Ch. Huber

Rapid reappearance of large granular lymphocytes (LGL) with concomitant reconstitution of natural killer (NK) activity after human bone marrow transplantation (BMT)

The frequency of large granular lymphocytes and their relationship to functional NK‐activity as assessed by the capacity to lyse the K562 tumour target was analysed in five allogenic and two autologous human bone marrow transplant recipients. Date revealed: (i) almost identical disappearence and reconstitution of both parameters further indicating that LGL represent effector cells of spontaneous lysis of K 562 targets; (ii) a long‐lasting suppression of the absolute numbers per ml of blood of both LGL and functional NK activity which we believe was not the consequence of reconstitution with immature effector cells but rather reflected immunosuppressive therapy; (iii) LGL exhibits the fastest reappearance rate subsequent to total body irradiation of all populations of circulating leucocytes.

Neopterin as a New Biochemical Marker in the Clinical Assessment of Ulcerative Colitis

In previous publications we showed that neopterin, a pyrazino-pyrimidin compound, represents a biochemical marker for the assessment of cellular immune responses. We thought that the evaluation of this marker molecule might enable insight into the activity of cellular immune responses underlying ulcerative colitis (UC). Evaluation of urinary neopterin excretion in 25 consecutive untreated UC patients revealed striking correlations between neopterin levels and the severity of disease: elevated levels were observed in 9 out of 9 patients with moderately severe to severe, in 3 of 4 with mild and in none of 12 patients with quiescent disease. Further evidence for a correlation between disease activity and neopterin excretion was obtained on the basis of long-term follow-up studies performed in 4 cases. These studies indicated normalization of neopterin levels when clinical remission was achieved. Thereafter, the relative significance of neopterin excretion for determination of clinical stage was assessed by linear correlation analyses and was compared with conventional clinical parameters such as anemia, number of motions per day, raised temperature, ESR and extent of bowel involvement. The logarithm of neopterin excretion and the extent of bowel involvement were the two single parameters most closely related to the clinical stage of ulcerative colitis. We, therefore, conclude that evaluation of neopterin excretion in ulcerative colitis patients represents a new and useful tool for the clinical monitoring of disease activity.

Human interferon-? induces expression of HLA-DR on keratinocytes and melanocytes

SummaryPrimary human epidermal cell cultures composed of keratinocytes and melanocytes were exposed to supernatants of phytohaemagglutinin (PHA)-stimulated T cells, various lymphokines and interferon-β, and checked for the emergence of HLA-DR antigen using immunofluorescence and immunoelectron microscopy. HLA-DR expression was induced by the supernatants and human recombinant interferon-γ (rIFN-γ), whereas recombinant α2, interleukin-2 and non-recombinant human interferon-β had no such effect. The threshold concentration of rIFN-γ required to induce this phenomenon was 10 IU/ml; no further increase of reaction intensity was observed using doses of more than 100 IU/ml. Maximum reaction intensity was achieved after 72 h of incubation; a minimum of 3 h of incubation with rIFN-γ followed by 72 h incubation in rIFN-γ-free medium proved sufficient to induce HLA-DR expression. The inductive effect of the supernatants and rIFN-γ could be completely abrogated by pretreatment with excess doses of the monoclonal antibody GZ4 specific for human IFN-γ. Keratinocytes and melanocytes reacted in an identical fashion both qualitatively and quantitatively in all experiments. These data indicate that IFN-γ possesses specific signal functions in the induction of HLA-DR expression on epidermal cells.

Interferon-alpha-2 in the treatment of idiopathic myelofibrosis.

