The antigen-binding fragment of human gamma immunoglobulin prevents amyloid ß-peptide folding into ß-sheet to form oligomers.

The amyloid beta-peptide (Aß) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aß are harmless to cells, Aß can aggregate into ß-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aß aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aß oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aß aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aß aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aß aggregation and its neuroprotective role.

[Effects and mechanism of action of synthetic peptide octarphin].

We have synthesized the peptide TPLVTLFK corresponding to the ß-endorphin fragment 12-19 (the name given by the authors - octarphin), and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol) and its binding to the murine peritoneal macrophages has been studied. [(3)H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [(3)H]octarphin is inhibited by unlabeled ß-endorphin and selective agonist of non-opioid ß-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM respectively) and not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met(5)]enkephalin (K(i) > 10 µM). Inhibiting activity of unlabeled analogs of octarphin is more then 100 times lower the unlabeled octarphin. Octarphin stimulates activity of murine immunocompetent cells in vitro and in vivo: at the concentration of 1-10 nM enhances the adhesion and spreading of peritoneal macrophages as well as their capacity to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro. Intraperitoneal administration of peptide at dose 20 µg/animal on day 7,3 and 1 prior to the isolation of cells increases activity of peritoneal macrophages as well as T- and B-spleen lymphocytes.

[Synthetic peptide immunorphin as an instrument for studying nonopioid beta-endorphin receptor].

Research results of the synthetic decapeptide SLTCLVKGFY (the author's term is immunorphin) corresponding to the 364-373 sequence of G heavy-chain human immunoglobulin are summarized. Special attention is paid to the interaction between immunorphin and a nonopioid (insensitive to the opioid antagonist naloxone) beta-endorphin receptor. Using radioligand analysis, data were found regarding the distribution and functions of a nonopioid beta-endorphin receptor in human and animal bodies and the binding characteristics of immunorphin with a nonopioid receptor.

Inherent anti-amyloidogenic activity of human immunoglobulin gamma heavy chains.

We have previously shown that a subpopulation of naturally occurring human IgGs were cross-reactive against conformational epitopes on pathologic aggregates of Abeta, a peptide that forms amyloid fibrils in the brains of patients with Alzheimer disease, inhibited amyloid fibril growth, and dissociated amyloid in vivo. Here, we describe similar anti-amyloidogenic activity that is a general property of free human Ig gamma heavy chains. A gamma(1) heavy chain, F1, had nanomolar binding to an amyloid fibril-related conformational epitope on synthetic oligomers and fibrils as well as on amyloid-laden tissue sections. F1 did not bind to native Abeta monomers, further indicating the conformational nature of its binding site. The inherent anti-amyloidogenic activity of Ig gamma heavy chains was demonstrated by nanomolar amyloid fibril and oligomer binding by polyclonal and monoclonal human heavy chains that were isolated from inert or weakly reactive antibodies. Most importantly, the F1 heavy chain prevented in vitro fibril growth and reduced in vivo soluble Abeta oligomer-induced impairment of rodent hippocampal long term potentiation, a cellular mechanism of learning and memory. These findings demonstrate that free human Ig gamma heavy chains comprise a novel class of molecules for developing potential therapeutics for Alzheimer disease and other amyloid disorders. Moreover, establishing the molecular basis for heavy chain-amyloidogenic conformer interactions should advance understanding on the types of interactions that these pathologic assemblies have with biological molecules.

A novel paradigm for dendritic cells as effectors of cartilage destruction.

