CD30 expression by CD8+ T cells producing type 2 helper cytokines. Evidence for large numbers of CD8+CD30+ T cell clones in human immunodeficiency virus infection.

A large panel of CD8+ T cell clones generated from peripheral blood lymphocytes (PBL) of healthy donors or human immunodeficiency virus (HIV)-infected individuals were assessed for both cytokine secretion profile and CD30 expression and release. The great majority of CD8+ T cell clones generated from healthy individuals showed the ability to produce interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), and none of them either expressed membrane CD30 or released substantial amounts of soluble CD30 (sCD30) in their supernatant. In contrast, high numbers of CD8+ T cell clones generated from HIV-infected individuals, which produced IL-4 (and IL-5) in addition to IFN-gamma or IL-4 (and IL-5) alone, expressed membrane CD30 and released detectable amounts of sCD30 in their supernatants. Indeed, CD30 expression appeared to be positively correlated with the ability of CD8+ T cell clones to produce IL-4 and IL-5 and inversely correlated with their ability to produce IFN-gamma, whereas no correlation between CD30 expression and production of IL-10 was observed. These data suggest that CD30 is a marker for CD8+ T cells that have switched to the production of type 2 helper cytokines.

Serum levels of the soluble form of CD30 molecule as a tumor marker in CD30+ anaplastic large-cell lymphoma.

PURPOSE: To determine serum levels of the soluble form of CD30 molecule (sCD30) in patients with Ki-1/CD30+ anaplastic large-cell lymphoma (ALCL), and to evaluate its correlation with clinical features at presentation and its possible role as a tumor marker to monitor response to treatment and subsequent follow-up. PATIENTS AND METHODS: sCD30 serum levels were measured with an improved commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kit in 24 patients with CD30+ ALCL at diagnosis and in 13 after treatment. RESULTS: Increased values (> 20 U/mL) at diagnosis were observed in 23 of 24 cases (median, 842.5 U/mL; range, 16 to 37,250) as compared with controls (P < .0001). These values were greater than those of 60 stage-matched cases of Hodgkin's disease (HD) (P < .0001). The highest median value was observed in patients with T-cell-type ALCL (1,690 U/mL), with a significant overall difference as compared with B- and null-cell types (P = .004). Phenotype maintained its significance when results were corrected for other parameters, such as age, sex, and stage (P = .03). sCD30 values returned to the normal range in complete remission (CR), but remained increased in one patient who only partially responded to treatment. Subsequent increases of sCD30 levels were recorded in four of four patients after relapse. CONCLUSION: sCD30 appears to be a new biologic serum tumor marker of possible use in the clinical setting of CD30+ ALCL.

Serum levels of soluble CD30 are elevated in the majority of untreated patients with Hodgkin's disease and correlate with clinical features and prognosis.

PURPOSE: To evaluate the serum levels of the soluble form of the CD30 molecule (sCD30) in patients with Hodgkin's disease (HD) to establish whether there is a correlation with clinical features at presentation and prognosis. PATIENTS AND METHODS: The sCD30 serum levels of 117 patients were measured at diagnosis with a commercial sandwich enzyme-linked immunoadsorbent assay (ELISA) test kit, and in 78 of these patients the sCD30 levels were also recorded during the follow-up period. RESULTS: sCD30 levels at diagnosis were increased (> 20 U/mL) in a high proportion of patients (87.2%; mean +/- SD, 108 +/- 134 v 5.3 +/- 5.7 U/mL in controls, P < .0001) and correlated with stage (stages I + II, 73 +/- 97 U/mL; III + IV, 162 +/- 165 U/mL; P < .0001), with presence of B symptoms (stage A, 69 +/- 82 U/mL; stage B, 162 +/- 171 U/mL; P < .0001), and, to some extent, with tumor burden (bulky presentation, 141 +/- 129 U/mL; nonbulky, 91 +/- 133 U/mL; P = .058). Patients with sCD30 levels greater than 100 U/mL at diagnosis had a significantly higher rate of poor outcome in terms of failure to achieve a complete remission (CR) or disease relapse after CR achievement. In fact, the event-free survival (EFS) duration of patients with sCD30 levels greater than 100 U/mL was significantly worse (P = .0016). Using multivariate analysis, an sCD30 level greater than 100 U/mL retained its significance after adjustment for other prognostic parameters. CONCLUSION: sCD30 in HD at presentation strictly correlates with clinical features. Serum levels greater than 100 U/mL at diagnosis entail a significantly higher risk of treatment failure, a factor that is independent of other prognostic parameters.

