Cell-type composition is an important indicator of health. We present Guided Topic Model for deconvolution (GTM-decon) to automatically infer cell-type-specific gene topic distributions from single-cell RNA-seq data for deconvolving bulk transcriptomes. GTM-decon performs competitively on deconvolving simulated and real bulk data compared with the state-of-the-art methods. Moreover, as demonstrated in deconvolving disease transcriptomes, GTM-decon can infer multiple cell-type-specific gene topic distributions per cell type, which captures sub-cell-type variations. GTM-decon can also use phenotype labels from single-cell or bulk data to infer phenotype-specific gene distributions. In a nested-guided design, GTM-decon identified cell-type-specific differentially expressed genes from bulk breast cancer transcriptomes.The online version contains supplementary material available at 10.1186/s13059-023-03034-4. Cell-type composition and its relative proportions in a tissue is an indicator of health. For example, several studies have shown that type 2 diabetes is characterized by reduced beta cell mass and number in pancreatic tissue , 2. In aOn the other hand, bulk RNA-seq has been the workhorse behind transcriptome research over the past decades. Its falling costs and ease of experimental setup make it an attractive tool to work with any organism . SeveralMost of the deconvolution approaches require a set of gene markers for each cell type of interest. These marker genes are derived from expert knowledge or differential expression analysis of purified samples of specific cell types. Early methods that leverage these marker genes could achieve good performance in deconvolving mixtures with highly distinct cell types such as blood . CIBERSOIn this study, we present a guided topic model for cell-type deconvolution (GTM-decon). As an overview of our analysis, we first benchmark GTM-decon on deconvolving simulated and real bulk data in comparison with the state-of-the-art deconvolution methods. We then train GTM-decon on pancreatic and breast scRNA-seq datasets to deconvolve bulk RNA-seq datasets from pancreatic and breast tissues, respectively. When applied to deconvolving cancer bulk transcriptomes, GTM-decon successfully identifies the cell type of origin for pancreatic and breast cancer datasets. Interestingly, the results for human pancreatic cancer are recapitulated using the CTS topics inferred from the mouse pancreas scRNA-seq data, postulating cross-species deconvolution as an option where it is difficult to obtain scRNA-seq data due to technical or ethical challenges. Furthermore, our GTM framework also enables the inference of phenotype-specific topic distributions from bulk RNA-seq data by using the phenotypes  from single cell or bulk RNA-seq data as a guide for the topic inference. We leverage this capability to distinguish basal from estrogen receptor (ER) positive (ER +) breast tumor samples, not only achieving high classification accuracy but also identifying the genes and pathways that segregate the cancer subtypes. By fine-tuning the inferred CTS topic distributions guided by the breast cancer subtypes, we deconvolve the average differential gene expression into CTS expression changes, thereby enabling discovery of the subtype-specific aberrance of the gene regulatory programs.three methodological contributions. As our first and the main contribution, GTM-decon is a marker-free method and automatically infers the contribution of each gene for each cell type in the form of CTS categorical distributions, which we define as “topics”  . The modUpon convergence, the expected values for kth row of K-1 rows to relatively low values. For example, suppose we have 5 cell types and 3 sub-topics per cell type. If cell Suppose we use L values that correspond to the labeled cell type and relatively low expected value for the rest of the entries. We experimented modeling each cell type using The data generative process is identical to the basic GTM-decon except having K cell types .g under topic k, we computed the difference of its topic score K-1 topics K cell types and T topics per cell topic, the test statistic for cell type k and gene g isAfter GTM-decon topic inference, marker genes for each cell type were identified using permutation test. Specifically, for gene p-value is computed as fraction of permutations, where the test statistic is greater than the observed value.The significance of the observed statistic is compared against the same statistic calculated from 100,000 permutations. The empirical N − 1 subjects as the training examples and deconvolving the held-out subject as the validation example. The results are shown in Additional file We evaluated the deconvolution accuracy by comparing the inferred cell-type proportions against the ground truth values for the datasets. First, we simulated bulk RNA-seq data using the scRNA-seq dataset. We summed the gene expression counts of each sample from the scRNA-seq data to represent the bulk data of that sample . The ground-truth cell-type proportions are the fraction of cells for each cell type. To avoid information bias in the evaluation, we performed leave-one-out cross-validation (LOOCV) by inferring topics from scRNA-seq data for a human pancreas   and mouse (GSE81433); b Peripheral blood mononucleocytes (PBMC) of healthy human (GSE132044); c human blood cells (HBC) of healthy humans (GSE149938); d post-mortem brain tissue of frontal cortex from adult human (GSE97930); e Breast tissue from healthy individuals (GSE113197). Apart from healthy individuals, scRNA-seq references from patients with a pancreatic cancer (CRA001160) and b breast cancer (GSE176078) were used to deconvolve cancer bulk RNA-seq datasets.In addition, we also used real bulk data with known cell-type proportion to benchmark our methods. To conduct different benchmarking and qualitative experiments, we used scRNA-seq datasets from several tissues for training GTM-decon , b PBMC from a cohort of 13 individuals (GSE107011), c PBMC from a cohort of 346 individuals (SDY67). For brain tissue, ground truth proportions from immunohistochemistry were available for a cohort of 41 individuals from the Religious Orders Study / Memory and Aging Project (ROSMAP) study (CortexCellDeconv). A unique dataset with both bulk RNA-seq data and scRNA-seq data from pancreas was accessed from E-MTAB-5060 and E-MTAB-5061, respectively, with the cell type proportions from scRNA-seq considered as ground truth values.Bulk RNA-seq datasets with known ground truth values were chosen for benchmarking  and brain prefrontal cortex (ROSMAP) bulk datasets, we used two independent single-cell references with the most closely matched tissues of origin  Pearson correlation coefficient (PCC), (2) Spearman correlation (SCC), (3) cross entropy (CE), and (4) residual mean squared error (RMSE) . Similarly, bulk RNA-seq data from the pancreas of a cohort of 89 normal and diabetic individuals was obtained from GSE50244.For the qualitative analyses on the cancer datasets, the bulk RNA-seq datasets for pancreatic cancer (PAAD) and breast cancer (BRCA) datasets from TCGA were downloaded from the GDC data portal (https://cibersortx.stanford.edu/) for Signature Matrix generation and Cell Fraction imputation. For BayesPrism, all genes were provided to the web portal (https://www.bayesprism.org/) for cell type composition estimation, with the metadata column for tumor status set to 0 for all cells.We compared GTM-decon with other state-of-the-art methods, including BSEQ-sc, BISQUE, MuSiC, CIBERSORTx, and BayesPrism. For BSEQ-sc, marker genes were selected from CellmarkerDB for the brain and immune datasets, while the built-in pancreatic marker genes were used for the Segerstolpe dataset. BISQUE requires at least two paired reference bulk and reference single-cell samples, which is the case for the Segerstolpe dataset. When using human blood cell (HBC) scRNA-seq data as a reference, in order to use BISQUE, artificial bulk samples were constructed using single-cell data. For the other 3 bulk datasets, since we used only one scRNA-seq reference, we left out BISQUE from the evaluation. For CIBERSORTx, all genes were provided to the web portal ((i)all: all genes without removal of any gene;(ii)pp: preprocessed genes to remove uninformative genes ;(iii)hvg: highly variable genes identified using the highly_variable_genes (HVG) function of scanpy [f scanpy .The gene expression profiles of scRNA-seq data were used as training data. Since the performance of GTM-decon may vary depending on how the scRNA-seq count data are processed, we explored different gene selection and transformations of the scRNA-seq data. Specifically, the following gene selection were considered:(i)raw count —scRNA-seq read counts per gene;(ii)normr —normalize counts  as counts divided by the sum of counts per cell multiplied by a scaling factor of 10,000 (a commonly used factor for scRNA-seq data [seq data ), and ro(iii)normr_log1p : log-transform normalized counts in (ii), and round the values to their nearest integer to make the input suitable for topic modeling.Furthermore, for each of these gene sets, the following transformation were considered:GSEA was performed using the fgsea package from R, on two different gene sets: (a) gene sets corresponding to specific cell types from CellMarkerDB ; (b) genp-value < 0.05 based on log-rank test.For TCGA datasets, after cell-type proportions are inferred using GTM-decon, survival analysis is performed using the survival and survminer R packages in order to assess if there is any correlation between survival probability of the cancer subjects and cell-type proportions for the cell types. Kaplan–Meier curves were generated for those cell types, where the association is deemed statistically significant at To identify phenotype-specific genes, we applied GTM-decon to directly infer phenotype-topics from the sparsified bulk RNA-seq data Fig. b. To evasettings .each phenotype d. For example, for In identifying phenotype-specific differentially expressed genes, we developed a way to leverage the CTS topics inferred from the scRNA-seq reference data by fine-tuning the CTS topics based on gene expression from the sparsified bulk RNA-seq data for different phenotypes Fig. c. Specifj, suppose his/her phenotype label is dK topics corresponding to the phenotype label d were set to 0.9, and the For subject mentclass2pt{minimg between phenotypes d and k were identified by subtracting the p-value < 0.05 by permutation test calculated from the 100,000 randomly shuffled p-value is the fraction of times the null statistic from the permutations is greater than the observed test statistic. As a reference, we compared our approach against the DE genes identified from the same samples using DESeq2 [From the learned matrix, the test statistic of a DE candidate gene g DESeq2 . We idenAdditional file 1. Supplementary Section S1 ; S2 ; S3 (Experimenting hyperparameters Additional file 2. Review history.