OBJECTIVE: Dendritic cells (DCs) are enriched in RA synovium and have been implicated in the pathogenesis of RA primarily through their ability to present autoantigen and activate T cells. However, whether DCs play an effector role in cartilage destruction is unknown. The aim of this study was to investigate whether DCs can induce collagen release from cartilage and the mechanism involved. METHODS: Human monocyte-derived DCs (mDCs) were activated with CD40 ligand (CD40L) to mimic DC-T-cell interaction, and supernatants were incubated with cartilage explants. Hydroxyproline was assessed as a measure of collagen release and collagenolytic activity was measured by a bioassay using tritiated collagen. TNF-alpha in DC supernatants was measured by specific ELISA. RESULTS: Supernatants from CD40L-activated mDCs, but not unstimulated mDCs, strongly induced the destruction of cartilage collagen. mDC supernatants did not contain collagenases but did induce collagenolytic activity in cartilage explants. Neutralization of TNF-alpha in mDC supernatants completely abolished collagenolysis. CONCLUSIONS: This study shows that mDCs, upon CD40-ligation, induce cartilage collagen degradation through an indirect mechanism via the production of TNF-alpha. Our data suggest a potential important role for mDC-derived TNF-alpha in RA, which is in line with the previously reported observations that DCs are a major source of TNF-alpha in early autoimmune lesions and that anti-TNF-alpha therapeutics effectively suppress joint damage in RA patients. We propose that DCs can act as effectors in cartilage destruction, adding a new aspect to the functional role of DCs in RA pathogenesis.

Effect of gamma-immunoglobulin on the asaccharolytic growth of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVES: A minimal medium is indispensable for examining the growth properties of the asaccharolytic bacterium, Porphyromonas gingivalis. The purpose of the present study was to improve the widely used KGB medium to support good growth of P. gingivalis. MATERIAL AND METHODS: Growth of P. gingivalis (W50, W83, and ATCC33277) in a minimal medium was monitored by measuring the optical density of the culture during incubation. RESULTS: W50, W83, and ATCC33277 grew poorly with bovine serum albumin as the sole carbon and nitrogen source, and alpha-ketoglutarate had little or no effect on this poor growth. In contrast, FeCl3 improved the growth of W83 and ATCC33277; however, the use of a high concentration of FeCl3 elicited black pigmentation of the cells. Bovine gamma-immunoglobulin greatly recovered the growth defect. None of alpha-ketoglutarate, citrate, or trace metal ions, when used to supplement KGB medium, was required for growth. We determined the optimal conditions for growth, and developed a new simple minimal medium for P. gingivalis (GA medium). Growth of ATCC33277 in GA medium was dependent on gingipains; Arg-gingipains and Lys-gingipain contributed comparably to proliferation of the bacterium. CONCLUSION: These data indicate that GA medium is currently the most reliable minimal medium for examining the growth properties of P. gingivalis.

Pathogenic autoantibodies in the lambert-eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission in which antibodies are directed against voltage-gated calcium channels (VGCCs). We studied the action of LEMS immunoglobulin G (IgG) on cloned human VGCCs stably transfected into human embryonic kidney cells (HEK293). All LEMS IgGs tested bound to the surface of the P/Q-type VGCC cell line and caused a significant reduction in whole-cell calcium currents in these cells. By contrast, only 2 out of 6 IgGs bound extracellularly to the N-type VGCC cell line, and none of the LEMS IgGs tested was able to reduce whole-cell calcium currents in these cells. We used this apparent specificity of LEMS IgG for the P/Q-type VGCC to investigate the action of these IgGs on model systems where a number of different VGCC populations exist in equilibrium. LEMS IgG caused a significant downregulation in the omega-agatoxin IVA-sensitive P/Q-type VGCCs of cultured rat cerebellar neurons, but this was accompanied by a concomitant rise in the "resistant" R-type VGCCs. By using the passive transfer model of LEMS, similar results were observed at the mouse neuromuscular junction, where a significant reduction in P/Q-type VGCCs was paralleled by an increase in L- and R-type VGCCs. These results demonstrate an unexpected plasticity in the expression of VGCCs in mammalian neurons and may represent a mechanism by which the pathogenic effects of LEMS IgG are reduced.

