Analysis of CFTR mRNA and Protein in Peripheral Blood Mononuclear Cells via Quantitative Real-Time PCR and Western Blot.

The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.

Genetic landscape of pediatric acute liver failure of indeterminate origin.

BACKGROUND AND AIMS: Pediatric acute liver failure (PALF) is a life-threatening condition. In Europe, the main causes are viral infections (12%-16%) and inherited metabolic diseases (14%-28%). Yet, in up to 50% of cases the underlying etiology remains elusive, challenging clinical management, including liver transplantation. We systematically studied indeterminate PALF cases referred for genetic evaluation by whole-exome sequencing (WES), and analyzed phenotypic and biochemical markers, and the diagnostic yield of WES in this condition. APPROACH AND RESULTS: With this international, multicenter observational study, patients (0-18 y) with indeterminate PALF were analyzed by WES. Data on the clinical and biochemical phenotype were retrieved and systematically analyzed. RESULTS: In total, 260 indeterminate PALF patients from 19 countries were recruited between 2011 and 2022, of whom 59 had recurrent PALF. WES established a genetic diagnosis in 37% of cases (97/260). Diagnostic yield was highest in children with PALF in the first year of life (41%), and in children with recurrent acute liver failure (64%). Thirty-six distinct disease genes were identified. Defects in NBAS (n=20), MPV17 (n=8), and DGUOK (n=7) were the most frequent findings. When categorizing, the most frequent were mitochondrial diseases (45%), disorders of vesicular trafficking (28%), and cytosolic aminoacyl-tRNA synthetase deficiencies (10%). One-third of patients had a fatal outcome. Fifty-six patients received liver transplantation. CONCLUSIONS: This study elucidates a large contribution of genetic causes in PALF of indeterminate origin with an increasing spectrum of disease entities. The high proportion of diagnosed cases and potential treatment implications argue for exome or in future rapid genome sequencing in PALF diagnostics.

Reversing the Inflammatory Process-25 Years of Tumor Necrosis Factor-α Inhibitors.

Immune-mediated inflammatory diseases, such as rheumatoid arthritis, psoriatic arthritis, peripheral and/or axial spondyloarthritis, Crohn's disease, and ulcerative colitis, are characterized by molecular and cellular changes in the immune system. Due to the systemic nature of these diseases, organs such as the liver or cardiovascular system are often affected by the inflammatory process. Tumor necrosis factor-α inhibitor therapy reduces the activation of pro-inflammatory signaling cascades, mitigates the chronic inflammatory process by restoring cellular balance, and alleviates clinical consequences, such as pain and tissue damage.

Elexacaftor - Tezacaftor - Ivacaftor treatment improves systemic infection parameters and Pseudomonas aeruginosa colonization rate in patients with cystic fibrosis a monocentric observational study.

Background & aims: The CFTR-modulating therapy Elexaftor - Tezacaftor - Ivacaftor (ETI) has been widely prescribed since its approval in 2020 in the European Union. The aim of this study was to methodically evaluate the effects of an ETI treatment on clinical, biochemical data and Pseudomonas colonization in order to demonstrate its efficacy. Methods: This prospective monocentric study comprised 69 patients diagnosed with cystic fibrosis aged at least 12 years and treated with ETI between September 2020 and November 2021. Clinical and laboratory data of each patient and study visit were collected before and after 24 weeks of ETI treatment. Follow-up status of Pseudomonas aeruginosa (PsA) colonization was assessed after one year of therapy by regularly determined sputum or throat swab samples. Results: Marked improvements biochemical markers of systemic inflammation as white blood cell count, levels of immunoglobulins A, G and M and albumin within 24 weeks of therapy were observed. ETI treatment proved to be effective as seen by amelioration of lung function and sweat chloride concentration. Assessment of PsA colonization status revealed a conversion from a positive to negative detection in 36% of the cases after one year of therapy. Conclusions: ETI treatment effectively improves systemic inflammation parameters and shows promising results in PsA status conversion.

TNF promotes DECTIN2 family C-type lectin receptor expression in human macrophages.