Passive immunotherapies have been used to treat severe respiratory infections for over a century, with convalescent blood products from recovered individuals given to patients with influenza-related pneumonia as long ago as the Spanish flu pandemic. However, passive immunotherapy with convalescent plasma or hyperimmune intravenous immunoglobulin (hIVIG) has not provided unequivocal evidence of a clinical benefit for severe respiratory infections including influenza and COVID-19. Efficacy trials, primarily conducted in late-stage disease, have demonstrated inconsistent efficacy and clinical benefit for hIVIG treatment of severe respiratory infections. To date, most serological analyses of convalescent plasma and hIVIG trial samples have focused on the measurement of neutralizing antibody titres. There is, however, increasing evidence that baseline antibody levels and extra-neutralizing antibody functions influence the outcome of passive immunotherapy in humans. In this perspective, findings from convalescent plasma and hIVIG trials for severe influenza, COVID-19 and respiratory syncytial virus (RSV) will be described. Clinical trial results will be discussed in the context of the potential beneficial and deleterious roles of antibodies with Fc-mediated effector functions, with a focus on natural killer cells and antibody-dependent cellular cytotoxicity. Overall, we postulate that treating respiratory viral infections with hIVIG represents a delicate balance between protection and immunopathology. The field of passive immunisation, or transfer of antibody to confer temporary protection, began in 1890 when Behring and Kitasato showed that a rabbit could be protected from tetanus by injecting serum from an immunized animal . This apNeutralization of virus by antibody fragment antigen-binding (Fab) domains prevents infection of cells by blocking viral attachment, fusion and entry. Blood products used in passive immunotherapy are rich in IgG, the major antibody isotype found in human plasma. The IgG fragment crystallizable (Fc) domain selectively binds to activating or inhibitory Fc gamma receptors (FcγR) on immune effector cells inducing anti-viral functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent phagocytosis (ADP). Fc-mediated functions may be especially important in passive immunotherapy to promote clearance of virus-infected cells and extracellular virus in an infected host  shuttling it into macrophages that become productively infected –7. IntriA systematic review and meta-analysis of the effectiveness of convalescent blood products for severe influenza and severe acute respiratory syndrome (SARS) concluded that CP may reduce mortality, but well-designed trials were necessary to establish efficacy . WithoutHistorical studies show that passive immunotherapy with convalescent blood products may be an effective treatment for severe influenza. During the Spanish flu pandemic, influenza-convalescent blood products were reported to reduce morbidity and mortality in patients with influenza-associated pneumonia and ARDS . A meta-A more recent randomised phase II clinical trial (n=98) found that immune plasma [hemagglutination inhibition (HAI)≥80] was safe but did not show a clear treatment benefit for severe influenza A or B based on respiratory normalisation by day 28 . A largeInconclusive results for CP led to renewed interest in hIVIG therapy for severe influenza. A small (n=35) randomised controlled trial compared hIVIG, prepared from H1N1pdm09 CP, to IVIG prepared before 2009 for hospitalised adults with influenza . The hIVA phase III clinical trial (n=308) used a hIVIG called Flu-IVIG to treat patients hospitalised with severe seasonal influenza A or B within 7 days of symptom onset . The FluA growing body of work supports a potential protective role for influenza-specific ADCC –26, but CP therapies were tested in subjects with mild and severe COVID-19, with few showing any clinical benefit –37. TranAnti-SARS-CoV-2 spike protein (S) IgG and NAb titre were the main serological parameters measured in COVID-19 CP trials, but ADCC and antibody-dependent NK cell activation were explored. The CP for COVID-19 Respiratory Illness (CONCOR-1) trial found that antibody content of CP modified its therapeutic effect. High ADCC activity in CP was independently associated with lower risk of intubation and death 30 days post-infusion , suggestStandardised hIVIG preparations with higher concentrations of anti-SARS-CoV-2 antibody may overcome CP variability and improve therapeutic efficacy. A small trial reported that COVID-19 hIVIG was safe, increased survival and reduced the risk of disease progression in subjects severely or critically ill with ARDS . A phaseAntibody-dependent NK cell activation and ADCC have been linked to SARS-CoV-2 protection in CP trials and cohort studies , 44, 45,Collectively, CP and hIVIG have not demonstrated significant impact on COVID-19 mortality or clinical outcome. However, these trials revealed key information about protective and pathological features of SARS-CoV-2 antibodies. Further insights can be gained into the balance of harmful and beneficial antibody profiles in COVID-19 by measuring antibody Fc-functionality in hIVIG trials.A hIVIG (RSV-IVIG) and mAbs against the RSV fusion (F) protein  have been approved for prophylactic use against RSV in high-risk infants and children –59. RSV-The Fc-mediated functions of RSV-IVIG have not been assessed, but detection of RSV-specific ADCC in the respiratory tract of infants during natural infection was first reported over 40 years ago . RSV-speIn the 1960s, a formalin-inactivated RSV vaccine caused vaccine-induced ADE in children , 74. ChiProphylactic use of NAbs is efficacious for targeted protection of at-risk individuals, but antibody-based therapies are being widely tested for treatment of existing viral infections. Passive immunotherapy for severe respiratory virus infection is most effective if administered early in the course of disease, within 4-5 days of symptom onset. At early stages of infection, infused antibodies may enhance anti-viral activity, including neutralization and Fc functions, to clear virus and infected cells. In late-stage disease, Fc-mediated functions may contribute to inflammation outweighing any beneficial effects from viral clearance. High NAb titre correlates with therapeutic efficacy, but the level of Fc functions required for protection with CP or hIVIG remains uncharacterised. Moving forward, in-depth serological analyses, along with other measures of immune function including T cell-mediated immunity, are required to dissect the utility or harm of Fc-mediated antibody functions when treatment is received relatively late in the course of disease.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.HV: Conceptualization, Writing – original draft. SK: Conceptualization, Writing – review & editing.


