Differences between the in vitro combinations of secretory component (SC) and immunoglobulin polymers (Ig) by enumeration of SC epitopes

The enumeration of the SC epitopes has been established on 125I-labelled free and combined SC, by binding to anti-SC coated beads, then by addition of 3H-labelled anti-SC Fab' fragments of various specificities. The number of moles of Fab' fragments found on the beads increases in relation to the introduced amount. The extrapolation to an infinite concentration of added Fab' fragments gives the maximal theoretical accessible number of SC epitopes. The number of hidden epitopes (cryptotopes) is established by subtracting the total number found on sIgA, IgA-SC and IgM-SC from those found for free SC. These values are confirmed with Fab' fragments specific for the inaccessible determinant of SC. There are 4 cryptotopes in the case of sIgA, 3 for IgA1-SC, 2 for dimer IgA-SC and only 1 for IgM-SC (polyclonal or monoclonal). Thus the in vitro combinations of SC with polyclonal IgA dimers are different from the in vitro combinations with polyclonal or monoclonal IgM. The structural implications of these differences are discussed.     

Anti-human interleukin 2 receptor monoclonal antibody isotypic switching: Chimeric rat-human antibodies

Univalent and bivalent (2Fab'-Fc) chimeric rat-human antibodies were constructed by chemical coupling (thioether bonds) of Fab' fragments from the 33B31 rat anti-human interleukin 2 receptor ( chain) monoclonal antibody to human IgG or Fc fragments. The purity of the chimeric antibodies obtained after purification was assessed by the sodium dodecyl sulfide-polyacrylamide gel electrophoresis method. The affinity of chimeric antibodies for the human interleukin 2 receptor was determined. The affinity of the 2Fab'-Fc was better (Kd = 0.87 nM) than that of the Fab'-IgG (Kd = 2.04 nM) or the Fab'-Fc chimera (Kd = 3.93 nM). Binding studies on a T-cell clone showed that the percentage of positive cells recognized by chimeric antibodies was comparable to that obtained with unmodified 33B3.1 IgG2a or its corresponding Fab' fragment. In addition, the inhibition of interleukin 2-induced cell proliferation and allogeneic proliferative response by chimeric antibodies was of the same magnitude as that obtained with the rat IgG2a anti-interleukin 2 receptor monoclonal antibody and Fab' fragments. This study shows the possibility of changing the isotype of monoclonal antibodies without important modification to their binding activity. These reagents may offer an alternative to unmodified monoclonal antibodies for therapeutic application.     

Specific binding characteristics of high affinity monoclonal antidigitoxin antibodies

The specificity of various monoclonal antidigitoxin antibodies was characterized using 6 cardiac glycoside analogs. Spleen cells from BALB/c mice, immunized with BSA- or KLH-digitoxin conjugates, were fused with NS1 myeloma cells, and antibody-producing hybrids were identified by radioimmunoassay. Twenty-one monoclonal antidigitoxin-specific antibodies were obtained, 10 of which were cloned and characterized for affinity and specificity. All the antibodies had a high affinity constant, ranging from 8.10(8) to 2.5.10(10) 1/M. On the basis of their binding specificities, the antibodies could be classified into 3 groups: the first contained 7 antibodies exhibiting high cross reactivity (42-100%) with digitoxigenin, whereas the second and third groups did not recognize this analog (cross-reactivity of 1%). In the former group, the absence of the sugar moiety only slightly affected the binding reaction, although for the two other groups, this structure did appear to be involved in antibody recognition. Changes in the functional groups of the hapten molecule led to considerable changes in the antibody-antigen reaction. For all the antibodies except one, saturation of the lactone ring considerably affected binding. These results demonstrated that monoclonal antibodies of different specificities with respect to both the steroid backbone and the sugar moiety of digitoxin can be induced using a digitoxin-protein conjugate.     

