Good manufacturing practice production of CD34+ progenitor-derived NK cells for adoptive immunotherapy in acute myeloid leukemia.

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).

IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion,  N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.

Pitfalls in cytokine measurements - Plasma TGF-ß1 in chronic fatigue syndrome.

BACKGROUND: Serum TGF-ß1 concentrations are reported to be elevated in chronic fatigue syndrome (CFS). However, measurement of circulating cytokines is a complex procedure and control of pre-analytical procedures is essential. The objective of the current study was to measure circulating TGF-ß1 concentrations in CFS patients compared to healthy controls, taking into account differences in pre-analytical procedures. METHODS: Two cohorts of female CFS patients were included. In both studies patients were asked to bring a healthy, age-matched control. At baseline, TGF-ß1 levels were measured in plasma and additionally P-selectin, a marker of platelet activity, was determined in a subgroup of participants. RESULTS: 50 patients and 48 controls were included in cohort I, and 90 patients and 29 controls in cohort II. Within the cohorts there were no differences in TGF-ß1 concentrations. However, between the cohorts there was a large discrepancy, which appeared to be caused by differences in g-force of the centrifuges used. The lower g-force used in cohort II (1361 g) caused more platelet activation, reflected by higher p-selectin concentrations, compared to cohort I (p < 0.0001), which was confirmed in a second independent experiment. There was a correlation between TGF-ß1 and p-selectin concentrations (r 0.79, p < 0.0001). CONCLUSION: These results demonstrate that control of pre-analytical procedures is an essential aspect when measuring circulating cytokines. No evidence for enhanced TGF-ß1 in patients with CFS was found.

[Report of the Dutch National Health Council on chronic fatigue syndrome]. / GR-rapport over chronische-vermoeidheidssyndroom.

In our opinion, the recent report of the Dutch National Health Council on myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) lacks balance: it is very critical on the quality of evidence regarding behavioural interventions, but lacks a critical attitude regarding the presumed somatic components of the disorder. Without solid evidence, the report coins ME/CFS as a severe multisystem disease, and it embraces the diagnostic criteria of the American Institute of Medicine. We underscore the remarks in the report that physicians should not be reluctant to make diagnosis in patients with the disorder, and that these patients should be approached with empathy and respect. Regarding a future research programme, there is need for a well-designed research agenda.

Postural orthostatic tachycardia is not a useful diagnostic marker for chronic fatigue syndrome.

BACKGROUND: Postural orthostatic tachycardia syndrome (POTS) is considered a diagnostic marker for chronic fatigue syndrome (CFS). OBJECTIVES: The aims of this study were to (i) compare POTS prevalence in a CFS cohort with fatigued patients not meeting CFS criteria, and (ii) assess activity, impairment and response to cognitive behavioural therapy (CBT) in CFS patients with POTS (POTS-CFS) and without POTS (non-POTS-CFS). METHODS: Prospective cohort study at the Radboud University Medical Centre in the Netherlands. Between June 2013 and December 2014, 863 consecutive patients with persistent fatigue were screened. Patients underwent an active standing test, filled out questionnaires and wore an activity-sensing device for a period of 12 days. RESULTS: A total of 419 patients with CFS and 341 non-CFS fatigued patients were included in the study. POTS prevalence in adult patients with CFS was 5.7% vs. 6.9% in non-CFS adults (P = 0.54). In adolescents, prevalence rates were 18.2% and 17.4%, respectively (P = 0.93). Adult patients with POTS-CFS were younger (30 ± 12 vs. 40 ± 13 years, P = 0.001) and had a higher supine heart rate (71 ± 11 vs. 65 ± 9 beats per min, P = 0.009) compared with non-POTS-CFS patients. Severity and activity patterns did not differ between groups. In patients with CFS, criteria for Systemic Exertion Intolerance Disease (SEID) were met in 76% of adults and 67% of adolescents. In these patients with CFS fulfilling the SEID criteria, the prevalence of POTS was not different from that in the overall CFS population. POTS-CFS adolescents had less clinically significant improvement after CBT than non-POTS-CFS adolescents (58% vs. 88%, P = 0.017). CONCLUSION: In adults with CFS, the prevalence of POTS was low, was not different from the rate in non-CFS fatigued patients and was not related to disease severity or treatment outcome. In POTS-CFS adolescents, CBT was less successful than in non-POTS-CFS patients. The evaluation of POTS appears to be of limited value for the diagnosis of CFS.

Defective trained immunity in patients with STAT-1-dependent chronic mucocutaneaous candidiasis.

