Renal aquaporin-4 associated pathology in TG-26 mice

Human immunodeficiency virus-associated nephropathy (HIVAN) is a leading cause of end-stage renal disease in HIV patients, which is characterized by glomerulosclerosis and renal tubular dysfunction. Aquaporin-4 (AQP-4) is a membrane bound water channel protein that plays a distinct role in water reabsorption from renal tubular fluid. It has been proven that failure of AQP-4 insertion into the renal tubular membrane leads to renal dysfunction. However, the role of AQP-4 in HIVAN is unclear. We hypothesize that impaired water reabsorption leads to renal injury in HIVAN, where AQP-4 plays a crucial role. Renal function is assessed by urinary protein and serum blood urea nitrogen (BUN). Kidneys from HIV Transgenic (TG26) mice (HIVAN animal model) were compared to wild type mice by immunostaining, immunoblotting and quantitative RT-PCR. TG26 mice had increased proteinuria and BUN. We found decreased AQP-4 levels in the renal medulla, increased endothelin-1, endothelin receptor A and reduced Sirtuin1 (SIRT-1) levels in TG26 mice. Also, oxidative and endoplasmic reticulum stress was enhanced in kidneys of TG26 mice. We provide the first evidence that AQP-4 is inhibited due to induction of HIV associated stress in the kidneys of TG26 mice which limits water reabsorption in the kidney which may be one of the cause associated with HIVAN, impairing kidney physiology. AQP-4 dysregulation in TG26 mice suggests that similar changes may occur in HIVAN patients. This work may identify new therapeutic targets to be evaluated in HIVAN.     

Mediation of platelet adhesion to fibrillar collagen in flowing blood by a proteolytic fragment of human von Willebrand factor

The effect of purified von Willebrand Factor (vWF) fragments, SpII (dimer of two 110 kd subunits) and SpIII (dimer of two 170 kd subunits) obtained with S aureus V-8 protease was tested upon platelet adhesion to collagen. Purified fibrillar human collagen coated onto cover slips was incubated with SpII, SpIII, or undigested vWF and exposed to reconstituted human blood in a parallel-plate perfusion chamber at a high shear rate. Platelet-collagen interactions were estimated using 51Cr-platelets and quantitative morphometry. When blood was reconstituted with citrated autologous plasma, SpIII and vWF strikingly enhanced platelet adhesion to collagen whereas SpII had no effect. When blood was reconstituted with human albumin and divalent cations, SpIII and vWF again promoted platelet adhesion to collagen. In conclusion, our data suggest that (1) SpIII, the N-terminal portion of vWF which binds to platelet membrane glycoprotein Ib, functionally substitutes for vWF in supporting platelet adhesion to collagen; (2) SpII, the C- terminal portion which binds to glycoprotein IIb/IIIa, has no such effect; (3) in addition to its platelet binding domain, SpIII contains another site for binding to collagen; and (4) the multimeric structure of vWF is not required for platelet adhesion to collagen.     

Profiling of Silk Sericin from Cocoons of Three Southern African Wild Silk Moths with a Focus on Their Antimicrobial and Antioxidant Properties

Silk sericin was extracted from the cocoons of three Southern African wild silk moth species, namely Gonometapostica, G. rufobrunnae (Lepidoptera: Lasiocampidae), and Argema mimosae (Lepidoptera: Saturniidae); these three sericin extracts were analysed to determine the relationship that exists between their chemical structures and their functional properties. The relationship was investigated by utilising several methods that include the determination of the amino acid composition, and characterisation of the secondary structures with Fourier transformation infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The antibacterial properties of these three sericin extracts were evaluated by an agar well diffusion assay with three Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis) as test microorganisms; and, lastly, the antioxidant properties of the three sericin extracts were determined using several scavenging methods that include the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS˙⁺), and the ferric reducing antioxidant power (FRAP) assay. The amino acid composition in the silk sericin extracts from G. postica, G. rufobrunnea, and Argema mimosa in terms of the polar/non-polar ratio (P/NP) was found to be 65:35, 56:44, and 59:41, respectively. The FTIR spectra of these three silk sericin extracts showed distinct major bands such as amide A (3265 cm⁻¹), amide B (3062 cm⁻¹), amide I (1644 cm⁻¹), amide II (1538 cm⁻¹), and amide III (1244 cm⁻¹). The XRD patterns of the silk sericin extracts revealed both amorphous and α-helical structures, with small crystalline regions. All three silk sericin extracts presented potent antibacterial efficacy against the three Gram-positive bacteria and were found to have excellent antioxidant activities against the tested free radicals.     

