Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

Abstract

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.

<p>Early adoption of the fluocinolone acetonide (FAc) intravitreal implant in patients with persistent or recurrent diabetic macular edema (DME)</p>

“…It is deemed to be clinically significant if there is thickening of the retina at or within 500 µm of the center of the macula, or if hard exudates at or within 500 µm of the center of the macula are associated with thickening of the adjacent retina (excluding residual hard exudates remaining after disappearance of retinal thickening).The exact pathogenesis of DME is not fully understood,4 but it is defined by an abnormal collection of extracapillary fluid due to blood–retinal barrier breakdown because of increased production of inflammatory mediators and vascular permeability factors and loss of endothelial tight junctions 5. Vascular endothelial growth factor (VEGF) is believed to be a key mediator, promoting angiogenesis and breakdown of the blood–retinal barrier, resulting in accumulation of plasma proteins such as albumin, which exert high oncotic pressure in the neural interstitium, leading to interstitial edema 4,6. In DME, upregulation of multiple inflammatory cytokines occurs, and the resultant inflammatory cascade produces diverse anatomical and biochemical changes in the eye 6…”

“…Chronic hyperglycemia, hypertension, and hyperlipidemia are implicated in the development of diabetic retinopathy and DME,4 so management of blood sugar/diabetes, blood pressure, and lipids should be optimized to reduce the risk and slow progression of diabetic retinopathy 1,2. Although this can substantially decrease the risk of vision loss, a significant proportion of patients with diabetes develop DME or proliferative changes that require DME-specific treatments, such as laser therapy, anti-VEGF agents, and corticosteroids 1,4…”

“…Intravitreal anti-VEGF monotherapy is also considered for DME associated with proliferative diabetic retinopathy 2. Although many patients gain significant benefit from anti-VEGF therapies licensed for DME, which include ranibizumab, bevacizumab (off-label), aflibercept, and pegaptanib, a considerable number do not,6 and macular swelling after intravitreal anti-VEGF may require repeat treatments 4…”

“…Panretinal photocoagulation aims to destroy areas where there are capillary nonperfusion and retinal ischemia 3. Although laser photocoagulation reduces the risk of further vision loss, improvement in vision only occurs in about 12% of patients after 3 years 1,4,6. Furthermore, side effects can include scotoma, altered color perception, night blindness, hemorrhage, transient visual loss, macular edema, and visual field defects 6,7…”

Grp94 in complexes with IgG is a soluble diagnostic marker of gastrointestinal tumors and displays immune-stimulating activity on peripheral blood immune cells

“…Previous works had stably demonstrated that when liberated in the extracellular milieu - as it also occurs in autoimmune diseases [27, 31] - Grp94 is never detected as a single protein, but is always found linked in big, stable complexes with IgG [26, 28]. To explore the possibility that Grp94-IgG complexes could also circulate in cancer patients, we first tested any single plasma sample with anti-Grp94 Abs in Western blotting (WB) (Figure 2A).…”