Patients with signal transducer and activator of transcription-1 (STAT1)-dependent chronic mucocutaneous candidiasis (CMC) and patients with STAT3-dependent hyper-immunoglobulin (Ig)E syndrome (HIES) display defects in T helper type 17 (Th17) cytokine production capacity. Despite this similar immune defect in Th17 function, they show important differences in the type of infections to which they are susceptible. Recently, our group reported differential regulation of STAT-1 and STAT-3 transcription factors during epigenetic reprogramming of trained immunity, an important host defence mechanism based on innate immune memory. We therefore hypothesized that STAT1 and STAT3 defects have different effects on trained immunity, and this may partly explain the differences between CMC and HIES regarding the susceptibility to infections. Indeed, while trained immunity was normally induced in cells isolated from patients with HIES, the induction of innate training was defective in CMC patients. This defect was specific for training with Candida albicans, the main pathogen encountered in CMC, and it involved a type II interferon-dependent mechanism. These findings describe the role of STAT-1 for the induction of trained immunity, and may contribute to the understanding of the differences in susceptibility to infection between CMC and HIES patients. This study could also provide directions for personalized immunotherapy in patients suffering from these immunodeficiencies.

Mast-cell interleukin-1ß, neutrophil interleukin-17 and epidermal antimicrobial proteins in the neutrophilic urticarial dermatosis in Schnitzler's syndrome.

BACKGROUND: Schnitzler's syndrome (SchS) is an autoinflammatory disease characterized by a chronic urticarial rash, a monoclonal component and signs of systemic inflammation. Interleukin (IL)-1ß is pivotal in the pathophysiology. OBJECTIVES: Here we investigated the cellular source of proinflammatory mediators in the skin of patients with SchS. METHODS: Skin biopsies of lesional and nonlesional skin from eight patients with SchS and healthy controls, and patients with cryopyrin-associated periodic syndrome (CAPS), delayed-pressure urticaria (DPU) and cold-contact urticaria (CCU) were studied. We studied in vivoIL-1ß, IL-17 and antimicrobial protein (AMP) expression in resident skin cells and infiltrating cells. In addition we investigated the in vitro effect of IL-1ß, IL-17 and polyinosinic-polycytidylic acid (poly:IC) stimulation on cultured epidermal keratinocytes. RESULTS: Remarkably, we found IL-1ß-positive dermal mast cells in both lesional and nonlesional skin of patients with SchS, but not in healthy control skin and CCU, and fewer in CAPS. IL-17-positive neutrophils were observed only in lesional SchS and DPU skin. In lesional SchS epidermis, mRNA and protein expression levels of AMPs were strongly increased compared with nonlesional skin and that of healthy controls. When exposed to IL-1ß, poly:IC or IL-17, patient and control primary human keratinocytes produced AMPs in similar amounts. CONCLUSIONS: Dermal mast cells of patients with SchS produce IL-1ß. This presumably leads to activation of keratinocytes and neutrophil influx, and further amplification of inflammation by IL-17 (from neutrophils and mast cells) and epidermal AMP production leading to chronic histamine-independent neutrophilic urticarial dermatosis.

Lotus petal perforator flap for reconstruction of an extended chronic presacral defect after radiotherapy and rectal cancer resection.

AIM: As a result of their extent and complexity, pelvic wounds after surgery for anorectal malignancy often require a multidisciplinary approach to accomplish closure. This report describes a successful reconstruction using the lotus petal perforator flap. METHOD: This flap is based on perforators of the internal pudendal artery and was partially depithelialized for plugging the defect. RESULTS: Wound healing was achieved after 12 days. CONCLUSION: The lotus petal flap is a relatively simple and successful choice for reconstruction of an extended chronic presacral defect after radiotherapy and rectal cancer resection.

Different patterns of Toll-like receptor 2 polymorphisms in populations of various ethnic and geographic origins.

Upon the invasion of the host by microorganisms, innate immunity is triggered through pathogen recognition by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are the best-studied class of PRRs, and they recognize specific pathogen-associated molecular patterns (PAMPs) from various microorganisms. A large number of studies have shown that genetic variation in TLRs may influence susceptibility to infections. We assessed the genetic variation of TLR2, which encodes one of the most important TLRs, in various populations around the globe and correlated it with changes in the function of the molecule. The three best-known nonsynonymous TLR2 polymorphisms (1892C>A, 2029C>T, and 2258G>A) were assessed in different populations from the main continental masses: Romanians, Vlax-Roma, Dutch (European populations), Han Chinese (East Asia), Dogon, Fulani (Africa), and Trio Indians (America). The 2029C>T polymorphism was absent in both European and non-European populations, with the exception of the Vlax-Roma, suggesting that this polymorphism most likely arose in Indo-Aryan people after migration into South Asia. The 1892C>A polymorphism that was found exclusively in European populations, but not in Asian, African, or American volunteers, probably occurred in proto-Indo-Europeans. Interestingly, 2258G>A was present only in Europeans, including Vlax-Roma, but at a very low frequency. The differential pattern of the TLR2 polymorphisms in various populations may explain some of the differences in susceptibility to infections between these populations.

