What is the comparative effectiveness of first-line targeted therapy, immunotherapy, and combination therapy in a real-world cohort of patients with metastatic clear cell renal cell carcinoma?In this propensity-matched cohort study of 5872 patients treated in real-world clinical practice, first-line immunotherapy and combination therapy were associated with improved overall survival compared with first-line targeted therapy.This study suggests that clinical trial findings demonstrating an overall survival benefit associated with first-line immunotherapy-based regimens compared with targeted therapy are generalizable to a broader population of patients. Clinical trials have shown an overall survival (OS) benefit associated with first-line immunotherapy (IT) and combination targeted therapy (TT) and IT regimens compared with TT among patients with metastatic clear cell renal cell carcinoma (RCC). Generalizability of these findings in a real-world cohort outside of a clinical trial setting is unclear.To assess the association of first-line TT, IT, and combination TT and IT regimens with OS in a real-world cohort of patients with metastatic clear cell RCC.This retrospective propensity-matched cohort study identified 5872 patients with metastatic clear cell RCC in the National Cancer Database from January 1, 2015, to December 31, 2017, who received first-line TT, IT, or combination TT and IT and were not treated on a clinical trial protocol. Patients were stratified by first-line systemic treatment. Statistical analysis was conducted from October 1 to December 1, 2020.The primary outcome was OS from the date of diagnosis to death or censoring at last follow-up. After 1:1:1 nearest-neighbor caliper matching of propensity scores, survival analyses were conducted using Cox proportional hazards regression and Kaplan-Meier estimates.P < .001; combination TT and IT group: HR, 0.74 ; P = .005). No survival difference was seen between the IT and combination TT and IT groups .The final study population included 5872 patients , and the matched cohort included 1437 patients (479 per treatment group). Patients in the IT and combination TT and IT groups were younger than those in the TT group, had fewer comorbid conditions , and were more often treated at academic centers . Both first-line IT and combination TT and IT were associated with improved OS compared with first-line TT for patients with metastatic clear cell RCC . Evaluating new treatments in comparative effectiveness studies is a critical part of validating clinical trial findings because patients enrolled in clinical trials tend to be younger and healthier than those encountered in real-world practice.12 Given the limited availability of effectiveness data for novel and increasingly used IT regimens for metastatic clear cell RCC, we sought to examine real-world survival outcomes for TT, IT, and combination TT and IT regimens using a generalizable cohort of patients with metastatic clear cell RCC.First-line management of metastatic clear cell RCC has recently shifted toward immunotherapy (IT), largely owing to the emergence of immune checkpoint blockade (ICB) therapies and clinical trial results demonstrating improved overall survival (OS) and progression-free survival compared with tyrosine kinase–inhibiting targeted therapy (TT).13 Trained data abstractors collect and submit data to the NCDB using standardized coding definitions as specified in the most recent Commission on Cancer Facility Oncology Registry Data Standards guideline.14 This study was conducted using deidentified data and was determined to be exempt by the H. Lee Moffitt Cancer Center and Research Institute institutional review board because the analysis exclusively involved deidentified patient data and is considered a secondary analysis of existing data. This study was reported in a manner consistent with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.15Cases of clear cell RCC were identified and abstracted from the National Cancer Database (NCDB) between January 1, 2015, and December 31, 2017. The NCDB includes more than 70% of cancer cases diagnosed in the United States, which are reported by member facilities of the Commission on Cancer. These facilities are not limited to academic centers, with more than 50% of participating facilities representing community cancer programs or comprehensive community cancer programs.16 Criteria for case inclusion were clinical stage IV metastatic clear cell RCC at diagnosis, age between 18 and 100 years, availability of complete staging and demographic data, and receipt of either IT, TT, or combination TT and IT as first-line treatments. Patients in the TT and IT groups received those therapies alone. Cases were excluded if NCDB codes indicated that the patient was treated on an experimental or blinded clinical trial protocol.The year 2015 was chosen as the earliest date for the analysis because it was the first year of US Food and Drug Administration (FDA) approval for an ICB agent  for the treatment of metastatic clear cell RCC.20 Immunotherapy was defined as the receipt of a first-line systemic IT regimen. Immunotherapy is defined in the NCDB using the SEER*Rx Interactive Drug Database.21 Thus, patients in the IT cohort may have received non-ICB regimens, such as interferon α or high-dose interleukin 2. Combination TT and IT was defined as concurrent receipt of both systemic TT and systemic IT as first-line therapy. Age was defined as the age at initial diagnosis. Comorbidities were measured according to the Charlson-Deyo method and scored as discrete count categories  per NCDB reporting standards.23 Overall survival was measured from the date of initial diagnosis to the date of death or censorship at last follow-up.Consistent with previously peer-reviewed and published NCDB studies regarding TT use in RCC, TT was defined as the receipt of single or multiagent systemic chemotherapy.2 test of independence was used to compare normally distributed categorical variables, and the Kruskal-Wallis test was used to compare nonnormally distributed categorical variables.Statistical analysis was conducted from October 1 to December 1, 2020. Patients were stratified into groups based on receipt of TT, IT, or combination TT and IT as first-line treatment. Baseline patient and tumor characteristics were abstracted from the data set. Analysis of variance testing was used to compare continuous variables, the χ25 Baseline variables used to formulate propensity scores included age, sex, race/ethnicity, Charlson-Deyo score, facility type, insurance status, year of diagnosis, cT stage, cN stage, and cytoreductive nephrectomy status. Univariable analyses were repeated in the postmatching cohorts to assess for resolution of statistically significant differences between groups.Propensity score matching using a 1:1:1 nearest-neighbor match without replacement was performed with a caliper width of 0.2 SDs of the propensity score distribution, as previously described.P ≤ .10 as covariates in a multivariable Cox proportional hazards regression for OS and, if none of the variables met these criteria, to then perform a univariable Cox proportional hazards regression for OS stratified by treatment group. Two Cox proportional hazards regressions were used, 1 with TT as the reference in the therapy group and 1 with IT as the reference, to allow for 3 direct pairwise comparisons between treatment groups. A sensitivity analysis was then performed using a multivariable Cox proportional hazards regression for OS, including all patient and tumor demographic variables as covariates. Kaplan-Meier estimates were used to generate survival functions, and a log-rank test for equality of survivor functions was used.The a priori design for the survival analysis was set to include any variables from the postmatching univariable analysis with 26 All other analyses maintained the standard definition of statistical significance as a 2-tailed α risk of .05 or less. All statistical analyses and data visualization were performed using the R program, version 4.0.2 . Propensity matching was performed using the MatchIt package for R, and survival analyses were performed using the survival and survminer packages for R.The primary outcome involved 3 comparisons, and to reduce the risk of making a type I error, the definition of statistical significance for the Cox proportional hazards regression was adjusted using Bonferroni correction and defined as a 2-tailed α risk of .0167 or less.We identified 569 685 patients in the NCDB with a malignant neoplasm of the kidney or renal pelvis, and after application of the inclusion and exclusion criteria, the final study population included 5872 patients, with 4755 patients (81%) having received first-line TT , 638 patients (11%) having received first-line IT , and 479 patients (8%) having received first-line combination TT and IT  . A flow The incidence of treatment with TT decreased during the period studied , and treatment with either IT or combination TT and IT increased   (P = .08). The χ2 testing of the Schoenfeld residuals associated with the Cox proportional hazards regression model confirmed that the proportional hazards assumption was not violated  . With thP = .006; combination TT and IT group: HR, 0.76 ; P = .01). With the IT group as the reference, no significant difference in OS was seen for patients who received combination TT and IT .As a sensitivity analysis, a multivariable Cox proportional hazards regression for OS was performed including all patient and tumor demographic variables as covariates .CheckMate 214, a landmark study comparing the dual IT regimen ipilimumab plus nivolumab with sunitinib as first-line therapy for metastatic clear cell RCC, showed a significant OS benefit for patients receiving ipilimumab plus nivolumab.7 In our analysis, the comparison between combination TT and IT and TT confirmed this result .KEYNOTE-426, another landmark study comparing the combination TT and IT regimen axitinib plus pembrolizumab with sunitinib as first-line therapy for metastatic clear cell RCC, showed a significant OS benefit for patients receiving axitinib plus pembrolizumab.6 Because the NCDB does not report progression-free survival as an outcome or PD-L1 positivity as a pathologic variable, these specific outcomes could not be evaluated in our analysis.JAVELIN Renal 101 compared the combination TT and IT regimen axitinib plus avelumab with sunitinib as first-line therapy for metastatic clear cell RCC and did not show a significant OS difference between groups in the overall cohort; however, it did show a significant improvement in progression-free survival between groups in both the overall population and programmed death ligand 1 (PD-L1)–positive group.5 and KEYNOTE-4267 were significantly higher than those identified in our analysis, across all treatment groups , so any use of ICB during the period studied (2015-2017) was either via clinical trial protocol or off-label practice.30 Nevertheless, for this reason, the category is labeled IT and not ICB. Likewise, specific information is lacking regarding subsequent lines of therapy beyond the first-line treatment. Finally, the NCDB does not include data on kidney function, body mass index, performance status, treatment-related toxic effects, response rates, progression-free survival, recurrence-free survival, PD-L1 status, or tumor mutational burden, all of which would have contributed to this analysis if available for study.There are several limitations to this analysis. Primarily, there is an inherent risk of selection bias involved in retrospective comparative effectiveness studies. Despite nearest-neighbor caliper matching of propensity scores resulting in 3 groups without statistically significant differences in baseline demographic characteristics, the risk of selection bias due to unmeasured confounding will always exist in studies of this nature. In addition, the analysis was not designed to correct for immortal time bias regarding treatment effect on survival. Follow-up for the survival analysis was short . In addition, the NCDB broadly categorizes systemic therapies such that the names of the medications are not available. Thus, patients labeled as having received IT may have received non-ICB regimens, such as interferon α, high-dose interleukin 2, or potentially misclassified non–immune-modulating monoclonal antibodies . These non-ICB IT regimens are seldom used as first-line therapy in the postcytokine era, and given that 20% of patients in this cohort received an IT-based regimen, with increasing incidence over time, we are reassured that most of these patients likely received ICB therapy.This analysis of a nationally representative real-world cohort demonstrated that both IT and combination TT and IT were associated with improved OS for patients with metastatic clear cell RCC compared with TT alone. These findings imply the broader generalizability of previously reported clinical trial outcomes.