A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants

Endogenous immunoglobulin in tissue sections pose a problem in immuno-histochemical techniques employing homologous antibody as primary reagents and enzyme-labelled anti-immunoglobulin for the development. A method for the blocking of endogenous immunoglobulin in human tissue sections by incubation with monomeric pepsin fragments (Fab') of rabbit anti-human immunoglobulin before applying monoclonal antibody was evaluated for the screening of human monoclonal antibody. It was initially demonstrated that Fab' rabbit anti-human IgM and anti-IgG could block endogenous immunoglobulin in human IgM and IgG producing tumors thereby abolishing the binding of subsequently applied peroxidase-labelled anti-IgM or anti-IgG. Frozen sections of human colo-rectal adenocarcinomas show a variable background staining caused by the endogenous immunoglobulin. The background completely disappeared in the IgM system by preincubation with Fab' anti-IgM while the background was clearly reduced but not abolished in the IgG system. A human hybridoma supernatant containing IgM reactive with colo-rectal adenocarcinoma could easily be screened on frozen sections using this method. This approach should be generally useful for the screening of human antibody on human tissue sections.     

Evidence for sex steroid receptors in feline brainstem.

Specific binding sites for estrogens, androgens, and progestins were found in cytosol from perfused cat brainstem with the use of a steroid binding assay. These sites resemble 'classical' steroid receptors in ligand specificity as well as in sedimentation properties (sedimentation constant 8-10S on sucrose density gradient). The presence of estrogen receptor in brainstem extracts was also confirmed by an enzyme immunoassay employing monoclonal antibodies directed against human estrogen receptor. This finding may be relevant to known influences of sex steroids on respiration.     

Design of targeted lipid nanocapsules by conjugation of whole antibodies and antibody Fab' fragments

Immunonanocapsules were synthesized by conjugation to lipid nanocapsules (LNC) of whole OX26 monoclonal antibodies (OX26 MAb) directed against the transferrin receptor (TfR). The TfR is overexpressed on the cerebral endothelium and mediates the transcytosis mechanism. Fab' fragments, known for their reduced interaction with the reticuloendothelial system, were also conjugated to LNC. This coupling was facilitated by the incorporation of lipid PEG(2000) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) into LNC shells by a post-insertion procedure, developed initially for liposome pegylation. An interfacial model using the dynamic rising drop technique helped determine the parameters influencing the DSPE-PEG(2000)-maleimide insertion and the quality of the anchorage. Heat was essential to promote both an important and stable adsorption of DSPE-PEG(2000)-maleimide onto LNC. OX26 MAb were thiolated to react with maleimide functions whereas thiol residues on Fab' fragments were used directly. The number of ligands per nanocapsule was adjusted according to their initial quantity in the coupling reaction mixture, with densities from 16 to183 whole antibodies and between 42 and 173 Fab' fragments per LNC. The specific association of immunonanocapsules to cells overexpressing TfR was thus demonstrated, suggesting their ability to deliver drugs to the brain.     

The use of Fab' fragments in a screening method for the detection of human anti-tumor monoclonal antibodies

The use of immunoperoxidase staining methods to detect human monoclonal antibodies that react with frozen tissue sections of human tumors is limited due to endogenous immunoglobulin present in the sections. The endogenous immunoglobulin produces a significant background staining which makes the detection of a specific reaction difficult and unreliable. A method of incubating the sections with papain fragments (Fab') of goat anti-human immunoglobulin was evaluated. It was demonstrated that Fab' fragments of goat anti-human polyvalent immunoglobulin can block endogenous immunoglobulin background staining after prolonged washing of tissue sections (20h) and thus facilitating the screening of monoclonal antibodies against frozen sections of tumor tissue.     

Demonstration of the existence of two distinct Fc receptors for IgG isotypes on guinea-pig macrophages by the use of monoclonal antibodies

As reported in a previous paper by the authors (J. Biochem. 99, 227-235, 1986), the Fab' of a monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea-pig peritoneal macrophages with a myeloma cells line, completely inhibits the binding of ovalbumin (OA)-complexed IgG1 antibody to macrophages, but only partially the binding of OA-complexed IgG2 antibody. Based on these results, it was proposed that the cells have at least two types of Fc receptor (FcR) for homologous IgG isotypes: FcR2 for IgG2 and FcR1.2 for both IgG2 and IgG1, and also that VIA2 IgG1 is anti-FcR1.2 antibody. Thereafter, complete inhibition of the binding of OA-complexed IgG2 antibody to macrophages occurred when the Fab' of another monoclonal antibody, VIIA1 IgG1 was added to the Fab' of VIA2 IgG1, whereas the former did not affect the binding of OA-complexed IgG1 antibody. This effect of the Fab' of VIIA1 IgG1 indicates that VIIA1 IgG1 is a monoclonal antibody capable of selectively blocking the binding of OA-complexed IgG2 antibody to FcR2. When the antigen of VIIA1 IgG1 was isolated by affinity chromatography on the F(ab')2 of the antibody coupled to Sepharose, it gave a single band with a mol. wt of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It moved slightly faster than the FcR1.2 with a mol. wt of 55,000, which was isolated by the use of VIA2 IgG1, and corresponded to the fast moving portion of the broad band of FcRs isolated with OA-complexed IgG2 antibody. These results strongly suggest that VIIA1 IgG1 is a monoclonal antibody to FcR2.     