Absence of anamnestic response after transfusion of washed red blood cells in haemophilia A patients with antibody to factor VIII

Washed red blood cells (WRC) have been used for replacing the blood loss in haemophilia A patients with antibody to Factor VIII. Levels of VIII coagulant activity (VIII:C), VIII coagulant antigen (VIII:CAg) and VIII related antigen (VIIIR:Ag) have been measured during the different steps of the preparation of WRC. The amount of VIII:CAg decreases very rapidly after washing and both are undetectable in the final product. These results were correlated with the absence of anamnestic response in 10 haemophilia A patients with inhibitor known as high responders.     

Prognostic value of anti-ADAMTS 13 antibody features (Ig isotype, titer, and inhibitory effect) in a cohort of 35 adult French patients undergoing a first episode of thrombotic microangiopathy with undetectable ADAMTS 13 activity

To study both the pathophysiologic and the prognostic value of ADAMTS13 in thrombotic microangiopathies (TMAs), we enrolled a cohort of 35 adult patients combining a first acute episode of TMA, an undetectable (below 5%) ADAMTS13 activity in plasma, and no clinical background such as sepsis, cancer, HIV, and transplantation. All patients were treated by steroids and plasma exchange, and an 18-month follow-up was scheduled. Remission was obtained in 32 patients (91.4%), and 3 patients died (8.6%) after the first attack. At presentation, ADAMTS13 antigen was decreased in 32 patients (91.4%), an ADAMTS13 inhibitor was detectable in 31 patients (89%), and an anti-ADAMTS13 IgG/IgM/IgA was present in 33 patients (94%). The 3 decedent patients were characterized by the association of several anti-ADAMTS13 Ig isotypes, including very high IgA titers, while mortality was independent of the ADAMTS13 inhibitor titer. In survivors, ADAMTS13 activity in remission increased to levels above 15% in 19 patients (59%) but remained undetectable in 13 patients (41%). Six patients relapsed either once or twice (19%) during the follow-up. High levels of inhibitory anti-ADAMTS13 IgG at presentation were associated with the persistence of an undetectable ADAMTS13 activity in remission, the latter being predictive for relapses within an 18-month delay.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

Specific von Willebrand factor-cleaving protease in thrombotic microangiopathies: a study of 111 cases

Retrospective studies of patients with thrombotic microangiopathies (TMAs) have shown that a deficient activity of von Willebrand factor (vWF)-cleaving protease is involved in thrombotic thrombocytopenic purpura (TTP) but not in the hemolytic-uremic syndrome (HUS). To further analyze the relevance of this enzymatic activity in TMA diagnosis, a 20-month multicenter study of vWF-cleaving protease activity was conducted in adult patients prospectively enrolled in the acute phase of TMA. Patients with sporadic (n = 85), intermittent (n = 21), or familial recurrent (n = 5) forms of TMA (66 manifesting as TTP and 45 as HUS) were included. TMA was either idiopathic (n = 42) or secondary to an identified clinical context (n = 69). vWF-cleaving protease activity was normal in 46 cases (7 TTP and 39 HUS) and decreased in 65 cases (59 TTP and 6 HUS). A protease inhibitor was detected in 31 cases and was observed only in patients manifesting TTP with a total absence of protease activity. Among the 111 patients, mean vWF antigen levels were increased and the multimeric distribution of vWF was very heterogeneous, showing either a defect of the high-molecular-weight forms (n = 40), a normal pattern (n = 21), or the presence of unusually large multimers (n = 50). Statistical analysis showed that vWF-protease deficiency was associated with the severity of thrombocytopenia (P <.01). This study emphasizes that vWF-cleaving protease deficiency specifically concerns a subgroup of TMA corresponding to the TTP entity.