Marco Fizzotti

Defective Expression of Fas Messenger RNA and Fas Receptor on Pulmonary T Cells from Patients with Asthma

During the past several years, considerable progress has been made toward a better understanding of the pathogenesis of allergic diseases [1]. Inflammation at the sites of target organs seems to be the pathologic hallmark of the disease process; among the various types of cells involved in tissue infiltration and damage, T lymphocytes are probably the main effector cells priming the local ongoing allergic immune response [2-5]. These cells are principally involved in the local recognition of aerodispersed allergens and act by secreting interleukin-4 and interleukin-5, the so-called T-helper type 2 (Th2) cytokines [6, 7], and thus determining local and systemic IgE synthesis and the mucosal recruitment of other inflammatory cells, such as neutrophils and eosinophils. The cell secreting interferon- or interleukin-2 (or both) is T-helper type 1 (Th1), and it is involved in the delayed hypersensitivity reactions [8]. Although our knowledge about the initiation of the allergic response has rapidly expanded in recent years [9], nothing is known about why the inflammatory response cannot be turned off in atopic persons. Because immune reactions in general are potentially dangerous in a physiologic setting [10], they must be carefully controlled (if the antigenic stimulus becomes too great) or extinguished (if the damaging agent is successfully eliminated). One important mechanism of lymphocyte control is programmed cell death (apoptosis), which occurs in every immune response [11, 12], is energy dependent, and is associated with endonuclease activation. The biochemical hallmark of apoptosis is the fragmentation of chromatin into oligonucleosomes with subsequent changes in nuclear structure (picnosis) and alterations in the surface membrane of the apoptotic cell; this allows the cell to be eliminated from the site of inflammation by phagocytosis without damage to the surrounding host tissue [12]. T cells have at least two apoptotic pathways: active death, which is antigen driven, and passive death, which occurs at the end of an immune response and is due to lymphokine withdrawal or other mechanisms [13]. These two forms of death are molecularly distinct because the cell surface molecule Fas/APO-1 (CD95), a 45-kD protein belonging to the tumor necrosis factor (TNF) receptor family [14], and TNF itself are major participants in active, but not passive, death. The cross-linking of Fas receptor with its recently identified ligand (Fas ligand) leads to the induction of apoptosis in lymphocytes [15] and provides a mechanism for the removal of antigen-activated T cells [16, 17]. This leads to resolution of the inflammatory response [18], protecting the host against the detrimental effects that would ensue if cell disintegration or necrosis were to occur. Spontaneous remission of respiratory allergic disease invariably following withdrawal of allergen [19, 20] underlies the mechanism of passive death. Because Fas and TNF are major participants in active antigen-driven death [13], we investigated whether T cells from the lungs of untreated persons with asthma express Fas receptor. We show that the persistence of allergen-specific T cells at the mucosal surfaces of atopic persons results from local defective surface expression of Fas receptor and subsequent impairment in active, allergen-driven cell death. Methods Study Participants Twelve patients (6 male and 6 female; age range, 8 to 54 years) with newly diagnosed, mildly symptomatic chronic asthma who underwent bronchoalveolar lavage were included in the study. At the time of recruitment, all patients had stable pulmonary function and an FEV1 at least 70% of that predicted for their age and height. None was receiving inhaled or oral corticosteroids, sodium chromoglycate, theophylline, or 2 agonists. All had increased airway responsiveness to methacholine (concentration producing a decrease of 20% from baseline in FEV1, <8 mg/mL); were atopic, as defined by positive skin prick tests done with purified Dermatophagoides pteronyssinus allergen extract (Neo Abello, Madrid, Spain); and had positive results on enzyme-linked immunosorbent assay (DPC Corp., Los Angeles, California) for circulating allergen-specific IgE. No patients smoked or had had an upper respiratory tract infection in the 8 weeks before the study. Ten age- and sex-matched normal nonsmoking persons served as controls. No control had a history of asthma, systemic illness, or recent respiratory illness or any evidence of airway hyperresponsiveness on methacholine challenge. All had normal results on pulmonary function tests; none had positive results on skin prick tests to a panel of allergens that included house dust mites and pollens. The study protocol was approved by the institutional review board for human studies of our university, and informed consent was given by each patient or by the patients relatives if the patient was a child. The