Use of inhaled steroids by pregnant asthmatic women does not reduce intrauterine growth

BACKGROUND: Inhaled steroids are recommended for the treatment of persistent asthma during pregnancy, but their potential effects on intrauterine growth have been inadequately evaluated. OBJECTIVE: The purpose of this study was to evaluate the association between maternal use of specific inhaled steroids and inhaled steroid dose during pregnancy and the incidence of infants who are small for gestational age (SGA) and mean birth weight. METHODS: Pregnant asthmatic women being treated with inhaled steroids were enrolled in the study before delivery by their managing allergists. Information regarding the specific inhaled steroid and daily dose used, requirement for oral steroids, occurrence of acute asthmatic episodes, maternal race, birth weight, gestational age, and congenital malformations was obtained for each patient. SGA was defined through use of a published normative sample of American births. RESULTS: A total of 474 women were enrolled in the study; of the 451 enrolled participants whose pregnancy ended in a singleton live birth, 396 (88%) completed the study. The incidence of infants with low birth weight, preterm births, and congenital malformations in this cohort was not greater than expected in the general population. The incidence of SGA was 7.1% (95% CI, 5.0% to 10.1%). No significant relationships between specific inhaled steroid or dose of inhaled steroid used and either SGA or mean birth weight were observed. CONCLUSION: These data suggest that the use of inhaled steroids by pregnant asthmatic women does not reduce intrauterine growth and supports the recommendation that inhaled steroids should be used in the management of persistent asthma during pregnancy.     

Bispecific F (ab')2 monomer prepared with anti-CD3 and anti-tumor monoclonal antibodies is most potent in induction of cytolysis of human T cells

Induction of cytolytic activity of peripheral blood mononuclear cells towards target cells was studied by preparing bispecific F(ab')2 which was composed of two Fab' fragments, one of which was derived from anti-CD3 monoclonal antibody and the other from anti-tumor monoclonal antibody. After reduction of the interchange disulfide bonds of these fragments by dithiothreitol, a thiol-disulfide interchain reagent, 5,5'-dithiobis-2-nitrobenzoic acid, was added to convert the free SH groups of one of the Fab' fragments to mixed disulfide derivatives. These were then coupled with the other Fab' fragment bearing free SH groups, producing a bispecific hybrid F(ab')2 monomer of high yield. The bispecific F(ab')2 monomers were able to render nonactivated peripheral blood mononuclear cells cytotoxic against natural killer-resistant tumor cell lines at doses as low as 1 microgram/ml. The polymeric forms of the F(ab')2 fragments prepared by use of cross-linking reagents such as N-succinimidyl-3-(2-pyridyldithiol)-propionate (SPDP) or S-acetylmercaptosuccinic acid anhydride (SAMSA) were less efficient for induction of cytolytic activity than the monomeric one. It may be feasible to use bispecific F(ab')2 monomers in cancer immunotherapy because of the ease of preparation, as well as the efficiency in inducing cytolytic activity and their high tissue permeability due to their small size (100-110 kDa). In addition, redirected cytolytic activity towards peripheral blood mononuclear cells by reticuloendothelial system cells, resulting from linking these two types of cells through Fc-Fc receptor interactions, does not occur.     

Metabolic clearance rates of androgens and oestrogens in ageing women

Using the constant infusion technique we have measured the metabolic clearance rates (MCR) for Δ⁴-androstenedione (A), testosterone (T), oestrone (E₁) and oestradiol (E₂) in a large group of postmenopausal women. Their mean ± SE age was 64.5 ± 1.6 yr, their ages ranged from 46–90 yr. When the MCRs for each steroid were related to age by linear regression analysis no significant correlation was found for any of the steroids. Similarly, when the women were grouped according to their age by decade, the mean MCR for each steroid showed no trend with increasing age.

