Otto Prümmer

HCV and HGV in B-cell non-Hodgkin's lymphoma

BACKGROUND/AIMS A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkins lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkins lymphoma. METHODS Serum samples from 69 patients with non-Hodgkins lymphoma were studied. Diagnosis of non-Hodgkins lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy. RESULTS Three of 69 (4.3%) patients with B-cell non-Hodgkins lymphoma were HCV-infected and nine non-Hodgkins lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkins lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins. CONCLUSIONS HCV seems to have no significance for the pathogenesis of non-Hodgkins lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkins lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Interferon-alpha antibodies in patients with renal cell carcinoma treated with recombinant interferon-alpha-2A in an adjuvant multicenter trial

BACKGROUND Prolonged therapy with interferon (IFN) may lead to the formation of IFN antibodies. METHODS Patients with renal cell carcinoma (n = 270) with advanced localized disease were randomized after complete tumor resection to receive treatment with adjuvant recombinant IFN-alpha-2a (rIFN-alpha 2a) (9 x 10(6) IU subcutaneously, three times per week for a maximum of 12 months) versus no treatment. Patients (IFN-treated group, 106 patients; control group, 97 patients) were monitored for the presence of rIFN-alpha 2a antibodies. RESULTS Of 86 IFN-treated patients observed for more than 2 months, 40 (47%) had IFN-alpha 2a-binding and 25 (29%) had IFN-alpha 2a-neutralizing antibodies developed within a median of 3 and 6 months, respectively. A distinct peak in binding antibody titers occurred at 6-9 months. Therapy-induced neutralizing antibodies were equally reactive with two other recombinant IFN-alpha-2 subtypes but poorly recognized natural IFN-alpha (IFN-alpha), recombinant IFN-alpha-1/alpha-8, and recombinant IFN-omega-1. The duration of remission and rate of relapse were independent of the antibody status, although neutralizing and most non-neutralizing antibodies correlated with a reduction in the IFN-induced increase in beta-2-microglobulin levels. CONCLUSIONS Patients treated with IFN-alpha 2a should be monitored for the presence and clinical relevance of IFN-alpha antibodies to determine those who could respond to alternative treatment.

Treatment-induced antibodies to interleukin-2

Interleukin-2 (IL-2) is a 15 kDa glycoprotein with proven activity as an immune stimulant in the treatment of malignant disorders, congenital and acquired immune deficiencies, infectious disorders, and as an adjuvant to vaccines. Both natural and recombinant type IL-2 preparations have been applied in clinical treatment trials and have turned out to be immunogenic, although to a varying extent. Enzyme immunoassays and western blotting are standard procedures for the detection of IL-2-binding antibodies, whereas the neutralizing capacity of these antibodies is frequently demonstrated by inhibition of IL-2-dependent cell growth in vitro. The rate of treatment-induced IL-2 antibodies has varied from 0% to 100% in reported trials and frequently exceeded 50% in patients exposed to recombinant IL-2, whereas natural type IL-2 appeared to be little immunogenic. Duration of treatment, cumulative IL-2 dose, and route of IL-2 administration are likely to determine both the rate of seroconversion as well as composition and properties of the anti-IL-2 antibodies. Interleukin-2 antibodies are polyclonal in nature and predominantly composed of IgM and IgG types. Frequently they react with both recombinant and natural IL-2 types. As a rule, neutralizing IL-2 antibodies are detected in serum samples with high IL-2-binding titers and are recognized later than their non-neutralizing predecessors. Neutralization in vitro, however, does not predict neutralization in vivo, and there are very rare patients with documented, antibody-mediated loss of response to IL-2 treatment. More frequently, IL-2 antibodies will limit the expression of IL-2-dependent proteins in vivo, but the opposite has also been observed. Although the precise mechanism of antibody induction by IL-2 is unknown, immunogenicity of some drug formulations rather than polyclonal B-cell activation appears to play a critical role. Approaches aiming at limiting the immunogenicity of IL-2 preparations are discussed, and strategies how to recognize and circumvent antibody-mediated IL-2 resistance are presented.Interleukin-2 (IL-2) is a 15 kDa glycoprotein with proven activity as an immune stimulant in the treatment of malignant disorders, congenital and acquired immune deficiencies, infectious disorders, and as an adjuvant to vaccines. Both natural and recombinant type IL-2 preparations have been applied in clinical treatment trials and have turned out to be immunogenic, although to a varying extent. Enzyme immunoassays and western blotting are standard procedures for the detection of IL-2-binding antibodies, whereas the neutralizing capacity of these antibodies is frequently demonstrated by inhibition of IL-2-dependent cell growth in vitro. The rate of treatment-induced IL-2 antibodies has varied from 0% to 100% in reported trials and frequently exceeded 50% in patients exposed to recombinant IL-2, whereas natural type IL-2 appeared to be little immunogenic. Duration of treatment, cumulative IL-2 dose, and route of IL-2 administration are likely to determine both the rate of seroconversion as well as composition and properties of the anti-IL-2 antibodies. Interleukin-2 antibodies are polyclonal in nature and predominantly composed of IgM and IgG types. Frequently they react with both recombinant and natural IL-2 types. As a rule, neutralizing IL-2 antibodies are detected in serum samples with high IL-2-binding titers and are recognized later than their non-neutralizing predecessors. Neutralization in vitro, however, does not predict neutralization in vivo, and there are very rare patients with documented, antibody-mediated loss of response to IL-2 treatment. More frequently, IL-2 antibodies will limit the expression of IL-2-dependent proteins in vivo, but the opposite has also been observed. Although the precise mechanism of antibody induction by IL-2 is unknown, immunogenicity of some drug formulations rather than polyclonal B-cell activation appears to play a critical role. Approaches aiming at limiting the immunogenicity of IL-2 preparations are discussed, and strategies how to recognize and circumvent antibody-mediated IL-2 resistance are presented.

