GM-CSF, ARA-C, VP-16 and idarubicin (GM-IVA), a short, and effective induction treatment for de novo AML, suitable for the elderly.

GM-IVA is a short and effective induction therapy of non M3 de novo AML including GM-CSF (300 mcg 12 hrs before starting therapy), Ara-C (250 mg/sqm c.i. x 3 days), VP16 (100 mg/sqm x 3 days) and idarubicin (12 mg/sqm x 3 days); it was followed by a fludarabine containing salvage protocol (FLANG). Patients <60 years of age achieving CR received 2 courses of FLANG and autologous or allogeneic BMT when possible. Patients >60 years of age in CR received a second course of GM-IVA. Twenty-one consecutive patients (mean age 64, range 29-85) entered the study. Three patients (14%) died during induction therapy. After one course of GM-IVA, CR was achieved in 12 patients (57%). Two further patients were salvaged with FLANG therapy so that the final CR rate was 14/21 (67%). In elderly patients the final CR rate (62%) is noteworthy, considering that 6 patients were >70 years of age and 3 were >80. All three patients >80 achieved CR (lasting 5 to 7 months). The median time of granulocyte and platelet recovery was 15 days. Our scheme was well tolerated. In the group of elderly patients 3 out of 14 died during induction (21%) and 4 life-threatening infections were observed (28%). The short duration of cytotoxic therapy and perhaps the use of G-CSF contributed to a reduction of the hospitalization period (median of 22 days), thus providing major savings on induction costs and allowing for better utilization of beds as well as significantly improving patients' quality of life.

Preseasonal specific immunotherapy with modified Phleum pratense allergenic extracts: tolerability and effects.

UNLABELLED: The preparation of chemically modified allergens, with a reduced IgE binding capacity (responsible for side effects with traditional immunotherapy) but with the same or greater immunogenic activity, is one of the paths followed to obtain better results with specific immunotherapy (IT). The aim of the study was to evaluate the tolerability and effects of an extract Phleum pratense, modified with glutaraldehyde and absorbed on aluminium hydroxide, in controlling the seasonal symptomatology induced by grass pollen in a group of 10 monosensitized patients, compared to a group of 10 similar patients not treated with specific IT but with drugs alone. The monitoring parameters were: 1) Clinical: a) symptomatology after specific conjunctival provocation test (pre and post seasonal) and during the natural exposure to the allergen b) drug consumption. 2) Immunological (peripheral blood eosinophils, total and specific IgE, total specific IgG). 3) Cytological, before, during and after the pollen season. CONCLUSIONS: In subjects treated with specific IT a) both the overall symptomatology and the drug consumption resulted significantly reduced compared to the controls (p = 0.045); b) the phlogistic infiltrate showed a tendency to decrease during the pollen season; c) the peripheral blood eosinophils, total and specific IgE and IgG did not show any significant variation compared to the controls; d) no systemic reactions occurred and there were only two slight local reactions.

Topical azelastine reduces eosinophil activation and intercellular adhesion molecule-1 expression on nasal epithelial cells: an antiallergic activity.

BACKGROUND: It is well known that allergen-specific nasal challenge (ASNC) is a fruitful tool with which to evaluate antiallergic activity exerted by a drug. Azelastine is a new antihistamine also available in topical form (i.e., nasal spray). OBJECTIVE: The aim of the study was to evaluate the effects of azelastine nasal spray on inflammatory changes after ASNC in both the early-phase reaction and the late-phase reaction. METHODS: The study had a double-blind, placebo-controlled, randomized, and parallel-group design. Twenty patients with pollen allergy were enrolled out of pollen season. ASNC was performed at baseline (TO) and after 1 week of washout (T7). At T7, 10 patients sprayed azelastine (1 puff) into their nostrils, and 10 patients used placebo. ASNC was performed after 30 minutes. The considered parameters (evaluated during early- and late-phase reactions) were: (1) clinical signs and symptoms, (2) cytologic assessment (neutrophils and eosinophils), (3) assay-of mediators (eosinophil cationic protein and myeloperoxidase), and (4) expression of intercellular adhesion molecule-1 (ICAM-1) on nasal epithelial cells. We focused our attention on ICAM-1 because it is the natural ligand of leukocyte functional associated antigen-1 and Mac-1, expressed on eosinophils. In addition, ICAM-1 is expressed on epithelial cells only on allergen exposure (both natural and experimental). RESULTS: Placebo did not exert any modification on the considered parameters. After azelastine administration, significant decreases in total symptom score, eosinophilic and neutrophilic infiltration, and ICAM-1 expression were observed during both early- and late-phase reactions. Furthermore, serum eosinophil cationic protein levels decreased during the late-phase reaction, whereas myeloperoxidase was not affected by the treatment. These findings were confirmed by the powerful Koch's split-plot statistical analysis. CONCLUSION: Azelastine exerts antiallergic activity, mainly affecting eosinophil function and downregulating ICAM-1 expression, on nasal epithelial cells.