There was no difference in the MCRs for A, T and E₁ of the post-menopausal women and a large group of pre-menopausal women. However, there was a significant decrease in the mean MCR for E₂ between the two groups which is probably related to the marked decrease in circulating E₂ in postmenopausal women. We conclude that for these steroids age, per se, does not appear to be a major determinant of the MCR.     

Studies on the Immunosuppressive Role of Steroid Hormones During Pregnancy

It has been recognised that steroids can exert a profound influence over immunological reactivity. The present study analyzes the role of steroid hormones -estrogen, progesterone and cortisol - and their involvement in immunoregulation during pregnancy. As is known, the endogenous levels of all the three hormones increase during pregnancy. When the steroid levels in pregnancy serum were correlated with the lymphocyte response to mitogen, no correlation was observed. The suppressive effect of pregnancy serum was found to have no correlation with its steroid content. In general, steroids did not seem to affect the maternal immune system as evidenced by the present study.     

Additive effect of steroids and cholesterol on the liposomal transfection of the breast cancer cell line T-47D

The efficiency of gene transfer is many times higher in viral than in liposomal transfection. One reason for this is an insufficient intracellular transport of the exogenous DNA into the nucleus in lipofection. Using liposomal transfection techniques for gene therapy is safer than viral approaches, so it would be of great importance to find solutions for their enhancement. We found a 4.51-fold increase in liposomal transfection of T-47D breast cancer cells by the addition of progesterone and a 2.81-fold increase by the addition of cholesterol. The transfection efficiency was measured as the activity of the delivered reporter gene luciferase. The addition of progesterone and cholesterol in combination led to a further enhancement of the transfection efficiency up to 13.72-fold. This additive effect could also be seen when we combined cholesterol with other steroids, but not by the combination of different steroids. All of these steroids alone had also the potential to increase liposomal transfection. Therefore we suggest that steroids and cholesterol enhance liposomal transfection by different mechanisms. Both substances have been shown to shift the exogenous DNA from the cytosol to the nucleus. Steroids normally act through intracellular steroid receptors, which migrate into the nucleus upon activation. The transfected DNA might be co-transported to the nucleus together with the migration of the activated steroid receptors. Even if cholesterol causes also an intracellular shift of DNA to the nucleus, its impact on the fluidity of cellular membranes or on the stability of lipoplexes in serum containing media could be mainly responsible for its effect. If the steroid enhanced liposomal transfection is dependent on the presence of steroid receptors, a specifically-enhanced gene delivery for a subgroup of gynecological tumours expressing high levels of steroid receptors could be possible.     

Steroids in kidney transplant patients

Any evaluation of steroids in kidney transplantation is hampered by individual variability in metabolism, the lack of clinically available steroid blood levels, and overall little attention to steroid exposure. Many feel that steroids were an essential part of chronic immunosuppression in past decades but may no longer be necessary in low-risk populations when our newer and more potent drugs are used. Potential differences in long-term outcome will be unapparent in short-term antibody induction studies in low-risk patients, particularly with low on steroid doses, as may have happened in the recent, well-done Astellas trial. In many studies, the evidence for the superiority of mycophenolate (MMF) and tacrolimus (TAC) was not as strong as the evidence for the benefit of steroids in the Canadian cyclosporine study. As the practice of steroid withdrawal has increased, we have not seen the improvement in long-term graft survival that many expected with our newer agents. Steroids have immunosuppressive effects even in doses that are low by historic standards, and side effects may not justify their abandonment.     

Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors.

