Immune Function Tests Research Articles

Management of cytomegalovirus infection after liver transplantation.

Cytomegalovirus (CMV) infection is one of the primary causes of morbidity and mortality following liver transplantation (LT). Based on current worldwide guidelines, the most effective strategies for avoiding post-transplant CMV infection are antiviral prophylaxis and pre-emptive treatment. CMV- IgG serology is the established technique for pretransplant screening of both donors and recipients. The clinical presentation of CMV infection and disease exhibits variability, prompting clinicians to consistently consider this possibility, particularly within the first year post-transplantation or subsequent to heightened immunosuppression. At annual symposia to discuss CMV prevention and how treatment outcomes can be improved, evidence on the incorporation of immune functional tests into clinical practice is presented, and the results of studies with new antiviral treatments are evaluated. Although there are ongoing studies on the use of letermovir and maribavir in solid organ transplantation, a consensus reflected in the guidelines has not been formed. Determining the most appropriate strategy at the individual level appears to be the key to enhancing outcomes. Although prevention strategies reduce the risk of CMV disease, the disease can still occur in up to 50% of high-risk patients. A balance between the risk of infection and disease development and the use of immunosuppressants must be considered when talking about the proper management of CMV in solid organ transplant recipients. The objective of this study was to establish a comprehensive framework for the management of CMV in patients who have had LT.

Live Rotavirus Vaccination Appears Low-risk in Infants Born to Mothers With Inflammatory Bowel Disease on Biologics

Biologic therapies in the context of inflammatory bowel disease and pregnancy lead to improved maternal and fetal outcomes. Placental transfer results in detectable drug concentrations in infants. Rotavirus infection results in diarrheal related hospitalizations; however, the live oral vaccine is not currently recommended in biologic exposed infants. The aim of this study was to assess the effect of maternal biologic therapies on the infant immune system and safety of live rotavirus vaccination in biologic-exposed infants. Biologic-exposed infants underwent standardized clinical assessments, drug concentration, and immune function testing (complete blood count, differential, immunoglobulin levels, extended B-cell and T-cell subset enumeration, Recent Thymic Emigrants, regulatory T-cell numbers, mitogen stimulation assays, and review of T-cell Receptor Excision Circles in the newborn screen). Rotavirus vaccine-specific adverse effects following immunizations up to 42 days post the last dose of the vaccine series were recorded. There were 57 infants born to 52 mothers with inflammatory bowel disease exposed to infliximab (n= 21), adalimumab (n= 19), vedolizumab (n= 10), and ustekinumab (n= 7) in the third trimester for a median of 39 weeks (interquartile range, 38-39 weeks) at delivery. Immunologic assessments validated for age were normal in all infants despite median infliximab concentrations of 6.1 ug/mL (range, 0.4-28.8 ug/mL), adalimumab concentrations of 1.7ug/mL (range, 0.7-7.9 ug/mL), ustekinumab concentrations of 0.6 ug/mL (range, 0-1.1), and undetectable for vedolizumab at 10.7 weeks (interquartile range, 9.4-12.4) of age. The live oral rotavirus vaccine series was provided to 50 infants with the first dose given at a median of 13weeks of age. No adverse effects following immunization were reported. Immune function testing was normal, and administration of live rotavirus vaccination appeared low-risk in biologic-exposed infants irrespective of circulating drug levels.

(1107) - Monotherapy in Heart Transplantation Proves to Be Safe While Followed by T-Cell Immune Function Testing

(1107) - Monotherapy in Heart Transplantation Proves to Be Safe While Followed by T-Cell Immune Function Testing

A237 ROTAVIRUS VACCINE SAFETY IN INFANTS EXPOSED TO BIOLOGICS IN UTERO IN AN INFLAMMATORY BOWEL DISEASE POPULATION: A PROSPECTIVE COHORT STUDY