Terfenadine exerts antiallergic activity reducing ICAM-1 expression on nasal epithelial cells in patients with pollen allergy.

BACKGROUND: Rhinoconjunctivitis caused by pollen allergy is characterized by typical signs and symptoms and mucosal infiltration by inflammatory cells during the pollen season. It has recently been demonstrated that the adhesion molecule system is deeply involved in cell-to-cell interaction during the inflammatory response which follows allergic reactions. OBJECTIVE: The aim of the present study (placebo-controlled, double-blind, randomized) was the evaluation of the antiallergic activity of Terfenadine in the model of the allergic rhinitis due to natural pollen exposure. METHODS: Two groups of patients with pollen allergy were enrolled in this study. Ten patients were treated with Terfenadine (120 mg/die) for 7 days and 10 with placebo. Evaluation criteria were: (a) clinical: signs and symptoms (recorded daily in a diary card by patients); (b) cytological: inflammatory cell count (neutrophils, eosinophils, metachromatic cells) from nasal lavage at T0 and T7; (c) immunocytochemical: ICAM-1/CD54 expression on nasal epithelial cells at T0 and T7; and (d) mediators dosage (ECP-MPO) on nasal lavage at T0 and T7. RESULTS: As opposed to the placebo group, patients treated with Terfenadine showed a significant improvement of both symptoms (P < 0.022) and signs P < 0.001), a significant reduction of inflammatory cells infiltrate (P < 0.005), of ECP levels (P < 0.002) and ICAM-1 expression on nasal epithelial cells (P < 0.005). CONCLUSIONS: In conclusion, these data demonstrate that Terfenadine exerts antiallergic activity since it is able to reduce inflammatory cell infiltrate and downregulates ICAM-1 expression.

Comparison of human trichinellosis caused by Trichinella spiralis and by Trichinella britovi.

The first documented report of an outbreak of human trichinellosis caused by Trichinella spiralis in Italy is described. Two family groups were involved. The source was wild boar meat products. The principal clinical features were fever (60%), myalgia (50%), and diarrhea (40%). The most useful laboratory indicators were eosinophilia (100%), elevated levels of creatine phosphokinase (CPK) (90%) and other muscle enzymes, parasite-specific IgG titers (100%), and anti-newborn larvae antibodies (30%). The levels of these responses correlated with the number of infective muscle larvae ingested, which was influenced by the length of time the ingested meat was cured. The clinical and biological features observed during human infection with T. spiralis appear to have been different from those reported during two outbreaks due to T. britovi, which occurred in southern Italy. The main distinctions between the two types of infections were a longer duration of parasite-specific IgG, increased CPK levels, and a more severe intestinal symptomatology in T. spiralis-infected patients than in those infected with T. britovi.

Cytolytic function of clonable T cells after human bone marrow transplantation.

We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.

Lymphokine production by T-cell clones after human bone marrow transplantation.

