Mechanisms for increasing the activity of polyreactive immunoglobulins in vivo

The mechanisms of increasing of polyreactive immunoglobulin (PRIG) activity in vivo are suggested. It was shown that at some conditions, which can be observed in organism in sites of inflammation, activity of serum PRIG could be enhance considerably. This enhancing might be induced either by reactive oxygen species, mainly by hydroxyl radicals, or by lipases. The increasing of PRIG activity could be not only a result of preexisting PRIG unblocking, but also a result of transformation specific antibodies into PRIG, caused by peroxide degradation or lipolysis of lipids, tightly but noncovalently linked to antibodies, either by hydroxyl radicals or lipase. It was also suggested that most (if not all) specific antibodies consist of not only polypeptide chains, but also some lipids. Deprived of these lipids antibodies loose their specificity and transform into polyreactive immunoglobulins.     

Infection, autoimmune diseases, atherosclerosis, infarct, stroke, cancerogenesis: what is in common

The problem of the transformation of specific antibodies into nonspecific polyreactive immunoglobulins (PRIG) was considered. On the basis of the obtained data the conclusion has been made that this process can occur not only in vitro, but also in vivo. The biological consequences of such a transformation of circulated specific antibodies into PRIG or increase of PRIG reactivity because of their unblocking are discussed. It was supposed that PRIG was able to play positive role in the defence of organisms against some infections. Meanwhile, disregulation of the PRIG production in vivo can, probably, lead to their participation in induction and/or in aggravation of some danger diseases, such as autoimmune diseases, atherosclerosis, infarct, stroke, and cancer. If so, farther investigation of PRIG involvement in these pathological processes could open new ways for their curing or even prophylaxis.     

Lipid-protein interactions and conformation of proteinaceous macromolecules

Analysis of the data as to the mechanism of specific antibody transformation into polyreactive immunoglobulins (PRIG) shows that for this transformation it is necessary and sufficiently to deprive antibodies of lipids, which are in norm tightly bound to the antibodies. Removal of these lipids by any methods (by treatment of antibodies with chaotropic ions, low/high pH, reactive oxygen species and lipases) leads to the loose by antibodies of their specificity and acquiring the ability to react with various non-related antigens, i.e. to their conversion into PRIG. Mathematical modeling of the PRIG--antigen interaction and values of thermodynamic characteristics of this process shows that antigen-binding domains of PRIG are in semi-melted state, thanks to what they can fit their structure to be complementary to structurally different antigens. Thus, we conclude that lipids bound to the so-called "hydrophobic pockets" of immunoglobulins (Ig) can stabilize the conformation of Ig and increase their rigidity, and removal of these lipids induce flexibility of Ig domains, responsible for interaction with antigens. It was presumed that lipids could exert the similar function of conformation stabilization not only in the case of antibodies, but also combining with some other proteins, for example, enzymes. Their removal could lead to the changing of protein conformation and loosing its biological activity. In this case the function of lipid removing and protein inactivation could exert cellular reactive oxygen species and cellular lipases and lipoxygenases.     

Polyreactive immunoglobulins recognize hydrophobic parts of proteins

It was shown that polyreactive immunoglobulins (PRIG), which are capable to interact non-specifically with various antigens, could differ from so called natural antibodies, which have also the capacity to react non-specifically with various antigens, including self-antigens. The main differences are that in contrast to the natural antibodies, which mainly interact, probably, with hydrophilic epitopes, PRIG recognise and bond preferentially to hydrophobic epitopes. We can consider this binding as a new type of interaction between immunoglobulins and antigens.     

Complement-binding and immuno-modulating properties of polyreactive immunoglobulins

New data concerning biological properties of polyreactive immunoglobulins (PRIG) were obtained as a result of treatment of mouse serum immunoglobulins by 4 M KSCN and are presented in the paper. In particular, the capacity of PRIG to bind C1q, the subunit of the first component of complement was studied. It was shown that PRIG's binding capacity to C1q is similar to that of intact immunoglobulins. Intravenous administration of PRIG into mice together with either sheep red blood cells or heat-inactivated staphylococcal bacteria did not affect the immune response to these antigens. Meanwhile, the same administration of PRIG together with the purified protein derivate of tuberculin resulted in 10-fold increase of mouse antibody response to PPD. These results demonstrate that PRIG can have some immuno-modulating properties concerning low-immunogenic antigens.     

