Platelet Thrombus Formation in eHUS is Prevented by Anti-MBL2

Epidemic Hemolytic Uremic Syndrome (eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C−/−;MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.

Aberrant Cortical Activity In Multiple GCaMP6-Expressing Transgenic Mouse Lines

Transgenic mouse lines are invaluable tools for neuroscience but as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically-encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, though rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

Human Leucocyte Antigen-G (HLA-G) and Its Murine Functional Homolog Qa2 in theTrypanosoma cruziInfection

Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression afterTrypanosoma cruziinfection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated theHLA-G3′ untranslated region (3′UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression (heart, colon, and esophagus) in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues duringT. cruziexperimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differentialHLA-G3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels ofQa2and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, andNOS-2) genes were induced only during the acuteT. cruziinfection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimentalT. cruziinfection.

The Carboxyl-Terminal Domains of ADAMTS13 Cause Substrate-Dependent Divergence of ADAMTS13 Activity.

ADAMTS13, a circulating metalloprotease that cleaves the Y1605-M1606 bond of VWF, is critical for preventing intravascular platelet thrombosis of thrombotic thrombocytopenic purpura. Unlike genetic deficiency of ADAMTS13 in patients, inactivation of ADAMTS13 created by homologous recombination causes either no or delayed-onset phenotypic abnormalities in mice, depending on the strains examined. In order to further understand the role of ADAMTS13 in VWF homeostasis, we investigated the structure and function of the enzyme in various mouse strains. As in human subjects, RT PCR analysis revealed that ADAMTS13 was expressed primarily in the mouse liver. However, the enzyme activity levels of mouse plasma ADAMTS13 segregated in two groups: a low group (C56BL/6J strain, 24%±10% of normal human plasma, N=21; and DBA/2J strain, 24%±7%, N=6), and a high group (FVB/NJ strain, 288%±60%, N=23; and 129×1/SvJ strain, 313%±39%, N=6). No such difference was detected when GST-His fusion or FRET form of VWF73 (D1596-R1668) peptide was used for activity measurement. Real-time analysis of RNA extracted from mouse livers showed that the ADAMTS13 transcript levels were similar between the two groups. Cloning of the mouse liver ADAMTS13 yielded a predominant 3.5kb instead of the expected 4.3kb species from the low-activity group. This truncated cDNA contained the first 23 exons of the full-length (FL) ADAMTS13 and an extraneous sequence derived from the long terminal repeat sequence of a previously described IAP-type retrotransposone located in intron 23 of affected mouse genome. Protein expression analysis of the cloned cDNA in HEK 293 and MDCK cells showed that both full-length and IAP-type ADAMTS13 proteins were secreted efficiently without evidence of polarity. Nevertheless, the IAP-type ADAMTS13 was only 10% as active as FL ADAMTS13 in cleaving VWF multimers, but was 50% – 70% as effective in cleaving human or mouse VWF73 peptides, suggesting that truncation and/or presence of IAP sequence at the C-terminus sequence of ADAMTS13 markedly impeded cleavage of VWF multimers. Crossbreeding between C57BL/6J and FVB/NJ strains of mice confirmed that the low-activity phenotype was linked to homozygous IAP genotype. In SDS agarose gel analysis, both C57BL/6J and FVB/NJ strains contained ultra large multimers similar to those observed in ADAMTS13-null mice (kindly provided by D. Motto and D. Ginsburg). Analysis of recombinant human ADAMTS13 proteins also showed that proteins truncated at the disintegrin or spacer domain were relatively more active in cleaving VWF peptide substrates than cleaving VWF multimers. In conclusion, the presence of an IAP-type retrotransposone in the ADAMTS13 gene enhances alternative splicing, resulting in predominant expression of IAP types of ADAMTS13 in the affected mouse strains. Nevertheless, ADAMTS13 does not regulate VWF multimer size, and deficiency of ADAMTS13 is phenotypically silent in the mice examined. The carboxyl-terminus sequence of ADAMTS13 may enhance interaction with VWF multimers and its subsequent cleavage. Clinically, ADAMTS13 assay results should be interpreted with caution when VWF peptide-based proteins are used as the substrate, because they yield higher activity values for ADAMTS13 proteins lacking the carboxyl terminus sequence.