T cells recovering after bone marrow transplantation (BMT) were analyzed for their phenotypic and functional features by two-color immunofluorescence and a high efficiency cloning technique. A predominance of cells co-expressing natural killer (NK)-related surface antigens, such as Leu 7 (CD57) and CD11b, was detected within both the CD4+ and CD8+ subsets from 5 months postgrafting onward. Such cells are virtually absent among normal circulating CD4+ cells and account for a minority (approximately 30%) of normal CD8+ cells. Postgrafting T cells representative of the whole range of NK-related antigen co-expression were selected from six patients for clonal analyses. In control subjects, 63% and 41% of the CD4+ and CD8+ clones, respectively, produced interleukin-2 (IL-2) whereas approximately 30% of either CD4+ or CD8+ control clones produced interferon (IFN)-gamma. At variance, and irrespective of their CD4+/CD8+ phenotype, lower proportions of BMT recipient-derived clones produced IL-2 (20% and 12%, respectively), whereas the majority of both CD4+ and CD8+ clones (75% and 71%, respectively) released high amounts of IFN-gamma. Purified populations of CD57+/CD11b+ v negative cells from two BMT recipients and two control subjects were cloned and subsequently evaluated for IL-2 and IFN-gamma production. CD57+/CD11b+ cell-derived clones were poor IL-2 producers in both normal subjects and BMT patients. In contrast, IL-2-producing clones were frequent (62% to 79%) among those derived from CD57-/CD11b- cells from normal subjects, whereas they were still represented at lower than normal proportions, ie, 25% to 41%, among clones generated from BMT recipients. CD57+/CD11b+ cells gave rise to comparably high proportions of IFN-gamma producing clones in both normal subjects and BMT recipients (approximately 80%). In contrast, IFN-gamma producing clones were approximately 25% to 50% of CD57-/CD11b- cell-derived clones in both normal subjects and BMT patients. Therefore, while the predominance of NK-related antigen-positive T cells may be predictive of poor IL-2 and high IFN-gamma production, the immune derangement in long-term BMT recipients is further enhanced by the finding that all T cells may be poor IL-2 producers. It is also suggested that IL-2 production is a preferential function of T cells that do not express CD57 and CD11b, whereas IFN-gamma production is attributable to T cells that express CD57 and CD11b.

Partial inefficiency of T cell receptors gamma/delta composed of a heavy (55-kD) gamma chain to mediate cell activation upon binding to specific monoclonal antibodies.

We analyzed CD8+ T cell receptor (TCR) gamma/delta+ (delta-TCS-1 reactive) cell clones expressing the 55-kD gamma chain for their susceptibility to triggering by monoclonal antibodies (mAbs) specific for TCR or CD3 molecules. Clones were derived by limiting dilution from CD3+, WT31- FACS-purified peripheral blood populations or CD4-CD8- thymocytes (a fraction of the latter cells expressing de novo CD8 surface antigen upon culture in IL-2). Clones were screened according to their reactivity with both anti-CD8 and delta-TCS-1 mAbs. Analysis of CD3-associated molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions which preserve the CD3/TCR association (1% digitonin) showed a predominant 55-60-kD molecule both under reducing and non-reducing conditions. All clones expressing the delta-TCS-1+ CD8+ surface phenotype derived from either thymus or peripheral blood lysed the Fc gamma receptor-bearing P815 target cells in the presence of anti-CD3 mAb. On the other hand, delta-TCS-1 mAb was poorly efficient in triggering the lytic machinery of these clones, while it induced target cell lysis by delta-TCS-1+ CD8- clones.

Human thymocytes expressing gamma/delta T-cell receptors.