Association of esophageal candidiasis and squamous cell carcinoma.

Chronic esophageal candidiasis is an infection that is mostly seen in immunocompromised conditions, among which is chronic mucocutaneous candidiasis (CMC). Recently an association between CMC and esophageal carcinoma has been reported. Here we present two patients with chronic esophageal candidiasis who developed esophageal squamous cell carcinoma and we discuss the etiologic role of Candida-induced nitrosamine production, the loss of STAT1 function and impaired tumor surveillance and T-lymphocyte function in the development of esophageal carcinoma.

Is extensive screening for cancer in idiopathic venous thromboembolism warranted?

BACKGROUND: Patients with a first episode of idiopathic venous thromboembolism (IVTE) have an estimated 10% incidence of cancer within 12 months after diagnosis. However, the utility of screening for cancer in this population is controversial. METHODS: In this prospective concurrently controlled cohort study, limited and extensive cancer screening strategies were compared. All 630 patients underwent baseline screening consisting of history, physical examination, basic laboratory tests and chest X-ray. In the extensive screening group abdominal and chest CT scan and mammography were added. Outcomes were incidence and curability of cancer, and cancer-related and overall mortality. RESULTS: In 12 of the 342 (3.5%) patients in the extensive screening group malignancy was diagnosed at baseline compared with 2.4% (seven of 288 patients) in the limited screening group. Extensive screening detected six additional cancers (2.0%; 95% CI, 0.74-4.3), of which three were potentially curable. During a median 2.5 years of follow-up, cancer was diagnosed in 3.7% and 5.0% in the extensive and limited screening groups, respectively. In the extensive screening group 26 patients (7.6%) died compared with 24 (8.3%) in the limited screening group; adjusted hazard ratio 1.22 (95% CI, 0.69-2.22). Of these deaths 17 (5.0%) in the extensive screening group and 8 (2.8%) in the limited screening group were cancer related; adjusted hazard ratio 1.79 (95% CI, 0.74-4.35). CONCLUSIONS: The low yield of extensive screening and lack of survival benefit do not support routine screening for cancer with abdominal and chest CT scan and mammography in patients with a first episode of IVTE.

Maternal micronutrient supplementation with zinc and ß-carotene affects morbidity and immune function of infants during the first 6 months of life.

BACKGROUND/OBJECTIVES: Micronutrient deficiencies are prevalent worldwide, and a major cause of infant death. Supplementation with multiple micronutrients during pregnancy might improve micronutrient status of the newborn, thereby reducing morbidity and death. Moreover, maternal supplementation might affect the newborn's immune development. Therefore, this study investigated the effects of maternal zinc and ß-carotene supplementation on the infant's morbidity and immune function during the first 6 months of life. SUBJECTS/METHODS: Mothers were supplemented during pregnancy with ß-carotene and/or zinc, in addition to iron and folic acid, in a randomized, double-blind controlled trial. Newborn infants (n=136) were followed up for 6 months. RESULTS: Infants born from mothers receiving zinc during pregnancy had significantly fewer episodes of diarrhoea than infants born from mothers not receiving zinc (0.2 and 0.4, respectively), but more episodes of cough (1.3 and 0.9 respectively) during the first 6 months. Maternal ß-carotene supplementation had no effect on infants' morbidity. Cytokine production in the newborns was affected by maternal zinc and ß-carotene supplementation, with zinc supplementation giving higher interleukin-6 production (16% higher), and ß-carotene supplementation leading to lower interferon-γ production (36% lower). CONCLUSIONS: This study shows that maternal supplementation with zinc and ß-carotene affected the newborn's immune development in specific ways, but only maternal zinc supplementation significantly affected morbidity in the infants. Addition of zinc to routine iron and folic acid supplements for pregnant women could be an effective way to reduce diarrhoeal disease during the first 6 months of life, albeit at the expense of more episodes of cough.

Comprehensive gas chromatography with Time of Flight MS and large volume introduction for the detection of fluoride-induced regenerated nerve agent in biological samples.

