Low dose recombinant interferon alfa treatment for classic Kaposi's sarcoma.

BACKGROUND: High doses of interferon alfa are used to treat Kaposi's sarcoma in patients with immunodeficiency, whereas low doses are generally ineffective. Following low-dose recombinant interferon alfa treatment for lymphoma, two patients showed a regression of their hematologic malignancy-associated Kaposi's sarcoma. This observation prompted us also to try low-dose interferon alfa treatment in uncomplicated classic Kaposi's sarcoma, and two additional patients were thus treated on an outpatient basis. OBSERVATIONS: Initial response was noted after 3 to 13 weeks of treatment. Remission was achieved after 4 to 6 months of low-dose interferon alfa treatment and its duration was 8 to 14 months. Recurrences were treated again and additional remissions were obtained after only 5 to 8 weeks of treatment. CONCLUSION: Low-dose interferon alfa treatment may represent an effective therapeutic modality for the treatment of patients with both lymphoma-associated and uncomplicated classic Kaposi's sarcoma.

Antitumor effects of human recombinant interleukin-6 on acute myeloid leukemia in mice and in cell cultures.

Interleukin-6 (IL-6) has been shown to inhibit growth and induce differentiation of several myeloid leukemia cell lines. In this work, two in vivo models of acute myeloid leukemia (AML) in mice have been used to test the therapeutic potential of recombinant human IL-6. In mice inoculated by a transplantable AML tumor, IL-6 injections inhibited the development of leukemia and increased survival. The effect was related to dose and length of treatment. In a model of radiation-induced leukemogenesis in SJL/J mice, administration of low-dose IL-6 for 10 days, 4 months after irradiation, reduced the incidence of leukemia observed during 1 year, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) increased the incidence of leukemia. In vitro liquid cultures of leukemic blood cells obtained from AML patients showed that IL-6 slowed growth and decreased the proportion of blasts with an increase in more mature myeloid elements in 72% of M1, M2, M4 AML cases. In contrast, GM-CSF less often produced differentiation but stimulated leukemic cell growth in liquid cultures, without synergism by IL-6.

Effects of endotoxin-associated protein on hematopoiesis.

Endotoxin-associated protein (EAP), a gram-negative bacterial cell wall component, was evaluated for its effects on hematopoietic colony formation in vitro. Colony-stimulating activity, induced by EAP on circulating and bone marrow progenitor cells, was found to be partially mediated by T cells and augmented by interleukin-3. The addition of anti-human interleukin-1 (IL-1) antibodies reduced EAP activity, suggesting that EAP may induce IL-1 production. However, EAP was shown to promote the growth of mature progenitor cells independently, unlike the effects of IL-1 on the hematopoietic system. These studies demonstrate that bacterial components other than lipopolysaccharide, such as EAP, may have hematopoietic activity.

Restoration of in vitro hematopoiesis in B-chronic lymphocytic leukemia by antibodies to tumor necrosis factor.

Hematopoiesis was evaluated in 15 B-CLL patients using the mixed colony formation assay. The mean growth of all types of colonies in B-CLL peripheral blood was significantly lower than that of 10 normal controls (p less than 0.05). To investigate whether TNF is the cytokine involved in the reduced growth of hematopoietic progenitors in B-CLL, neutralizing anti-TNF antibodies (anti-TNF Abs) were added to the cultures. Anti-TNF Abs optimized in vitro hematopoiesis in 11 out of 15 B-CLL patients and a significant growth increase in all types of colonies was noted as compared to baseline cultures (p less than 0.05). In patients with stage IV disease, the increase in both mixed and erythroid colonies was more prominent than in patients with earlier disease stages. This optimization of growth was also observed in normal control cultures containing accessory cells. However, high TNF levels were measured in conditioned media from CLL patients and suppressed normal bone marrow hematopoietic progenitors growth. In contrast no TNF was detected in normal conditioned media. It is concluded that TNF and other cytokines, among them IL-3, play a role in the regulation of hematopoietic function in some B-CLL cases. These findings may have clinical applicability.

Interferon beta 2/interleukin-6 and interleukin-3 synergize in stimulating proliferation of human early hematopoietic progenitor cells.