Fc gamma RII (CDw32) on monocytes is capable of triggering both phagocytosis and lysis of chick red blood cells (CRBC) coated with antibody of the appropriate isotype. In this report we describe the production and characterization of a mouse monoclonal IgG1 antibody specific for Fc gamma RII and compare its activity in binding studies, tissue distribution and redirected cellular cytotoxicity (RCC), with the previously identified anti-Fc gamma RII antibodies KB61 and IV.3. Immunohistochemical and flow cytometry analyses demonstrated that AT10 binds very strongly to Fc gamma RII on normal monocytes, but only weakly to that expressed on lymphocytes. This pattern does not correspond to the staining seen with either KB61 or IV.3, and appears to give an intermediate profile. The binding constant (Ka) for the Fab' fragment of AT10 was calculated at 5.3 x 10(8) M-1, four times higher than that for KB61 (1.4 x 10(8) M-1). Bispecific F(ab')2 antibodies were constructed from Fab' fragments of AT10 or KB61 thioether-linked to Fab' from an anti-CRBC monoclonal antibody. These bispecific derivatives directed monocyte cytotoxicity against CRBC as efficiently as either a monoclonal or polyclonal anti-chick erythrocyte antibody. The bispecific F(ab')2 antibodies had a distinct advantage over the conventional reagents, in that they were not blocked in the presence of human Fc gamma at 3.5 mg/ml (a concentration comparable with that provided by IgG in serum). Therefore, bispecific derivatives constructed with the high affinity anti-Fc gamma RII antibody, AT10, may be used as therapeutic reagents for targeting tumour cell lysis in vivo.     

Motivations for Anabolic Steroid use Among Bodybuilders

Steroid use is increasing, in parallel with rising concerns about body image. This study aimed to uncover bodybuilders' motivations for using steroids using 135 questionnaires completed by readers of two bodybuilding magazines. The analyses reveal a polarization of beliefs about steroids between users and non-users. Steroid users were less likely to be concerned about the physical side effects, and many believed that steroids are not harmful in moderation, and that only 'ignorant people' criticize steroid use. Their main motivations for using steroids were: wanting to excel at competitive bodybuilding; wanting to be more muscular; and feelings of enhanced confidence. The fact that steroid users in the sample were 'stacking' dangerously high levels of steroids (up to 15 steroids at a time) reveals the need for a detailed understanding of the motivations for steroid use in order to inform the development of effective harm minimization messages.     

Large scale preparation of immunotoxins constructed with the Fab' fragment of IgG1 murine monoclonal antibodies and chemically deglycosylated ricin A chain

In this report, we describe a method for the preparation of large amounts (grams) of immunotoxins (ITs) consisting of Fab' fragments of murine IgG1 monoclonal antibodies conjugated to chemically deglycosylated ricin A chain (dgA). The preparation of Fab' and dgA chain and the purification of the Fab'-dgA IT were accomplished by gel filtrations and affinity chromatography utilizing six Pharmacia Bioprocess columns (Sephadex G-25M, Sephacryl S-200HR and Blue Sepharose CL-4B) integrated into a semi-automatic chromatography system controlled by a Pharmacia C3-process controller. The final Fab'-dgA ITs were highly purified, potent, sterile and low in endotoxin concentration.     

The effects of anabolic steroids and endurance training on systolic time intervals in the dog

The effects of administration of anabolic steroids (SG, n = 6), endurance training (EG, n = 7) and a combination of steroid administration (Methandienone R) and training (ESG, n = 7) on systolic time intervals (STI) in the dog heart were compared with control dogs (CG, n = 7) by a catheterization technique. Isoproterenol infusion was used as a positive inotropic load. Physical performance was checked with submaximal exercise test, a 30 beats min-1 decrease in heart rate response was considered a sufficient training effect. During isoproterenol infusion, exercise was found to enhance the pump performance of left ventricle and increased stroke volume was related to shortened pre-injection period (PEP) (P less than 0.05), whereas steroid administration failed to induce any significant differences in PEP or PEP/LVET (LVET = left ventricular ejection time) when compared with CG. However, when steroid administration was combined with endurance training, both PEP and PEP/LVET were lengthened significantly in comparison with EG (P less than 0.05 and P less than 0.01, respectively). In conclusion, it seems that the anabolic steroids affected the STIs only, when their administration was combined with endurance training.     

Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay

A simple method to increase the overall sensitivity of immunoblot assays is described. The method is based on fixation of the primary monoclonal antibody-antigen complex on nitrocellulose paper by a divalent crosslinker, glutaraldehyde. The fixation of the antibody-antigen complex significantly increases the sensitivity of the assay. Unexpectedly, the glutaraldehyde treatment also improves the signal/noise ratio by decreasing non-specific binding of secondary antibodies and/or tertiary reagents. Thus, a significant overall improvement of immunoblot assays is achieved by this method.