The concentration of cerebrospinal fluid total protein (CSF-TP) is important for the diagnosis of neurological emergencies. Recently, some Western studies have shown that the current upper reference limit of CSF-TP is quite low for older patients. However, little is reported about the concentration of CSF-TP in the older Asian population. In this study, we retrospectively analyzed the CSF-TP concentrations in healthy older Japanese volunteers. CSF samples in 69 healthy Japanese volunteers (age range: 55–73 years) were collected by lumbar puncture, and the data of CSF were retrospectively analyzed. The mean (standard deviation) CSF-TP was 41.7 (12.3) mg/dL. The older group (≥65 years old) had higher CSF-TP concentration than the younger group (55–64 years old). The 2.5th percentile and 97.5th percentile of CSF-TP were estimated as 22.5 and 73.2 mg/dL, respectively, which were higher than the current reference range in Japan (10–40 mg/dL). Conclusions: The reference interval of CSF-TP in the older population should be reconsidered for the precise diagnosis of neurological emergencies. Worldwide, the concentration of cerebrospinal fluid total protein (CSF-TP) is used for the diagnosis of various life-threatening neurological emergencies, such as infectious, inflammatory, and immune diseases of the central/peripheral nervous systems; thus, a valid reference interval of CSF-TP concentrations is crucial for precise diagnosis and treatment.The current gold standard, the reference range of 15–45 mg/dL (0.15–0.45 g/L) for lumbar CSF-TP dates back to 1938, and it has been adopted for a wide range of population for almost a century . HoweverHere, we revisit the CSF-TP levels in a healthy older population in Japan. In our facilities, CSF sampling in healthy volunteers has been performed as part of clinical trials. Therefore, we retrospectively analyzed the clinical laboratory data of CSF in healthy older Japanese volunteers and assessed whether the CSF-TP level linearly increased with age.This study was approved by SOUSEIKAI Hakata Clinic Institutional Review Board . This single-center retrospective study was designed to analyze the de-identified CSF laboratory data from October 2013 to March 2019 at SOUSEIKAI Mirai Hospital Clinical Research Center. The study was conducted in compliance with the Declaration of Helsinki and, Ethical Guidelines for Medical and Health Research Involving Human Subjects, Japan.All CSF sampling procedures were carried out as a part of four clinical trials from October 2013 to March 2019 at SOUSEIKAI Mirai Hospital Clinical Research Center. Each study protocol was reviewed and approved by SOUSEIKAI Hakata Clinic Institutional Review Board. All CSF samples were collected by a lumbar puncture by experienced in-house anesthesiologists after a thorough explanation of the procedure to the subjects and after written informed consent was given. The collection volumes of CSF samples were 6–12 mL depending on the study protocols. The participants were healthy Japanese adults with no existing neurological symptoms or signs, no abnormal laboratory findings that indicated inflammatory diseases, and no medical treatments including using medical drugs. The total protein concentration, glucose levels, and the cell numbers in the CSF were measured using the pyrogallol red method, glucokinase method, and Fuchs-Rosenthal method, respectively, at a commercial laboratory, LSI Medience Corporation .The results are shown as mean ± standard deviation (SD). The difference of CSF-TP concentrations between males and females and the difference between age groups (55–64 years vs. ≥ 65 years) were analyzed by the Wilcoxon rank-sum test. The lower and upper reference limits of the CSF-TP were estimated to be the 2.5th percentile and the 97.5th percentile, respectively . Accordit-tests. To be used as historical controls, the studies should have included younger study subjects and have data availability regarding the mean CSF-TP concentrations, SD, and the number of subjects.Our study population was a relatively older population and did not include the individuals younger than 55. Therefore, in the supplementary analysis, we compared our CSF-TP concentrations to previous published data ,12,16,17p-values < 0.05 were considered as statistically significant. Statistical analyses were performed using JMP Pro 15  and Graph Pad Prism 9 .Two-tailed The data of 69 Japanese healthy older volunteers were analyzed. Thirty-seven (54%) were male. The age range was 55–73 years, and the mean age was 62.8 ± 5.2 . The mean CSF-TP concentration in all participants was 41.7 ± 12.3 mg/dL, which is higher than the general upper reference limit in Japan (40 mg/dL) . The disp = 0.08]  = 0.51 (−0.06 to 1.07),  = 0.08] C, the ol = 0.08] . In the  = 0.08] .The mean glucose value in CSF was 59.9 ± 5.5 mg/dL , which iIn the present study, the 2.5th and 97.5th percentiles of CSF-TP concentration in the Japanese older population was higher than the current lower and upper reference limit respectively in Japan, and the older population had a higher CSF-TP concentration than the younger population.CSF protein concentration is known to increase in various clinical situations such as a tumor, inflammation, bleeding, or injury in the central nervous system. It is also well accepted that the protein concentration in the CSF is correlated with serum protein concentrations, reduced CSF turnover, the blood–brain barrier (BBB), or blood–CSF barrier functions ,19,20,21In this study, to investigate the CSF-TP levels in healthy older volunteers in Japan, we collected and reviewed CSF-TP data from 69 healthy Japanese volunteers aged 55 to 73 years old from clinical study data. From the analysis, we found that both the 2.5th and 97.5th percentiles of CSF-TP concentration in the study population was higher than that of the general lower and upper reference limits in Japan . Forty five percent of our population had higher CSF-TP concentration than the general upper reference limit in Japan. Even using an upper reference limit of 45 mg/dL which has been used worldwide, 31% of the volunteers had higher CSF-TP concentrations, which is consistent with the previously mentioned report from a group of Western older adults ,9. Some The concentrations of CSF-TP are known to differ according to the neuroaxis ,10,13. AMales have been reported to have a slightly higher CSF-TP concentration than females ,16. Our This study has some limitations. First, the number of volunteers we analyzed was relatively small compared with previous extensive studies ,3,4,6. HOne of this study’s strengths is that our data are based on the data from healthy volunteers who had no existing neurological symptoms, abnormal laboratory findings, or medical treatments, although neurological diseases had not been excluded by imaging tests such as magnetic resonance imaging. Due to the invasiveness and the potential risks following the CSF sampling, it is challenging to collect CSF samples in healthy volunteers when they have no clinical symptoms. Thus, most reports on CSF data were based on patients who needed to undergo CSF examinations or lumbar anesthesia. Although patients who had neurological diseases had been excluded from the analyses when published , there mThe current reference limits of CSF-TP, particularly the upper reference limit, may not be reflecting the physiological conditions of older adults. Re-evaluating the reference interval of CSF-TP for older populations should be considered to increase the precision of diagnosing neurological emergencies, as well as to safely assess clinical study data.


