Primary membranoproliferative glomerulonephritis (MPGN) is a rare glomerulopathy characterized by complement dysregulation. MPGN progresses rapidly to kidney failure when it is associated with nephrotic syndrome. We assessed the effects of C5 convertase blockade in patients with MPGN and terminal complement activation.
Prospective off-on-off-on open-label clinical trial.
Consenting patients with immune complex-mediated MPGN (n=6) or C3 glomerulonephritis (n=4) with sC5b-9 (serum complement membrane attack complex) plasma levels>1,000ng/mL and 24-hour proteinuria with protein excretion>3.5g identified from the Italian Registry of MPGN and followed up at the Istituto di Ricerche Farmacologiche Mario Negri IRCCS (Bergamo, Italy) between March 4, 2014, and January 7, 2015.
Anti-C5 monoclonal antibody eculizumab administered during 2 sequential 48-week treatment periods separated by one 12-week washout period.
Primary outcome was change in 24-hour proteinuria (median of 3 consecutive measurements) at 24 and 48 weeks.
Median proteinuria decreased from protein excretion of 6.03 (interquartile range [IQR], 4.8-12.4) g/d at baseline to 3.74 (IQR, 3.2-4.4) g/d at 24 weeks (P=0.01) and to 5.06 (IQR, 3.1-5.8) g/d (P=0.006) at 48 weeks of treatment, recovered toward baseline during the washout period, and did not significantly decrease thereafter. Hypoalbuminemia, dyslipidemia, and glomerular sieving function improved during the first treatment period. 3 patients achieved partial remission of nephrotic syndrome and all had undetectable C3 nephritic factors before treatment. Mean measured glomerular filtration rate was 69.7±35.2 versus 87.4±55.1 and 75.8±42.7 versus 76.6±44.1mL/min/1.73m2 at the start versus the end of the first and second treatment periods, respectively, among all 10 study participants. Unlike C3, sC5b-9 plasma levels normalized during both treatment periods and recovered toward baseline during the washout in all patients.
Single-arm design, small sample size.
Eculizumab blunted terminal complement activation in all patients with immune complex-mediated MPGN or C3 glomerulonephritis and nephrotic syndrome, but persistently reduced proteinuria in just a subgroup.
Registered in the EU Clinical Trials Register with study no. 2013-003826-10.

BACKGROUND: Mucous membrane pemphigoid (MMP) is a vesiculobullous, autoimmune disease that occurs primarily in older women. The objective of this study was to perform a retrospective analysis of the intraoral clinical signs, symptoms and diagnostic findings of MMP.
METHODS: The charts of 729 patients in a university-based dental referral practice were reviewed.
RESULTS: Of 729 patients, a clinical diagnosis of MMP was rendered in 29 cases. Of these cases, 93 percent had only oral lesions at the time of presentation, whereas 7 percent had lesions at other sites in addition to the oral cavity. Sixty-eight percent were female and 83 percent of the patients were over 50 years at onset. Common sites of involvement were gingiva and buccal mucosa. Sixty-three percent exhibited erosive or ulcerative lesions. Thirty-five percent showed clinical evidence of epithelial separation. Eighty-eight percent of biopsied patients had histopathologic findings consistent with MMP. Seventy-seven percent of patients exhibited IgG and C3 in the basement membrane region, consistent with pemphigoid. Eighty-two percent of the 29 patients who had two or more lesions were treated with topical corticosteroids.
CONCLUSIONS: The intraoral sites most commonly affected by MMP are the gingiva and buccal mucosa. Routine histopathologic evaluation is an effective diagnostic tool when used in conjunction with direct immunofluorescence.

