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Background: The wide diffusion of multicomponent collection in donor apheresis has led to the yielding of different components, such as plasma-reduced platelet-pheresis at high PLT concentration. We investigated whether this collection modality could induce more PLT activation compared to standard plateletpheresis., Study Design and Methods: Forty-one plateletpheresis collections (20 Trima and 21 Spectra LRS Turbo v.7.0, COBE) were evaluated. Donor, procedure, and product data were recorded. ADP, collagen, and U46619 (a thromboxane-A2 analog)-induced PLT aggregation was investigated in basal (donor) and final (plateletpheresis unit) samples. The expression of PLT activation marker P-selectin (CD62P) was studied using flow cytometry in basal and final samples. In all cases, P-selectin was investigated in final samples after stimulation with ADP to assess for a possible further release of the antigen. Four additional plateletpheresis procedures were performed in donors from Group A, using the traditional, nonplasma-reduced program., Results: Plateletpheresis obtained by means of the Trima device showed a lower response to in-vitro induced PLT aggregation and a higher percentage of P-selectin-expressing PLT when compared to products obtained using the Spectra device. Moreover, P-selectin release after ADP stimulation was reduced in plateletpheresis units obtained using the Trima device. These differences disappeared when a nonplasma-reduced collection program was used. In-vivo evaluation did not detect any difference between plateletpheresis obtained by means of the two cell separators., Conclusions: Plateletpheresis units obtained by means of multicomponent collection show a higher degree of PLT activation compared to traditional plateletpheresis procedures when high-concentration plasma-reduced products are collected. Randomized clinical studies are needed to assess the real impact of these findings in terms of in-vivo efficacy of plasma-reduced plateletpheresis units.

Lymphocyte subsets were determined in 20 packed red blood cell units (PRC) before and after filtration (FPRC) with the Pall Leukotrap RC inline filter system; 10 units were prepared by low spin and platelet rich plasma (PRP) removal (Group A) and 10 with high spin, plasma and buffy-coat (BC) removal (Group B). Flow cytometry was employed for white blood cell (WBC) enumeration and phenotype analysis. Median WBCs in prefiltered units was 2.08 x 10(9) (Group A) vs. 0.8 x 10(9) (Group B) (p < 0.0001). Five Group A and three Group B filtered units had WBC counts above the limit of detection (LD), median values being 25.59 and 3.08 x 10(3), respectively. Whereas CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte subsets were assessable in 20-40% of Group A units, inline filtration of Group B units lowered lymphocytes below the LD of the present study. Post-filtration CD19+ lymphocytes were below the LD in all the 20 units.

In recent years peripheral blood stem cell (PBSC) collection for allogeneic or autologous transplantation has experienced an increased use in the onco-hematological setting. The latest generation cell separators allow a satisfactory and safe PBSC collection. Nevertheless, as in all therapeutic apheresis procedures, patients may experience procedure-related side-effects, mainly vasovagal reactions or symptoms related to hypocalcemia and/or hypomagnesemia. We investigated electrolyte changes in 18 patients, with a median age of 46 years (range 7-62), undergoing PBSC collection from January to April 1998. A significant decrease in total calcium in the final sample (9.65 +/- 0.7 mg/dL) with respect to the basal one (9.2 +/- 0.6 mg/dL, P < 0.05) was observed; also ionized calcium decreased markedly from the first sample drawn at +30 minutes: 1.22 +/- 0.14 vs. 1.03 +/- 0.15 mmol/L (P < 0.05), and a highly significant difference emerged when basal value were compared to the final value: 1.22 +/- 0.14 vs. 0.94 +/- 0.13 mmol/L (P < 0.0001). Similar findings affected potassium concentration: 4.1 +/- 0.4 vs. 3.3 +/- 0.3 mEq/L (P < 0.0001). Three out of eighteen patients (16.7%) reached a final potassium level <3.0 mEq/L, and eight out of eighteen (44.5%) showed a potassium concentration decrease >20% with respect to the basal value. A mild metabolic alkalosis occurred during the procedure: pH increased from 7.35 +/- 0.02 to 7.43 +/- 0.028 (P < 0.001), and plasma bicarbonate concentration increased from 27.48 +/- 2.21 to 32.44 +/- 2.52 mmol/L (P < 0.01). Sodium and chloride did not differ in the final sample with respect to the basal sample. None of our patients experienced clinically relevant side effects related to severe electrolyte changes (i.e., >20% with respect to the basal value). Because our current therapeutic schedules include patients older than 50 years in the PBSC collection and transplantation program and since it is well known that subclinical myocardial disease may occur in up to 4% of middle-aged males, we suggest that patients aged 50 or older undergoing PBSC collection procedures be carefully monitored in order to identify significant electrolyte variation, especially if they present with low serum potassium levels. However, further investigation of larger patient series are needed to determine the clinical relevance of serum potassium changes during apheresis.

