Rotational dynamics of immunoglobulin G antibodies anchored in protein A soluble complexes

The rotational dynamics of rabbit IgG anti-dansyl antibodies anchored in staphylococcal protein A (SpA) soluble complexes were studied by both steady-state and nanosecond fluorescence spectroscopy. To aid in the interpretation of the anisotropy data, the results of recently reported hydrodynamic and electron microscopic studies of IgG-SpA complexes were used to calculate global tumbling times of the various complexes and to estimate the steric hindrance of the antibody Fab segments. The anisotropy decays, fitted to the sum of two exponentials, indicated that the Fab arms of antibodies bound to SpA by their Fc regions exhibit considerable flexibility. For the different IgG-SpA mixtures examined, changes in the IgG preexponential anisotropy weighting factors, fS and fL, and the short rotational correlation time, phi S, were relatively small. On the other hand, the long rotational correlation time, phi L, increased systematically when the percentage of larger IgG-SpA complexes in a mixture was increased. The greatest restriction of Fab flexibility was observed for antibodies anchored in the exceptionally compact IgG4-SpA2 complexes. Available electron microscopic data suggest that increases in phi L correlate with increased steric hindrance of the antibody segments. Both native and hinge-disulfide-cleaved IgG experienced similar percentage increases in phi L when bound in SpA complexes. In agreement with our earlier interpretation, the results of this study provide rather striking evidence that phi L mainly represents flexible motions of the Fab segments and not global tumbling: the phi L-values of IgG bound in the various SpA complexes ranged from 101 to 162 nsec, whereas the calculated global tumbling times of the different complexes ranged from about 300 to 3000 nsec.     

Correlation between the structure of IgG and its Fc-fragment and their ability to interact with staphylococcal protein A

Interaction between 7S monomers of rabbit IgG, dimers of molecules of this protein, IgG with disulfide bond ruptured in the hinge region of the molecule, and various fragments of IgG molecules, on the one hand, and protein A ofStaphylococcus aureus, on the other hand, were investigated by the passive hemagglutination inhibition test. Only the Fc-fragment of the rabbit IgG molecule obtained with papain was shown to bind protein A. Activity of the Fc-fragment on a molar basis was shown to be only one-sixth of that of native IgG. After repair of the disulfide bond between the chains in the hinge region of the IgG molecule, its ability to bind protein A was reduced by two-thirds. The binding activity of IgG was increased on a molar basis twelvefold as a result of its spontaneous dimerization. It is concluded from the results that the structural organization of the Fc-fragment of the IgG molecule correlates with its ability to interact with protein A.Laboratory of Immunochemistry, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 318–320, March, 1980.     

Mouse monoclonal antibodies at the red cell surface--I. Generation of EAC4 and interaction with C1

C1 and C1 activity measurements were performed with EA and EAC4 prepared with rabbit anti-Forssman IgG or IgM and were compared to measurements with EA and EAC4 prepared with mouse monoclonal IgG2b and IgM anti-DNP antibodies on cells coupled with TNP: the amount of TNP per cell was optimal for antibody activity. No differences were found in the ability of EAC4 made with poly- or monoclonal IgM to measure C1 activity; in contrast, monoclonal IgM was capable of activating only about 30% of C1 when compared to activation by polyclonal IgM. Monoclonal vs polyclonal IgGs behaved in a similar manner but they were detecting only 50% of C1 or C1 activity when compared to IgM of the appropriate class. It was concluded that monoclonal antibodies were capable of generating EAC4 intermediate, and that the ability of monoclonal antibodies in the EAC4 complex to bind C1 and to detect C1 activity is not significantly different from that of polyclonal antibodies but that monoclonal antibodies are less efficient in activating C1 than polyclonal antibodies.     

Early effects of corticosteroids on basophils, leukocyte histamine, and tissue histamine

The comparative effect in 11 atopic subjects of a single intravenous injection of methylprednisolone on sequential studies of blood eosinophils, basophils, leukocyte sensitivity to antigen for histamine release, leukocyte histamine content, and skin histamine was examined. No significant changes occurred in any parameter after placebo treatment. In contrast, 4 hr after intravenous treatment with steroid there were significant decreases in mean eosinophil counts (-95%), basophil counts (-72%), and histamine content of 1 X 10(7) leukocyte samples (-62%). Temporal changes in the latter paralleled alterations in circulating basophil levels. No significant changes occured in the antigen histamine release sensitivity, or the total skin histamine. Studies over a longer period after steroids in 4 subjects showed eosinophil and basophil levels at a nadir at 8 hr, remaining suppressed for 24 hr, and returned to pretreatment levels by 72 hr. Results suggest that corticosteroids induce a prominent decrease in leukocyte histamine due to a depletion of basophils without a decrease in histamine content per basophil, and that skin tissue histamine stores remain unchanged by such treatment.     

