The signaling function of IDO1 incites the malignant progression of mouse B16 melanoma.

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene (Ido1WT ) or a mutated variant lacking the catalytic, but not signaling activity (Ido1H350A ). As compared to the Ido1WT -transfected counterpart (B16WT), B16-F10 cells expressing Ido1H350A (B16H350A) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16H350A cells, also detectable in vivo, were found to be accompanied by a reduction in tumor-infiltrating CD8+ T cells and an increase in Foxp3+ regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule.

Lombardy diagnostic and therapeutic network of thrombotic microangiopathy.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA) requiring urgent treatment. Standardization of its diagnosis and optimal management is challenging. This study aimed to evaluate the role of centralized, rapid testing of ADAMTS13 in patients experiencing acute TMAs requiring plasma-exchange (PEX) and to estimate the incidence of TTP in a large Italian Region. METHODS: We perfomed a cohort study in the frame of the project "Set-up of a Lombardy network for the study and treatment of patients undergoing apheresis", including 11 transfusion centers in the Region. Consecutive patients referred from 2014 to 2016 with acute TMAs requiring PEX were enrolled. Centralized ADAMTS13 activity testing was performed at the Milan Hemophilia and Thrombosis Center within 24 h. RESULTS: Forty-three TMA patients (44 events) were enrolled, of whom 35 (81%) had severe ADAMTS13 deficiency. Patients with severe ADAMTS13 deficiency were younger, mainly women, with a higher prevalence of autoimmune disorders and a lower prevalence of cancer. Clinical and laboratory characteristics of patients with and without severe ADAMTS13 deficiency largely overlapped, with a lower platelet count being the only baseline marker that significantly differed between the two patient groups (ADAMTS13 activity < 10% vs ≥ 10%: median difference of -27 × 109/l, 95% CI - 37 to - 3). PEX treatment was initiated in all patients, but soon discontinued in cases without severe ADAMTS13 deficiency. In this group, the mortality rate was higher and no episode exacerbations or relapses within 6 months occured. The estimated average annual incidence of acute acquired TTP events was 1.17 [0.78-1.55] per million people. CONCLUSIONS: Severe ADAMTS13 deficiency distinguished two groups of patients with largely overlapping clinical features but different treatment and disease course. This study provides a feasible model implemented in a large Italian region for the practical clinical approach to TMAs and underlines the importance of urgent ADAMTS13 activity testing for an accurate differential diagnosis and therapeutic approach.

T cell receptor beta chain gene rearrangements in lymphoproliferative disorders of large granular lymphocytes/natural killer cells.

Twelve cases of T gamma LPD (lymphoproliferative disorders of Fc gamma receptor-bearing T cells) involving an expansion of large granular lymphocyte/natural killer (LGL/NK) cells were investigated for the expression of LGL/NK-associated markers and for T beta gene rearrangement. All the cases selected were classified as T gamma LPD on the basis of morphology, function, and phenotype of the circulating cells. 10 to 12 cases displayed clonal rearrangements of the T beta locus and expression of the T3 antigen, whereas the 2 remaining cases displayed the germline configuration of the T beta gene and no expression of the T3 antigen. T8, Mol, B73.1, and N901 antigens were variably expressed among both T beta+T3+ and T beta-T3- T gamma LPD cases. We suggest that individual T gamma LPD cases represent the clonal expansion of cells frozen at different stages of differentiation/activation within an individual hematopoietic LGL/NK lineage.

An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals. Expression of vector-derived ADA restored immune functions, as indicated by the presence in reconstituted animals of human immunoglobulin and antigen-specific T cells. Retroviral vector gene transfer, therefore, is necessary and sufficient for development of specific immune functions in vivo and has therapeutic potential to correct this lethal immunodeficiency.

HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

Lymphokine-activated killer activity of tumor-associated and peripheral blood lymphocytes isolated from patients with ascites ovarian tumors.

Peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) were isolated from 36 patients with advanced ovarian adenocarcinoma and peritoneal effusions for study of lymphokine-activated killer activity. PBLs and TALs cultured in vitro for 3-5 days in the presence of interleukin-2 (IL-2, supernatant of the MLA 144 gibbon cell line, or human recombinant IL-2) expressed higher levels of cytotoxicity as compared to cells cultured in medium alone, against natural killer (NK)-susceptible (K562) or NK-resistant targets (Daudi and the human ovarian carcinoma cell line SW626). When ovarian tumor cells, freshly isolated from carcinomatous ascites or surgical specimens, were used as target cells in the cytotoxicity assay, 8 of 14 PBLs and 5 of 7 TAL preparations lysed the autologous tumor after treatment with IL-2, while no spontaneous reactivity was observed in any of the 14 patients tested. Although levels of lysis were usually relatively low, these data demonstrate that PBLs and TALs from ovarian cancer patients (TALs usually exhibiting low NK activity) when stimulated in vitro by IL-2 acquire some cytotoxic potential against the autologous tumor.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA-deficient SCID.

