To evaluate the effectiveness of 4-hand reduction for obturator hernia with the guidance of sonography (FROGS) as a new treatment strategy for obturator hernia. Since November 2019, FROGS was performed for all patients with obturator hernia at our emergency department. We retrospectively compared the clinical data of 20 patients who underwent FROGS (after FROGS group) to those of 23 patients who did not (before FROGS group). All patients except one were female. The male-to-female ratio, age, duration of symptoms, lesion site, and predisposing factors did not significantly differ between groups. However, the diameter of the prolapsed bowel and the body mass index of the after FROGS group were significantly larger and lower, respectively. Manual reduction was successfully performed for all 20 patients in the after FROGS group, and bowel resection was avoided for all 20 cases. However, 14 patients in the before FROGS group underwent manual reduction; of these, only one was successfully treated using a method other than FROGS, and 8 patients underwent bowel resection. There were no significant differences between groups in terms of postprocedural complications or mortality within 30 days of hernia presentation. The FROGS technique was safe and reproducible and could be used as the first choice of treatment for patients with obturator hernia. Previously, determining a definite preoperative diagnosis has been difficult; however, it has become much easier with the use of advanced imaging techniques.–5 Obturator hernia usually develops in elderly, thin, and multiparous women who often have comorbidities and other debilitating conditions that can preclude them from undergoing emergency surgery. Therefore, the postoperative mortality rate after emergency surgery is high.,7 However, after the diagnosis is determined, emergency surgery is usually performed because delaying treatment can lead to intestinal ischemia and subsequent perforation. Previous reports have described successful manual reductions of obturator hernias.–11 However, these were case reports with no statistical confirmation of the efficacy of manual reduction. Therefore, until recently, there has been no controversy regarding whether manual reduction (with or without elective surgery) or emergency surgery was more effective. Kamihata et al described a new repositioning maneuver for incarcerated obturator hernia called 4-hand reduction for obturator hernia with the guidance of sonography (FROGS) in Japan. This simple technique was created based on previous reports to treat obturator hernias in the emergency department. To the best of our knowledge, this is the first report to statistically demonstrate the efficacy of a noninvasive manual reduction technique, FROGS, as a new treatment strategy for obturator hernias and includes 6 more cases than the previous study.Obturator hernias are rare forms of abdominal hernias that account for < 1% of all abdominal hernias.The clinical data of all patients with an obturator hernia who were admitted to the Department of Surgery at the Kurashiki Central Hospital between April 2016 and December 2021 were retrospectively reviewed. We have implemented FROGS in the emergency department as the first choice of treatment since November 2019. We compared the clinical data  of patients who underwent FROGS (after FROGS group) with those of patients who did not (before FROGS group). All procedures performed during this study were in accordance with the ethical standards of the institutional research committee (3658) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained for the academic use of photographs of the patients.–11 This technique requires 2 surgeons, which is why it is referred to as the 4-hand technique. Before initiating this procedure, the presence of an obturator hernia  must be confirmed. As the surgeon confirms the hernial sac, the assistant supports the patient to keep the hip joint flexed and rotated externally so that the hernial sac can be visualized  while the right hand pushes the hernial sac. The assistant places the hip and knee joints in the flexed position.Step 2: The assistant slightly rotates the hip joint laterally, thus widely opening the hernia orifice . Continuous variables are expressed as the mean ± standard deviation, and categorical variables are expressed as absolute numbers and percentages. All variables were analyzed using Student’s A total of 43 patients with obturator hernias were admitted to the Department of General Surgery at Kurashiki Central Hospital between April 2016 and December 2021. Before the FROGS technique was implemented, 23 patients had been admitted (before FROGS group). After November 2019, 20 patients were admitted; all of them underwent FROGS as the first treatment choice (after FROGS group). The demographic characteristics of all the patients are summarized in Table P = .033) (Table P = .049)  Table  and the 9) Table .P < .001) (Table P < .001) . In the before FROGS group, 14 patients underwent manual reduction; of them, only one was successfully treated (success rate of 7.14%) using a method other than FROGS. The success rate of manual reduction was significantly higher in the after FROGS group than in the before FROGS group (Table P < .001) (Table All 7 elective surgeries performed in both groups involved laparoscopic repair (TAPP repair). However, of the 21 patients who underwent emergency surgeries, only 4 (19.0%) underwent laparoscopic TAPP repair. The rate of TAPP repair was significantly higher for patients who underwent elective surgery (1) Table .P = .003) (Table P = .028) (Table P = .645) (Table P = .177)  Table . The len8) Table  There weWe statistically evaluated the effectiveness of FROGS as a new treatment strategy for obturator hernia. Currently, manual reduction with FROGS has a success rate of 100%. The results showed that patients can avoid emergency surgery and bowel reduction without increasing the risk of postprocedural complications or mortality within 30 days of hernia presentation because of FROGS. Concomitant illnesses, such as chronic obstructive pulmonary disease, constipation, and kyphoscoliosis, could result in increased intraperitoneal pressure and facilitate the growth of the hernial sac. Lean and elderly women are usually affected because they have less preperitoneal fat and several comorbidities that favor the pathogenesis of this condition.Obturator hernia is a rare type of abdominal hernia caused by the loss of preperitoneal fat and lymphatic tissues that normally overlie the obturator canal and results in a space around the obturator vessels and nerves. mentioned that “an obturator hernia needs a laparotomy, not a diagnosis.” Many surgeons believe that emergency laparotomy is the optimal treatment for possible incarcerated obturator hernias. However, recent advances in imaging techniques, such as computed tomography and ultrasonography, have improved the correct preoperative diagnosis rate, thus making less invasive approaches feasible.–4 Some studies have described less invasive therapeutic strategies, such as elective surgery after successful manual reduction.–7 In these studies, elective surgeries were demonstrated to be associated with a higher rate of TAPP repair implementation. The decision to undertake laparoscopic approaches less frequently during emergency surgeries could be attributed to the smaller peritoneal cavity resulting from a dilated intestine.Laparotomy was once the standard approach for the treatment of obturator hernia because the preoperative diagnosis is difficult because of its rarity and the signs and symptoms are nonspecific. Ziegler et al showed that emergency surgery for complicated inguinal hernias is burdened by high morbidity and mortality rates for elderly patients. For patients with asymptomatic or minimally symptomatic inguinal hernias, watchful waiting is recognized as an acceptable option.,18 Although these studies have mainly focused on inguinal hernias, the results could be applied to obturator hernias as well.This study aimed to evaluate the efficacy of FROGS as the first choice of treatment for incarcerated obturator hernias. Patients with an obturator hernia often have comorbidities, thus making them unsuitable for emergency surgeries. They should be treated with less invasive therapy in an elective setting, if possible. Additionally, some patients and their families do not consent to surgery. Ceresoli et al a watchful approach could be a choice based on these results and the literature.During our study, emergency surgeries for asymptomatic or minimally symptomatic patients were avoided because of the implementation of FROGS. Some of these patients underwent safe elective TAPP repair; however, others did not because of comorbidities or family wishes. Irrespective of whether elective surgery was performed, bowel resection was avoided for all patients. Additionally, the short-term prognoses for the after FROGS and before FROGS groups were comparable, and FROGS facilitated a shorter hospital stay. Although the balance between the risks of elective surgery and the risks of the watchful approach is still a topic of debate in the absence of specific recommendations for elderly patients,Although patients who do not undergo elective surgery are still at risk for recurrence, FROGS can be reproducible in cases of recurrence because of its high success rate. Therefore, we believe that FROGS could be the first choice of treatment for any case of obturator hernia incarceration. The mechanism of FROGS is unclear and may be complex. We believe that the complex coordinated movements of the muscles around the obturator canal may have an important role. The driving pressure generated by these movements helps surgeons to reduce the hernial sac. Furthermore, at some point during this maneuver, the obturator canal may be maximally relaxed . By repeating FROGS, we can eventually find this particular point.It is difficult to decide whether bowel resection is necessary, especially if FROGS is implemented. No patient in the after FROGS group required a bowel resection. The longest duration from symptom onset to hernia presentation was 72 hours. Based on these results, we can argue that if there is no obvious evidence of strangulation or ischemia, manual reduction using FROGS is acceptable within 72 hours.The limitation of this study is its relatively small sample size. Further observations and analyses are necessary to confirm the effectiveness of FROGS. Nevertheless, manual reduction with FROGS was found to be safe and reproducible and can be the first choice of treatment for patients with obturator hernias. FROGS can be used instead of emergency surgery as a less invasive method to treat patients with obturator hernias while preserving the bowel.Based on the retrospective data of 43 patients, which included those of 20 patients who underwent FROGS (after FROGS group) and 23 patients who did not (before FROGS group), FROGS was found to be safe and reproducible and can be the first treatment choice for obturator hernias instead of emergency surgery. More observations and analyses are necessary to further discuss the scope of the indications for FROGS because the sample size was relatively small. However, we believe our results are quite promising.We would like to thank Editage for English proofreading.All authors contributed to the study conception and design. YT and KK performed the material preparation, data collection, and data analysis. YT wrote the first draft of the manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.Conceptualization: Yoshio Nagahisa, Kazuyuki Kawamoto.Writing – original draft: Yuki Togawa.Writing – review and editing: Kyohei Kamihata.Writing – review and editing: Yoshio NagahisaWriting – review and editing: Yuki Togawa.