TNF blockade constitutes an effective therapy for inflammatory bowel disease, yet increases the risk for infection, including active tuberculosis. The DECTIN2 family C-type lectin receptors MINCLE, MCL, and DECTIN2 sense mycobacterial ligands and activate myeloid cells. In mice, upregulation of DECTIN2 family C-type lectin receptor after stimulation with Mycobacterium bovis Bacille Calmette-Guérin requires TNF. Here, we investigated whether TNF controls inducible C-type lectin receptor expression in human myeloid cells. Monocyte-derived macrophages were stimulated with Bacille Calmette-Guérin and the TLR4 ligand lipopolysaccharide, and expression of C-type lectin receptor was analyzed. Bacille Calmette-Guérin and lipopolysaccharide strongly upregulated messenger RNA expression of DECTIN2 family C-type lectin receptor but not of DECTIN1. Bacille Calmette-Guérin and lipopolysaccharide also induced robust production of TNF. Recombinant TNF was sufficient to upregulate expression of DECTIN2 family C-type lectin receptor. Blocking TNF with the TNFR2-Fc fusion protein etanercept abrogated, as expected, the effect of recombinant TNF and impaired induction of DECTIN2 family C-type lectin receptor by Bacille Calmette-Guérin and lipopolysaccharide. Flow cytometry confirmed upregulation of MCL at the protein level by recombinant TNF and showed inhibition of Bacille Calmette-Guérin-induced MCL by etanercept. To investigate the impact of TNF on C-type lectin receptor expression in vivo, we analyzed peripheral blood mononuclear cells of patients with inflammatory bowel disease and observed downregulation of MINCLE and MCL expression after therapeutic TNF blockade. Together, TNF is sufficient to upregulate DECTIN2 family C-type lectin receptor in human myeloid cells and contributes to this process after encounter with Bacille Calmette-Guérin or lipopolysaccharide. Impaired C-type lectin receptor expression in patients receiving TNF blockade may dampen the sensing of microbes and defense against infection.

Increase of liver stiffness and altered bile acid metabolism after triple CFTR modulator initiation in children and young adults with cystic fibrosis.

BACKGROUND: Novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies (elexacaftor/tezacaftor/ivacaftor-ETI) promise clinically significant and sustained improvements for patients with cystic fibrosis (CF). In this study, we investigated the impact of ETI therapy on liver stiffness and bile acid metabolism in a cohort of children and young adults with CF. METHODS: A prospective observational study (NCT05576324) was conducted from September 2020 to November 2021 enrolling CF patients naive to ETI. Standard laboratory chemistry, sweat test, lung function, share wave velocity (SWV) derived by acoustic radiation force impulse imaging (ARFI) and serum bile acid profiles were assessed before and 6 months after induction of ETI therapy. RESULTS: A total of 20 patients (10 aged <20 years) completed the study. While lung function and BMI improved after ETI therapy, ARFI SWV increased in CF patients <20 years of age (from 1.27 to 1.43 m/s, p = 0.023). Bile acid (BA) profiles revealed a decrease in unconjugated (5.75 vs 1.46, p = 0.007) and increase in glycine-conjugated derivatives (GCDCA) (4.79 vs 6.64 p = 0.016). There was a positive correlation between ARFI SWV values and GCDCA (r = 0.80, p < 0.0001). Glycine-conjugated BA provided high diagnostic accuracy to predict increased ARFI measurements (AUC 0.90) and clinical (Colombo) CFLD grading (AUC 0.97). CONCLUSIONS: ARFI SWV and bile acid profiles provide evidence for early increase in liver stiffness and altered bile acid metabolism in young CF patients after initiation of ETI and may serve as synergistic measures for detection of hepatic complications during ETI therapy.

A Modified Surgical Technique for Kidney Transplantation in Mice.

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

Mucosal Mast Cell Distribution in the Gastrointestinal Tract of Children: A Preliminary Study for Establishing Reference Values.