Effective anti-infective strategies are of crucial importance in patients with multiple myeloma undergoing high-dose therapy followed by autologous stem cell transplantation (HDT/ASCT). We compare, for the first time, antibiotic prophylaxis versus granulocyte-colony stimulating factor (G-CSF) support as anti-infective strategies in this specific setting, including 353 individual cases of HDT/ASCT. We show similar efficacy in preventing infectious complications regarding antibiotic prophylaxis and G-CSF. Furthermore, we demonstrate that G-CSF support is associated with a shorter duration of inpatient stay and a lower rate of emerging multidrug resistant bacteria, especially vancomycin-resistant Enterococcus faecium. Therefore, G-CSF support should be the preferred anti-infective strategy in patients with multiple myeloma receiving HDT/ASCT.p < 0.001, hazard ratio (HR) = 16.22), and hospitalization  compared to antibiotic prophylaxis. Rates of febrile neutropenia, need of antimicrobial therapy, transfer to intensive care unit, and death, were similar between the two groups. Furthermore, antibiotic prophylaxis was associated with a significantly increased risk for the development of multidrug resistant bacteria especially vancomycin-resistant Enterococcus faecium compared to G-CSF support (odds ratio (OR) = 17.38, p = 0.01). Stop of antibiotic prophylaxis as an anti-infective strategy was associated with a reduction in overall resistance rates of bacterial isolates. These results indicate that G-CSF support should be the preferred option in MM patients undergoing HDT/ASCT.We compare, in this manuscript, antibiotic prophylaxis versus granulocyte-colony stimulating factor (G-CSF) support as anti-infective strategies, in patients with multiple myeloma (MM), undergoing high-dose therapy followed by autologous stem cell transplantation (HDT/ASCT). At our institution, antibiotic prophylaxis after HDT/ASCT in MM was stopped in January 2017 and replaced by G-CSF support in March 2017. Consecutive MM patients who received HDT/ASCT between March 2016 and July 2018 were included in this single-center retrospective analysis. In total, 298 patients and 353 individual cases of HDT/ASCT were evaluated. In multivariate analyses, G-CSF support was associated with a significantly shortened duration of severe leukopenia < 1/nL ( In patients with multiple myeloma (MM), infections are a leading cause of morbidity and mortality. Compared to the general population, the risk of developing an infection is increased seven-fold, attributable to the malignant disease itself and the administered therapy . DiseaseThe administration of antibiotic prophylaxis, mainly fluoroquinolones, following HDT/ASCT was shown to decrease the risk of febrile neutropenia and septicemia ,7,8. HowThe application of granulocyte-colony stimulating factor (G-CSF) after HDT/ASCT represents an alternative anti-infective strategy. G-CSF shortens the duration of neutropenia, but no consistent clinical benefit in this specific setting, in terms of development of febrile neutropenia, infections, and need of empirical antimicrobial therapy, could be demonstrated so far ,13,14,15To our knowledge, no study directly comparing antibiotic prophylaxis versus G-CSF support as anti-infective strategies, after HDT/ASCT in MM, has been published so far. Therefore, the aim of the present study was to compare antibiotic prophylaxis with ciprofloxacin or cotrimoxazole versus G-CSF support, with respect to blood count recovery, infectious complications, and emerging MDR bacteria in patients with MM, undergoing HDT/ASCT.pneumocystis jirovecii pneumonia (PCP) with cotrimoxazole thrice weekly was implemented, beginning in March 2017. All patients received antiviral prophylaxis against herpes simplex and varicella zoster virus reactivation with acyclovir. Patients with prior hepatitis b infection (anti-HBc antibody positive) received antiviral prophylaxis with lamivudine, entecavir, or tenofovir. Detailed information about the anti-infective strategies during the period March 2016 to July 2018 are shown in At our institution, a tertiary referral and transplant center, antibiotic prophylaxis with daily ciprofloxacin or cotrimoxazole after HDT/ASCT in MM was stopped in January 2017, and replaced by G-CSF support with filgrastim in March 2017. Prophylaxis against 2, or dose-reduced in case of renal insufficiency followed by ASCT, as inpatients at our institution between March 2016 and July 2018, were included into this single-center retrospective analysis. Patients receiving HDT/ASCT as part of the first-line treatment or at relapse were included. In the case of tandem HDT/ASCT, both HDT/ASCT were evaluated separately. MM patients who were transplanted in an outpatient setting were excluded.Consecutive MM patients who received HDT with melphalan, 200 mg/mData were collected by review of medical records regarding duration of leukocytes <1/nL, duration of platelets <20/nL, need of platelet transfusions, duration of inpatient stay, development of mucositis, febrile neutropenia and infections, detection of relevant pathogens, need of empirical or targeted antimicrobial therapy, new detection and type of MDR bacteria, transfer to intermediate or intensive care unit, death, hospital readmission after discharge, and response after ASCT. The duration of platelets <20/nL was defined by the time from the first day when platelets declined <20/nL to the first day when platelets rose ≥20/nL, regardless of administered platelet transfusions. The number of neutrophil granulocytes are not assessed at our institution in patients with leukocytes <1/nL. Therefore, the development of febrile neutropenia was defined by leukocytes <1/nL plus the occurrence of fever (ear temperature once ≥38.3 °C or ≥38.0 °C measured twice within twelve hours) [Data regarding the absolute number of blood cultures sent for microbial analyses and the number of positive blood cultures, as well as the resistance rates to amoxicillin/clavulanate, cefuroxime axetil, ciprofloxacin, and cotrimoxazole were extracted from the database of the laboratory information system , retrospectively. All bacterial species tested for the respective antimicrobial substance were included in the analyses to calculate overall resistance rates. Equally, data were extracted regarding isolates and resistance rates from all tested materials.Enterococcus faecium (VRE). The impact of the strategies (I)–(III) was adjusted for age at ASCT, ASCT at relapse versus ASCT in first-line treatment, response before ASCT, and transfused stem cell amount.The collected data of the HDT/ASCT inpatient stay were analyzed regarding differences between the conducted anti-infective strategies using the chi-square test. Multivariable mixed effect models were applied to consider potentially repeated measurements due to tandem ASCT per patient. Multivariable mixed effect Cox regression analyses were conducted to assess the impact of the regimes (I)–(III) on the duration of leukocytes <1/nL and platelets <20/nL. The Cox proportional hazards model was applied to allow patients who were released before leukocytes were ≥1/nL or before platelets were ≥20/nL and, therefore, were censored at the end date of inpatient stay. Furthermore, there were no competing events during the observation period. The impact of the anti-infective strategies on the duration of inpatient stay was assessed by multivariable linear mixed effects regression analysis. Furthermore, a multivariable mixed effects logistic regression approach was applied to analyze the effect of the anti-infective strategies on the need of carbapenems, and a new detection of vancomycin-resistant Cases of death within 30, 100, and 180 days after ASCT in the three groups of anti-infective strategy were assessed and respective mortality rates calculated.(I)Antibiotic prophylaxis: median duration of inpatient stay in days x standard rate per day on general ward (EUR 450) + median duration of inpatient stay in days x costs ciprofloxacin/cotrimoxazole per day (EUR 0.12/EUR 0.14).(II)G-CSF support: median duration of inpatient stay in days x standard rate per day on general ward (EUR 450) + duration from ASCT to leukocytes ≥1/nL in days x costs G-CSF (EUR 5.75) + median duration of inpatient stay in days x cost cotrimoxazole (EUR 0.07)/2.A cost analysis was conducted examining the costs of an ASCT inpatient stay depending on the application of antibiotic prophylaxis or G-CSF. Because expensive drugs as reserve antibiotics were not needed during the inpatient stay in our study cohort and transfers to the intermediate or intensive care unit were similar in the groups, the cost analysis was simplified to the costs of antibiotic prophylaxis or G-CSF and the standard rate on general ward per day. These rates include especially costs for medical personnel, equipment and materials, food and laboratory examinations, and are around EUR 450 per day. For the costs of ciprofloxacin, cotrimoxazole, and G-CSF the purchase price our institution had to pay in June 2021 was used. For G-CSF, the dose for an adult weighing up to 70 kg was used. Costs of inpatient stay were determined as:www.r-project.org/ accessed on 15 April 2021). Figures regarding resistance rates of blood culture and all tested isolates were done with GraphPad Prism Version 5.0 .All results were assessed on the 5% significance level. R version 3.6.2 was used for performing the statistical analysis  antibiotic prophylaxis in 151 HDT/ASCT and (II) G-CSF support in 150 HDT/ASCT. As antibiotic prophylaxis, ciprofloxacin was administered in 60 (39.7%), cotrimoxazole daily in 86 (57.0%), and another antibiotic prophylaxis in five (3.3%) cases. In 52 HDT/ASCT, (III) no prophylaxis was applied.2 was administered in >90% of patients in all groups. Further characteristics at HDT/ASCT in the entire cohort and in the groups (I)–(III) are shown in The median age at HDT/ASCT was 61 years. The standard melphalan dose of 200 mg/mp < 0.001, hazard ratio (HR) = 16.22, 95% confidence interval (95% CI) = 10.88–24.18; p < 0.001, estimate = −0.19, 95% CI = −0.25–−0.12; The median time from leukocytes <1/nL to ≥1/nL was 9 days in patients receiving (I) antibiotic prophylaxis compared to 6 days in patients receiving (II) G-CSF support. In multivariate analyses, G-CSF support was associated with a significantly shortened time to leukocytes ≥1/nL compared to antibiotic prophylaxis (p = 0.15). Mucositis was present in (I) 90.1% (136/151) and (II) 90.0% (135/150).The rate of febrile neutropenia was similar in the groups (I) antibiotic prophylaxis  and (II) G-CSF support . The most common infection in both groups was FUO ((I) 76/151; 50.3% versus (II) 80/150; 53.3%). Respiratory infections were slightly more frequent in patients receiving (I) antibiotic prophylaxis  compared to (II) G-CSF support . Rates of septicemia were similar ((I) 22/151; 14.6% and (II) 25/150; 16.7%) A. There Escherichia coli, Clostridioides difficile, Pseudomonas aeruginosa, influenza virus, and parainfluenza virus. In total, four patients developed PCP, two patients  who had received prophylaxis with cotrimoxazole and two patients  who had not received prophylaxis.During the inpatient stay, a relevant pathogen was detected in blood, urine, or stool culture, bronchoalveolar lavage, or smear of the throat or another region in (I) 27.1% (41/151) and (II) 28.7% (43/150). The most common pathogens were p = 0.54; The need for empirical or targeted antimicrobial therapy was similar in the groups (I) antibiotic prophylaxis and (II) G-CSF support B. MultivA transfer to