RUNX1-RUNX1T1 and one donor-derived MDS with deletion 5q. A review of the cytogenetic profiles of previously reported DDMN indicates a significant contribution of therapy-related myeloid neoplasms. Cases with direct evidence of donor- or recipient-dependent factors are rare; a role of direct transfer of leukemic cells, genomic instability of the donor, abnormal gene methylation in donor cells, proleukemic potential of abnormal stromal niche, and the role of immunological surveillance after transplantation has been observed. The role of additional potential pathogenetic factors that are without clinically observed evidence are also reviewed.Donor-cell derived myeloid neoplasm (DDMN), a rare complication after allogeneic hematopoietic cell transplantation (HCT), is of interest for its potential to reveal donor-derived and host-derived factors that contribute to the pathogenesis of leukemia. The accurate diagnosis of donor-derived leukemias has been facilitated by the more frequent use of molecular techniques. In this study, we describe three additional cases of DDMN; the first reported case of donor-derived chronic myelomonocytic leukemia (CMML), one acute myeloid leukemia (AML) with t;  Donor-derived myeloid neoplasm (DDMN) is an infrequent complication encountered after allogeneic hematopoietic cell transplantation (HCT) and occurs as a result of the oncogenic transformation of apparently normal donor hematopoietic cells in the transplant recipient. Since the first report of donor-derived leukemia in 1971, an increasing number of cases have been reported in the literature over the past six decades, though a complete catalog of these cases does not exist. Reporting of this intriguing entity has accelerated in recent years due to the more frequent use of molecular techniques. DDMN is the subject of considerable interest for providing unique insights into the mechanisms of donor-derived and host-derived factors of leukemogenesis. In this study, we describe three new cases of DDMN. We analyze the cytogenetic spectrum of previously reported DDMN and discuss individual cases that provide an insight into contributing factors from either the donor’s or the recipient’s perspective. We review the current understanding of DDMN pathogenesis.Three cases of donor-derived myeloid neoplasm (DDMN) were identified in the Pathology Archives of the Mayo Clinic in Arizona. The diagnosis and subclassification were based on the 2016 World Health Organization Classification of Myeloid Neoplasms [Peripheral blood (PB) smears were stained with Wright stain. Bone marrow (BM) aspirate smears and biopsy touch imprint preparations were stained with Wright-Giemsa. BM biopsies were fixed in zinc-formalin and stained with hematoxylin eosin (H and E). Evaluation of morphologic dysplasia and blast count was performed on PB and BM aspirate smears. BM cellularity was evaluated on BM core biopsy specimens.RPN1), 3q26.2 (MECOM) dual color/double fusion; 3q25.32(MLF1), 5q35.1(NPM1) dual color/double fusion; 5p15.2(D5S630), 5q31 (EGR1), region gain or loss; 6p23(DEK), 9q34(NUP214) dual color/double fusion; 7CEN(D7Z1), 7q31(D7S486) region gain and loss; 8p11.2(KAT6A), 16p13.3 (CREBBP) dual color/double fusion; 8q22(RUNX1T1), 21q22(RUNX1), dual color/double fusion; 8CEN(D8Z2), 8q24.1(MYC), region gain and loss; 9q34(ABL1), 22q11.2(BCR), dual color/double fusion; 11q23, 13q34(LAMP1), region gain and loss; 15q24.1 (PML), 17q21(RARA), dual color/double fusion; 16p13(MYH11), 16q22(CBFB), dual color/double fusion; 17p13(TP53), 17CEN(D17Z1), region gain and loss; 20q12(D20S108), 20qter, region gain and loss. FISH of sex chromosomes was performed using the X centromere (CEPX) and Y centromere (CEPY) probe set. Cytogenetic abnormalities were classified according to the International System for Human Cytogenetic Nomenclature [Karyotype analysis was performed in duplicates on fresh anticoagulated BM aspirate specimens in a complete tissue culture medium. Chromosome preparations, including harvesting and GTW banding, were performed using standard methods. FISH was performed using the Mayo Clinic MDS/AML panel including the following probes and probe strategies: 3q21.ASXL1, BRAF, BCOR, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SRSF2, TERT, TET2, TP53, U2AF1, WT1, and ZRSR2. This NGS panel has a sensitivity of 5–10% variant allele fraction (VAF) with a minimum depth coverage of 250x for single-base substitutions and for insertion/deletion events.DNA was extracted from PB or BM aspirate sample and, following library preparation by hybrid capture, subjected to NGS with post-sequencing analysis of tumor-associated mutations, including the following mutations: TP53 mutation. He was treated with a 7 + 3 induction and mitoxantrone, etoposide, and cytarabine (MEC) chemotherapy, followed by a matched sibling donor PB hematopoietic cell transplantation (HCT) from his brother. He received post-transplant azacitidine and was well until 28 months after transplantation when he developed macrocytic anemia and thrombocytopenia. BM morphologic examination showed 23% small to intermediate-sized blasts with occasional Auer rods and subtle morphologic dysplasia. The cytogenetic study demonstrated t. The donor, the patient’s 64-year-old brother, was asymptomatic at the time of the onset of DDMN. The donor’s BM biopsy was normal, with a normal male karyotype and no mutations detected by NGS myeloid mutation panel. A 71-year-old man was diagnosed with an AML with myelodysplasia-related changes (AML-MRC) characterized by a complex hypodiploid karyotype including deletions of 3p, 5q, and 17p, with one additional metaphase representing a hypo-tetraploid subclone, and a known TP53 mutation. He was treated with decitabine and venetoclax, but soon venetoclax had to be withheld due to infectious complications. He died 5 months after the relapsed MDS/AML (58 months after his transplantation). The patient received 7 + 3 induction and four cycles of high-dose cytarabine chemotherapy. He achieved remission at 7 months after the onset of donor-derived AML, with a normal karyotype and 100% donor chimerism detected in a bone marrow sample. Two years after his donor-derived AML, he developed a relapse of his original AML-MRC, with recurrent complex cytogenetics and A 59-year-old man with a history of T-lymphoblastic leukemia/lymphoma (T-ALL), status post an allogeneic HSCT from his brother, presented with leukocytosis and splenomegaly 8 years after allo-HSCT. Complete blood counts (CBC) showed hemoglobin of 12.0 g/dL, total leukocyte count of 275,000 cells/μL and platelet count of 316,000/μL. The peripheral blood smear was notable for absolute monocytosis , neutrophilia, basophilia (1%), and granulocytic left shift with 2% circulating blasts. BM examination showed hypercellular marrow with myeloid predominance, 2% blasts, increased monocytes, and small monolobated megakaryocytes. Flow cytometric analysis was performed on the diagnostic bone marrow aspirate and showed 2% myeloid blasts with an aberrant immunophenotype as follows: CD5, CD13, CD33, CD34, CD117, and HLA-DR positive. Monocytes were increased, comprising 25% of the total analyzed cells and expressed CD13, CD14, CD15, CD33, CD64, HLA-DR, and aberrant CD56. Lymphoid populations showed no immunophenotypic abnormalities.aureus infection, and he died 12 months after the diagnosis of DDMN  was rendered. The donor was reported healthy at the onset of DDMN. The patient was treated with hydroxyurea. He had a complicated clinical course with polyneuropathy, chronic alcohol dependence, and methicillin-resistant Staphylococcus  of DDMN .A 59-year-old male with a history of an allogeneic HSCT 24 years prior to AML presented with macrocytic anemia. The donor was an unrelated matched female. BM showed normocellular bone marrow with many small round megakaryocytes and dyserythropoiesis without an increase in blasts. Flow cytometry performed on the bone marrow aspirate revealed a small myeloid blast population with an unremarkable immunophenotype. Lymphocytes included polyclonal B-cells and T-cells without immunophenotypic abnormalities.A cytogenetic study performed on a bone marrow sample showed that 20 metaphases were 46, XX, and fourteen of them had additional 5q deletion. The 5q region had a segment of unidentified additional material on it, resulting in the effective deletion of 5q. The diagnosis of a donor cell-derived MDS with deletion 5q was rendered. He was treated with lenalidomide and responded well. The patient is currently alive and well, 32 months after the diagnosis of DDMN. At present, he is off lenalidomide and continues to have mild macrocytic anemia .A review of the literature identified 82 cases of DDMN, including our current three cases. The demographic, clinicopathological features, and transplant characteristics are summarized in A review of the cytogenetic results revealed that the most common cytogenetic finding in DDMN was a normal karyotype (28.0%), and the most common abnormal karyotype was monosomy 7 (17.2%). Complex karyotypes, MLL rearranged, and t were represented in 6.1% of cases each, respectively, followed by t (4.9%), deletion 5q (4.9%), and the remaining abnormalities were rarely observed, represented by either one or two cases in the cohort , t, inv(3), inv(16), t, t, t, add 21, del(20q). In some cases, a cytogenetic result was not reported. Our three new cases represent an AML with t, a CMML with a normal karyotype, and an MDS with deletion 5q abnormality. (Cytogenetic findings of 82 cases of DDMN are shown in Few cases reported findings that provide direct insight into the pathogenesis of DDMN . The traDonor cell-derived leukemia (DDL) was first recognized in 1971, but for many years, the paucity of reported cases suggested it to be a rare phenomenon. The majority of evidence in the literature is still anecdotal, and the exact incidence is not known. Based on several reviews, the incidence of DDL has been estimated to be between 0.12% and 5% ,17,18,19In this paper, we report three new cases of DDMN, including a donor cell-derived chronic myelomonocytic leukemia, a subtype that has not been reported before. The other two cases, an AML with t8;21) and a case of MDS with deletion of 5q, has been reported previously. Only one of the three cases had an opposite-sex transplant. Chimerism analysis has confirmed the donor cell origin of all three cases. Discussion of the myeloid donor cell-derived neoplasms separated from their lymphoid counterparts is justified because myeloid disorders share a cytogenetic classification and often occupy a continuum of disease progression. Since myeloid neoplasms are subclassified along the cytogenetic guidelines, it is informative to review the spectrum of cytogenetic abnormalities in the donor cell-derived cohort. Notably, the single most common cytogenetic affiliation is represented by monosomy 7, an abnormality known to be associated with therapy-related myeloid neoplasm (t-MN)  and a ca. The various hypotheses postulated for the mechanism leading to leukemia usually separate along the lines of donor-derived and recipient-derived factors. Several cases of donor-cell leukemia provide evidence that undetected leukemia cells in the donor at the time of the donation are capable of establishing leukemia in the recipient environment. Accidental transplantation of AML, MDS, CML, cutaneous T-cells lymphoma, and various B-cell lymphomas have been reported ,22,23,24CEBPA and RUNX1 gene mutations [TP53 defect), increase the lifetime risk of MDS/AML [TEL-AML1, and 0.2% AML-ETO [Alternatively, healthy donor HSCs may carry genetic defects predisposing to malignant potential. Since 80% of donor cell leukemias involve related donors, familial cancer predisposition syndromes characterized by germline mutations of tumor suppressor or DNA repair genes, genomic instability, and oncogenic mutations, are of great interest. Hereditary predisposition to MDS/AML has been described with utations . Chromos MDS/AML . In thes MDS/AML . Another MDS/AML . With an MDS/AML , and lat MDS/AML . Potenti AML-ETO , raising AML-ETO .Recently there has been a shift in emphasis moving away from the role of the stem cells and toward the microenvironment in understanding the pathogenesis of donor-cell leukemia . Due to In addition, impaired immune surveillance in the recipient microenvironment may promote the evolution of a donor cell neoplasm. T-cells remain quantitatively and qualitatively suppressed for long periods after HSCT . The T-cTelomere shortening may also have implications in donor-cell leukemia. Telomeres, the noncoding regions of DNA flanking the end of chromosomes, play a critical role in chromosome integrity. Several studies have documented significant telomere shortening following HSCT ,35,36. TDue to the sporadic nature of reported cases, lack of sufficient follow-up information in many of the cases, and the variations in treatments, it is difficult to assess the prognosis of DDMN in comparison with de novo leukemia. Most cases are reported by case reports of small case series, and at the time of the report, the patient is alive. Overall, the prognosis is predicted to be poor, in line with other secondary leukemias. Based on observations of the case series of European Blood and Marrow Transplantation (EBMT), six of nine patients entered remission after chemotherapy . Long-laDonor-derived leukemias pose a set of ethical questions in relationship to the physician’s responsibility to inform recipients. The afterlife of stem cell donation is not known for unrelated donors, and even sibling donors may not be aware of the clinical outcome in the recipient. Though feedback of adverse events in recipients is encouraged to donor registries, this may be practically not feasible due to the absence of centralization and due to international donations. Further complicating the issue is the observation that most donors do not develop leukemia themselves . The feeDDMN is a rare complication of allo-HCT and holds promise as a model to study in vivo leukemogenesis. With the evolution of new molecular techniques for chimerism analysis, the detectability of DDMN became easier. Donor cell-derived neoplasms should be considered for myeloid diseases that develop in the post-transplant period, especially if clinically different from the original disease. The etiology is multifactorial, and the relative contribution of each of the genetic and transplant-related factors is still largely unknown. Further molecular and immunological investigations would help clarify the pathogenesis. There is a need for large multicentric studies to ensure more inclusive reporting and comprehensive analysis of cases.