OBJECTIVES: The physiological number and distribution of mast cells (MCs) in the pediatric gastrointestinal (GI) tract is not well defined and reference values of normality are missing. To define a physiological and disease defining cut-off, a systematic histological exploration of MC distribution from the esophagus to the rectum in healthy as well as in patients with gastrointestinal food allergies (GFA) was performed. METHODS: Nine pediatric subjects that exhibited unremarkable histopathological evaluations or underwent endoscopy for surveillance reasons after a previous polypectomy of single colonic juvenile polyps served as reference cohort. In all of these subjects, a chronic inflammatory disease (eg, inflammatory bowel disease, celiac disease) or allergy was excluded. In addition, a group of 15 patients with gastrointestinal complaints suspected to be caused by a GFA were investigated. Immunohistochemistry was performed from all biopsies using CD117 (c-Kit) as a reliable marker to identify MCs in the lamina propria. RESULTS: There were distinct differences of MC counts in all parts of the pediatric GI tract. The highest counts of MCs in both symptomatic patients and control cohort, were found in the duodenum, terminal ileum, cecum and ascending colon. The lowest counts were found in the esophagus. Significant disparities between GFA and healthy subjects were found in the gastric corpus (22.1 ±â€Š4.0/ high power field [HPF] vs 32.0 ±â€Š10.1/HPF; P = 0.034) and ascending colon (44.8 ±â€Š10.4/HPF vs 60.4 ±â€Š24.3/HPF; P = 0.047). CONCLUSIONS: Mucosal MC counts in the pediatric GI tract are higher than previously reported, with a considerable overlap between healthy and GFA patients. These results provide detailed information on distribution and numbers of MCs in pediatric allergic patients while allowing estimates of physiological values in childhood for the first time. With regard to diagnostic procedures in GFA further laboratory parameters have to be integrated.

Reactivations of Latent Viral Infections Are Associated with an Increased Thr389 p70S6k Phosphorylation in Peripheral Lymphocytes of Renal Transplant Recipients.

Reactivations of BK polyoma virus (BKPyV) and human cytomegalovirus (HCMV) frequently cause life- and graft-threatening complications after renal transplantation. Both viruses are dependent on the mTOR pathway for replication. In this study we investigated the association of viral replication with mTOR activity in peripheral lymphocytes of renal transplant recipients. A flow-cytometry based assay for the measurement of Thr389 p70S6k phosphorylation, a surrogate marker of the mTOR pathway was established. Forty-eight adult renal transplant recipients were recruited to measure p70S6k activity in their peripheral blood mononuclear cells. This data set in conjunction with information concerning previous replication of BKPyV and HCMV was examined for correlations. Episodes of BKPyV replication were significantly associated with increased p70S6k phosphorylation in CD4+ T lymphocytes (p = 0.0002) and CD19+ B lymphocytes (p = 0.0073). HCMV infection of patients with a high-risk HCMV constellation of donor and recipient (D+/R-) was associated with increased p70S6k phosphorylation in CD19+ B lymphocytes (p = 0.0325). These associations were found to be independent of the trough levels of the immunosuppressive drugs. Conclusion: P70S6k phosphorylation in peripheral lymphocytes is associated with BKPyV reactivations and to a lesser extent with HCMV infections in renal transplant recipients.

Distribution and Cytokine Profile of Peripheral B Cell Subsets Is Perturbed in Pediatric IBD and Partially Restored During a Successful IFX Therapy.

BACKGROUND: The role of B cells in inflammatory bowel disease (IBD) is ambiguous, as B cells may have both pathogenic and protective functions in IBD. We studied B cell subsets before and after initiation of an anti-tumor necrosis factor alpha (anti-TNFα) therapy in pediatric IBD. The aim of the study was to examine the behavior of B cells in pediatric IBD patients undergoing an anti-TNFα therapy and, more specifically, to clarify their association with a successful or an unsuccessful infliximab (IFX) treatment. METHODS: A total of N = 42 pediatric IBD patients (Crohn disease, n = 30; ulcerative colitis, n = 12) for whom an anti-TNFα therapy with and without a concomitant azathioprine (AZA) medication was administered were recruited. Fourteen healthy age-matched children served as control patients. Blood samples were collected before initiation of the anti-TNFα therapy, before the fourth infusion at the end of the induction phase, and after 6 and 12 months under therapy maintenance. Flow cytometry (CD20, CD27, CD38, CD138) and intracellular staining (interleukin 10 [IL10], TNFα, granzyme B) were performed. Responders to successful IFX therapy were classified exhibiting a fecal calprotectin level of below 100 µg/g or achieving levels of <10% of the baseline value at initiation than at the end of the 12-month follow-up period. RESULTS: Before initiation of anti-TNFα therapy, flow cytometry revealed increased percentages of naïve B cells whereas transitional B cells were reduced compared with those in the healthy control patients. The IL10-producing B cells of both ulcerative colitis and Crohn disease patients were reduced at the initiation of IFX therapy, whereas TNFα-producing transitional CD24hiCD38hi B cells in ulcerative colitis patients were increased compared with those in healthy control patients. After 12 months of therapy, we detected a significant increase of IL10-producing transitional B cells in responding patients.The IFX trough levels in the responding patients showed a significant increase until 6 months after IFX initiation, attaining mean values of 9.9 µg/mL, whereas the IFX dosage was significantly lower than that in the nonresponding patients. The IFX trough levels in AZA-treated patients reached earlier therapeutic levels than in patients without AZA comedication, whereas during the course of the IFX therapy, comedication with AZA had no significant effect on the outcome. CONCLUSIONS: Attaining a normalization of IL10 production among CD24hiCD38hi B cells after 12 months of therapy may represent additional information about the reconstitution of a patient's immune system in responding patients. The achievement of an IFX trough level of ~10 µg/mL at 6 months of treatment is associated with a successful anti-TNFα therapy. In addition, AZA comedication supports an earlier achievement of therapeutic IFX trough levels.

Association of clinical factors with postoperative complications of esophageal atresia.

BACKGROUND: Esophageal atresia (EA) with or without tracheoesophageal fistula (TEF) remains one of the most common gastrointestinal neonatal malformations. Even though postoperative management is standardized, it differs between hospitals and disease-associated clinical factors that may play a role in outcome have not yet been assessed in detail. METHODS: In this monocentric retrospective study, data of 43 patients with EA between 2010 and 2018 were analyzed. Analysis includes assessment of the clinical background, surgical technique, postoperative management including application of continuous muscle relaxation (CMR), influence of coagulation parameters such as factor XIII and incidence of complications. RESULTS: 21 patients (49%) were preterm infants with birth weights between 490 and 2840 g (median 1893 g). Only 35% (n = 15) presented without any concomitant malformations. Within the entire study population, representing Vogt II, IIIb and IIIc, we observed an association between the development of a postoperative pneumothorax and anastomotic failure (AF) (p = 0.0013). Furthermore, pneumothorax was associated with anastomotic stenosis (AS) in Vogt IIIb patients (p = 0.0129). CMR (applied since March 2017 in 7 patients in an attempt to prevent anastomotic problems due to high complication rates) and coagulation factor XIII did not significantly correlate with postoperative outcome. CONCLUSION: Appearance of pneumothorax was correlated with postoperative complications. These children should be monitored carefully in closer scheduled gastroenterological follow-up esophago-gastro-duodenoscopies. CMR and factor XIII substitution did not reduce anastomotic leakage but should be tested within an enlarged study population.

Idiopathic Mast Cell Activation Syndrome With Associated Salicylate Intolerance.

Idiopathic mast cell activation syndrome can be a rare cause for chronic abdominal pain in children. It remains a diagnosis by exclusion that can be particularly challenging due to the vast variety of possible clinical manifestations. We present a 13-year-old boy who suffered from a multitude of unspecific complaints over a long period of time. In this case, an assessment of mast cell-derived metabolites and immunohistochemical analysis of bioptic specimen was worthwhile. After ruling out, primary (oncologic) and secondary causes for mast cell activation, pharmacologic treatment adapted to the patient's salicylate intolerance resulted in a major relief of symptoms.

Diversity of disorders causing neonatal cholestasis - the experience of a tertiary pediatric center in Germany.