the intermediate or intensive care unit was a rare event with (I) eight (5.3%) versus (II) nine (6.0%) patients. Two patients died during the inpatient stay, one in the group (I) antibiotic prophylaxis and one in the group (II) G-CSF support, leading to an overall fatality rate of 0.6%. One patient died due to pulmonary sepsis, the other patient due to sepsis and paralytic ileus.Pseudomonas aeruginosa and VRE plus Escherichia coli with combined resistance to third-generation cephalosporins and fluoroquinolones) were observed. Three MDR bacteria led to a clinically relevant infection. Two patients developed septicemia due to Escherichia coli and one patient developed urinary tract infection due to VRE. In multivariate analyses, antibiotic prophylaxis was associated with a significantly increased risk for the acquisition of VRE compared to G-CSF . However, comparing the total duration of inpatient stay (duration of inpatient stay plus duration of a possible readmission), G-CSF support was still associated with a significantly shortened total duration of inpatient stay compared to antibiotic prophylaxis . Patients receiving (III) no prophylaxis more frequently required antibiotic treatment, especially with carbapenems  69/151; 45.7% and (II) 65/150; 43.3%)  no prophylaxis after HDT/ASCT at our institution. The rate of febrile neutropenia was higher in group (III) no prophylaxis  compared to (I) antibiotic prophylaxis  and (II) G-CSF support . However, in multivariate analyses, the impact was not statistically significant  B. Multivn = 1) and (II) 1.33% (n = 2). The 180-day mortality rate was (I) 0.66% (n = 1) and (II) 3.33% (n = 5). There were no cases of death in group (III). Due to low numbers of deaths, only descriptive analyses were conducted.At the first regular follow-up outpatient visit (median 46 days after ASCT), response was ≥very good partial remission in (I) antibiotic prophylaxis 74.0%, (II) G-CSF support 74.5% and (III) no prophylaxis 76.9%. In total, six patients died within 180 days after ASCT. Within 30 days after ASCT, one case of death occurred in group (I) . The 100-day mortality rate was (I) 0.66% . Because transfers to the intermediate or intensive care units were similar in the groups, the associated costs were not considered as well as the costs of very rare hospital readmissions. In our simplified cost analysis, median costs of inpatient stay in patients receiving antibiotic prophylaxis with ciprofloxacin was EUR 8552 compared to EUR 8553 in patients receiving cotrimoxazole. The median cost per inpatient stay in the G-CSF support group was EUR 7708 €.The resistance rates of blood culture isolates, as well as of all tested isolates to amoxicillin/clavulanate, cefuroxime-axetil, ciprofloxacin, and cotrimoxazole in the entire hematology department and transplant unit, are shown in To our knowledge, this is the first study comparing the anti-infective strategies—antibiotic prophylaxis versus G-CSF support after HDT/ASCT in a large cohort of MM patients. Antibiotic prophylaxis and G-CSF support showed similar rates of febrile neutropenia, pathogen detection, need of antimicrobial therapy, transfer to intermediate or intensive care unit, and death, reflecting equal efficacy in preventing infectious complications. In contrast, G-CSF support enables a lower rate of MDR bacteria and a shorter duration of inpatient stay, accompanied with lower costs.At this time, there are very few studies comparing G-CSF support and antibiotic prophylaxis in cancer patients undergoing chemotherapy. Recently, a prospective randomized trial was presented at ASCO 2020, comparing ciprofloxacin prophylaxis with G-CSF support during adjuvant chemotherapy in patients with early stage breast cancer, showing lower rates of febrile neutropenia and a trend for reduced hospitalizations in patients receiving G-CSF . A systeThere is no study available comparing antibiotic prophylaxis with G-CSF support in the context of HDT/ASCT in MM. Previously published studies show comparisons of antibiotic prophylaxis plus G-CSF vs. antibiotic prophylaxis or G-CSF and antibiotic prophylaxis vs. control ,12,14,25Our analysis suggests similar efficacy of antibiotic prophylaxis and G-CSF support in preventing infections. Previously published studies investigating antibiotic prophylaxis plus G-CSF support in MM patients receiving HDT/ASCT show lower rates of febrile neutropenia and septicemia, reflecting that this strategy might be even more effective ,11. HoweG-CSF support enables a shortening of the duration of severe neutropenia and hospitalization ,13,15. TSince the expiry of patent protection, prices for G-CSF have dropped significantly. Large hospitals being able to buy in bulk nowadays pay less than EUR 10 per daily dose of G-CSF for a normal weight adult. In contrast, hospital bed occupancy costs, including  medical personnel, equipment and materials, food, etc., regularly run up to several hundred euros per day. Since highly expensive antimicrobial drugs, such as reserve antibiotics, were not required in our study cohort during the inpatient stay, and cost-intensive transferals to intermediate or intensive care units were very rare events occurring at similar frequency in the G-CSF and antibiotic prophylaxis groups, our cost analysis was simplified to assess the costs of inpatient hospital days plus administered G-CSF versus antibiotic prophylaxis. Our cost analysis demonstrates that the total costs of an inpatient stay are mainly determined by the duration of inpatient stay. Therefore, a shortening of inpatient stay due to application of G-CSF is accompanied with cost reduction.We were further able to demonstrate that there is an increased risk of acquiring VRE in patients receiving no prophylaxis that is even similar to patients receiving antibiotic prophylaxis and might be explained by the high necessity of empirical or targeted antibiotic therapy. Infections caused by MDR bacteria constitute a worldwide threat. According to a recent analysis of the European Antimicrobial Resistance Surveillance Network data, there were 671,689 infections with antibiotic-resistant bacteria detected in countries of the EU and the European Economic Area in 2015, leading to 33,110 cases of death . CompareOur evaluation of resistance rates of bacterial isolates in the entire hematology department and transplant unit showed extremely high resistance rates, questioning the benefit of antibiotic prophylaxis. Furthermore, our analysis is able to confirm the association between antibiotic use and development of resistance. While omitting antibiotic prophylaxis with ciprofloxacin or cotrimoxazole led to a reduction of resistance rates, resistance against cotrimoxazole rose again after wide reintroduction as PCP prophylaxis.In fact, in our analysis, G-CSF support reduced the risk of emerging MDR bacteria compared to antibiotic prophylaxis. Similarly, Maakaron et al. were able to demonstrate significant higher rates of MDR bacteria in MM patients receiving levofloxacin plus filgrastim after HDT/ASCT compared to lymphoma patients receiving only filgrastim after HDT/ASCT .Antibiotic prophylaxis not only increases the risk of acquiring MDR bacteria, but also impacts the balance of our gut microbiota. Because there is an intensive interaction between our microbiota and immune system, a dysbiosis of the microbiota affects our immune system, too. For example, gut bacteria produce short-chain fatty acids that are able to down-regulate the NF-κB pathway, leading to an inhibition of tumor progression. Consequently, a dysbiosis of the gut microbiota due to antibiotic prophylaxis may impact response to cancer treatment and patient outcome . In our Our study has some limitations, including its retrospective nature and consecutive minor heterogeneity regarding baseline characteristics between the groups of anti-infective strategies. Furthermore, HDT/ASCT without prophylaxis were conducted from January to March 2017 during influenza season, which might be a confounder. A major strength of our study, however, is the large cohort size of consecutively and uniformly treated patients.To conclude, antibiotic prophylaxis and G-CSF support have similar efficacy in preventing infectious complications following HDT/ASCT in MM. Furthermore, G-CSF support enables a lower rate of emerging MDR bacteria and a shorter duration of inpatient stay, accompanied with cost reduction. Therefore, G-CSF support might be a preferable anti-infective strategy in MM patients receiving HDT/ASCT. Prospective randomized controlled trials are warranted to help identify optimal strategies to prevent infectious complications in these patients.


Cryptococcal infections of the central nervous system (CNS) are common opportunistic infections in immunocompromised hosts. They can occur in immunocompetent hosts, and this has been documented in isolated case reports. Rapid neurological deterioration can be seen, particularly with hydrocephalus, and diagnosis can be difficult without a high index of suspicion. Treatment arms include prolonged antifungal therapy and cerebrospinal fluid (CSF) diversion procedures.We present a case of a middle-aged immunocompetent male, who presented with an acute confusional state and papilledema. An urgent computed tomography (CT) and magnetic resonance imaging (MRI) revealed obstructive hydrocephalus, and an external ventricular drain was placed. CSF samples were collected, and analysis revealed cryptococcal infection. He was treated with antifungal therapy but failed external ventricular drain challenging. A ventriculoperitoneal shunt was placed after negative CSF studies were obtained.While uncommon, cryptococcal meningitis in immunocompetent hosts can present with obstructive hydrocephalus. It can result in rapid neurological decline and death. Emergent CSF diversion and antifungal therapy are the primary treatment modalities. CSF diversion may be permanently required in some cases. Cryptococcal infections of the central nervous system (CNS) are common opportunistic infections in immunocompromised hosts, particularly in the tropics . It typiWe discuss an immunocompetent patient who presented with signs of intracranial hypertension and hydrocephalus, with investigations revealing cryptococcal meningitis. The need for permanent CSF diversion needs to be tailored to each patient individually, and we report on our experience in managing one such case.A 57-year-old male presented to the emergency department with a progressively worsening confusional state and headaches for three days duration. His relatives noted that his memory had dramatically worsened over the previous 24 hours, and his consciousness was fluctuating. He had no fever, vomiting, or seizures. Past medical history was negative for immunosuppression or any significant comorbidities. He did not have a history of bird handling nor prolonged exposure to birds.Physical examination revealed a lethargic appearing patient, with a Glasgow Coma Scale (GCS) of 13 (E3 M6 V4). His pupils were 4 mm bilaterally and sluggishly reactive to light. Cranial nerve examination was grossly intact, but papilledema was noted on fundoscopy. Motor and sensory examination of his limbs were normal aside from brisk reflexes (3+). He had no clonus and both plantars were downgoing.A brain computed tomography (CT) scan was done which revealed acute hydrocephalus. This was immediately followed by a magnetic resonance imaging (MRI) of the neuraxis, which revealed dilation of all ventricles and stenosis at the floor of the fourth ventricle Figure . HyperinThe patient