The performance of a hydraulically expanded joint between tubesheet and titanium tube was analyzed using a finite element numerical calculation. The connection strength of Q345R tubesheet and TA2-Q345R clad tubesheet was studied using a tight expansion method. The results proved that the residual contact pressure and pullout force of the tight expansion joint of TA2-Q345R clad tubesheet were greater than those of the Q345R tubesheet. However, the residual contact pressure of the expanded joint without a groove for the TA2-Q345R tubesheet and the pullout force failed to meet the requirement of connection strength. Hence, the groove was employed on the contact surface. The influences of groove position and groove width on the connection strength of the expanded joint with grooves in tubesheet hole were studied. The results show that the residual contact pressure of the clad tubesheet of grooving in the cladding layer was higher than that of grooving in the base layer. The effect of the position of groove in the cladding layer and base layer on the residual contact pressure could be neglected. A wider groove led to a higher residual contact pressure, which increased significantly when the groove width was 4 mm. Titanium is an alloy with low density, high strength, and strong corrosion resistance, which is widely used in the chemical industry, marine ships, aviation, medical instruments, automotive industry, livelihood supplies, and other fields ,2. BecauThe joint between the tube and tubesheet is the key part of the shell and tube heat exchanger, which is also prone to failure. Expansion is an important type of connection between the tube and tubesheet, which can be divided into hydraulic expansion, mechanical expansion, and explosion expansion. Among them, hydraulic expansion is a uniform flexible expansion method. The expansion pressure is easy to control and does not damage the inner wall surface of the expansion; thus, the joint is of good quality.o and t are the outside radius and thickness of the tube, respectively. The residual contact stress of titanium–steel clad tubesheet with a groove in the base layer was calculated by numerical simulation  of the steel heat exchange tube of tight expansion is 2 MPa; thus, the pullout force should satisfy F > 7.23 kN according to Equation (2). For titanium expansion joints, the pullout force decreases with the time after the expansion due to the elastic hysteresis effect. A previous study [According to GB/T151-2014 “Heat Exchanger” , the allus study  gave thepc* value at the sealing ring was much higher than that of the Q345R tubesheet, and the pc* value of the middle and shell side of the two kinds of tubeplates was similar.Three axial paths A–A′, B–B′, and C–C′ in  were takpc* on the circumferential paths D–D′ (tube side) and E–E′ (shell side) on two sealing rings. The results show that the pc* of the shell side of the two tubesheets was low, and the pc* at some point on the TA2-Q345R clad tubesheet was zero, indicating that, with the rebound of the TA2 tube after unloading, the sealing ring at some positions on the contact surface of the shell side gradually disappeared. When the heat exchanger is in service, the shell side medium would leak along the contact surface and extend to the pipe side, which would damage the contact surface and cause hidden dangers to the safe operation of the equipment. Therefore, the method of ungrooved hole tight expansion was not suitable for the titanium–steel clad tubesheet, and it was necessary to groove the tubesheet hole for strength expansion to improve the joint connection strength. w, groove distance s, and groove spacing b on the connection strength and sealing performance are also investigated. The groove dimensions are listed in In the standard GB/T 151-2014, the structure and dimension of the circumferential grooves in clad tubesheet holes are as shown in w1 or w2 was 8 mm; when both the cladding layer and the base layer were grooved, the base layer groove width w2 was 8 mm, and the cladding layer groove width w1 was 6 mm. When a single groove was in the base layer or the cladding layer, the groove width pc* than those without grooved holes, and pc* was positively correlated with expansion pressure. Lines A and B coincided, indicating that grooving in the base layer or the cladding layer had little effect on the average residual contact pressure. When there was a groove in the cladding layer, the grooving in the base layer significantly improved pc*, and the double grooving in the base layer was best.F almost increased with pi, and pi had a weak influence on F when the number of grooves was two or three. Under the same expansion pressure, the relationship of pullout force at different grooving locations was as follows: F for one groove in the cladding layer and two grooves in the base layer > F for one groove in the cladding layer and one groove in the base layer > F for one groove in the cladding layer only > F for one groove in the base layer only > F for no grooving. The pullout force of grooved joints was greater than 28.92 kN; therefore, when the strength expansion with a groove width of 8 mm was applied to titanium tubes, even when considering the decline in the pullout force caused by elastic hysteresis, it could still meet the requirements of the standard for the pullout force when the equipment is in service. In addition, during grooved expansion, the pullout force drop caused by elastic hysteresis is smaller than it is in the joint without groove [As shown in t groove ; thus, tpc*. With the increase in groove width, pc* showed an overall upward trend. When the groove width was 10 mm, pc* approached the maximum, and the optimal groove width for uniform expansion was generally 8–10 mm [pc* increased significantly and peaked earlier. In this paper, the expansion pressure of 290–320 MPa was also studied. The results show that a larger pc* was obtained when the groove width was 4 mm. 8–10 mm . It is wpc* of the outer wall of the tube at the corresponding groove corner increased significantly. When the groove width was 6 mm and 8 mm, the titanium tube and the bottom of the groove contacted, and the corresponding position of the tube outer wall generated residual contact pressure. At the same time, “bottoming” led to a decrease in the titanium tube and groove edge line contact pressure, and the overall effect was a slight decrease in pc*. When the groove width was 10 mm, with the increase in contact area between the titanium tube and the groove bottom, the contact was more sufficient, and the pc* increased. When the groove width was greater than 10 mm, pc* showed a slight downward trend.The reasons were as follows: in the expansion process, the external surface of the tube and the grooved structure always contacted from the edge of the groove, followed by the bottom of the groove, and finally filled the side of the groove. When the groove width was 2 mm, a small part of the titanium tube “sank” into the groove, the concentrated line contact pressure was generated between the tube wall and the groove edge, and the residual contact pressure after unloading increased compared with that without grooving. When the width of the groove increased to 4 mm, the “sinking” effect was greater. However, because the slot width was smaller, the middle of the depression was not in contact with the bottom of the groove, and the titanium tube was close to the edge of the groove. At this time, the pc*. The elastic modulus of titanium was small, and the titanium tube could easily “sink” into the groove. Before contact with the bottom of the groove, the titanium tube contacted the edge line of the groove. The groove edge produced a high radial force on the outer surface of the titanium tube, which corresponded to a large As shown in pc*, where w1 was 6 mm and the width of the single groove in the base layer w2 was 10 mm. As depicted in pc* showed an upward trend. In engineering practice, the distance from the single groove in the base layer to the interface can be appropriately increased to improve the performance of the expanded joint.pc*. Here, the width of the clad groove w1 was 6 mm, the width of the double groove in the base layer w2 was 10 mm, and the distance from the first groove in the base layer to the interface was 4 mm. As can be seen from the figure, the double groove space b had little influence on pc*. When the spacing was 6 mm, pc* exhibited the highest value. pc* and pullout force F in the TA2-Q345R tubesheet expanded joint were analyzed. The following conclusions could be drawn:(1)pc* and F in the cladding layer were higher than in the base layer. The connection strength of the TA2-Q345R clad tubesheet joint was better than that of the Q345R tubesheet joint.During tight expansion, the clad tubesheet and the tube were both made of titanium, the rebound after the expansion was consistent; thus, (2)pc* and F could be obtained.In the case of single groove, the residual contact pressure of grooving in the cladding layer was equivalent to that in the base layer, while the pullout force of grooving in the cladding layer was higher than that in the base layer. When the number of grooves in the base layer was two, the optimal (3)The expansion performance of the joint was gradually enhanced with the increase in groove width within the range of 2–14 mm. When the groove width was 4 mm, the residual contact pressure increased significantly due to the higher radial force of the groove edge. The position of the groove had no obvious effect on the joint performance in the base layer or in the cladding layer.In this study, the finite element method was used to explore the influence of different expansion parameters on the performance of the expanded joint between a titanium–steel tubesheet and a titanium tube. The difference between a Q345R tubesheet and TA2-Q345R tubesheet in tight expansion was analyzed, and the effects of groove position and width on residual contact pressure