Inmunoglobulina G endovenosa: características de un medicamento elaborado en la Argentina / Intravenous immunoglobulin G: of a preparation manufactured in Argentina

Antecedentes: las preparaciones de inmunoglobulina G endovenosa (IgG EV) son utilizadas como terapia efectiva en diversas patologías: inmunodeficiencias primarias y secundarias, enfermedades autoinmunes e inflamatorias, enfermedades infecciosas y enfermedades alérgicas, entre otras. Este medicamento debe cumplir con tres premisas relevantes: tolerancia clínica, actividad biológica plena y seguridad viral. Objetivos: informar las características de una inmunoglobulina G endovenosa eleborada en nuestro país (Inmunoglobulina G endovenosa-UNC), con plasma de Argentina, Uruguay y Chile, con relación a su eficacia terapéutica, tolerancia clínica y seguridad viral. Materiales y métodos: se analizaron todos los parámetros recomendados por la Organización Mundial de la Salud y la Farmacopea Europea en 10 lotes de Inmunoglbulina G Endovenosa-UNC. Resultados: todos los parámetros evaluados en Inmunoglbulina G Endovenosa-UNC se encontraron dentro de los límites establecidos por la Farmacopea Europea y la Organización Mundial de la Salud. Conclusiones: los resultados de los parámetros físico-químicos y biológicos, evaluados en este trabajo, demuestran que la Inmunoglobulina G Endovenosa-UNC, eleborada en la Argentina, es de igual calidad que los más avanzados estándares internacionales de este producto (AU)

[Influence of intravenous-immunoglobulin on T lymphocyte subsets, NK cell and some cytokines in mice with experimental viral myocarditis].

This study sought to gain an insight into the immunological pathogenesis of viral myocarditis and the mechanism of therapeutic action of intravenous-immunoglobulin (IVIG) on the disease, BALB/c mice were randomized into four groups: normal control group; "myocarditis model group", inoculated intraperitoneally with CVB3; "IVIG protection group", injected intraperitoneally with CVB3 and IVIG; and "IVIG treatment groups", injected intraperitoneally with CVB3 and IVIG. The amount of T cell in different T cell subsets and the activities of NK cell, IL-1 and IL-2 were assayed using spleen specimen while peripheral blood was used to measure TNF activity. The results showed: that in comparison with normal control, all spleen T cell subsets decreased in amount in myocarditis model group, so did its NK cell activity while its IL-1, IL-2 and TNF activities increased significantly. When compared with myocarditis model group, however, both IVIG protection group and IVIG treatment group showed significant elevation of NK cell activity and T cell subsets but reduced IL-1, IL-2 and TNF activities with no remarkable change in T cell subsets. The results of suggest that the immunological injury mediated by T cells plays a critical role in the pathogenesis of viral myocarditis. The protective and therapeutic effects of IVIG on the murine CVB3 myocarditis and the related immunological evidence of its actions may indicate the prospect that IVIG will become a potent and safe treatment for viral myocarditis in human.

[Polyvalent hyperimmune immunoglobulins prevent the infection of monocytic type cells by human cytomegalovirus]. / Les immunoglobulines polyvalentes hyperimmunes préviennent l'infection des cellules de type monocytaire par le cytomégalovirus humain.

Monocyte/macrophage cell types can be infected with Human Cytomegalovirus (HCMV) could be a reservoir and a vehicle for virus spread in infected patients. We developed a model to study the effects of antiviral molecules on these cells. The monocytic-like cell line THP-1 and the human diploïd cells MRC-5 were used. THP-1 cells were cultivated with a phorbol 12-myristate 13-acetate (PMA) for 24h prior to the infection. We studied infection of these cells with reference strain (AD-169). A cell free virus suspension of HCMV was preincubated with hyperimmune polyvalent immunoglobulins. The infection of the cells was studied on the basis of immune detection of viral immediate early antigens (IEA) in nucleus 24h after culture. Our results showed that hyperimmune polyvalent immunoglobulins have been able to neutralize fibroblasts and THP-1 cells infection, whereas control antibodies did not inhibit the infection of the cells. This is the first report of the use of monocytic-like cells for testing the efficiency of anti-CMV molecules.