Severe combined immunodeficiency (SCID) caused by deficiency of the enzyme adenosine deaminase (ADA) is the first genetic disorder considered for human somatic cell gene therapy. ADA-SCID patients can be cured by HLA-matched sibling donor bone marrow transplantation. Alternative transplantation strategies as well as enzyme replacement are being tested in those patients who do not have a suitable matched sibling donor. Some ADA-SCID patients may not be candidates for cytoablation due to infectious damage to the lung or liver, or may have a milder phenotype that does not justify the risks associated with haploidentical bone marrow transplantation. Replacement therapy with PEG-ADA has resulted in improvement in growth, a variable increase in the number of peripheral blood lymphocytes, and a decrease in the incidence of severe infections. Another approach to the treatment of severe genetic diseases is now represented by somatic cell gene therapy. We and others have conducted experiments in vitro and in vivo that have documented that T-lymphocytes are suitable vehicles for gene transfer. Although the pluripotent stem cell remains the ideal target cell for somatic cell gene therapy of disorders of the hematopoietic system, the use of T-lymphocytes as gene therapy vehicles is specifically indicated for ADA-deficient patients where they represent the affected cells. Furthermore, the selective engraftment of T-cells only, following bone marrow transplantation, has resulted in reconstitution of cellular and humoral immunity. A model for the functional analysis in vivo of the human immune system has been utilized for the preclinical evaluation of this approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunomodulation of neutrophil chemotaxis in rheumatoid arthritis using levamisole and methisoprinol.

The inflammatory process of rheumatoid arthritis is characterized by an alteration in neutrophil function and an accompanying increase in the number of these cells within the joint space. Both inhibition of peripheral neutrophil chemotaxis and the paradoxic and concomitant deleterious effects of overreactive synovial neutrophils are expressions of an imbalance within the immune system. Levamisole and methisoprinol have been found to improve the alterations in cellular function and immune response. We studied the in vitro effect of these drugs on neutrophil function in four patients with rheumatoid arthritis whose basal chemotaxis was seriously inhibited. Levamisole and methisoprinol improved neutrophil chemotaxis by inducing, in monocytes incubated with these drugs, the production of an important chemotactic factor effective on the altered neutrophils. We suggest that the use of these drugs is beneficial in the treatment of rheumatoid arthritis.

Pharmacological effect of hyaluronic acid (HA) on phagocytes: hypothesis for an HA-induced monocyte chemotactic factor for neutrophils.

The connection between hyaluronic acid and phagocyte function is not well documented in the literature. Its action may either inhibit or enhance polymorphonuclear neutrophil (PMN) function, depending on its concentration. Studies were conducted to verify the effect of hyaluronic acid on phagocytes, both directly using hyaluronic acid and indirectly via a mediated route using the medium from a hyaluronic acid monocyte incubation. Determinations were made of phagocytosis, reduction of nitroblue tetrazolium, superoxide production, and chemotaxis before and after incubation with hyaluronic acid. Chemotaxis of PMNs was used to evaluate the chemotactic action of a medium in which monocytes had been incubated with hyaluronic acid. This method resulted in progressive improvement in the chemotactic index. The authors conclude that the monocytes incubated with hyaluronic acid produce a chemotactic factor for neutrophils.

Decrease of CD4+CD45+ T-cells in chronic-progressive multiple sclerosis.

Circulating lymphocyte subpopulations defined by anti-CD45 and other more common T-cell-specific monoclonal antibodies were analysed in 77 patients with multiple sclerosis and 38 healthy controls. A selective decrease of CD4+CD45+ cell percentages and absolute numbers in chronic-progressive patients was found; in 13 out of 26 patients this subpopulation was less than 11% CD4+CD45+ cells. Similarly, the whole CD45+ cell subset, as well as CD45+ cells expressed as percentages of CD4+ cells, were significantly reduced in chronic-progressive multiple sclerosis. CD4+CD45+ cells, commonly termed "inducer of suppression" T-lymphocytes, did not correlate with percentages or numbers of CD8+ cells. It is concluded that suppressor inducer T-cells act on the CD8+ subset function rather than reducing CD8+ cell numbers. Since CD4+CD45+ cells represent an early stage of lymphocyte maturation (naive T-cells), an under-representation of this subpopulation in active multiple sclerosis might reflect an increased conversion of naive cells into memory cells. This concept may be relevant for a better understanding of the disease pathogenesis.

Relationship between pancreatic and serum amylase in basal conditions and during stimulation in man. Report of a new intraoperative technique.

The correlation between serum amylase and pancreatic amylase secretion was studied in two patients. Both patients underwent sphincterotomy, and the pancreatic duct was cannulated with a polyethylene tube. The tube was left in place for 15 to 21 days. The rate of amylase secretion over 7 days was studied in response to (1) a standard meal, (2) duodenal acidification, (3) scalar doses of cholecystokinin, (4) scalar doses of secretin, and (5) scalar doses of secretin with simultaneous infusion of cholecystokinin. Blood samples were collected during the tests to measure serum amylase. No significant correlation was shown between blood concentration and output of amylase in any of the tests. Our findings show that under normal conditions serum amylase levels are not influenced by pancreatic secretion and suggest that serum amylase concentration is not related to pancreatic exocrine secretion.