Thermal injury quantitatively and qualitatively alters hematopoiesis, including monocyte-macrophage lineage changes, resulting in altered mononuclear cell function. These bone marrow cells (BMCs) ultimately become fixed tissue macrophages (e.g., Kupffer cells). To study the effects of thermal injury on macrophage-hepatocyte interactions, rat BMCs were isolated 24 hours after burn injury, and myelopoiesis was induced by 7-day culture in granulocyte-macrophage colony-stimulating factor. Separate cultures included inflammatory mediators with growth factor function (IL-6 or PGE2). Cultured cells were incubated up to 96 hours with isolated normal hepatocytes (+/- lipopolysaccharide stimulation). The 96-hour exposure to postburn BMCs produced less of the acute phase proteins (APPs), C3 and transferrin, but more cytotoxicity as measured by 1-lactate dehydrogenase release. Sham BMCs cultured with added IL-6 caused higher APP release and minimal cytotoxicity, whereas burn BMCs stimulated lower APP release and retained cytotoxicity. In conclusion, myeloid cells regulate APP synthesis differently after thermal injury and may become more cytotoxic to hepatocytes.

This study established a murine model for the study of mycotic mastitis. The mammary glands of BALB/c mice were inoculated on the fifth day of lactation with graded doses (10(4), 10(5) and 10(6) cells) of a pathogenic strain of Candida krusei isolated from bovine mastitis. The animals were killed 1, 2, 3, 4 or 5 days after inoculation. In the infected mammary glands, the pathological reaction consisted of primary infiltration with heterophils and mononuclear cells, focal necrosis, formation of microabscesses, epithelial hyperplasia and some fibrosis. The severity of the changes was dose-dependent and increased with time after infection. An increase in the plasma concentrations of complement factors C1, C3c, C4 and C5, factor B and alpha-2-macroglobulin suggested that an acute phase response and activation of the complement system had occurred as a result of the infection.

We have reported a transmission electron microscopic study of the two C3 convertases of human complement and their precursors. The corresponding proteins and complexes of the classical and alternative pathway appear very similar. Cofactors C3b and C4b are nearly indistinguishable and display a characteristic but highly irregular substructure. C2 and Factor B are globular with diameters of 85 +/- 8 A and 80 +/- 8 A and both consist of three discrete globular domains each approximately 40 A in diameter. Bb and C2a each contain two domains connected by a short linker segment. Both domains of Bb and one domain of C2a are 42 A in diameter (28 kd), while the second domain of C2 is 47 A in diameter (39 kd). Attachment of the enzymatic subunits to cofactors occurs through one domain only.

The formation of classical C3 convertase of complement and its regulation by C4b-binding protein (C4bp) were studied using two different approaches: (a) the analysis was first carried out in fluid phase; a soluble stabilized C3 proconvertase could be assembled from C4b (or C4b-like C4) and iodine-treated C2 in the presence of Ni2+ ions. Upon activation of this complex by C1s, a C3 convertase C4b(C4b-like C4)-C2a was generated which was able to cleave purified C3. C4bp dissociated both C3 proconvertase and C3 convertase, but its effect was more important on C3 convertase. (b) A model system of phospholipid vesicles has been developed to study the assembly of the C3 convertase on a membrane. Among different phospholipid mixtures tested, P-glycerol/P-choline vesicles were found most effective for C4b binding. Optimal conditions were determined for C4b fixation on these vesicles; bound C4b participated in the formation of a functional membrane-associated C3 convertase. C4bp was found to bind to phospholipid vesicles with a higher affinity than C4b; it was able to dissociate the vesicle-associated C3 convertase.

