments must be carefully designed because of the handicap of sperm and seminal fluid integrity. First, experimental set-ups must disentangle the paternal effects that occur through seminal fluid that arise independently of sperm. The use of animal models, such as genetically-modified males that can only produce and transfer seminal fluid can help to control sperm-borne genetic or epigenetic effects on offspring. For example, conducting a double-mating assay where a female mates with two males in turn, but receives sperm from only one of them, could help to unravel the sources of phenotypic variance in offspring explained by seminal fluid or sperm-borne factors. In addition, the confounding genetic effects of both sexes, and their interactions must also be controlled while testing for Sfc-mediated paternal effects, especially in light of the evidence for paternal genetically-driven maternal effects. If an effect on the phenotype of the offspring is observed when only the seminal fluid composition is manipulated while all other sources of variation are held constant, then it can be assumed that the effect is caused by Sfcs. To control for such effects while estimating environmental paternal effects on offspring phenotype, a quantitative epigenetic approach can be very useful to improve the study design that has been recently highlighted ,20,198.Given their role in reproduction, the importance of Sfcs has recently been highlighted in the context of assisted reproductive technology and has already begun to improve the process of in vitro fertilization in humans ,199 and


Systemic lupus erythematosus is a chronic and autoimmune disease that causes inflammation in connective tissue such as cartilage and the lining of blood vessels in which the immune system attacks its tissues. The aetiology factors are unknown but are linked to environmental, genetic, and hormonal factors; they can affect the joints, skin, brain, lungs, kidneys and blood vessels. We here report the case of a 30-year-female patient, who came to the dermatology ward with the complaints of flat red rash across the cheek and bridge of the nose, fluid-filled lesion all over the body, pain and itching, burning associated with the lesion, pedal oedema, oral ulcer, she was apparently alright four days back when she developed fluid-filled lesion which was sudden in onset and gradually progressive, the lesion occurred after ingestion of drug for fever. Initially, blood, electrocardiogram (ECG), chest X-ray investigation done, ultrasound sonography test (USG) done, which was suggestive of mild hepatosplenomegaly with the dilated portal vein and minimal free fluid in the pouch of Douglas, raised cortical echotexture of kidneys, skin biopsy done, and it shows atrophied epidermis, histopathological features suggestive of erythema multiform. The patient was referred to the dermatology and medicine department for further management.