study was conducted according to local ethical committee guidelines and the principles of the Declaration of Helsinki. Collection of Bronchoalveolar Lavage Fluid Fiberoptic bronchoscopy was performed as reported elsewhere [21]. Lavage of the right middle lobe was done with three consecutive 20-mL aliquots of prewarmed physiologic saline. Recovered bronchoalveolar lavage cells were spun immediately at 200 g for 10 minutes at 4C. After resuspension, cytospins were prepared for differential cell counts. Reagents and Flow Cytometry Immunophenotyping was done simultaneously on pulmonary and peripheral blood T cells from patients and controls by using the following monoclonal antibodies in various combinations: phycoerythrin-conjugated anti-CD3 (OKT3, Ortho, Raritan, New Jersey), which recognizes up to 90% of T cells bearing the or T-cell receptor heterodimer; anti-CD4 and anti-CD8 (OKT4 and OKT8, Ortho), which stain helper/inducer and cytotoxic T-cell subsets; anti-CD25 (anti-interleukin-2 receptor, Becton-Dickinson, Mountain View, California) and anti-HLA-DR, which recognize, respectively, the interleukin-2 receptor chain and the major histocompatibility complex class II molecule (both referring to activated T cells); anti-CD45R0 (Immunotech, Marseille, France), a common leukocyte antigen present on activated/memory T cells; fluorescein-conjugated anti-Fas receptor (Immunotech); and phycoerythrin-conjugated anti-TCR [T-cell receptor] delta1 (T-Cell Sciences, Cambridge, Massachusetts), a pan-reactive T-cell reagent. For staining, 5000 to 10 000 cells were resuspended in 50 L of saline, incubated at 4C for 30 minutes, washed, and analyzed by flow cytometry (FACScan, Becton-Dickinson). For analysis of two-color cytofluorometric data, an electronic gate was set on the lymphocyte population based on the forward-angle versus the right-angle light scatter histogram. Quadrant markers in fluorescence histograms were set by using matched isotype controls. The Lysis II program (Becton-Dickinson) was used to optimize gating of lymphocytes and to provide an objective way to exclude both debris (noncellular events due to particulate matter) and other cells from the lymphocyte gate. Separation of Enriched T-Cell Populations Bronchoalveolar lavage mononuclear cells from patients and controls were separated into sheep erythrocyte rosette-enriched (containing T cells) and rosette-depleted subsets [22]. On the basis of their reactivity with the anti-CD3 monoclonal antibody OKT3 (>98% by immunofluorescence), the rosetted mononuclear cells were considered to be highly purified T-cell subsets and were used for Fas messenger RNA (mRNA) polymerase chain reaction (PCR) analysis and apoptotic cell death experiments. Modulation of Surface Fas Receptor on Pulmonary T Cells T-cell-enriched populations from patients and controls were cultured in vitro on 24-well plastic plates (Kostar, Denmark) coated with anti-CD3 monoclonal antibody for 24 hours in the presence of recombinant human interleukin-2 (50 U/mL; Serotec, Oxford, United Kingdom) or interleukin-4 (400 IU/mL; Serotec). At the end of the culture time, cells were analyzed for surface expression of Fas molecule by double-color immunofluorescence. Evaluation of Fas-Induced Programmed Cell Death Enriched T lymphocytes derived from bronchoalveolar lavage were washed and resuspended in RPMI-1640 (Gibco, Grand Island, New York) supplemented with 10% fetal calf serum, 2 mmol of L-glutamine per L, 10 mmol of HEPES per L, 50 U of penicillin per L, and 50 g of streptomycin at 106 cells/mL. All samples were incubated for 18 hours with medium alone or with anti-Fas IgM (a specific antibody to the Fas receptor that can induce apoptosis of Fas+ target cells), washed, centrifuged at 200 g for 10 minutes, and dissolved in hypotonic lysing buffer (100 mmol of NaCl per L, 10 mmol of Tris per L, 1 mmol of EDTA per L, 1% sodium dodecyl sulfate, 200 g of proteinase K per mL, pH of 7.5). A standard DNA electrophoresis assay was used for qualitative evaluation of apoptotic cells, as described elsewhere [23]. Quantitative analysis of spontaneous and anti-Fas-induced programmed cell death was done by cytofluorometry with a fluorochrome solution containing propidium iodide [23]. Fas Messenger RNA Expression Expression of mRNA was detected by reverse transcription PCR. With the guanidinium isothiocyanate acid phenol method, RNA was isolated from bronchoalveolar lavage-enriched T cells from patients, controls, and the U937 cell line (as a positive control) [24]. First-strand complementary DNA synthesis was done by using total RNA, random hexadeoxynucleotide primers, and Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Life Technologies, Milano, Italy), as described elsewhere [25]. Primers for Fas and -actin PCR amplification were designed according to previously published sequences [25]. After amplification, one tenth of the PCR product was run on 2% agarose gel and stained with ethidium bromide. Statistical Analysis Patients were stratified according to age. Pair

