Static and dynamic compression application and removal on the intervertebral discs of growing rats

Fusionless implants are used to correct pediatric progressive spinal deformities, most of them spanning the intervertebral disc. This study aimed at investigating the effects of in vivo static versus dynamic compression application and removal on discs of growing rats. A microloading device applied compression. 48 immature rats (28 d.o.) were divided into two groups (43d, 53d). Each group included four subgroups: control (no surgery), sham (device installed without loading), static (0.2 MPa) and dynamic compressions (0.2 MPa ± 30% with 0.1 Hz). In 43d subgroups, compression was applied for 15 days. In 53d subgroups, compression was followed by 10 days without loading. Disc heights, nucleus/annulus volumetric proportions and nucleus proteoglycan contents were analyzed using one‐way ANOVA and post‐hoc Tukey comparisons (p < 0.05). Disc heights of 43d and 53d static and dynamic loading rats were lower than shams (p < 0.05). Volumetric proportions remained similar. At 43d, nucleus proteoglycan contents increased in both static and dynamic loading rats. However, at 53d, static loading rats had lower proteoglycan content than dynamic loading rats (p < 0.05). Disc structure is altered following static compression removal, but nucleus proteoglycan content remaining elevated in dynamic group. Dynamic fusionless implants would better preserve disc integrity. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:290–298, 2016.     

Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex–mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex–mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.The MHC class I–like neonatal Fc receptor (FcRn), a heterodimer comprising a heavy chain and β₂-microglobulin light chain, is the major regulator of IgG and albumin homeostasis.¹ Perinatally, FcRn mediates the transfer of IgG from mother to offspring, across the placenta in primates and trans-intestinally in suckling rodents. Throughout life, FcRn protects IgG and albumin from catabolism, explaining the unusually long t_1/2 and high serum levels of these proteins. IgG and albumin taken up by cells by pinocytosis bind strongly to FcRn at pH 6.0–6.5 in endosomes. FcRn-bound ligands are then recycled to the plasma membrane, where they dissociate at pH 7.4, whereas IgG and albumin not bound to FcRn are targeted to lysosomes for degradation. FcRn is thought to promote some autoimmune diseases because it protects pathogenic IgG from degradation. For instance, Fcrn^−/− mice are resistant to passive transfer of arthritis by K/BxN sera and autoimmune skin pathology induced by antibodies targeting autoantigens at the dermal–epidermal junction, although this protection can be overcome by excess autoantibodies.^2–4In kidneys, FcRn is expressed in podocytes and proximal tubular epithelial cells.⁵ Overall, renal FcRn reclaims albumin but facilitates elimination of IgG.⁶ Tubular FcRn mediates IgG transcytosis.⁷ Podocytes use FcRn to clear IgG from the glomerular basement membrane (GBM).⁸ IgG accumulates in the glomeruli of aged Fcrn^−/− mice due to impaired clearance of IgG from the GBM, and saturating this clearance mechanism by excess ligand potentiates the pathogenicity of nephrotoxic sera in wild-type mice. Podocyte FcRn has been postulated to be involved in the clearance of immune complexes (ICs) present in pathologic conditions such as membranous nephropathy.⁵ Expression of FcRn in human podocytes is increased in various immune-mediated glomerular diseases.⁹ Given its role in IgG and albumin handling in the kidneys and systemically, FcRn can be expected to influence the development of immune-mediated kidney diseases at multiple levels. This conjecture awaits experimental verification.To determine the role of FcRn in IgG-mediated glomerular disease, we asked how FcRn deficiency alters the course of disease in mice immunized with the NC1 domain of α3 type IV collagen (α3NC1). We chose this antigen because of its reported ability to induce disease in C57Bl/6 (B6) mice,¹⁰ corroborated in pilot studies (Supplemental Figure 1). Fcrn^−/− mice are hypoalbuminemic due to impaired albumin recycling,¹¹ and also exhibit reduced urinary albumin excretion.