Reversible metastatic pulmonary calcification in a patient with multiple myeloma

A-52-year-old patient presented with a 2-year history of multiple myeloma, recurrent episodes of hypercalcemia, and extensive bone involvement. She developed pulmonary infiltrates, initially misdiagnosed as interstitial pneumonia. High-resolution computed tomography and bone scintiscanning indicated pulmonary calcification, which was confirmed by a transbronchial biopsy. Cytostatic treatment of multiple myeloma in combination with repetitive i.v. administration of bis-phosphonates over a period of 6 months led to a significant improvement of clinical symptoms. Regression of pulmonary infiltrates was demonstrated by chest radiograph and computed tomography. There are only a few reports on pulmonary calcification in patients with multiple myeloma; the condition was associated mostly with progressive disease, kidney failure, adult respiratory distress syndrome and bad prognosis. In our patient isolated calcification of the lungs without involvement of other organ systems was successfully treated. These findings suggest that interstitial pulmonary calcinosis in multiple myeloma can be reversed by normalization of serum calcium levels using bisphosphonates combined with cytostatic treatment.

Capillary leak syndrome associated with elevated IL-2 serum levels after allogeneic bone marrow transplantation

SummaryThe pathophysiological mechanisms involved in the development of a spontaneous systemic capillary leak syndrome (CLS) are unknown. In contrast, CLS is a well-known side effect of high-dose interleukin-2 (IL-2) therapy in solid tumors. We report on a patient who developed CLS with high serum levels of endogenous IL-2 under immunosuppressive therapy for chronic graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation (BMT). Generalized edema persisted for 10 weeks. The condition resolved after antibiotic therapy of a septic shock withβ hemolyzing streptococci group A. Thus, a latent infection may alter cytokine homeostasis and may cause CLS in BMT patients.

Spontaneous interferon-α antibodies in a patient with pure red cell aplasia and recurrent cutaneous carcinomas

SummaryHigh-titered spontaneous interferon (IFN) antibodies were detected in a patient with pure red cell aplasia (PRCA), a benign mediastinal tumor, and recurrent cutaneous carcinomas. The circulating IFN antibodies reacted broadly with various human IFN-α subtypes (20–140×103 neutralizing units/ml serum) but not with IFN-β or IFN-γ, and they neutralized the antiviral activity of the patients endogenous IFN-α. The IFN-αbinding activity was restricted to the IgG1 subclass in a nonmonoclonal manner. Whereas the PRCA repeatedly responded to immunosuppression with high-dose cyclosporin A (CSA) and CSA plus plasmapheresis, IFN antibody production continued during treatment with cyclophosphamide and CSA. Serological analysis revealed past infection with parvovirus B19 and persistent B19 IgM titers. Antibody-mediated impairment of the IFN-α system might have favored the development of both PRCA and the various cutaneous carcinomas in this patient.

Sensitive antiproliferative neutralization assay for the detection of neutralizing IFN-α and IFN-β antibodies

Abstract Antibodies to interferon (IFN) may compromise IFN treatment in some patients. In tumor therapy, a critical function of type I IFNs is their antiproliferative effect. For the quantification of neutralizing IFN antibodies we have developed an antiproliferative neutralization assay (APA) based on the reduction of IFN-mediated growth inhibition of Daudi cells by IFN-α and IFN-β antibodies. Proliferation was quantified by [3H]thymidine incorporation, and the neutralizing potency of IFN antibody-positive sera was expressed as the neutralizing titer inhibiting 50% of the antiproliferative activity of 10 IU/ml of IFN (NT50). The APA is easy to perform, reproducible, and more sensitive than a well-established antiviral neutralization assay (AVA). All 30 sera with recombinant IFN-α2a-binding antibodies proved to be neutralizing antibody-positive in the APA whereas seven were scored antibody-negative or uninterpretable in the AVA. The APA is recommended as a second or third line assay for the estimation of the neutralizing potency of spontaneous or treatment-induced IFN-α-and IFN-β-specific antibodies.