Primary central nervous system (CNS) germ cell tumors (GCTs) are rare neoplasms predominantly observed in the pediatric and young adult populations. A mixed GCT including immature teratoma exhibiting growing teratoma syndrome is presented. The pathogenesis of growing teratoma syndrome remains unclear, and its treatment strategy has not been established. GCTs are often located within the ventricles, causing hydrocephalus, which sometimes improves after removal of the tumor due to restoration of cerebrospinal fluid (CSF) flow. On the other hand, even if the flow route of CSF from the third ventricle to arachnoid granulations on the brain surface quadrigeminal cistern is restored after removal of the tumor, hydrocephalus may not improve.A case whose intractable hydrocephalus improved after penetrating the aqueductal membrane via endoscopy is described. An 11-year-old boy was treated for pineal intracranial growing teratoma syndrome (IGTS). The tumor grew rapidly in a short period, and hydrocephalus progressed despite endoscopic third ventriculostomy (ETV). Although the obstruction was removed by radiation, chemotherapy, and total tumor resection, the hydrocephalus did not improve. Endoscopic membrane perforation was performed because a membrane-like structure was seen at the entrance of the cerebral aqueduct on magnetic resonance imaging. The hydrocephalus improved immediately after the operation, and the patient’s consciousness disturbance also improved significantly.The purpose of this report is to update the current knowledge and standards of management for patients with growing teratoma syndrome, as well as to drive future translational and clinical studies by recognizing the unmet needs concerning hydrocephalus. CNS germ cell tumors (GCTs) are rare and account for 3.1% of primary brain tumors in Japan . The neuThe timeline from the time of admission is shown in Fig. Because it was considered that obstruction of CSF flow was restored by tumor removal via the occipital transtentorial route, the clamp test was performed several times to remove the extraventricular CSF drainage tube. However, hydrocephalus did not improve Fig. d, and thThe etiology of IGTS is unknown because its incidence is low. Radiation therapy may play a role, but establishing a relationship between IGTS and radiation therapy is difficult , 8. ChemThere are several typical surgical approaches to pineal tumors, including OTA, the supra-cerebellar infra-tentorial approach, and the transventricular approach using an endoscope. In the present case, OTA was the most suitable approach because the tumor had grown rapidly, and a wide view was required to completely remove the large tumor.The most important point is that a good outcome can be obtained without performing ventriculoperitoneal shunting by restoration of normal CSF perfusion to treat the hydrocephalus that persisted after IGTS removal.The membrane that formed at the cerebral aqueduct entrance after tumor removal may have been formed by physical obstruction due to tumor growth, stagnation of CSF flow, inflammation due to surgery, and fibrosis associated with radio-chemotherapy . AccordiWe hypothesize that hydrocephalus after tumor surgery is often caused by impaired absorption of arachnoid granulations, but evaluation of the cause of hydrocephalus is important. Imaging modalities such as MRI CISS are sometimes useful to identify the obstruction point, and this type of imaging is also effective for pre- and postoperative evaluation because it can evaluate the flow of CSF . PhysiciPreviously, CSF was thought to be produced in the choroid plexus, circulate unidirectionally through the arachnoid granulation, and be absorbed. However, CSF is currently thought to move bidirectionally between the CSF cavity and the interstitial fluid of the surrounding brain tissue. . MoisturSchroeder et al. reported that endoscopic aqueduct plasty is effective for membranous occlusion of the cerebral aqueduct , and it Although the effectiveness of shunting has been established, Mallucci et al. reported that about 20% of all types of shunts require shunt revision, a rate that is not low [Although a few case reports of IGTS have been published, this is the first report to describe hydrocephalus after performing effective surgery for IGTS. It was suspected on MRI that a membrane had formed, and that spinal fluid perfusion was impaired. However, it was doubtful that perforating the membrane was enough to improve the hydrocephalus, and it was discussed among the medical team many times. Finally, a challenging approach was attempted to obtain a good outcome without shunting, considering that the patient was a child and his future growth and life. It was very novel that it was possible to achieve this, and it is necessary to accumulate more cases and reconsider the necessity of shunt insertion in the future.In closing, a case of IGTS for which determination of the optimal timing of excision was difficult was presented. Although IGTS basically requires excision, neoadjuvant therapy before excision may eliminate malignant components and prevent dissemination at the time of excision. Thus, the timing of removal should be carefully considered in each case. Establishing normal CSF circulation is important when hydrocephalus occurs, because creating one route from the third ventricle to the posterior part of the brain surface was not effective in the present case.


Generally, successful testis transplantation has been considered to require immune suppression in the recipient to avoid rejection of the transplanted tissue. In the present study, we demonstrate in medaka that allogeneic adult testicular tissue will engraft in adult recipients immediately after partial castration without the use of immunosuppressive drugs. The allografted testes are retained in the recipient’s body for at least 3 months and are able to produce viable sperm that yield offspring after natural mating. Some recipients showed a high frequency (over 60%) of offspring derived from spermatozoa produced by the transplanted testicular tissue. Histological analyses showed that allografted testicular tissues included both germ cells and somatic cells that had become established within an immunocompetent recipient testis. The relative simplicity of this testis transplantation approach will benefit investigations of the basic processes of reproductive immunology and will improve the technique of gonadal tissue transplantation.The online version contains supplementary material available at 10.1186/s40851-022-00195-1. Gonadal or germline transplantations have been used for investigations of reproductive biology/immunology and have also been successfully applied for selective breeding in livestock and aquaculture, species conservation, and fertility treatment. A variety of allogeneic or xenogeneic transplantation protocols for gonadal tissues or germ cells have been developed and used to create potentially superior broodstocks, as insurance against the accidental death of vital broodstocks and for maintenance of threatened breeds and species –6. One oTestis allografting is a possible alternative approach for germline transplantation that can be easily performed, but the potential for immunorejection of donor cells and tissues remains with this method. A few sites in the body display “immune privilege”, in which an antigenic response is not elicited by the presence of transplanted cells. The testes are known to have immune privilege and are more likely to accept transplanted tissues (immune privilege site) and also to be a source of donor cells (immune privilege tissue) . A similOryziaslatipes) for our analyses, as they spawn daily, are amenable to gene editing, and a surgical method for gonadectomy has been established . S. S30]. Sdeadend gene knockdown fish [Methods for allogeneic or xenogeneic transplantation of SSCs, which are abundant in the testis, have been developed in many species. The methods for germ cell transplantation in teleosts can be classified into three approaches : primordown fish , 50. Cytown fish , 42, 43.own fish . In the Cryopreservation methods for the whole testis have been developed in medaka . The comWe demonstrated the feasibility of allografting testicular tissue into immunocompetent recipients whose genetic background was distinctly different to those of the donors; functional engraftment was achieved after partial castration of the recipient without use of immunosuppressive treatments or chemical castration of the recipient. Further studies are required to improve our understanding of the immunological responses after testicular transplantation, and the results of such studies will be of value for aquaculture.Additional file1: Figure S1. The protein level of GFP in the germ cells is relatively low compared to that of the surrounding somatic cells.  Representative images from the IHC analysis using an anti-GFP antibody visualized by DAB staining (a) or fluorescent detection (b). (a) Left panel shows an image of the testis that consistently expressed GFP with beta-actin (actb-GFP hetero). Right panel shows an image of the testis of d-rR (recipient) strain. Scale bar, 100 μm. (b) Upper and lower panels show an image of actb-GFP hetero and d-rR testis, respectively. Left and middle panels show images of DAPI (blue) and GFP (green), respectively, in the same section; right panel shows the merged image. The GFP signal in germ cells was faint in the fluorescent observation. Scale bar, 50 μm.


