Role of human leukocyte interferon-alpha in the treatment of patients with polycythemia vera.

Eighteen patients with polycythemia vera who were less than 60 years old received human leukocyte interferon-alpha subcutaneously at a starting dose of 3 MU three times a week. The interferon dose was escalated to 6 MU three times a week if it was well tolerated and disease was not controlled after 3 months of treatment at the lower dose. Hematologic response was defined as complete if the hematocrit was maintained at less than 45% in the absence of phlebotomy and partial if the hematocrit was kept at 45% to 50%, associated with a 50% or greater reduction of phlebotomy requirements; no response was defined as a response less than a partial response. Complete disease control was achieved in 11 patients, with partial control in a further six cases. One patient failed to respond. Median duration of response was 16 months (range 5 to 43 months), with 15 patients still under treatment. Therapy with human leukocyte interferon-alpha significantly improved (p <.01) phlebotomy requirements, the degree of splenomegaly, pruritus scores, iron stores and mean red cell volume values, and platelet and leukocyte counts. Interferon treatment did not produce remarkable side effects and no patient withdrew from the study because of intolerance. We conclude that subcutaneous human leukocyte interferon-alpha is an effective and well-tolerated therapy in the management of polycythemia vera-associated myeloproliferation and pruritus in patients less than 60 years old.

Serum levels of tumour necrosis factor-alpha predict response to recombinant human erythropoietin in patients with myelodysplastic syndrome.

We measured pretreatment serum levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in 25 patients with myelodysplastic syndrome receiving recombinant human erythropoietin (rhEPO) at dosages up to 300 U/kg thrice weekly for 12 weeks. Both TNF-alpha and IL-1 beta levels were measured using commercially available enzyme-linked immunoassays. A complete response (CR) was defined as a rise in untransfused haemoglobin concentrations of at least 2 g/dl or a 100% decrease in RBC transfusion requirements over the treatment period; a partial response (PR) was an increase in untransfused haemoglobin values of 1-2 g/dl or a decrease in RBC transfusion requirements equal to or greater than 50%; no response (NR) was defined as a response less than a PR. After 12 weeks of rhEPO treatment, four patients showed a CR, five patients a PR, and 16 patients NR. Serum levels of both TNF-alpha (80.5 %/- 64.8 vs 8.1 +/- 4.2 ng/l, P < 0.001) and IL-1 beta (60.4 +/- 49.9 vs 8.9 +/- 4.7 ng/l, P < 0.001) were higher in MDS patients than in a group of 28 normal controls. Responders (CR + PR) showed significantly lower serum levels of TNF-alpha than non-responders (21.6 +/- 26.2 vs 106.3 +/- 60.8 ng/l, P < 0.001), whereas IL-1 beta concentrations between those who benefited from therapy and unresponsive cases were not significantly different (39.8 +/- 48.9 vs 73.4 +/- 48.2 ng/l, P = 0.120). It is noteworthy that TNF-alpha levels were within the normal range in all responsive patients but one, whereas all non-responders presented elevated cytokine concentrations. No relationship was found between TNF-alpha or IL-1 beta values and haemoglobin levels, transfusion requirement, serum EPO or ferritin concentrations. We conclude that pre-treatment TNF-alpha levels might help to select those MDS patients who are most likely to benefit from rhEPO treatment.

Efficacy and safety of human leucocyte interferon-alpha treatment in patients younger than 60 years of age with polycythaemia vera.