SummaryWe investigated the effect of human recombinant DNA-derived IFN-alpha-2 given in a dose of 1–2×106 units daily by subcutaneous injection to five patients with advanced idiopathic myelofibrosis (IM). Transfusion dependent anemia and symptomatic splenomegaly were taken as inclusion criteria for this pilot study. Two patients succumbed, one and three months after starting interferon-treatment because of pneumonia and traumatic cranial injury, respectively. While on IFN-treatment no improvement of cytopenia or reduction of splenomegaly was seen in four of the patients. In one patient, however, the requirement for erythrocyte transfusions decreased from 5 to 1.7 monthly upon IFN-treatment. After two, four and six months respectively IFN-treatment had to be stopped in these cases because of progressive thrombocytopenia and/or neutropenia. These observations suggest, that IFN-alpha might be of only marginal value in the treatment of advanced idiopathic myelofibrosis.

A biological approach to optimize interferon treatment in hairy cell leukemia.

Progress with the clinical application of interferons to neoplastic diseases has been slow and complicated by the need for attention to a new spectrum of therapeutic and toxic effects manifested by the interferons. In this report, we present a new approach to define clinically effective but atoxic doses of interferon-alpha for treatment of hairy cell leukemia. In order to find in vivo biologically active interferon doses, the biochemical marker neopterin was selected as a means to assess a cellular interferon response in vivo. Subcutaneous administration of minimal doses of recombinant interferon-alpha-2 (5-8 X 10(5) U/day), which induced maximum neopterin release in serum and urine, proved to be clinically effective: Eight of nine patients responded to this dose regimen. This response rate was comparable to that of a conventional dose schedule (3 X 10(6) U/sqm/day) which was also applied to nine patients (eight responders). Whereas no difference in the clinical efficacy between the two therapeutic strategies could be established, toxicity was clearly confined to the conventional dose regimen. These preliminary results suggest that at least in hairy cell leukemia the therapeutic dose range of interferon can be separated from the toxic.

Neopterin, ein neuer biochemischer Marker zur klinischen Erfassung zellulärer Immunreaktionen

SummaryActivated T-lymphocytes represent crucial effector cells. They are pathogenetically involved into various disease states such as allograft rejection or viral infection. So far their assessment is laborious and rarely possible in clinical routine. In this review article we present the compount neopterin as a new biochemical marker for the in vivo and in vitro detection of activated T-lymphocytes. Our main finding was that in vitro as well as in vivo stimulation of T-lymphocytes with foreign and chemically or virally modified autologous cells in invariably associated with increased neopterin production. It thus appeared that neopterin might represent a potential marker for biochemical monitoring of diseases caused by or associated with T-lymphocyte activation. Our clinical experience with neopterin determination in allograft rejection and in infectious or autoimmune states strongly support this view. We conclude that evaluation of neopterin represents a useful and simple tool for the biochemical monitoring of immunological and/or malignant states.Activated T-lymphocytes represent crucial effector cells. They are pathogenetically involved into various disease states such as allograft rejection or viral infection. So far their assessment is laborious and rarely possible in clinical routine. In this review article we present the compount neopterin as a new biochemical marker for the in vivo and in vitro detection of activated T-lymphocytes. Our main finding was that in vitro as well as in vivo stimulation of T-lymphocytes with foreign and chemically or virally modified autologous cells in invariably associated with increased neopterin production. It thus appeared that neopterin might represent a potential marker for biochemical monitoring of diseases caused by or associated with T-lymphocyte activation. Our clinical experience with neopterin determination in allograft rejection and in infectious or autoimmune states strongly support this view. We conclude that evaluation of neopterin represents a useful and simple tool for the biochemical monitoring of immunological and/or malignant states.