Among people who inject drugs, frequent injecting and experiencing withdrawal are associated with facilitating others’ first injections. As these factors may reflect an underlying substance use disorder, we investigated whether first-line oral opioid agonist treatment  reduces the likelihood that people who inject drugs help others initiate injecting.We used questionnaire data from semi-annual visits between December 2014–May 2018 on 334 people who inject drugs with frequent non-medical opioid use in Vancouver, Canada. We estimated the effect of current first-line OAT on subsequent injection initiation assistance provision  using inverse-probability-weighted estimation of repeated measures marginal structural models to reduce confounding and informative censoring by time-fixed and time-varying covariates.By follow-up visit, 54–64% of participants reported current first-line OAT whereas 3.4–6.9% provided subsequent injection initiation assistance. Per the primary weighted estimate (n = 1114 person-visits), participants currently on first-line OAT (versus no OAT) were 50% less likely, on average, to subsequently help someone initiate injecting . First-line OAT was associated with reduced risk of subsequent injection initiation assistance provision in participants who, at baseline, injected opioids less than daily  but not in those who injected opioids daily .First-line OAT seemingly reduces the short-term likelihood that people who inject drugs facilitate first injections. However, the extent of this potential effect remains uncertain due to imprecise estimation and observed heterogeneity by baseline opioid injecting frequency. InjectiPReventing Injecting by Modifying Existing Responses (PRIMER) study is a consortium of existing cohorts across North America  that aims to identify interventions that might reduce the likelihood that people who inject drugs provide injection initiation assistance ; the AIDS Care Cohort to Evaluate exposure to Survival Services (ACCESS) study; and the At-Risk Youth Study (ARYS). This period corresponds with VIDUS/ACCESS/ARYS survey cycles 18–24 and overlaps with prior studies of OAT and injection initiation assistance provision in Vancouver  (Measures section).We included VIDUS/ACCESS/ARYS participants who attended a visit in each of the first two survey cycles involving PRIMER questions, i.e., cycles 18 (December 2014-May 2015) and 19 (June 2015-November 2015). Hereafter, we refer to the first and second of these visits by date as a participant's ‘look-back’ and ‘baseline’ visits, respectively. We then restricted to those participants who, at their baseline visit, reported ever injecting drugs and frequent  non-medical opioid use via injection and/or non-injection over the past six months. These criteria were intended to select participants with injecting experience (at-risk for providing injection initiation assistance) and a probable opioid use disorder (presumably OAT-eligible) . To faciendix 1) . Last, wAppendix 1), or (3) June 2018 (administrative censoring), whichever came first.Each participant contributed up to 5 follow-up visits from December 2015-May 2018, beginning with their first semi-annual visit after baseline and ending with the last visit they attended before (1) their first missed outcome measurement , (2) their first visit where they reported recent OAT with an alternative opioid agonist medication ; t; t-1 and t-2 respectively, with t had their treatment at visit t-1 been set, possibly contrary to their observed exposure, to t had everyone  been on first-line OAT at visit t-1 versus had everyone not been on any medication for opioid use disorder at visit t-1, conditional on everyone having the same treatment status at visit t-2  weight, t=(1–5); k  . Application of the IPTC weights creates a pseudo-population where treatment selection and censoring are statistically independent of measured time-independent and time-varying covariates featured exclusively in the denominator of the weights (t=(1–5) (Appendix 2). With a rare outcome and more common exposure, weighting to control for confounding facilitates model convergence and reduces overfitting versus traditional covariate adjustment  by measured time-independent and time-varying covariates, we fit model 1 using inverse probability-of-treatment-and-censoring (IPTC) weights . Informaistories . Formalls, 0=no) ; Vi0 rep weights . In othejustment . While ojustment . To expl2.5We assessed the joint impact of unmeasured confounding and non-ignorable censoring by prior outcome response  (Appendix 4), we: (1) randomly sampled sensitivity and specificity (bias parameters) from the beta distributions describing our uncertainty in their values; (2) used the selected parameter values to adjust person-visit observations for exposure misclassification; (3) re-fit model 1 (unweighted) in the bias-adjusted dataset; and (4) repeated steps (1)-(3) for 10,000 iterations   based on the lagged value of a participants’ bias-corrected exposure (Appendix 4).We conducted quantitative bias analyses to explore the impact of self-reported treatment misclassification bias on the direction and magnitude of our effect estimate. First, we assumed self-reported exposure (current OAT at visit endix 3) . Using aigure 1) . The medigure 1) . Second,Appendix 5) . All bias analysis results (median RR with 95% SI) were compared with the conventional, unweighted estimate.We followed the same step-by-step approach as the exposure-only misclassification bias analysis to adjust for suspected outcome underreporting (endix 5) . Without2.6t-1  , we inveisit t-1 :log.33.1At baseline, a lower proportion of participants on first-line OAT (versus those not on OAT) reported recent provision of injection initiation assistance, homelessness, overdose, daily injecting of opioids or non-opioids, and any public injecting over the past six months. Over 80% of participants on OAT at baseline reported current treatment at their look-back visit versus only 19% of participants not on OAT at baseline.3.2Overall, 50 (15%) participants were censored before their first semi-annual visit after baseline. The other 284 (85%) participants contributed 1114 follow-up visits (median [IQR]=5 [3–5] visits per participant) . Of the 3.3t=4), the most frequently observed treatment history among uncensored participants was reporting current first-line OAT at every visit  followed by never reporting current OAT .The proportion of participants reporting recent injection initiation assistance provision was low, irrespective of follow-up visit (range=3.4–6.9%). In total, there were 50 follow-up visits where recent injection initiation assistance provision was reported across 36 unique participants .t-1 on the risk of providing injection initiation assistance in the following six months , conditional on treatment status at visit t-2. Based on the primary IPTC-weighted estimate, participants on first-line OAT at a given visit were, on average, 50% less likely than participants not on OAT to report helping someone initiate injecting in the following six months . Weighting minimally impacted estimation, as the IPTC-weighted and unweighted estimates  are of comparable magnitude and precision. Compared to the IPTC-weighted estimate, the IPT-weighted estimate was further from the null  whereas the IPC-weighted estimate was closer to the null . Non-parametric percentile bootstrapping generated 95% CI of similar width to the robust 95% CI (Appendix Table 1).t-1 to the IPTC weights yielded an equivalent short-term treatment effect estimate  to the primary IPTC-weighted analysis.Adding recent injection initiation assistance provision at visit In a post hoc analysis, current first-line OAT (versus no OAT) was associated with a substantial reduction in the risk of providing injection initiation assistance within the next six months in participants with less-than-daily baseline opioid injecting  but not in participants with daily baseline opioid injecting .4In this longitudinal cohort study, we evaluated whether first-line OAT  in people who inject drugs with suspected opioid use disorder—characterized by frequent non-medical opioid use at baseline—reduces their short-term likelihood of helping others initiate injection drug use. According to our primary inverse-probability-weighted estimate, participants currently on first-line OAT were 50% less likely, on average, to help someone initiate injecting in the following six months versus participants not on medication for opioid use disorder; however, the corresponding 95% CI was imprecise, exhibiting compatibility with both a stronger beneficial effect (77% relative risk reduction) and weak, likely negligible detrimental effect (11% relative risk increase). Notably, the magnitude and precision of the estimated treatment effect were relatively consistent across unweighted and alternatively-weighted secondary analyses exploring violations of the no weight model misspecifications, no unmeasured confounding, and ignorable censoring assumptions underlying our inferences. Additionally, adjustments for self-reported outcome and treatment misclassification yielded slightly more protective effect estimates.Our study is the first to use incidence data to demonstrate support for an effect of first-line OAT on subsequent injection initiation assistance provision in people who inject drugs with frequent non-medical opioid use. Despite intentional differences in design and analysis, the direction and magnitude of our primary effect estimate fits with prior cross-sectional associations involving the same Vancouver-based cohorts and outcome (recent injection initiation assistance provision) . Using VWhile the direction and magnitude of our primary estimate offer support for a protective treatment effect, the imprecision of the corresponding 95% CI represents a key limitation that could be due to several factors. First and foremost, precision was clearly limited by the low number of outcomes contributing to the analysis as a result of our small sample size and suspected underreporting of injection initiation assistance provision because of stigma and poor recall . PrecisiAnother explanation for the uncertainty in our primary result is treatment effect heterogeneity across subgroups constituting the overall study population . SupportThere are other study limitations worth noting. The generalizability of our results to the intended target population (Vancouver-based people who inject drugs with opioid use disorder) is questionable, as the study cohort represents a convenience sample in which the prevalence of current first-line OAT (range=54–64%) far exceeds prior estimates of current OAT enrollment in North American people who inject drugs with opioid use disorder (range=11–34%) . RelatedOur findings suggest that first-line OAT may meaningfully reduce the short-term likelihood that people who inject drugs with frequent non-medical opioid use help others initiate injecting, particularly among individuals with less-than-daily opioid injecting. However, the extent of this presumed effect remains uncertain due to imprecise estimation and observed heterogeneity by baseline opioid injection frequency. Future investigations on this topic should prioritize recruiting a larger cohort, consider effect modification by baseline opioid injection frequency (and previous OAT history), and evaluate emerging alternative OAT medications, including in people who inject opioids daily given their increased risk of providing injection initiation assistance.ZB, ACT, LR, and DW formulated the research question and hypothesis. ZB conducted the statistical analyses and wrote the initial draft. All authors contributed to the design, interpretation of the results, and manuscript revisions.None.This study was approved by the University of California San Diego Human Research Protection Program, the University of British Columbia/Providence Health Care and University of Toronto Research Ethics Boards (#27433).Not applicable.Data sharing and privacy agreements for VIDUS/ACCESS/ARYS prohibit the investigators from making the data set publicly available. Partial code has been provided in the Supplemental Materials.The authors declare that they have no conflicts of interest.


Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, characterized by a highly invasive nature and therapy resistance. Combination of menadione and ascorbic acid (AA+MD) exerts strong ROS-mediated anti-GBM activity in vitro. The objective of this study was to improve AA+MD anti-GBM potential by modulating the activity of Akt and c-Jun N-terminal kinase (JNK), molecules with an important role in GBM development. The effects of Akt and JNK modulation on AA+MD toxicity in U251 human glioblastoma cells were assessed by cell viability assays, flow cytometry, RNA interference and plasmid overexpression, and immunoblot analysis. The AA+MD induced severe oxidative stress, an early increase in Akt phosphorylation followed by its strong inhibition, persistent JNK activation, and U251 cell death. Small molecule Akt kinase inhibitor 10-DEBC enhanced, while pharmacological and genetic Akt activation decreased, AA+MD-induced toxicity. The U251 cell death potentiation by 10-DEBC correlated with an increase in the combination-induced autophagic flux and was abolished by genetic autophagy silencing. Additionally, pharmacological JNK inhibitor SP600125 augmented combination toxicity toward U251 cells, an effect linked with increased ROS accumulation. These results indicate that small Akt and JNK kinase inhibitors significantly enhance AA+MD anti-GBM effects by autophagy potentiation and amplifying deleterious ROS levels. Glioblastoma multiforme (GBM) is a grade IV astrocytoma originating from neuroepithelial glial cells and represents the most lethal primary brain tumor in adults. Despite multimodal therapy comprised of surgery, postoperative radiotherapy, and chemotherapy with alkylating agent temozolomide, the median overall survival of GBM patients is ~14.6 months ,3. This Targeting the members of signaling pathways altered in GBM using small molecule inhibitors has gained notable attention as a strategy to suppress GBM progression and overcome its resistance to conventional therapy. Numerous studies in cell lines and animal GBM models show potent anti-GBM effects of PI3K/Akt/mTOR and MAPK/JNK inhibitors, exerted mainly through proliferative suppression, apoptosis induction, autophagy impairment, or by restricting the tumor-initiating potential of GBM cells ,14,15,16®) has gained an orphan drug status for the treatment of stage III and IV bladder cancer and has shown promising results in patients with advanced prostate cancer who had failed standard therapy . I. I37]. Ireatment c. These reatment d. Finallreatment e. AltogeTo evaluate the selectivity of investigated combinations towards GBM cells, we conducted experiments on human lung fibroblasts MRC5. CV and MTT tests showed that the viability of MRC5 cells was not significantly affected by the applied treatments a,b. The The present study demonstrates the capacity of small kinase Akt and JNK inhibitors, 10-DEBC and SP, to potentiate the toxicity of MD and AA+MD, well-known oxidative stress generators, towards GBM cells in vitro. The mechanisms underlying the enhancing effect of 10-DEBC include an Akt attenuation-mediated increase in deleterious ROS and cytotoxic autophagy levels, resulting in augmented cellular demise. SP-related JNK inhibition exacerbates AA+MD and triggers MD’s anti-GBM effect through enhancement of ROS levels and U251 cell death, independently of autophagy modulation.+PI+ cells, has been shown to strongly support necrotic over the other types of cell demise [Potentiation of ROS-mediated U251 cell death by Akt and JNK attenuation observed here is in accordance with the ability of other Akt inhibitors MK-2206 , perifosine , Akt inhibitor IV (ATP-competitive), and JNK inhibitors AS602801 and SP (ATP-competitive) to increase irradiation-, temozolomide-, gefitinib-, and vincristine-induced death of various GBM cell lines in vitro ,16,42,43l demise . Thus, il demise ,28. Howel demise ,47.Both Akt and JNK have been involved in GBM tumorigenesis, invasiveness, and resistance to antineoplastic agents and radiation and enhanced activity of these protein kinases in response to conventional therapy is reported in established and primary GBM cell lines ,51,52,53Autophagy induction by MD and AA+MD observed here is in accordance with the results of our previous study, showing ROS-triggered AMPK/mTOR/ULK1-dependent autophagy in MD- and AA+MD-treated cells . Pre-treThe small molecule kinase inhibitors 10-DEBC and SP applied alone in our study did not affect U251 cell viability but sensitized U251 GBM cells to the sub-toxic concentration of MD. Simultaneous treatment resulted in a moderate decrease in U251 cell viability, suggesting an additive rather than synergistic interaction between these molecules. Regardless of the nature of their interaction, our results suggest that co-treatment of GBM cells with MD and Akt or JNK inhibitors may be proposed as a potential anti-GBM strategy. Furthermore, the previous hypothesis has a good basis in the fact that small molecules and MD can cross the blood−brain barrier ,72. In aThe treatment-induced necrosis observed in our study, potentiated in the presence of the small molecule inhibitors, may represent a desirable therapy-induced GBM cell death pattern bearing in mind that cells with defective apoptotic pathways confer a resistant phenotype to GBM . Althoug


Nature Medicine reported on the Phase I clinical trial of a mitochondria-targeting anti-cancer agent IACS-01059 in patients with acute myeloid leukemia (AML) and solid tumors [A recent paper published in d tumors . Interesd tumors . A follod tumors . Of noted tumors  the BH3 d tumors  or immund tumors , as welld tumors . These fd tumors , resulteNature Medicine [The results of the IACS-010759 Phase 1 clinical trial [+) that anchors biologically active agents at the interface between the inner mitochondrial membrane (IMM) and the mitochondrial matrix [+-tagged anti-cancer agents of which mitochondrially targeted tamoxifen (MitoTam)   appearedBased on research and pre-clinical results, we launched the Phase I/Ib trial of MitoTam with patients with untreatable, metastatic solid tumors, including some 10 types of cancer . In Phas+ tagging of small molecules with anti-cancer efficacy and molecular target within mitochondria , is a pmitocans ,18.