Twenty-seven cancer patients underwent peripheral blood stem cell apheresis during hematopoietic regeneration following induction high-dose cyclophosphamide (7 g/sqm; HD-CTX). Among these patients, eleven were also treated with granulocyte-macrophage colony stimulating factor (rhGM-CSF) for 14 days after HD-CTX. We describe technique, peripheral blood cell yields and side effects of 76 leukaphereses performed using a continuous-flow blood cell separator COBE 2997. Leukaphereses, carried out through 2-5 consecutive days per patient, were started significantly earlier in rhGM-CSF treated patients. In comparison to patients receiving HD-CTX only, administration of rhGM-CSF resulted in a significantly higher yield per leukapheresis of mononuclear cells and granulocyte-macrophage colony forming units (CFU-GM) (two-fold and eight-fold, respectively). Procedures were completed and well tolerated in all cases. Minor side effects were unfrequent. Our results suggest that rhGM-CSF accelerates hematopoietic recovery after HD-CTX and facilitates large-scale collection of peripheral blood stem cells utilizable for autologous transplantation.

Circulating haematopoietic progenitors from 36 cancer patients were collected by continuous-flow leukapheresis during the phase of rapid haematopoietic recovery after pancytopenia induced by high-dose cyclophosphamide and then cryopreserved for autologous transplantation. 20 of the patients also received intravenous infusion of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) for 7, 10 or 14 days after cyclophosphamide. 106 leukapheresis procedures were done for 2-5 consecutive days. Leukapheresis was started significantly earlier in patients receiving rhGM-CSF. In these patients, yields of peripheral blood elements (leucocytes, mononuclear cells, haematopoietic progenitors and platelets) were significantly higher than in controls treated with cyclophosphamide only. In particular, the mean number of granulocyte-monocyte colony-forming cells was 43.88 X 10(4) vs. 6.16 X 10(4) per kg patient body weight per leukapheresis. Side-effects of leukapheresis were limited to central venous catheter occlusion and fever in 4% and 2% of all procedures, respectively.

In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.

Evidence for heterogeneity of several biological features of human malignant melanoma (Me) like morphology, cytogenetics, oncogenes activation, antigenic expression, metastatizing capacity and procoagulant activity are briefly reviewed in an attempt to distinguish findings related to primary vs. metastatic lesions. In our own studies monoclonal antibodies were used to study expression of MHC class I, class II products and of Me-associated antigens (MAA) on primary and metastatic Me cells. High expression of class I antigens was found in a high percentage of both primary and metastatic tumors, whereas DR and MAA showed a significant variation (from 3 to 90% of cells) in expression both in primary and in metastatic Me. When autologous cell-mediated immune responses were evaluated, it was found that Me cells from primary tumors but not those from lymph node metastases were able to stimulate autologous lymphocytes to proliferate and become cytotoxic for autologous Me. Clonal analysis of cytotoxic lymphocytes was then carried out in order to see whether the lack of lymphocytes reactivity to metastatic cells was due to the absence or to a low frequency of cytotoxic cells in the unstimulated PBL. CTL clones cytotoxic for autologous Me (Auto-Me) cells were indeed isolated. Three classes of CTL clones were identified: 1) one which is cytotoxic for Auto-Me; 2) a second one which lyse Auto-Me and allogeneic Me; and 3) a third one which is cytotoxic for Auto-Me and allogeneic normal and neoplastic cells. Metastatic Me cells, however, had the ability to suppress the stimulation of autologous PBL by alloantigens or IL-2. This effect was dose-dependent and was not due to absorption of IL-2 by Me cells. Since it has been reported that Me cells express class II MHC antigens, we investigated whether there was any correlation between autologous immune responses and DR expression on Me cells. Autologous lymphocytes stimulation was found to occur only with DR+ Me cells from primary lesions, whereas metastatic cells, either DR+ or DR-, did not stimulate autologous PBL. Moreover, the suppressive effect of metastatic Me cells was associated with their expression of DR antigens. The modulation of DR antigens on Me cells by Interferon-gamma correlated positively with their suppressive capacity. Thus, it appears that primary Me can behave differently from the metastatic one in their interactions with the immune system of autologous host. These findings suggest that DR antigens on Me cells may have an important role in the regulation of autologous immune responses.