Mechanisms of interaction of polyreactive immunoglobulins and protein antigens

New features of interaction between polyreactive immunoglobulins (PRIG) and protein antigens were considered. It was shown that unlike specific antibodies, recognizing mainly hydrophilic epitops of proteins and interacting against them with high affinity according to the mechanisms "lock-and-key" and/or "induced fit", PRIG recognized and nonspecifically bound to hydrophobic patches of protein antigens. On this reason it is possible to prevent or markedly diminish PRIG-antigen interaction using the reagents that have high affinity to hydrophobic regions of proteins and therefore are capable to block these regions. The obtained data are in a good agreement with the former data concerning the kinetic and thermodynamics characteristics of PRIG-antigen interaction described by us earlier.     

Polyreactive immunoglobulins and natural antibodies are different substances

It was shown that the polyreactive immunoglobulins of intact animal or human sera and the natural antibodies of these sera have different properties. Polyreactive immunoglobulins interact non-specifically with various antigens and this interaction is strongly dependent on an exposure of hydrophobic sites by antigens and, probably, by polyreactive immunoglobulins. Tween 20 and low temperature can substantially suppress this reaction. Various non-related soluble antigens can inhibit the binding of PRIG to any immobilized denatured antigen with similar efficiency. In contrast, natural antibodies interact specifically with appropriate antigens and this interaction can be suppressed only by the same or serologically similar competing antigens. Intact sera contain appreciable amount of polyreactive immunoglobulins, apparently much higher concentration than the concentration of natural antibodies. Biological functions of polyreactive immunoglobulins still remain unknown.     

Met-enkephalin-induced release into the blood of a factor causing postural asymmetry

Met- and Leu-enkephalin applied subarachnoidally into the rostral portion of a transected spinal cord (at the T6-T7 level) induce postural asymmetry of the hind limbs in rats, Met-enkephalin being predominantly responsible for the flexion of the right, and Leu-enkephalin of the left, hind leg. The blood serum of rats injected with Met-enkephalin contains a factor which, when administered subarachnoidally into the caudal portion of the transected spinal cord, is capable of inducing the hind limb postural asymmetry--predominantly, with the right leg flexion. This factor is inactivated by papain and differs from Met- and Leu-enkephalin in chromatographic properties. Apparently, Met-enkephalin induces the release of a peptide factor into the blood, from the brain or organs innervated by the neurons lying above the cut. It is then carried with the blood to the hind limbs and effects the hind limb postural asymmetry.     

Effects of prostaglandins E2 and I2, and arachidonic acid on vascular reactivity to norepinephrine in isolated rat mesenteric artery,hind limb and splenic artery

The effects of prostaglandins E2(PGE2) and I2(PGI2), arachidonic acid, and indomethacin on the vascular reactivity to norepinephrine were tested in three different isolated rat vascular beds (mesenteric artery, hind limb and splenic artery) perfused with the Krebs bicarbonate solution. In these vascular beds PGE2 (0.25 – 16 ng/ml), PGI2 (0.1 – 100 ng/ml), arachidonic acid (0.1 – 10 μg/ml) or indomethacin (5 – 25 μg/ml) in the perfusate did not change the basal pressure. In the splenic artery, both PGE2 and PGI2 attenuated the vascular response to norepinephrine in a dose-related manner. In the mesenteric vascular bed and the hind limb, however, PGE2 potentiated the vascular response to norepinephrine, while PGI2 attenuated this response. Arachidonic acid, a prostaglandin precursor, potentiated the vasoconstrictor response to norepinephrine in the mesenteric artery and the hind limb, whereas in the splenic artery, attenuation of the response to norepinephrine occurred. In these three vascular beds, indomethacin, a prostaglandin synthetase inhibitor, attenuated the vascular response to norepinephrine. In the mesenteric artery and the hind limb, PGE2 and not PGI2 reversed the effect of indomethacin, while in the splenic artery, neither PGE2 nor PGI2 reversed the inhibitory effect of indomethacin. These results suggest that, at least in the rat mesenteric artery and the hind limb where the modulating effect of arachidonic acid is similar to that of PGE2, PGE2 and not PGI2 is a primary endogenous prostaglandin in determining the vascular reactivity to norepinephrine.     