Cyberknife radiosurgery is a frameless stereotactic robotic radiosurgery which has shown to deliver better treatment outcomes in the treatment of advanced head and neck (H&N) carcinomas, especially in previously irradiated and recurrent cases. The aim of the study was to perform a systematic review of the available data on the outcomes of Cyberknife radiosurgery for treatment of head and neck cancer and to evaluate its collective outcomes. This systematic review was registered with the university with the registration no. FRP/2019/63 and was approved by the Institutional Review Board (RC/IRB/2019/132). Literature search was performed in the following: PubMed, Science direct, SciELO, MyScienceWork, Microsoft Academ EMBASE, Directory of Open Access Journals, and Cochrane databases with the keywords “Cyberknife,” “oral cancer,” “oropharyngeal cancer,” and “head and neck cancer” and data was extracted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The records identified were 147 manuscripts. Excluded articles included 5 duplicate articles, 33 abstracts, 101 full text articles due to being off-topic, case reports, review, non-English, 1 survey, and 2 other articles containing data extracted from a main study which was already included. A total of 5 articles were evaluated for qualitative synthesis. The mean dose of Cyberknife radiosurgery delivered for previously irradiated recurrent H&N carcinoma patients was 34.57 Gy, with a mean sample size of 5 studied during the period of 2000 to 2016. The available evidence from the systematic review indicates that Cyberknife can be an efficacious treatment option for recurrent previously irradiated H&N carcinoma, especially for nonresectable tumors. There is paucity of homogenous data and studies in this arena; hence, meta-analysis could not be performed. Further standardized studies are essential, especially where the treatment of H&N carcinoma is considered. The concept of Cyberknife radiosurgery was invented by a Stanford neurosurgeon, Dr. John Adler, and came into effective practice by 1900. Cyberknife is a stereotactic radiosurgical unit capable of irradiating the tissues using ionizing radiation. The ability of beam shaping using multilamellar collimators and three-dimensional conformation of the target tissue with submillimeter accuracy make this radiosurgical system stand out from the other radiosurgical units which has accuracy in millimeters. This highly precise dose delivery enables minimal damage to the surrounding structures.Cyberknife is used for the treatment of benign and malignant lesions as well as intracranial and extracranial lesions, including whole body radiosurgery.Cyberknife has also been widely used in the treatment of head and neck (H&N) cancers and lesions. However, no systematic reviews have been conducted to determine its efficiency. Hence, this systematic review was conducted to provide valuable information on the treatment efficacy and analyze the outcomes of the treatment with Cyberknife.The research proposal was registered with the university with the registration no. FRP/2019/63 and was approved by the Institutional Review Board (RC/IRB/2019/132). A meticulous literature search was made in the following major search engines: PubMed, Science direct, SciELO, MyScienceWork, Microsoft_Academ EMBASE, Directory of Open Access Journals, and Cochrane databases with the keywords “Cyberknife,” “oral cancer,” or “oropharyngeal cancer,” and “head and neck cancer.” This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Gross tumor volume (GTV) was defined as the volume of visible tumor, as evaluated from imaging studies.Complete response (CR) was defined as disappearance of all target lesions.Partial response (PR) was defined as 30% decrease in the sum of the longest diameter of target lesions.Progressive disease (PD) represented 20% or greater increase in the sum of the longest diameter of target lesions.Stable disease (SD) represented targets where there is neither sufficient shrinkage to be termed as PR nor sufficient increase to qualify it as PD.Overall survival (OS) represents the period from the start of reirradiation radiotherapy to the date of demise from any cause or till the last follow-up.From identification to eligibility phase, all authors  individually reviewed and compiled data with the guidance and supervision of C.S. Database search contributed to 141 articles and a Google search for additional data retrieved 6 articles, with a total number of 147 manuscripts in the identification phase. Five duplicates were removed, and 33 abstracts were removed during screening. For assessment of eligibility, 101 articles were excluded due to the following reasons: off topic—76, case reports—10, reviews—9, non-English articles—6. Further, three articles were excluded: 1 survey and 2 articles which projected outcomes from one main study, which was already included for final phase of systemic analysis including 5 articles .After careful scrutiny of the published data, C.S. evaluated the articles to be included in the qualitative synthesis, with a total of 5 articles included for data extraction on the outcomes of Cyberknife radiotherapy for (H&N) cancer treatment, comprising 275 patients studied during 2000 to 2016. Data was extracted and cross-evaluated by all the authors. Details of the included studies with their sample sizes and demographics are presented inIn this study, we evaluated the treatment efficacy of Cyberknife for the treatment of H&N cancers on 275 patients between the years 2000 to 2016. The outcomes assessment for CK treatment of H&N cancer was assessed on locoregional tumor control, overall survival rate at 1 year and 2 years, median survival rate, mean follow-up period, and development of major complications during the period of follow-up. Cyberknife treatment was found to have a good loco regional tumor control,Radiotherapy for the management of cancers were used as one of the methods of primary management in the early 20th century due to limitations of anesthesia and surgical outcomes. However, prior to 1948, radiotherapy administration was limited to radium implants and orthovoltage radiation with insufficient depth of penetration into the tissues and increase in severity of skin damage.Gammaknife was invented in 1972, and it is currently one of the prominent radiotherapy delivery units. However, it requires skeletal fixation, which can cause pain, thereby restricting flexible and highly precise conformal radiotherapy.Cyberknife radiotherapy can be used for complex tumors such as around the spine in which surgical outcomes may be undesirable, cases of recurrent tumors with proximity to vital structures, as a concurrent or adjuvant for remaining inaccessible tumor after surgery, as a palliative therapy, and also in cases of previously irradiated cases of H&N cancers with or without concurrent radiotherapy.In the present systematic review, we also found results similar to the previously quoted 2017 study by Mahboubi et al for vestibular schwannomas using Cyberknife. All the patients analyzed in this study were previously irradiated/treated H&N carcinoma patients with recurrence at the time of Cyberknife radiotherapy. The mean survival rate was 10.9 months after Cyberknife radiotherapy. Out of 275 patients, 11 patients experienced CBOS. However, it was not mentioned that whether the interval between previous radiotherapy and Cyberknife treatment in these 11 patients were less than 30 months or more than 30 months. CBOS is a life-threatening conditions seen in patients with H&N cancer.Recent data from the International Agency for Research on Cancer (IARC) based on the 2018 report on lip and oral cavity cancers and associated death from 185 countries imparts light on the newly diagnosed cancer cases per year and that it could be considered to carry a high proportion of H&N cancers.There were several precincts in this study. The outcomes reported in the published studies have heterogenous methodology and parameters of assessment hindering the meta-analysis in all variables. Low samples size in majority of these studies was also a limiting factor. Association of outcome variables to previous treatment methodologies and its interval were not available. Selection bias was found to be common in many of the studies. All the studies included were not retrospective cohort studies. The factors listed above pose an inherent limitation in drawing conclusions regarding Cyberknife treatment in H&N cancer patients. Nevertheless, evaluation of the available data suggests that Cyberknife treatment for H&N cancers offers patients significant control on distant metastatic progression with preservation of vitality of adjacent vital structures and less toxicity. This study is the first study to systematically review the treatment efficacy of Cyberknife radiotherapy on previously irradiated recurrent H&N cancer.The mean dose of Cyberknife radiosurgery delivered for previously irradiated Recurrent H&N carcinoma patients was 34.57 Gy, with a mean sample size of 51 studied during the period of 2000 to 2016. Cyberknife treatment limits the occurrence of distant metastatic progression, thereby increasing the overall survival rates. The available evidence from the systematic review indicates that Cyberknife can be an efficacious treatment option for recurrent previously irradiated H&N carcinoma, especially for nonresectable tumors. There is paucity of homogenous data and studies in this arena. Further standardized studies are essential, especially where the treatment of H&N carcinoma is considered.


C4A) gene expression are linked to excessive synapse elimination. In two independent cohorts, we show that cerebrospinal fluid (CSF) C4A concentration is elevated in patients with first-episode psychosis (FEP) who develop schizophrenia . We show that the CSF elevation of C4A in FEP-SCZ exceeds what can be expected from genetic risk variance in the C4 locus, and in patient-derived cellular models we identify a mechanism dependent on the disease-associated cytokines interleukin (IL)−1beta and IL-6 to selectively increase neuronal C4A mRNA expression. In patient-derived CSF, we confirm that IL-1beta correlates with C4A controlled for genetically predicted C4A RNA expression . These results suggest a role of C4A in early schizophrenia pathophysiology.Postsynaptic density is reduced in schizophrenia, and risk variants increasing complement component 4A ( C4) genes. Here the authors show that C4A is elevated in individuals with schizophrenia.Schizophrenia risk has been associated with the complement component 4 ( C4) genes. Distinct from mouse, human C4 is encoded by two closely related genes, C4A and C4B, then typically present in multiple copy numbers (CNs) per genome and gene isotype. SCZ risk attributed to this locus can largely be explained by genetically predicted C4A RNA expression, and elevated C4A expression has been confirmed in SCZ postmortem brain tissue1. During brain development, microglia utilize complement signaling for selective removal of supernumerary synapses by complement receptor 3 (C3R)-dependent phagocytosis3. In line with the observed decrease in synapse density in SCZ5, excessive microglial synapse elimination has been observed in SCZ-derived in vitro models6. C4A CNs also correlate with complement deposition in patient-derived models, as well as complement-dependent microglial engulfment of synaptic structures6. Notably, C4B CNs, not linked to SCZ risk1, do not influence neuronal complement deposition or synapse elimination in these models6. More recently, these in vitro findings were also confirmed in vivo using mouse models overexpressing mouse C47 or human C4A8.Schizophrenia (SCZ) is a highly heritable and polygenic brain disorder with the strongest associated SCZ locus located close to the complement component 4 , collected from two independent cohorts of patients with first-episode psychosis (FEP), hypothesizing that patients with FEP who develop SCZ (FEP-SCZ) would display elevated CSF concentrations of C4A, although not C4B, as compared to matched healthy controls (HCs) . As polygenic risk scores have been shown to discriminate FEP-SCZ cases from FEP-nSCZ cases10, we hypothesized that patients with FEP-SCZ would also display elevated CSF C4A concentrations as compared to patients with FEP-nSCZ, given that this within patients comparison would be less prone to confounding related to patient status.Despite the accumulating evidence from experimental models linking Cs) Fig. . Taking n = 29) or not . The three groups (HCs and two patient groups) displayed no significant differences in terms of demographics and clinical characteristics (Supplementary Table P (FEP-SCZ vs. HCs) = 0.025, adjusted P (FEP-SCZ vs. FEP-nSCZ) = 0.037); Fig. P (FEP-SCZ vs. HCs) = 0.550, adjusted P (FEP-SCZ vs. FEP-nSCZ) = 0.098; Fig. P = 0.931), then suggesting that the elevation in C4A levels in patients with FEP-SCZ were not due to confounding by antipsychotic drugs.In the first cohort  we included 44 patients with FEP and 20 age- and sex-matched HCs. Patients with FEP were stratified depending on if they subsequently developed SCZ  and 21 HCs. The FEP-nSCZ group included more smokers (64%) than the HC group (34%) but smoking was unrelated to CSF C4A or C4B concentration . Otherwise, the three groups did not display any major differences in terms of demographics and clinical characteristics (Supplementary Table P (FEP-SCZ vs. HCs)=0.047, adjusted P (FEP–SCZ vs. FEP-nSCZ)=0.001); Fig. P (FEP-SCZ vs. HCs)=0.999, adjusted P (FEP-SCZ vs. FEP-nSCZ)=0.708; Fig. P = 0.193).To replicate our initial finding, we used an independent FEP cohort , comprising of 31 patients with FEP , we observed elevated CSF C4A concentrations in patients with FEP-SCZ as compared to both patients with FEP-nSCZ (57% increase) and HCs (51% increase), while CSF C4B concentrations were not significantly different between the groups  and were used for exploratory analyses of correlations between CSF C4A concentrations and disease phenotypes. After controlling for multiple testing, we observed no significant correlations between CSF C4A concentration and scores on the positive, negative, and general psychopathology scale of the Positive and Negative Syndrome Scale (PANSS), or performance in the key domains of the MATRICS Consensus Cognitive Battery (MCCB)  = 0.036, habituation 3: r = 0.43, CI: 0.14–0.65 P(adjusted)=0.036; habituation 5: r = 0.35, CI: 0.05–0.60 P(adjusted)=0.040, habituation 6: r = 0.39, CI: 0.09–0.63 P(adjusted)=0.036, habituation 9: r = 0.37, CI:0.07–0.61 P(adjusted)=0.036, habituation 10: r = 0.39, CI: 0.09–0.62 P(adjusted)=0.036, and habituation 14: r = 0.37, CI: 0.07–0.60 P(adjusted)=0.036; Supplementary Table 13.Clinical data regarding severity and symptom profiles were available for the KaSP cohort (B) Table . We thenC4A CNs and C4A RNA expression in the brain1, previous studies suggest that upregulation of C4A expression in SCZ exceeds what can be expected from genetic risk variance in the C4 locus14. To test this at the protein level, we used droplet digital PCR (ddPCR) to measure C4A CNs and HERV insertions in patients with FEP that had available DNA and CSF C4A protein levels in the GRIP cohort (n = 22 FEP and n = 21 HCs). We observed a moderate correlation between C4A CNs and CSF C4A protein levels , as well as between genetically predicted C4A RNA expression1 and CSF C4A protein levels  then remained significantly elevated in patients with FEP-SCZ as compared to patients with FEP-nSCZ and HCs =0.039 and 0.031 respectively, P (FEP-SCZ vs. FEP-nSCZ)=0.029 and 0.020 respectively; Supplementary Fig. C4 locus contributes to the increase in CSF C4A protein levels in SCZ. However, CSF C4B protein levels were not significantly different when adjusting for copy numbers (as well as predicted C4B RNA expression) between FEP-SCZ and HC or FEP-nSCZ −1beta and IL-6 both have repeatedly been shown to be elevated in CSF of patients with SCZ18, and we therefore explored their influence on neuronal C4A expression. To this end, we generated stably inducible neurogenin 2 (NGN2) neural progenitor cells from induced pluripotent stem cell (iPSC) of four patients and differentiated these cells to cortical excitatory neurons. These derived cultures were then stimulated for 24 hours with either IL-1beta (1 ng/ml), IL-6 (1 ng/ml), or a combination of IL-1beta and IL-6  stimulated cultures from the same patients  that had been genotyped for C4 haplotypes (ddPCR) and had equal CNs of C4A and C4B (two CNs for each gene). The significant increase in relative C4A mRNA expression after IL1beta/IL-6 stimulation remained when only analyzing cultures from this line (VEH: median (fold change) = 1.0, CI:0.66-1.5, IL-1beta: median = 0.98, CI:0.50-1.7, IL-6: median = 2.1, CI:1.1-2.4, IL1beta/IL-6: median=2.5, CI:2.0-3.5, Kruskal-Wallis H test: adjusted P (VEH vs. IL-1beta) = 0.999, adjusted P (VEH vs. IL-6)=0.285, and adjusted P (VEH vs. IL1beta/IL-6) = 0.004), while no significant increase was observed for C4B mRNA expression after IL-1beta/IL-6 stimulation  = 0.999, adjusted P (VEH vs. IL-6) = 0.411, and adjusted P (VEH vs. IL1beta/IL-6) = 0.094).Recent brain co-expression network analysesL-6 Fig. . We obsents Fig. , while nion Fig. , then suC4A RNA expression using whole genome sequencing data1, we observed a significant correlation between IL-1beta and CSF C4A concentration  were recently shown to inversely correlate with prefrontal cortical thickness determined by Magnetic Resonance Imaging (MRI), as well as to predict synapse loss in dementia and Alzheimer’s disease20. Thus, we measured these three markers in CSF from participants in the KaSP cohort . After adjusting for age, sex, and diagnosis, we observed significant negative correlations in CSF between C4A and neuronal pentraxin 1  = 0.026), neuronal pentraxin 2  = 0.026), and the neuronal pentraxin receptor  = 0.040), see Fig. As C4A CNs on microglial synapse engulfment in patient-derived models6, we show that C4A protein levels in patient-derived CSF inversely correlate with previously validated proxies of synapse density. This emphasizes the in vivo relevance of C4A for synapse density in SCZ and supports the notion from human experimental studies that C4A CNs influence SCZ risk by causing excessive synaptic pruning. Although the elevation of CSF C4A protein levels in patients with FEP who develop SCZ was modest, it was still larger than expected from previous genetic studies. Thus, on protein level we corroborate previous findings from gene expression analyses that C4A CNs do not fully explain the observed C4A RNA upregulation in SCZ14. Using a patient-derived cellular model, we show that IL-1beta/IL-6 induces neuronal C4A mRNA expression. As patients with SCZ as a group display elevated levels of these cytokines in CSF21, the increase in C4A levels per C4A CNs can therefore be expected to be more pronounced in SCZ as compared to HCs and partly explain the remaining elevation in patients with FEP-SCZ after adjusting for C4A CNs. This hypothesis is supported by our CSF analyses, where we show that IL-1beta levels correlate with CSF C4A levels per C4A CNs, suggesting in vivo relevance. The results are in line with studies showing that immune activation in SCZ, including increased levels of pro-inflammatory cytokines such as IL-1beta and IL-6, are linked to a more pronounced cortical thinning and worsen cognitive performance24. Further, IL-1beta has been proposed as a biomarker also for several neurocognitive phenotypes beyond SCZ26, as well as in suicidal behavior28. Further studies are warranted to evaluate if a mediation by induction of C4A mRNA expression and excessive synaptic pruning contributes to these phenotypes.This study demonstrates that C4A protein concentrations are specifically elevated in the CSF of patients with FEP who subsequently develop SCZ, strengthening the notion of a role for this complement component in the early pathophysiological events of SCZ. Furthermore, in line with the effect of 29, and from a mechanistic perspective habituation largely relies on synaptic plasticity. Thus, our data suggest that patients with SCZ and a high C4A CN burden may exhibit more difficulties with habituation due to inappropriate neuronal wiring. Notably, we observe no significant correlations with performance measurements across the domains in the MATRICS test battery. This is at odds with previous studies reporting inverse associations between genetically predicted C4A expression (imputed from common variants) and performance in tests measuring memory aspects and processing speed31. While it is likely to assume that the use of CSF protein levels reduced non-differential bias, the obvious reduction in sample size  made our sample less suitable for studying the influence of the C4 locus on cognition due to limited power.Utilizing detailed clinical data regarding severity and symptom profiles we also study within-subject correlations to CSF C4A levels. We observe a clear inverse correlation between CSF C4A concentration and habituation of the startle response. Patients with SCZ as a group display decreased habituation of the startle response35, and C4 levels in plasma do not significantly correlate with total C4 levels in CSF36. In the current study, we separate C4A and C4B levels in CSF and in line with previous experimental and genetic studies we observe that only C4A is increased in first episode patients with SCZ. A recent study reported elevated total C4 concentrations in 32 patients with chronic SCZ spectrum disorder (median duration of illness of 14 years) with ongoing antipsychotic medication36. Thus, we also measured a peptide corresponding to total C4 levels in CSF but unlike the previous study of chronic patients with schizophrenia we did not observe a significant elevation of total C4 levels in CSF of patients with FEP  and the Institutional Review Board of Partners HealthCare . Informed consent was obtained from all included subjects (CSF sampling and skin biopsies for deriving iPSCs).KaSP Cohort: Karolinska Schizophrenia Project (KaSP) is a Swedish multidisciplinary research consortium investigating the pathophysiology of FEP and SCZ. Patients who seek health care for psychotic symptoms for the first time are recruited at psychiatric emergency wards and in- or outpatient facilities at the psychiatric clinics located in Stockholm, Sweden. Exclusion criteria are ongoing or previous prescription of an antipsychotic for more than 30 days, severe somatic and neurological diseases, current substance abuse (except nicotine use), or autism spectrum disorder. These patients are excluded by clinical examination, medical history, routine laboratory tests, including screening for drugs and MRI scans. All KaSP study participants were white. A baseline diagnosis is established based on a structured clinical interview of the Diagnostic and Statistical Manual of Mental Disorders IV, DSM-IV  at the time of inclusion. For the subdivision of patients with FEP into a FEP-SCZ and a FEP-nSCZ group we used data from a 1.5-year follow-up (median time to follow-up: 1.6 years). The same diagnostic procedure as at baseline was used. Patients who did not receive a SCZ diagnosis at follow-up received a diagnosis of delusional disorder, unspecified psychosis, acute and transient psychotic disorder, schizoaffective disorder, or major depressive disorder.HCs were recruited by advertisement and matched on age and sex. Eligibility was determined by medical history, routine laboratory tests, clinical examination, and an MRI examination, as evaluated by an experienced neuroradiologist at the MR Centre, Karolinska University