Abstract Background Biologics, the mainstay of therapy in patients with moderate-to-severe inflammatory bowel disease (IBD), undergo placental transfer during the second trimester of pregnancy and may remain detectable in the blood of exposed infants for up to 12 months. Due to concerns regarding the risk of immunosuppression and adverse events following immunization with live attenuated vaccines in infants exposed to biologics in utero, current guidelines recommend avoiding live vaccines in the first 6 to 12 months of life. Aims This study investigates whether the live rotavirus vaccine can safely be administered in infants exposed to biologics in utero. Methods We conducted a single-centre prospective cohort study, which included infants exposed to biologics in utero and born to mothers with IBD between October 1, 2020 and June 30, 2022. Infants were included in the study if they were assessed by Paediatric specialists at the Special Immunization Clinic (SIC) at SickKids Hospital (Toronto, Ontario). Mothers were followed by Gastroenterologists at the Special Pregnancy IBD Clinic at Mount Sinai Hospital (Toronto, Ontario). Data were collected on biologic agent exposure history, infant immune function testing, SIC recommendations for rotavirus vaccination, rotavirus vaccine series completion, and adverse events after immunization. Adverse events following immunization included fevers, vomiting, diarrhea, and/or intussusception within 42 days following immunization. Results 22 infants were included in the study. Of the 22 infants, 5 (22.7%) were exposed to infliximab in utero, 10 (45.5%) were exposed to adalimumab, 3 (13.6%) were exposed to vedolizumab, and 3 (13.6%) were exposed to ustekinumab. Of the 22 infants, 18 (81.8%) were recommended to receive the rotavirus vaccine by the SIC. 1 (0.05%) was not recommended to receive the vaccine due to neutropenia and low CD54 count on lymphocyte testing. The recommendation given to 3 infants is unknown. Of the 18 infants who were recommended to receive the vaccine, 16 (88.9%) received the vaccine and had no adverse events following immunization. 1 (6%) infant did not receive the rotavirus vaccine despite it being recommended due to parental choice. The rotavirus vaccination status and response in 1 infant recommended to receive the vaccine, is unknown. Conclusions Administration of the rotavirus vaccine in biologic-exposed infants did not result in adverse events in our study cohort. Although guidelines currently advise against live vaccinations in infants exposed to biologics in utero, specialist assessment of infant immune function can help encourage safe live vaccination uptake. Funding Agencies None

Methods for Analysis of Nanoparticle Immunosuppressive Properties.

Adverse drug effects on immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of drug withdrawal from the market is attributed to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is T cell dependent antibody response (TDAR). This method involves a 28-day in vivo study evaluating the animal's antibody titer to a known antigen (Keyhole Limpet Hemocyanin; KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human lymphocyte activation (HuLA) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of nanoparticles. The antigen-specific lymphocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLA assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme.

Eight-year chronic wound caused by Tarlov’s cyst: a case report

BackgroundTarlov’s cyst is often underdiagnosed since it is difficult to identify without imaging assistance. Herein, we report the case of a young girl who presented with an 8-year history of chronic osteomyelitis of bilateral proximal phalanges and metatarsal bones caused by a Tarlov’s cyst that did not contain a nerve root. The chronic wound in the forefoot is an unusual presentation and resulted from the Tarlov’s cyst accompanied with tethered conus syndrome.Case presentationA 10-year-old Asian girl presented with an 8-year history of chronic osteomyelitis of bilateral proximal phalanges and metatarsal bones. She received sequestrectomy five times, however the immune function tests were all normal. A neurological examination revealed diminished sensation and a slapping gait pattern. Magnetic resonance imaging (MRI) demonstrated a lobulated cyst at the right aspect of the sacrum (S) 1 to sacrum (S) 3 canal near the dorsal root ganglion. Tethered conus syndrome was highly suspected. She received laminectomy of lumbar (L) 5 and S1–S2, which led to the diagnosis of a right S1–S3 epidural cyst. The final diagnosis from the histopathological examination was a right sacral Tarlov’s cyst. The clinical conditions of diminished sensation and slapping gait pattern greatly improved after successful surgical treatment.ConclusionIn children who present with a recalcitrant chronic wound in the forefoot accompanied with a slapping gait pattern and foot hypoesthesia to pain, aggressive imaging examinations such as spine MRI should be arranged for further evaluation, especially in immunocompetent children.

ALTERED PROFILES OF EXTRACELLULAR MITOCHONDRIAL DNA IN IMMUNOPARALYZED PEDIATRIC PATIENTS AFTER THERMAL INJURY.