The CD4 molecule belongs to the phenotypic repertoire of most cases of acute myeloid leukemia.

In the present study fresh leukemic cells obtained from 23 patients with acute myeloid leukemia (AML; FAB subtypes: three M1, five M2, two M3, five M4, eight M5) were investigated for the membrane expression of the CD4 molecule by cytofluorimetric analysis with an anti-CD4 monoclonal antibody (mAb). In 15 cases the presence of the CD4 mRNA was also investigated using Northern blot analysis. Membrane expression of the CD4 molecule was demonstrated in 19 out of 23 cases, and it was found to be weaker than in CD4+ lymphocytes and monocytes obtained from normal controls. Full-length CD4 mRNA was detected in 12 out of 15 (80%) cases, and AML cells positive for CD4 mRNA expression also expressed the CD4 antigen. Since the CD4 molecule expressed by T cells is associated with p56lck, a member of the src family of intracellular tyrosine kinases, we investigated whether the CD4 molecule expressed by myeloid blasts is also associated with a tyrosine kinase activity. In vitro kinase assays performed on anti-CD4 immunoprecipitates from lysates of myeloid leukemia cells from four CD4+ cases were negative for the presence of a tyrosine kinase activity. This finding was not due to the lack of expression of members of the src family since we were able to detect at least p60src and p59fyn in myeloid leukemia cells. According to our results, the CD4 molecule seems to belong to the phenotypic repertoire of most AML, irrespective of their FAB subtypes. However, in myeloid blasts this molecule is not associated with a tyrosine kinase activity as it occurs in T lymphocytes.

Alpha (p55) and beta (p75) chains of the interleukin-2 receptor are expressed by AML blasts.

In the present study, we have investigated the leukemic cells obtained from 16 patients with acute myeloid leukemia (AML) at diagnosis for the membrane expression of p55 (alpha) and p75 (beta) interleukin-2 receptor (IL-2R) chains using specific monoclonal antibodies (mAbs), as well as for the presence of their transcripts using Northern blot analysis. In addition, immunoprecipitation of the p75 membrane molecule with TU27 and Mik-beta 1 mAbs was carried out in selected cases. The p75 IL-2R beta transcripts were detected in all cases, whereas the membrane p75 molecule was demonstrable by flow cytometry in three cases. However, data from the immunoprecipitation analysis suggest that the lack of the p75 IL-2R detection by flow cytometry might be caused by the low density of molecules per cell rather than the fact that the specific mRNA is not translated into the p75 surface molecule. In addition, a consistent membrane positivity with an anti-p55/CD25 mAb, present on fresh uncultured blasts in 37.5% of the cases, became detectable after short-term culture in 75% of cases. In each individual case, a strict correlation was found between membrane CD25 reactivity and the expression of p55 mRNA. Taken together, our data suggest that the expression of both alpha (p55) and beta (p75) IL-2R molecules is a common feature of leukemic cells in AML, and provide new arguments for reassessing the possible role of IL-2 in leukemic growth.

Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation.

In the present study, we explored the suitability of a new cell fixative (ORTHO PermeaFix, OPF) for the detection by flow cytometry of intracellular molecules while preserving the cell surface immunoreactivity, scatter features and morphology. The effect of OPF was investigated on whole blood of ten normal donors, and on separated blasts of 17 leukemic patients. OPF fixation for 45 min to 24 h maintained the morphology of lymphoid cells with minimal cellular distortion and scatter changes, and only slightly modified cell surface immunoreactivity. For at least 1 week following fixation, the cells were still suitable for immunostaining with monoclonal antibodies that recognize the main lymphoid populations. These included CD3, CD4 and CD8 for T-cell subsets, CD19 and CD16 for B lymphocytes and NK cells, and CD45 for leukocyte common antigen (LCA). The OPF fixation of leukemic cells allowed the simultaneous detection of nuclear TdT in conjunction with membrane CD19, and with membrane and/or cytoplasmic CD22 in common-ALL, as well as with cytoplasmic CD3 in T-ALL cases. Our findings suggest that with the introduction of this new fixative into the routine laboratory service, a number of convenient and practical arrangements can be made which increase the efficiency of immunodiagnosis. Small laboratories with no inhouse flow-cytometric facilities can now accumulate OPF-treated whole blood samples for at least 3-4 days and send these to reference laboratories. In addition, the immunodiagnosis of acute leukemia is greatly facilitated by combination staining for membrane and intracellular antigens both at diagnosis and when the analysis of minority populations is warranted for detecting minimal disease.

