Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.

Recent advances in the management of cystic fibrosis (CF) target underlying defects in the CF transmembrane conductance regulator (CFTR) protein, but efficacy analyses remain limited to specific genotype-based subgroups. Patient-derived model systems may therefore aid in expanding access to these drugs. Brushed human nasal epithelial cells (HNEs) are an attractive tissue source, but it remains unclear how faithfully they recapitulate human bronchial epithelial cell (HBE) CFTR activity. We examined this gap using paired, brushed HNE/HBE samples from pediatric CF subjects with a wide variety of CFTR mutations cultured at the air-liquid interface. Growth and structural characteristics for the two cell types were similar, including differentiation into mature respiratory epithelia. In electrophysiologic analysis, no correlation was identified between nasal and bronchial cultures in baseline resistance or epithelial sodium channel (ENaC) activity. Conversely, robust correlation was demonstrated between nasal and bronchial cultures in both stimulated and inhibited CFTR activity. There was close correlation in modulator-induced change in CFTR activity, and CFTR activity in both cell types correlated with in vivo sweat chloride measurements. These data confirm that brushed HNE cell cultures recapitulate the functional CFTR characteristics of HBEs with fidelity and are therefore an appropriate noninvasive HBE surrogate for individualized CFTR analysis.

Detection of CFTR function and modulation in primary human nasal cell spheroids.

BACKGROUND: Expansion of CFTR modulators to patients with rare/undescribed mutations will be facilitated by patient-derived models quantifying CFTR function and restoration. We aimed to generate a personalized model system of CFTR function and modulation using non-surgically obtained nasal epithelial cells (NECs). METHODS: NECs obtained by curettage from healthy volunteers and CF patients were expanded and grown in 3-dimensional culture as spheroids, characterized, and stimulated with cAMP-inducing agents to activate CFTR. Spheroid swelling was quantified as a proxy for CFTR function. RESULTS: NEC spheroids recapitulated characteristics of pseudostratified respiratory epithelia. When stimulated with forskolin/IBMX, spheroids swelled in the presence of functional CFTR, and shrank in its absence. Spheroid swelling quantified mutant CFTR restoration in F508del homozygous cells using clinically available CFTR modulators. CONCLUSIONS: NEC spheroids hold promise for understanding rare CFTR mutations and personalized modulator testing to drive evaluation for CF patients with common, rare or undescribed mutations. Portions of this data have previously been presented in abstract form at the 2016 meetings of the American Thoracic Society and the 2016 North American Cystic Fibrosis Conference.

Exposure to hypomethylating agent, 5-azacytidine, may improve iCasp9 suicide gene therapy for treating GvHD in allografts.

Anti-tumor cellular immunotherapies that implement a suicide gene system can limit potential undesirable effects. In a haplo-identical bone marrow transplant clinical trial, over 90% of iCaspase-9-expressing cells were eradicated after AP1903 exposure, and signs of graft-versus-host disease disappeared. Nevertheless, low numbers of genetically modified T cells survived this treatment. We studied genetically modified cell lines (GMCL) that carried a dual iCaspase-9/ΔCD19 DNA construct (ΔCD19=truncated CD19). With AP1903 exposure, a low percentage of cells (1.47±0.67%; n=5 replications) persisted in vitro. Repeated exposures to increasing AP1903 doses generated low (GMCLLR) and high AP1903-responders (GMCLHR), which expressed different levels of surface ΔCD19 and intracellular iCaspase-9. Compared with GMCLHR, GMCLLR exhibited higher methylation of 5'-long-terminal repeat (LTR) promoters, both in the number of sequences with at least one methylated CpG (16 vs 51.5%, respectively) and in the number of CpG islands (1.2 vs 8.9%, respectively). Four days of 5-azacytidine exposure reduced methylation and increased ΔCD19 and iCaspase-9 expression. Interestingly, LTR demethylation restored GMCLLR sensitivity to AP1903 by 24.3-fold (1.8 vs 43.8%) without affecting GMCLHR. We showed that 5'-LTR-methylation inhibited transgene expression and caused AP1903 hypo-responsiveness. Treating with a hypomethylating agent restored AP1903 sensitivity. This approach can be applied in further clinical trials to improve iCaspase-9 response if low response is detected.