¹² As a control for this potential confounder, we used FCRN-humanized mice, which have normal serum albumin because human FcRn recycles mouse albumin but not mouse IgG.¹³All mice immunized with α3NC1 developed circulating mouse IgG anti-α3NC1 antibodies, which reached the maximum titer about 6 weeks later and gradually declined thereafter. At all times, the levels of mouse IgG anti-α3NC1 antibodies in sera from Fcrn^−/− mice and FCRN-humanized mice were approximately 50%–70% lower than those in wild-type mouse sera (Figure 1A). The results were similar for mouse IgG1, IgG2b, and IgG2c anti-α3NC1 antibodies (Supplemental Figure 2). Wild-type B6 mice immunized with α3NC1 started developing progressive albuminuria 8–10 weeks later (Figure 1B). By week 14, the urinary albumin creatinine ratio increased approximately 100-fold, and hypoalbuminemia developed (Figure 1C). Urinary albumin excretion in Fcrn^−/− mice and FCRN-humanized mice immunized with α3NC1 was not significantly higher than in adjuvant-immunized control mice. No mice developed renal failure (Supplemental Figure 3).Open in a separate windowFigure 1.FcRn ablation reduces serum levels of mouse IgG anti-α3NC1 antibodies and prevents the development of albuminuria in α3NC1-immunized mice. (A) The left panel shows circulating mIgG anti-α3NC1 antibodies from C57Bl6 wild-type mice (○), Fcrn^−/− mice (□), FCRN-humanized (hFCRN) mice (◇), and the control CFA group (△), which are assayed by indirect ELISA in plates coated with α3NC1 (100 ng/well). Mouse sera are diluted 1:5000. The right panel shows the significance of circulating mIgG anti-α3NC1 antibody differences among groups at week 12, as assessed by one-way ANOVA followed by Bonferroni post tests for pairwise comparisons. (B) The left panel shows that the urinary albumin creatinine ratio (mean±SEM) time course is monitored in C57Bl6 wild-type mice (○), Fcrn^−/− mice (□), and hFCRN mice (◇) immunized with α3NC1 (n=5–8 mice in each group, from two separate experiments). Mice in the control group (△) are immunized with adjuvant alone (n=9). The right panel shows the urinary albumin creatinine ratio (mean±SEM) at 14 weeks, when mice are euthanized. The significance of differences among groups is assessed by one-way ANOVA followed by Bonferroni post tests for pairwise comparisons. (C) The left panel shows SDS-PAGE analysis of serum (0.5 µl/lane) and urine samples (2 µl/lane) from CFA-immunized control mice (a) and α3NC1-immunized wild-type mice (b), Fcrn^−/− mice (c), and hFCRN mice (d) collected at week 14. The right panel presents a densitometric analysis of the relative levels of albumin in mouse serum samples showing that α3NC1-immunized wild-type mice developed hypoalbuminemia. *P<0.05 by two-tailed t test versus CFA-immunized wild-type mice; **P<0.01; ***P<0.001. ns, not significant; WT, wild type.At 14 weeks after α3NC1 immunization, kidneys examined by light microscopy showed mild glomerular pathology, with few crescents and relatively little inflammation (Figure 2A), similar to α3NC1-immunized DBA/1 mice with comparable albuminuria.