Background: Thermal injury is a major cause of morbidity and mortality in the pediatric population worldwide with secondary infection being the most common acute complication. Suppression of innate and adaptive immune function is predictive of infection in pediatric burn patients, but little is known about the mechanisms causing these effects. Circulating mitochondrial DNA (mtDNA), which induces a proinflammatory signal, has been described in multiple disease states but has not been studied in pediatric burn injuries. This study examined the quantity of circulating mtDNA and mtDNA mutations in immunocompetent (IC) and immunoparalyzed (IP) pediatric burn patients. Methods: Circulating DNA was isolated from plasma of pediatric burn patients treated at Nationwide Children's Hospital Burn Center at early (1-3 days) and late (4-7 days) time points postinjury. These patients were categorized as IP or IC based on previously established immune function testing and secondary infection. Three mitochondrial genes, D loop, ND1, and ND4, were quantified by multiplexed qPCR to assess both mtDNA quantity and mutation load. Results: At the early time point, there were no differences in plasma mtDNA quantity; however, IC patients had a progressive increase in mtDNA over time when compared with IP patients (change in ND1 copy number over time 3,880 vs. 87 copies/day, P = 0.0004). Conversely, the IP group had an increase in mtDNA mutation burden over time. Conclusion: IC patients experienced a significant increase in circulating mtDNA quantity over time, demonstrating an association between increased mtDNA release and proinflammatory phenotype in the burn patients. IP patients had significant increases in mtDNA mutation load likely representative of degree of oxidative damage. Together, these data provide further insight into the inflammatory and immunological mechanisms after pediatric thermal injury.

Second allogeneic hematopoietic stem cell transplantation with reduced-intensity conditioning and donor changes in relapsed hematological malignancies after the first allogeneic transplant

Objective: The purpose of this study was to assess the safety and efficacy of a second allogeneic hematopoietic stem cell transplantation (allo-HSCT) with reduced-intensity conditioning (RIC) in patients with hematological malignancies who had relapsed after the first allo-HSCT. Methods: Between April 2018 and June 2021, 44 patients with hematological malignancies (B-ALL 23, T-ALL/T-LBL 4, AML15, and MDS 2) were enrolled and retrospectively examined. Unrelated donors (n=12) or haploidentical donors (n=32) were used. Donors were replaced in all patients for the second allo-HSCT. Hematological and immunological germline predisposition genes and hematopoietic and immune function tests were used to select the best-related donor. Total body irradiation (TBI) /fludarabine (FLU) -based (n=38), busulfan (BU) /FLU-based (n=4), total marrow irradiation (TMI) /FLU-based (n=1), and BU/cladribine-based (n=1) were the RIC regimens used. For graft versus host disease (GVHD) prevention, cyclosporine, mycophenolate mofetil, short-term methotrexate, and ATG were used. Eighteen (40.9%) of 44 patients with gene variations for which targeted medications are available underwent post-transplant maintenance therapy. Results: The median age was 25 years old (range: 7-55). The median interval between the first and second HSCT was 19.5 months (range: 6-77). Before the second allo-HSCT, 33 (75%) of the patients were in complete remission (CR), whereas 11 (25%) were not. All patients had long-term engraftment. The grade Ⅱ-Ⅳ GVHD and severe acute GVHD rates were 20.5% and 9.1%, respectively. Chronic GVHD was found in 20.5% of limited patterns and 22.7% of severe patterns. CMV and EBV reactivation rates were 29.5% and 6.8%, respectively. Hemorrhage cystitis occurred in 15.9% of cases, grade Ⅰ or Ⅱ. The 1-yr disease-free survival (DFS), overall survival (OS), and cumulative recurrence incidence (RI) rates of all patients were 72.5% (95% CI, 54.5%-84.3%), 80.6% (95% CI, 63.4%-90.3%), and 25.1% (95% CI, 13.7%-43.2%), respectively, with a median follow-up of 14 (2-39) months. There were eight deaths (seven relapses and one infection). The rate of non-relapse mortality (NRM) was only 2.3%. The CR patients' 1-yr RI rate was significantly lower than the NR patients (16.8% vs 48.1%, P=0.026). The DFS rate in CR patients was greater than in NR patients, although there was no statistical difference (79.9% vs 51.9%, P=0.072). Univariate analysis revealed that CR before the second allo-HSCT was an important prognostic factor. Conclusion: With our RIC regimens, donor change, and post-transplant maintenance therapy, the second allo-HSCT in relapsed hematological malignancies after the first allo-HSCT is a safe and effective treatment with high OS and DFS and low NRM and relapse rate. The most important factor influencing the prognosis of the second allo-HSCT is the patient's illness condition before the transplant.