The clinical relevance of Routine Outcome Monitoring (ROM) to formulate longitudinal evaluations of treatment appropriateness/efficacy and to assist decision making aimed at improving the quality of person-centered interventions has been poorly implemented in forensic psychiatry, also in Italy. Indeed, very few assessment instruments have been developed in this crucial field.As reliable ROM instruments are lacking, the aim of the current investigation was to examine psychometric properties  of the Parma Scale   in an Italian sample of forensic psychiatric patients.Participants were male adult offenders with mental disorder recruited within the Parma REMS (“Residence for the Execution of Security Measure”) or the Parma Penitentiary Institute (PPI). Exclusion criteria were known moderate/severe intellectual disability or any other medical condition inducing inability to express a valid consent for participating in the research. The Pr-Scale includes 20 items divided into 3 main domains: “Historical”, “Clinical”  and “Treatment Plannining". To test psychometric properties of the Pr-Scale, we examined interrater reliability, short-term (1-week) test-retest reliability and internal consistency. As measure of concurrent validity, a correlation analysis of Pr-Scale item scores with corresponding HKT-R  item subscores was performed. Finally, we examined the Pr-Scale sensitivity to measure scores’ longitudinal changes over a 3-month treatment follow-up period.60 male adult patients were recruited in this study. Our findings showed good to excellent interrater and test-retest reliability, concurrent validity and internal consistency for the Pr-Scale. Pr-Scale scores also display a moderate to large changeability over time . Statistically significant correlations of Pr-Scale item scores with the corresponding HKT-R scores were found. Across the 3-month follow-up period, we observed statistically significant sensitivity values in measuring longitudinal changes for the Pr-Scale total score and Pr-Scale domain and item subscores.Our results support the clinical use of the Pr-Scale in different forensic psychiatric settings  as reliable ROM instrument.None Declared


Emerging immune checkpoint blockade (ICB) therapies have been shown to have promising therapeutic efficacy in treating solid tumors. A phase III clinical trial reported that the combination of ICB and chemotherapy lengthened progression-free survival in patients with metastatic PD-L1+ TNBC3; however, most patients had primary resistance or acquired resistance to ICB. Thus, the intrinsic mechanisms underlying ICB resistance are still under investigation4.Triple-negative breast cancer (TNBC) is the most difficult type of breast cancer to treat. TNBC is defined by the lack of expression of three receptors: estrogen receptor (ER); progesterone receptor (PR); and human epidermal growth factor receptor 2 (HER2). Chemotherapy is currently first-line treatment for TNBC; however, due to the high heterogeneity of TNBC, most patients eventually develop chemotherapy resistance, which is associated with a poor prognosis5. Chemotherapy not only has a limited killing effect on CSCs TNBC, but also promotes the generation of drug-resistant CSCs.CSCs are sub-population of tumor cells with self-renewal and high tumorigenicity properties, and are considered to be the main reason for tumor heterogeneity. Studies have indicated that in many solid tumors, including TNBC, CSCs are responsible for tumor metastases and resistance to chemotherapy, ultimately leading to tumor relapse. Moreover, non-CSC tumor cells can adapt to changes through cellular reprogramming, such as the epithelial-to-mesenchymal transition (EMT) and regenerating CSCs7. CSCs secrete soluble mediators to modulate the immune cells in the TME: CCL2 recruit macrophages, MDSCs and Treg cells; CSF1 polarize tumor-associated macrophages (TAMs); TGFβ promote immunosuppressive myeloid cell activity, T-cell and NK cell inhibition, Treg cell polarization and support the CSC phenotype. CSCs also upregulate PD-L1 to inhibit T and NK cell activity and promote EMT in patients with TNBC8. CSCs also downregulate the surface expression of adhesion molecules, such as CD103 ligand and E-cadherin, through EMT to evade CD8+CD103+ memory T lymphocytes9. A recent study reported that ICB therapy selects enriched CSCs with reduced antigen processing/presentation machinery (APM) to facilitate ICB resistance in patients with TNBC10. TNBC tissues and cell lines are characterized by CD44+CD24− and/or increased aldehyde dehydrogenase (ALDH) activity compared to other breast cancer subtypes. Chemotherapy and ICB currently used in clinical practice enrich tumor stem cells, thereby causing drug resistance and recurrence11. Because CSCs contribute to intra-tumoral heterogeneity and promote therapy resistance, successful treatment against CSCs is required to eradicate tumors; however, due to the similarity between CSCs and stem cells, avoiding on-target/off-CSC toxicity remains challenging.CSCs evade immune surveillance by actively interacting with immune cells in the tumor microenvironment (TME) by secreting soluble mediators and receptor/ligand direct interactions to promote the immunosuppressive TME and tumor stemnessFigure 1A, bi-specific T cell engager antibodies (BiTEs) are typical TCEs, which have achieved great success in hematomas; the curative effect is comparable to chimeric antigen receptor (CAR)-T cell therapy. TCE therapy has also shown promise in redirecting polyclonal T cells to kill poorly immunogenic CSCs12; however, the function of TCEs relies exclusively on TAAs expressed by tumor cells. Tumor cells may have primary or acquired resistance to TCEs due to TAA expression heterogeneity or loss. Tri-specific TCEs, the next generation of TCEs, incorporate a T cell binding motif and two TAA binding motifs . Tri-specific TCEs target more than one TAA on tumor cells to overcome TAA heterogeneity or loss during treatment. Tri-specific TCEs targeting CD3, epidermal growth factor receptor (EGFR), and epithelial cellular adhesion molecule (EpCAM) were developed to specifically kill EGFR and/or EpCAM-expressing colorectal cancer cells13. This tri-specific TCE has enhanced tumor targeting ability and better therapeutic efficacy than the corresponding bi-specific controls. Tri-specific TCEs simultaneously engage TAAs to promote T cell binding and subsequent tumor lysis; however, tri-specific TCEs may also enable T cells to kill normal cells upon binding to TAAs, which are expressed on normal cells. This on-target/off-tumor toxicity significantly hinders TCE applications. The trophoblast cell-surface antigen 2 (Trop2), carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5), erythropoietin-producing hepatocellular carcinoma receptor A10 (EphA10), EpCAM, EGFR, and mesothelin are potential TAAs for TNBC; however, it is difficult to identify TAAs that are specifically expressed on tumor cells, especially CSCs. The goal of the next generation of bi-specific antibodies is to improve tumor-targeting specificity of TCEs to increase therapeutic efficacy, while minimizing on-target/off-tumor toxicity.TCEs bind to tumor-associated antigens (TAAs) on tumor cells and CD3 on T cells to redirect polyclonal T cells to eliminate tumor cells independent of the antigen specificity of the T cell receptors (TCRs), co-stimulation receptor activation, or cytokine stimulation. As shown in via circulating blood. In healthy tissues, peptide-masked TCEs are largely intact. The masked peptide prevents antibody from binding to the target due to the shielding, preventing T cells activation and avoiding side effects on healthy tissues. In addition, masked TCEs have a long half-life and continue to circulate in the blood. The masked TCEs are inactive until activated by TME-associated proteases. The tumor-related protease cleaves the substrate linker when the masked TCEs reach the TME, thereby releasing the masked peptide and facilitating TCE selective bind to the target antigen in the tumor tissue. The activated TCEbind the antigen and CD3 on the tumor surface, thus activating T cells to kill the tumor. One study developed an anti-idiotypic anti-CD3 single-chain variable fragment (scFv)-masked TCE to block the anti-CD3 fragment antigen-binding (Fab) region, which is intact in the systemic circulation and healthy tissues, thus the anti-CD3 Fab region cannot redirect T cells to kill TAA-expressing cells14. The tumor-specific protease-cleavable linker between the mask and anti-CD3 Fab region is preferentially unmasked and the activated TCE in the TME specifically kills TAA-expressing cells . Thus, the masked TCE strategy enhances the specificity and safety of TCE therapy and expands the selectable TAAs.Novel TCEs that are specifically activated in the TME have been developed using protease response peptide masking technology to avoid on-target/off-tumor toxicity. The peptide is connected to the antigen recognition region of the TCEs by a TME-associated protease cleavable linker. The peptide acts as a space shield to mask the antigen recognition ability of TCEs. Peptide-masked TCEs enter body tissues 15. Conventional TCE therapy can hardly reverse an immunosuppressive TME. Thus, overcoming an immunosuppressive TME within TNBC is the next challenge for successful TCE therapy. Moreover, the ultimate goal of immunotherapy is to generate long-lasting antitumor reactions that require the collaboration of innate