Bone Marrow Structure and Its Possible Significance for Hematopoietic Cell Renewal

The authors review the progress made during the last quarter of a century in the fields of hematopoietic cellular proliferation and differentiation in relation to the bone marrow structure and the microenvironment provided by the marrow stroma in which unlimited self-renewal occurs. The marrow is conceived of as an organ in which the stroma originates from local mesenchymal elements which form a vascularized and innervated matrix, seeded later by blood-borne stem cells. Transplantation studies using total-body-irradiated dogs show that stem cells derived from the marrow, as well as those from the blood and from the fetal liver, are able to repopulate a marrow rendered aplastic by irradiation. By grafting equal numbers of GM-CFU from peripheral blood and bone marrow, a faster hemopoietic reconstitution is provided by blood-derived stem cells. The most efficient stem cells in the long range are those derived from fetal liver. Bone marrow and peripheral blood GM-CFU differ in some in vitro characteristics such as radiation sensitivity. These peripheral blood cells are more radiosensitive than those derived from the marrow. Autografting of bone marrow mononuclear cell fractions obtained by velocity sedimentation techniques demonstrates that the fraction of small mononuclear cells holds a repopulating potential similar to that of circulating blood stem cells. The cells collected in fraction 2 of a discontinuous albumin gradient contain most of the blood stem cells and repopulate the marrow without causing GVHD, while cells collected in fractions 3 and 4 contain a minimal amount of stem cells and cause severe GVHD.

Fetal liver transplantation in the dog. I. Restoration of hemopoiesis with cryopreserved fetal liver cells from DLA-identical siblings.

Fetal liver cells (FLC) were obtained from beagle fetuses 52 days postconception, and were cryopreserved prior to transplantation into ten sibling recipients that had previously been exposed to total-body irradiation delivered in 3 fractions of 6 Gy each at 4 days, 2 days, and 2 hr before grafting. Donors and hosts were genotypically identical for dog leukocyte antigens (DLA)-A, B, and D. A rapid and lasting engraftment was achieved in all animals following the transfer of 0.2x108 to 1.6x108 mononuclear FLC/kg body weight, which were equivalent to 0.9x104 to 19.8x104 granulocyte/macrophage progenitor cells (CFU-GM)/kg. Between days 14 and 20 posttransplant pretreatment levels were detected for blood granulocytes, between days 23 and 28 for circulating platelets, and between days 35 and 40 for the erythrocyte count and hemoglobin concentration. Increasing the number of CFU-GM transfused resulted in an accelerated granulocyte and platelet recovery. Bone marrow cells were of donor origin throughout the observation interval, but declining proportions of host lymphocytes circulated in the peripheral blood during the initial recovery phase. In two dogs, skin alterations that might indicate slight graft-versus-host disease (GVHD) were noted following days 20 and 70, respectively. Six recipients had to be sacrificed due to inanition, probably secondary to radiation-induced pancreatic insufficiency two to three months after grafting. The results of this study indicate that cryopreserved FLC are highly effective in restoring hemopoiesis in DLA-compatible sibling dogs. Transplantation of canine FLC may prove valuable in analyzing mechanisms pathogenetically related to graft rejection or to the development of GVHD following the transfer of T-cell-depleted hemopoietic grafts at a preclinical stage.

Fetal liver transplantation in the dog. II. Repopulation of the granulocyte-macrophage progenitor cell compartment by fetal liver cells from DLA-identical siblings.

The restoration of the granulocyte-macrophage progenitor cell (CFU-GM) compartments in blood and bone marrow, and the recovery of blood monocytes were followed for up to one year in ten beagles that had been exposed to fractionated (3x6 Gy) total-body irradiation before being transfused with cryopreserved fetal liver cells (FLC) from sibling donors that were genotypically matched for dog leukocyte antigens. Grafts contained 0.2–1.6x108 mononuclear cells and 0.9–19.8x104 CFU-GM/kg body weight. Numbers of circulating monocytes rose parallel to granulocyte numbers after day 6 and became normal by day 18 posttransplant. In bone marrow aspirates, low numbers of CFU-GM were detected on day 3 and their incidence per 105 mononuclear cells was normal after day 14. Circulating CFU-GM were present in significant numbers by day 7 and their elevated concentration per milliliter of blood after day 14 continued for one year. Dextran sulfate injection mobilized normal numbers of CFU-GM into the blood early after transplantation, and spontaneously circulating CFU-GM in a later phase did not differ from blood progenitors of normal animals with respect to radiation sensitivity and sedimentation velocity. Thus, FLC transplantation effected a rapid restoration of granulopoiesis and monocytopoiesis, which was reflected at both the level of mature blood cells and the compartments of CFU-GM in blood and bone marrow, underlining the high repopulating capacity of fetal liver stem cells.