A phase-I study of the recombinant, non-mutagenized interleukin 2 (rIL2, BioleukinTM) was performed in 12 melanoma patients (Pts). From 100 to 800 micrograms/m2 of rIL2 were administered by i.v. bolus injection, TID for 4-8 days. Side-effects included fever, malaise, low serum K+ and Ca++ values, electrocardiographic abnormalities, leukopenia and thrombocytopenia. No major organ toxicity and no significant fluid retention were observed at the administered doses. Treatment induced a rapid depletion of peripheral blood lymphocytes (PBL) with a rebound (2-6 times the pre-treatment values), 24-48 hr after rIL2 discontinuation. PBL obtained between the 5th treatment day and the 2nd post-treatment day showed: (a) enhanced proliferation (II/12 Pts) with stimulation indexes of 6-52; (b) increased cytotoxicity against autologous tumor cells (2/2 Pts), allogeneic melanomas (5/7 Pts), the Daudi (5/6 Pts) and K562 cell lines (7/12 Pts); and (c) increased expression of IL2 receptors (8/12 Pts) and of DR antigens (6/12 Pts). Lymphocytes collected 1-2 days after treatment and activated in vitro with rIL2 showed a more rapid development of tumor cytotoxicity, with an earlier loss of activity. Spontaneous proliferation, autologous or allogeneic tumor cytotoxicity and expression of IL2 receptors obtained after in vivo treatment with rIL2 were significantly weaker than those induced during in vitro stimulation. No major objective responses were detected in these patients.

The conditions of a linear assay for 4-methylumbelliferyl-alpha-D-N-acetyl-neuraminic acid (4-MU-NeuAc) sialidase activity in human lymphocytes, granulocytes, platelets and red cells plasma membranes have been determined. Lymphocytes and granulocytes have the same pH curve with two maximums at pH 4.0 and 4.8; however lymphocytes show a higher specific activity and a higher Km value. Sialidase activity detected in red cells plasma membranes has again a double-peak pH curve with maximums at pH 4.2 and pH 4.6, and specific activity levels less than one/30 compared to the other blood cells preparations. Platelets have a sialidase activity of the same magnitude as granulocytes, with an optimum pH at 4.2.

Previous work has shown that infusion of autologous helper-enriched, alloactivated lymphocytes in melanoma patients may induce, in addition to other mild signs of toxicity, a transient but sharp elevation of blood pressure. To avoid such a disturbing symptom, the in vitro protocol of peripheral blood lymphocyte activation has been modified. In the present study we show that such a modification has led to a lower toxicity of autologous lymphocyte infusion in 4 melanoma patients; in particular, hypertension was no longer observed. In addition, an immunologic monitoring was carried out in these patients. In 1 of 4 patients the treatment enhanced the in vitro cytotoxic activity of peripheral blood lymphocytes against autologous tumor cells. Other parameters such as NK activity and T4/T8 ratio did not show significant trends. The possible implications of these findings for clinical trials of adoptive immunotherapy with lymphocytes are discussed.

From January 1987 to February 1988, 15 stage IV melanoma patients were treated with two courses of bolus injection of rIL-2 plus LAK cell infusions at the National Cancer Institute of Milan. The original treatment regimen included a first course of rIL-2 administration (400 micrograms/m2 bolus injection 3 times a day [TID] for 4 days) and a second course of rIL-2 administration (800 micrograms/m2 bolus injection TID for 7 days) separated by 4 consecutive daily leukaphereses. Autologous lymphokine activated killer (LAK) cells were reinfused into each patient on three occasions during the second period of rIL-2 administration. Due to the appearance of grade III-IV neurological, hepatic and cardiopulmonary toxicity, 7 patients discontinued dosing before the end of treatment, one patient desired to be withdrawn and one patient died from rapidly progressive disease, although complications of rIL-2 administration may have contributed to her death. Only 6 patients completed the schedule without evidence of major intolerance, even though the planned dose during the second course of rIL-2 was reduced to 400 micrograms/m2. The complete duration of treatment ranged from 11 to 19 days. The total dose of rIL-2 injected ranged from 12.6 to 30.4 mg. The number of infused LAK cells ranged from 15.5 x 10(9) to 60 x 10(9)/patient. Two of the 14 evaluable patients showed a minor anti-tumor response. In 5 patients new metastases in other sites were documented from 2 to 5 months after completion of dosing. No apparent association was found between progression of the disease (or the appearance of new metastases) and the total dose of rIL-2 injected, the number of LAK cells administered or the number of days of treatment. By December 1988, all patients had died of their disease in a period ranging from 3 to 14 months from the last injection of rIL-2. The lack of significant clinical responses in this study and the high toxicity of this treatment lead us to conclude that at least as far as melanoma patients are concerned, adoptive immunotherapy with rIL-2 plus LAK cells (as described here) is not a justifiable treatment option unless new evidence presents itself.

A new procedure for activation of peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL2) is described. PBL obtained by leukapheresis were subjected to NH4Cl (ACK) treatment to clear erythrocyte contamination; Ficoll separation was not performed. PBL were subsequently seeded in 10-floor multitrays (Cell FactoryTM, CF), gasified and incubated at 37 degrees C for 3-4 days in a humidified 5% CO2 atmosphere. This procedure achieved an activation (evaluated as cytotoxicity and proliferation) comparable with that obtained by culturing PBL in small flasks. Optimal activation of PBL was achieved in CF even in the presence of granulocyte contamination of up to 40%. It was also possible to freeze, thaw and recover most of the frozen cells and their cytotoxic activity. With this procedure therefore large quantities of lymphokine activated killer cells (LAK) can be easily produced to be used in adoptive immunotherapy trials.

A phase I study was carried out to test the feasibility and toxicity of infusing large numbers of autologous, alloactivated helper lymphocytes into patients with metastatic melanoma. Patient peripheral blood lymphocytes (Pt-PBL) obtained by lymphopheresis and expressing the helper phenotype BT5/9 were separated and stimulated for 48 or 72 h with a pool of PBL from four to six healthy donors. Patients were then infused with such activated lymphocytes over a 2-3 h period. A total of 4 phereses and infusions (2/week for 2 weeks) were carried out for each cycle in each patient. Of the five patients treated, two received a second round of infusions. Infusion of autologous PBL stimulated in vitro for 48 h caused chills, fever, headache, and increased blood pressure. All symptoms disappeared in 2-3 h and were easily controlled by appropriate therapy. When lymphocytes were given after 72 h of allostimulation, no or very mild toxicity was observed. Serum chemistry, coagulation, autoimmunity, and urine analysis showed no gross abnormalities during therapy or follow-up of the patients. Immunological parameters (OKT4/OKT8 ratio, NK activity and cytotoxic T cell activity to autologous melanoma) were evaluated before starting the therapy, during its course and during the 3 to 6 months follow-up. The OKT4/OKT8 ratio increased significantly but transiently soon after the first course of infusions in one of the two patients tested. NK activity increased after 75-100 days in the three patients tested and in one of them it was high even after 180 days. No correlation between NK activity and prognosis was apparent. Cytotoxicity to autologous tumor was assessed in two patients, only of one of whom exhibited an increased activity from 75 to 180 days, which was associated with a prognosis better than that of the negative patient. Five patients were treated: two had progressive disease, two had stable disease for 5 and 6 months, respectively. In the first of these patients, a new cycle of lymphocyte infusions was carried out which caused a measurable reduction of lung tumor nodules whose growth, however, resumed 4 months later. This patient died 14 months after the onset of therapy. The fifth patient had a partial regression of pulmonary and intracranial metastases after therapy, but eventually died 3 months later. These results indicate that infusion of a high numbers of autologous, allostimulated helper PBL is a feasible and safe procedure, which could therefore be used in future studies of adoptive immunotherapy of cancer.

Starting from mucosal cells, isolated from rat small intestine, subcellular fractionation was carried out. Four fractions were prepared and characterized by marker enzymes and electron microscopic examination: nuclei plus microvilli, mitochondria, microsomes and supernatant. Thiamine-pyrophosphokinase activity was localized mainly in the supernatant fraction, with minor amount in mitchondria and microsomes. On the contrary thiamine-pyrophosphatase activity was present only in the particulate fractions (especially in microsomes), being completely absent from the supernatant.

The authors summarise their experience with the new "early granulocyte transfusion" scheme obtained in cancer patients. The data, based on three trials, clearly point out )1 the importance of early granulocyte transfusion as compared to the traditional one; 2) the efficacy of early granulocyte transfusion in pediatric age and among adult patients in those with good regenerating hemopoietic system.

We performed HLA typing in 96 couples affected by recurrent abortion "sine causa". We matched these patients with 124 fertile couples and 204 individuals random paired. No significant difference in HLA sharing was demonstrated in the three study groups. The statistical analysis denoted significant differences in regard to HLA A3, A24, B12, and DR- comparing patients and normal members of fertile couples. The frequencies of HLA Cw5, Cw6 and DR2 were different in patients when compared with their partners.