and adaptive immune cells. TCEs redirect T cells to kill TAA-expressing tumor cells, but has a limited effect on innate immune cells. Therefore, a better therapeutic strategy is needed to reverse the immunosuppressive microenvironment and activate innate immune cells to cooperate with TCE therapy to involve the entire immune circulation against cancer; however, the combination of multiple immunomodulators often leads to increased toxicity. There is currently a lack of targeted immunotherapy for TNBC CSCs.Among solid tumors, especially the ‘immunologically cold’ TNBC, the immunosuppressive TME and the lack of sufficient tumor-infiltrating lymphocytes (TILs) leads to TCE primary or acquired resistance. The immunosuppressive immune cells  in the TME inhibit T cell recruitment and activation. High levels of PD-L1 expression in TNBC inhibit T cell activation and promote T cell exhaustionNext-generation TCEs should be designed to have additional functionalities to overcome an immunosuppressive TME, including blocking the immune checkpoint, recruiting T cells and dendritic cells (DCs) to the TNBC TME, and activating innate and adaptive immune cells. A TME responsive flexible platform/delivery system integrates multiple functionalities to enable the delivery of immunomodulatory therapeutics, such as ICB immunoagonist,and TCEs in the TME that reverse the immunosuppressive microenvironment to promote TCE activity is needed, which would be more precise, potent, and safer than the systemic administration of individual immunomodulators and TCEs.16. In addition, chemotherapy selectively enriches CSCs with repressed type I IFN signaling, while activation of type I IFN intrinsically reduces CSC properties and reverses EMT17. Moreover, IFN signaling increases the APM of tumor cells, making tumor cells visible to the immune system. Type I IFN also activates innate and adaptive immune cells in the TME, including increased DC maturation, antigen presentation, cross-priming, and recruiting additional T cells to facilitate the generation of long-lasting antitumor immune memory reactions. Therefore, activating the IFN pathway inhibits CSCs and reverses the immunosuppressive TME. Recent findings demonstrated that delivery of a stimulator of IFN gene (STING) agonists in tumor tissues upregulate the transcription of genes that encode type I IFNs, and proinflammatory cytokines and chemokines, which leads to the accumulation and infiltration of CD8+ T-cells and DC maturation, while repression of STINGs in TNBC promotes immune evasion and resistance to ICB18. Moreover, preclinical studies with STING agonists have shown promising antitumor efficacy. Thus, STING agonists may act synergistically with TCEs to better inhibit CSCs, reverse the immunosuppressive TME, and generate antitumor immune memory reactions. The metabolic instability, limited tumor targeting ability, and systemic toxicity of STING agonists hinder clinical application19. Both TCEs and STING agonists have on-target/off-tumor toxicity upon systemic administration. Thus, the combined administration of STING agonists and TCEs may cause unacceptable toxicity.CSCs, which are enriched in TNBC and contribute to intratumor heterogeneity, are characterized by an increased expression of multiple immunosuppressive pathways20. PAMAM works as a flexible platform to integrate multiple functionalities without sophisticated protein engineering compared to tri-specific antibodies. We used mPEG with a weakly acidic responsive linker as a mask to shield TriTNEs, which were largely intact in systemic circulation but preferentially unmask TriTNEs in the weakly acidic TME to minimize off-target toxicity while expanding the targets for TAA selection. Taking advantage of the PEG mask, CD44 and PD-L1 were chosen as TAAs. These proteins have been reported to be overexpressed on TNBC cells, and both are important for CSC maintenance and immune escape. We chose an anti-PD-L1 antibody to target and block PD-L1. The oligosaccharide Hyaluronic acid (HA) has modest affinity for CD44 and preferentially targets TNBC cells with high CD44 expression rather than CD44-expressing normal cells. This dual TAA-targeting strategy improves selectivity and binding affinity to tumor cells and preferably overcomes TAA heterogeneity and PD-L1-mediated immune escape.Taking advantage of nanotechnology, we adopted a modular design of a multifunctionality platform using a poly(amidoamine) [PAMAM] dendrimer with an ultrasmall size (∼4 nm), adjustable positive charge, and many cavities as substrates to create a TME-responsive Methoxy Poly(ethylene glycol) (mPEG) masked CD44×PD-L1×CD3 tri-specific T-cell nanoengager (PmTriTNE) loaded with a negatively-charged STING agonist [c-di-AMP (CDA)] to overcome the immunotherapeutic resistance of TNBC20. TriTNE targets CD44 and PD-L1, which are enriched in both non-CSCs and CSCs of TNBC  to avoid TAA heterogeneity. CDA activates the CSC IFN pathway to intrinsically increase CSC immunogenicity and reverse CSC properties. CDA also stimulates DC maturation and tumor antigen cross-priming, subsequently recruiting CD8+ T and NK cells. TriTNEs block PD-L1 on tumor cells and enables CD8+ T cells to directly kill tumor cells. Then, dead cancer cells release damage-associated molecular pattern(DAMP) and tumor antigens to recruit and activate innate and adaptive immune cells, thus achieving multilayer control of non-CSCs and CSCs in TNBC and generate long-lasting antitumor immunity. PmTriTNE@CDA eliminated TNBC in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mice, suggesting a new strategy for overcoming immunotherapy resistance in TNBC. PmTriTNE@CDA was designed to integrate various immune functions into one nanoplatform, thereby enhancing efficacy without compromising safety. Because CSCs contribute to inter-tumoral heterogeneity and immune escape across multiple solid tumor types, this proof-of-concept nanoplatform could broaden the reach of the TCE strategy to many types of cancer and pave the way for new CSC-targeted therapies and rational combination approaches.We determined that the TME-responsive PEG mask provides a half-life extension and enables the selection of TAAs that are also expressed in normal cells


Nature, Park and colleagues have explored an innovative approach for improving adoptive neuron transfer, with possible implications for Parkinson´s Disease (PD) and other neurodegenerative diseases.1 By co-transplanting autologous regulatory T (Treg) cells, they managed to reduce surgical trauma-induced neuroinflammation, enhance the safety of cell replacement therapy by inhibiting proliferation of non-dopaminergic cells and improve therapeutic outcomes.In a paper published in substantia nigra (SN) and their projections into the striatum, leading to severe motor and non-motor symptoms. Recent advancements in stem cell technology raise the possibility that PD could eventually be treated by implanting dopaminergic neurons generated from human induced pluripotent stem cells (iPSc) and embryonic stem cells (ESC). However, significant limitations of this therapeutic approach are the low survival rate of transplanted neurons. Further, potential proliferation of unwanted cell populations gives rise to safety concerns  dopaminergic neurons. This was linked to needle-induced neuroinflammation. By co-transplanting autologous regulatory T cells (Treg), they then managed to reduce surgical trauma-related inflammation and improve therapeutic outcomes.To overcome these limitations, Park et al. generated human midbrain dopaminergic progenitor (mDAP) cells, using human ESC, or skin-derived iPSC from a patient with PD as starting material. To induce lesions in the striatum of rodents, they stereotactically injected 6-hydroxydopamine (6-OHDA) into the SN. Subsequent intra-striatal injection of mDAP initially resulted in a very poor survival of implanted tyrosine hydroxylase-expressing  at two weeks after transplantation and in vitro, while almost all TH- grafted cells survived. Mitigating neuroinflammation to prevent neuronal cell death thus appeared essential for successful cell transfer, even in autologous settings. Co-injecting CD4+CD25+FoxP3+ Treg, which are potent tolerogenic and anti-inflammatory cells required for immune homeostasis and tissue repair, was a logical, and successful next step. Still, while correct synapse formation by transplanted human iPSC-derived neurons and endogenous neurorepair processes require time, co-injected Treg have a limited lifespan. This may explain why the functionally impressive rescue in an amphetamine-induced rotation model in Fischer344 rats, observed at 20 weeks after implantation of human patient-derived mDAP cells, required additional daily treatment with cyclosporine A (CsA). Nevertheless, CsA treatment alone was significantly less effective than CsA treatment combined with adoptively transferred Treg.This study demonstrates that the surgical procedure of inserting a needle into the rodent striatum itself triggers profound neuroinflammation. Under inflammatory conditions, TH- cells, which also emerge from the injected iPSC- (or ESC-) derived mDAP. Still, some of the described findings imply further direct effects of Treg on the iPSC-derived dopaminergic neurons.This clearly indicates that Treg exert additional effects on top of CsA-induced immunosuppression. Moreover, beneficial effects of co-injected Treg were not only found in immunocompetent Fischer344 rats, or in mice