Recovery of functional fitness, lung function, and immune function in healthcare workers with nonsevere and severe COVID-19 at 13 months after discharge from the hospital: a prospective cohort study

ObjectivesThis study aimed to evaluate the recovery of functional fitness, lung function, and immune function in healthcare workers (HCWs) with nonsevere and severe COVID-19 at 13 months after discharge from the hospital. MethodsThe participants of “Rehabilitation Care Project for Medical Staff Infected with COVID-19” underwent a functional fitness test (muscle strength, flexibility, and agility/dynamic balance), lung function test, and immune function test (including cytokines and lymphocyte subsets) at 13 months after discharge. ResultsThe project included 779 HCWs (316 nonsevere COVID-19 and 463 severe COVID-19). This study found that 29.1% (130/446) of the HCWs have not yet recovered their functional fitness. The most affected lung function indicator was lung perfusion capacity (34% with diffusion capacity for carbon monoxide-single breath <80%). The increase of interleukin-6 (64/534, 12.0%) and natural killer cells (44/534, 8.2%) and the decrease of CD3+ T cells (58/534, 10.9%) and CD4+ T cells (26/534, 4.9%) still existed at 13 months after discharge. No significant difference was found in the HCWs with nonsevere and severe COVID-19 regarding recovery of functional fitness, lung function, and immune function at 13 months after discharge. ConclusionThe majority of Chinese HCWs with COVID-19 had recovered their functional fitness, lung function, and immune function, and the recovery status in HCWs with severe COVID-19 is no worse than that in HCWs with nonsevere COVID-19 at 13 months after discharge from the hospital.

Dynamic Immune Function Changes Before and After the First Radioactive Iodine Therapy After Total Resection of Differentiated Thyroid Carcinoma.

The effects of total thyroidectomy or radioactive iodine therapy on immune activation and suppression of the tumor microenvironment remain unknown. We aimed to investigate the effects of these treatments on the immune function in patients with differentiated thyroid carcinoma (DTC). Our cohort included 45 patients with DTC treated with total thyroidectomy and radioactive iodine therapy (RAIT). Immune function tests were performed by flow cytometry at 0, 30, and 90 days post-RAIT. Both the percentage and absolute number of circulating regulatory T cells were significantly lower in the postoperative DTC compared to the healthy controls. Notably, the absolute number of multiple lymphocyte subgroups significantly decreased at 30 days post-RAIT compared to those pre-RAIT. The absolute counts of these lymphocytes were recovered at 90 days post-RAIT, but not at pre-RAIT levels. Additionally, the Th17 cell percentage before RAIT was positively correlated with thyroglobulin (Tg) levels after RAIT. The tumor burden might contribute to increased levels of circulating Tregs. In conclusion, RAIT caused transient radiation damage in patients with DTC and the percentage of Th17 cells before RAIT could be a significant predictor of poor prognosis in patients with DTC.

Abstract 255: Peroxidasin deficiency recruits pro-healing macrophages into the liver and inhibits NAFLD progression to HCC

