Biotherapy (Dordrecht, Netherlands)最新文献

Type I IFNs constitute a family of proteins exhibiting high homology in primary, secondary, and tertiary structures. They interact with the same receptor and transmit signals to cellular nucleus through a similar mechanism, eliciting roughly homogeneous biological activity. Nevertheless, the members of that family, IFN alpha species, IFN beta and IFN omega, due to local differences in the structure sometime show distinct properties. From the reported data it results that even minute changes or differences in the primary sequences could be responsible for a significant variety of biological actions, thus inducing to the hypothesis that Type I IFNs, rather than to be the result of a redundant replication during the evolution play definite roles in the defense of living organisms to foreign agents.

Several assays have been used for detection of antibodies against cytokines. The choice of assay is greatly dependent on the intended goal, e.g. detection of naturally occurring antibodies or therapy induced antibodies. The different assays can be grouped in 2 categories. The interference or indirect assays are based on the detection of the test sample interference with the biological activity, with detection of the cytokine in EIA or with binding to cellular receptors. In direct assays cytokine antibodies are detected by binding to solid phase fixed cytokines, followed by incubation with a secondary enzyme-labelled anti-human Ig antibody or by binding to 125I-labelled cytokines in RIA.

Low amounts of high-affinity autoantibodies to various cytokines have been detected in sera from healthy donors. Their levels, although highly variable, are increased in the circulation of patients subjected to cytokine therapy or suffering from a variety of immunoinflammatory diseases. It has been suggested that these autoantibodies play a regulatory role in the intensity and duration of an immune response. The antibodies may prevent the binding of a cytokine to its specific cell surface receptor thereby neutralizing its biological activity in vivo. They may also act as carrier proteins preventing the rapid elimination of a cytokine from the circulation and thus increase its bioactivity. Additionally or alternatively, autoantibodies may modulate cytokine-induced intracellular signal transduction pathways or trigger complement-mediated cytotoxicity towards cells carrying membrane-bound cytokines. The autoantibodies may exert their regulatory role in compliance with the other factors that control cytokine activity, including soluble cytokine receptors, cell surface decoy receptors, and receptor antagonists. Although not favored by many investigators, a less sophisticated role for naturally occurring anti-cytokine autoantibodies should be considered as well. Recent evidence has shown that autoantibodies are generated at a high frequency as part of a response to foreign antigens. These antibodies are produced by B cells arising from the process of somatic mutation. Thus anti-cytokine autoantibodies may be the result of a "leaky" B cell response triggered by immunoinflammatory processes. High-titered autoantibodies induced by cytokine therapy are of clinical concern since their occurrence is often associated with the loss of response to treatment. Moreover, they may also neutralize endogenously produced cytokines with possible pathological consequences. In this paper we have reviewed the available information on the biological and clinical significance of both naturally occurring and therapeutically induced anti-cytokine autoantibodies in animals and man with the emphasis on antibodies directed to interferons.

It is well known that natural and recombinant proteins can cause antibody formation in the host. We have studied the incidence of binding and neutralizing antibodies in carcinoid patients (n = 327). All together 204 patients received interferon-alpha 2b (Intron-A), median does 15 MU range 9-35 MU/week subcutaneously and 51% of the patients developed binding antibodies by immunoassay and 17% showed positive neutralization assay but high titer antibodies (> 800 NU/ml) were only found in 4% of the patients. The median time until the development of binding antibodies was 26 months and neutralizing antibodies 25 months. Twenty-nine patients received interferon-alpha 2a (Roferon), median does 18 MU/week subcutaneously and 45% developed binding antibodies, 38% had positive neutralization assay and 28% presented high titer antibodies. Binding and neutralizing antibodies occurred at the same time after median six months of treatment. Patients treated with Wellferon (n = 45) and leukocyte interferon (n = 48), median dose of 15 MU/week subcutaneously did not develop any neutralizing antibodies. The majority of the interferon-alpha 2 antibodies were of the IgG isotype. The clinical relevance of the development of high titer neutralizing antibodies was evaluated in the patients. All together 17 patients developed high titer neutralizing antibodies and of these 12 patients showed loss of antitumor response measured as increased level of tumor markers and of tumor progression. In nine of these patients a switch to human leukocyte interferon reinstituted an antitumor response. Neutralizing antibodies against recombinant interferon-alpha 2a and 2b might occur in patients with carcinoid tumors. The incidence of high titer neutralizing antibodies is significantly higher in patients treated with interferon-alpha 2a compared to interferon-alpha 2b. A significant number of patients lost the antitumor effect during development of neutralizing antibodies at high titers, but human leukocyte interferon can be used as rescue treatment.