Combining Aspergillus mitochondrial and ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients.

The combination of two quantitative Aspergillus PCR assays, targeting a mitochondrial and a ribosomal target (AfQPCR), has proved effective for diagnosing invasive aspergillosis (IA) in hematology patients with risk factors and a positive galactomannan antigen (GM). The aim of the present study was to assess the performance of systematic AfQPCR for IA screening in at risk patients in a hematology intensive care unit (ICU). The study was performed in the hematology ICU at Besançon University Hospital from March 2012 to December 2013. GM detection (Platelia Aspergillus, Biorad, France) and AfQPCR were performed on the same serum sample, twice a week, in all patients with risk factors for IA. Risk factors and clinical, radiological, and biological data were prospectively recorded using the information sheet from the French network for the surveillance of Invasive Fungal Infection. Thirty-two patients were diagnosed with proven, probable, or possible IA according to the 2008 EORTC/MSG criteria. Sixteen patients had a positive AfQPCR: 9/16 had a positive GM at the same time (GM index >0.5), 4/16 had a positive GM before the AfQPCR and 3/16 had a negative GM at the time of the positive AfQPCR. Screening at risk patients using both AfQPCR and GM on the same serum sample is very feasible in a routine clinical setting. Our results confirm the usefulness of combining biomarkers for an early IA diagnosis.

[Mucositis after allogeneic stem cell transplantation: Risk factors, clinical consequences and prophylaxis]. / La mucite post-allogreffe de cellules souches hématopoïétiques: facteurs de risque, conséquences cliniques et prévention.

AIM: Oral mucositis is a very common complication of allograft. However, preventive treatments are still limited. The objective of this study is to identify risk factors for onset of oral mucositis in patients undergoing allogeneic hematopoietic stem cells transplantation (HSCT), to measure clinical consequences and to study their evolution according to type of prevention. PATIENTS AND METHODS: All patients undergoing HSCT in hematology unit of CHU Besançon between January 2009 and August 2010 were included, and received according to their choice, either the standard protocol: solution of sodium bicarbonate 1.4% associated with chlorhexidine-chlorobutanol (Eludril(®)) (n=49), or the experimental treatment by the ionic solution, Caphosol(®) (n=42). RESULTS: The overall incidence of severe mucositis and mucositis is respectively 69% and 36%. In multivariate analysis, a myeloablative conditioning (OR=11.1) and prevention of GVHD (graft-versus-host disease) including methotrexate (OR=7.5) appear such as the two significant mucositis risk factors. The presence of mucositis resulting in a significant increase in the incidence of febrile aplasia (P=0.008) and the use of opioid analgesics and parenteral nutrition (P<10(-3)). The risk of acute gastrointestinal GVHD is also increased in severe mucositis (P=0.01). The duration of post-transplant hospitalization is not changed. The type of prevention does not influence the incidence of mucositis (P=0.11). CONCLUSION: The consequences of mucositis are significant and the risk factors identified. The interest of the ionic solution Caphosol(®) seems limited, the incidence of mucositis is not decreased by this prevention.

Diagnosis and management of nocardiosis after bone marrow stem cell transplantation in adults: lack of lymphocyte recovery as a major contributing factor.

Hematopoietic cell transplantation (HCT) is a curative treatment for hematological malignancies. This therapeutic approach is associated with a profound immune deficiency and an increased rate of opportunistic infections. Nocardiosis is a rare bacterial infection occurring mainly in patients with deficient cell-mediated immunity, such as AIDS patients or transplant recipients. Diagnosis of nocardiosis can be challenging, as signs and symptoms are non-specific. Routine prophylaxis with trimethoprin/sulfamethoxazole (TMP/SMZ) does not prevent the risk of infection. Between May 2001 and December 2009, five cases of nocardiosis were diagnosed from the 366 allogeneic HCT recipients in our centre. Four patients developed a disseminated nocardiosis within the first year after HCT. The fifth patient presented a localized cutaneous nocardiosis. In disseminated cases, median total CD4+ T-cells were below 100 cells/µL. Naive CD4+ CD45RA+/RO- T-cells were almost undetectable. CD8(+) T-cells and NK cells were below the normal range and CD19+ B-cell reconstitution was completely deficient. In a localized case, we observed a lack of naive thymic emigrants CD4+ CD45RA+/RO- T-cells.