Abstract Introduction: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix (ECM) proteins. ECM stabilization influences the speed of tissue recovery. Here, we used a mouse model of nonalcoholic fatty liver disease (NAFLD) to study the function of peroxidasin (PXDN), a peroxidase that uses H2O2 to cross-link collagen IV, during liver fibrosis progression to hepatocellular carcinoma (HCC). Method: Pxdn-/- and Pxdn+/+ mice were fed with a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) for 16 weeks to create a NAFLD-HCC preclinical model. Liver histology, collagen content, flow cytometry, immunostaining of immune cells, RNA-seq, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A. Results: Using Fe-PyC3A as an MRI contrast agent, we detected a higher content of ROS in Pxdn-/- livers (healthy) that was not necessarily directly toxic but could activate hypoxia-related molecular pathways. Genome-wide expression analysis of liver tissue and differential gene expression (DGE) combined with Gene Ontology (GO) analysis identified significant upregulation of genes associated with hypoxia and TNFα signaling pathways already in Pxdn-/- sham livers (without injury). In addition, we observed an upregulation of genes involved in the innate immune response, leukocyte activation, and chemotaxis. After 16 weeks of CDAHFD, gross analysis of collected liver showed no HCC nodule formation in Pxdn-/- mice while 60% of the WT mice had HCC tumors. Collagen deposition showed less collagen accumulation in Pxdn-/- mice. Flow cytometry of macrophages showed Pxdn-/- mice had increased pro-healing M2 macrophages recruitment in early- and mid-stage NAFLD (4 weeks and 8 weeks on CDAHFD) compared to WT controls. In addition, we observed a significant decrease in the number of CD3+ T cells and CD8+ cytotoxic T cells in the late-stage of NAFLD in Pxdn KO mice. DGE analysis revealed that IL-12 is highly expressed in Pxdn-/- injured livers. Additionally, multiple other T cell-related molecules such as IL-10, IL-6, CCL2, IL-7, and CD4 were elevated in Pxdn-/- injured liver. Elevation of these cytokines is an indicator for higher recruitment of pro-healing and anti-HCC macrophage to the site of injury. Conclusion: Our findings demonstrate that PXDN deficiency is associated with the induction of the hypoxia and TNFα signaling pathways and the recruitment of pro-healing and anti-HCC macrophages to the liver. This results in significantly decreased collagen stabilization during liver fibrosis and accelerates fibrosis reversal. In addition, recruited macrophages-controlled T cell response and inhibited HCC formation in Pxdn-/- mice. Citation Format: Mozhdeh Sojoodi, Stephen C. Barrett, Derek J. Erstad, Shadi Salloum, Shijia Zhu, Tongqi Qian, Selene Colon, Eric Gale, Veronica Clavijo Jordan, Yongtao Wang, Shen Li, Michael Lanuti, Lawrence Zukerberg, Peter Caravan, Yujin Hoshida, Raymond T. Chung, Gautam Bhave, Georg M. Lauer, Bryan C. Fuchs, Kenneth K. Tanabe. Peroxidasin deficiency recruits pro-healing macrophages into the liver and inhibits NAFLD progression to HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 255.

Peroxidasin Deficiency Re-programs Macrophages Toward Pro-fibrolysis Function and Promotes Collagen Resolution in Liver.

Background & AimsDuring liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix proteins. Extracellular matrix stabilization influences the speed of tissue recovery. Here, we studied the expression and function of peroxidasin (PXDN), a peroxidase that uses hydrogen peroxide to cross-link collagen IV during liver fibrosis progression and regression.MethodsMouse models of liver fibrosis and cirrhosis patients were analyzed for the expression of PXDN in liver and serum. Pxdn-/- and Pxdn+/+ mice were either treated with carbon tetrachloride for 6 weeks to generate toxin-induced fibrosis or fed with a choline-deficient L-amino acid-defined high-fat diet for 16 weeks to create nonalcoholic fatty liver disease fibrosis. Liver histology, quantitative real-time polymerase chain reaction, collagen content, flowcytometry and immunostaining of immune cells, RNA-sequencing, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A.ResultsIn human and mouse cirrhotic tissue, PXDN is expressed by stellate cells and is secreted into fibrotic areas. In patients with nonalcoholic fatty liver disease, serum levels of PXDN increased significantly. In both mouse models of liver fibrosis, PXDN deficiency resulted in elevated monocyte and pro-fibrolysis macrophage recruitment into fibrotic bands and caused decreased accumulation of cross-linked collagens. In Pxdn-/- mice, collagen fibers were loosely organized, an atypical phenotype that is reversible upon macrophage depletion. Elevated ROS in Pxdn-/- livers was observed, which can result in activation of hypoxic signaling cascades and may affect signaling pathways involved in macrophage polarization such as TNF-a via NF-kB. Fibrosis resolution in Pxdn-/- mice was associated with significant decrease in collagen content and improved liver function.ConclusionPXDN deficiency is associated with increased ROS levels and a hypoxic liver microenvironment that can regulate recruitment and programming of pro-resolution macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest a novel pathway that is involved in the resolution of scar tissue.