Expression of T-cell receptors of gamma/delta type characterizes a small subset of peripheral T lymphocytes which is homogeneously composed of cytolytic cells and, in most instances, lack CD4 and CD8 differentiation antigens. By the use of anti-TCR gamma/delta MAbs it is possible to identify two distinct subsets of TCR gamma/delta+ cells that are characterized by a C gamma 1 or C gamma 2-encoded forms of gamma-chain, respectively. While the BB3 MAb-reactive (C gamma 1 encoded) cell subset is prevalent in peripheral blood (PB), these cells represent less than 10% in TCR gamma/delta+ thymocyte populations. In thymus, the majority of cells was found to react with delta-TCSI (or A13) MAbs. Culture of CD4-8- thymocytes (highly enriched in TCR gamma/delta+ cells) in IL-2 resulted in the de novo expression of CD8 surface antigen and of non MHC-restricted cytolytic activity. Cloning of CD4-8- thymocytes resulted, for the most part, in CD3+ TCR gamma/delta+ cells. Moreover, the majority of clones expressed the unusual delta-TCSI+ CD8+ phenotype and lysed the NK-sensitive K562 target cells. Analysis of the immunoprecipitated TCR molecules showed the existence of the (rare) heavy (55 kDa) form of gamma-chain. A redirected killing assay using murine P815 target cells and appropriate "stimulatory" antibodies was further employed for functional analysis of thymus-derived TCR gamma/delta+ clones. While anti-CD3 MAbs efficiently triggered the cytolytic activity of all clones irrespective of their phenotype, MAbs directed to TCR gamma/delta induced efficient lysis only of BB3+ or delta-TCSI+CD8 clones, but not of delta-TCSI+ CD8+ clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Human peripheral blood lymphocytes bearing T cell receptor gamma/delta. Expression of CD8 differentiation antigen correlates with the expression of the 55-kD, C gamma 2-encoded gamma chain.

We analyzed the CD3-associated molecules present on peripheral blood-derived TCR-gamma/delta+ clones that express CD8 surface antigens. Clones were derived by limiting dilution from CD3+WT31- FACS-purified populations derived from several donors. Eight of greater than 300 TCR-gamma/delta+ clones analyzed expressed CD8 and reacted with delta-TCS-1 mAb. Cell numbers suitable for more detailed analyses could be obtained from four clones, including one derived from thymus. Analysis of CD3-associated TCR molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions that preserve the CD3/TCR association (1% digitonin) showed a predominant 55-60-kD molecule both under reducing and nonreducing conditions. On the other hand, the delta-TCS-1-reactive molecules immunoprecipitated from 25 CD3+ delta-TCS-1+ CD8- clones, in all instances, displayed a 40-44-kD mol mass. In two-dimensional PAGE, TCR-gamma molecules precipitated from delta-TCS-1+ CD8+ clones appeared more acidic than those of BB3+ or delta-TCS-1+ CD8+ clones. Southern analysis confirmed that this type of non-disulphide-linked TCR-gamma/delta is also coded for by the C gamma 2 gene segment.

Clonal analysis of CD4-CD8- human thymocytes expressing a T cell receptor gamma/delta chain. Direct evidence for the de novo expression of CD8 surface antigen and of cytolytic activity against tumor targets.

CD4-CD8- human thymocytes were obtained by treating total thymocyte suspensions with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. The resulting cell populations contained virtually no CD4+, CD8+ or WT31+ cells and 17-65% CD3+ cells. In addition, analysis of cell reactivity with delta-TCS-1 mAb (specific for the C gamma 2-encoded, nondisulfide-linked form of TcR gamma/delta), revealed the presence of a variable proportion of delta-TCS-1+ cells (the % of delta-TCS-1+ cells were lower than the percentage of CD3+ cells). Upon culture in recombinant interleukin 2 (IL2, in the presence of irradiated mononuclear cells), CD4-CD8- thymocytes underwent extensive proliferation. In addition, a progressive increase of CD8+ cells (but not of CD4+ or WT31+ cells) could be detected. Cells also progressively acquired cytolytic activity against K-562 or fresh melanoma cells. Fresh CD4-CD8- thymocytes were cloned under limiting dilution conditions. The cloning efficiencies were relatively high (1/3 cells); in addition, virtually all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31-delta-TCS-1+ surface phenotype. About half of the clones analyzed were CD8+, whereas none expressed CD4 antigens. We conclude that (a) only delta-TCS-1-reactive, TcR gamma/delta+ cells can be isolated from CD4-CD8- thymocytes cultured in IL2, and (b) the expression of CD8 antigen and of cytolytic activity reflects a true in vitro phenotypic change of CD8-, noncytolytic precursors (and not the preferential growth of few contaminating cells).