^14,15 Electron microscopy showed extensive subepithelial IC deposits surrounded by an expanded GBM and effacement of podocyte foot processes in α3NC1-immunized B6 mice, whereas Fcrn^−/− mice had fewer subepithelial deposits (Figure 2B, Supplemental Figure 4). Immunofluorescence staining showed granular capillary loop deposition of mouse IgG, exogenous antigen, C3, and C5b-9, more intense in wild-type mice than in Fcrn^−/− mice and FCRN-humanized mice (Figure 2, Ca–Cp, Supplemental Figure 5). A loss of nephrin staining, indicative of podocyte injury, occurred in α3NC1-immunized B6 mice but not in Fcrn^−/− mice or FCRN-humanized mice (Figure 2, Cq–Ct).Open in a separate windowFigure 2.FcRn deficiency reduces formation of pathogenic subepithelial ICs. (A) Light microscopic evaluation of kidneys from adjuvant-immunized control mice (a) and α3NC1-immunized wild-type mice (b) and Fcrn^−/− mice (c) revealed few pathogenic changes and the absence of glomerular inflammation (periodic acid–Schiff staining). (B) Transmission electron microscopy shows normal GBM (arrow) and podocyte foot processes in control mice (a), extensive subepithelial electron dense deposits (arrowhead), thickened GBM, and podocyte foot process effacement in α3NC1-immunized wild-type mice (b), and fewer IC deposits in the Fcrn^−/− mice (c). (C) Immunofluorescence analysis of kidneys from adjuvant-immunized control mice (a, e, i, m, and q) and α3NC1-immunized wild-type mice (b, f, j, n, and r), FcRn^−/− mice (c, g, k, o, and s), and hFCRN mice (d, h, l, p, and t) evaluate the deposition of mouse IgG (a–d), exogenous α3NC1 antigen stained by mAb RH34 (e–h), mouse C3c (i–l), C5b-9 (m–p), and nephrin staining (q–t) at 14 weeks. Wild-type mice exhibit linear-granular GBM deposition of mouse IgG and granular GBM deposition of exogenous antigen, C3, and C5b-9, which are attenuated in Fcrn^−/− mice and hFCRN mice and essentially absent in control mice. Compared with control mice, α3NC1-immunized wild-type mice but not Fcrn^−/− or hFCRN mice exhibit a loss of nephrin staining, indicative of podocyte injury. WT, wild type; EM, electron microscopy, PAS, periodic acid–Schiff. Original magnification, ×400 in A; ×2850 in B; ×200 in C.Because B6 mice immunized with bovine GBM NC1 hexamers have normal kidney function and histology despite linear GBM deposition of IgG autoantibodies binding to mouse α345(IV) collagen (Supplemental Figure 1), the question arises as to what causes proteinuria in α3NC1-immunized mice. Because the clinical presentation, morphology, and effector mechanisms depend on where ICs are localized in the capillary wall, we compared IgG distribution in α3NC1-immunized mice and mice injected with anti-α3NC1 antibodies modeling anti-GBM autoantibodies. The distribution and relative abundance of mouse IgG, as imaged by immunoperoxidase immunoelectron microscopy and stochastic optical reconstruction microscopy (STORM), a method for super-resolution fluorescence microscopy, were concordant. In α3NC1-immunized mice, IgG deposition was abundant in the areas of expanded GBM and especially in regions corresponding to the subepithelial dense deposits seen by routine electron microscopy. By contrast, in mice injected with α3NC1-specific anti-GBM mAb, the IgG was confined to an ultrastructurally normal GBM that lacked subepithelial deposits (Figure 3).Open in a separate windowFigure 3.Localization of IgG by high-resolution imaging. The localization of mouse IgG in glomerular capillary walls of wild-type mice immunized with α3NC1 (A, C–E), or intravenously injected with anti-mouse α3NC1 IgG mAb 8D1 (B, F–H) is determined by immunoperoxidase electron microscopy (A and B) and STORM imaging (C–H). In A, the GBM is irregularly thickened, and abundant electron dense peroxidase reaction product is present in discontinuous, subepithelial patterns beneath broadly effaced podocyte foot processes (arrows). In B, the peroxidase reaction product is diffusely present throughout the GBM (arrowhead), but less abundant compared with A. Electron dense deposits are absent, and podocyte foot process architecture appears normal. (C–E) By STORM imaging, anti-agrin (blue) identifies both normal and thickened areas of the GBM, both of which contain dense accumulations of mouse IgG throughout (red). The electron microscopy correlation in E shows GBM staining with respect to the podocytes and endothelial cells. (F–H) IgG mAb 8D1 (red) is present in the GBM, which shows no evidence of thickening. CL, capillary lumen; EM, electron microscopy En, endothelium;Po, podocyte.Subepithelial ICs, a hallmark of human membranous nephropathy (MN), form when IgG antibodies bind to podocyte antigens, such as phospholipase A2 receptor (PLA2R) and neutral endopeptidase (NEP), or to planted antigens, such as cationic BSA.