Origin of the soluble interleukin-2 receptor in the serum of patients with hairy cell leukemia.

High levels of soluble IL-2 receptor (sIL-2R) are detectable in the serum of HCL patients. To determine the cell source of this molecule, we evaluated the presence of sIL-2R in the supernatants obtained from in vitro cultures of leukemic (hairy cell, HC) and non-leukemic lymphocytes from six untreated HCL patients and from an additional four patients under therapy with rIFN-alpha 2. We demonstrated that cultured HCs at resting conditions were able to spontaneously release the sIL-2R, whereas control enriched B cells did not. This phenomenon was present only when culturing HCs recovered from patients observed at the time of diagnosis but was not observed during treatment with rIFN-alpha 2. Following activation in vitro with a series of different stimulatory agents including BCGF, phorbol myristate acetate, and anti-human IgM antibody, cultured HCs increased their capability to shed the IL-2R molecules. On the other hand, the release of sIL-2R from enriched T cell populations from HCL patients did not significantly differ from the value obtained in controls. Taken together, these findings provide evidence that leukemic B cells represent the main source of sIL-2R in HCL patients and further emphasize the importance of evaluating this parameter as a relevant marker for monitoring the effectiveness of rIFN-alpha 2 therapy.

IL-8 mRNA expression and IL-8 production by acute myeloid leukemia cells.

Purified leukemic cells from 30 acute myeloid leukemia (AML) cases at diagnosis were investigated for the presence of interleukin 8 (IL-8) mRNA by Northern blot analysis. IL-8 specific transcripts were detected in uncultured blasts in 14/30 cases, 10/14 from patients with M4-M5 and 4/14 from cases with M0-M3 morphology. The transcript expression was associated with the detection of IL-8 molecule in blast cells by immunostaining performed on cytospin preparations. After 24-hour culture, a strong up-regulation or the appearance in cases negative before culture of IL-8 mRNA was observed in all cases tested, and culture supernatants contained high amounts of IL-8. Our data demonstrate that leukemic cells in AML are equipped with the functional apparatus for IL-8 production. Since IL-8 displays a wide range of biological activities, including the regulation of some membrane molecules relevant to adhesion and migration processes, its production by AML blasts might be of relevance for the pattern of leukemic growth.

Leukemic cells in hairy cell leukemia and B cell chronic lymphocytic leukemia release soluble TNF receptors.

The two tumor necrosis factor receptors (TNF-Rs) exist in their soluble form in different biological fluids. In this study we investigated the concentrations of soluble tumor necrosis factor receptors (sTNF-Rs) in the culture supernatants of leukemic cells and in the serum obtained from 33 patients: 12 with hairy cell leukemia (HCL) and 21 with B cell chronic lymphocytic leukemia (B-CLL). In seven patients with HCL, sTNF-Rs were also evaluated following in vivo treatment with interferon-alpha (IFN-alpha). Purified leukemic cells from patients with HCL and B-CLL spontaneously released sTNF-R75 but not sTNF-R55. The levels of sTNF-R75 were higher in supernatants obtained from cultured hairy cells than from cultures of B-CLL cells. The shedding of sTNF-R75 was further increased both in HCL and B-CLL subjects by some B cell-related stimuli, including BCGF, PMA, SAC and was partially inhibited by IFN-alpha in patients with HCL. Sera from HCL patients presented increased levels of both sTNF-Rs with respect to normal controls. Treatment of HCL patients with IFN-alpha resulted in a decrease in serum levels of sTNF-Rs, particularly sTNF-R75. These findings suggest that leukemic cells account for the increased serum levels of sTNF-R75 observed in patients with HCL and B-CLL, but not for sTNF-R55.

High serum level of the soluble form of CD30 molecule in the early phase of HIV-1 infection as an independent predictor of progression to AIDS.