Recently, several methods have been developed to verify exposure to nerve agents. Most of these methods, such as the fluoride reactivation technique and the analysis of inhibited phosphonylated butyrylcholinesterase (BuChE), are based on mass spectrometry. The high specificity of the mass spectrometer might also imply a disadvantage, because the acquisition mass, i.e. the identity of the analyte must be known beforehand in order to direct the MS analysis in the most sensitive mode. In real cases, the identity of the nerve agent is not always known beforehand and the mass spectrometer should be operated in a scanning mode, with the consequence that sensitivity of the method will be lower. Comprehensive GC, or GC x GC, is a technique which offers enhanced separation. The implied larger selectivity of the GC separation allows mass spectrometry to be conducted in a less specific, scanning, mode. By the use of this configuration, the identity of the nerve agent does not have to be known beforehand but can be traced. In order to be able to detect lower concentrations and assess lower exposure levels, a large volume injection technique was developed allowing sample sizes up to 100 microL. The technique was tested with plasma samples that had been inhibited with various nerve agents. Subsequently, the cholinesterase-bound nerve agent was regenerated by the fluoride reactivation technique. Using the newly developed comprehensive GC-MS method it was possible to detect nerve agent at an exposure level of 1% BuChE inhibition, which is approximately 70 pg nerve agent/mL. These low exposure levels cannot be verified with a cholinesterase (ChE) activity assay. Moreover, the identity of the regenerated nerve agent was verified by the mass spectrum that was generated by the TOF mass spectrometer. This paper presents a technique able to deliver full-scan data on the analysis of nerve agents in biomedical samples at relevant exposure levels (1% BuChE inhibition). This full-scan data meets for a large part the forensic requirements that are in place for the analysis of biomedical samples in the context of alleged use of Chemical Warfare Agents.

A prospective cohort study on the absolute risks of venous thromboembolism and predictive value of screening asymptomatic relatives of patients with hereditary deficiencies of protein S, protein C or antithrombin.

BACKGROUND: Absolute risks of venous thromboembolism (VTE) in protein S-, protein C-, or antithrombin-deficient subjects are mainly based on retrospective data. Screening asymptomatic relatives of these patients is disputed, though studies addressing this issue have yet to be conducted. METHODS: We prospectively followed 382 relatives of 84 probands. Participants were assessed for other thrombophilic defects and occurrence of exogenous risk factors (i.e. surgery/trauma/immobilization, malignancies, use of systemic estrogens, and pregnancy/puerperium). After screening, deficient subjects were advised to use thromboprophylaxis during exogenous risk factors; use of oral contraceptives was discouraged. RESULTS: Overall annual incidence of VTE was 1.53% (95% CI, 1.00-2.34) in deficient vs. 0.29% (0.13-0.64) in non-deficient relatives; adjusted hazard ratio, 7.0 (95% CI, 2.7-18.0). Annual incidence of unprovoked VTE was 0.95% in deficient vs. 0.05% in non-deficient subjects; age-adjusted hazard ratio, 22.3 (P = 0.003). In contrast, annual incidence of provoked VTE was 0.58% vs. 0.24%; age-adjusted hazard ratio, 2.8 (P = 0.08). Fifty-five (37%) deficient and 80 (34%) non-deficient subjects experienced 91 and 143 exogenous risk factors, respectively, during which six vs. five VTEs (6.6% vs 3.5% per risk-period) occurred, despite the higher compliance with recommended thromboprophylaxis use in deficient (51%) vs. non-deficient (22%) subjects. In deficient subjects all provoked VTEs occurred when thromboprophylaxis was not used. CONCLUSIONS: Protein S, protein C or antithrombin deficiencies confer high absolute risk of VTE. Screening and subsequent augmentation of thromboprophylaxis use may result in reduction of provoked VTE, whereas risk of unprovoked VTE could not be affected by screening.

Milder clinical hyperimmunoglobulin E syndrome phenotype is associated with partial interleukin-17 deficiency.

Mutations in the signal transducer and activator of transcription 3 (STAT3) were reported to cause hyperimmunoglobulin E syndrome (HIES). The present study investigates T helper type 17 (Th17) responses triggered by the relevant stimuli Staphylococcus aureus and Candidia albicans in five 'classical' HIES patients, and a family with three patients who all had a milder HIES phenotype. We demonstrate that patients with various forms of HIES have different defects in their Th17 response to S. aureus and C. albicans, and this is in line with the clinical features of the disease. Interestingly, a partial deficiency of interleukin (IL)-17 production, even when associated with STAT3 mutations, leads to a milder clinical phenotype. We also observed defective Th17 responses in patients with the 'classical' presentation of the disease but without STAT3 mutations. These data demonstrate that defective IL-17 production in response to specific pathogens can differ between patients with HIES and that the extent of the defective Th17 response determines their clinical phenotype.