The fluid-phase interaction between factor B and an activated form of C3 (C3b or C3(H2O)) is fundamental to the formation of the alternative complement pathway C3 convertase. The present study reports on the thermodynamic parameters that govern these interactions. The extrinsic fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS) and factor B were found to act as competitive ligands in binding to C3b. Thus, complex formation between C3b or C3(H2O) and factor B could be monitored by the quenching of C3b/C3(H2O)-dependent ANS fluorescence upon the addition of B. Under physiological conditions (0.5 mM Mg2+, 37 degrees C, mu = 0.15), the Ka governing the binding of C3b to B was 2.5 X 10(6) M-1, whereas the interaction of C3(H2O) with factor B was of 5-fold lower affinity. Both reactions were endothermic, with the van\'t Hoff enthalpy being approximately +16.0 kcal mol-1 in each case. Thus, a large positive entropy change provides the net driving force in these interactions. Although Ka increased at higher Mg2+ concentrations, this was not an enthalpy-mediated phenomenon. Taken together, these data are consistent with hydrophobic interactions being dominant in C3b.B or C3(H2O).B complex formation. The enhancement of complex formation by Mg2+ and concomitant increase in delta S suggests that the metal ion plays a role in increasing the number of hydrophobic contacts.

N-Acetyl-aspartyl magnesium glutamate (Rhinaaxia, NAAGA) is a topically active antiallergic dipeptide. The compound acts in two different ways. On the one hand NAAGA inhibits the mast cell-degranulation, on the other hand this compound blocks the activation of the C3-convertase, subsequently followed by a blocked cleavage of the fragments C3a and C5a, respectively. 20 patients suffering from pollinosis were treated for 2 weeks according to a randomized double-blind placebo-controlled study. Besides subjective complaints nasal obstruction was objectively documented via rhinomanometria. 9 out of 10 patients under placebo had to use the rescue drug tritoqualine, a histidine decarboxylase inhibitor, compared to none in the verum group (p less than 0.01). After 14 days of treatment with NAAGA the nasal peak flow rate increased by 21.5 l/min and 21.8 l/min in the tritoqualine/placebo group, respectively (not significant). Nasal obstruction improved statistically significantly after 7 and 14 days of treatment in both groups. Tolerance was reported to be good in either group.

The inhibitory activity of the sodium salt of the anti-inflammatory peptide N-acetyl-aspartyl-glutamic acid (NAAGA) on activation of the classical and alternative pathways of human complement was compared with that of the clinically used magnesium salt of NAAGA (NAAGA-Mg). Sodium salt of NAAGA (NAAGA-Na) inhibited both pathways of activation in a dose-dependent manner at concentration ranging from 1 to 10 mM by acting on formation and/or function of the C3 convertases as shown by the inhibitory capacity of the peptide on the release of the C3 cleavage fragment C3b and C3a. NAAGA-Na was as effective as NAAGA-Mg in inhibiting classical pathway activation at concentration above 10 mM. NAAGA-Na was more effective than NAAGA-Mg in inhibiting the alternative pathway since the sodium salt did not interfere with Mg-dependent formation of the alternative pathway C3 convertase.

In an effort to understand the development and control of autoantibody production, we studied the affinity of autoantibody to the alternative pathway C3/C5 convertase (C3 nephritic factor (C3NeF)) and its autoanti-idiotypic antibodies, Ab2 alpha and Ab2 beta. These were isolated and purified from newborns, normal adults, and patients with membranoproliferative glomerulonephritis. In all cases, both IgG and IgM C3NeF were available for study. The affinity of IgG and IgM C3NeF for their natural Ag (10(8) liters/mol) as well as for the internal image of that Ag displayed on Ab2 beta was high (10(10) liters/mol). Furthermore, the affinity of IgG C3NeF was nearly 100-fold higher in patients than in newborns, whereas there were no significant changes with IgM C3NeF. By contrast, there were not differences in the affinity of IgG Ab2 alpha (which does not display any likeness to the native Ag) from normal adults and patients to any C3NeF isolate. There was, however, a progressive increase in affinity between both Ab2 alpha preparations and IgG C3NeF from newborns, adult normal subjects, and patients, implying an alteration in C3NeF to account for the changes in affinity. These data suggest that Ag-driven affinity maturation occurs with autoantibody but may not occur within the idiotypic network. These data also indicate that as autoantibody affinity matures, it appears to modify its idiotype, perhaps in an effort towards autoregulation.