Lambert-Eaton myasthenic syndrome: immunoglobulin G inhibition of Ca2+ flux in tumor cells correlates with disease severity.

We compared the effects of Lambert-Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) obtained from patients with and without small-cell lung carcinoma (SCLC) on voltage-gated (K+-stimulated) 45Ca2+ flux in cell lines derived from a human SCLC (MAR10) and from a rat pheochromocytoma (PC12) and related these to electromyographic indexes of clinical severity. Control IgG was obtained from patients with other neurological disorders or healthy individuals. Inhibition of Ca2+ flux by LEMS IgG was time and dose dependent. The flux was significantly reduced in MAR10 cells grown in either SCLC-LEMS IgG (0.38 nmol/10(6) cells; p less than 0.001) or non-SCLC-LEMS IgG (0.35 nmol/10(6) cells; p less than 0.001), compared with that in MAR10 cells grown in control IgG (0.7 nmol/10(6) cells). Similar significant reductions were also observed in PC12 cells. The reduction in amplitude of the resting compound muscle action potential in the LEMS patients correlated positively (r = 0.70; p = 0.007) with the inhibition of Ca2+ flux in MAR10 cells by their IgG. These results strongly support the view that IgG autoantibodies that can inhibit Ca2+ flux in SCLC cells are responsible for the disorder of transmitter release at motor nerves in SCLC-associated LEMS.

Effect of ibuprofen and dexamethasone on Kupffer cell complement receptor function after endotoxemia and the phagocytosis of erythrocytes.

Depression of Kupffer cell complement receptor (CR) function is associated with several states of depressed host defense. This study was carried out to determine if ibuprofen and dexamethasone, which decrease the mortality rate following endotoxemia, could prevent the depression of CR function caused by endotoxemia and the phagocytosis of antibody-coated erythrocytes (EIgG). The depression of CR function caused by endotoxin was completely prevented by the administration of ibuprofen or dexamethasone. Thus, the ability of these drugs to prevent the depression of macrophage function may contribute to their salutory effects during endotoxin shock. In contrast to the effect with endotoxemia, the depression of CR function caused by the phagocytosis of EIgG was not modified by pretreatment with ibuprofen or dexamethasone. Additional studies demonstrated that the depression of CR function caused by EIgG was probably not due to EIgG in the blood or bound to Kupffer cells, interfering with the receptor probe for access to the CR. This study has shown that ibuprofen and dexamethasone can prevent the depression of CR function caused by endotoxin but not the depression caused by the phagocytosis of EIgG. These results suggest that different mechanisms mediate the depression of CR function caused by endotoxin and the phagocytosis of EIgG.

Lambert-Eaton myasthenic syndrome IgG depletes presynaptic membrane active zone particles by antigenic modulation.

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that can be transmitted from human to mouse with immunoglobulin G (IgG). Electrophysiological studies indicate that LEMS IgG acts on presynaptic voltage-sensitive calcium channels, probably reducing their number, and freeze-fracture electron microscopy demonstrates that LEMS IgG has an effect on the presynaptic active zone particles, which represent putative voltage-sensitive calcium channels. The active zone particles, normally arranged in double parallel rows, move closer together, form clusters, and are reduced in number. The morphological data suggest modulation of the active zone particles crosslinked by LEMS IgG. If this were the case, then only divalent LEMS IgG and F(ab')2 should alter the deployment of active zone particles and monovalent Fab should be without effect. To test this hypothesis, mouse diaphragms were exposed to control and LEMS IgG and IgG fragments in organ culture for 24 hours and then studied by quantitative freeze-fracture electron microscopy. Divalent LEMS IgG and F(ab')2 aggregated and depleted the active zone particles, whereas monovalent Fab had no effect. The findings reconfirm that the active zone particles are targets of LEMS IgG and are direct evidence for modulation of the particles by LEMS IgG. The findings are in harmony with parallel electrophysiological studies of the effects of LEMS IgG fragments on transmitter release in the same diaphragm muscles (Lang et al, J Physiol 1987;390:173P).