Interferons (IFNs) and tumor necrosis factors (TNFs) in T cell-mediated immune responses against alloantigens. I. influence on the activation of resting and antigenprimed T cells

The aim of this study was to investigate the influence that endogenous IFNs released in response to antigenic or viral stimuli has on recognition of alloantigens in MLC. Results indicated that both the magnitude and the kinetics of response can be modified by IFNs. Neutralizing antibodies with specificity for IFN-gamma inhibit early and enhance late proliferative responses in MLC. Addition of physiological concentrations of IFN-gamma enhanced both early and peak proliferation, whereas IFN-alpha markedly inhibited alloantigen-induced lymphocyte proliferation. Further experiments revealed that IFN effects in MLC are not caused by direct interaction with responder cells: pretreatment with IFNs neither failed to alter their subsequent proliferative reactivity, nor did it influence production of IL 2 in MLC. IFN-gamma mainly affected MLC responses by direct interaction with stimulator cells. These influences on hemopoietic and non-hemopoietic stimulator cells were complex and could not simply be explained on the basis of an altered expression of class II MHC antigens. When induced by IFN-gamma to maximally express class II antigens, pbmnc, LCL or homogeneous populations of macrophages showed a marked deficiency to induce primary or secondary proliferative T cell responses. Resting unsensitized or sensitized T cells were not stimulated by class II MHC antigens constitutively expressed or induced by IFN-gamma on cell types other than dendritic cells or LCL. Class II antigens on the former cells were, however, readily recognized by T helper blasts, and this process involved the T4 epitope of the T cell receptor. IFN-gamma treatment also influenced the intrinsic suppressive capacity of macrophages or keratinocytes without involving prostaglandin synthesis or inducing expression of IL 2 receptors on non T cells.

Die Differenzierung menschlicher Blutlymphocyten mit serologischen und autoradiographischen Methoden

SummaryUsing125I labelled monospecific antisera against human,μ-,γ-,α-,κ- andλ-chains we investigated the percntage of surface Ig-bearing lymphocytes in patients suffering from primary or acquired hypogammaglobulinaemias. Two patients with sex-linked hypogammalobulinaemia, 2 with IgA defects, 4 with transitory hypogammaglobulinaemia and 13 patients with secondary hypogammaglobulinaemia (7 with multiple myeloma, 2 with M. Waldenström, 4 with chronic lymphocytic leukaemia) were studied.In healthy adult controlsκ-chains were found in the average on 18.5%.λ-chains on 2.8%,μ-chains on 12.1%,γ-chains on 10.5% andα-chains on 3.9% of blood lymphocytes. Comparable results were obtained in children, in patients suffering from transitory hypogammaglobulinaemia and in those with IgA-defects. In contrast, lymphocytes bearing Ig-determinants were almost absent in both cases with sex-linked hypogammaglobulinaemia (normal values were obtained in both parents and the siblings investigated).In patients with multiple myeloma, despite a low level of at least one immunoglobulin in the serum, normal or only slightly reduced numbers of lymphocytes with Ig-determinants were observed. In patients with M. Waldenström IgM-bearing lymphocytes were markedly increased, but the other Ig classes were represented in normal numbers despite reduced serum levels. In patients with chronic lymphocytic leukaemia the disagreement between very high numbers of lymphocytes bearing at least L-chain determinants on their surfaces and the Ig serum levels was striking. The results are discussed in regard to a maturation defect of B-lymphocytes in hypogammaglobulinaemias.ZusammenfassungUnter Verwendung125I markierter monospezifischer Antiseren gegen menschlicheμ-,γ-,α-,κ-undλ-Ketten bestimmten wir den Prozentsatz Ig-tragender Blutlymphocyten bei Patienten mit primären und symptomatischen Hypogammaglobulinämien. Zwei Patienten mit geschlechtsgebundenen Hypogammaglobulinämien, 2 mit IgA-Defekten, 4 mit transitorischen Hypogammaglobulinämien und 13 mit symptomatischer. Hypogammaglobulinämien (7 multiple Myelome, 2 M. Waldenström und 4 chronische Lymphadenosen) wurden untersucht.Bei erwachsenen Kontrollen fanden wir im Mittelκ-Ketten an 18,5%,λ-Ketten an 2,8%,μ-Ketten an 12,1%,γ-Ketten an 10,5% undα-Ketten an 3,9% der Blutlymphocyten. Prinzipiell ähnliche Ergebnisse wurden bei gesunden Kindern, bei Patienten mit transitorischen Hypogammaglobulinämien und solchen mit IgA-Defekten erhalten. Im Gegensatz dazu fehlten Ig-tragende Lymphocyten in beiden Fällen mit geschlechtsgebundener Hypogammaglobulinämie weitgehend. Normale Werte wurden bei ihren Eltern und bei den untersuchten Geschwistern erhalten.Trotz einer deutlichen Verminderung zumindest eines der Serum-Ig fanden sich bei Patienten mit multiplen Myelomen normale oder lediglich leicht herabgesetzte Zahlen Ig-tragender Lymphocyten. Bei Kranken mit M. Waldenström waren IgM-tragende Lymphocyten stark vermehrt. Trotz verminderter Serum-Ig Spiegel waren die anderen Ig-Klassen an der Zelloberfläche in etwa normaler Häufigkeit nachzuweisen. Diese Diskrepanz war bei Patienten mit chronischer Lymphadenose besonders ausgeprägt, bei denen eine extreme Vermehrung Ig-tragender Lymphocyten mit normalen oder verminderten Serum-Ig-Spiegeln einherging. Die Ergebnisse werden im Hinblick auf eine Störung der B-Zellreifung diskutiert.