BACKGROUND AND OBJECTIVE: Rapidly establishing the cause of neonatal cholestasis is an urgent matter. The aim of this study was to report on the prevalence and mortality of the diverse disorders causing neonatal cholestasis in an academic center in Germany. METHODS: Clinical chemistry and cause of disease were retrospectively analyzed in 82 infants (male n = 42, 51%) that had presented with neonatal cholestasis to a tertiary medical center from January 2009 to April 2013. RESULTS: Altogether, 19 disorders causing neonatal cholestasis were identified. Biliary atresia was the most common diagnosis (41%), followed by idiopathic cases (13%), progressive familial intrahepatic cholestasis (PFIC, 10%), cholestasis in preterm infants (10%), α1AT deficiency, Alagille syndrome, portocaval shunts, mitochondriopathy, biliary sludge (all 2%), and others. Infants with biliary atresia were diagnosed with a mean age of 62 days, they underwent Kasai portoenterostomy ~66 days after birth. The majority of these children (~70%) received surgery within 10 weeks of age and 27% before 60 days. The 2-year survival with their native liver after Kasai procedure was 12%. The time span between Kasai surgery and liver transplantation was 176 ± 73 days. Six children (7%), of whom three patients had a syndromic and one a non-syndromic biliary atresia, died prior to liver transplantation. The pre- and post-transplant mortality rate for children with biliary atresia was ~12 and ~17%, respectively. CONCLUSION: Neonatal cholestasis is a severe threat associated with a high risk of complications in infancy and it therefore requires urgent investigation in order to initiate life saving therapy. Although in the last 20 years new causes such as the PFICs have been identified and newer diagnostic tools have been introduced into the clinical routine biliary atresia still represents the major cause.

Pulse oximetry is insufficient for timely diagnosis of hepatopulmonary syndrome in children with liver cirrhosis.

OBJECTIVE: To prospectively investigate the prevalence of hepatopulmonary syndrome (HPS), the importance of pulse oximetry in diagnosing HPS, and the longitudinal course after liver transplantation in children with cirrhosis referred for liver transplantation. STUDY DESIGN: Fifty-six patients aged 1-17 years (mean age, 4.6 ± 5.0 years) with liver cirrhosis were screened for HPS by hyperemic capillary blood gas (CBG) analysis and contrast-enhanced transthoracic echocardiography. Eleven patients were excluded owing to conditions that can produce cardiopulmonary dysfunction, including 5 with cystic fibrosis, 1 with pulmonary arterial hypertension, and 5 with an intracardial shunt. HPS was classified in accordance with the European Respiratory Society Task Force criteria on pulmonary-hepatic disorders. Patient groups were compared for biochemical and clinical characteristics. RESULTS: Eighteen children (40%) with cirrhosis were intrapulmonary vasodilatation (IPVD)-positive and had a pulse oximetry oxygen saturation level >98%. Two of these patients (11%) exhibited moderate HPS with an elevated alveolar arterial oxygen gradient >15 mm Hg and PaO2 <70 mm Hg; they died before undergoing liver transplantation. The sensitivity and specificity of CBG analysis for detecting elevated alveolar arterial oxygen gradient in children with IPVD was 94% and 53%, respectively. HPS was associated with late hepatoportoenterostomy (P < .04). Liver transplantation led to resolution of HPS in all patients. CONCLUSION: IPVD is frequent in children with liver cirrhosis (40%). Pulse oximetry is insufficient for timely HPS diagnosis. Pathological CBG analysis data indicate IPVD in the majority of cases, but are imprecise in children aged <2 years. Contrast-enhanced transthoracic echocardiography and CBG analysis are recommended for evaluation of HPS in children with cirrhosis, regardless of liver synthesis capacity and clinical chemistry data.

Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients.