Early 4-hydroxyperoxycyclophosphamide (4-HC) resistant hematopoietic progenitor cells (pre-colony-forming units, pre-CFU) were evaluated by a two-step liquid culture system, (earlier progenitors), pre-CFU, as well as by the conventional semi-solid mixed colony assay (later progenitors) for their growth response to interleukin-6 (IL-6), interleukin-3 (IL-3), and a combination of both factors. The effect of the IL-6/IL-3 combination was compared to that of IL-1/IL-3. IL-3 alone proved less effective in supporting earlier pre-CFU cells than later progenitor cells. In a previous work IL-6 promoted the growth of early multipotential progenitor cells circulating in hairy cell leukemia (HCL) patients. IL-6 alone did not stimulate growth of either early or later normal progenitor cells. However, a significant synergistic effect was obtained when IL-6 and IL-3 were added together (p less than 0.05). IL-6/IL-3 synergism was more potent than IL-1/IL-3 in promoting growth of colonies. The previously described synergistic effect of IL-1/IL-3 seems to be independent of IL-6. Thus, our results suggest that the multi-functional cytokine IL-6, may be of use in shortening the engraftment time in bone marrow transplantation.

Spinal epidural compression in chronic lymphocytic leukemia.

Spinal epidural compression is a rare neurologic complication in patients with lymphoma. It occurs mostly in those with intermediate-grade to high-grade malignancy disease. This type of neurologic involvement has not been described in chronic lymphocytic leukemia (CLL). A patient with a long, stable CLL course developed spinal epidural compression and consequently died. The frequency of spinal epidural compression in lymphoma, according to the histologic subtypes and the considerations in making the right choice of therapy are discussed in light of the presented case.

Granulocytic macrophage colony stimulating factor restores in vitro growth of granulocyte-macrophage bone marrow hematopoietic progenitors in dyskeratosis congenita.

In vitro 14-day cultures of bone marrow from a patient with dyskeratosis congenita showed virtually no growth of colonies. The addition of recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) promoted a significant increase in the number of GM colonies (CFU-GM). Interleukin 3 also increased GM colony formation but to a lesser extent. GM-CSF may have a therapeutic implication for pancytopenia in dyskeratosis congenita.

Stem cells: a tree model.

A tree model is presented in order to illustrate the hypotheses that earlier stem cells will respond differently than later progenitors to the same stimuli. Experimental data and decision analysis tools are shown to demonstrate the concept.

Characterization of lympho-myeloid-erythroid-megakaryocytic stem cells in peripheral blood of hairy cell leukemia patients.

Circulating stem cells with lympho-myeloid-erythroid differentiative capacity have been described in the peripheral blood of hairy cell leukemia (HCL) patients (Michalevicz R. & Revel M. (1987) Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in Hairy Cell Leukemia. Proc. natn. Acad. Sci. U.S.A. 84, 2307.) The aim of the present work was to enrich the progenitors and characterize their antigenic and growth properties. Peripheral blood (PB) from HCL patients was stained with antibodies (BI3C5 (CD34) and/or My10 as well as RFB7 (CD20) and RFT12 (CD7) and sorted using flow cytometry (FCM) into positive and negative fractions. Peripheral blood cells from several patients showed 4-16% cells positive for the BI3C5/My10. The positive fraction contained all the colony-forming cells and LGEM/LG/LGM colonies were enriched approximately ten-fold as compared to the unsorted population. No colony was found in the negative fraction. All colony forming cells were in the RFB7 and RFT12-negative fractions. Thus, circulating stem cells with lymphoid/myeloid potential can be isolated from PB of HCL patients.

Game theoretic analysis of interferons effect on hematopoietic progenitors growth.

The effects of recombinant alpha, beta and gamma interferons (IFN), alone or in combinations were studied in a case of essential thrombocythemia, using the mixed colony formation assay. This assay allows growth of multipotent (CFU-Mix), and unipotent granulocytic-macrophage (CFU-GM), erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors. The bone marrow precursors were cultured in the presence of each type of IFN at 100 U/ml and all possible combinations performed. The results were analysed using the Shapley formula, a game theoretic approach. It is concluded that IFN alpha would be the best candidate for reducing megakaryocytic progenitors while growth of other hematopoietic precursors would be stimulated. These types of analytical biological experiments controlled by using six permutations and calculation of the Shapley value for the three types of IFNs are suggested as a fair approach for a rational choice.

Recombinant interferon alpha-C for advanced hairy cell leukemia. An Israeli multicenter study.