Clinical utility of fractionating erythrocytes into "Percoll" density gradients.

Two rapid methods for fractionating the RBC into five or nine layers of increasing density are reported. These procedures have been used to monitor the decline of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activity during the process of red cell aging in normal subjects and in beta-thal carriers, to study transfused patients with G6PD and pyruvate kinase (PK) deficiency and to test the effects of inositol hexaphosphate (IHP) encapsulation on RBC subpopulations.

Mitomycin C, 5fluorouracil and folinic acid in combination with alpha 2b interferon for advanced colorectal cancer.

AIMS AND BACKGROUND: This study was conducted to investigate the activity and toxicity of 5fluorouracil folinic acid+mitomycin C combined with alpha 2b interferon in advanced colorectal cancer based upon recent studies suggesting a possible biochemical modulation of 5fluorouracil by interferon. PATIENTS AND METHODS: Between June 1990 and April 1991 25 previously untreated patients with advanced colorectal carcinoma were treated with mitomycin C 10 mg/m2 iv bolus on day 1, 5fluorouracil 375 mg/m2 on days 1 to 4 and folinic acid 200 mg/m2 on days 1 to 4 every 4 weeks, combined with alpha 2b interferon 3 million U day continuously. RESPONSE: Of the 25 patients entered into the study, 20 were evaluable for response as 5 patients withdrew due to toxicity (grade 3-4 thrombocytopenia in 4 cases and fatigue in 1). No complete response was recorded, 6 patients had partial remission (30%; 95% confidence interval, 10% to 50%), 4 experienced no change and 10 showed progressive disease. The toxicity of this regimen was significant, particularly myelosuppression. CONCLUSIONS: This combination showed a significant toxicity and low response rate compared with other 5fluorouracil based regimens in advanced colorectal cancer.

A phase II study of mitomycin C, vindesine and cisplatin combined with alpha interferon in advanced non-small cell lung cancer.

AIMS AND BACKGROUND: MVP chemotherapy (mitomycin C, vindesine or vinblastine, cisplatin) is one of the most commonly used regimens for advanced non-small cell lung cancer (NSLCLC). Experimental data suggest a synergistic cytotoxic activity of alpha-interferon (alpha-IFN) when combined with cisplatin, mitomycin C and vinca alkaloids. In an effort to improve MVP chemotherapy activity, we have combined this regimen with alpha-IFN. PATIENTS AND METHODS: Thirty-five patients with advanced NSCLC (19 stage IV) were treated with the MVP regimen (mitomycin C, 8 mg/m2; vindesine, 3 mg/m2, cisplatin, 75 mg/m2, all on day 1) plus alpha-2a-IFN, 3x10(6) U from day 1 to 7. The cycles were repeated every 28 days. RESULTS: There were no complete responses and 18 partial responses, for an overall response rate of 51%. Median time to treatment failure was 6 months (range, 1-18), and the median survival was 9.5 months (range, 1-32). WHO grade 3 toxicity was recorded in up to 8% of patients, flu-like syndrome was a common complaint; one toxic death occurred. CONCLUSIONS: The combination yielded a level of response comparable to that of other cisplatin-based regimens. Larger randomized trials are needed to assess the role of alpha-IFN combined with chemotherapy in advanced NSCLC.

[Importance of hyaluronic acid in the modulation of neutrophil migration]. / Importanza dell'acido ialuronico nel modulare la migrazione dei neutrofili.

The relationship between JA and phagocyte function has often been reported in the literature. The action of JA may either inhibit or stimulate PMNs function depending on the concentration. On the basis of this experience, the efficacy of JA action, both directly and mediated after incubation was studied. In particular phagocytosis, NBT, superoxide production and chemotaxis were studied in basal conditions and after incubation with hyaluronic acid. In particular chemotaxis was also performed to assay the chemotactic action of the medium in which the monocytes were incubated with JA and the technique was found to produce a distinct progressive improvement in the chemotactic index. In conclusion, it is hypothesised that monocytes incubated with JA produce a chemotactic factor for PMNs.

Chronic insomnia in workers poisoned by inorganic mercury: psychological and adaptive aspects.

Insomnia is one of the symptoms of inorganic mercury poisoning (IMP). The objective of this study is to analyze the chief psychological aspects in the adjustment of workers with chronic insomnia associated with IMP. For this purpose the Preventive Clinical Interview and the Ryad Simon Operational Adaptive Diagnostic Scale (Escala Diagnóstica Adaptativa Operacionalizada-EDAO) were utilized. Fifteen subjects with mean age of 40 years (10 males and 5 females) were studied. Nine were diagnosed with High Adaptive Inefficacy, five with Moderate Inefficient Adaptation and only one with Mild Inefficient Adaptation. Impairment occurred in four adaptive sectors: affective relationship, social-cultural, productivity and organic. Adaptive efficiency indicated that in all the 15 subjects studied the adaptive solutions were frustrating and led to psychic suffering and/or environmental conflict confirming the severity of the involvement in chronic IMP.