Immune Function Testing and Long-Term Immune-Related Outcomes among Heart Transplant Recipients

<h3>Purpose</h3> The Cylex ImmuKnow (IK) assay, a rapid quantitative assessment of T-cell-mediated function, is used to monitor immunosuppression among heart transplant (HT) recipients. It is unclear whether the IK assay can aid in characterizing the long-term risk of adverse immune-related events among HT recipients. <h3>Methods</h3> We conducted a single-center, retrospective, cohort study of all single organ HT recipients from 2014 to 2018. The IK assay divided subjects into low, moderate, or high immune response categories at 6-month, 1-, and 2-year intervals. IK assay categorization was correlated to the primary composite outcome which included acute cellular rejection, antibody-mediated rejection, development of donor-specific antibodies, coronary allograft vasculopathy, and death over 2 years of follow-up. <h3>Results</h3> 134 single organ HT recipients were included (median age 56.5 years, 70% male). On an immunosuppressive regimen of tacrolimus, prednisone, and mycophenolate mofetil, most subjects had low and moderate immune response levels at 6 months (53% low vs 37% moderate vs 10% high). A higher proportion of subjects exhibited moderate immune response at 1 (44% low vs 47% moderate vs 9% high) and 2 years (29% vs 60% vs 11%). The composite outcome occurred in 14% of subjects by 6 months, 35% by 1 year, and 36% by 2 years. There was no difference in the rate of the composite outcome or its individual components between the immune response groups at any of the time points (Fig. 1B). The relative change in an individual's IK score between timepoints was not associated with the composite outcome. <h3>Conclusion</h3> On standard immunosuppression, a majority of HT recipients exhibit low to moderate immune response per the IK assay. The IK assay reflects expected increases in immune function with reduced immunosuppression. IK assay-based immune response categorization does not appear to aid in identifying HT recipients at risk for adverse immune-related events in the long-term. The results are limited by the sample size and low event rates.

Long-Term Effects of Monotherapy with Low Dose Tacrolimus

<h3>Purpose</h3> Monotherapy with tacrolimus alone (TAC-A) was found to be safe and effective in the TICTAC Trial. However, in that trial, tacrolimus levels were maintained at 10-12 ng/mL over the first year which resulted in a higher serum creatinine level compared to those left in combination. We have been using TAC-A at lower doses in patients who have developed leukopenia or thrombocytopenia due to immunosuppression or a natural state. The presence of cytomegalovirus infections and other viruses excluded those patients from being administered monotherapy. It has not been established whether low dose TAC-A is safe and effective. <h3>Methods</h3> Between 2010 and 2017 we assessed 151 heart transplant patients who are maintained on monotherapy with TAC-A. Patients were maintained on a low level of TAC-A between 4-7 ng/ml. These patients were assessed for subsequent 1-year rejection, subsequent 3-year survival, 3-year freedom from non-fatal major adverse cardiac events (NF-MACE: MI, new CHF, PCI, ICD implant, stroke), cardiac allograft vasculopathy (CAV, epicardial disease with angiographic stenosis>30%), and cardiac dysfunction (LVEF ≤ 40%). Furthermore, periodic testing was done with the T-cell immune function test to assess whether these patients were adequately immunosuppressed. <h3>Results</h3> Patients who were maintained on TAC-A had comparable outcomes compared to patients left in combination immunosuppression (see table). The T-cell immune function test showed that these patients had adequate immunosuppression (average was 276 ng/mL with a range of 56-794 ng/mL and standard deviation of 137.1 ng/mL) with the therapeutic range being 200-550ng/ml. Patients had an average of 11 Tacrolimus lab tests (range: 4-46). <h3>Conclusion</h3> Low dose TAC-A appears safe and effective in select patients after heart transplantation guided by T cell immune function testing. Further studies with larger number of patients will be needed to confirm these findings.

Clinical immunity and medical cost of COVID-19 patients under grey relational mathematical model