OBJECTIVE: To determine the serum levels of the soluble form of the CD30 (sCD30) activation molecule in the early phase of HIV-1 infection, and to investigate the possible correlation with evolution to AIDS. METHODS: sCD30 values were determined by an enzyme-linked immunosorbent assay (ELISA) on serum samples collected at the time of the first evidence of HIV-1 infection in 110 individuals with a median follow-up of 56 months (range, 12-88 months), at the A1 (74 cases) or A2 (36 cases) stages of the 1993 revised Centers for Disease Control and Prevention classification. The data were evaluated using established clinical and immunological parameters, including circulating CD4+ T-cell count. The controls were 110 blood donors and 51 HIV-1-negative subjects belonging to groups at risk for HIV-1 infection. RESULTS: Elevated sCD30 levels (> 20 U/ml) were found in 83.6% of HIV-1-infected cases and in 47% of at-risk seronegatives. Data analysis revealed that HIV-1-infected patients with higher sCD30 levels (> 35 U/ml) experienced faster disease progression (P = 0.0002). This was also the case in patients at the earliest stage (A1) of HIV infection (P = 0.0027). In these latter cases the predictive value of sCD30 was independent of the initial absolute number of circulating CD4+ lymphocytes. CONCLUSIONS: Serum levels of sCD30 are increased in the large majority of patients in the early phase of HIV-1 infection and represent an indicator of progression to AIDS independent of other prognostic parameters.

Shedding of the soluble form of the CD8 complex by CD8+/HLA-DR+ cells in HIV-1-infected patients.

High levels of the soluble form of the CD8 molecule (sCD8) are detectable in the serum of HIV-1-infected patients. To investigate the mechanisms accountable for the release of this molecule we evaluated the presence of sCD8 in the supernatants obtained from in vitro cultures of highly purified CD8 cells isolated from 20 HIV-1-infected patients. At resting conditions cultured CD8 cells from HIV-1-infected patients released low amounts of sCD8; no statistically significant differences were observed between unstimulated cultures from HIV-1-seropositive patients and from HIV-1-seronegative subjects at risk for HIV-1 infection or normal healthy controls. Following in vitro activation of highly purified CD8 cells with a series of stimulatory agents, including phorbol myristate acetate, phytohemagglutinin (PHA) and recombinant interleukin-2, CD8 cells of HIV-1-infected patients significantly increased the shedding of sCD8. By expressing the results of activation-related release index (ARRI = sCD8 levels detected in the cultures with stimulatory agent/sCD8 levels detected in the unstimulated cultures), significantly higher values were observed upon PHA stimulation in HIV-1-infected patients than in control subjects. In order to identify the cell subset responsible for the enhanced release of sCD8 by PHA-stimulated cultures, we correlated the amounts of sCD8 detected in the supernatants with the phenotypic profile of CD8+ cells recovered from the cultures. A significant relationship was demonstrated between the percentage of CD8+/HLA-DR+ lymphocytes and sCD8 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-interferon activated cytotoxic lymphocytes in hairy cell leukemia patients: evaluation of cytotoxic events.

To further define the mechanisms responsible for the alpha-interferon (alpha-IFN) efficacy in the treatment of hairy cell leukemia (HCL), experiments were carried out to specify the cytotoxic events taking place following this type of therapy. Although an increased natural killer (NK) activity was demonstrable after alpha-IFN treatment, evidence has been provided that hairy cells were not specifically lysed either by fresh autologous/allogenic NK lymphocytes or by lymphokine activated killer (LAK) cells. This property could not be induced in vitro by alpha-IFN or by interleukin-2 (IL-2). Our data favour the hypothesis that the increase of NK cell activity observed following alpha-IFN therapy has not a direct antineoplastic effect but is likely to be of relevance for a non-specific enhancement of the host immune system. In alpha-IFN treated HCL this latter property may account for the better resistance to infections which usually represents the major cause of mortality in these patients.

Naturally-occurring anti-G-CSF antibodies produced by human cord blood B-cell lines infected with Epstein-Barr virus.

INTRODUCTION: Naturally occurring antibodies (auto-Abs) recognizing human granulocyte-colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. MATERIALS AND METHODS: Mononuclear cells from cord blood samples of different newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. RESULTS: Six different, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD5-, CD10-, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing lambda clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. CONCLUSION: The secreted Abs did not affect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing.