^16–18 Subsequent expansion of the GBM, complement activation, and podocyte injury by C5b-9 cause proteinuria. Although it is unexpected, formation of subepithelial ICs in α3NC1-immunized mice may be explained by exogenous α3NC1 deposited in glomeruli acting as a planted antigen.¹⁹ Alternatively, anti-α3NC1 antibodies in complex with α3NC1 antigen may act as surrogate antipodocyte antibodies, because α3NC1-containing ICs bind to podocytes.²⁰ After four immunizations with α3NC1 monomers, B6 mice and DBA/1 mice eventually develop crescentic GN by 26 and 10 weeks, respectively.^10,14 The combination of subepithelial ICs and crescentic anti-GBM antibody GN was most recently described in a series of eight patients with circulating anti-α3NC1 autoantibodies but undetectable anti-PLA2R autoantibodies.²¹In contrast to wild-type B6 mice, congenic Fcrn^−/− mice and FCRN-humanized mice did not develop albuminuria after α3NC1 immunization. Their resistance to proteinuria was associated with lower serum titers of anti-α3NC1 IgG antibodies and reduced glomerular deposition of IgG, antigen, C3, and C5b-9. Because C5b-9 is an essential mediator of podocyte damage and proteinuria by subepithelial ICs,^22,23 reduced complement activation potentially explains the attenuated glomerular pathology in FcRn-deficient mice. The resistance of FCRN-humanized mice indicates that FcRn promotes IC-mediated glomerular disease due to its interaction with IgG rather than albumin. We propose that FcRn promotes the development of subepithelial ICs and subsequent glomerular injury primarily by maintaining higher serum levels of pathogenic IgG (Supplemental Figure 6). However, we cannot formally exclude a possible pathogenic role of podocyte FcRn, whose stimulation by ICs may induce maladaptive signaling.⁹ Future studies in mice with podocyte-specific ablation of FcRn would address this possibility.Our findings identify FcRn as a potential target for therapeutic intervention in IC-mediated glomerular diseases, typically treated with nonspecific immunosuppressants that are toxic and sometimes ineffective. More specific therapies include ablation of B cells by rituximab. In patients with idiopathic MN who respond to rituximab therapy, serum levels of anti-PLA2R IgG autoantibodies decline over a period of many months, and their disappearance is followed by resolution of proteinuria.²⁴ The slow decline in proteinuria is problematic for patients already suffering from complications of nephrotic syndrome, who would benefit from ancillary therapies that reduce pathogenic IgG antibodies more rapidly. This may be achieved by inhibiting FcRn.One implementation of this concept is therapy with high-dose intravenous Ig (HD-IVIG). HD-IVIG accelerates the degradation of IgG by saturating FcRn,²⁵ one of the mechanisms that explain the beneficial effects of HD-IVIG therapy in some autoimmune diseases.³ In pregnant women with circulating anti-NEP alloantibodies mediating antenatal MN, treatment with HD-IVIG reduces the titers of IgG alloantibodies by approximately 30% within 2–3 weeks.²⁶ However, HD-IVIG is inefficient, because large amounts of IgG (1–2 g/kg) cause relatively modest reductions in pathogenic IgG titers. Specific FcRn inhibitors recapitulate this activity of HD-IVIG more effectively at lower doses. By reducing pathogenic IgG levels, function-blocking anti-FcRn mAbs ameliorate experimental myasthenia gravis in rats,²⁷ and engineered IgG “Abdegs” that bind with high affinity to FcRn ameliorate arthritis transferred by K/BxN serum.