The Tox Gene Encodes Two Proteins with Distinct and Shared Roles in Gene Regulation.

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection-associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

NLRP3 Inflammasome Mediates Immune-Stromal Interactions in Vasculitis.

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In-Hospital Outcomes of Rotational Atherectomy in High-Risk Patients With Severely Calcified Left Main Coronary Artery Disease: A Single-Center Experience.

BACKGROUND: Severe coronary artery calcification is a challenge for percutaneous coronary intervention (PCI), particularly in left main coronary artery disease (LM-CAD). Rotational atherectomy (RA) is a useful tool for modification of calcified plaque prior to PCI. We report our experience with RA for severely calcified LM-CAD. METHODS: From January 2008 to January 2017, all patients who underwent RA-assisted LM-PCI were evaluated. The study population included both protected and unprotected LM-CAD patients. Clinical characteristics and in-hospital outcomes were collected retrospectively. In-hospital outcomes included post-PCI myocardial infarction, stroke, death, emergency coronary artery bypass graft surgery, and urgent repeat PCI. Angiographic success was defined by residual stenosis <20% and presence of TIMI 3 flow. RESULTS: Fifty-five consecutive patients who underwent RA-assisted PCI of LM-CAD were identified (mean age, 73.0 ± 10 years; 64% male). Mean left ventricular ejection fraction was 37.5 ± 15.7%. Fifty-one patients (93%) had multivessel disease and 39 patients (71%) underwent RA-assisted LM-PCI with use of a mechanical support device. The median largest burr size used was 1.5 mm. The mean number of LM stents implanted was 0.95 ± 0.3. The mean LM stent diameter and length were 3.7 ± 0.3 mm and 15.8 ± 7.5 mm, respectively. Intravascular ultrasound was used to assess vessel size and stent apposition in 20 patients (36.0%). Angiographic success was obtained in all patients (100%). CONCLUSION: Despite technical challenges, RA of the LM coronary artery can be performed safely and is associated with a high rate of angiographic success.

A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help.

More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope-derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope-derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

Echocardiography in Pregnancy: Part 2.

The prevalence of pregnant women with cardiovascular heart disease is increasing. Transthoracic echocardiography is safe during pregnancy, and it is an important diagnostic tool in pregnant women with established heart disease in order to monitor ventricular and valvular anatomy and function. In addition, it can be used to delineate cardiac anatomy in complex congenital heart disease and help stratify maternal risk during pregnancy. This review will focus on the use of echocardiography in the diagnosis and management of pregnant women with common congenital lesions and with prosthetic valves.

Impaired Left and Right Ventricular Systolic and Diastolic Function in Response to Exercise in Patients with Diastolic Dysfunction.

BACKGROUND: Diastolic dysfunction (DD) is associated with exercise intolerance. To evaluate mechanisms of exercise intolerance in patients with DD, we performed bicycle stress echocardiography. METHOD AND RESULTS: Doppler measurements were performed at baseline, during exercise and recovery in 26 patients (58 ± 11 years) with DD and normal left ventricular ejection fraction and 6 normal controls (53 ± 5 years). Compared to controls, patients achieved similar target heart rates but lower METs (5.5 ± 2 vs. 9.8 ± 3, P < 0.01) and a higher peak pulmonary artery pressure (PAP) at peak exercise (50 ± 13 vs. 32 ± 7 mmHg, P < 0.01) despite similar PAP at rest and similar mean blood pressure at peak exercise (110.4 ± 18.0 vs. 106.9 ± 11.4 mmHg, P = NS). In patients versus controls, mitral E/E' was similar at baseline (10 ± 3 vs. 8 ± 1.3, P = NS) but higher at recovery (11 ± 2 vs. 7 ± 2, P < 0.05), % mitral filling time was shorter at baseline, onset, and peak exercise, whereas % LV and RV ejection time was similar to controls throughout exercise. Compared to controls, lateral mitral annular S' (11.8 ± 2.5 cm/sec vs. 14.9 ± 1.9 cm/sec, P < 0.02) and tricuspid annular S' (14.8 ± 4.1 cm/sec vs. 19.4 ± 4.0 cm/sec, P < 0.05) were lower at peak exercise in patients. CONCLUSION: Diastolic filling time is reduced at rest and stress while LV ejection time increases normally during exercise in DD. There is a reduced systolic reserve in LV and RV during exercise in DD. These mechanisms contribute to exercise intolerance and elevation of left atrial and PAP in patients with DD.

Molecular evolution of hypoallergenic hybrid proteins for vaccination against grass pollen allergy.

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.