that had been immunologically humanized with peripheral blood mononculear cells from the iPSC donor. . Treg were also protective in highly immunodeficient NSG mice and in vitro, when neurons were just exposed to proinflammatory cytokines such as IFN-γ, TNF-α, or IL1-β. Accordingly, effects of Treg must go beyond suppression of host inflammatory immune responses. Given that the safety of cell replacement therapy is of utmost importance, it is intriguing to note that Park et al. found that Treg inhibit the proliferation of undesirable cells. Indeed, the authors demonstrated that Treg-derived TGF-β limits the proliferation of TH2 (ii) activation of the neuroprotective Rac1/Akt signaling pathway in dopaminergic neurons when CD47 on Treg binds to Signal regulatory protein α (SIRPα) on neurons, (iii) release of brain-derived neurotrophic factor (BDNF), of amphiregulin, TGF-β and IL-10, and (iv) polarization of astrocytes and microglia towards neuroprotective phenotypes characterized by secretion of BDNF and glial cell-derived neurotrophic factor (GDNF).3 Local administration of Treg, as performed in this study, allows for all these effects. In the MPTP mouse model for PD, adoptive i.v. transfer of Treg or in vivo Treg induction by treatment with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) also protected nigral dopaminergic neurons - even without co-administration of mDAP. In a clinical phase I trial, treatment with rhGM-CSF (sargramostim) increased Treg frequency and function. Remarkably, this translated into improved scores on the Unified Parkinson’s Disease Rating (UPDRS-III) Scale and magnetoencephalography-recorded cortical motor activities in PD patients (NCT01882010).3In this context, known regeneration-promoting mechanisms of Treg include (i) secretion of the growth regulatory protein CCN3, which in turn promotes differentiation of oligodendrocyte precursor cells and myelin regeneration,+ and activated CD8+ T cells into the SN and activation of microglia were found to precede the symptomatic loss of dopaminergic neurons.4 An increased frequency of effector memory T cells in peripheral blood correlates with more severe motor function impairments. A direct link with Treg was established when peripheral blood Treg from PD patients were found to possess reduced immunosuppressive capability and disease-associated functional deficits, which further contributes to pathological inflammation. Epidemiologically, a population-based case-control study including 48,295 PD patients and 52,324 controls found that intake of immunosuppressants like azathioprine and mycophenolate greatly reduced the risk of developing PD.5 As these medications shift the balance between effector and regulatory T cells, this study suggests that an early immunomodulatory intervention might interfere with the development of PD. Still, current symptomatic treatments for PD hardly modulate ongoing immune alterations.These remarkable effects of Treg in PD concur with recent clinical observations, which suggest that the persistent neuroinflammation observed in PD patients is no mere response to neurodegeneration and cell death. Instead, accumulation of pro-inflammatory pathological α-Synuclein, priming and expansion of α-Synuclein-specific T cells, infiltration of Th1-skewed CD4In the present study, however, Park et al. used a model that resembled an advanced stage of disease, where Treg alone might not have been effective.  Combining human mDAP, Treg and CsA, they observed a functional repair of the lesioned rat brain within 20 weeks, suggesting that adoptively transferred xenogeneic neurons survived and showed sufficient plasticity to integrate into pre-formed neural networks. In summary, this highlights the potential of a combined transplantation of stem cell-derived dopaminergic midbrain neurons and autologous anti-inflammatory Treg as a putative treatment strategy for PD patients. With this combined approach, Park et al. have successfully addressed crucial limitations of stem cell-based therapy. Importantly, as other post-mitotic neurons such as GABAergic neurons were similarly affected by needle trauma-induced neuroinflammation, similar strategies could also enhance the treatment of various other (neuro)degenerative diseases.


Several studies suggested that SARS-CoV-2 was already spreading worldwide during the last months of 2019 before the first outbreak was detected in Wuhan, China. Lombardy  was the first European region with sustained SARS-CoV-2 transmission and recent investigations detected SARS-CoV-2-RNA-positive patients in Lombardy since late 2019.We tested for anti-SARS-CoV-2 IgG all serum samples available in our laboratory  that we received within the framework of measles/rubella surveillance from measles and rubella virus-negative patients.Thirteen of 235 samples (5.5%) were IgG-positive. The positivity rate increased starting in 2019 and was significantly different from the expected false positive rate from 2019 onwards. Additionally, in 2019 the percentage of IgG-positive patients was significantly lower among SARS-CoV-2 RNA-negative patients (3/92) compared to SARS-CoV-2 RNA-positive patients . The highest percentage of IgG positivity in the pre-pandemic period was recorded during the second half of 2019. This coincided with an increase in negativity for measles and a widening of the peak of the number of measles discarded cases per 100,000 inhabitants, indicating a higher-than-normal number of measles-negative patients experiencing fever and rash. This also coincided with the first patient positive for SARS-CoV-2 RNA ; this patient was also positive for anti-SARS-CoV-2 IgG and IgM.Although the number of samples was low and one cannot conclusively establish that the virus started circulating in Lombardy around September 2019, our findings should stimulate similar research investigating the possibility of undetected SARS-CoV-2 pre-pandemic circulation.The online version contains supplementary material available at 10.1186/s12985-023-02117-9. Lombardy  was the Italian region with the highest COVID-19 clinical burden in early 2020 –5. In ouAs World Health Organization (WHO)-accredited Subnational Reference Laboratory for measles and rubella surveillance in Lombardy  we regularly receive samples from patients with fever and rash for molecular and serological diagnosis of measles and rubella . Part ofIn the same study, we also evaluated whether SARS-CoV-2 infections could be detected in the same patients by identifying antibodies directed against this virus through ELISA (enzyme-linked immunosorbent assay) . While sThe study included all available serum samples (N = 235) collected within the framework of measles/rubella surveillance from measles and rubella negative patients between March 2017 and March 2022. Patients were 112 females (47.7%) and 123 males (52.3%), the median age was 22 (range 0–95 years), and samples were collected 0–23 days  after rash onset.Sera were retrospectively tested for anti-SARS-CoV-2 IgG using the semi-quantitative Anti-SARS-CoV-2 IgG ELISA  test according to manufacturer’s instructions. Positive samples were those with a sample extinction to calibrator extinction ratio logit function) was performed to identify independent variables predictors of dependent variable positivity (IgG+). Statistical analyses were performed using Stata version 17 (StataCorp).Numeric variables were reported as the median (IQR). Proportions were reported in percentages for dichotomous count variables, excluding borderline samples (ratio 0.8–1.1). Observed proportions were compared to the expected proportions of false positive results , using the test on the equality of proportions and computed Clopper-Pearson 95% confidence intervals. Other proportions were compared with Fisher’s exact tests and, in all cases, two-sided p values < 0.05 were considered statistically significant. A logistic regression analysis (Thirteen out of the 235 tested samples (5.5%) were IgG-positive and three 1.3%) were borderline. Figure .3% were During the years 2017–2018, the number of measles/rubella discarded cases per 100,000 surveilled inhabitants fluctuated and the percentage of measles/rubella negative suspected cases varied depending to measles epidemic trends  and observed that the difference between these two proportions was statistically significant for the year 2019, including when considering the first and second half separately, but not for the years 2017 and 2018. Additionally, in 2019, the percentage of IgG-positive patients was significantly lower among SARS-CoV-2 RNA-negative patients than among SARS-CoV-2 RNA-positive patients.The semi-quantitative method we used showed that the strength of the signal increased in 2021–2022. Although weakly reacting antibodies could indicate a false positive signal, highly reacting antibodies identified in a few patients symptomatic in later phases of the pandemic (2021–2022) could have been determined by re-exposures or vaccination. Indeed, three of the five strongly positive samples were collected after COVID-19 mass vaccination campaigns had started and all five were sampled after at least one year of virus circulation.The highest percentage of IgG positivity during the pre-pandemic period was during the second half of 2019, with three positive samples in September and October. This coincided with an increase in negativity for measles and a widening of the peak of the number of discarded cases per 100,000 inhabitants, which followed a measles epidemic that ended in July. In other words, while measles virus stopped circulating, there was still a higher-than-normal number of patients experiencing fever and rash. This is consistent with the observed increase in measles discarded rate for the year 2019 . NoticeaThis study has some limitations. Firstly, the low number of available samples limits our findings and the power of our statistical analyses. Secondly, the investigated period is limited to 3 pre-pandemic years and 3 pandemic years with a variable number of samples in each period. However, the population type was consistent across the years of the study. Additionally, confirmation assays, such as neutralization, was not performed and, while the specificity of the used assay is high, the kit is characterized by a low sensitivity  and some cases may have gone undetected. Nonetheless, although this study cannot conclusively establish when the virus started circulating in Lombardy, it should serve the purpose of stimulating further research as evidence for undetected SARS-CoV-2 pre-pandemic circulation continues to accumulate.Below is the link to the electronic supplementary material.Supplementary Material 1


Endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA) has proven to be highly accurate in lung cancer diagnosis and staging. However, its efficacy in diagnosing lymphoma, especially Hodgkin’s lymphoma, remains controversial, mainly due to the need for larger biopsies for definitive diagnosis. This case study presents a 53-year-old HIV-positive man with a controlled viral load, who presented with a large left hilar mass and a left upper lobe nodule, both showing significant uptake on positron emission tomography scans. The patient underwent bronchoscopy with bronchoalveolar lavage, EBUS-TBNA using an Olympus™ Vizishot 2 needle , and EBUS-guided transbronchial forceps biopsies (TBFB) of a left hilar lymph node using a 1.8 mm Boston Scientific™ forceps . The EBUS-TBNA revealed granulomas, while the subsequent EBUS-guided TBFB revealed nodular lymphocyte-predominant Hodgkin's lymphoma. EBUS-TBFB may be a promising technique for obtaining larger tissue samples and enhancing diagnostic yield in cases of mediastinal lymphadenopathy with suspected lymphoma. Endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA) is a widely used procedure and has been validated for the diagnosis and staging of lung cancer and the diagnosis of sarcoidosis. Despite its established role in lung cancer staging, its diagnostic accuracy in lymphoma remains a subject of debate due to certain disadvantages, such as lesser core tissue sampling and inferior negative predictive value . This arA 53-year-old man with a medical history of obstructive sleep apnea, pulmonary nodule, and HIV infection presented with PET-positive left hilar mass Figure  and leftTM  and wedged into the LUL bronchus. BAL was performed, and the fluid was sent for cultures, cytology, and antigen testing. EBUS-TBNA was then performed on the station 11L lymph node using a 22-gauge Vizishot 2 needle . A tract was formed by pushing the sheath in and dilating the area. Additionally, six transbronchial biopsy (TBBX) samples were obtained from the same lymph node under ultrasound guidance using 1.8 mm forceps. The lymph node TBNA revealed granulomatous inflammation. The lymph node biopsy using transbronchial forceps revealed nodular lymphocyte-predominant Hodgkin's lymphoma of intrathoracic lymph nodes, indicating HIV-associated Hodgkin's lymphoma. Figures The patient underwent bronchoscopy with bronchoalveolar lavage (BAL) of the LUL. The bronchoscope was introduced through an iGELDuring histopathological examination of the tissue samples, specific cells exhibited weak staining for PAX5, a critical transcription factor associated with B-cell development. This weakened staining pattern strongly suggests a decrease in the expression of B-cell markers, a hallmark characteristic of classic Hodgkin's lymphoma . On immuEBUS-TBFB provides a minimally invasive and accurate method for obtaining tissue samples from mediastinal lymph nodes, aiding in the diagnosis and staging of various pulmonary conditions, including Hodgkin's lymphoma. In scenarios where EBUS-TBNA fails to yield a diagnosis, as in our case, EBUS-TBFB can be a safe and effective alternative to improve diagnostic accuracy when performed in conjunction with EBUS-TBNA.In a recent investigation, Pathak et al. studied a cohort of 30 patients, eight patients had a history of lymphoma, one patient had a history of squamous cell carcinoma, and one patient had a history of chronic lymphocytic leukemia with 25 of them undergoing EBUS-TBFB. The overall diagnostic yield of EBUS-TBFB was found to be 73%. However, when EBUS-TBFB was combined with EBUS-TBNA, the diagnostic yield significantly improved to 86% (26 out of 30 patients). While TBFB provided additional diagnostic information beyond TBNA in some cases, the study was not powered to thoroughly evaluate the diagnostic yield. These findings suggest that combining EBUS-TBFB with EBUS-TBNA may enhance diagnostic accuracy in the evaluation of patients with suspected lung pathology [Mehta et al. aimed to assess the effectiveness and safety of a novel technique called EBUS-TBFB when conventional EBUS-TBNA with rapid on-site evaluation (ROSE) failed to provide a diagnosis. The study included 30 consecutive patients with enlarged mediastinal lymph nodes and negative EBUS-TBNA ROSE results. EBUS-TBFB successfully obtained adequate tissue samples in all cases where both EBUS-TBNA and EBUS-TBFB were performed. Particularly, in patients with non-diagnostic EBUS-TBNA ROSE, EBUS-TBFB yielded positive diagnostic results in eight out of 30 patients 27%), of which six were diagnosed with tuberculosis, one with aspergillosis, and one with Hodgkin’s lymphoma. For patients with a negative EBUS-TBNA ROSE, EBUS-TBFB proved to be a safe and valuable technique, contributing to the diagnostic yield. The findings indicate that EBUS-TBFB is a valuable addition to EBUS procedures, especially in benign causes of mediastinal lymph node enlargement [%, of whiIn their study, Diab et al. compared the diagnostic yield of EBUS-TBNA and EBUS-TBFB individually with their combined approach in cases of mediastinal lymphadenopathy with unknown etiology. The combined technique provided a definitive diagnosis in 31 out of 35 cases 88.6%), accurately identifying both Hodgkin's and non-Hodgkin's lymphomas in 90% of cases without the need for further invasive procedures. Additionally, the combined approach successfully confirmed all cases of granulomatous inflammation. The study concludes that the safe and high diagnostic yield of the combination of EBUS-TBFB and EBUS-TBNA makes it particularly effective for diagnosing mediastinal adenopathy of unknown origin, especially in lymphoma cases. The larger samples obtained from TBFB also improved sensitivity in detecting granulomatous disease and provided specimens for clinical trials in malignancy cases when needle aspirates were insufficient [8.6%, accRecently, there has been evidence supporting the high diagnostic success and safety of EBUS-TBFB for mediastinal lymph nodes. However, a rare technical issue was observed during the procedure, where a rupture of the steering band prevented the closure of forceps jaws in a subcarinal lymph node. This problem could lead to severe complications. The report highlights the importance of addressing such complications during transbronchial sampling of mediastinal lesions and presents a solution to this specific issue, along with other procedure-related complications reported in the literature .In another study, combining bronchoscopic tissue forceps biopsy (BBX) and EBUS-TBNA improves diagnostic yield in lung lesion evaluation. In 115 patients, the combined approach provided a definitive diagnosis in 93% of cases, with 81% being malignant. Seventy-eight patients underwent BBX and EBUS-TBNA of the lymph node(s) only; malignancy was diagnosed in 63 (81%) patients; 28 (36%) had malignant cells in both specimen types, 19 (24%) had malignant cells only in the BBX, and 16 (21%) had malignant cells only in the EBUS-TBNA. In the remaining 15 patients who underwent BBX and EBUS-TBNA of the lymph nodes only, 10 (67%) were diagnosed with either granulomatous disease or evidence of infection, and five (33%) with malignancy on subsequent procedures. EBUS-TBNA was especially valuable when BBX results were negative, increasing diagnostic yield by 18% and assisting in tumor staging. Additionally, the combination allowed for more tissue sampling for immunohistochemistry and molecular testing, facilitating personalized management in a minimally invasive manner [Our case demonstrates the importance of performing EBUS-TBFB in cases of mediastinal adenopathy of unknown etiology. Had we not performed EBUS-TBFB, EBUS-TBNA alone would have led us to a misdiagnosis of sarcoidosis in this case.EBUS-TBFB provides a minimally invasive and accurate method for obtaining tissue samples from mediastinal lymph nodes that may aid in the diagnosis and staging of various pulmonary conditions, including Hodgkin's lymphoma, non-Hodgkin’s lymphoma, and sarcoidosis. In scenarios where EBUS-TBNA fails to yield a diagnosis, EBUS-TBFB can be a safe and effective alternative to improve diagnostic accuracy when performed in conjunction with EBUS-TBNA. Further randomized controlled prospective trials are needed.