²⁸To assess the translational potential of our findings, we asked whether pharmacologic blockade of human FcRn can reproduce the effects of genetic FcRn deficiency. To this end, FCRN-humanized and Fcrn^−/− mice were passively immunized with human IgG containing anti-α3NC1 (Goodpasture) autoantibodies. To inhibit human FcRn, we used a lysine analog of SYN1436 (Figure 4A),²⁹ a peptide that binds with subnanomolar affinity to human FcRn, thus preventing IgG binding.³⁰ In vivo, SYN1436 reduces IgG levels in cynomolgus monkeys by 80%.³⁰ Serum anti-α3NC1 autoantibodies in FCRN-humanized mice treated with anti-FcRn peptide, but not with control peptide, sharply decreased to the same levels as in Fcrn^−/− mice (Figure 4B), and were no longer detected after 4 days. In mice, human IgG elicits murine anti-human IgG antibodies, forming ICs that can deposit in glomeruli, as shown in active serum sickness models. Glomerular deposition of ICs containing human IgG was abolished in mice treated with anti-FcRn peptide, but not with control peptide (Figure 4C). Linear GBM deposition of human anti-GBM IgG was not observed, because the epitopes recognized by Goodpasture autoantibodies are completely inaccessible in the mouse GBM.³¹ These results provide proof of concept that therapies targeting human FcRn effectively lower serum levels of pathogenic human IgG autoantibodies, which could be beneficial in patients with IgG-mediated kidney diseases. Because FcRn also mediates the trans-placental transfer of IgG from mother to the fetus, FcRn inhibition may be particularly attractive for preventing antenatal MN caused by maternal anti-NEP alloantibodies.Open in a separate windowFigure 4.Pharmacologic blockade of human FcRn accelerates the catabolism of human IgG autoantibodies in FCRN-humanized mice. (A) Structure of a peptide that binds with high affinity to human FcRn, competitively inhibiting its interaction with human IgG (top). The control peptide (bottom) containing D-amino acids does not bind to human FcRn. Pen, Sar, and NMeLeu denote penicillamine, sarcosine, and N-methyl-leucine, respectively. (B) Serum level of human IgG anti-α3NC1 antibodies in FCRN-humanized mice treated with anti-FcRn peptide (▪) or control peptide (●) and in Fcrn^−/− (▲) mice sera (n=3 in each group) is analyzed by indirect ELISA in plates coated with α3NC1 (100 ng/well). Mouse sera are diluted 1:500. (C) Kidney deposition of human IgG (a and b) and mouse IgG (c and d) in FCRN-humanized mice treated with control peptide (a and c) or anti-FcRn peptide (b and d) is evaluated by direct immunofluorescence staining. Treatment with anti-FcRn peptide prevents the glomerular deposition of ICs containing human IgG.     

Ovarian cancer--diagnostic dilemmas and unusual therapeutic response

The aims of this paper are both to highlight some dilemmas concerning the diagnosis of primary epithelial ovarian carcinoma mainly in its early stages and to underline the capricious responses of this type of malignancy to an otherwise well coded modern management. This study is based on the analysis of the records of 78 patients with ovarian carcinoma admitted for diagnosis and surgical treatment to the Department of General Surgery of Craiova C.F.R. Clinic from 1993 through 2003. The results of this analysis are difficult to interpret due to loss to follow up (in terms of response rates) of some of our 78 operated on ovarian carcinoma patients who went on with their platinum-based chemotherapy (following surgical cytoreduction) under the supervision of different Oncology Departments nationwide. Nevertheless, it is worth mentioning that most of this study patients (71.9%) presented with advanced-stage (III and IV) ovarian carcinoma which sometimes seemed quite confusing by its clinical polymorphism but its prognosis was very much related to both the degree of surgical cytoreduction accomplished and tumor sensitivity to chemotherapy. Finally, although this study does not allow us to draw firm conclusions it is an attempt to share out our current perception on the primary epithelial ovarian cancer management.