consented to immediate CSF diversion via an external ventricular drain, and CSF samples were sent for analysis. The patient had immediate improvement postoperatively, with the restoration of GCS to 15/15, and recovery of memory. His headaches ceased and there was no further papilledema. Postoperative CT showed resolution of hydrocephalus and good placement of the external ventricular drain (EVD).3 , with neutrophilia being predominant. PCR testing for cryptococcus neoformans was positive, consistent with cryptococcal ventriculitis. Quantitative cryptococcal antigen titres were not available in our setting. The patient’s serum white cell count was 14,000/ml , and his C-reactive protein (CRP) was elevated at 48 mg/L . HIV testing was negative.CSF analysis revealed glucose of 23 mg/dL , with a concurrent serum glucose of 102 mg/dL . The CSF protein was 350 mg/dL , and CSF white cell count was elevated at 480/mm3.Antifungal therapy was commenced, with amphotericin 50 mg IV once daily and fluconazole 80 mg IV once daily. This was continued for four weeks until repeat CSF studies showed resolution of infection, with a negative PCR test for cryptococcus and normal parameters of glucose, protein, and white cell count. The EVD was kept in situ during this time and handled with an aseptic technique to avoid contamination. CSF studies were performed every 48 hours to ensure that no additional infection developed. After 14 days, repeat CSF studies were negative for cryptococcus, and the CSF glucose was 75 mg/dL (serum 99 mg/dL), CSF protein was 35 mg/dL and the CSF leucocytes were 2/mmAt this time, the EVD was challenged, by clamping it for a period of 24 hours and assessing the patient’s tolerance of this. After eight hours, he became disoriented to time and place, and a CT scan revealed hydrocephalus. This confirmed both clinical and radiological evidence of a lack of ventricular compliance. A decision was made for ventriculoperitoneal shunting as the definitive method of CSF diversion, and this was performed successfully. While endoscopic third ventriculostomy may have been an option, in this case, that procedure is not offered in our setting due to lack of equipment. The patient recovered neurologically and was discharged on long-term antifungal therapy with fluconazole for a one-year duration. At a three-month follow-up, he remains neurologically intact, with no evidence of intracranial hypertension.The causes of hydrocephalus are multiple and varied, and fungal meningitis is an occasional etiological factor. Cryptococcal meningitis is an opportunistic infection that is known to primarily affect immunocompromised patients. It is the most common cause of meningoencephalitis in sub-Saharan Africa and Southeast Asia, with mortality rates ranging from 20% to 60% . Up to 2PathophysiologyCryptococcus neoformans is responsible for the majority of infections and tends to cause more severe disease, whereas C. gattii is associated with a milder clinical course [There are two main species of cryptococci that affect humans. l course . It is il course .Hydrocephalus can arise from impaired CSF absorption and outflow when there is the involvement of the arachnoid villi. Fungal polysaccharide accumulates in these villous spaces, and further contribute to this . AdditioIntracranial hypertension due to cryptococcal meningitis has been recorded in both immunocompetent and immunodeficient patients. The exact mechanism of this is unclear, and one series by Cherian et al. did not discover any predisposing factors. Ventricular morphology was not related to the development of intracranial hypertension .Clinical presentationThis is consistent with signs and symptoms of infection and intracranial hypertension. These may include fever, cognitive impairment, gait disturbance, headaches, vomiting, papilledema, seizures, and decreased consciousness ,6. It isEvaluationInitial assessment should be with a non-contrast CT scan of the brain to confirm hydrocephalus. This should be followed by an MRI of the brain to determine whether the hydrocephalus is obstructive or communicating and if a lumbar puncture is contraindicated. Along with hydrocephalus, cryptococcomas, nodules, dilated perivascular spaces, pseudocysts, and enlarged choroid plexuses can be seen . ConfirmManagementThere are two major arms of management - antifungal therapy and CSF diversion. Antifungal therapy is usually commenced with amphotericin B and fluconazole, for a minimum of two weeks. The goal is three consecutive negative CSF studies . Oral flEVD has the advantage of the ease of CSF sampling and the capability to challenge the ventricles and avoid shunting. However, they may remain in situ for lengthy periods and cause superimposed ventriculitis, as well as be misplaced, malpositioned, and require repeated trips to the operating theater. Early VP shunting reduces these risks but may not guarantee a superior outcome ,10. The OutcomesThe timing of CSF diversion is dependent on the patient’s symptomology. It was noted by Tang that patients who presented with acute signs and symptoms of hydrocephalus had better outcomes when shunted, compared to those who presented with symptoms for over a week’s duration ,12. AnotCryptococcal meningitis in immunocompetent patients remains a rare phenomenon. It can lead to obstructive hydrocephalus, intracranial hypertension, and acute neurological deterioration, resulting in death if not rapidly treated. CSF diversion in the form of serial lumbar punctures, external ventricular drainage, endoscopic third ventriculostomy, and VP shunting have all been shown to be successful in these patients. Further series with larger patient numbers need to be done, to assess the best management protocols for these patients.


We report a child with Fanconi anemia who, after hematopoietic stem cell transplantation (HSCT) complicated by acute graft-versus-host disease (GVHD), underwent orthotopic liver transplantation (OLT). Approximately 1 month after OLT, the presence of third-party genetic material from the liver donor was noted and in the next few weeks, the chimerism assessment revealed 100% liver donor leukocytes in the peripheral blood. The rapidly progressing GVHD with gut involvement resulted in patient’s death 6 months after OLT. The liver can act as a clinically significant source of hematopoietic stem cells, and the liver donor’s young age must be emphasized as potentially predisposing to this phenomenon. Transfer of OLT hematopoietic stem cells may not have clinical significance unless the patient is not immunocompetent or develops liver-transplantation associated GVHD, that can result in lymphocyte mediated elimination of original hematopoiesis. Patients with preexisting immunity disorder (such as primary or secondary immunodeficiency) might require intensified immunosuppressive therapy in peritransplant period as a prevention of liver-transplantation associated GVHD. Close monitoring of hematopoietic chimerism after OLT is warranted in patients at risk, because cytopenia or OLT hematopoiesis can reflect subclinical GVHD and further studies are necessary to elucidate this phenomenon. In patients with end-stage liver failure due to hepatic graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), orthotopic liver transplantation (OLT) is the therapy of choice. OLT for this indication is very rare, and since the first attempts, only approximately one hundred cases have been described in adults and children –4. AmongAt the time of the study, mononuclear cell (MNC) and T-lymphocyte chimerism was evaluated. The MNC isolation procedure employed from blood or bone marrow was a Ficoll-Hypaque gradient. Ficoll-Hypaque solution has a specific gravity of 1.077 at room temperature and is denser than lymphocytes, and monocytes, but less dense than granulocytes and RBCs, allowing for the successful separation of these cell populations. T-lymphocytes were enriched by positive selection using a magnetic cell separation technique with CD3 magnetic beads and MiniMACS separator  according to manufacturer instruction. All results of posttransplant chimerism studies were based on short tandem repeat fragment analysis of variable number short nucleotide repeats using the AmpFlSTR SGM PLUS amplification kit  with a sensitivity of 1%.2, cyclophosphamide 2 × 20 mg/kg b.w. and anti-thymocyte globulin  3 × 20 mg/kg b.w., and GVHD prophylaxis consisted of cyclosporin A (CsA) and mycophenolate mofetil (MMF). The posttransplant course was uneventful with typical but mild complications . Successful engraftment with complete donor chimerism was observed on posttransplant day +12. The patient was discharged home on day +28 but was readmitted 3 days later with BK virus-related hemorrhagic cystitis and cutaneous manifestation of microangiopathy. Antiviral treatment with cidofovir was administered, and CsA was stopped. The girl developed grade III acute GVHD with skin, gut and liver involvement and was treated with corticosteroids and MMF. Despite a good response in the skin and gut, liver injury was observed, with elevation of aminotransferases  and gamma-glutamyl transpeptidase activity  and jaundice with a maximum total bilirubin concentration of 29.7 mg/dl.A 13-year-old girl with Fanconi anemia was referred for allogeneic HSCT from 10/10 HLA-matched unrelated donors due to bone marrow failure. The patient was prepared according to the modified GEFA03 protocol: busulfan 4 × 0.5 mg/kg b.w., fludarabine 6 × 30 mg/mMultimodal immunosuppressive treatment with etanercept, basiliximab, ruxolitinib, plasmapheresis and extracorporeal photopheresis (ECP) was administered, but laboratory results showed that liver failure had progressed. Liver biopsy revealed vanishing bile duct syndrome – a typical histological picture related to end-stage liver GVHD. In view of the biopsy findings and Model of End-Stage Liver Disease (MELD) score of 19 points, the patient was placed on the liver transplant list. Eleven months later and 16 months after HSCT, she underwent a liver transplant from a deceased donor. Shortly after OLT, CMV replication was observed, and the patient was treated with ganciclovir, foscarnet and human cytomegalovirus immunoglobulin. Approximately 1 month after OLT, trilineage cytopenia with severe neutropenia not responding to filgrastim was noted. Bone marrow (BM) biopsy revealed aplasia with erythrophagocytosis, and secondary hemophagocytic syndrome related to CMV infection was initially suspected. The patient was transferred to the stem cell transplantation unit for hematological treatment. At that time, decreased bone marrow donor chimerism was recorded, and the patient was treated for 3 days with ATG, at a cumulative dose of 60 mg/kg b.w.In hematopoietic chimerism studies, the presence of third-party genetic material was noted, and using frozen liver donor lymph node samples, the new genetic profile was identified to be compatible with the liver donor material. HLA typing confirmed complete HLA incompatibility between the patient  and the liver donor .The first chimerism measurement after ATG therapy revealed 78% liver donor MNCs in the peripheral blood, and the percentage increased up to 100% in the next few weeks. Bone marrow chimerism revealed 36% HSCT donor cells and 64% OLT donor cells and single chimerism assessment in the nucleated cells showed 84% OLT donor cells .6 cells/kg b.w. were infused without conditioning as a stem cell boost but without any effect. One week later, the patient experienced severe abdominal pain with vomiting and bloody diarrhea, hyperbilirubinemia and elevated GGTP