A Six-Dye Staining Schedule for Sections of Mesquite and Other Desert Plants

Materials are fixed in FPA (formalin, 2; propionic acid, 1; 70% ethanol, 17). Paraffin sections on slides are brought to 50% ethanol and stained as follows: (1) in Bismarck brown Y, a 0.02% solution in 0.1% aqueous phenol, 10-30 min; wash 30 sec in 0.7% acetic acid, and wash in distilled water 20-30 sec; (2) in crystal violet, 1% in 70% ethanol alkalinized with 1 drop of 1 N NaOH per 100 ml, 12-35 min; wash 30-60 sec in tap water to remove excess stain, and rinse 0.5 sec in 70% ethanol; then mordant in I₂-KI, 1% each in 70% ethanol, 40 sec, and rinse in 70% ethanol 2-5 sec; (3) in a mixture containing 0.4% acid fuchsin and 0.6% crythrosin B in 70% ethanol about 0.5 sec; rinse in 70% ethanol 5-15 sec to remove excess red; dehydrate in 70%, 95%, and absolute ethanol, 2-3 sec each; (4) in fast green FCF, 0.5% in a mixture of equal parts of methyl cellosolve, absolute ethanol, and clove oil, 5-15 sec; rinse in a mixture of clove oil, 10 ml; absolute ethanol, 100 ml; and methyl cellosolve, 10 ml, 5-7 sec; (5) in orange G, 0.75 gm in a mixture of clove oil, 40 ml; absolute ethanol, 40 ml; and methyl cellosolve, 60 ml, 5-30 sec; rinse clean in a 1:1 mixture of xylene and absolute ethanol, 5-20 sec Complete the clearing in pure xylene, 3 changes, 1.5 min in each, and apply a cover glass with synthetic resin. Slides are agitated in all steps except Bismark brown Y, crystal violet, and the xylenes. Contrast and staining intensity are adjusted by varying staining times in the dye solutions.     

India Ink-Gelatin Vascular Injection of Skeletal Tissues

Dogs under sodium pentothal anesthesia were given 100 mg of heparin intravenously and, 15 min later, exsanguinated. A mixture consisting of Higgins India ink #4415, 1 part, and a 14% solution of calfskin gelatin, 1 part, was injected (at 37° C) into the femoral artery of a disarticulated hindlimb. A pump-type 5 ml hypodermic syringe was connected with a gravity-feed reservoir that contained the injection mass, with a filter in the outflow from the reservoir. Veins severed by the disarticulation of the limb permitted a free flow of the injection fluid out of the vascular tree. After injection of about 400 ml of fluid by pumping the syringe, the limb was chilled (—20° C) to solidify the gelatin. Specimens from cleaned bones were sawed out and fixed in Zenker-formalin solution for 24 hr, then decalcified with 6% sulfosalicylic acid. The technique was completed by embedding in celloidin and sectioning. Blood vessels in both cortical and trabecular regions were completely filled by the injection mass.     

Effects of hexapeptide, a compound analogous to insulin B chain fragment B-21-26(DP-432) on the glucose uptake into the perfused hind limb of rats

DP-432 is a synthetic new peptide analogous to insulin B-chain fragment B21-26. It has been reported that this hexapeptide shows insulin potentiating action besides insulin-like activities in adipose tissue and diaphragm of rats. The perfusion of the hind limb of rats was performed according to the procedure of Ruderman with some modifications. The medium was Krebs-Ringer bicarbonate buffer containing 0.5% bovine albumin and 8.3 mM glucose. The erythrocytes were omitted from the medium. In this experimental procedure it was shown that the glucose uptake into the hind limb of rats increased by infusion of DP-432 (100 microgram/ml). The amount of glucose uptake into the hind limb responded to insulin doses (50, 100, 1000 muU/ml). DP-432 had insulin-like activity of 100 muU/ml. However, DP-432 itself did not have any potentiation of insulin activity on the muscle.     