This study was to explore the performance of immune function and compositions of hospitalization cost for patients with COVID-19 as well as the application of a grey relational mathematical model (GRMM). A total of 100 COVID-19 patients diagnosed by nucleic acid test and chest CT examination in our hospital were collected in this study. They were divided into 2 groups: non-severe group (mild and moderate patients, n = 57 cases), and severe group (severe and critical patients, n = 43 cases) based on the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7) published by the World Health Organization (WHO). The general clinical data, blood routine indexes, cellular immune and humoral immune function test indexes, and the composition of hospitalization costs of the two groups of patients were collected and analyzed. The results showed that the average age, proportion of males, smoking history, and the number and proportion of patients in the non-severe group were smaller than those in the severe group (P < 0.05); the severe group had significantly more shortness of breath patients than the non-severe group (P < 0.05). Compared with the non-severe group, the number of white blood cells (WBC), the number and proportion of neutrophils, and the count of neutrophils/lymphocytes in the severe group increased obviously (P < 0.05), and the number of lymphocytes and the proportion of monocytes decreased dramatically (P < 0.05); the number and proportion of CD3+, CD4+, CD8+, and CD19+ in the severe group were much lower in contrast to those in the non-severe group (P < 0.05), while the ratio of CD4+/CD8+ was greatly higher in contrast to that of non-severe patients (P < 0.05). Compared with the non-severe group, the bed fee, laboratory test fee, diagnosis fee, and medicine fee of the severe group were increased observably (P < 0.05). The changes in hospitalization cost of patients in the severe group was related to bed fees, laboratory fees, and expenses of proprietary Chinese medicines, while the hospitalization cost of patients in the severe group was related to bed fees, laboratory fees, and examination fees. The results revealed that elderly COVID-19 patients with basic diseases were prone to develop severe disease, immune cell depletion may be one of the reasons for the development of severe patients, and the medical insurance policy greatly reduced the hospitalization costs of COVID-19 patients.

Challenges of non-clinical safety testing for biologics: A Report of the 9th BioSafe European Annual General Membership Meeting

ABSTRACT New challenges and other topics in non-clinical safety testing of biotherapeutics were presented and discussed at the nineth European BioSafe Annual General Membership meeting in November 2019. The session topics were selected by European BioSafe organization committee members based on recent company achievements, agency interactions and new data obtained in the non-clinical safety testing of biotherapeutics, for which data sharing would be of interest and considered as valuable information. The presented session topics ranged from strategies of in vitro testing, immunogenicity prediction, bioimaging, and developmental and reproductive toxicology (DART) assessments to first-in-human (FIH) dose prediction and bioanalytical challenges, reflecting the entire space of different areas of expertise and different molecular modalities. During the 9th meeting of the European BioSafe members, the following topics were presented and discussed in 6 main sessions (with 3 or 4 presentations per session) and in three small group breakout sessions: 1) DART assessment with biotherapeutics: what did we learn and where to go?; 2) Non-animal testing strategies; 3) Seeing is believing: new frontiers in imaging; 4) Predicting immunogenicity during early drug development: hope or despair?; 5) Challenges in FIH dose projections; and 6) Non-canonical biologics formats: challenges in bioanalytics, PKPD and biotransformation for complex biologics formats. Small group breakout sessions were organized for team discussion about 3 specific topics: 1) Testing of cellular immune function in vitro and in vivo; 2) MABEL approach (toxicology and pharmacokinetic perspective); and 3) mRNA treatments. This workshop report presents the sessions and discussions at the meeting.

Immune Function Testing and Donor-Specific Antibody Production among Heart Transplant Patients

De novo donor-specific antibody (DSA) production has been correlated to the development of antibody-mediated rejection after heart transplantation. It is unclear whether the Cylex ImmuKnow (IK) assay - a rapid, quantitative assessment of T-cell-mediated immune function designed to monitor immunosuppression response - can be a useful tool in overall immunosuppression and identification of patients at risk for de novo DSA production. We hypothesize that, among heart transplant recipients, increased immune function activity as measured by the IK assay would correlate with de novo DSA production. We conducted a single center cohort study including all consecutive single organ heart transplant recipients from 2014 to 2018. The IK assay categorized subjects into low, moderate, or high immune response levels at 6, 12, and 24-month intervals. De novo DSA production based on human leukocyte antigen antibody testing was correlated to IK immune response levels at these time points. 122 single organ heart transplant recipients were included. High immune response levels were noted in 10%, 9%, and 12% of subjects at 6, 12, and 24-months, respectively. Compared to baseline, de novo DSA production was noted in 21%, 23%, and 25% of subjects at the respective time intervals. 6-month IK category did not correlate with DSA production at 6 (r = -0.03, p=0.9), 12 (r = -0.03, p=0.9), or 24 months (r = 0.15, p=0.3). Similar findings were noted at 12 and 24 months. Being in a high IK category at any interval was not associated with de novo DSA production at any time point (p>0.05). Univariate analysis of demographic factors, induction agents used, and co-morbidities (including EMV or CBV infection status) did not reveal significant associations with de novo DSA production. High T-cell-mediated immune response levels, a possible indicator of under-suppression, as measured by the IK assay were not associated with de novo DSA production after heart transplantation.