The presence of microchimerism in peripheral blood of solid organ transplant recipients has been postulated to be beneficial for allograft acceptance. Kinetics of donor cell trafficking and accumulation in pediatric allograft recipients are largely unknown. In this study, we implemented SNPs of the HVRs I and II of mitochondrial DNA to serve as molecular genetic markers to detect donor-specific cell chimerism after pediatric renal transplantation. Serial dilution of artificial chimeric DNA samples showed a linear correlation coefficient of R > 0.98 and a detection sensitivity of 0.01% with high reproducibility. Longitudinal semiquantitative analysis of donor-specific SNPs was then performed in peripheral blood mononuclear cells samples up to two yr post-transplant. Quantity of donor-specific cell chimerism in peripheral blood was highest in the early post-transplant period reaching values of ~10% after liver-kidney and 2.8% after renal transplantation. From one wk after transplantation, renal transplant patients exhibited an amount of donor-specific mtDNA ranging from 0.01% to 0.1%. We developed a highly accurate, sensitive, and rapid real-time quantitative PCR method using sequence-specific primers and fluorescent hydrolysis probes for the detection of at least 0.01% donor-specific cells in the recipient's peripheral blood after renal transplantation.

Modulation of T-cell activation by malignant melanoma initiating cells.

Highly immunogenic cancers such as malignant melanoma are capable of inexorable tumor growth despite the presence of antitumor immunity. Thus, only a restricted minority of tumorigenic malignant cells may possess the phenotypic and functional characteristics needed to modulate tumor-directed immune activation. Here we provide evidence supporting this hypothesis. Tumorigenic ABCB5(+) malignant melanoma initiating cells (MMICs) possessed the capacity to preferentially inhibit IL-2-dependent T-cell activation and to support, in a B7.2-dependent manner, induction of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Compared with melanoma bulk cell populations, ABCB5(+) MMICs displayed lower levels of MHC class I, aberrant positivity for MHC class II, and lower expression levels of the melanoma-associated antigens MART-1, ML-IAP, NY-ESO-1, and MAGE-A. Additionally, these tumorigenic ABCB5(+) subpopulations preferentially expressed the costimulatory molecules B7.2 and PD-1, both in established melanoma xenografts and in clinical tumor specimens. In immune activation assays, MMICs inhibited mitogen-dependent human peripheral blood mononuclear cell (PBMC) proliferation and IL-2 production more efficiently than ABCB5(-) melanoma cell populations. Moreover, coculture with ABCB5(+) MMICs increased the abundance of Tregs, in a B7.2 signaling-dependent manner, along with IL-10 production by mitogen-activated PBMCs. Consistent with these findings, MMICs also preferentially inhibited IL-2 production and induced IL-10 secretion by cocultured patient-derived, syngeneic PBMCs. Our findings identify novel T-cell modulatory functions of ABCB5(+) melanoma subpopulations and suggest specific roles for these MMICs in the evasion of antitumor immunity and in cancer immunotherapeutic resistance.

Cutting edge: Vascular endothelial growth factor-mediated signaling in human CD45RO+ CD4+ T cells promotes Akt and ERK activation and costimulates IFN-gamma production.

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-gamma in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

P2Y2 receptor polymorphisms and haplotypes in cystic fibrosis and their impact on Ca2+ influx.