Evaluation of invasive aspergillosis risk of immunocompromised patients alternatively hospitalized in hematology intensive care unit and at home.

UNLABELLED: Contrary to hospital exposure, little is known about the indoor fungal exposure of hematology patients at home. The aim of our study was to investigate the mold exposure of hematology patients both at home and at hospital to assess their invasive aspergillosis (IA) risk. Fungal exposure was assessed by quantifying opportunistic molds at hospital during hospitalization and in homes of 53 hematology patients. IA was diagnosed in 13 of 53 patients and invasive fungal infection (IFI) in one patient. In hospital, no opportunistic species, or low levels of opportunistic species, were found in 98% of weekly controls. Only 2% of hematology intensive care unit (ICU) controls showed a high level of Aspergillus fumigatus spores in corridor air. Five patients IA were hospitalized during these periods. Seven dwellings of 53 (5/14 dwellings of patients with IA/IFI and 2/39 dwellings of non-IA patients) had a percentage of A. fumigatus and Aspergillus flavus to total mold (significant predictor variable of IA/IFI in our study, general linear model, P-value = 0.02) as high as 15%. Maintaining a 'zero Aspergillus' goal at hospital is essential, and establishing specific and individually opportunistic mold monitoring at home could help to further reduce the IA risk through continuous surveillance. PRACTICAL IMPLICATIONS: This study emphasizes the fact that preventive measures should not be aimed only at the hospital setting: among patients diagnosed with invasive aspergillosis/invasive fungal infection (IA/IFI), 5 of 14 (36%) were exposed to opportunistic fungal species at home exclusively. Moreover, four of these five patients were living in homes having the highest percentage of Aspergillus fumigatus and Aspergillus flavus (>15%), one of which had 48% of A. fumigatus. Therefore, our work supports the need for a counselor to carry out an environmental survey in patients' homes.

Diagnosis and treatment of digestive cryptosporidiosis in allogeneic haematopoietic stem cell transplant recipients: a prospective single centre study.

Digestive cryptosporidiosis (DC) can mimic GVHD after allogeneic haematopoietic stem cell transplantation (HSCT), thus requiring a reduction of immunosuppressive drugs and a specific therapy, whereas GVHD requires an intensification of immunosuppression. We systematically searched for cryptosporidiosis by light microscopy, immunochromatography and PCR in HSCT recipients who presented with at least one episode of diarrhoea. Of 115 consecutive patients allografted between July 2006 and November 2008, we analysed stools in 52 of 56 patients meeting these criteria. We identified Cryptosporidium parvum in 5 of the 52 patients (9.6%) at a median of 503 days (range 20-790) after HSCT. In those five patients, the median CD4+ cell and B lymphocyte counts were 60/mm3 (0-234) and 0/mm3 (0-96), respectively. Two patients died of invasive fungal infections. In the other three patients, diarrhoea disappeared after a median of 5 weeks following onset of bitherapy with azithromycine and nitazoxanide; they were still alive 433, 380 and 1179 days after the DC diagnosis. DC is probably under diagnosed after HSCT because it is difficult to detect during the asymptomatic phase. Early bitherapy and reduction of immunosuppression seem efficacious. In our series, DC has a seasonal pattern and is promoted by profound T lymphopenia.

False-positive Aspergillus real-time PCR assay due to a nutritional supplement in a bone marrow transplant recipient with GVH disease.

PCR screening for circulating DNA, especially when combined with antigen testing, has shown promise for the definitive diagnosis of invasive aspergillosis. False positives for Aspergillus real-time PCR assays have been described in several reports, but no sources of fungal DNA contamination could be clearly identified. We report a false-positive case for both galactomannan (GM) antigenemia and Aspergillus PCR due to nutritional supplement intake in a bone marrow transplant recipient with digestive graft-versus-host disease. Our case report also suggests that fungal DNA can pass into the serum from the intestinal tract in the same way as fungal GM. Clinicians should be aware of this possibility, so that the administration of costly, unnecessary antifungal treatments with potential adverse side-effects can be avoided.

Peripheral T-cell expansion and low infection rate after reduced-intensity conditioning and allogeneic blood stem cell transplantation.