Thirteen patients with advanced hairy cell leukemia were treated with a new subspecies of interferon (IFN): recombinant IFN (rIFN)-alpha-C. The timing of the peripheral hematologic remission of individual blood elements was similar to that reported for other interferons, and 60% of patients attained a complete peripheral hematologic remission at 9 months. Two patients relapsed despite a good initial response to IFN. No anti-IFN antibodies could be detected in their sera. In vitro studies of colony formation from the peripheral blood of all responding patients showed that rIFN-alpha-C did not inhibit the growth of colonies but favorably affected the maturation of their elements towards monocytes, granulocytes, and erythroid elements. The relapsing patient examined initially experienced a similar beneficial in vitro response which paralleled his in vivo improvement. During relapse, rIFN-alpha-C inhibited both the colony formation and the myelomonocytic differentiation in the in vitro cultures. These findings may suggest that the acquired resistance to IFN in our patient could be due either to an acquired stem cell maturation arrest in response to IFN or to emergence of a new clone indifferent to IFN-alpha-C differentiation effect.

Interferon-beta induced remission in a hairy cell leukemia patient resistant to interferon-alpha.

Two patients with advanced hairy cell leukemia and pancytopenia responded successfully to intramuscular injections of recombinant interferon-beta, with normalisation of blood counts. One of the patients had developed resistance to interferon-alpha. The in-vivo response was predicted by in-vitro studies showing a beneficial effect of IFN-beta on erythropoiesis in cell cultures derived from these patients.

Recombinant interleukin 2 and anti-Tac influence the growth of enriched multipotent hemopoietic progenitors: proposed hypotheses for different responses in early and late progenitors.

Experiments were designed to evaluate the effect of recombinant IL-2 on growth of hemopoietic precursors from different sources (normal cord blood and bone marrow, and PB from CGL patients). For this purpose, combined cell sorting techniques and multipotent colony forming cell assays were used. A monoclonal antibody BI-3C5, which recognizes an antigen present on early lympho-myeloid cells as well as on all colony forming cells (CFU-GEMM assay), was used to enrich the studied populations. Double colour immunofluorescence techniques were performed to analyse the expression of Tac antigen on early progenitors. The results showed that rIL-2 had a stimulatory effect on growth of enriched progenitors from the three sources and surprisingly that addition of anti-Tac did not abolish this effect. On the contrary, anti-Tac enhanced even more growth of these sorted BI-3C5 precursors, suggesting a ligand action of the antibody. More interestingly, a low percentage of cord cells (1 in 1000) expressed both BI-3C5 and Tac antigens. The vast majority of cells did not concomitantly express both markers. The double labelled cells had a lymphoid-like morphology, high nucleus/cytoplasmic ratio and 2-3 nucleoli. The results will be discussed focusing on early and late "stem" cell growth.

Osteoclast-like cells grow in cultures of multipotent hematopoietic progenitors in thrombocytopenia and absent radii (TAR) syndrome.

Cultures of bone marrow multipotent hematopoietic progenitors were performed in a case of TAR syndrome and normal bone marrow. It was found that clones of large multinucleated cells formed after 2 weeks of cultures from the TAR bone marrow but not in that of control subjects of the same age. Those cells contained a tartrate-resistant acid phosphatase activity and were negative for both monocytes and megakaryocytic markers. The data suggest that the multinucleated cells have several characteristics of osteoclasts. It was also found that growth of multipotent and megakaryocytic colonies was reduced when compared with normal control subjects (36 and 24%, respectively). This new finding of osteoclast or osteoclast-like cells derived from cultures of bone marrow hematopoietic precursors in TAR syndrome is discussed in relation to the lack of megakaryocytic characteristics in this disorder.

Fenbufen induced pure red cell aplasia in rheumatoid arthritis.

Pure red cell aplasia occurred after fenbufen administration in a patient with severe rheumatoid arthritis. In vitro studies were performed to determine the pathogenesis of the selective red cell aplasia. No cellular or humoral inhibitory mechanisms were demonstrated on growth of erythroid and multipotent bone marrow progenitors. Also, no direct effect of fenbufen alone or in combination with IgG and/or patient serum was found. It is possible that a metabolite of the drug formed from its metabolism was responsible for the aplasia and that the target marrow cell precursor affected is later than both erythroid bone marrow progenitors (BFU-E and CFU-E) and therefore not apparent in our studies. Recovery upon cessation of fenbufen suggests its implication in the pure red cell aplasia.

Reduced production of tumor necrosis factor by mononuclear cells in hairy cell leukemia patients and improvement following interferon therapy.