OBJECTIVE: Activation of P2Y2 receptors in airway epithelia by ATP and UTP stimulates a Ca2+-regulated Cl- channel, which regulates Cl- secretion in cystic fibrosis (CF). We hypothesized that genetic alterations in the P2Y2 receptor may act as disease modifiers in CF and thus analyzed the coding region of this gene for polymorphisms in 146 CF patients and 64 healthy controls. We also assessed the impact of the genetic variants on Ca2+-influx of P2Y2-null cells transfected with several P2Y2 receptor haplotypes. RESULTS: We identified three frequent nonsynonymous P2Y2 receptor polymorphisms: Leu46Pro; Arg312Ser and Arg334Cys, of which only Arg312Ser was significantly more common in CF: Arg = 0.80, Ser = 0.20 (CF) vs. Arg = 0.72, Ser = 0.28 (controls), P < 0.05; for Leu46Pro, Leu = 0.92, Pro = 0.08 (CF) vs. Leu = 0.96, Pro = 0.04 (controls), P = 0.65 and for Arg334Cys, Arg = 0.79, Cys = 0.21 (CF) vs. Arg = 0.84, Cys = 0.16 (controls), P = 0.79. The most frequent haplotype was Leu46Leu/Arg312Arg/Arg334Arg (28% in CF, 31% in controls) but 6% of CF patients and none of the controls had Leu46Leu/Ser312Ser/Arg334Cys or Leu46Leu/Arg312Arg/Cys334Cys. To assess function of the receptor haplotypes, we stably transfected 1321N1 (P2Y-null) cells to similar levels of mRNA expression with Leu46Leu/Arg312Arg/Arg334Arg (wild-type), Leu46Leu/Ser312Ser/Arg334Arg and Leu46Leu/Arg312Arg/Cys334Cys and measured ATP-stimulated transient Ca2+-influx. Cells expressing the homozygous Cys334 variant had significantly increased Ca2+-influx compared to wild-type (P<0.01). The increase in Ca2+-influx was more pronounced in cells carrying the homozygous Ser312 variant than in cells with the other two genotypes (P<0.01). CONCLUSIONS: These data indicate that P2Y2 receptor gene haplotypes influence intracellular Ca2+-release. Such genetic variants might therefore represent modifiers of Cl- secretion or of response to P2Y2 agonist therapy in CF.

Human adenosine 2B receptor: SNP discovery and evaluation of expression in patients with cystic fibrosis.

OBJECTIVES AND METHODS: We analyzed the adenosine 2B (A2B) receptor gene, which consists of two exons, for single nucleotide polymorphisms (SNPs) and tested the hypothesis that coding sequence polymorphisms in the gene contribute to disease state in patients with cystic fibrosis (CF). Using PCR and restriction fragment length polymorphism (RFLP) analysis, we assessed 53 American subjects of mixed ethnicity, 64 European Caucasian control subjects and 148 Caucasian patients with CF for A2B SNPs. RESULTS: We identified one SNP in the 5' untranslated region (UTR) and seven SNPs in the open reading frame. One SNP was identified in the coding region of a German patient with CF but in none of the American subjects. No other SNPs were found in CF patients. A nonsynonymous SNP was identified in exon 2 of the German controls with an allelic frequency of 11%. The US subjects were of mixed ethnicity and most frequently (10.4%) had a SNP in the 5'UTR; 7 coding SNPs occurred in <3%. All SNPs other than the Leu96Phe and Gly136Arg variants were found only in African-Americans. Of the 26 African-Americans who were genotyped, 42% were hypertensive but the study group was too small to show an association between blood pressure status and SNP expression. CONCLUSIONS: The data show that SNPs in the A2B receptor gene are much more frequent in African-Americans than in Caucasians and that German Caucasians, but not African-Americans, express Gly136Arg. None of the SNPs identified in A2B receptors are likely to be modifiers in CF.

Use of polymorphisms in the noncoding region of the human mitochondrial genome to identify potential contamination of human leukemia-lymphoma cell lines.

The availability of the complete sequence of human mitochondrial DNA (mtDNA) has proven extremely useful in phylogenetic studies, forensic science and the determination of chimerism after allogeneic stem cell transplantation. In this study, we could demonstrate that the analysis of mtDNA polymorphisms is a quick and reliable method to identify contamination of human hematopoietic cell lines. This assay is based on PCR-sequencing of three hypervariable segments of the control region of mtDNA (hypervariable region (HRV) 1, 2 and 3). All three regions contain a large number of single-base polymorphisms. mtDNA was isolated according to standard laboratory procedures and amplified by PCR. Subsequently products were sequenced and evaluated with a semiautomated DNA sequencer system. So far, 21 human leukemia-lymphoma (LL) cell lines and nine other human cell lines were screened for contamination by other cell lines applying this method. We conclude that analysis of mtDNA polymorphisms is a quick, reliable and inexpensive method to detect intra - and interspecies cross-contamination and for the authentication of human LL cell lines. In comparison to other methods (cytogenetics, fluorescence in situ hybridization or immunophenotyping), this technique is less laborious and time consuming.