Dose-dependent effects of the 5-HT1A receptor agonist 8-OH-DPAT on sleep and wakefulness in the rat

SUMMARY  Sleep and wakefulness were studied in rats following administration of a selective 5-HT_1A agonist (8-OH-DPAT), a non-selective 5-HT_1A antagonist [(-) pindolol] and a combination of 8-OH-DPAT and (—) pindolol.
8-OH-DPAT (1.0–4.0 μg) injected into the dorsal raphe nucleus increased slow-wave sleep and decreased wakefulness. Administration of the 5-HT_1A agonist by subcutaneous route induced biphasic effects such that low doses (0.010 mg kg^-1) decreased wakefulness and increased slow-wave sleep while higher doses (0.375 mg kg^-1) induced opposite effects. REM sleep was suppressed and REM latency was increased, what could be tentatively ascribed to a non-specific effect (hypothermia). (-) Pindolol (1.0–4.0 mg kg^-1) induced an initial increase of wakefulness and a decrease of NREM sleep and REM sleep. Thereafter, NREM sleep showed a marked increase while REM sleep remained depressed. Pretreat-ment with (—) pindolol reversed the effects of both small and large doses of 8-OH-DPAT on slow-wave sleep and wakefulness.
The opposite effects, observed on the waking EEG after activation of either serotonin autoreceptors or postsynaptic 5-HT_1A receptors with adequate doses of 8-OH-DPAT, tend to indicate an active role for the 5-HT_1A receptor in the control of the waking state.     

LGR5 is a biomarker for stratification of HER-2 positive breast cancer patients and personalized treatment

VEGFR and HER2 are both important transmembrane proteins associated with several types of cancer. Overexpression of these 2 proteins had long been thought to contribute to cancer progression and poor outcomes, thus, therapies targeting HER-2 and VEGFA signaling pathways have been applied in recent years.     

The relative sensitivities of heart rate and T-wave amplitude to stress: Comments on,and some alternative interpretations of,Penzien et al.'s results

Although the joint measurement of heart rate (HR) and T-wave amplitude (TWA) in experiments manipulating psychological processes is a sound and fruitful approach, Penzien Hursey, Kotses and Beazel's (1982) interpretation of their results may be questioned on two grounds: (a) Wether the process being manipulated between their groups was really the degree of stress; and (b) whether the degree of threat or aversiveness is really indexed more reliably by HR changes than by changes in TWA. This note questions these two assumptions, and also offers an alternative vagal interpretation of the Penzien et al. (1982) results.     

Colostral antibody-mediated and cell-mediated immunity contributes to innate and antigen-specific immunity in piglets

Immunoglobulins and immune cells are critical components of colostral immunity; however, their transfer to and function in the neonate, especially maternal lymphocytes, is unclear. Cell-mediated and antibody-mediated immunity in sow blood and colostrum and piglet blood before (PS) and after (AS) suckling were assessed to investigate transfer and function of maternal immunity in the piglet. CD4, CD8, and γδ lymphocytes were found in sow blood and colostrum and piglet blood PS and AS; each had a unique T lymphocyte profile. Immunoglobulins were detected in sow blood, colostrum, and in piglet blood AS; the immunoglobulin profile of piglet serum AS mimicked that of sow serum. These results suggest selectivity in lymphocyte concentration into colostrum and subsequent lymphocyte transfer into the neonate, but that immunoglobulin transfer is unimpeded. Assessment of colostral natural killer activity and antigen-specific proliferation revealed that colostral cells are capable of influencing the innate and specific immune response of neonatal pigs.     

The molecular interaction between the glutamatergic, noradrenergic, dopaminergic and serotoninergic systems informs a detailed genetic perspective on depressive phenotypes

The glutamatergic pathway has been consistently involved in the physiopathology of depressive disorder. However a complete dissection and integration of its role in the context of other known mechanisms is lacking. We summarized and integrated the evidence of various levels of interaction between glutamatergic and monoaminergic pathways (see videos). We identified six molecular pathways, some of which with specific regional distribution within the brain. From the six pathways we identified the key proteins and their coding genes, we then provided a detailed list of possible candidates with practical suggestions for association studies planning.     