Quantitation of gene expression by means of HPLC analysis of RT-PCR products

A method for the quantitative analysis of specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent detection of products by means of ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) on alkylated micropellicular polystyrene-divinylbenzene particles has been developed. For RT-PCR, we used the EZrTth RNA PCR kit (Perkin Elmer). This method allows reverse transcription and amplification of specific target mRNA in a single reaction tube. RT-PCR products were analyzed qualitatively and quantitatively by means of IP-RP-HPLC and UV detection at 254 nm. The enzymatic amplification combined with chromatographic separation and UV detection permitted high precision (and intra assay CV < 10%), with good practicability (two pipetting steps only). A total RNA preparation of a tissue that highly expressed the sequence of interest and that was stored in multiple aliquots, was diluted to give a standard curve. This external standard curve was used to define when samples have to be diluted, i.e., when the signal is in the plateau phase of amplification. The validity of the method is demonstrated with the example of human retinoic acid receptor mRNA.

DNA-synthesizing T and non-T cells in chronic lymphocytic leukemia

ZusammenfassungBei 21 Patienten mit chronischer lymphatischer LeukÄmie (CLL) und 8 Normalpersonen wurde die Anzahl der DNA-synthetisierenden peripheren Blutlymphozyten autoradiografisch untersucht. Die Lymphozyten wurden dabei mit Hilfe des EN-Rosettentests in T-Zellen und Nicht-T-Zellen differenziert. Es zeigt sich eine normale T-Zellproliferation und eine erhöhte Nicht-T-Zellproliferation im Stadium O-I. In den fortgeschritteneren Krankheitsstadien erfÄhrt die Proliferation für T- und Nicht-T-Lymphozyten eine signifikante Steigerung. Die Proliferation der T-Zellen ist im Stadium IV auf das ca. 20fache, die der Nicht-T-Zellen auf das ca. 50fache gesteigert. Auf die Möglichkeit zu prognostischen Aussagen wird hingewiesen.SummaryIn 21 patients with chronic lymphocytic leukemia (CLL) and in 8 hematologically normal persons the number of DNA-synthesizing peripheral blood lymphocytes was investigated by autoradiographic techniques. The lymphocytes were differentiated by En-rosette tests into T and non-T lymphoid cells. The results show a normal number of proliferating T lymphoid cells and an increased number of proliferating non-T lymphoid cells in clinical stages O-I. Stages III–IV demonstrate a significant increase of the proliferation rate of both T and non-T lymphoid cells. The possible pathogenetic factors and the prognostic value of these results are discussed.