Evaluation of plasma muscle enzyme activity as an indicator of lesion characteristics and prognosis in horses undergoing celiotomy for acute gastrointestinal pain

Background

In horses undergoing celiotomy for acute gastrointestinal pain, identification of variables correlating with lesion severity and location, and survival provide veterinarians and owners with information that aids in making informed decisions regarding appropriate treatment. Muscle enzyme activity is often increased in horses undergoing celiotomy for acute gastrointestinal pain and it is not known if muscle enzyme activity increase is specific to lesion type or impacts prognosis for survival. The objective of this study was to evaluate the relationship of pre-operative increase in muscle enzyme activities with intestinal lesion characteristics, specifically lesion location (large versus small intestine) and whether it was strangulating versus nonstrangulating, and case survival in horses undergoing celiotomy for acute gastrointestinal pain.

Methods

Records of 241 horses undergoing exploratory laparotomy for colic were reviewed retrospectively. Evaluation of preoperative plasma aspartate aminotransferase (AST), creatine kinase (CK), sorbitol dehydrogenase (SDH), and gamma-glutamyltransferase (GGT) activities, fibrinogen and glucose concentrations, and hematocrit (HCT) and their association with gastrointestinal lesion characteristics and survival was performed.

Results

Pre-operative increase in plasma CK and AST activity, and HCT and decrease in plasma bilirubin concentration were significantly associated with presence of lesions resulting in intestinal ischemia. Increase in plasma CK activity and HCT were significantly associated with a decreased probability of survival to hospital discharge. Plasma GGT and SDH activity, and glucose and fibrinogen concentration were not significantly associated with survival or severity of disease in multivariate analysis.

Conclusions

Plasma muscle enyzme activity may be useful as a prognostic indicator in equine colic cases. Given that increases in plasma CK and AST activity were significantly associated with nonsurvival and the presence of intestinal ischemia, preoperative increase in these enzyme activities could assist in identification of disease severity and prognosis of horses undergoing celiotomy for acute gastrointestinal pain. Further study is indicated to elucidate the etiology of increased muscle enzyme activity in horses with surgical colic disease observed in this preliminary study.

     

High‐resolution array‐CGH in patients with oculocutaneous albinism identifies new deletions of the TYR,OCA2, and SLC45A2 genes and a complex rearrangement of the OCA2 gene

Oculocutaneous albinism (OCA) is caused by mutations in six different genes, and their molecular diagnosis encompasses the search for point mutations and intragenic rearrangements. Here, we used high‐resolution array‐comparative genome hybridization (CGH) to search for rearrangements across exons, introns and regulatory sequences of four OCA genes: TYR, OCA2, TYRP1, and SLC45A2. We identified a total of ten new deletions in TYR, OCA2, and SLC45A2. A complex rearrangement of OCA2 was found in two unrelated patients. Whole‐genome sequencing showed deletion of a 184‐kb fragment (identical to a deletion previously found in Polish patients), whereby a large portion of the deleted sequence was re‐inserted after severe reshuffling into intron 1 of OCA2. The high‐resolution array‐CGH presented here is a powerful tool to detect gene rearrangements. Finally, we review all known deletions of the OCA1–4 genes reported so far in the literature and show that deletions or duplications account for 5.6% of all mutations identified in the OCA1–4 genes.     

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.     

Proteomic Analysis of C2C12 Myoblast and Myotube Exosome-Like Vesicles: A New Paradigm for Myoblast-Myotube Cross Talk?

Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.     

3-amino-4-sulfonylpyridinone acetamide and related pyridothiadiazine thrombin inhibitors

We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-substituent, and forms a hydrogen bond to the insertion loop tyrosine OH. We also describe a related series of fused bicyclic dihydrothiadiazinedioxide derivatives, of which one had improved pharmacokinetics in dogs after oral dosing.     

A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker

Expression profiles represent new molecular tools that are useful to characterize the successive steps of tumor progression and the prediction of recurrence or chemotherapy response. In this study, we have used quantitative proteomic analysis to compare different stages of colorectal cancer. A combination of laser microdissection, OFFGEL separation, iTRAQ labeling, and MALDI-TOF/TOF MS was used to explore the proteome of 28 colorectal cancer tissues. Two software packages were used for identification and quantification of differentially expressed proteins: Protein Pilot and iQuantitator. Based on ～1,190,702 MS/MS spectra, a total of 3138 proteins were identified, which represents the largest database of colorectal cancer realized to date and demonstrates the value of our quantitative proteomic approach. In this way, individual protein expression and variation have been identified for each patient and for each colorectal dysplasia and cancer stage (stages I-IV). A total of 555 proteins presenting a significant fold change were quantified in the different stages, and this differential expression correlated with immunohistochemistry results reported in the Human Protein Atlas database. To identify a candidate biomarker of the early stages of colorectal cancer, we focused our study on secreted proteins. In this way, we identified olfactomedin-4, which was overexpressed in adenomas and in early stages of colorectal tumors. This early stage overexpression was confirmed by immunohistochemistry in 126 paraffin-embedded tissues. Our results also indicate that OLFM4 is regulated by the Ras-NF-κB2 pathway, one of the main oncogenic pathways deregulated in colorectal tumors.     

Identification of a sudden cardiac death susceptibility locus at 2q24.2 through genome-wide association in European ancestry individuals

Sudden cardiac death (SCD) continues to be one of the leading causes of mortality worldwide, with an annual incidence estimated at 250,000–300,000 in the United States and with the vast majority occurring in the setting of coronary disease. We performed a genome-wide association meta-analysis in 1,283 SCD cases and >20,000 control individuals of European ancestry from 5 studies, with follow-up genotyping in up to 3,119 SCD cases and 11,146 controls from 11 European ancestry studies, and identify the BAZ2B locus as associated with SCD (P = 1.8×10⁻¹⁰). The risk allele, while ancestral, has a frequency of ∼1.4%, suggesting strong negative selection and increases risk for SCD by 1.92–fold per allele (95% CI 1.57–2.34). We also tested the role of 49 SNPs previously implicated in modulating electrocardiographic traits (QRS, QT, and RR intervals). Consistent with epidemiological studies showing increased risk of SCD with prolonged QRS/QT intervals, the interval-prolonging alleles are in aggregate associated with increased risk for SCD (P = 0.006).     

Regulation of T cell priming by lymphoid stroma

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues.