As the coronavirus disease 2019 (COVID-19) pandemic grows throughout the world, it is imperative that all approaches to ameliorating its effects be investigated, including repurposing drugs that show promise in other diseases. We have been investigating an approach to multiple disorders that involves recruiting the innate immune system to aid the body’s healing and regenerative mechanism(s). In the case of West Nile Virus encephalitis and potentially COVID-19, the proposed intervention to stimulate the innate immune system may give the adaptive immune response the necessary time to develop, finish clearing the virus, and provide future immunity. Furthermore, we have found that GM-CSF-induced recruitment of the innate immune system is also able to reverse brain pathology, neuroinflammation and cognitive deficits in mouse models of Alzheimer’s disease and Down syndrome, as well as improving cognition in normal aging and in human patients with cognitive deficits due to chemotherapy, both of which exhibit neuroinflammation. Others have shown that GM-CSF is an effective treatment for both bacterial and viral pneumonias, and their associated inflammation, in animals and that it has successfully treated pneumonia-associated Acute Respiratory Distress Syndrome in humans. These and other data strongly suggest that GM-CSF may be an effective treatment for many viral infections, including COVID-19. Furthermore, in AD, some inflammatory proteins expressed in the brain, specifically α1-antichymotrypsin and apolipoprotein E (ApoE), especially ApoE4, clearly promote and, indeed, are necessary for amyloidogenesis that leads to cognitive decline in humans and/or animal models4. The finding that the introduction of pro-inflammatory molecules, such as lipopolysaccharide, into mouse models of AD exacerbated amyloid pathology and cognitive deficits also suggested that inflammation contributed essentially to the disease, possibly by a self-perpetuating feed-forward mechanism5. This idea was reinforced by the finding that people with rheumatoid arthiritis, who routinely take non-steroidal anti-inflammatory drugs (NSAIDS) to control the swelling and inflammation in their joints, have a much lower risk of developing AD, which suggested that their NSAID use was protective against, and might be used to treat, AD7.There is much debate about how to best treat disorders that include an inflammatory component. For example, in Alzheimer’s disease (AD), inflammation is evidenced by the activation of microglia and astrocytes, the consequent over-expression of inflammatory cytokines and acute phase proteins in the brain, and by the presence of inflammatory markers in the blood9. Indeed, steroids have been used in some human patients with WNV infection13. In respiratory diseases, an overactive innate immune reaction, termed a ‘cytokine storm’ may arise and has been treated with high dose steroids. Furthermore, use of minocycline to suppress microglial activation and promote expression of anti-inflammatory cytokines reduces neuronal cytotoxicity and mortality in WNV-infected mice15. Minocycline, has also been suggested as a treatment for AD based on experiments in cell culture and animal models, but its effects were complex and resulting changes to the peripheral and CNS immune system remain unclear17.In the case of viral diseases, including West Nile Virus (WNV) encephalitis, bacterial and viral pneumonia, Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV-1), Middle East Respiratory Syndrome-coronavirus (MERS-CoV), the development of inflammation has been cited as evidence that an effective treatment should include anti-inflammatory drugs, including steroids20, and minocycline also failed in a recently-completed trial in participants with early AD21. In WNV and Japanese Encephalitis Virus infected mice,reducing microglia in the brain by blocking the receptor of Macrophage Colony Stimulating Factor (MCSF) with PLX56229 resulted in increased viral load and mortality, suggesting that microglia, traditionally considered to be an indication of a dangerous pro-inflammatory milieu may, in fact be beneficial in fighting the virus infection.Despite the logical and preclinical data-based arguments in favor of using anti-inflammatory drugs for treatment of inflammation-associated disorders, clinical trials showed that NSAIDs were unsuccessful and potentially detrimental in AD and mild cognitive impairment participants22. Recent studies report increased plasma levels of chemokines and cytokines, including CCL-2, CCL-3, RANTES, INFγ, IL-1β, IL-2, IL-6, IL-8, IL-10, G-CSF, IP10, TNFα, and others, in severe cases of COVID-19 as compared to mild cases24, while decreased levels of lymphocytes are also commonly found in severe cases of SARS and COVID-19 patients30. Thus, it is unclear whether treatment with broad-acting immunosuppressant drugs to combat the increased cytokine and chemokine signaling would also exacerbate leukopenia, resulting in inhibited clearance of the virus and repair of tissue damage. Because no controlled trials using NSAIDs or steroids in COVID-19 patients have yet been reported, the CDC has taken a cautious stand, stating that “Currently, there are no data suggesting an association between COVID-19 clinical outcomes and NSAID use” and that “34; however, the benefit of corticosteroid use cannot be determined based upon uncontrolled observational data. By contrast, patients with MERS-CoV or influenza who were given corticosteroids were more likely to have prolonged viral replication, receive mechanical ventilation, and have higher mortality39. Therefore, corticosteroids should be avoided unless indicated for other reasons, such as management of chronic obstructive pulmonary disease exacerbation or septic shock.”Corticosteroids have been widely used in hospitalized patients with severe illness in China40Such alternative, even opposing, views of the role of inflammation is particularly striking in recent discussions about treating COVID-19. For example, it is proposed that inflammation in COVID-19-associated SARS should be suppressed because the cytokine storm is thought to play a major role in the pulmonary damage that so greatly increases morbidity and mortality in SARS , a feature of infection by both SARS-CoV-1 and SARS-CoV-243 or the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF)44. The hypothesis that blocking IL-6 or GM-CSF will be beneficial is based in part on the finding that IL-6 and GM-CSF and/or the cells that express them are increased in the blood of COVID-19 patients41 and on the beneficial results froma preliminary single-arm trial of an IL-6 receptor blocker, which reportedly lowered fever and improved oxygenation in COVID-19 patients43. Lowering IL-6 or other inflammatory cytokines in COVID-19 patients is reasonable, but the timing may be critical as early treatment may be detrimental. A multi-arm study including both early and later stage patients is essential to determine the clinical relevance of such intervention42, especially for anti-IL-6 therapies, such as tocilizumab and sarilumab, as IL-6-mediated inflammation has been shown to play a critical role in effective cellular and humoral immunity against viral infections48. Thus, if anti-IL-6 therapies are used too early to treat patients with COVID-19, this strategy could potentially inhibit viral clearance and/or leave the patients susceptible to recurrent infection with SARS-CoV-2.As another, more targeted approach, it is suggested that inflammation may be best prevented or reduced by using specific monoclonal antibodies to block either the IL-6 receptorincreasing the levels of GM-CSF. This innate immune system stimulant has been approved by the FDA as recombinant human GM-CSF (rHuGM-CSF/sargramostim/Leukine®) for almost 30 years as a therapy to increase white blood cell numbers by stimulating the division and differentiation of hematopoietic stem cells49. Approved indications for such treatment include reducing the incidence of severe and life-threatening infections after chemotherapy, accelerating myeloid reconstitution following hematopoietic cell transplantation (HCT), and treating cases of acute radiation syndrome. The finding of low white blood cell counts in patients with COVID-1923 suggests that the ability of GM-CSF to increase select leukocyte populations, particularly phagocytes, may be of therapeutic value in COVID-19, especially given GM-CSF’s ability to treat life-threatening infections, and as data from Wuhan, China have shown that half of all COVID-19 patients who died had evidence of secondary infections50. This concept is further supported by a previous study that found that the endogenous levels of GM-CSF were significantly higher in bronchoalveolar lavage fluid from patients with ARDS due to trauma or sepsis whosurvived compared to those who died51, in contrast to the potential deleterious effects of GM-CSF that were inferred from the study cited above41.As an alternative to treatment approaches designed to block pro-inflammatory cytokine responses, we suggest that COVID-19-associated SARS may be better prevented or treated by instead increasing the activation and numbers of macrophages and microglia, in particular byThese results suggest to us that GM-CSF may be beneficial in the treatment of WNV infection and the inflammation that results, and by implication, other viral infections, potentially including COVID-19. In preliminary experiments, we have found that GM-CSF treatment improves survival in mice infected by footpad injection with the TX02 strain of WNV, from approximately 35% in untreated animals to 80% survival in mice treated with GM-CSF starting the day after viral inoculation (p=0.0034) . These findings are particularly important, as there are currently no effective treatments for WNV encephalitis, and the long safety history of rHuGM CSF/sargramostim in treating leukopenia and preventing life-threatening infections suggests that these preliminary findings, if confirmed, could quickly be translated into human trials.61, which, like COVID-19, induce a cytokine storm that can lead to respiratory distress and multi-organ failure. Notably, some of these studies showed that inhaled GM-CSF was particularly efficacious58. Inhaled GM-CSF has also been used successfully in humans off-label as a treatment for pulmonary alveolar proteinosis65, and successfully as compassionate treatment for pneumonia-associated ARDS66.Furthermore, the potential benefit of GM-CSF as a pulmonary therapy has been shown directly in several other animal models in which GM-CSF treatment was able to both protect against and treat bacterial and viral pneumonias67 and the fact that GM-CSF is well known to stimulate the mobilization of MSCs from the bone marrow70 provide further support for the argument that GM-CSF administration may effectively treat COVID-19.The recent report that injection of mesenchymal stem cells (MSCs) reverses clinical and peripheral manifestations of COVID-1977. Additionally, in studies of SARS-CoV-1 and MERS-CoV, the presence of viral neuroinvasion has been identified in both animal studies79 and in post-mortem human brain samples81. Significantly, GM-CSF can easily cross the blood-brain barrier82, and our work with GM-CSF in both animals and humans indicates that it can ameliorate CNS disorders of various kinds that include an inflammatory component. For example, GM-CSF/sargramostim treatment is associated with improved cognition in:1) mouse models of Alzheimer’s disease83, which has since been replicated by others84, 2) a mouse model of Down syndrome , 3) aged wild-type control mice83 and , which has also been replicated by others85, 4) a retrospective study of leukemia patients after immune system chemoablation and HCT, who acquire chemotherapy-associated cognitive impairment86, and 5) mild-to-moderate Alzheimer’s disease participants, based on one measure, the Mini-Mental State Exam , all of which are CNS disorders. These findings are relevant to COVID-19 because all of these disorders induce inflammation9 and yet still benefit from treatment with GM-CSF, whichincreases, rather than decreases, the number and activation of microglia, the resident phagocytes of the CNS that are often cited as indicators of a dangerous pro-inflammatory milieu that must be suppressed to achieve successful treatment. Additionally, we have found that GM-CSF treatment reverses astrogliosis, which is also associated with CNS disorders and has been reported to be reversed by GM-CSF treatment that ameliorates spinal cord injury and inhibits glial scar formation88. Furthermore, multiple studies have shown that there is a very high prevalence of cognitive impairment in ARDS survivors, from between 70–100% at hospital discharge, to 46–80% at one year, and persisting in 20% at five years91, which strongly argues for extended treatment with GM-CSF/sargramostim, given its ability to improve cognition in several animal models and reverse cognitive impairment associated with chemotherapy and neurodegeneration.It is also relevant that the unusually rapid onset of COVID-19-associated SARS after infection with the SARS-CoV-2 virus may be due to infection and inflammation in the central nervous system (CNS), including in the brain stem, which could contribute to respiratory failure, and neurological symptoms have been reported in COVID-19 patients, such as hypogeusia, hyposmia, neuralgia, dizziness, headache, ataxia, epilepsy, and others92. We hope that sites in the U.K., the U.S., and elsewhere will be invited to join that study, and welcome comments and data at:https://medschool.cuanschutz.edu/GMCSF-COVIDBecause GM-CSF/Sargramostim has been safely and routinely used as a subcutaneous injection treatment and, off-label, as an inhalation treatment, we suggest that this long-FDA-approved drug should be tested for its ability to improve recovery in COVID-19 patients and/or after resolution of infection in severe ARDS survivors to ameliorate potential cognitive deficits. Although compassionate use is possible, it is more important that, either, or a combination, of these two routes of administration in a randomized, pilot trial be used to properly assess the possibility that the innate immune system stimulant, GM-CSF, may reduce morbidity and mortality from COVID-19, as the studies discussed above suggest. Indeed, while an earlier version of this manuscript was being reviewed, a trial of sargramostim in COVID-19 patients with ARDS was announced in Europe using both inhaled/nebulized and/or injected administrations93.If GM-CSF/sargramostim proves to be beneficial as a treatment for COVID 19, the fact that the U.S. Department of Health and Human Services maintains a stock of rHuGM-CSF/sargramostim to be used in the event of a nuclear accident may provide short-term resourcesNo data is associated with this article. et al. draws parallels from their finding that GM-CSF ameliorates intrinsic and viral neuro-inflammation induced in the course of Alzheimer's disease and West Nile Virus infection, respectively. The authors propose that the application of GM-CSF may have utility for the treatment of COVID-19. Neurotropism for SARS-CoV-2 and other respiratory coronaviruses has been documented, although this is not discussed in the present commentary as a relevant source of targetable neuroinflammation by GM-CSF administration.The commentary of Potter Supporting a beneficial role of GM-CSF, there is an abundance of studies that supplementation of GM-CSF in the airway enhances resistance to viral and bacterial pneumonia and, furthermore, that therapeutic GM-CSF inhalation can stabilize critical illness by macrophages increasing the odds for survival. These beneficial effects are observed locally at a high GM-CSF dosage. It is well-established that the effects of GM-CSF on myeloid cell behavior, activation, and interactions interfacing innate and adaptive immunity are concentration- and source-dependent. Homeostatic functions of GM-CSF at local sites, such as surfactant metabolism by alveolar macrophages may contribute to anti-inflammatory scavenging and repair of tissue damage at a time of need. On the other hand, activated lymphocytes unloading their GM-CSF payload in inflammatory immune synapses during immune remediation against an infectious agent could deliver substantial collateral damage and tissue destruction. To this end, there is an abundance of studies that GM-CSF contributes to adverse inflammation in multiple sterile and non-sterile inflammatory models, autoimmune disease models, and in chronic inflammatory disease caused by chronic environmental exposures such as smoking. Buttressing a detrimental contribution of GM-CSF in the development of COVID-19, there are a plethora of clinical trials underway to neutralize GM-CSF signaling. This, however, is evolving against a backdrop of disappointing data that IL-6 blockade in the Sanofi and Regeneron trials were not effective for the treatment of COVID-19. The article could be updated with this latest information, although the effects of GM-CSF in such trials remains to be seen. The challenge in designing immuno-modulatory therapies for COVID-19 is the systemic nature of the syndrome. Timing, disease stage, manifestation, and severity, dosage, and route of administration are critical parameters. Significant research effort is, therefore, required to inform the decision making process for GM-CSF supplementation or blockade gave the diverse functions of resident myeloid cell populations in different tissues. A more discerning argument on the compartmental utility of  GM-CSF in preventing cognitive sequelae of COVID-19 is thus warranted.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?PartlyReviewer Expertise:Mechanisms of infectious and noninfectious inflammatory syndromes of the lung, surfactant protein immune functions, GM-CSF in pulmonary homeostasisI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. et al. discuss the relationship of this disease—and particularly, the hyperinflammatory syndrome manifest in the most severe cases—to other disorders. While it may not be surprising to SARS-CoV-2 infections placed vis-à-vis with other viral infections such as West Nile Virus (WNV), some readers may be shocked to see the comparison extended to Alzheimer’s disease (AD). But this is not an unreasonable stretch. The inflammatory hypothesis of AD has been around since the late 1980s, when we and others began to report the elevation of immune-system markers such as interleukin-1 and complement proteins1'2'3'4. We were reluctant to refer to these changes as “inflammation” at the time, as the brain’s partial privilege from immune sequelae5 puts a damper on thecalor, dolor, and rubor classically associated with peripheral inflammation. Nevertheless, the cytokines most closely associated with inflammation now appear to drive every element of AD pathology, as we proposed in the “cytokine cycle”6 (which a more poetic turn of phrase might have rendered “cytokine cyclone” and thus provided the first such “storm”).In an interesting perspective on immune system-based strategies to combat COVID-19 and its accompanying cytokine storm, Potteret al. touch on this in reference to the effects of lipopolysaccharide in mouse models of AD, though they could have helped their case by including the intriguing finding that such stimuli can attenuate amyloid pathology7. Many current therapeutic attempts are aimed at manipulating inflammatory pathways in AD, including the application of GM-CSF  by Potter’s group. The strange career of COVID-19 and the varied effects it has on multiple organ systems suggests a similar need for targeted manipulation of immune responses. The elaboration of cytokines during neurodegeneration must be presumed to arise from some program that would be otherwise appropriate and adaptive, and several findings indicate that it is modulation rather than wholesale suppression of immune mediators that will be beneficial in AD. Potter8. It is possible that this reflects attempts by the innate immune system to pick up the slack left by virus’s targeted suppression of acquired immunity via an exquisite ability to suppress interferon-dependent responses9. This provides one more similarity to AD via the changes in immune systems brought about by advanced age, which invariably results in lymphocyte decline and the elevation of inflammatory cytokines as a component of the senescence-associated secretory phenotype. Evidence clearly indicates that SARS-CoV-2 promotes inflammation by augmenting actions of the innate immune system. In addition to the cytokine storm, the life-threatening form COVID-19 sometimes takes is associated with other inflammatory markers such as C-reactive protein, ferritin, and D-dimers, as well as elevation of the neutrophil:lymphocyte ratioet al. acknowledge this evidence of an overactive innate response in their criticism of treatments that would suppress the entire immune system, such as steroids and NSAIDS. This latter is also cited in relation to the ADAPT trial of naproxen or celecoxib in AD, halted due to untoward side effects of celecoxcib. However, it is interesting to note, as the authors do, that nondemented subjects who took naproxen for two or three years of the trial had reduced rates of developing AD. Such findings fit well with a VA database retrospective study of ibuprofen and naproxen use among almost 50,000 clinically and neuropathologically confirmed Alzheimer patients and 200,000 non-AD patients. The odds of being in the AD group was reduced from 1.03 to 0.56—i.e., more than 40 percent—among those who took ibuprofen for 5 years or longer10. Potteret al. suggest that the success of sargramostim to treating some forms of leukopenia means it will boost lymphocyte actions leukopenia. However, the difference in CD4+ lymphocytes produced by sargramostim treatment was just 22 percent, and this was only after 6 months of treatment11. The vast majority of leukocytes that accumulate after GM-CSF/sargramostim administration are neutrophils, with a considerable number of eosinophils, monocytes, and macrophages; this would seem to be precisely contraindicated in COVID-19. Indeed, Zhou et al. (2020), cited by Potter et al., suggest suppression of GM-CSF among other strategies to restrain the innate immune system. Less compelling are the prospects for effectively guiding the immune system through a SARS-CoV-2 attack with GM-CSF. Potter Thus, the logical argument for GM-CSF in SARS or COVID-19 is one that should raise red flags; there is a chance that it could worsen outcomes. Improved outcomes with GM-CSF in the WNV model is encouraging. However, it should be noted that WNV is in an entirely different phylum from coronaviruses. Thus, the WNV data may serve best as justification for conducting preclinical studies with a relevant coronavirus model. In the end, it is imperative that no stone is left unturned in the quest for effective treatments for COVID-19 while we suffer without a vaccine. For regardless of how different that acute disease might be from AD and related disorders, there is every reason to believe that the inflammatory ravages triggered by SARS-CoV-2 will set in motion cyclones and cycles that will increase the rates of chronic neurodegeneration.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?PartlyAre all factual statements correct and adequately supported by citations?PartlyAre the conclusions drawn balanced and justified on the basis of the presented arguments?PartlyReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. presents the provocative hypothesis that targeted stimulation of certain immune populations by the administration of GM-CSF could ameliorate certain neuroinflammatory conditions, such as Alzheimer's or West Nile Virus infection, or pulmonary conditions such as COVID.This opinion article by Potter There are many references drawn together to support this suggestion. However, there is insufficient clarity in the arguments to introduce the subject to readers unfamiliar with the literature. Of largest concern is the casual mixing of very different immunomodulators, without discussion of the various mechanisms of action. The authors discuss steroids, NSAIDS, minocycline, monoclonal antibody therapies, and an experimental therapy called PLX5622 without an adequate introduction to the very different pathways inhibited by these modulators. Additionally, the authors do not always distinguish between the different cell types affected. For instance, they comment that GM-CSF might help COVID by improving the "leukopenia" noted in severe cases. However, it is LYMPHOPENIA that marks the most severe cases of COVID, and systemic GM-CSF administration will not help that at all. In terms of GM-CSF, the authors do not clearly distinguish between the likely different mechanisms of action when GM-CSF is administered systemically vs via inhalation. It is unlikely, for instance, that inhaled GM-CSF stimulates demargination of PMNs to the same extent that subQ does. Finally, the combined discussions of auto-neuro-inflammation (e.g. Alzheimer's), WNV neuroinflammation, and pulmonary inflammation becomes confusing. In short, this article would be vastly improved by separating the topic into multiple subheadings, and addressing one sub-topic  at a time. The information is good. The references are solid. The proposal of using GM-CSF therapeutically to modify inflammation is exciting. But the disorganization of this article currently obscures its strengths, and it should be rewritten for clarity.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?PartlyAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?PartlyReviewer Expertise:Pulmonary innate immunityI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


In the original article, there was an error. Although the publication by Christina Papachristou et al. 2004 was referred to within the text and in the Reference section, the authors would like to rephrase some sentences in the section of The Materials and Methods and The Data Analysis subsection to give proper credit to the above mentioned work and to make it clear that some of the information was reproduced from this work and to insert a reference in the proper place.Materials and Methods section, paragraph 1:A correction has been made to the “This study was conducted on consecutive 193 living-liver donors who underwent partial hepatectomy for LDLT, during the period between April 2003 and January 2013 at the National Liver Institute Menoufeyia University, Egypt. All donors were interviewed. During the interview, it was made clear that participation would be confidential and would therefore not affect any treatment they or their loved ones were currently receiving. Each interview lasted for approximately 30 min. Two reviewers assessed each case independently and resolved any disagreement by discussion. The interviews were structured, using mainly open questions, encouraging the donors to express themselves freely and reflect on their intentions to donate. The questionnaire were reproduced from a study made by Papachristou et al. (5). Open questions were used. The potential donors were encouraged to express their intentions for donation (5).”Materials and Methods section, subsection Data Analysis, paragraph 2:A correction has been made to the “Reproduced from the previously mentioned study made by Papachristou et al. (5) we classified our potential donors into:The altruistic donor: the well-being and the life of the recipient are of utmost priority.The relationship-oriented donor: the emotional donor–recipient relationship and its maintenance are the main motives for donation.The moral donor: ethical principles are of high priority to the donor. Religious or spiritual background is of great importance.The self-interested donor: the donor's feelings, expectations, and personal profit are of priority. The donation is an attempt to take control over a stressful situation, to reduce anxiety and fear of loss. The maintenance of the self-image is a strong motivation. The donation may be seen as a personal challenge.The ambivalent donor: the motivation for donation is not clear. The relationship to the recipient is controversial, and the advantages and disadvantages of the surgery cannot be estimated.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Tumor biopsy is essential for the definitive diagnosis of central nervous system (CNS) lymphoma. However, the biopsy procedure carries the risk of complications such as bleeding, convulsions, and infection. Cerebrospinal fluid (CSF) β2‐microglobulin (β2‐MG), soluble IL‐2 receptor (sIL‐2R), and interleukin‐10 (IL‐10) are known to be useful diagnostic biomarkers for CNS lymphoma. The C‐X‐C motif chemokine ligand 13 (CXCL13) was recently reported to be another useful biomarker for CNS lymphoma. The purpose of this study is to establish a diagnostic algorithm that can avoid biopsy by combining these diagnostic biomarkers. In the first, we conducted a case‐control study (n = 248) demonstrating that the CSF CXCL13 concentration was significantly increased in CNS lymphoma patients compared with various other brain diseases (AUC = 0.981). We established a multi‐marker diagnostic model using CSF CXCL13, IL‐10, β2‐MG, and sIL‐2R from the results of the case‐control study and then applied the model to a prospective study (n = 104) to evaluate its utility. The multi‐marker diagnostic algorithms had excellent diagnostic performance: the sensitivity, specificity, positive predictive value, and negative predictive value were 97%, 97%, 94%, and 99%, respectively. In addition, CSF CXCL13 was a prognostic biomarker for CNS lymphoma patients. Our study suggests that multi‐marker algorithms are important diagnostic tools for patients with CNS lymphoma. A comparison of the cerebrospinal fluid (CSF) concentrations of CXCL13 between CNS lymphomas and other CNS diseases. The CSF CXCL13 levels of CNS lymphoma were significantly higher than those of the other diseases. The CXCL13 expression levels increased in the CNS lymphoma specimens compared with the other tumor specimens. The multi‐marker prediction algorithms based on CSF CXCL13, IL‐10, sIL‐2R, and β2‐MG had excellent diagnostic performance.  AICAkaike information criterionAUCarea under the ROC curveBICBayesian information criterionBLR‐1Burkitt's lymphoma receptor 1CSFcerebrospinal fluidCXCL13C‐X‐C motif chemokine ligand 13CXCR5C‐X‐C chemokine receptor type 5GBMglioblastomaIL‐10interleukin‐10iNPH, idiopathic normal pressure hydeocephalus; MRImagnetic resonance imagingMTXmethotrexateNHLnon‐Hodgkin lymphomaOSoverall survivalPCNSLprimary central nervous system lymphomaPFSprogression‐free survivalRMSRroot mean squared errorROCreceiver operating characteristicSCNSLsecondary central nervous system lymphomasIL‐2Rsoluble IL‐2 receptorβ2‐MGβ2‐microglobulin1Central nervous system lymphoma (CNS lymphoma) is an aggressive extranodal non‐Hodgkin lymphoma (NHL) and is found in approximately 4% of all brain tumors.Tumors frequently occur in the corpus callosum, cerebellum, and in the cerebral white matter near the lateral ventricles.Tumor biopsy is needed to confirm the diagnosis of CNS lymphoma. However, the biopsy procedure has a certain rate of complications such as bleeding.CXCL13 is strongly expressed in the follicles of the spleen and lymph nodes, and promotes the migration of B‐lymphocytes.This study established useful CSF multi‐marker prediction algorithms to diagnose CNS lymphoma. We first evaluated the diagnostic utility of CSF CXCL13 in patients with CNS lymphomas. We then used a logistic regression model to construct multi‐marker prediction algorithms based on four CSF markers of CXCL13, IL‐10, β2‐MG, and sIL‐2R using 143 case‐control study patients. Next, we validated the diagnostic performance of the multi‐marker prediction algorithms applied to the patients in a prospective study. We confirmed that CSF CXCL13 is a useful diagnostic marker for patients with CNS lymphomas. The multi‐marker prediction algorithms based on CSF CXCL13, IL‐10, sIL‐2R, and β2‐MG had excellent diagnostic performance with high sensitivity and specificity. Our study suggests that the CSF multi‐marker prediction algorithm can help diagnose CNS lymphoma with surgical difficulties and be used for treatment planning.22.1To evaluate the diagnostic ability of the CSF CXCL13 levels in patients with CNS lymphomas, 248 patients treated at the Department of Neurosurgery of the Kobe University from January 2004 to December 2015 were retrospectively examined. The patients included 66 CNS lymphoma cases and 182 controls: 66 cases of primary central nervous system lymphoma (PCNSL), 59 cases of glioblastomas (GBMs), 36 cases of other gliomas, 17 cases of germ cell tumors, 13 cases of metastatic brain tumors, 14 cases of other tumors, 14 cases of multiple sclerosis, 10 cases of idiopathic normal pressure hydrocephalus (iNPH), and 19 cases of other brain diseases. Detailed patient data are shown in Table 2.2To verify the diagnostic ability of the multi‐marker predictive algorithms based on the results of the case‐control study, we conducted a prospective study at the Department of Neurosurgery of the Kobe University from January 2016 through December 2018. The patients who were preoperatively suspected of having CNS lymphomas by MRI were included. Patients with symptoms of increasing intracranial pressure were excluded because of a risk of lumber puncture. In addition, patients with severe brain shift because of tumor and/or peritumoral edema were also excluded. A total of 104 patients were enrolled in this study. To examine the CSF samples, the patients underwent lumbar puncture after informed consent was obtained and their CXCL13, IL‐10, β2‐MG, and sIL‐2R levels were preoperatively analyzed in combination with routine biochemical examinations including protein and lactate dehydrogenase. Serum samples were also obtained from 67 patients. For the pathological diagnosis, brain biopsies or tumor removals were performed after CSF examination. This prospective study was approved by the ethical review board of our institution (No. B190031).2.3The CSF and blood samples were immediately centrifuged and stored at −70°C. The CSF and serum concentration of CXCL13 were measured using a Human BLC ELISA Kit  and plate reader . The CSF concentration of IL‐10 was measured using a human IL‐10 ELISA kit  and a plate reader . The CSF concentration of sIL‐2R was measured using a sandwich ELISA test kit  in a fully automated EIA analytical instrument  at an SRL company (SRL). The CSF concentration of β2‐MG was measured using a latex agglutination turbidimetric immunoassay. The limits of the tests for the quantification of CXCL13, IL‐10, sIL‐2R, and β2‐MG levels were 1 pg/mL, 2 pg/mL, 50 U/mL, and 200 μg/L, respectively.2.4Antibodies to CXCL13 and CXCR5 were purchased from Boster Biological Technology and Abcam. Archived paraffin blocks from the department of pathology of our hospital were used. The sections were heated in 0.01 mol/L citrate buffer (pH 6.0) for 15 min by autoclaving .2.52) was administered as an intravenous infusion over 4 hours. MTX‐based chemotherapy was administered for 1‐4 cycles in each patient. In whole brain irradiation, both eyes were included in the RT field, and the radiation dose was typically 36 Gy (1.8 Gy × 20 fractions). The boost irradiation dose for the tumor was 10‐20 Gy. Follow‐up MRI scans were conducted every 3‐6 months or if clinically necessary. Progression‐free survival (PFS) was determined from the onset of treatment until relapse, disease progression, or the last follow‐up evaluation. Overall survival (OS) was determined from the onset of treatment until the last follow‐up evaluation or death from any cause.There are a total of 93 patients with PCNSL; the case‐control study had 66 patients and the prospective study had 27 patients. For the survival analysis, we selected the newly diagnosed patients with PCNSL treated with chemotherapy and/or radiotherapy. Thirty patients were excluded and 63 patients were analyzed. Of the 63 patients with PCNSL, high‐dose MTX‐based chemotherapy + radiotherapy (RT) was administered to 49 patients. MTX (3‐6 g/m2.6R2) was used to measure how close the data were to the fitted regression line. Akaike information criterion (AIC), the Bayesian information criterion (BIC), and root mean squared error (RMSE) were used as measures of lack of fit  and the area under the ROC curve (AUC) was used as a discrimination measure. The AIC and BIC penalized model complexity, favoring simpler models, while the RMSE and AUC focused on the predictive ability itself, favoring more complex models. To evaluate the diagnostic ability of the multi‐marker predictive algorithms indicated as best according to the aforementioned indexes , the sensitivity and specificity terms were provided. The logistic regression analyses were conducted using JMP 11 statistical software (JMP Institute). All of the analyses were supported by biostatisticians at the Kobe university clinical and translational research center.The CSF data from 143 patients examining all four markers  of the case‐control study were used to establish the multi‐marker predictive algorithms. Logistic regression analysis was applied to generate the diagnostic algorithms and obtain the best sensitivity/specificity to predict disease presence. The coefficient of determination  curves were created and the area under the curve (AUC) was calculated to evaluate the diagnostic accuracy. Survival was estimated using Cox's proportional hazards model and the Kaplan‐Meier method, and the significance was determined using the log‐rank and Wilcoxon's test. A P < .05 was considered statistically significant. The statistical analysis was conducted using JMP 11 software (JMP Institute). All of the analyses were supported by biostatisticians at the Kobe university clinical and translational research center.The Mann‐Whitney 33.1The case‐control study analyzed 248 patients. Of these, 66, 59, 36, 17, 13, 14, 14, 10, and 19 had CNS lymphomas, glioblastomas, other gliomas, germ cell tumors, metastatic tumors, other tumors, multiple sclerosis, iNPH, and other diseases, respectively. The mean concentrations of CSF CXCL13 in CNS lymphoma and non‐CNS lymphoma were 1483 ± 731 and 55 ± 209 pg/mL, respectively. The mean concentrations of CSF CXCL13 in glioblastomas, other gliomas, germ cell tumors, metastatic tumors, other tumors, multiple sclerosis, iNPH, and other diseases were 21 ± 214, 24 ± 45, 38 ± 77, 176 ± 390, 4 ± 3, 36 ± 72, 14 ± 31, and 233 ± 498 pg/mL, respectively. The CSF CXCL13 levels in CNS lymphoma were significantly higher than in the other CNS diseases Figure . The ROC3.2To examine the CXCL13 expression, immunohistochemical examination was conducted using the case‐control study specimens. As shown in Figure 3.3P = 1/1 + Exp(−Z)CNS lymphoma probability: Z = −9.26 + CXCL13 × (0.0037) + IL‐10 × (1.87)2‐marker model (CXCL13 + IL‐10): Z = −10.72 + CXCL13 × (0.0038) + IL‐10 × (1.69) + β2‐MG × (0.0011)3‐marker model 1 (CXCL13 + IL‐10 + β2‐MG): Z = −9.30 + CXCL13 × (0.0037) + IL‐10 × (1.87) + sIL‐2R × (0.00062)3‐marker model 2 (CXCL13 + IL‐10 + sIL‐2R): Z = −10.79 + CXCL13 × (0.0039) + IL‐10 × (1.71) + β2‐MG × (0.0013) + sIL‐2R × (−0.0052)4‐marker model (CXCL13 + IL‐10 + β2‐MG + sIL‐2R): In addition with CSF CXCL13, we examined the CSF concentrations of IL‐10, β2‐MG, and sIL‐2R in 143 patients in the case‐control study Figure . To cons3.4We conducted the prospective study of 104 patients to evaluate the aforementioned multi‐marker diagnostic models. The patients’ pathological diagnoses and CSF data are listed in Table 3.5Of the 104 patients in the prospective study, the serum concentrations of CXCL13 were measured in 67 Figure . The mea3.6In the 14 patients with CNS lymphomas, the CSF concentrations of CXCL13 were measured after the completion of therapy Figure . The CSF3.7> 1500 pg/mL, n = 32) and low CXCL13 group . As shown in Figure P = .009, Wilcoxon: P = .017) and shorter OS  than the 4‐marker model (99%). In the present study, it was not able to conclude which of the 2‐marker model or the 4‐marker model has the higher diagnostic accuracy; however, the 4‐marker model may be more sensitive and specific to various diseases and steroid administration, and should be verified in many cases in the future.Given that CNS lymphoma cells express CXCR5, it is tempting to speculate about the role of CXCL13 in the pathogenesis of CNS lymphoma. The CXCL13/CXCR5 signal is associated with mitogen‐activated protein kinase (MAPK) pathway, which participates in lymphocyte activation.Recently, several reports have shown that circulating tumor DNA (ctDNA) is useful for the diagnosis of CNS lymphoma. MYD88 and CD79B mutations were detected in 38% ~ 76% and 30% ~ 83% of patients with PCNSL, respectively.Our study has several limitations. First, the size was relatively small, and it was conducted at only one institution. In particular, the number of neuroinflammatory disease cases was small. Second, in prognosis analysis, individual treatment did not follow the same protocol. Most of the patients received both RT and chemotherapy; however, some patients received RT or chemotherapy alone. In addition, several patients were treated with corticosteroids before CSF sampling. The effects of corticosteroids are unknown; however, the CSF concentrations of CSF CXCL13, IL‐10, β2‐MG, and sIL‐2R seemed to be decreased by corticosteroids. Nevertheless, our prospective study demonstrated the diagnostic utility of the CSF multi‐marker predictive algorithms.In conclusion, CSF CXCL13 is a useful diagnostic biomarker for patients with CNS lymphomas. The CSF CXCL13 level is a useful prognostic factor for patients with CNS lymphoma. In addition, we found that the combined use of CSF CXCL13, IL‐10, sIL‐2R, and β2‐MG demonstrated excellent diagnostic performance. In order for our diagnostic algorithms to be usable in the clinical setting, they need to be validated in large prospective cohort studies. When the diagnostic value of these combinations of CSF markers is confirmed, risk of biopsy could be avoided and treatment can be started earlier for patients with CNS lymphoma.All authors have no conflict of interest to declare.TS conceived this work, performed CSF/serum analyses, and wrote the paper. MM performed CSF/serum analyses and wrote the paper. KT and MK performed statistical analyses. SN, HT, MN, YF, KS, and MK collected and stored the CSF/serum samples. TH and TI performed pathological diagnosis. EK supervised the whole study.This study was approved by the ethical review board of our institutions (No. 1312) (No. B190031).Figure S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Angiostrongylus cantonensis is the most common cause. However, the EM definition varies in the literature, and its relation to parasitic meningitis (PM) remains unclear.Eosinophilic meningitis (EM) is a rare clinical syndrome caused by both infectious and noninfectious diseases. In tropical pacific countries, 3 in the CSF or blood eosinophilia (>16% of WBCs) without CSF eosinophils. In total 679 patients were enrolled: 7 (1.03%) had ≥10% CSF eosinophilia, 20 (2.95%) had ≥10/mm3 CSF eosinophilia, and 7 (1.03%) had >16% blood eosinophilia. The patients with ≥10% CSF eosinophilia were significantly younger (p = 0.017), had a lower body temperature (p = 0.036) than patients with ≥10/mm3 CSF eosinophilia among whom bacterial pathogens were detected in 72.2% (13/18) of those who were tested by culture and/or PCR. In contrast, the characteristics of the patients with >16% blood eosinophilia resembled those of patients with ≥10% CSF eosinophilia. We further conducted serological tests and real-time PCR to identify A. cantonensis. Serology or real-time PCR was positive in 3 (42.8%) patients with ≥10% CSF eosinophilia and 6 (85.7%) patients with >16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia.Adult and adolescent patients of 13 years old or above with suspected central nervous system (CNS) infections with abnormal CSF findings were prospectively enrolled at a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. Patients with EM or suspected PM (EM/PM) were defined by the presence of either ≥10% eosinophils or an absolute eosinophil cell counts of ≥10/mmA. cantonensis. The eosinophil percentage is a more reliable predictor of parasitic EM than absolute eosinophil count in the CSF. Patients with PM may present with a high percentage of eosinophils in the peripheral blood but not in the CSF.The etiology of PM in northern Vietnam is  Angiostrongylus cantonensis, which is a rat lungworm parasite, is the most common cause in tropical Asia. Previous papers have defined EM as CSF eosinophils ≥10% or CSF eosinophils ≥10/mm3. However, the relationship of EM to parasitic meningitis (PM) remains unclear. This prospective study enrolled 679 patients with suspected CNS infection who were admitted to a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. The characteristics of patients with ≥10% CSF eosinophilia resembled those of patients with >16% blood eosinophilia without CSF eosinophils, whereas those of patients with ≥10/mm3 CSF eosinophilia were comparable with those of patients with typical bacterial meningitis. Serology or real-time PCR for A. cantonensis was positive in 3 out of 7 patients with ≥10% CSF eosinophilia and 6 out of 7 patients with > 16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia. The percentage, in contrast to the absolute eosinophil count in CSF, is reliable for predicting parasitic EM. Patients with PM may present with eosinophilia in the peripheral blood but not in the CSF.Eosinophilic meningitis (EM) is a rare meningitis accompanied by eosinophils in the CSF and caused by multiple etiologies.  Angiostrongylus cantonensis larvae were identified in the CSF [A. cantonensis, Gnathostoma spinigerum, cysticercosis (Taenia solium) and Toxocara canis [Eosinophilic meningitis (EM) is a rare clinical syndrome characterized by meningeal inflammation and eosinophilic pleocytosis in the cerebrospinal fluid (CSF) ,2. The f the CSF . Since t the CSF –6. There the CSF ,2,5. Howra canis . Therefora canis .3 in the CSF [3 of 20 or more, of which 10% or more are eosinophils [The definition of EM varies. Many EM publications have followed the definition originally suggested by Kuberski ; the pre the CSF . However the CSF . Further the CSF ; patientinophils ,12.A. cantonensis reached 76.6% and 80.2%, respectively. This group investigated the presence of PM with a blood serological test without lumbar puncture. Schulte et al. [In addition, some EM papers have reported that 30–80% of patients with meningitic angiostrongyliasis have accompanying blood eosinophilia ,12,13. Se et al.  also repTo our knowledge, however, few studies have systematically attempted to delineate the clinical implication of various definitions of EM to date with the objective of identifying PM ,16. We bThis study was approved by the independent ethics committees of the Institute of Tropical Medicine, Nagasaki University , Nagasaki, Japan, Bach Mai Hospital and the National Institute of Hygiene and Epidemiology as part of a “Collaborative Study on Emerging and Re-emerging Infectious Diseases in Vietnam” , Hanoi, Vietnam. Written informed consent was obtained from all patients prior to enrollment. For those who were unconscious, a parent or guardian was asked to provide informed consent, and the data were analyzed anonymously.Between June 2012 and May 2014, we conducted a prospective observational study of undiagnosed febrile illness in the Infectious Disease Department of Bach Mai Hospital, which is the largest government referral medical center in Hanoi covering patients in northern Vietnam, as published previously .Patients were enrolled according to the following criteria: 1) age ≥ 13 years, 2) axillary temperature > 37.5°C (any time from onset to admission), and 3) lumbar puncture due to suspected CNS infection by the admitting physician. The exclusion criteria were patients with a clinically definitive diagnosis , patients with hepatitis-related disease , and patients with microbiologically identified infectious diseases . Regarding fever criteria, even if patients did not have fever at admission, they were enrolled as long as they had had fever at any time point from onset to admission. Patients and samples to be enrolled were determined on the following morning of each admission day.3. At Bach Mai Hospital, we defined EM/PM cases in three ways: 1) the eosinophils accounted for ≥ 10% of the total WBCs in the CSF and the absolute number of eosinophils was ≥ 10/mm3 in the CSF, 2) the percentage of eosinophils was < 10% of the total WBC count in the CSF and the absolute number of eosinophils was ≥ 10/mm3 in the CSF, or 3) the absolute number of eosinophils was < 10/mm3 in the CSF and the percentage of eosinophils was > 16% of the peripheral blood WBC count.We defined abnormal CSF as CSF protein > 0.4 g/l and CSF absolute WBC count > 5/mm3. After identifying patients with suspected EM or PM, we conducted a retrospective chart review to obtain further clinical information using hospital records.We prospectively collected epidemiological data , and biological blood and CSF data. We also collected initial blood samples (plasma and buffy coat) and initial CSF samples after admission. At the hospital, the WBC type and number of cells in the CSF sample were confirmed by Giemsa staining when the total WBC count was greater than 10/mmA. cantonensis, Toxocara canis, Paragonimus spp., and Strongyloides stercoralis. According to internal data of Miyazaki University, the sensitivity / specificity of parasite ELISA was 90.0 / 99.1 for strongyloidiasis, 97.1 / 97.4 for paragonimiasis, 97.3 / 74.6 for larva migrant syndrome due to Toxocara or Ascaris infections. But the sensitivity / specificity of A. cantonensis has not yet been fully established due to lack of positive confirmed cases in Japan. The current ELISA for testing antibodies to A. cantonensis was based on a previously described method [A. cantonensis antigen was prepared from fourth-stage larvae recovered from the brains of experimentally infected rats. The sensitivity and specificity of this test were reported as 100% and 66.8%, respectively [We tested initial blood plasma samples of suspected EM/PM patients and 20 control patients for anti-parasite antibodies in enzyme-linked immunosorbent assay (ELISA). The antigen tested were those of d method , in whicectively . The 20 A. cantonensis, which is the most common parasite specie causing EM in Asia, using CSF samples from EM/PM patients fulfilling either of the three criteria. First, we prepared 200 mm3 of the CSF samples, as previously described [3 of elution buffer. We performed TaqMan Real-time PCR for A. cantonensis with positive and negative controls for each assay.We conducted real-time PCR analyses for A. cantonensis, we followed the protocol of Qvarnstrom et al. [A. cantonensis. We cut 3 worms into small pieces and extracted whole DNA from the worms using a QIAmp DNA Mini kit  with 100 mm3 of elution buffer. The upper limit of dilution of the positive control was 100,000 times for detection by TaqMan real-time PCR for A. cantonensis.For the real-time PCR for m et al. , using TG. spinigerum, we designed two sets of oligonucleotide primers to amplify a 144-bp fragment of the first internal transcribed spacer (ITS1) gene of G. spinigerum and a 115-bp fragment of the second internal transcribed spacer (ITS2) gene of G. spinigerum, based on Primer3 <https://primer3plus.com/cgi-bin/dev/primer3plus.cgi> with GenBank accession no. AB181155. The primers targeting ITS1 were Gspi-ITS1F (5’-CATCGGCTCTGATCTTCGCT-3’) and Gspi-ITS1R (5’-AGACACCAACGGATGCTGTT-3’); the primers targeting ITS2 were Gspi-ITS2F (5’-CATTCATCGAGCGGCAAGTG-3’) and Gspi-ITS2R (5’-GCGTACGCACCTCGATAAGA-3’). After confirming that the G. spinigerum positive control showed a single band by conventional PCR with each of the two sets of primers, using GoTaq Flexi DNA Polymerase , we performed SYBR Green Real-time PCR for G. spinigerum using each of the two sets of primers with Power SYBR Green PCR Master Mix  and a 7500 Real Time PCR System . The positive control for G. spinigerum was its whole genome, which was kindly provided by the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University. The whole-genome DNA concentration was 3100 ng/mm3. The upper limit of the positive control dilution was 1,000,000 times for detection by SYBR Green Real-time PCR.For the real-time PCR for We showed the demographic and clinical characteristics of each EM/PM group with those of the other patients using frequencies and percentages for categorical values and the median and interquartile range (IQR) for continuous variables. When we were comparing CSF eosinophils ≥ 10% group with other definition group individually or comparing the EM/PM criteria not fulfilled group with each definition group individually, categorical variables were compared by Fisher’s exact test, and continuous variables were compared by the Mann-Whitney nonparametric test. We calculated the odds ratio (OR) with 95% confidence intervals using logistic regression analysis. In addition, we calculated p-values among the 4 groups by Kruskal-Wallis test for continues variables and Chi-square test for categorical variables. Statistical analysis was conducted using STATA version 15 . All tests were two-tailed, and p < 0.05 was considered statistically significant.During the study period, from June 2012 to May 2014, 7,505 patients were admitted to the department, and 2,458 patients were hospitalized with undiagnosed febrile illness. Among them, 679 patients underwent lumbar puncture and were enrolled. Abnormal CSF was found in 431 (63.5%) patients. Blood samples of all patients (100%) were available, and CSF samples of 473 patients (69.7%) were available for this study.3, and 20 (2.95% of 679 patients) had an absolute eosinophil count in the CSF ≥ 10/mm3 but eosinophils not accounting for ≥ 10%. Seven (1.03% of 679 patients) had eosinophils accounting for > 16% of the peripheral blood WBCs but no eosinophil in the CSF. The remaining 397 were non-EM/PM patients, who did not fulfill any EM/PM definition criteria. This included 22 patients who were diagnosed with TB meningitis as defined by positive result of TB-PCR test using CSF sample. There were no patients who had < 10/mm3 eosinophils in the CSF and eosinophils accounting for ≥ 10% of the total WBC count in the CSF.  had eosinophils accounting for ≥ 10% of the total WBC count in the CSF, all of whose absolute eosinophil counts in the CSF were ≥ 10/mmSF. Figs .3, IQR 60–600, ranging from 6 to 31820) and elevated protein concentration  in their CSF.Among the 431 patients with abnormal CSF, the median (IQR) age was 44 (30–57) years old, male sex was predominant , the median (IQR) duration of fever before admission was 6.5 (3–11) days, most patients had a headache , and 33.4% (n = 143) had a Glasgow Coma Score (GCS) of ≤14. All patients had mild leukocytosis  and tended to have a longer fever duration . In addition, compared with the non-EM/PM group, the CSF eosinophil ≥ 10/mm3 group had a significantly shorter duration of fever prior to admission (p < 0.001). The median body temperature in the CSF eosinophils ≥ 10% and blood eosinophils > 16% groups was almost the same, whereas the body temperature of the CSF eosinophils ≥ 10/mm3 group was significantly higher than that of the CSF eosinophils ≥ 10% group (p = 0.036). Consciousness disorder (GCS < 15) was significantly higher in the CSF eosinophil ≥ 10/mm3 group  compared with that in the non-EM/PM group.The characteristics and laboratory findings of patients are summarized according to each definition of EM/PM in 3 group was unique. The WBC counts, neutrophil percentages and CSF protein levels were higher, and the CSF glucose concentration was lower in the CSF eosinophil ≥ 10/mm3 group than the other groups. In this group, 19 (95%) patients had neutrophils accounting for more than 80% of the CSF WBCs, though in the CSF eosinophil ≥ 10% and blood eosinophil > 16% groups, no patient had neutrophils dominant in their CSF. Nineteen (95%) patients with CSF eosinophils ≥ 10/mm3 had more than 1.00 g/l CSF protein, and 6 (30%) patients had less than 0.04 g/dl CSF glucose, whereas none of patients with the CSF eosinophils ≥ 10% and blood eosinophils > 16% had less than 0.04 g/dl CSF glucose. In the CSF of the eosinophil ≥ 10/mm3 group, out of 18 patients who underwent CSF culture or CSF bacterial PCR, 13 patients (72.2%) showed positive results: Streptococcus suis (n = 9), and S. pneumoniae (n = 4). In the CSF eosinophils ≥ 10% group, only 1 out of 7 patients (14.3%) showed a positive result of CSF bacterial PCR, which was Neisseria meningitidis. There was a significantly higher prevalence of CSF culture or bacterial-positive PCR results in the CSF eosinophil ≥ 10/mm3 group  compared with the non-EM/PM group.Comparisons of the four groups showed a significant difference in all laboratory findings except for blood culture result. This was because the characteristics of the CSF eosinophils ≥ 10/mm3 had few eosinophils and increased numbers of neutrophils in their peripheral blood.Concerning the peripheral blood findings, both the median blood eosinophil percentages and eosinophil counts of the CSF eosinophil ≥ 10% group were lower than those of the blood eosinophil > 16% group. Patients with CSF eosinophils ≥ 10/mmA. cantonensis. Of the 54 patients whose samples were tested by serology, 7 patients had positive results; 2 patients were from the CSF eosinophil ≥ 10% group, and 5 patients were from the blood eosinophil > 16% group. None of the 20 patients with CSF eosinophil counts ≥ 10/mm3 or the 20 control patients had any antiparasitic antibodies.Regarding the serological test for the 4 parasites using blood samples, the samples were positive for only A. cantonensis: 2 of the 5 CSF samples from the CSF eosinophil ≥ 10% group and 1 of 3 CSF samples from the blood eosinophil > 16% group. None of the 17 CSF samples from the CSF eosinophil ≥ 10/mm3 group was positive. None of the 25 CSF sample fulfilling any of the three EM/PM definition criteria and the 20 CSF sample from same control patients for the ELISA was positive in the SYBR Green Real-time PCR for G. spinigerum.From the 34  patients fulfilling any of the three EM/PM definition criteria , only 25  stored CSF samples were available. Of these samples, 3 were positive in the TaqMan Real-time PCR for A. cantonensis among the CSF eosinophil ≥ 10% and blood eosinophil > 16% groups. Only 1 patient with eosinophils ≥ 10% of the CSF WBCs showed both positive serology and real-time PCR results for A. cantonensis. The other 2 real-time PCR-positive patients had negative serological tests.3 but CSF eosinophil percentages < 10% were consistent with those of bacterial meningitis patients. Further serological and real-time PCR results indicated that there might be a non-negligible number of patients with PM without eosinophils in the CSF or fulfilling any of the previously defined EM criteria.This prospective descriptive study included all patients with a suspected CNS infection at the largest referral medical center in Hanoi, northern Vietnam. This is the first study focusing on the implication of various EM definitions in the context of diagnosing PM. Our results indicate that the characteristics of patients with CSF eosinophil counts ≥ 10/mmWe defined these EM/PM criteria because first, the criteria of CSF eosinophils ≥ 10% was most commonly used, followed by the absolute number of eosinophils ≥ 10/mm3, and by peripheral blood eosinophils >16% in previous publications ,8,15. Se3 in addition to eosinophilia ≥ 10% of the WBCs in the CSF. If the same definition was applied herein, the prevalence would be 3.98%. These previous studies did not specifically discuss the differences in patient characteristics according to each EM definition.In our study, the prevalence of EM patients with CSF eosinophils accounting for ≥ 10% of the WBC count was 1.03% among total patients with a suspected CNS infection. This prevalence is higher than that previously reported in the southern and middle regions of Vietnam, which was 0.6% among 1241 CNS-infection patients  and 0.693 but not ≥ 10% of the WBCs in the CSF were more likely than those in the other groups to have bacterial meningitis because their clinical characteristics tended to present as acute, associated with a reduced level of consciousness, an increased number of neutrophils, an increased level of protein and a reduced level of glucose in the CSF. Therefore, the eosinophils ≥10 cells/mm3 criterion should be carefully interpreted. In fact, this EM criterion was not included in the diagnosis of EM caused by parasites in recent papers from Thailand, Vietnam and Laos [In endemic areas, the majority of patients fulfilling the EM criteria are more likely to have a parasitic infection ,4,5. Howand Laos –13,16,223, which were predominantly neutrophils (n = 2) or lymphocytes (n = 5).Intriguingly, the clinical characteristics and laboratory results of patients with blood eosinophils accounting for > 16% of the WBCs were similar to those of EM patients with CSF eosinophils accounting for ≥ 10% of the WBCs, with the exception of CSF eosinophilia. None of the patients in this group had any eosinophils in their CSF, though they had an abnormally high number of cells in the CSF with a median of 420 (IQR 310–540) cells/mmA. cantonensis: 2 (28.6%) patients from the CSF eosinophils ≥ 10% group and 5 (71.4%) patients from the blood eosinophils > 16% without CSF eosinophils group. In contrast, none of the 20 patients with CSF eosinophil counts ≥ 10/mm3 nor the 20 control patients had any anti-parasite antibodies. Subsequent real-time PCR analyses identified 3 patients positive for A. cantonensis and no patient positive for G. spinigerum, indicating that EM in these patients was due to A. cantonensis. Interestingly, one of the PCR-positive patients had blood eosinophils > 16% of the WBCs without CSF eosinophils. This finding, together with the highest seroprevalence among patients with blood eosinophils > 16% and their clinical characteristics being compatible with a parasitic infection, raises the hypothesis that patients in this group may have genuine PM. To date, many papers have reported that patients with A. cantonensis-induced EM have blood eosinophilia [A. cantonensis infection-induced PM [A. cantonensis infection in clinically suspected patients with blood eosinophilia using real-time PCR. According to the previously published study [A. cantonensis is an important clue to diagnose angiostrongyliasis. However, none of our patients positive for serology or real-time PCR mentioned exposure history.Our serological results identified 7 patients with antibodies against duced PM . Howevered study , exposurA. cantonensis larvae, an increase in blood eosinophil percentage was found to precede the CSF eosinophil percentage increase in mice, which are nonpermissive hosts, with a 14-day lag [A. cantonensis in humans, it is plausible that there might be a lag between CSF and peripheral blood eosinophil responses among PM patients. In fact, the presence of low percentage or no eosinophils in the CSF during early stage of angiostrongyliasis has been previously reported [A. cantonensis, only 5 had CSF eosinophilia in the initial lumbar puncture [A. cantonensis-induced EM within the USA [3 with 5% eosinophils, but later his CSF showed high eosinophilia, reaching 21% of the CSF WBCs. However, to our knowledge, no study has performed both PCR and serology tests for diagnosing A. cantonensis on multiple meningitis patients without CSF eosinophils. In our study patients, three, whose CSF data were followed up, did not show CSF eosinophilia despite multiple lumbar punctures. However, at least 3 patients were prescribed albendazole during admission and reported significant clinical improvements, such as a reduction in headache severity.According to a recent study of the pharmacodynamic effects of helminth-derived molecules using mouse models and soluble antigens of -day lag . Althougreported ,26. Trev the USA . His firA. cantonensis ELISA and real-time PCR was not yet established. Development of serological diagnosis of helminth infection remains difficult. Several studies attempted to establish serological tests to diagnose A. cantonensis infection [A. cantonesis because the presence of parasite bodies cannot be demonstrated in the majority of cases thus in most studies, positive cases were indirectly diagnosed by clinical symptoms and clinical histories [et al. reported that 37 CSF samples (67.8%) was positive among 57 CSF samples of patients with CSF eosinophils ≥ 10%, using the same real-time PCR assay [The sensitivity and specificity of the current nfection . Howeveristories –31. SimiCR assay . But thiThe main limitation of this study is that our study population biased toward febrile patients since the inclusion criteria included a history of fever anytime from onset to admission. Nevertheless, this was necessary in the current study as it primarily aimed to provide useful information to clinicians working in infectious disease wards to improve clinical judgment and management of patients suspected with meningitis. The latest literature review reported that around 45% of adult patients with EM/PM are afebrile , therefoA. cantonensis identification, which has not been validated in the context of northern Vietnamese population. We do not fully know the baseline sero-prevalence of people at a various degree of high risk of exposure to these parasites thus we need carefully interpret the positive results with this serology test. Forth, it is possible that patients with negative results in the real-time PCR or serological tests may have had CSF eosinophilia due to other infectious or noninfectious causes, which we did not aggressively investigate. However, regarding noninfectious causes, among the patients with CSF eosinophils ≥ 10% or blood eosinophils > 16% patients, any drug allergy was not confirmed, and neoplasms were unlikely because of their long-term clinical courses. Tuberculosis may also cause EM or even blood eosinophilia but none of EM/PM suspected patients was diagnosed as tuberculous meningitis as defined by PCR test with their CSF samples. One of patients with blood eosinophils > 16% group was positive Toxocara spp. serology in Bach Mai Hospital, whose serological test for A. cantonensis was also positive in this study. There is also a possibility of G. spinigerum infection because its infection in swamp eels and in human at Vietnam were reported [G. spinigerum was positive. Furthermore, a possibility of false negative result of the real-time PCR test for A. cantonensis cannot be excluded due to insufficient sensitivity.Our study has several other limitations. First, this was a single referral hospital-based study with a limited number of EM/PM patients. We could not have sufficient statistical power to demonstrate the significance of clinical features and sensitivity and specificity of the real-time PCR. However, Bach Mai Hospital is the biggest tertiary hospital in northern Vietnam and our study patients were identified as a consequence of screening a substantial number of patients with suspected CNS infections and CSF results. Collaborative studies are necessary for further investigation of such a rare clinical syndrome. A community-based study could provide a broader perspective on the epidemiology of EM/PM and a different prevalence of EM/PM. Since lumbar puncture was not required as a routine investigation for all patients with a headache, many mild cases of EM/PM may be treated as a nonspecific headache. Second, while general information was prospectively collected, the detailed clinical information of EM/PM patients was collected retrospectively from medical records. In particular, the information of three patients with high blood eosinophilia but CSF eosinophil percentage <10% was missing. Third, we used a serological test for reported ,34 and t3 without high CSF eosinophilia more than 10% can be bacterial meningitis. Therefore, the percentage is more reliable than the absolute eosinophil count in the CSF for predicting PM. Despite the lack of exposure history to the parasite, clinical features, serology and real-time PCR suggest that the most likely etiology of EM seems to be A. cantonensis in this area. Our results re-confirmed previously reported findings that PM due to A. cantonensis infection may have CSF eosinophils less than 10% or even subsequent CSF eosinophils with no CSF eosinophils at the beginning. Diagnosing PM is challenging.In conclusion, in northern Vietnam, the prevalence of EM was 1.03% among patients with a history of fever, suspected of having CNS infection if the definition of EM is eosinophils ≥ 10% of CSF WBC. Patients with CSF eosinophils ≥ 10/mmS1 Strobe Checklist(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Tg value of BHE reaches 64.8%). PVC plasticized with BHE displays greater thermal stability than that of PVC or PVC plasticized with DOP materials. BHE improves the thermal stability and flexibility of PVC materials. As a plasticizer, BHE displays lower solvent extractability and greater volatilization resistance than DOP. Acute oral toxicity indicates that BHE has toxic doses of 5 g/kg, suggesting that BHE is non-toxic.The aim of this study was to develop a facile one-pot reaction for the synthesis of biomass-based hyperbranched poly(ether)s end-capped as acetate esters (BHE) for use as a sustainable, safe and feasible plasticizer for flexible poly(vinyl chloride) (PVC) materials. BHE is completely miscible with PVC but shows weaker plasticizing effect than dioctyl phthalate (DOP) (EΔ Poly(vinyl chloride) (PVC) is a thermoplastic resin derived from radical polymerization of vinyl chloride, which is widely used in construction materials, such as pipes, fittings, panels, flexible roofing masks, wire and cable layers, because of its low cost and excellent performance . Althoug50) of dioctyl phthalate (DOP) in rats and mice has been reported to be 25 g/kg and 30 g/kg, respectively [Studies on the toxicity of phthalates have been carried out for more than half a century. The lethal dose 50 , triethyl citrate, glycidol, methanol, acetic anhydride, chloroform, sodium hydroxide, magnesium sulfate and tetrahydrofuran (THF) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. PVC paste resin (degree of polymerization (DP) = 1300 ± 100, K value = 69.7–73.0) was provided by All Hanwha, Korea. All chemicals were used as received without further purification.Partially deprotonated (10%) triethyl citrate  was equipped in a reaction bulb under a nitrogen atmosphere, and 16 g of glycidol (12 equiv) was slowly dropped in the reaction bulb at 75 °C for 4 h. Then, the mixture was stirred at 100 °C for another 4 h to finish the reaction. The product was dissolved in methanol to obtain yellowish viscous BHP. Excess acetic anhydride was mixed with BHP and stirred at 130 °C under a nitrogen atmosphere for 3 h to finish the reaction. The product was dissolved in chloroform. BHE was obtained after washing with sodium hydroxide solution and distilled water, drying with magnesium sulfate and removing water and chloroform with reduced pressure distillation. PVC paste resin (3.0 g) was mixed with 30 wt %, 40 wt %, 50 wt % or 60 wt % BHE in 60 mL of THF solvent and stirred using a magnetic stirrer for 20 min at 50 °C . The obt1H and 13C nuclear magnetic resonance (1H NMR). The FT-IR spectra were investigated using a Nicolet iS10 FTIR  Fourier transform infrared spectrophotometer. 1H and 13C NMR were performed using an AV-300 NMR spectrometer  at a frequency of 400 MHz. CDCl3 was used as solvent, and tetrametnylsilane (TMS) was used as an internal standard in the process. The infrared absorption peak of ester groups in plasticized PVC materials was also detected via FT-IR according to the above method.BHP and BHE were characterized with FT-IR spectra and 2 atmosphere (50 mL/min). The heating rate was 10 °C/min.The thermal stability of PVC materials and plasticizers was investigated using a TG209F1 thermal gravimetric analyzer (TGA) instrument  in NgT, which was investigated using a dynamic thermomechanical analysis (DMA) Q800  measurement with a frequency of 1 Hz in a dual cantilever mode under N2 atmosphere. The temperature ranged from −20 to 120 °C at a heating rate of 3 °C/min. The plasticizing efficiency of BHE was calculated according to Equation (1) [The plasticizing efficiency of BHE was evaluated by tion (1) ,19:(1)EΔThe plasticizing performance of PVC materials was evaluated via tensile strength and elongation at break, which were detected according to GB/T 1040.1-2006  and at room temperature using an E43.104 Universal Testing Machine  [1 is the initial weight of PVC materials, and W2 is the final weight of test PVC materials. The extraction loss data were collected using the average value of five test samples. The solvent resistance of PVC materials was investigated according to the American Society for Testing Materials ASTM D5227) and relevant papers ,22. PVC 7 and relThe volatility of BHE was evaluated by placing plasticized PVC samples in a convection oven at 70 °C for 24 h and cooling them to room temperature in a desiccator for 2 h. The weight changes were measured before and after the heating.The acute oral toxicity test was carried out by Ningbo Entry-Exit Inspection and Quarantine Bureau Technical Center according to Chinese National Standards (GB/T 21603-2008) and relevant papers ,23. The −1; after the ring-opening polymerization, the broad –OH group FT-IR band appears at 3431 cm−1 and is stronger than that of triethyl citrate, indicating that the ring-opening polymerization occurred and that more hydroxyl groups were generated [1H NMR and 13C NMR of BHP and BHE were investigated; they are shown in 1H NMR of triethyl citrate, new peaks  and the temperature of peak thermal degradation rate (Tp) are 255.1 °C and 302.4 °C. There are two degradation stages for BHE; Td and Tp for BHE are 187.9 °C and 284.8 °C. It is obvious that the thermal stability of DOP is better than that of BHE in the low-temperature range. However, DOP was almost completely degraded at 311 °C and only produced 1.01% char residue at 600 °C, while BHE stopped degrading at 384.8 °C and produced 15.89% char residue at 600 °C. A high char residue yield is a good indicator of the improvement of the thermal stability of BHE [y of BHE ,28,29.Low-toxicity or non-toxic plasticizers can be used in a wide range of PVC products, such as food packaging materials, toys and medical equipment, to replace reproductive toxic phthalate plasticizers . Test regT), the temperature of peak heat release rate (Tp) and char residue, are summarized in d of SD is 217.0 °C lower than that of PVC. Char residue for SD is lower than for PVC, and only 5.07% of its initial mass is left. For PVC plasticized with BHE samples, thermal stability is improved with the further addition of BHE because BHE is thermally stable in the high-temperature range and produces more char residue than DOP; the char residue has the effect of covering and isolating heat sources on PVC materials, which further suppresses thermal degradation. Normally, PVC thermally degrades easily as a result of structural defects such as head-to-head units, tertiary chlorines at branched carbons and chlorine atoms adjacent to internal double bonds . The addgT. The polymer presents flexibility at temperatures above gT and becomes hard below gT. The lower gT of plasticized PVC materials shows excellent plasticizing properties. In our case, the gT of PVC and plasticized PVC material was detected via DMA. gT values are summarized in gT. It can be observed that there is only one tan theta peak for these PVC materials, which indicates that PVC and plasticizers, including DOP and BHE, are completely compatible with PVC, and there is no phase separation between PVC and plasticizers because strong hydrogen bonds between H atoms in PVC and carbonyl group in BHE result in better miscibility and only one gT [gT of PVC is 82.5 °C, and the gT decreased with the addition of BHE. When the content of BHE was 60 wt % of PVC, the gT of PVC material reached 28.3 °C, while the gT of the SD sample reached −1.2 °C, which illustrates that the plasticizing efficiency of BHE was lower than that of DOP. The EΔgT value of BHE reached 64.8%. The plasticizing efficiency of plasticizers is closely related to their chemical structure and physical properties. Plasticizers with a lower relative molecular mass, less of a ring-like chemical structure, more ether chains and short alkyl chains have higher plasticizing efficiency ,34. The y one Tg . The Tg −1 to 1732.95 cm−1 when the content of BHE reaches 60 wt % of PVC, which demonstrates that the hydrogen bond was formed, as seen from In order to further investigate the plasticizing mechanism and the relationship between miscibility and hydrogen bonds in a PVC/plasticizer system, the FT-IR of PVC plasticized with different content of BHE was detected. The mechanical properties of plasticized PVC materials were also investigated because the excellent compatibility of PVC and plasticizer causes a high elongation at break and low tensile strength. Plasticizers with a high molecular weight and a high branching degree usually have better solvent extraction resistance, which has a positive effect on keeping the properties of PVC materials long-term stability and decreases the risk of plasticizer toxicity in humans. In our case, solvent extraction resistance of BHE in different solvents and volatilization resistance were detected and compared with those of DOP. The results are shown in Tg value of BHE reached 64.8%). BHE showed more solvents and volatilization resistance than DOP. Furthermore, PVC-plasticized BHE showed better thermal stability than PVC and PVC-plasticized DOP materials. The cardiac, liver, lung and spleen indices of experimental animals did not show abnormal changes in the acute oral toxicity test. The acute oral toxicity test indicated that BHE had toxic doses of 5 g/kg, suggesting that BHE is non-toxic. This excellent performance makes BHE extremely attractive as bio-based, non-toxic and feasible plasticizers for flexible PVC materials with various applications in different fields.The synthesis of biomass-based hyperbranched poly(ether)s end-capped as acetate esters (BHE) was accomplished in a facile one-pot reaction of triethyl citrate, glycidol and acetic anhydride. BHE was completely miscible with PVC, though it presented a lower plasticizing effect than DOP (EΔ


In vivo assays further confirmed ASPM ablation markedly blocked tumor growth in vivo compared with control. In addition, a co-expression was found between ASPM and CDK4 in human tumor tissues. Taken together, our data provides strong evidence that ASPM promotes lung squamous cell carcinoma proliferation in vitro and in vivo, and indicates its potential role as a LSCC therapeutic target.Lung cancer is a type of malignant tumor with high morbidity and mortality. Due to its complicated etiology and clinical manifestations, no significant therapeutic advance has been made. Lung squamous cell carcinoma (LSCC) is the most common type of lung cancer. To combat this disease, novel therapeutic targets are badly requirement. ASPM  is involved in multiple cellular or developmental processes, such as neurogenesis and brain growth. ASPM is also reported widely expressed in multiple tumor tissues and involved in the development and progression of several cancers including lung cancer. However, the potential role on ASPM on LSCC is still unclear. In this study, we reported that ASPM was related to the poor prognosis of patients with lung squamous cell carcinoma. Our results further showed that ASPM depletion dramatically inhibited the proliferation of LSCC cells, consistent with the obviously decreased of cyclin D1(CCND1) and cyclin dependent kinases 4 (CDK4) expression.  Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to human health Abnormal spindle-like microcephaly-associated protein (ASPM), a type of microtubule associated centrosome protein, plays roles in multiple cellular or developmental processes, including the regulation of neurogenesis and brain growth Recently, ASPM is reported widely expressed in multiple tumor tissues and involved in the development and progression of several cancers in vitro and suppressed tumor growth in mice. Thus, ASPM could represent a novel therapeutic target to combat lung squamous cell carcinoma.Herein, we demonstrated that ASPM was positively associated with the poor prognosis of patients with LSCC. ASPM ablation obviously blocked proliferation Anti-ASPM , Anti-β-actin . Anti-CDK4 , Anti-CCND1 CCND1 .Forward, 5'-GGGAAAGGCAAATGGAAAAC-3'; and,Reverse, 5'- CCCAAGGCCATACAAGTGTT-3'.The quantitative PCR primer sequences of ASPM were as follows:Forward, 5'-CGACCACTTTGTCAAGCTCA-3'; and,Reverse, 5'-GGTTGAGCACAGGGTACTTTATT-3'.The quantitative PCR primer sequences of GAPDH were as follows:The shRNA clone for ASPM was purchased from Addgene, and the targeted sequences of ASPM were as follows: 5'- CCGGTCCTGTCTCTCAGCCACTT-3'.A total of 82 human lung squamous cell carcinoma tissues were obtained from the patients receiving surgical treatment in Tianjin medical university cancer institute & hospital. Meanwhile, the clinical pathological characteristics, such as patient ages at diagnosis, genders, tumor stage, tumor size, and recurrence of patients were recorded.To explore the potential correlations between ASPM expression, CDK4 expression and lung squamous cell carcinoma, immunohistochemical assays were then performed. In brief, sample sections were fixed with 4% PFA at room temperature and subsequently blocked with 2% BSA for 30 minutes at room temperature. Tumor and normal tissue slides were then incubated with anti-ASPM antibody at room temperature for 2 hours. After washed with PBS for 4 times, slides were incubated with biocatalytic secondary antibody for 1.5 hours, and diaminobenzidine was used as a chromogen substrate.Subsequently, the score criteria were as follows. Briefly, intensity score, 0 means negative staining, 1 represents for weak staining, 2 means modest staining and 3 means strong staining. The proportion of cells with strong staining. For the staining analysis, the staining intensity was multiplied by the proportion of staining cells. Overall score of 5 to 100, 101 to 300 was classified into low-level, and high-level expression, respectively. For the results, ASPM nuclear staining was scored by using 4-point scales . According to the distribution of scores, the ASPM was divided into high (2-3+) and low (0-1+) expression groups respectively. Sections that exhibited complete absence of staining or staining in less than 5% of the cells were thought as negative staining.2 incubator.NCI-H520 and SK-MES-1 human LSCC cells were bought from ATCC. Both NCI-H520 and SK-MES-1 cells were cultured in RPMI-1640 and EMEM culture medium, respectively, and supplemented with 10% of fetal bovine serum (FBS). Both cell lines were maintained at 37 °C in a 5% COin vitro and animal assays.The ASPM shRNA plasmids were transfected into LSCC cells by Invitrogen Lipofectamine® 3000 . The specific shRNA above to target ASPM, and scrambled sequence was used as negative control. 100,000 cell per well in six-well plates according to the manufacturer's protocol, 3 groups were set, including: ASPM depletion group, which was transfected with shRNA targeting ASPM; Negative Control group, which was transfected with scrambled sequence; and Mock group was treated without any transfection (data not shown). Silence-efficiency was measured by quantitative PCR and immunoblot after 48 hours' transfection. These reduced cells were used to explore the links between ASPM and cell proliferation and cell invasion. Then the ASPM stable depletion cell lines were screened and used for the Total RNA was extracted from NCI-H520 and SK-MES-1 cells by Trizol reagent (Invitrogen), respectively. Then the total RNAs was reverse-transcribed by M-MLV reverse transcriptase (Promega). Meanwhile, total RNAs were reverse transcribed to produce cDNA by cDNA synthesis system including dNTP-mix, primer-mix, 5×PrimeScript buffer, DTT and DEPC water. Quantitative PCR was subsequently conducted using SYBR mixture (Takara), and the relative expression level of ASPM was normalized to the expression of GAPDH.Protein samples were extracted from LSCC cells and tumor tissues and the expression of proteins, including ASPM and β-actin was detected through SDS-PAGE. After transmembrane, the PVDF membranes were blocked with 5% dry milk in TBST then incubated with the primary antibodies for the detection of targeted proteins for 2 hours. Then the membranes were incubated with HRP-conjugated secondary antibodies for 45 minutes at room temperature. Signals were visualizal with an ECL kit.For colony formation assay, 1000 cells were added into a 6-well culture plate and transfected with control or ASPM shRNA plasmids and then maintained for 2 weeks. Cells were subsequently fixed with 4% PFA and stained with 0.1% crystal violet at room temperature for 30 minutes and washed with PBS twice. Cell numbers were manually counted.For MTT assays, LSCC cells were plated in 96-well plates at a density of 500 cells each well, transfected with shRNA plasmids and cultured for 2 days. Cells were then incubated with MTT for 4 hours and removed the culture medium. Subsequently cells were washed with PBS buffer. MTT was extracted by 200 μL DMSO and the absorbance value at a wavelength at 570 nm was detected with a microplate reader.Nude BalB/c mice  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . All animal assay procedures were approved by our Institutional Animal Care and Use Committee (IACUC). 6 NCI-H520 cells were implanted into athymic nude mice. 2 weeks later, tumor was isolated, photographed, and the tumor volume was measured every week. Then the tumor growth curves were calculated and compared.Briefly, NCI-H520 cells were infected with control or ASPM shRNA plasmids to stable deplete the expression of ASPM. Approximately 1 x102 tests. Associations of survival and tumor progression and ASPM expression were estimated by Kaplan-Meier method and log-rank tests. Data are shown as the mean ± standard deviation (SD) in vitro and animal experiments. Student's t-test was used for statistical comparisons. χ2 tests, and correlation analysis (Pearson and Spearman) were performed to analyze the associations between ASPM and CDK4. A value of P<0.05 was thought statistically significant.Data were analyzed with SPSS 22.0 software . For the immunohistochemistry experiments, associations between ASPM, CDK4 expression and the clinicopathological features were evaluated using χASPM, interestingly, was known involved in the progression of mutiple tumors. Furthermore, ASPM was also reported to be associated with the poor prognosis of patients with lung adenocarcinoma. Thereby, to explore the potential function of ASPM in the development of lung squamous cell carcinoma, another large type of lung cancer, several assays were all performed. Firstly, ASPM expression in tumor tissues of lung squamous cell carcinoma patients who underwent surgical resection was examined by immunohistochemistry assays. 82 surgical samples were divided into two groups according to the staining intensity of ASPM Figure A. Data sP=0.024) and clinical stage (P=0.021)  rate and progression-free survival (PFS) rate Figure C. In conTo explore the potential regulatory mechanism of ASPM in the progression of lung squamous cell carcinoma, the shRNA specifically targeted ASPM was transfected into two types of LSCC cells, NCI-H520 and SK-MES-1 cells, to knockdown the expression of ASPM. Quantitative PCR (Figure Abnormal cell proliferation is a key factor in the development and progression of tumors. To explore the potential role of ASPM in the proliferation of lung squamous cell carcinoma, colony formation assays were then performed. As expected, we found that knockdown of ASPM dramatically blocked the proliferation of NCI-H520 and SK-MES-1 cells Figure A. SimilaSince ASPM could significantly affect the proliferation capacity of LSCC, we hypothesize that it might affect proliferation through the regulation of cell cycle. And our results proved that ASPM depletion resulted in an obvious increase of G1 stage and an obvious decrease of S/G2stage Figure A. We tesin vitro.Taken together, data indicated the involvement of ASPM in the regulation of LSCC cell proliferation in vivo.According to the previous data, ASPM ablation led to the defect of proliferation capacity of LSCC cells, we then explored the potential function of ASPM in the growth and progression of LSCC in vivo assay, NCI-H520 cells were infected with control or ASPM shRNA lentivirus and subsequently injected into nude mice. After two weeks, tumors were the formation, and the volume was detected every week. Representative photos of tumors were taken and showed in Figure To perform the in vivo.Taken together, these results revealed that ASPM promotes the progression of LSCC As ASPM affects cell cycle and the related proteins, the relationship between ASPM and CDK4 can be explored in human LSCC tissues. In our study, we used the immunohistochemistry to observe the association between the expression of ASPM and CDK4 in tumor tissue by uninterrupted slicing. The typical staining was shown Figure A. The reLung cancer is a malignant tumor with high morbidity and mortality in vitro and in vivo, and further found that ASPM expression was associated with tumor size and clinical stage, indicating a tight link between ASPM and LSCC. Whether ASPM plays a similar role in lung adenocarcinoma deserves further study.It is known that ASPM is widely expressed in different tissues and affects the occurrence and development of multiple types of tumors As cell cycle regulators, CCND1 and CDK4 play critical roles in regulating the cell cycle of normal and tumor cells As we know, ASPM, which is located at the spindle poles in metaphase of mitotic, is involved in the organization and orientation of spindles, and its deletion or mutation could lead to the disorder of cell cycle


Epidermal growth factor receptor (EGFR) amplification and EGFR variant III  are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors  and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma. Moreove and 45% , 25. OneEGFRvIII variant, which has been shown to be a potential therapeutic target [EGFRvIII variant. The EGFR3 amplicon is not located in a specific gene region affected by recurrent splicing variants or deletions but is located between exon 25 and the C-terminal region. The qualitative discrepancy between EGFR1 and EGFR2 amplicons for detecting the gene copy number from the same tumor is probably due to breakpoint variability of the EGFRvIII variant. The EGFR1 amplicon is located at the start of intron 1 in an area containing various breakpoints for the EGFRvIII splicing variant [One of the other benefits of the dPCR assay developed in our work is its potential economic cost compared to FISH. For one patient, the cost decreases from 40 to 50% when using only the EGFR2/EGFR3 assay. Moreover, in contrast to FISH, dPCR allows the simultaneous detection of the c target . Lower c variant . The EGF variant , 28.EGFR amplification was 100% when comparing dPCR and EGFR-targeted NGS. The major advantage of NGS resides in the fact that a single assay may detect somatic point mutations and multiple CNVs. However, the cost of a single NGS assay remains high, which currently hampers its routine use. Moreover, it has also been reported that multiple CNVs may be easily detected with the LNA-probe hydrolysis dPCR method without any proportional cost increase, for example, the concomitant detection of other amplicons located on MET, PDGFRA, KIT, AKT1 or CDKN2A homozygous deletion [EGFR amplification detection in glioblastoma sample, the ideal comparison tissue should have been healthy brain tissue, which is virtually impossible to obtain in daily practice [HMBS reference gene is not altered in tumor samples.NGS-based CNV identification using panels dedicated to glioblastoma has been demonstrated to be as sensitive as FISH or CGH array , 29. In deletion . Moreovepractice , 32. TheAt last, our results are based on experiments using LNA-probes provided by Roche®. The experimental procedure is not restricted to specific manufacturer’s probes and could easily be used with other manufacturers’ LNA-probes provided that these probes are designed to be used at a hybridization temperature of 56 °C.EGFR amplification and EGFRvIII variant and may be used routinely in patients treated for glioblastoma.In conclusion, our results highlight that the dPCR assay using LNA-hydrolysis probes allowed the simultaneous detection of the Additional file 1: Supplementary Table. Details of EGFR amplicons by NGS.Additional file 2: Supplementary Data. Results of LD-RT-PCR for EGFRvIII detection.Additional file 3.Additional file 4: Supplementary Material: cost evaluation of FISH method.