activity. Treatment with oral budesonide capsules, intravenous methylprednisolone at a dose of 2 mg/kg b.w. and etanercept was administered, but due to rapid clinical progression, basiliximab and ECP therapy were added. Endoscopic examinations revealed diffuse esophagitis, erosive gastritis and duodenitis with histological confirmation of acute GVHD. The rapidly progressing signs of GVHD with gastrointestinal tract functional obstruction led to the performance of surgical gastrojejunal anastomosis with the Roux-Y loop, which caused partial but transient improvement. As a consequence of fulminant GVHD progression with multiorgan failure, the patient died 21 months after HSCT.Due to a continuous decrease in HSCT donor chimerism and progressive hematopoietic failure, 3 months after OLT, CD34-enriched peripheral blood stem cells from the same BM donor at a total of 5.3 × 10Liver transplantation is feasible in end-stage liver disease HSCT survivors, but is associated with worse outcome than in general population. According to the multicenter survey conducted by Burdick et al. in patients after hematopoietic stem cell transplantation in childhood, the overall survival rate at 1 and 5 years after OLT was 74 and 63.2%, respectively (9–1010 cells)  . Liver tThe unusual clinical course in the reported patient was associated not only with liver donor GVHD but also with expansion of liver donor hematopoiesis, revealing an unknown phenomenon in which the mature liver can act as an organ containing hematopoietic stem cells. To fully understand the meaning of reported chimerism values it must be noted, that the MNC product of Ficoll-Hypaque isolation from healthy individuals contains on average 30% monocytes and 60–70% lymphocytes . LymphocIt can be hypothesized that both transfer and durable engraftment of OLT donor hematopoiesis were caused by a combination of a few uncommon factors. First, the transplant recipient was in a state of profound immunosuppression, which helped the passenger lymphocytes eliminate the recipient’s hematopoiesis and prevented the recipient’s immune system from eliminating the liver-derived hematopoietic stem cells. Second, the liver was collected from a 10-year-old donor, which might have had a physiologically higher ability to shield and transfer hematopoietic stem cells. The liver is the most important blood-producing organ in fetal life, with hematopoietic activity expiring at birth, except for rare pathological conditions, such as osteopetrosis or osteomyelofibrosis. Nevertheless, it is probable that the liver environment can act as a temporary niche harboring hematopoietic stem cells even without the ability to provide a functional hematopoietic site. There are scanty and historical data regarding the presence of such cells in an adult liver and of their reactivation after liver transplantation , 13. OnlThis case unravels the fact that the liver can act as a clinically significant source of hematopoietic stem cells, and the liver donor’s age factor must be emphasized as potentially predisposing to the expansion of liver donor hematopoiesis. Transfer of OLT hematopoietic stem cells may not have clinical significance unless the patient is not immunocompetent or develops liver-transplantation associated GVHD, that can result in lymphocyte mediated elimination of original hematopoiesis. Patients with preexisting immunity disorder (such as primary or secondary immunodeficiency) might require intensified immunosuppressive therapy in peritransplant period as a prevention of liver-transplantation associated GVHD. This stands in opposition to the recommendation on minimizing immunosuppression maintenance, suggested by Brockmann et al. but intensive immunosuppression should be used until the risk of transplantation associated GVHD subsides . AccordiThe datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.TJ and MR: data collection, analysis, and manuscript preparation and acceptance. BR, RR-K, KG, and KB-K: data collection, molecular studies, and manuscript acceptance. MK-D, PC, PK, JT, MS, EG, and KK: data collection, patient care, and manuscript acceptance. MU: concept, analysis, and final acceptance. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Capillary self-alignment technique can achieve highly accurate and fast alignment of micro components. Capillary self-alignment technique relies on the confinement of liquid droplets at receptor sites where hydrophobic–hydrophilic patterns are widely used. This paper reports a low-cost microsecond pulse laser micromachining method for fabrication of super hydrophilic–super hydrophobic grooves as receptor sites for capillary self-alignment of microfibers. We investigated the influence of major manufacturing parameters on groove sizes and wetting properties. The effects of the width (20 µm–100 µm) and depth (8 µm–36 µm) of the groove on the volume of water droplet contained inside the groove were also investigated. We show that by altering scanning speed, using a de-focused laser beam, we can modify the wetting properties of the microgrooves from 10° to 120° in terms of the contact angle. We demonstrated that different types of microfibers including natural and artificial microfibers can self-align to the size matching super hydrophilic–super hydrophobic microgrooves. The results show that super hydrophilic–super hydrophobic microgrooves have great potential in microfiber micromanipulation applications such as natural microfiber categorization, fiber-based microsensor construction, and fiber-enforced material development. Capillary self-alignment technique can achieve highly accurate and fast alignment of micro components ,2,3,4. MIn this paper, we study the influences of fabrication parameters of the grooved surfaces on capillary self-alignment of microfibers, and we show that by altering scanning speed using a de-focused laser beam, we can modify the wetting properties of the microgrooves from 10° to 120° in terms of the contact angle. We design and fabricate micro grooves with different sizes and wetting properties of shapes matching the microfibers. We investigate the key fabrication parameters including the number of scanned lines, number of scans, scanning speed, and their effects on the geometry and wetting properties of the microgrooves. We also study the influences of the resulted width and depth of the groove on the volume of water droplet confined inside the grooves. Finally, we demonstrate that diverse types of microfibers, including natural and artificial microfibers, can self-align to super hydrophilic–superhydrophobic microgrooves.In this paper, we use a low-cost microsecond pulse laser micromachining method to fabricate super hydrophilic–super hydrophobic grooves for capillary self-alignment of microfibers. The grooves are fabricated on silicon substrates. First, a super hydrophobic coating  is sprayed on a silicon substrate and dried at room temperature for 24 h. The substrate is then patterned with a UV laser marking machine HGTECH LU-5  with a 5 W power and a 355 nm wavelength. The robotic manipulation system for performing capillary self-alignment test is shown in Capillary self-alignment relies on the droplet confinement inside the grooves. There are two ways to confine the liquid droplet inside the groove. One is based on the large wetting contrast for planar patterns, and the other method utilizes sharp edges to pin the liquid according to Gibbs’ criterion . In our The size of the grooves plays an important role in capillary self-alignment of microfibers. A series of experiments were conducted to investigate the key manufacturing parameters and their effect on groove size. The grooves with different width were fabricated by adjusting the number of scanned lines. A single scanned line has a width of about 20 µm. Parallel lines were machined to create grooves of varying widths. The spacing between parallel scanned lines is set to 20 µm. The depth of the grooves is determined primarily by the number of scans. To fabricate the groove with a different depth, by repeatedly scanning over the same location, the depth of the groove can be adjusted. To fabricate grooves with varying wetting properties, we first set the laser out of focus, reducing the intensity of the laser beam significantly compared with a focused laser beam. Then, we adjusted the scanning speed to change the morphology of the surfaces. The groove structures fabricated at different scanning speeds of 100 mm/s, 1500 mm/s, 4000 mm/s, and 7000 mm/s are shown in Given the pulse frequency of the laser beam x-axis represents the width and the depth of the groove, and the y-axis represents the maximum volume of the droplet confined inside the groove, which consists of a mean of 5 repetitions with the standard derivation. The orange squares connected with dotted lines in Droplet confinement is key for capillary self-alignment; we experimentally studied the influence of the size of the groove on the droplet confinement. We tested the grooves of sizes ranging from 20 µm to 100 µm in width, 8 µm to 36 µm in depth, all of which were 3 mm long. The water contact angles in the groove and on the substrate are about 10° and 155°, as mentioned earlier. The volume of the droplet confined inside the groove is defined as the maximum volume of the droplet before spreading out. A series of droplets was dispensed at the groove, at a rate of one droplet per 25 milliseconds, with the volume of 92 pl per droplet  until the droplet spreads out from the groove to the substrate. We investigated both the relationship between the maximum confined volume as the function of the width and the depth. The depth of the groove was fixed at 8 µm for tests of different widths, and the width of the groove was fixed at 40 µm for the tests of different depths. Each test was repeated 5 times. To demonstrate that the fabricated hydrophilic–super hydrophobic grooves can be used to align microfibers, we carried out capillary self-alignment tests using various types of microfibers including both natural and artificial fibers, e.g., dog tail grass, dandelion seeds, legs of ants, and glass fibers. The microfibers have diameters ranging from 15 µm to 200 μm and the length of around 3 mm. The fabricated super hydrophilic–super hydrophobic microgrooves match the shape of the microfibers, having the width of 20–220 μm, depth of 24 μm, length of 3 mm, and the water contact angle of 10°. This paper reports a simple and low-cost laser micromachining method for fabrication of super hydrophilic–super hydrophobic grooves for capillary self-alignment of microfibers. We investigated key manufacturing parameters and its effect on the sizes and wetting properties of the grooves. We studied the influences of the width (20 µm–100 µm) and the depth (8 µm–36 µm) on the volume of water droplet confined inside the groove. The results reveal that the groove’s width and depth are proportional to the number of scanned lines and scans, respectively. We further demonstrated that, by adjusting the scanning speed of a de-focused laser beam, we can change the microgrooves’ wetting properties from 10° to 120° in terms of the contact angle. We demonstrated that diverse types of microfibers, including both natural and artificial microfibers, can self-align to super hydrophilic–superhydrophobic microgrooves. The results suggest that super hydrophilic–super hydrophobic microgrooves have a lot of potential in microfiber micromanipulation applications such as natural microfiber categorization, fiber-based microsensor construction, and fiber-enforced material development.