Complete Resolution of Life-Threatening Bleomycin-Induced Pneumonitis After Treatment With Imatinib Mesylate in a Patient With Hodgkin's Lymphoma: Hope for Severe Chemotherapy-Induced Toxicity?

Introduction Bleomycin-induced pneumonitis (BIP) is a scarcely manageable pulmonary toxicity that occurs in approximately 20% of patients who are affected with cancers that are treated with bleomycin-containing regimens. Generally, the clinical picture is extremely complicated and therapy is based on steroids. The overall response rate to such therapy is limited. Recent insights in BIP pathogenesis have indicated that a key feature is deregulated mechanisms of tissue repair that are driven by profibrotic cytokines. Here we describe a patient with lifethreatening BIP who was completely cured with imatinib mesylate (IM) after steroids and all other therapies had failed.

Role of T-helper type 2 cytokines in down-modulation of Fas mRNA and receptor on the surface of activated CD4+ T cells: molecular basis for the persistence of the allergic immune response

The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation‐induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL‐2), IL‐4, IL‐5, transforming growth factor β1, interferon γ, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). In vitro exposure of purified CD4+ lymphocytes to allergen yielded only transient up‐regulation of surface Fas but did not influence susceptibility to Fas‐mediated cell death. T‐helper type 2 cytokines (IL‐4, IL‐5, and GM‐CSF) had a dose‐dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure.—Spinozzi, F., Agea, E., Fizzotti, M., Bassotti, G., Russano, A., Droetto, S., Bistoni, O., Grignani, F., Bertotto, A. Role of T‐helper type 2 cytokines in down‐modulation of Fas mRNA and receptor on the surface of activated CD4+ T cells: molecular basis for the persistence of the allergic immune response. FASEB J. 12, 1747–1753 (1998)

Interphase FISH for Y Chromosome, VNTR Polymorphisms, and RT-PCR for BCR-ABL in the Monitoring of HLA-Matched and Mismatched Transplants

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.

An acute hepatitis resembling autoimmune hepatitis occurring during imatinib therapy in a gastrointestinal stromal tumor patient.