Increased levels of soluble interleukin-2 receptor in non-Hodgkin's lymphomas. Relationship with clinical, histologic, and phenotypic features.

In this study the authors investigated the serum levels of the released soluble form of interleukin-2 receptor (sIL-2R) in patients with non-Hodgkin's lymphomas (NHLs). Data were evaluated in relationship to the morphologic and immunophenotypic heterogeneity of NHL at diagnosis and in progressive advanced diseases. Increased sIL-2R levels were found in most cases, when compared with levels observed in healthy controls. No obvious statistical correlation has been observed between sIL-2R values in different NHL subtypes as defined by current classifications. On the other hand, major significance was related to the extent of the disease. Very high values, comparable to those observed in hairy cell leukemia, were observed in a number of large cell NHLs complicating low-grade B-cell lymphoproliferations and in a single case of T-cell Kil+ NHL. The authors' findings suggest that the detection of sIL-2R in NHL may represent a good marker in improving risk assignment of single cases and/or for monitoring remissions and exacerbations during the treatment of cases with very high levels at diagnosis. Nevertheless, the observed overlap between groups on an individual case basis can render the clinical application of this marker problematic.

Soluble molecules as biological markers in Hodgkin's disease.

Hodgkin's disease (HD) is characterised by a complex architectural and functional derangement of involved tissues. The interactions between neoplastic cells and the heterogeneous microenvironment lead to the expression and release of different cellular messengers [cytokines, soluble (s) forms of cytokine receptors and other membrane-associated molecules] which can be detected in the circulation and evaluated as biological markers. We and others investigated several of these molecules looking for their possible role as diagnostic or prognostic parameters in patients with HD. We update here the results of serum determination of sIL-2Ralpha, sCD8, sICAM-1, sTNFRs, and sCD30 in a large series of cases from our institution. We found that their levels are generally increased at presentation and during the active phase of the disease. They correlate with stage and clinical aggressiveness and have some prognostic implication. However, we were unable to demonstrate a prognostic usefulness for their detection, with the exception for sCD30 which was found to directly correlate with disease spread and burden at presentation and, most importantly, to have an independent prognostic significance. The prognostic significance of sCD30 might derive from a crucial involvement of this molecule in the pathophysiology of HD.

Circulating levels of soluble CD30, a marker of cells producing Th2-type cytokines, are increased in patients with systemic lupus erythematosus and correlate with disease activity.

OBJECTIVE: To measure the levels of serum soluble CD30 (sCD30), a marker of cells producing T helper 2(Th2)-type cytokines, in systemic lupus erythematosus (SLE) and undifferentiated connective tissue disease (UCTD), and to determine its value in assessing disease activity. METHODS: Serum levels of sCD30 were measured by ELISA in 21 patients with SLE, in 17 patients with UCTD and in 40 normal donors. Disease activity was evaluated according to the ECLAM scoring system. RESULTS: sCD30 values were 53.84 +/- 58.24 U/mL in SLE, 22.65 +/- 9.82 U/mL in UCTD and 5.3 +/- 5.7 in normal controls (p < 0.0005 SLE vs controls; p < 0.05 SLE vs UCTD). sCD30 levels were directly related to the disease activity (p < 0.002). CONCLUSION: These data support a relationship between the Th2-type immune response and the pathogenesis of SLE, and suggest that sCD30 can be used as a simple marker for the evaluation of disease activity.

Empiric therapy of infections in hematologic malignancies: a prospective, randomized trial.

Patients with hematologic malignancies were randomly assigned to receive cefuroxime (group A) or tobramycin plus ampicillin (Group B) during 86 febrile episodes. In both regimens carbenicillin was added during neutropenia (71% of all episodes: groups C and D). The most common type of infection was pneumonia (48% alone; 72% with other sites involved), which accounted for a high fatality rate (15%); the highest rate occurred during septicemia with pneumonia (50%). The overall response rate to initial therapy was 63% without significant differences among the four regimens. The worst prognosis was observed in neutropenic patients without granulocyte recovery. When initial and cross-over trials were combined, there were favorable outcomes in 90% of all cases. Cefuroxime alone seems to be as effective as tobramycin plus ampicillin in the treatment of infections in hematologic malignancies. No side effects could be attributed to the cefuroxime-containing regimens.