Dynamics of the interaction of polyreactive immunoglobulins with immobilized antigens]

The kinetic of polyreactive immunoglobulins (PRIG) and immobilized antigen interaction was examined at different temperatures. It was shown that this process can be described by the so-called "competitive" model, and the relatively simple method for the rate constant determination for this process was developed. According to the "competitive" model PRIG molecule could be either in "active" or in "inactive" state and dynamic equilibrium exist between "active" and "inactive" molecules which strongly depend on incubation temperature. Only "active" PRIG can interact with antigens, and this is the reason of strong temperature dependence of PRIG-antigen interaction. The data also show that the mechanism of PRIG-antigen interaction differ from that of antibody-antigen interaction.     

Demyelinating Antibodies to Myelin Oligodendrocyte Glycoprotein and Galactocerebroside Induce Degradation of Myelin Basic Protein in Isolated Human Myelin

Abstract: Although the specificity of multiple sclerosis (MS) brain immunoglobulins (lgs) remains unknown, the incubation of these lgs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.     

Effect of prostaglandin E2 on vascular reactivity to norepinephrine in isolated rat mesenteric artery,hind limb and splenic artery

The effects of prostaglandin E2 (PGE2) and indomethacin on the vascular reactivity to norepinephrine were tested in three different isolated rat vascular beds (mesenteric artery, hind limb and splenic artery) perfused with the Krebs bicarbonate solution. In these vascular beds PGE2 (0.1–64 ng/ml) or indomethacin (0.1–96 μg/ml) in the perfusate did not change the basal pressure. In the mesenteric vascular bed and the hind limb, PGE2 dose-dependently potentiated the vascular response to norepinephrine, whereas PGE2 dose-dependently inhibited the vascular response to noreinephrine in the splenic artery. In these three vascular beds indomethacin in the perfusate dose-dependently attenuated the vascular response to norepinephrine. In the mesenteric artery and the hind limb PGE2 restored the effect of indomethacin, but in the splenic artery PGE2 did not restore the inhibitory effect of indomethacin. These results indicate that the modulating effect of exogenously administrated PGE2 on the vascular action to norepinephrine varies in different vascular beds. It is also suggested that the contribution of endogenous PGE2 synthesized in the vascular wall to the vascular reactivity to norepinephrine is, as well as the effect of exogenous PGE2, different in different vascular beds.     

Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cancer: novel therapeutic strategies that exploit the TNF-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor pathway

Cancer is a widespread disease, with half of all men and one-third of all women in the United States developing cancer during their lifetime. The efficacy of many cancer treatments including radiotherapy, chemotherapy and immunotherapy is due to their ability to induce tumor cell apoptosis. Recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is currently being developed as a cancer therapeutic since it selectively induces apoptosis in a variety of transformed cells, but not in most normal cells. Agonistic monoclonal antibodies (mAbs) specific for human death-inducing TRAIL receptors (DR4 or DR5) are also being actively pursued. Importantly, in experimental mice, synergistic anti-tumor effects have been observed with a combination treatment of agonistic mAb against DR5 together with either IL-21 or agonistic mAbs against CD40 and CD137. Together, these findings suggest that antibody-based therapies that cause tumor cell apoptosis and promote T cell memory or function may be effective in fighting cancer.     

Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections

Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.     

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.     

Hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus

A series of hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus was obtained and their cultural properties were characterized. HEMA-12 and HEMA-24 secreted monoclonal IgG2b antibodies, HEMA-101 secreted monoclonal IgG1 antibodies, HEMA-31, HEMA-9 and HEMA-11 secreted monoclonal IgG2 antibodies. According to the results of the indirect immunofluorescence test, the titer of specific immunoglobulins in the culture fluid was 1:16-1:32, but sometimes reached 1:64-1:128. The titer of antibodies in ascitic fluid amounted to dilutions of 10(4)-10(5). Hybridomas were cloned by the method of ultimate dilutions. The injection of 5-15 million HEMA cells into the abdominal cavity of BALB/c mice induced the formation of ascitic tumors in the animals within 7-11 days and the accumulation of ascitic fluid in a volume of 1-4 ml. Hybridomas, found to be capable of passage from mouse to mouse, underwent 5-16 passages during the term of observation.