A 65-year-old man was admitted to our hospital with a 6-month history of increasing abdominal size. His medical history was unremarkable. On physical examination, a mass was felt in the right abdomen. Laboratory tests were within normal range. In particular, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 21 and 22 U/dL, respectively (normal range 40 U/dL). Computed tomography scan showed a 16-cm peritoneal mass without evidence of hepatic involvement. A gastrointestinal stromal tumor (GIST) was diagnosed and imatinib (IM) therapy (400 mg/d) was started. One month later, abdominal computed tomography scan showed initial shrinkage of this mass, but an asymptomatic progressive increase of AST and ALT values was observed. IM was withdrawn on the hypothesis of a rare toxicity due to IM. Nevertheless, AST and ALT went up to 418 and 1056 U/dL, respectively (Fig. 1). All other liver function tests were within normal range, and no viral infection could be detected. Alcohol and other drug abuse were excluded. Antinuclear antibodies and M2 fraction of antimitochondrial antibodies were positive. A liver biopsy showed no tumoral cells but interface hepatitis with piecemeal necrosis (Fig. 2). This picture was consistent with an autoimmune hepatitis (AH). Ursodeoxycholic acid (1200 mg/d) and prednisone (1 mg/kg/d) were begun. Six weeks later, imatinib was given at the dose of 400 mg/d. The aminotransferase serum levels decreased progressively (Fig. 1) and prednisone, as well as ursodeoxycholic acid, was suspended after 6 months. One month later, a liver biopsy showed a complete resolution of the inflammatory disease (Fig. 3). IM therapy was continued, and the patient was well at his last follow-up visit. Hepatotoxicity (mild transaminitis) is observed in 10% of patients treated with imatinib, and a grade 3 or 4 elevation in liver function tests affects fewer patients. Imatinib has been occasionally reported to cause hepatotoxicity with a common histologic signature: a centrolobular hepatic necrosis with lymphoplasmacytic infiltration and interface hepatitis. In one case, antinuclear antibodies were elevated while autoantibodies were either normal or not evaluated in other reports. None of the largest published series examine the 3% to 5% reported G3-4 hepatotoxicity, and this is the first case, to our knowledge, of AH described during imatinib therapy in GIST. The association of GIST, IM, and AH may have been coincidental, but the chronological sequence raises the suspicion of a causal relationship. Less clear is the pathogenesis of this phenomenon. A first hypothesis is that IM itself could have been the triggering event. In a relatively “young” drug, rare side effects become evident in the long run. However, we suggest this is an unlikely mechanism. Indeed, autoimmune drug (eg, phenytoin) side effects require drug withdrawal, and readministration From the *Medical Oncology Unit, †Gastroenterology Unit, and ‡Pathology Unit, Institute for Cancer Research and Treatment, Candiolo, Italy. Reprints: Giovanni Grignani, MD, Medical Oncology Unit, Institute for Cancer Research and Treatment, Str. Provinciale 142, 10060 Candiolo, Italy. E-mail: ggrignani@ircc.mauriziano.it. Copyright

Biochemical and immunological responses of hairy cell leukemia patients to interferonβ

SummaryTen hairy-cell leukemia patients were treated with interferon β (IFN-β) at a dose rate of 2 × 106 IU/m2 × 5 days for 4 weeks (induction therapy) and, thereafter, at the same dose three times a week for 11 months (maintenance therapy). The effect of this treatment on serum neopterin, β2-microglobulin, (2′–5′)oligoadenylate [(2′–5′)An] levels, intracellular (2′–5′)An values and human Mx protein synthesis was analysed. There were significant rises in serum neopterin and (2′–5′)An levels during both induction and maintenance, whereas β2-microglobulin levels rose only during induction. Rises in intracellular (2′–5′)An were documented mainly during induction, but they were not significantly higher than pretherapy values. IFNβ provoked an increase in human Mx protein synthesis over the entire induction — maintenance period, but was only significantly higher than baseline during induction. All markers proved useful for monitoring the effects of IFNβ dose schedules, but were not predictive of clinical outcome. Natural killer activity and IFNγ production, which were initially defective, followed a different trend from that of the other factors studied, in that increases were documented only late in the course of therapy when the disease was already in remission.

Post-Transplant Cyclophosphamide and Tacrolimus–Mycophenolate Mofetil Combination Prevents Graft-versus-Host Disease in Allogeneic Peripheral Blood Hematopoietic Cell Transplantation from HLA-Matched Donors

Allogeneic hematopoietic cell transplant (HCT) remains the only curative therapy for many hematologic malignancies but it is limited by high nonrelapse mortality (NRM), primarily from unpredictable control of graft-versus-host disease (GVHD). Recently, post-transplant cyclophosphamide demonstrated improved GVHD control in allogeneic bone marrow HCT. Here we explore cyclophosphamide in allogeneic peripheral blood stem cell transplantation (alloPBSCT). Patients with high-risk hematologic malignancies received alloPBSCT from HLA-matched unrelated/related donors. GVHD prophylaxis included combination post-HCT cyclophosphamide 50 mg/kg (days +3 and +4) and tacrolimus/mofetil mycophenolate (T/MMF) (day +5 forward). The primary objective was the cumulative incidence of acute and chronic GVHD. Between March 2011 and May 2015, 35 consecutive patients received the proposed regimen. MMF was stopped in all patients at day +28; the median discontinuation of tacrolimus was day +113. Acute and chronic GVHD cumulative incidences were 17% and 7%, respectively, with no grade IV GVHD events, only 2 patients requiring chronic GVHD immunosuppression control, and no deaths from GVHD. Two-year NRM, overall survival, event-free survival, and chronic GVHD event-free survival rates were 3%, 77%, 54%, and 49%, respectively. The graft-versus-tumor effect was maintained as 5 of 15 patients (33%) who received HCT with evidence of disease experienced further disease response. A post-transplant cyclophosphamide + T/MMF combination strategy effectively prevented acute and chronic GVHD after alloPBSCT from HLA-matched donors and achieved an unprecedented low NRM without losing efficacy in disease control or impaired development of the graft-versus-tumor effect. This trial is registered at clinicaltrials.gov as NCT02300571.