APOB, FGA, FGG, SERPINC1, APOA1, and F2) involved in RBP4 regulation. A pathway enrichment analysis for RBP4 indicated complement and coagulation cascades, metabolic pathways, antibiotic biosynthesis pathways, peroxisome proliferator-activated receptor signaling pathways, and pyruvate metabolism pathways. These results suggest that RBP4 may be a novel biomarker for HCC prognosis, and an indicator of low immune response to the disease.Recent research has indicated that metabolically related genes play crucial roles in the pathogenesis of hepatocellular carcinoma (HCC). We evaluated the associations between novel biomarkers and retinol-binding protein 4 (RBP4) for predicting clinical HCC outcomes, hub-related genes, pathway regulation, and immune cells infiltration. Bioinformatic analyses based on data from The Cancer Genome Atlas were performed using online analysis tools. RBP4 expression was low in HCC and was also down-regulated in pan-cancers compared with normal tissues. RBP4 expression was also significantly different based on age (41–60 years old versus 61–80 years old), and low RBP4 expression levels were associated with advanced tumor stages and grades. Higher RBP4 expression was associated with better overall survival time in HCC patients, and we identified a deletion-mutation rate of 1.4% in RBP4. We also identified ten co-expressed genes most related to RBP4 and explored the relationships between six hub genes ( Hepatocellular carcinoma (HCC) can occur after years of chronic liver disease and comprises more than 90% of primary liver cancers. HCC is the second leading cause of cancer-related deaths worldwide and is responsible for nearly 800,000 deaths annually ,2. AssocCarcinogen-mediated molecular mechanisms are not only involved in the genesis of cancer but also metabolically in the tumor-associated microenvironment . MetabolRBP4 gene and hypermethylation [Retinol binding protein 4 (RBP4) is a 21-kDa protein belonging to the lipocalin family and is a retinol transporter in the blood ,13. RBP4hylation . RBP4 mahylation . A compahylation ,27.In the present study, we performed bioinformatic analyses to explore any relationships between RBP4 expression and HCC patient survival using data from tumor and normal tissue samples. We explored differentially expressed genes, co-expressed genes, hub genes, and immune cell infiltration related to RBP4 in HCC. These explorations may provide novel insights into RBP4 associated function role in HCC initiation, progression, prognosis. HCC prognosis prediction.http://ualcan.path.uab.edu), an online resource, was used for in-depth analyses of gene expression data using TCGA-Liver Hepatocellular Carcinoma (TCGA-LIHC) datasets. Student’s t-tests were applied, and we considered P-values<0.05 to be significant.All the data used in this study  were obtained from The Cancer Genome Atlas (TCGA). UALCAN  (http://https://www.proteinatlas.org/). Data from two patients (IDs: 1846 and 2556) were analyzed for the expression of RBP4.We used RBP4 protein level data from the tissue microarrays represented in the Human Protein Atlas  .Possible correlations between RBP4 expression levels and clinicopathological features were also assessed. Clinical prognostic features, such as overall survival (OS), recurrence-free survival (RFS), disease-specific survival (DSS), and progression-free survival (PFS) were compared with RBP4 expression levels, and the results were displayed using Kaplan−Meier plots   and LinkedOmics [RBP4 gene mutations, co-expressed genes, and mutations related to protein expression. Genomic profiles included mutations, putative copy number alterations from TCGA-LIHC data, and mRNA expression Z-scores (RNA Seq V2 RSEM) using a Z-score threshold setting of ± 2.0. In addition, we determined the top 10 genes co-expressed with RBP4 using Spearman's rank-order correlation, with R > 0.7.The online CBioPortal site (kedOmics  were useGene ontology (GO) enrichment analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and protein–protein interaction (PPI) networks were visualized using Network Analyst 3.0.https://cistrome.shinyapps.io/timer/), a comprehensive platform for the statistical analyses of immune cell purity and infiltration of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells into tumors, to assess RBP4-related immune cell infiltration. Additionally, we explored the expression of RBP4 across pan-cancer through TIMER.We used the Tumor IMmune Estimation Resource  in normal tissue P<0.001, A,B. For P=0.003, P=0.007, To further explore the relationship between RBP4 expression and clinical information, we applied the HCC tumor tissues transcriptional profiles, and examined possible subgroup associations with RBP4 expression based on patient age, gender, race, tumor grade, TNM staging, and lymph node metastasis. The results demonstrated that RBP4 expression in the younger group (41–60 years of age) was lower than that in the elder group (61–80 years of age) P=0.003, A, and CaP = 1.8e-0, P = 1.5e-05, P = 7.4e-10, P = 2.8e-07, To investigate prognoses for clinical survival, the OS analyses showed that RBP4 overexpression was associated with longer survival time (hazard ratio (HR) = 0.35, 95% confidence interval (95% CI): 0.25–0.5,  A. The diRBP4 gene expression and other differentially expressed genes, we examined the positive and negative correlations for other gene expressions to determine significance. A volcano chart analysis showed the positively (right) and negatively (left) correlated genes that were differentially expressed , but SERPINC1 levels were not significantly different between normal and tumor tissues (P>0.05). Furthermore, we assessed patient OS for the six hub genes, and the high expression levels of APOB, F2, FGG, FGA, SERPINC1, and APOA1 were associated with better prognoses, and coincided with the OS results for RBP4 expression and prognosis  and there was no significant difference between APOBE expression and other immune cells infiltration . SLC27A5 has been reported to participate in the transport of fatty acids, in the metabolism of bile acids, and to act as a tumor suppressor in HCC progression [We also identified the 10 top co-expressed genes for RBP4 (gression . The dowgression . AZGP1 hgression . LDHD hagression . In the RBP4 mutations were negatively correlated with immune cell infiltration, indicating that RBP4 expression is closely related to immune cell bioactivity in preclinical and clinical models. RBP4 expression has been reported to be closely related to immune cell infiltration, especially during inflammation [Cells of the immune system are essential regulators for tumor microenvironment homeostasis. We determined that ammation . RBP4 caammation . RBP4 isammation . RBP4 haammation  (i.e., TIn conclusion, these links between RBP4 and HCC malignancy, metabolic regulation, and immune cell infiltration support the idea of a protective role for RBP4 in HCC, and for its use in predicting HCC outcomes.


The creation of a testis organoid  with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air–liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications. The establishment of an in vitro testis organoid system from dissociated testis cells has become a hot topic for research in regenerative medicine and reproductive biotechnology ,2,3. TesThe range of in situ studies and manipulations that can be performed during the early embryonic development of testis tissue is extremely limited. A suitable testis organoid system can provide an accessible in vitro model for investigations into the effects of potential factors that can adversely affect testicular morphogenesis. The goal of an ideal testis organoid system is to reconstruct an artificial tissue that is structurally and functionally similar to intact testis tissue. Three criteria have been proposed by Edmonds and Woodruff (2020) to evaluate the testis organoid formation in a given culture system ,15. ThesSince the late 20th century, a wide range of testis organoid culture systems have been introduced to recapitulate de novo testis organogenesis ,16. DespGdnf) is lethal to neonates [Testis organoids have tremendous potential to be used in basic and clinical research into etiology of infertility, strategies to preserve fertility potential of immature individuals, and restoration of male fertility ,25,26. Kneonates ; hence, neonates , and henneonates .Testes were collected through aseptic castration of 1-week-old Yorkshire-cross piglets at our University’s swine center. The testes were kept in Dulbecco’s phosphate-buffered saline , containing 1% antibiotics solution , and were transported on ice. In the laboratory, the testes were rinsed three times with DPBS prior to processing. The tunica albuginea and excess connective tissue containing collagen fibers, fibroblasts, and fibrocytes, were first removed from the testis tissues. The remaining tissue was mainly testis parenchyma that contained testis tubular and interstitial components, which was further processed for testis cell isolation. Testes were also collected from 1- and 4-week-old piglets to be used as reference tissue for histological assessments.w/v antibiotics  at 37 °C for 15 min. Fetal bovine serum  was added to stop the digestion. To remove cell clumps and undigested tissue segments, the suspension was vortexed for another 30 s and filtered through a 40 µm filter . The filtrate cell suspension was then centrifuged at 500× g at 16 °C for 5 min to form a cell pellet and the supernatant was removed. The cell pellet was then re-suspended with 20 mL of the lysis buffer and placed at room temperature (RT) for 30 min for erythrolysis. The lysis buffer contains 156 mmol/L ammonium chloride , 10 mmol/L potassium bicarbonate , and 0.1 mmol/L disodium ethylenediaminetetraacetate  in sterile distilled water. The cell-lysis buffer mixture was then centrifuged at 500× g at 16 °C for 5 min and the supernatant was removed. Lastly, the cell pellet was washed with DPBS and re-suspended in 5 mL of DMEM supplemented with 10% knockout serum replacement . The cell number and viability of the resultant cell suspension was assessed using the trypan blue exclusion assay.Testis cell isolation was performed using methods previously established in our laboratory (including a three-step enzymatic digestion) ,31. BrieThe resultant testis cells were either used fresh for organoid formation or cryopreserved using our previously published protocol , with mi2 at 37 °C for 24 h to allow cells to form cell spheroids. A stable culture environment was provided, and the handling of culture plates was minimized during cell spheroid formation. The cell spheroids were then cultured on small blocks (1 × 1 × 0.5 cm) of 1.5% agarose gel bedding  immersed in DMEM supplemented with 10% KSR  for efficient tubulogenesis and germ cell maintenance in the organoids.The isolated testis cells were first cultured in U-bottom 96-well plates  placed in an incubator with 5% CO 10% KSR . NotablySamples were fixed in Bouin’s solution for 4 h, then rinsed with and stored in 70% ethanol. The samples were processed using standard histological preparations, embedded in paraffin, and sectioned at 5 μm thickness. The sections were deparaffinized and stained with hematoxylin and eosin (H&E) or used for immunohistochemistry (IHC). The H&E staining was performed using previously described protocols . Histoloh as the chromogen . Masson’s trichrome (MT) and periodic Schiff-methenamine (PASM) staining were also performed using an automated staining platform. Histological analysis of slides was performed using a light microscope equipped with digital photomicrography .We performed immunohistochemistry (IHC) and tissue-specific staining to identify major cell types, cell orientations, and structural components of the organoids. Primary antibodies included anti-UCHL1 antibody , anti-GATA-4 antibody , anti-CYP17A1 antibody , anti-α-SMA , and anti-vWF . Secondary antibodies included the HRP-labeled anti-mouse/rabbit secondary antibody  and anti-goat secondary antibody . Detailed information for the antibodies has been listed in Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the organoids. The fixation, embedding, and sectioning of samples for TEM were performed using our previously reported protocols . BrieflyIn intact testis, testosterone secretion by Leydig cells is induced by luteinizing hormone (LH). To investigate the release of testosterone by the organoids’ Leydig cells in the culture media and their LH-responsiveness, we supplemented the organoid culture media with LH  starting at day six of culture until the end of experiments (4 weeks). The organoid culture media were collected every other day for the measurements of testosterone levels (ng/mL) using testosterone ELISA kits . Samples were also collected from organoid culture media that did not receive LH supplementation for comparison.p < 0.05 is considered significant. The data from tubular relative areas and germ cell ratios were analyzed using two-way analysis of variance (ANOVA) with Tukey’s post-hoc test. Percentages were transformed (using Arcsin function) prior to ANOVA. The data from testosterone levels were analyzed by Welch’s ANOVA with Games–Howell post-hoc test. The data were log transformed prior to ANOVA for normalization.All quantitative data were obtained from a minimum of three independent experiments using separately isolated testis cells. Statistical analyses were performed using SPSS . All data are expressed as means ± SEM. When the isolated neonatal porcine testis cells were deposited in low-attachment U-bottom wells and allowed to settle for 24 h, they formed a coherent cell spheroid in each well A. HistolTo test whether organoids can also be generated from cryopreserved testis cells, frozen-thawed testis cells from 1-week-old piglets were used to form organoids using the same culture system. Similar to fresh cells, the organoids derived from cryopreserved testis cells also formed tubular structures as early as 1 week after culture  . A significant increase in the tubular relative area was observed at least at one time point for each group over the length of culture. Such an increase was observed at week 2 for both 1.0 × 106 and 0.6 × 106 cell density groups, whereas it was observed at weeks 3 and 4 for the 0.8 × 106 group (p < 0.05).To examine the effect of cell density on in vitro tubulogenesis of organoids, we quantified the relative area of tubular cross-sections in organoids resulting from different cell densities (1.0 × 10rganoid) D. The tup < 0.05). In comparison, the tubular relative area of the 10% FBS group was lower than other groups, regardless of the time of culture (p < 0.05). A significant increase in the tubular relative area was observed at least at one time point during the culture for every group. Such an increase was observed at week 2 of culture in all groups, and at week 3 for the 5% KSR + 10% FBS group (p < 0.05). In this media, the tubular diameters measured at 1 and 4 weeks of culture were 47 ± 1 μm (n = 3) and 54 ± 0.9 μm (n = 3), respectively.To examine the effects of media supplementation on in vitro tubulogenesis of the organoids, the relative area of tubular cross-sections was quantified over time in different supplementation groups  A. The tup > 0.05). Since the total number of cells was relatively lower in the 10% FBS group than other groups, the relative germ cell numbers appeared to be numerically (but not significantly) higher than other groups at weeks 1, 2, and 4. Moreover, although a slight numeric decrease in the number of germ cells was observed for all groups over time, no statistical differences were detected (p > 0.05).To examine the effects of media supplementation on germ cell numbers in organoids, the relative number of germ cells in the organoids were quantified over time of culture B. SurpriTo identify the cell types and structural components of the organoids, we performed IHC and tissue-specific staining. The organoids displayed a compartmentalized architecture resembling the tubular and interstitial compartments in the native testis tissue A,B. Notap > 0.05). However, starting on day 9 of culture and through the end of this experiment (day 30), testosterone levels in the LH-supplemented group were significantly higher than the control group (p < 0.05), suggesting that Leydig cells in the organoids can be induced by LH to release more testosterone.Based on TEM imaging, numerous lipid droplets were observed within the Leydig cells in week 1 organoids A, while We have previously reported that de novo testis tubulogenesis can be induced by the in vivo implantation of primary testis cell aggregates, derived from neonatal piglets, under the skin of immunodeficient mice to form a compartmentalized testis tissue ,37. In tIn the present study, we also showed that frozen-thawed neonatal porcine testis cells can generate testis organoids similar to organoids derived from fresh testis cells. Similar results were also reported by Sakib et al. 2019b) using a microwell culture system 19b using,43 in si3 to 2.0 × 106 cells per organoid have been reported [6 cells per organoid which caused central necrosis in the resultant organoids, while using 0.5 × 106 cells/organoid resulted in delayed or impaired tubulogenesis, and the organoids were too small to be handled for histological processing. Therefore, mid-range cell densities including 0.6 × 106, 0.8 × 106, and 1.0 × 106 cells/organoid were investigated further for their organoid formation and tubulogenic efficiency. In the present study, we evaluated tubulogenic efficiency by quantifying the relative area occupied by viable tubular cross-sections within a testis organoid. Therefore, a high tubular relative area indicates superior tubular reconstruction which was provided by 0.8 × 106 testis cells per organoid as an optimal cell density to generate a porcine testis organoid.The initial number of cells used to form a testis organoid  has varied among different studies. In general, a higher cell density is expected to promote intimate cell-to-cell interactions, which should improve de novo testis organogenesis. However, high cell densities also increase the size of organoids hence causing poor perfusion of oxygen and nutrients to the center of the organoid, eventually leading to central necrosis. On the other hand, while lower cell density is beneficial in improving nutrient/oxygen perfusion in the organoids , organoireported ,20,38. IFurthermore, there has been no consensus on the most suitable culture media supplementation for formation of testis organoids . For serNext, IHC and tissue-specific staining were performed to determine the cell types and structural components of the newly established testis organoids. Our findings showed that the testis organoids consisted of tubular and interstitial compartments resembling native testis tissue. Namely, testis tubular compartments were formed by Sertoli cells, PTMCs, and the peritubular basement membrane and gonocytes were almost exclusively observed within these tubules. Also, reconstruction of the inter-tubular interstitial compartments containing Leydig cells and collagen fibers was observed in testis organoids. As described earlier, native testis tissue is comprised of both interstitial and tubular compartments; each compartment contains mixed cell populations and distinctive spatial orientation. The cellular and acellular components in both compartments play a crucial role in the SSC niche to regulate SSC proliferation and/or differentiation . TherefoDesert hedgehog (Dhh) null mice [Yokonishi et al. (2013) observed irregular architecture of the reconstructed testis tubular structures in mouse testis organoids; the tubular structures had uneven diameter and a maze-like configuration . In addiull mice . Also, tull mice . TherefoOne of the most exciting findings in the present study was the formation of vascular structures in the testis organoids. During vascular formation, single-layered endothelial-cell tubes are first generated as immature vasculature (nascent vessels). Subsequently, this immature vasculature specializes and develops into microvessels such as capillaries, arterioles, and venules. Capillaries are constructed by endothelial cells and pericytes, and surrounded by a basement membrane; in comparison, arterioles and venules have an additional coverage of smooth muscle cells that are responsible for vasoconstriction and vasodilation . In our In vitro maturation of fetal Leydig cells was reported using an air–liquid interface culture system . To our Moreover, testosterone is a crucial hormone in supporting testis development and spermatogenesis, especially spermatogenic differentiation during meiosis and spermiogenesis stages ,73,74; h6 cells/organoid and a combined supplementation of 5% KSR + 10% FBS in media were deemed optimal for improving tubulogenesis of the organoids. Furthermore, the number of germ cells (gonocytes) did not significantly decrease over the 4 weeks of culture in any of the media supplementation groups. Our testis organoids consist of tubular and interstitial compartments resembling the innate testis tissue. Importantly, the cell types and their orientations also closely correspond to the innate testis tissue, including the formation of testis tubular compartments by Sertoli cells, PTMCs, and peritubular basement membrane which also encompass germ cells. Formation of the inter-tubular interstitial compartments containing Leydig cells, collagen fibers, and most notably vascular structures was also observed in the organoids. Furthermore, the testis organoids have endocrine functionality of testosterone secretion and LH-responsiveness. Therefore, these findings provide a robust, accessible, and tunable model for a wide range of basic research and diagnostic applications. This includes exploring the contribution of individual cell types, the effects of various matrix materials, and potential consequences of known or unknown factors to gain insights into the testis development. As such, this testis organoid system can prove valuable in embryology and reproductive biology research, pharmaco-toxicology testing, and regenerative medicine.We established a new testis organoid culture system using neonatal porcine testis cells by first forming cell spheroids in low attachment culture wells followed by long-term culture in an air–liquid interface system. Both fresh and cryopreserved testis cells were fully capable of forming testis organoids comprising testis cord-like structures, which could be maintained for at least 4 weeks. A testis cell density of 0.8 × 10