An increase in circulating B cells and B cell activation markers in peripheral blood is associated with cigarette smoking in a male cohort in Bangladesh

In a cohort of approximately 200 Bangladeshi men, equally divided into smokers and non-smokers and equally divided by exposure to high and low levels of drinking water arsenic, we examined ex vivo a series of immune markers and immune function tests in peripheral blood mononuclear cells (PBMC). These immune parameters included PBMC cell surface markers (CSM) for B, T, monocytes, and NK cells, activated T and B cell markers, cytokine production in vitro, and analysis of CD4 subsets (Th1, Th2, Treg, and Th17 cells). We found that the effects of cigarette smoke were quite different than those associated with arsenic or polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Cigarette smoking was associated with a significant increase in the number of PAH-DNA adducts as well as an increase in urinary levels of 1-hydropxypyrene (1-OHP). After correcting for arsenic exposure and PAH-DNA adducts, we found that cigarette smoking was associated with an increase in the percentage of CD19+ B cells, as well as the percentage of activated B cells (CD19+, HLA-DRbright cells) found in PBMC. These findings demonstrate activation of the immune system during chronic exposure to cigarette smoke, which is a known risk factor for autoimmune diseases.

Immune function testing in sepsis patients receiving sodium selenite

PurposeWe examined in a longitudinal study the role of sodium selenite in sepsis patients in strengthening the immune performance in whole blood samples using immune functional assays. Materials and methodsThis was a sub-study from a randomized, double blinded multicenter clinical trial (SISPCT) registered with www.clinicaltrials.gov (NCT00832039) and with data collected at our center. Full blood samples were incubated with various recall antigens and the supernatants were measured for their cytokine concentrations as markers for immune response. Data from days 0, 4, 7, 14, and 21 (from sepsis onset) were analyzed using a generalized least squares model in R to appropriately take the longitudinal structure and the missing values into account. ResultsFrom the 76 patients enrolled in the study at our center, 40 were randomized to selenium therapy and 36 to placebo. The analyses of immune response assay data showed no statistical difference between the selenium and placebo groups at each of the time points. There was however an overall dampening of cytokine release, which tended to recover over time in both groups. ConclusionSelenium has long been an adjuvant therapy in treating sepsis. Recently, it was proven to not have beneficial effects on the mortality outcome. Using data from our center in this sub-cohort study, we identified no relative improvement in cytokine release of stimulated blood immune cells ex vivo from patients with selenium therapy over a three-week period. This offers a potential explanation for the lack of beneficial effects of selenium in sepsis patients.

Correlation of ImmuKnow Assay Levels with Rejection and Infection after Heart Transplantation

Purpose Cylex Immune Function Test (IFT)(ImmuKnow®) assesses T-cell activity by measuring ATP levels. It is a useful assay that measures global immune response in patients post heart transplantation (HTx). However, its role in guiding medical therapy is still limited. We aim to study the association of IFT with HTx rejection and infection. Methods We investigated all patients who received HTx from 4/2014 - 8/2018. All IFT levels measured were divided into 3 groups: rejection phase (within 1 month before rejection episode, ISHLT 1990 ≥ grade 1B), infection phase (within 1 month of infection requiring admission), other stable phase, and analyzed these IFT values. Rejection episodes within 1 month post-HTx were excluded. Results 156 HTx patients were included (mean age 54 years, 74% male). The mean follow-up period from HTx was 2.1 ± 1.4 years, over which 757 IFT values were collected. 25 IFT levels were assessed in rejection phase, 123 in infection phase and 611 were in stable phase. Mean IFT in infection phase was significantly lower compared to stable phase (166 ± 143 vs 264 ± 180 ng ATP/mL, p Conclusion Infection was associated with lower IFT levels compared to stable post-transplant levels. IFT may be a valuable tool in the detection of infection post-HTx and can guide reduction in immunosuppression. However, its role in predicting rejection needs further evaluation.