In 16 patients with hairy cell leukemia (HCL) there was a marked reduction in the production of cytotoxins (CTXs) by peripheral blood mononuclear leukocytes in response to stimulation in vitro by phytohemagglutinin (PHA), 4 beta-phorbol-12-myristate-13-acetate (PMA), or Sendai virus. CTX yields of 23.5 +/- 21.5 U/ml, 15 +/- 18 U/ml, and 12.1 +/- 12.1 U/ml were obtained in response to PHA, PMA, and Sendai virus, respectively, as compared with corresponding yields of 207.3 +/- 93.1, 154 +/- 37.4, and 205.2 +/- 62.4 in healthy controls. The extent of reduced production of CTXs appeared to be correlated with the severity of the disease. Systemic interferon (IFN) administered to four patients caused CTX production to improve in response to PHA (147.5 +/- 55.1 U/ml compared with pretreatment values of 14.1 +/- 6 U/ml, P less than 0.05). However, CTX production in response to Sendai virus remained low. The extent to which CTX production by hairy cell leukemia mononuclear cells was reduced was proportionate to the observed decrease in monocyte counts. However, the degree to which CTX production improved after IFN treatment was significantly greater than the observed increase in monocyte counts. The major CTX induced by PHA in mononuclear cells of healthy donors and of IFN-treated HCL patients was identified as tumor necrosis factor-alpha.

Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in hairy cell leukemia.

In vitro 14-day cultures of peripheral blood mononuclear cells from hairy cell leukemia patients consistently showed the presence of hematopoietic stem cells giving rise to multilineage colonies containing a high proportion of lymphoid cells associated with the myeloid and erythroid progenitors. These stem cells are not the hairy cells but appear to be pluripotent lymphomyeloid primitive stem cells persisting in this leukemia. Interferon alpha c or beta 1 did not inhibit the growth of these colonies, as they did the growth of colonies of normal hematopoietic progenitors, but markedly decreased the ratio of lymphoid to myelomonocytic cells, by increasing the formation of monocytes and other nonlymphoid cell types in these multilineage colonies. Interferon gamma did not have the same effects on differentiation.

JM, a Thy-ALL cell line produces both inhibitory and stimulatory lymphokines for bone marrow progenitor cells.

Normal and malignant T cells as well as T-cell hybridomas have frequently been reported to produce factors which stimulate the growth of committed hemopoietic progenitors. One previous report described a lymphokine produced by a T-cell clone which inhibited hemopoietic progenitor cell proliferation. We now describe the simultaneous production of two activities by a Thy-ALL cell line (JM), a sub-line of Jurkat. Two sets of culture conditions were used: the Fauser & Messner and Iscove's assays. We have been able to separate both inhibitory and stimulatory factors for the growth of multipotent and committed bone marrow progenitors (CFU-GEMM, BFU-E, CFU-E and CFU-GM). The stimulatory factor has an apparent mol. wt of less than 30,000 and the inhibitor an apparent mol. wt of 65-80,000. The growth promoting activity for BFU-E and CFU-GEMM could replace that of phytohemagglutinin stimulated leucocyte conditioned medium (PHA-LCM). We do not know if the production of both activities is due to the malignant phenotype or if there is a normal counterpart to JM that could produce both inhibitory and stimulatory factors.

Effect of platelet-derived growth factor on enriched populations of haemopoietic progenitors from patients with chronic myeloid leukaemia.

The effect of pure platelet-derived growth factor and fresh serum on the in-vitro growth of purified haemopoietic progenitors from the peripheral blood of 12 patients with CML was studied. Purified haemopoietic progenitors were prepared using Percoll separation followed by cell sorting with the monoclonal antibody BI.3C5. Both pure PDGF at a concentration of 20 ng/ml and fresh serum significantly increased the numbers of BFU-E (p less than 0.01) and CFU-GEMM (p less than 0.014), but not the CFU-GM. That the PDGF effect was not mediated to any significant extent via prostaglandins, was shown by the lack of inhibitory effect of indomethacin on the growth of purified progenitor cells in the presence of fresh serum. Increased amounts of pure PDGF were required to give maximal stimulation of purified CML peripheral blood progenitors compared to normal bone marrow progenitors. These results show that CML progenitors are capable of responding to PDGF. Whether the quantitative difference in response is due to a reduced proportion of mesenchymal cells in CML peripheral blood compared to normal marrow, or whether CML progenitors are most likely already stimulated by autocrime PDGF or other growth factors remains to be elucidated.