Inherited resistance to N- and B-tropic murine leukemia viruses in vitro. Effect of the Fv-1 locus on the rescue of a replication-defective and transformation-defective murine sarcoma virus in the Fv-1 congenic strains SIM.S and SIM.R.

An in vitro assay for MuLV (murine leukemia virus) using mouse embryo cultures derived from the congeneic strains SIM.S and SIM.R differing at the Fv-1 locus and containing the genome of a replication- and transformation-defective (R^?T^?) MuSV (murine sarcoma virus) isolate is described. Studies on the interrelationship of MuLV and MuLV using this focus induction assay indicated that rescue of the defective MuSV was subject to Fv-1 restriction of the superinfecting MuLV helper. Titration patterns of N-, B-, and NB-tropic MuLV on these SIM.S and SIM.R “R^?T^?” cells were all linear and parallel over the range of dilutions used. The kinetics of MuLV infection appeared to be single hit using both focus and XC-plaque assays in both permissive and nonpermissive host cells. MuSV rescued by N-, B-, or NB-tropic MuLV showed no strict host range specificity. Host range of these rescued MuSV was determined by the degree of restriction of coinfecting MuLV. Results of these studies using both normal and MuSV(R^?T^?)-infected SIM.S(Fv-1^n/n) and SIM.R(Fv-l^b/b) cell lines suggest that Fv-1-mediated restriction acts solely by restricting MuLV replication and thereby inhibiting focus formation by the defective MuSV genome in Fv-1 nonpermissive cells.     

Three polyethylene glycol dependent methods for the detection of circulating immune complexes in pathological sera: Comparison with the Raji cell method

A composite method using polyethylene glycol (PEG) and different markers for detecting circulating immune complexes (CIC) is described. The markers used are bovine conglutinin (RK-BA), C1q (C1q-BA) and IgG, IgM quantitation of PEG precipitate (RID-Ig). A composite scoring system is used in interpreting results from individual assays. The sensitivity of multiple PEG methods (MPM) was determined in 418 serum samples and compared with Raji cell assay in 204. Correlations between individual assays, viz., RK-BA, C1q-BA, RID-Ig and Raji cell test in several disease conditions including rheumatoid arthritis, glomerulonephritis, post-renal transplantation, maintenance haemodialysis, multiple sclerosis and normal pregnancies were computed. The relative discriminatory ability of a single PEG technique to differentiate normal from pathological sera in these disease states was observed in comparison with the composite PEG index. This index gives an improved assessment of abnormal sera, is simple and sensitive and has some advantages over biological techniques such as the Raji cell assay.     

Short- and long-term effects of cromolyn sodium on the airway reactivity of asthmatics

To study the effects of cromolyn sodium on the airway reactivity of asthmatics, we performed a randomized, double-blind, crossover study in which 11 atopic asthmatics inhaled 20 mg of cromolyn sodium or a matching placebo four times daily for 4 wk each, while in their allergy season. Bronchial challenges consisting of either eucapnic hyperventilation with frigid air or methacholine were performed before, in the middle, and at the end of each treatment period. Stimulus-response relationships were assessed with the forced expiratory volume in I sec (FEV1). The level of ventilation (VE) and the provocative dose of methacholine (meth) required to reduce the FEV1 20% from control were recorded as the PD20VE and PD20meth, respectively. There were no significant differences in the baseline FEV1 on any study day. The short-term administration of cromolyn brought about a significant increase In PD20VE prior to both the placebo and active phases of the study. Placebo had no effect on airway reactivity. After 2 wk of cromolyn, PD20VE rose significantly and stayed elevated during the course of the study. Neither the short- nor long-term administration of cromolyn had any effect on the responsiveness to methacholine. These results demonstrated that long-term therapy can attenuate the responsiveness to naturally occurring asthmogenic stimuli even when nonspecific reactivity is unchanged.     