Response to intermediate and standard doses of IFN-β in hairy-cell leukaemia

Abstract Thirteen hairy-cell leukaemia patients were treated with IFN-β (6 × 10 6 IU/m 2 ) for 7 days, alternate weeks, for three cycles. IFN-β was then continued at the same dose twice a week for 24 weeks. Treatment was discontinued in 2 non-responders and 2 partial responders (1 haem PR, 1 path PR) because of complications unrelated to IFN. The objective response in the nine patients who completed therapy was 66% (1 CR, 3 path PR and 2 haem PR); 2 patients achieved MR. Responses lasted from 5 to 45+ months. Four newly diagnosed patients and 3 in relapse after discontinuation of IFN-β therapy (6 × 10 6 IU/m 2 ), were treated with a lower dose of IFN-β (2 × 10 6 IU/m 2 ). The objective response to this dose was 57% (3 path PR, 1 haem PR). Another patient obtained MR. No patient has relapsed 6–12 months after therapy discontinuation. IFN-β was well tolerated, especially at the lower dose and no chronic toxicity was observed. Therefore IFN-β may be suggested as an alternative treatment for HCL.

Modulation of NK activity by thymic hormones:in vitro effects of thymostimulin

Plastic-adherent depleted or not depleted peripheral mononuclear blood cells (PMBC) from healthy donors showed enhanced lytic activity against51Cr-labelled K562 target cells when exposed to thymostimulin (TS), 1 μg ml-1, for 3 h, washed and incubated in TS-free medium before testing for natural killer (NK) cytotoxicity. No modification of NK cell activity was seen when effector cells were treated with placebo (splenic extract). The NK boosting activity of TS was lost when effector cells were treated for 3 h immediately before the performance of the cytotoxic test or when this thymic extract was added directly to the mixture of effector and target cells during the lytic phase of51Cr release assay.

Natural-killer-stimulatory effect of combined low-dose interleukin-2 and interferon β in hairy-cell leukemia patients

The association of low doses of interleukin-2 (IL-2; 5 IU/ml) and interferon β (IFNβ; 10 IU/ml) induced an additive or synergic stimulatory effect on natural killer (NK) activity (32%) in peripheral blood samples from hairy-cell leukemia patients, both those with active disease and those in remission. The synergic NK stimulatory effect was more commonly found in samples from patients with active disease, while the additive effect was more frequent in the patients in remission. The IL-2/IFNβ combination provoked a nonadditive nonsynergic NK-stimulatory effect in a further 19.8% samples. The targets of the IL-2/IFNβ combination were typical NK cells, as shown by the fact that there was increased cytotoxicity (synergic, additive or nonadditive nonsynergic) against the K562, but not the Daudi cell line in peripheral blood mononuclear cell samples treated with the combination of the two cytokines. When CD16+/CD56+ or CD57+/CD16+/CD56+ cells were removed, the NK-stimulatory effect was lost. The fact that the NK-cell-enhancing activity of the IL-2/IFNβ combination was reduced when Percoll fractions 2 and 3 were used, but still persisted in 66% of tests, may have been due to cytotoxicity being higher in the untreated fractions 2 and 3 than in the untreated unfractionated samples. One of the factors responsible for the NK-stimulatory effect appears to be the capacity of the IL-2/IFNβ combination to trigger an increase in IFNγ synthesis. If similar experiments give like results in samples from patients suffering from other B-cell lymphoproliferative, or HIV-associated disorders, all of which are characterized by a deficiency in NK activity, it should be possible to use low-dose IL-2/IFNβ to treat these disorders and, perhaps, residual neoplastic disease without exposing the patient to undue toxicity. Further, by testing other combinations one should be able to identify the lowest IL-2 and IFNβ doses that would effectively boost the additive or synergic effect in a greater number of cases.