OBJECTIVES: To evaluate the therapeutic activity and toxicity of human leucocyte interferon-alpha (lIFN-alpha) in patients with polycythaemia vera (PV) aged less than 60 years. DESIGN: An open clinical study. SETTING: Department of Medical Sciences, Regina Apostolorum Hospital, Albano Laziale, and Chair of Haematology, University of Rome 'Tor Vergata', S. Eugenio Hospital, Rome, Italy. SUBJECTS: Fourteen patients with PV and aged < 60 years who had active disease as indicated by the need for phlebotomy and/or cytoreductive therapy. INTERVENTIONS: lIFN-alpha administered subcutaneously at the starting dose of 3 MU thrice weekly. The interferon dose could be escalated to six MU thrice weekly if it was well tolerated and disease was not controlled after three months of treatment at the lower dose. MAIN OUTCOME MEASURES: Change in phlebotomy requirements, spleen size, pruritus score and haematological parameters after 6 months of treatment. Evaluation of lIFN-alpha side effects. RESULTS: Complete or partial disease control was achieved in 13 patients. Six patients achieved a complete response (CR) and four a partial response (PR) after 3 months of therapy. Dose escalation in partial or nonresponders resulted in two patients switching from a status of PR to CR, and three other patients achieving a partial response after being unresponsive to the lower dosage. Human leucocyte interferon-alpha therapy significantly improved (P < 01) phlebotomy requirements, the degree of splenomegaly, pruritus scores, iron stores and MCV values, and platelet and leucocyte counts. A mild flu-like syndrome (low-grade fever, nausea and myalgias) appeared during the early phase of therapy in the majority of patients, but no patient had to discontinue lIFN-alpha because of intolerance. CONCLUSIONS: Subcutaneous human leucocyte interferon-alpha appears an effective and well tolerated therapy in the management of PV-associated myeloproliferation and pruritus in patients aged less than 60 years.

Response to recombinant human erythropoietin in patients with myelodysplastic syndromes.

Recombinant human erythropoietin (rhEPO) at pharmacological doses was used to improve anemia and reduce the transfusional requirements of 43 patients with myelodysplastic syndrome (MDS). rhEPO was given by s.c. injection three times per week for 12 weeks. The EPO dose was started at 150 IU/kg and was increased to 300 IU/kg if after 6 weeks there was no or suboptimal erythroid response. Responses were defined as being a complete response (CR), partial response (PR), or no response (NR). A CR was considered a rise in untransfused hemoglobin concentrations of at least 2 g/dl or a 100% decrease in RBC transfusion requirements over the treatment period. A PR was defined as an increase in untransfused hemoglobin values of 1-2 g/dl or a decrease in RBC transfusion requirements equal to or greater than 50%. NR was defined as responses less than a PR. Patients who responded to therapy were continued on rhEPO at the same dose for 6 additional months. An objective response (CR and PR) was observed in 7 of 42 (16.7%) assessable cases after 6 weeks of treatment at the dose of 150 IU/kg. Dose escalation (300 IU/kg) in nonresponders resulted in another six patients attaining a rise in hemoglobin concentrations. The final response rate was 13 of 41 (31.7%); 4 patients became transfusion independent. Therapy was tolerated well, with no relevant side effects. MDS progression was seen in one case. An elevated bone marrow erythroid infiltration (erythroid index) and detectable pretreatment circulating erythroid progenitors (burst-forming units-erythroid) were the best predictors of hemoglobin response when we controlled for other variables. These data suggest that rhEPO has a role in the treatment of certain patients with MDS, particularly in those with a high erythroid index and detectable circulating erythroid burst-forming units.

Serum interleukin-10 in plasma-cell dyscrasias.

Serum levels of interleukin-10 (IL-10) were measured by enzyme-linked immunosorbent assay in 115 patients with multiple myeloma (MM) in various phases of the disease (68 at diagnosis, 22 in plateau phase, 22 in relapse), in 71 individuals with monoclonal gammopathy of undetermined significance (MGUS), and in 53 normal volunteers. Detectable levels of serum IL-10 were found in 24 myelomas (20.9%), in 7 cases of MGUS (9.9%), and in 4 normal subjects (7.5%) (P = NS, chi2 test). In patients with MM, cytokine was detected with a comparable frequency in all pathologic stages and phases of the disease: 4/19 in stage I, 6/26 in stage II, 5/23 in stage III, 4/22 in plateau phase, and 5/25 in progressing or relapsed disease. IL-10 concentrations did not differ significantly between controls and patients with plasma-cell dyscrasia, between patients with MGUS and those with MM, between early vs. advanced MM, or between patients in different phases of the disease. In 36 patients with MM in whom IL-10 was measured serially, no significant changes were observed over the course of the disease. Also, when comparing the outcomes of individuals with detectable or undetectable IL-10 in single stages or in the whole myeloma group, no differences were revealed. Our results do not support an apparent involvement of IL-10 in the pathogenesis of MM in vivo. However, further studies are required to define the exact role of this cytokine within the complex cytokine network of this neoplastic disorder.

Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target.

This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells. The effect of Q was dose-dependent at concentrations ranging between 1 and 10 microM. Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123). Our results indicate that Q and 3-OMe Q (3',4',7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner. Moreover, 10 microM Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay. In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anti-cancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.

DNA ploidy pattern in human chronic liver diseases and hepatic nodular lesions. Flow cytometric analysis on echo-guided needle liver biopsy.

BACKGROUND: Significantly elevated fractions of diploid hepatocytes and reduction in the polyploid populations have been reported in human and experimentally induced hepatocellular carcinomas (HCC). This study was conducted to determine how these changes are related to conditions that often precede HCC, such as chronic hepatitis, cirrhosis, and premalignant focal nodules in cirrhotic livers. METHODS: Ultrasound-guided needle biopsy specimens of the liver were obtained from patients with chronic hepatitis, cirrhosis, or ultrasonographically diagnosed nodules within cirrhotic livers; biopsy specimens also were taken from patients without hepatic disease. DNA flow cytometry was performed on isolated nuclei to determine the percentages of diploid, tetraploid, and octaploid hepatocytes; the S-phase fraction for each diploid peak and the diploid/polyploid (tetraploid + octaploid) ratio also were calculated. Part of each specimen was reserved for evaluation of hepatocyte binuclearity. RESULTS: Chronically hepatitic (18 patients) and cirrhotic (18 patients) livers showed significantly increased diploid/polyploid ratios, with respect to normal livers, that were significantly correlated with decreases in hepatocyte binuclearity. This trend was even more marked in euploid nodules (4 premalignant and 5 malignant), in which the S-phase fractions were significantly higher than those of normal liver; aneuploidy was found in 6 of 11 malignant and 2 of 6 premalignant nodules.

In vitro expansion of CD34+ cells mobilized with chemotherapy and G-CSF.

Hemopoietic CD34+ progenitors were isolated by immunomagnetic method from normal bone marrow (BM) or from peripheral blood (PB) of patients with non-Hodgkin's lymphoma treated with chemotherapy and granulocyte colony-stimulating factor (GCSF). Aliquots were seeded in long-term cultures (LTC) on bone marrow-derived stromal layers; non-adherent and adherent clonogenic content of the cultures was assayed weekly. The final recovery and the clonogenic efficiency of the CD34+ cells were slightly higher in PB samples than in BM controls. In long term cultures PB cells sustained hemopoiesis as much as BM cells; at week 3 and 4 PB total mononuclear cells and CD34+ cells showed a non-adherent cell recovery higher than the respective BM controls. Furthermore, PB CD34+ cells were expanded in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF alone or combined with interleukin 3 (IL3), stem cell factor (SCF), interleukin 1 (IL1), interleukin 6 (IL6). The combination of GM-CSF, IL3, SCF, IL1 and IL6 produced the maximum increase of both mononuclear cells (30-fold) and granulocyte-macrophage colony forming units (CFU-GM) (4.6-fold) after 7 days of cultures; yet after 14 days a strong decrease of the CFU-GM occurred. These data suggest that G-CSF following chemotherapy mobilizes both early and committed hemopoietic progenitors.

Tamoxifen-induced immune-mediated platelet destruction. A case report.

Drug-induced immunologic thrombocytopenia, a fairly common disorder, is characterized by drug-dependent antiplatelet antibodies that destroy circulating platelets in the presence of the provoking drug or its metabolites. The development of reliable methods for the detection of platelet-bound immunoglobulins causing in vivo platelet destruction, such as the use of monoclonal antibodies tagged with fluorescein and flow cytofluorimetric analysis, has ushered in a new era to differentiate between immune and non-immune thrombocytopenias. A severe thrombocytopenia developed in an elderly female patient treated with tamoxifen, a non-steroidal anti-estrogen drug, after surgery for breast cancer. A tamoxifen-dependent platelet antibody was detected in the patient's serum and linked on the platelet membranes. This antibody reacted only in the presence of the offending drug and showed platelet specificity. Withdrawal of drug restored platelet count to normal levels.

Increased polymorphonuclear leukocyte Fc gamma-dependent chemiluminescence in active psoriasis is not related to FcRIII (CD16) receptor expression.

The role of the two main serum opsonins (IgG and C3b) in the induction of polymorphonuclear leukocyte chemiluminescence was studied in a group of psoriatic patients. Chemiluminescence was stimulated with zymosan opsonized by fresh plasma (IgG- and C3b-dependent chemiluminescence) or by complement-depleted plasma (IgG-dependent chemiluminescence). While C3b-dependent chemiluminescence was similar in patients with chronic or active forms of psoriasis, IgG-dependent chemiluminescence was significantly increased in patients with active disease. However, FcR-III expression, evaluated by means of flow cytofluorimetry, was similar in the different groups of patients studied. The discrepancy between Fc-receptor (CD16) expression and IgG-dependent chemiluminescence is, therefore, indicative of modifications that occur in psoriatic neutrophils that do not involve FcIII-receptor expression.

Markers of eosinophilic inflammation in allergic diseases.

The paper reviews personal studies aimed at identifying clinical markers of eosinophil inflammation in allergic diseases. Preliminary data on the use of flow cytometry as a method to detect eosinophil activation through phenotypic activation markers are reported. The concept of eosinophil releasibility is introduced on the basis of in vitro eosinophil activation and mediator release by different stimuli. Data showing an increase of serum levels of ECP mainly in subjects with sensitivity to perennial allergens and related to histamine bronchial reactivity are discussed in view of their practical impact in diagnosing and monitoring of allergic diseases and asthma.

Evaluation of a new whole blood cytometric lymphocyte transformation test for immunological screening.

Lymphocyte transformation test (LTT) is usually performed by 3H-thymidine (3H-TdR) incorporation of phytohemagglutinin stimulated lymphocytes. This test presents a lack of standardization as well as a great variability of results making it difficult to compare the results carried out by different laboratories. Flow cytometry (FCM) can precisely evaluate cell kinetics by measuring the DNA content of stained nuclei. We tested a new ready-to-use kit for the blastogenic response of mitogen stimulated lymphocytes in FCM for clinical immunological screening (Blastest, Ylem). Separated peripheral blood lymphocytes from the same donors were also tested with the 3H-TdR method at 72h and results compared to FCM. Our results show a significant correlation between data obtained from FCM and 3H-TdR method. We show that there are no differences in results between different cytometers if CVs are low. Flow cytometry is more precise and reproducible and makes a better standardization feasible, it also offers a simpler and faster way for routine LTT enabling a wider clinical use.

Interleukin 2 proliferative response by cord blood mononuclear cells of term and preterm neonates.

The results of previous studies on the proliferative response of resting cord blood mononuclear cells (CBMC) of term neonates to human recombinant interleukin 2 (hrIL-2) are contrasting. Some authors have reported a good and others a poor response. In our study we have obtained a significant reactivity, compared with the response of resting peripheral blood mononuclear cells (PBMC) of adult subjects, of resting CBMC to varying quantities of hrIL-2 in the absence of any known mitogenic or antigenic stimuli. Most responding cells was CD3 positive. Both CD4-positive and CD8-positive cells responded. However, we observed a more marked increase of the percentage of the CD4-positive T cells and a clear reduction of the percentage of the CD21-positive cells (B-lymphocytes) testing CBMC rather than PBMC of the adult subjects. The percentage of the NK cells was reduced in both the categories of subjects. Moreover, we have examined the reactivity to hrIL-2 of CBMC of preterm neonates. The results showed that this response is low. The peculiar level and kinetic of CBMC proliferative response to hrIL-2 are discussed.

Effects of dexamethasone on the growth and epidermal growth factor receptor expression of the OVCA 433 ovarian cancer cells.

We studied the correlation between dexamethasone (Dex) induced growth effects and modulation of epidermal growth factor receptor (EGFR) expression in OVCA 433 ovarian cancer cells. These cells express specific high and low affinity 125I-EGF binding sites and are growth stimulated by EGF. Dex exhibits mitoinhibitory effects by recruiting OVCA 433 cells in the G0-G1 phase of the cycle, but increases the number of both the high and the low affinity EGFR in a dose dependent manner. The maximal EGFR expression increase occurs after 24 h of Dex treatment consistently with Northern blot studies. The mitogenic activity of EGF in OVCA 433 cells is not affected by the presence of Dex. Moreover Dex growth inhibition occurs in JA1 cells, an ovarian cancer cell line which expresses unfunctional EGFR and which is unresponsive to EGF. Our results indicate that the Dex induced growth effects occur independently of EGFR expression.