Interactions between Fab and Fc regions in liganded immunoglobulin G: A study employing the antigenic polymorphism and multivalency of the human red-cell membrane

Previous studies indicated that the inter-y chain disulphide bonds restrict segmental flexibility within the Fc region of human IgG; the present study suggests that there are also non-covalent hinge-region restrictions of flexibility in the liganded molecule. Staphylococcus aureas protein A-Sepharose-passed F(ab′)₂ fragments from IgG fractions of blood-group antisera were tested in parallel with the parent antibodies in haemagglutination tests. At molar-equivalence in protein, the titre of F(ab′)₂ from incomplete antibodies was 2- to 3-fold greater than the titre of the intact antibody (p < 0.001). Increased activity occurred whether enzyme or albumin techniques were used to convert the incomplete antibodies to agglutinins, and was particularly striking with Kell blood-group antibodies where F(ab′)₂ fragments were weak saline agglutinins. Antiglobulin tests, using anti-κ, revealed small differences in intrinsic antigenbinding activity between IgG and F(ab′)₂ fractions, but these differences were either of inverse relationship or insufficient to account for the increased agglutinating activity of F(ab′)₂ compared with the whole molecule. By contrast, with IgG saline agglutinins [anti-κ (anti-glycophorin A^M) and anti-B], the apparent freedom introduced by peptic removal of Fc, or by reduction of interchain disulphide bonds, was inimical to agglutination, as would be predicted from other studies (Hornick & Karush, 1972; Crothers & Metzger, 1972). These results show that the Fe region imposes limitations on full segmental flexibility of Fab regions in liganded IgG.A comparison of the intrinsic and functional antigen-binding activities of IgG anti-M and anti-B confirmed that both antibodies engage in substantial monogamous bivalency. That the relative monogamous bivalency of F(ab′)₂ and IgG anti-M was unaffected by an increased mean separation of M sites on the cell surface suggests that it is predominantly dimers of glycophorin a^M, in situ, which generate the monogamous bivalency and that, on M/N red cells, some glycophorin-A^M molecules are associated as homologous dimers. For IgG anti-A and anti-B, the known decreased affinity with A₂ and A₁B red cells can be explained by the fewer opportunities for monogamous bivalency. Using the increased flexibility in F(ab′)₂ and reduced-alkylated IgG anti-D as molecular probes for spatial relationships between D antigens, (i) a marked deviation from an average separation of antigens was found on normal red cells under conditions of agglutination, and (ii) an early effect of trypsin-treatment (< 2 min) which permits limited redistribution of D sites was detected.Mildly reduced IgG3 exhibited significantly greater flexibility than reduced IgG1, presumably reflecting the extended γ3 hinge region. Unreduced IgG3, however, did not show the increased Fab-region flexibility shown by F(ab′)₂ fragments on the IgG1 sub-class. In reduced IgG molecules, Cγ2 and Cγ3 domains retained much of their native surface topography.     

Trk and cAMP-dependent survival activity of adenosine A2A agonist CGS21680 on rat motoneurons in culture

The survival activity of adenosine A_2A agonist CGS21680 on motoneurons in culture through the transactivation of neurotrophin receptor TrkB has been reported previously; however, since adenosine A_2A receptor belongs to a Gs-protein-coupled receptor, we investigated the involvement of the cAMP pathway in the survival activity of CGS21680 using purified motoneurons in culture. CGS21680 alone showed only small survival activity, but the activity was significantly enhanced by the addition of a phosphodiesterase inhibitor, IBMX. This survival activity was partially inhibited by a protein kinase A inhibitor H89 or a neurotrophin receptor tyrosine kinase inhibitor K252a, and was completely inhibited by their combination. These results indicate that the survival activity of CGS21680 on motoneurons is exerted by the mixed effect of the adenylate cyclase–cAMP–PKA pathway and transactivation of Trk neurotrophin receptor. Under conditions in which the maximum survival of motoneurons was supported by sufficient concentrations of brain-derived neurotrophic factor (BDNF), a TrkB ligand, the addition of 100 μM AMPA for 3 days led to significant cell death. Treatment with CGS21680 and IBMX protected motoneurons from the toxicity of AMPA, further supporting the presence of a TrkB-independent pathway of CGS21680 activity and suggesting a novel therapeutic approach to motoneuron diseases such as amyotrophic lateral sclerosis.     