Two subsets of human T lymphocytes expressing gamma/delta antigen receptor are identifiable by monoclonal antibodies directed to two distinct molecular forms of the receptor.

Two mAbs directed to the TCR-gamma/delta were analyzed for their pattern of reactivity with CD3+WT31- cell populations or clones. In normal individuals, the BB3 mAb reacted with approximately 2/3 of peripheral blood CD3+WT31- lymphocytes, whereas delta-TCS-1 stained approximately 1/3 of such cells. In addition, the sum of the percentages of BB3+ and delta-TCS-1+ cells approximated the percentages of peripheral blood CD3+WT31- lymphocytes in seven normal donors tested. Also, in peripheral blood-derived polyclonal CD3+WT31- populations, cultured in IL-2, cells reacting with one or another mAb accounted for the whole cell population. On the other hand, only delta-TCS-1-reactive cells, but not BB3+ cells, could be detected in unfractionated as well as in CD4-8-thymocyte populations. Analysis of peripheral blood-derived CD3+WT31- clones showed that 70% of 72 clones analyzed reacted with BB3 mAb, but not with delta-TCS-1 mAb. On the other hand, delta-TCS-1 mAb stained the remaining BB3- clones. Five clones expressing medium-low amounts of CD8 antigen were BB3- delta-TCS-1+. Both types of clones lysed the Fc gamma receptor-bearing P815 target cell in the presence of anti-CD3 mAb (but not of mAb directed against HLA-DR, CD7 molecules, or TCR-alpha/beta). In this cytolytic assay, BB3 mAb induced target cell lysis only by BB3+ clones, whereas delta-TCS-1 mAb was effective only with delta-TCS-1+ clones. The CD3-associated surface molecules expressed by BB3+ or delta-TCS-1+ clones were analyzed after cell surface iodination and immunoprecipitation with the corresponding anti-TCR mAb or with anti-CD3 mAb (in digitonin-containing buffer). In SDS-PAGE, molecules immunoprecipitated from 13 BB3+ clones displayed, under nonreducing conditions, a molecular weight of 80 kD (in some cases, a minor 38-kD band could be detected). Under reducing conditions, two major components of 44 and 41 kD (and a minor component of 38 kD) were detected. On the other hand, TCR molecules immunoprecipitated from 11 different delta-TCS-1+ clones appeared as a diffuse band of 41-44 kD, both under reducing and nonreducing conditions (under non-reducing condition, an additional 38-kD band was present). Therefore, BB3+ cells express a disulphide-linked form of TCR-gamma/delta whereas delta-TCS-1+ cells express a non-disulphide-linked form.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-2-induced proliferation of CD4-CD8- human thymocytes. In vitro expression of CD3 and CD8 antigens and cytolytic activity.

Human thymocytes lacking both CD4 and CD8 differentiation antigens were prepared by treating total thymocyte suspensions with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies and complement. The resulting populations contained less than 2% CD4+, CD8+ or WT31+ cells and variable percentages (less than 20%) of CD3+ cells. These cell populations were cultured in recombinant IL-2 in the presence of peripheral blood mononuclear cells as feeder cells. Cells underwent extensive proliferation accompanied by a progressive increase of CD3+ and CD8+ cells. On the other hand, appearance of neither WT31+, alpha/beta-positive T cell receptor (TCR), nor CD4+ cells could be observed in several independent experiments. Functional analyses revealed the appearance and the progressive increase of cytolytic activity against the natural killer (NK)-sensitive K562 cells as well as the NK-resistant fresh melanoma cells. Experiments of T cell cloning indicated that both the expression of CD8 and CD3 antigens and the appearance of cytolytic activity were consequent to cell maturation occurring at the level of CD4-CD8- non-cytolytic cell precursors. In these experiments, more than 30% of cells underwent clonal expansion and all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31- surface phenotype. The expression of CD8 was variable, whereas no CD4+ clones could be obtained. Cells expressing such surface phenotype are known to belong to the TCR gamma-positive T lymphocyte subset lacking the typical alpha/beta TCR and thus appear to be the only T cell type capable of in vitro proliferation and maturation under easily reproducible culture conditions.