Peripheral blood stem cell transplantation after reduced-intensity conditioning (RIC-PBSCT) regimen is an alternative to conventional regimens with less immediate toxicity. Since immune recovery is of crucial importance for the control of infections, we retrospectively studied the recovery of T-, B- and NK cell subsets in 20 consecutive patients undergoing RIC-PBSCT. We also studied the thymic output using T-cell receptor excision circle assay. Engraftment was rapid and few infectious complications were seen: three early (before 2.5 months) cases of asymptomatic cytomegalovirus reactivation, two late Gram-negative bacterial infections and no fungal infection. While CD4+ T-cell reconstitution was slow, CD8+ T-cell counts were close to normal values at 4 months. Median CD19+ B-cell counts reached normal values at 11 months. Rapid CD56+ NK cell reconstitution was noticed as early as 1.5 months. Low T-cell receptor excision circle numbers and preponderance of memory-type subsets among T cells further suggested that CD8+ T-cell reconstitution resulted predominantly from peripheral expansion and that thymic-dependent reconstitution was severely impaired. In conclusion, large peripheral T-cell expansion may compensate for late thymic-dependent lymphopoiesis, and may, with other factors such as NK and B-cell reconstitution and careful antiinfectious prophylaxis, help limit the incidence of severe infections after RIC-PBSCT.

Polyvisceral arteritis in chronic graft-versus-host disease: antiphospholipid-negative thrombotic syndrome mimicking polyarteritis nodosa.

A case of polyarteritis is reported in an 18-year old woman, occurring 2 years after an allogeneic bone marrow transplant. The clinical manifestations were similar to those of polyarteritis nodosa (PAN) with a wide range of organs involved including life-threatening cardiac and mesenteric problems requiring plasmapheresis and intravenous immunoglobulin (IgIV).

Adoptive transfer of ex vivo activated memory T-cells with or without cyclophosphamide for advanced metastatic melanoma: results in 36 patients.

Autolymphocyte therapy (ALT) is the infusion of autologous peripheral blood mononuclear cells (PBMC) activated ex vivo by a cytokine-rich supernatant (T3CS) generated from a previous autologous lymphocyte culture using low doses of the anti-CD3 mitogenic monoclonal antibody. The mechanism of action is enhancement of a recall response by CD45RO+ (memory) T-cells (ALT cells) to host tumour without dependence on exogenous interleukin (IL)-2. The existence of anti-tumour-specific T-cells in melanoma patients has been well described, and efforts to utilise them therapeutically have achieved modest tumour response rates. However, few long-term survival data have been reported. From 1986 to 1992, we treated 36 patients with disseminated melanoma using ALT alone (26 patients) or adoptive chemoimmunotherapy using ALT and cyclophosphamide (CY) (10 patients). Over this time period, the cell activation method evolved from using cytokine supernatants derived from a one-way allogeneic mixed lymphocyte culture (MLCS), to the current practice of utilising anti-CD3 and autologous cytokines (T3CS). There were 21 men and 15 women, average age 57 years, range 30-82. 27 had failed prior therapies and 9 had no prior therapy. A total of 161 infusion of ALT cells were given: 65 with cells activated in MLCS and 96 with T3CS. There were no grade 3 adverse events, and an approximate 20% incidence of grades 1 and 2 reactions to ALT-cell infusions. Transient cytopenias were seen in patients receiving CY. Sixty-one per cent (22/36) of patients received the planned six ALT-cell infusions, while 39% did not due to progressive disease. In 33 evaluable patients, there were four complete responses, four partial responses and 6 patients with stable disease (SD). Responding patients and those with SD had prolonged survival compared to historical controls when matched for number of organ systems involved. Ex vivo depletion of CD45RO+ T-cells revealed preferential lysis of autologous and HLA-A-matched melanoma targets that was dependent on these memory T-cells. These data suggest that adoptive cellular therapy using ex vivo activated memory T-cells with and without CY is active, has low toxicity, is tumour-specific and can result in clinical benefit in patients with disseminated melanoma.

Adoptive chemoimmunotherapy using ex vivo activated memory T-cells and cyclophosphamide: tumor lysis syndrome of a metastatic soft tissue sarcoma.