Diagnosis and management of neuromyelitis optica spectrum disorders - An update

Neuromyelitis optica (NMO) and Neuromyelitis optica spectrum disorders (NMOSD) are a group of autoimmune conditions characterized by inflammatory involvement of the optic nerve, spinal cord and central nervous system. Novel evidence showed a key role of autoantibodies against aquaporin-4 immunoglobulin G (AQP4 IgG) in the pathogenesis of NMOSD and, recently, new classification and diagnostic criteria have been adopted to facilitate an earlier identification and improve the management of these conditions. Diagnosis of NMOSD is currently based on clinical, neuroimaging and laboratory features. Standard treatment is based on the use of steroids and immunosuppressive drugs and aims to control the severity of acute attacks and to prevent relapses of the disease. This review gives an update of latest knowledge of NMOSD and NMO, emphasizing the novel diagnostic criteria and both current and future therapeutic approaches.     

The nociceptive and anti-nociceptive effects of bee venom injection and therapy: A double-edged sword

Bee venom injection as a therapy, like many other complementary and alternative medicine approaches, has been used for thousands of years to attempt to alleviate a range of diseases including arthritis. More recently, additional theraupeutic goals have been added to the list of diseases making this a critical time to evaluate the evidence for the beneficial and adverse effects of bee venom injection. Although reports of pain reduction (analgesic and antinociceptive) and anti-inflammatory effects of bee venom injection are accumulating in the literature, it is common knowledge that bee venom stings are painful and produce inflammation. In addition, a significant number of studies have been performed in the past decade highlighting that injection of bee venom and components of bee venom produce significant signs of pain or nociception, inflammation and many effects at multiple levels of immediate, acute and prolonged pain processes. This report reviews the extensive new data regarding the deleterious effects of bee venom injection in people and animals, our current understanding of the responsible underlying mechanisms and critical venom components, and provides a critical evaluation of reports of the beneficial effects of bee venom injection in people and animals and the proposed underlying mechanisms. Although further studies are required to make firm conclusions, therapeutic bee venom injection may be beneficial for some patients, but may also be harmful. This report highlights key patterns of results, critical shortcomings, and essential areas requiring further study.     

Binding and activation of C1 by cell bound IgG: Activation depends on cell surface hapten density

We have investigated the binding and activation of C1 by IgG-anti methotrexate antibody at cell surfaces. Under conditions where variation in cell surface hapten density had no effect on binding of IgG. the number of C1 (or its active form, C1) bound by the IgG was independent of hapten density. The ability of the C1 binding IgG complex to activate C1, however, was decreased with decreasing density of the hapten. The decreased ability to activate bound C1 was paralelled by decreased ability to activate the hemolytic sequence in whole complement. The results were interpreted to mean that binding of C1 was the result of aggregation (doublet formation) by IgG while activation of the bound C1 depended on changes induced in the IgG molecule by straddling hapten molecules at varying distances.     

Neurochemical pathways involved in the protective effects of nicotine and ethanol in preventing the development of Parkinson's disease: potential targets for the development of new therapeutic agents

In this short review, neurochemical targets are identified where nicotine, and possibly ethanol, may interact to prevent the occurrence of Parkinson's disease. These are (a) the nicotinic acetycholine receptors present in the nigrostriatal area or on the surface of microglia, (b) monoamine oxidases and (c) inducible nitric oxide synthase. If such induced changes can be verified in clinical studies, this may help in the design of new therapeutic drugs which may be of relevance to diminish the incidence and perhaps the progression of the debilitating condition of Parkinson's disease.     

Non-competitive antagonists of NMDA and AMPA receptors decrease seizure-induced c-fos protein expression in the cerebellum and protect against seizure symptoms in adult rats

The aim of the present study was to examine the role of ionotropic glutamate receptors in the cerebellum during generalized seizures. Epileptic neuronal activation was evaluated through the immunohistochemical detection of c-fos protein in the cerebellar cortex. Generalized seizures were precipitated by the intraperitoneal injection of 4-aminopyridine. The animals were pretreated with the NMDA receptor antagonists MK-801 (2?mg/kg), amantadine (50?mg/kg), and the AMPA receptor antagonist GYKI 52466 hydrochloride (50?mg/kg). Two hours after 4-aminopyridine injection, the number of c-fos immunostained cell nuclei was counted in serial immunohistochemical sections of the cerebellar vermis. The number of c-fos immunostained cell nuclei in the granular layer decreased significantly in animals pretreated with the glutamate receptor antagonists compared to the untreated animals having convulsion. We can conclude that mossy fiber stimulation exerts its seizure-generating action mainly through the ionotropic glutamate receptors of the mossy fiber synapses. Both NMDA and AMPA receptor antagonists are effective in reducing glutamate-mediated postsynaptic effects in the cerebellar cortex.