Hospital, Solna. The Mini International Neuropsychiatric Interview (MINI), performed by either a resident or a specialist in psychiatry, was used to exclude previous or current psychiatric illness. Further exclusion criteria were former or current use of illegal drugs, first-degree relatives with psychotic illness or bipolar disorder, as well as neurologic disease and/or severe somatic disease. All participants (patients and controls) were free from any form of substance abuse evaluated with Alcohol Use Disorders Identification Test (AUDIT) and the Drug Use Disorders Identification Test (DUDIT) at the time of the study. All eligible subjects with available data were included in the present study.r = 0.15 CI: −0.25–0.50, P = 0.454) or C4B levels , as well as to a SCZ- vs. a non-SCZ-related diagnosis . Patients who did not receive a SCZ diagnosis at the diagnosis evaluation around 2 years after baseline received a diagnosis of bipolar disorder, brief psychotic disorder, substance-induced psychotic disorder , or a psychotic disorder not otherwise specified.GRIP cohort: The Gothenburg research and investigation on psychosis (GRIP) is a study collecting data from patients with schizophrenia spectrum disorders at the psychosis clinic, Sahlgrenska University Hospital, Mölndal, Sweden. The initial diagnostic assessments are performed by treating clinicians at outpatient tertiary-care units, as well as the inpatient units, in Sahlgrenska University Hospital (Sweden). Further clinical information is obtained from interviews at tertiary-care units and investigation units, and when needed supplemented with information from clinical records. To secure diagnoses for research, a series of case conferences are then held (for this study in between October 2017–March 2018). A minimum of two board-certified psychiatrists participates in these case conferences where a consensus diagnosis is established according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). The diagnosis is based on structured interviews, medical records , and when needed supplemented with information from the treating psychiatrist. The participating research clinicians are blinded to the results of the CSF analyses. The median time from inclusion in the study  to the establishment of a research consensus diagnosis on the case conference was here 1.8 years. For patients with FEP, the median time from first contact with psychiatric care to referral for further investigation of psychotic symptoms at the tertiary unit  was 2 years. This time period, as well as time to a SCZ diagnosis within the study period, was unrelated to CSF C4A  22 G spinal needle inserted at the L4–5 interspace with collection of 18 mL of CSF. Not more than 1 h after collection, CSF was centrifuged at 3500 rpm for 10 min divided into 10 aliquots and stored at –80 °C until analysis. All CSF samples were individually analyzed.GRIP cohort: Subjects fasted overnight, and the lumbar puncture was performed between 10.45 and 11.15 am. A non-cutting (atraumatic) 22 G spinal needle was inserted into the L4/L5 interspace and about 15 ml of CSF was collected, gently inverted to avoid gradient effects, centrifuged for 10 min at 3500 rpm at 20 degrees, not more than 30 min after sampling. CSF was then divided into 0.5 ml aliquots that were stored at −80 °C pending analysis. All CSF samples were individually analyzed.The protein concentration of the CSF samples was measured in triplicates using Micro BCA Protein Assay Kit  prior to sample preparation. The measurements were performed in a 96-well plate  and fluorescence read by plate reader FLUOstar Omega. . Sample preparation was performed on the Agilent AssayMAP Bravo Platform. Fifty µg protein of each CSF sample was used for analysis. Depending on the protein concentration of the samples, the appropriate volume of each sample was transferred to a 96-well plate (Greiner G650201) and 200 mM triethylammonium bicarbonate buffer  manually added to a final volume of 50 µL. The Bravo platform was used for the following steps; addition of 50 µL TEAB buffer and 50 µL 3.3% solution of sodium deoxycholate  for dissociation of proteins . Proteins were reduced by the addition of 10 µL 80 mM tris(2-carboxyethyl) phosphine  and the reaction was carried out for 1 h at 55 °C. Alkylation was carried out by the addition of 15 µL 120 mM iodoacetamide  for 30 min in the dark at room temperature. One µg trypsin  was added to each sample and incubated overnight at 37 °C. The CSF samples were divided into four groups for sample preparation, and they were labeled with #1, #2, #3, and #4. In group #3, the protein concentration was lower than in the other groups and the starting volume for each sample was therefore more than 50 µL. To these samples, 200 mM triethylammonium bicarbonate buffer was manually added to a total volume of 100 µL and additional liquid handling steps were carried out on the Bravo platform. The trypsin digestion was quenched by the addition of 30 µL 10% FA  and the SDC pellet was removed by filtering the samples through a polypropylene filter plate  with hydrophilic PVDF membrane (mean pore size 0.45 µm). The tryptic peptide concentration was detected using the Pierce Quantitative Colorimetric Peptide Assay . Each sample was spiked with a mixture of heavy isotope-labeled peptide standards (AQUA QuantPro peptides from Thermo Fisher Scientific) before LC-MS analysis.m/z. Precursors were fragmented by higher energy collision dissociation (HCD) and detected in Orbitrap detector with a resolution of 60,000. The normalized collision energy (NCE) for HCD was 25%. The values for the AGC target and maximum injection time were 5 × 104 and 118 ms, respectively. The peptide inclusion list is shown in Supplementary Table The LC-MS analysis was performed on a Tribrid mass spectrometer Fusion equipped with a Nanospray Flex ion source, coupled to an EASY-nLC 1000 ultra-high pressure liquid chromatography (UHPLC) system . The tryptic CSF peptides spiked with AQUA peptides, were loaded on to an Acclaim PepMap 100 C18 precolumn  and separated on an Acclaim PepMap RSLC column  with the flow rate 300 nL/min. The column was kept at 40 °C using a Phoenix S&T Nano LC column heater . Solvent A  and solvent B  were used for the nonlinear gradient. The percentage of solvent B was 1% for the first 3 min and increased to 30% in 50 min and then to 90% in 5 min which was kept for 7 min to wash the column. The tryptic CSF peptides spiked with AQUA peptides, were introduced into the mass spectrometer via a stainless-steel Nano-bore emitter  with the spray voltage of 2 kV and capillary temperature 275 °C. The Orbitrap Fusion was operated in parallel reaction monitoring (PRM) mode. MS2 precursors were isolated with a quadrupole mass filter set to a width of 1.2 The linear range of each heavy peptide was measured in pooled CSF digest. A mixture of heavy AQUA peptides was spiked at five different concentrations into pooled CSF samples. The concentration range was adjusted for each individual peptide according to its expected endogenous signal. Each sample was analyzed three times, and the peak areas of heavy peptides were plotted against the spiked heavy peptides concentrations Fig. .Fig. 5ReH test followed by post-hoc tests: adjusted P[FEP-SCZ vs. HCs] = 0.038, and adjusted P[FEP-SCZ vs. FEP-nSCZ] = 0.00003), while C4B levels in the KaSP cohort remained similar across groups . Similar results were also obtained by imputing values with half of the limit of detection in the GRIP cohort  and in the KaSP cohort .All the raw data generated on the Fusion MS were imported to Skyline v4.1  for data analysis. Peak integration was done automatically by the software and was manually inspected to confirm correct peak detection. Peak identities were confirmed by measured transitions (dotp) and also between endogenous and corresponding heavy peptides (rdotp). The best 3–5 transitions were selected for the quantification and was performed by matching light and heavy peak area ratios. All calculations were performed using Microsoft Excel. For quality control, we also measured a peptide shared for C4A and C4B . Finally, two subjects did not display detectable C4A (in the GRIP chort) and C4B (in the KaSP cohort) peptide levels. We decided to initially exclude these subjects (one sample from the KaSP cohort and one sample from the GRIP cohort) in the main analyses. However, elevated levels of CSF C4A in FEP-SCZ remained in the GRIP cohort when including all values  in the GRIP cohort were measured from genomic DNA isolated from blood samples6. C4A-HERV+, C4A-HERV−, C4B-HERV+, and C4BHERV− CNs were determined based on imputations. A detailed protocol for imputations can be found on Github: https://github.com/freeseek/imputec4. For distributions of C4 CNs in the samples see Supplementary Fig. C4A, or C4B, CNs displayed no association to age  or sex .Using ddPCR, CNs of C4 structural elements  in the KaSP cohort were imputed from MHC genotypes computed from whole genome sequencing (WGS) data. HapMap3 CEU reference haplotype panel, from Sekar et al.1, was used to impute pre-annotated C4 haplogroups, using Beagle (version 3.3), with subsets of SNPs in the extended MHC locus (chr6: 25–34 Mb).The Measurement and Treatment Research to Improve Cognition in Schizophrenia Consensus Cognitive Battery was used to evaluate cognitive function in the KaSP cohort. This battery contains 10 tests that measure 7 cognitive domains: Speed of processing ; Attention/vigilance ; working memory ; verbal learning ; Visual learning ; reasoning and problem solving  and social cognition . One psychologist (HFB) administered all the tests.Electromyography (EMG) was recorded from the orbicularis oculi using two 20-mm disk Ag/AgCl electrodes positioned below and lateral to the eye. A 40 × 50 mm ground electrode was implanted behind the ear on the bone. EMG activity was filtered (1- to 1000-Hz notch filter and 60-Hz notch filter) and digitized at a rate of 1 kHz. Acoustic stimuli were supplied via headphones using a Psylab Stand Alone monitor and a tone generator . The EMG signal was amplified with a Grass A.C. Amplifier  and acquired by using commercially available hardware/software (BioPac) on a laptop computer (Hewlet Pacard Compaq 6715b).37. The next three blocks each consisted of 15 trials: 4–5 pulse-alone trials and 5 prepulse-pulse trials with the ISI of interest and 5 prepulse-pulse trials with other ISIs . Processing of the EMG recording was done offline with a 100-Hz high-pass filter and baseline correction by using a 100 ms prestimulus baseline. Response onset was defined by the first crossing from baseline within a 20–120 ms window after stimulus onset. The difference of the most positive peak and most negative in a 20–150 ms window after pulse onset was used to calculate the peak response amplitude. The following startle measures were examined; (1) reactivity, or the magnitude of response (startle amplitude (SA), (2) PPI or the percentage of change in startle magnitude to prepulse + pulse versus pulse-alone trials ((pulse − prepulse + pulse) / pulse) * 100), (3)29 habituation or the decrement in reactivity with repeated stimulus administration. The primary outcome measures of PPI were calculated for 30, 60, and 120 ms ISI conditions, respectively, as percent change in response amplitudes [% PPI = /mean startle alone × 100]. Startle response was calculated by comparing the mean startle magnitude of the middle five pulses during the first trial .All subjects (KaSP) were tested using the same startle system. Subjects were instructed to relax and keep their eyes open and directed against a position marked on the wall, during the test. Throughout the session, background noise was set to 70 dB. The session consisted of 60 trials with two conditions: pulse-alone trials, consisting of a 40 ms, 115-dB pulse of white noise and