Neutrophils were intra-cellularly "loaded" with the chemotherapeutic agent, doxorubicin applying a variety of incubation conditions in order to identify parameters which maximize chemotherapeutic incorporation, while simultaneously preserving optimal viability and chemotactic responsiveness. Doxorubicin "loaded" neutrophils (DLN) were produced in triplicate at different combinations of incubation conditions such as temperature (4 degrees C, 37 degrees C); duration (0, 1, 2 hours); and doxorubicin concentration (20, 40, 60 micrograms/ml). Chemotactic responsiveness of rinsed DLN preparations was subsequently assessed against the neutrophil peptide chemotactic agent, formyl methionyl leucyl phenylalanine (fMLP, 10(-6) M) utilizing a modified 96-well Boyden chemotactic chamber apparatus. Viable, fMLP-responsive DLN preparations were subsequently detected with MTT vitality staining reagent. At sub-physiological incubation temperatures (4 degrees C), profound declines in the viability of DLN preparations were detected when simultaneously incubated with doxorubicin formulated at concentrations greater than 10 micrograms/ml. In contrast, DLN preparations incubated at 37 degrees C displayed diminished viability only when incubated with doxorubicin formulated at a concentration of 60 micrograms/ml. Viable DLN populations were subsequently evaluated to determine their ability to exert in vitro cytotoxic activity against monolayer populations of human mammary carcinoma (HTB-19) propagated in a tissue culture environment. The lethal effect which DLN preparations inflicted towards HTB-19 populations was substantially greater than was observed with an equivalent population of untreated neutrophils. Maximal in vitro cytotoxic activity was detected with DLN preparations produced at 37 degrees C in the presence of doxorubicin formulated at a concentration of 40 micrograms/ml. In contrast, DLN preparations produced at an incubation temperature of 37 degrees C, and a doxorubicin concentration of 20 micrograms/ml displayed relatively lower levels of in vitro cytotoxic activity against HTB-19 monolayer populations. The degree of in vitro cytotoxic activity exerted against HTB-19 monolayer populations by DLN preparations was directly influenced by the duration of the challenge period. Maximal in vitro cytotoxic activity was observed when HTB-19 monolayer populations were challenged with DLN preparations for a period of 96-hours duration at 37 degrees C. Challenge periods of 48-hours duration produced levels of in vitro cytotoxic activity which were substantially lower than those observed for challenge periods of 96-hours duration. Optimal in vitro cytotoxic activity was recognized when DLN preparations were allowed to establish direct contact with HTB-19 monolayer populations at an estimated DLN:HTB-19 cellular ratio of approximately 5:1 (37 degrees C, CO2, 6%). Significantly less in vitro cytotoxic activity was recognized when DLN preparations were only permitt

The occurrence of antibody formation in patients administered biologically active human proteins as biotherapy for different diseases emphasizes the importance of establishing a common method of reporting neutralizing antibody levels for such cytokines. For quantitative neutralization bioassays, the preferred expression of the neutralizing potency of an antiserum is a titer, that is, the dilution of serum that reduces 10 Laboratory Units (LU)/ml of the cytokine to 1 Laboratory Unit/ml, the endpoint of most bioassays. This 10-to-1 LU/ml expression, which has been recommended by the World Health Organization for recording the results of interferon neutralization by the constant interferon method (with varying dilutions of serum) can also be used with the constant antibody method (with varying concentrations of interferon). For various reasons, interferon doses in International Units (IU)/ml should not be used for the neutralization test. Should the interferon concentration vary, intentionally or otherwise, from the intended dose of 10 LU/ml, a simple calculation allows expression of the neutralizing potency as the recommended reduction of 10-to-1 LU/ml as follows: the titer to be reported is the reciprocal of the antibody dilution (achieving the endpoint), multiplied by the interferon concentration (measured in that day's titration) minus one, divided by 9. This index of neutralization is the preferred method to represent the neutralizing potency of polyclonal and monoclonal antibodies and should make the results from different laboratories more readily interpretable and enable comparison.

In this paper, we examine the effects of SPG, which is a well known BRM, both in vivo and in vitro on the neutrophilic ROS production and the serum opsonic activity by the chemiluminescence technique using luminol as a probe. To investigate the in vivo effects, SPG was administered to 12 healthy male volunteers and two phases of enhancement of the neutrophilic ROS production and the serum opsonic activity was observed. In vitro, the addition of SPG showed a dose-dependent suppression. To investigate the mechanisms in these contradictory effects of SPG, supernatants of a lymphocytes culture medium in the presence of SPG with or without mitogen were added to the neutrophils. The addition of supernatants at a lower concentration of SPG (0.01 mg/ml) with mitogens showed significant preventive effects on the neutrophilic ROS production for the duration of incubation. This suggests that cytokines derived from lymphocytes may contribute to the in vivo effects of SPG. SPG can play an important role in the host's defense against microbe infections by enhancing it's effect on non-specific immunity when administered in vivo.

It has been reported that immunologic function is deteriorated in head and neck cancer patients by primary therapies such as surgery, irradiation and chemotherapy or tumor itself. As previously described by us, immunologic dysfunction in such patients may be recovered by treatment with BRMs. In the present study, we investigated the effects of BRMs on survival of patients who had primarily been treated in our clinic. Fifty-one patients (23 patients; Stage I or Stage II, 28 patients; Stage III or Stage IV) were treated with BRMs (BRM group), and 49 patients (22 patients; Stage I or Stage II, 27 patients; Stage III or Stage IV) were employed as controls (Control group). The results obtained were as follows: (1) In patients of all Stages, the survival period was significantly (p < 0.05) longer in BRM group than in Control group; (2) The survival periods of patients of Stage I or Stage II were not different between the groups; and (3) The survival period of BRM group was significantly (p < 0.05) longer than that of Control group in patients of Stage III or Stage IV. There were observed more patients in BRM group who survived for a prolonged period. These results suggest that BRMs may be useful for recovering immunologic function in head and neck cancer patients particularly of Stage III or Stage IV who usually receive multimodality therapy.

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.