Adoptively transferred immune cells in combination with chemotherapeutic agents form the basis for adoptive chemoimmunotherapy (ACIT) of neoplastic disease. Autolymphocytes (ALT-cells) are ex vivo activated peripheral blood lymphocytes (PBL) from tumor-bearing hosts (TBH) that consist primarily of tumor-specific CD45RO+ (memory) T-cells. These ALT-cells combined with cimetidine (CIM) as autolymphocyte therapy (ALT), have previously been demonstrated to be a safe and active form of outpatient adoptive immunotherapy (AIT) in human TBH with metastatic renal cell cancer (RCC). We have previously described an effective ACIT protocol using ALT and cyclophosphamide (CY) for patients with relapsed and refractory non-RCC solid tumors. We now report a case of a patient with a metastatic gastric leiomyosarcoma to the liver, who developed a clinical picture consistent with a tumor-lysis syndrome (TLS), following salvage therapy for his tumor with ACIT using ALT and CY. TLS is a well-known complication resulting from the treatment of rapidly proliferating hematopoietic tumors such as Burkitt's lymphoma and acute lymphocytic leukemia. TLS has also been rarely described in chronic lymphocytic leukemia, as well as certain solid tumors such as breast cancer, small cell lung cancer, and medulloblastoma. However, there have been no previous reports of TLS occurring either secondary to immunotherapy or in sarcomas. The nature of these unusual findings is discussed.

Altered cytokine activity in adjuvant inhibition of autoimmune diabetes.

The destructive insulitis that develops in disease-prone NOD mice is characterized by a high frequency of IFN-gamma-producing cells in the lesion. Complete Freund's adjuvant (CFA) challenge of young, disease-prone animals inhibits the development of diabetes but does not alter the frequency with which pancreatic insulitis develops. However, the non-destructive lesions that develop in syngeneic islets transplanted to the kidney capsule of NOD mice following CFA therapy differ in cytokine production from the destructive lesions that develop in control animals transplanted with NOD islets. Non-destructive lesions are characterized by a high frequency of IL4-producing cells and a relatively low frequency of IFN-gamma-producing cells.

Adoptive chemoimmunotherapy for the treatment of relapsed and refractory solid tumors using ex vivo activated memory T cells (autolymphocyte therapy) and cyclophosphamide.

Autolymphocytes (ALT cells) are ex vivo activated peripheral blood lymphocytes (PBL) from tumor-bearing hosts (TBH) that consist primarily of tumor specific CD45RO+ (memory) T cells. These ALT cells combined with cimetidine as autolymphocyte therapy (ALT) have previously been demonstrated to be a safe and active form of outpatient adoptive immunotherapy (AIT) in human TBH with metastatic renal cell cancer (RCC). To determine activity of ALT in human TBH with therapy-resistant solid tumors other than RCC and whether it was feasible to combine ALT with chemotherapy, we studied 21 patients with relapsed or primary refractory solid tumors following a study protocol of adoptive chemoimmunotherapy (ACIT) using ALT and cyclophosphamide. Twenty patients were evaluable. Five responses were seen, including two complete responses (CRs) and three partial responses (PRs). ACIT activity was noted in relapsed TBH who had responded to their previous chemotherapy and/or radiation therapy. The toxic effects of this ACIT study were minimal with no treatment-related morbidity or mortality. It appears that in some relapsed but not primary refractory solid tumors, ACIT using ALT (CD45RO+ T cells and cimetidine) together with cyclophosphamide has definite antitumor activity associated with little or no toxic effects. Further studies of ACIT in solid tumors other than RCC are justified.

Coordinate production by a T cell hybridoma of gamma interferon and three other lymphokine activities: multiple activities of a single lymphokine?

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.

[Joint pain crises and chronic inflammatory-reactive phenomena: a unitary approach with proglumetacin]. / Crisi algiche articolari e fenomeni flogistico-reattivi cronici: un approccio unitario con la proglumetacina.

Nineteen patients with acute joint pain and 12 patients with chronic rheumatic disorders were treated with 450 mg/day of proglumetacin for a period ranging between 15 and 30 days. The overall results revealed a satisfactory response to treatment, and tolerance was rated as "good" or "very good" in all patients.