prepulse-pulse trials, consisting of a 40 ms 115 dB pulse  preceded by a 16-dB (above background) prepulse of 20 ms duration (white noise). The ISI between the prepulse and the pulse tested were 30, 60, and 120 ms, respectively. A 3-min acclimation period with 70 dB white noise started the session. The immediately following block consist of 15 pulse-alone trials, used for the calculation of habituation using the latent curve modeling6.iPSCs from four chronic patients  were used in this study. The patients were recruited from outpatient units at the Massachusetts General Hospital (MGH). All individuals signed a written informed consent before participating in the study, as approved by the Institutional Review Board of Partners HealthCare. All relevant ethical regulations were followed when performing the study. A structured psychiatric evaluation was performed using the Structured Clinical Interview for DSM-IV and the Mini-International Neuropsychiatric Interview by a single psychiatrist with at least 5 years of clinical experienceFollowing subcutaneous injection of lidocaine 1%, a 3.0 or 4.0 mm punch tool was used to obtain a dermal biopsy sample from the non-dominant forearm. Biopsies were collected into a transport medium of DMEM (Gibco) with 1% penicillin–streptomycin–glutamine . Upon receipt of samples, they were washed twice in PBS with 10% penicillin–streptomycin–glutamine. Two 60-mm tissue culture-treated plates were used, one to cut up the biopsy and the other one to plate the pieces of biopsy. A scalpel was used to cut the biopsy into small pieces and then to grid the dish where the pieces of biopsy were plated (dermis side down). The dish was placed in an incubator at 37° for 15 min in order for the skin to adhere to the dish. Medium of DMEM with 10% FBS and 1% penicillin–streptomycin–glutamine was finally gently added over the pieces of skin.The collected fibroblasts were then reprogrammed (mRNA reprogramming) and the resulting iPSC colonies stabilized and expanded under xeno-free conditions by Stemiotics . Briefly, stable iPSCs were expanded in NutriStem XF medium  and on biolaminin 521 LN-coated (BioLamina) plates to at least passage 3. iPSCs were then purified using MACS with anti-TRA-1–60 MicroBeads (Miltenyi Biotec) on LS columns according to the manufacturer’s instructions. All fibroblasts and iPSCs were screened and found negative for Mycoplasma; they stained positive for octamer-binding transcription factor 4  and TRA-1–60 Fig. .NGN2 expressing stable NPC lines using TALEN-based plasmids as previously described6. Briefly, the doxycycline-inducible NGN2 AAVS1 knock-in plasmid, based on an AAVS1 SA-2A-puro-pA donor , was generated by replacing the puromycin resistance gene with the neomycin resistance gene and cloning a cassette containing the Tet-On 3G promoter driving the human NGN2 complementary DNA followed by P2A, a zeocin resistance gene, the BGH polyA, CMV early enhancer/chicken β actin (CAG) promoter driving Tet-On 3G and BGH polyA. The AAVS1-targeting TALENs are based on hAAVS1 1R TALEN and hAAVS1 1L TALEN  and were generated by golden gate assembly. Plasmid maps can be provided upon request. NPCs were collected with Accutase and counted. For each transfection, 4 × 106 cells were pelleted at 300 g and then resuspended in 100 μL prewarmed nucleofector solution ; 4 μg of NGN2 plasmid and 1.5 μg each of 1R and 1L plasmids were added directly to the resuspended cells, or negative control without plasmid, followed by nucleofection  according to the manufacturer’s protocol using program B-016. After nucleofection, cells were plated onto Geltrex-coated six-well plates in neural expansion medium . Stable lines were expanded by selection with the addition of 125 μl mL−1 G418  over 8–10 d or until the negative transfection control cells completely died.We then generated NGN2 neurons were then generated as described earlier6. Briefly, NPCs were plated as single cells on poly-L-ornithine/laminin coated 24-well polypropylene plates at 2.5 × 105 cells/500 μL in 50% neurobasal medium  and 50% DMEM/F12 (ThermoFisher Scientific) with 1× neural induction supplement (Thermo Fisher Scientific). On the following day, the medium was replaced with neuronal differentiation medium , 1× N-2 supplement (ThermoFisher Scientific), 10 ng mL−1 brain-derived neurotrophic factor  and 10 ng m L−1 recombinant human NT-3 ). Day 2 cells were differentiated in neuronal differentiation medium enriched with 1× B-27 (ThermoFisher Scientific) and 2 μg m L−1 doxycycline . This medium was maintained for the entire differentiation process, unless noted. On days 3–6, cells were incubated with the same medium with the addition of 5 μg m L−1 zeocin (ThermoFisher Scientific). For neuronal maturation, 11 days after seeding, astrocyte conditioned medium  was added to the culture. Assays were then performed at day 25.NGN2 neurons were cultured on cover slides coated with poly-L-ornithine/laminin. Cells were fixed in 4% paraformaldehyde in PBS for 15 min. Following permeabilization in PBS containing 0.1% Triton-X100 for 5 min, the slides were blocked with blocking solution  for 1 hour at room temperature. Primary antibodies against MAP-2 , Beta III Tubulin , Oct4 , TRA 1-60  in blocking solution were added and incubated overnight at 4 degrees C. Cells were then washed with PBS and incubated with the corresponding Alexa Fluor-conjugated secondary antibodies (1:500) for 1 h at room temperature. The following secondary antibodies were used: goat anti-chicken , goat anti-mouse  and goat anti-rabbit .NGN2 neurons were treated with either VEH, 1 ng/mL IL-1β  or 1 ng/mL IL-6 , alone or in combination for 24 hours. Following stimulations of cells, total RNA was extracted using Trizol® Reagent (Invitrogen) according to the manufacturer’s protocol. The quantity and purity of total RNA was measured using a NanoDrop® 1000 spectrophotometer (Thermo Scientific). Complementary DNA was synthesized using High-Capacity RNA-to-DNATM Kit (Applied Biosystems) following the manufacturer’s protocol. qPCR was performed with TaqMan Gene Expression Assays (Applied Biosystems) by using the StepOnePlus Real-time PCR system (Applied Biosystems). Each 20 μL PCR reaction contained 5 μL of cDNA, 300 nM of forward and reverse primer, 250 nM of probe and 10 μL of TaqMan PCR master mix (Applied Biosystems). Primers for C4A and C4B reactions were as follows (forward: GCAGGAGACATCTAACTGGCTTCT and reverse: CCGCACCTGCATGCTCCT). Specific probes for C4A (FAM-ACC CCT GTC CAG TGT TAG-MGB) and C4B (FAM-ACC TCT CTC CAG TGA TAC-MGB) were used. ∆Ct values were obtained by comparison to Ct values of the housekeeping gene  for each technical replicate. Fold changes (2 − ∆∆Ct) were then generated by normalization to the average ∆Ct value for VEH treated wells per plate. C4B mRNA could not be detected in non-stimulated cultures from one subject (not genotyped) and these cultures were excluded from quantification of C4B mRNA expression after stimulations. In one culture, C4A and C4B mRNA expression were also ~100× larger than in the other cultures and this well was excluded (see Source data). All other wells that passed QC  were analyzed by qPCR and included in the statistical analyses.After 25 days, mature IL-1beta and IL-6 were previously measured as part of the Novex Human Ultrasensitive Cytokine 10-Plex Panel  on the Luminex immunoassay platform. In contrast to the manual provided with the kit, an additional calibration curve point was included to extend the assay dynamic range in the low concentration range. 50 µl sample was diluted with 50 µl of sample diluent, the final sample dilution factor was 1:2. The assay was processed as recommended by the kit instructions. The measurement was performed on a FM3D instrument . Data analysis was performed using MasterPlex QT 2010 . Standard curves were generated using weighted (1/y²) 5-parameter logistics. The analyte concentrations in the samples were calculated based on the standard curves. In this study, only IL-1beta and IL-6 were initially analyzed (based on our in vitro data). Upon request from one of the reviewers, data on all cytokines were then extracted from the database for post-hoc analyses. Seven more cytokines were detected in more than 10 subjects  but none of them displayed a significant association with C4A . Briefly, in a flowrate of 0.3 mL/min, sample injection of 40 µL and a 30 min gradient was employed for separation on a Hypersil Gold reversed phase column . The two mobile phases (A and B) were composed of 0.1% formic acid in water (v/v) and 0.1% formic acid/84% acetonitrile in water (v/v), respectively. Electrospray settings were the following: gas temperature 220 °C, gas flow 15 L/min, nebulizer 40 psi, sheath gas temperature 200 °C, sheath gas flow L/min, capillary voltage in positive mode 3500 V and, nozzle voltage at 500 V. iFunnel settings were the following in positive mode: high-pressure RF 200 V and low-pressure RF 160 V. Individually optimized collision energies and cell accelerator voltages were used for each peptide. The scheduled multiple reaction monitoring method used a delta retention time of 0.8 min for each peptide. Quality control samples, consisting of pooled CSF samples obtained from the Neurochemistry Laboratory at Sahlgrenska University Hospital, Mölndal, Sweden, were injected at regular intervals to monitor the performance of the assay over time. The collection and use are in accordance with the Swedish law on biobanks in healthcare (2002:297). Skyline 20.1 (MacCoss Lab Software) was used for peak visualization and inspection. Peak adjustment was performed if required for optimal peak area calculation. Relative peptide concentrations were obtained by summing all measured fragment peak areas for each peptide and dividing that by the sum of the fragment peak areas of the corresponding IS, followed by multiplication of the amount of IS added per µL of CSF.Preparation of samples and LC-MS/MS analyses were performed as previously described6 (an estimated correlation coefficients of at least 0.5 for C4A (or C4B) CNs versus corresponding RNA expression in 101 subjects, and an odds ratio of 1.4 (P = 2 × 10−5) comparing C4A RNA expression in 35 SCZ subjects and 70 HCs). According to the pre-defined analytical plan, a significant increase in C4A (or C4B) concentration in the discovery cohort  was to be followed-up in an independent cohort then again measuring C4A, as well as C4B, with significance set at P < 0.05 . Potential confounders were to be tested on group levels (in each cohort). If this indicated any evidence of between group differences , indicated variables were to be studied in relation to CSF C4A or C4B concentration. If this revealed an association with P < 0.05, we were to perform an adjusted analysis. The exception to this rule was (1) antipsychotics, here we before analyses decided to compare levels between medicated and non-medicated patients, and even if we observed no indication of altered levels in medicated vs. unmedicated we were to perform stratified/adjusted analyses, and (2) as post-hoc analyses, we also performed adjusted analyses  to control for possible residual confounding. For group comparisons, CSF C4A and C4B concentrations (or C4A and C4B RNA in experiments) in the main analyses were analyzed using Kruskal-Wallis H tests followed by post-hoc tests. Continuous variables were analyzed by Spearman’s or Pearson’s correlation analyses  as indicated. Experimental data were analyzed using Kruskal–Wallis H test followed by post-hoc tests. Data were analyzed using R for Mac OS (version 4.2.1), IBM SPSS statistics (version 28.0.0.0), and Graphpad prism 9 for macOS (version 9.3.1).Available sample size for the initial analysis in the discovery cohort was deemed adequate based on the data presented by Sekar et al.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary