MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Novel mutants of NAB corepressors enhance activation by Egr transactivators.

The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserved domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transactivators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, we have identified several point mutations in NAB corepressors that interfere with their ability to bind to the Egr1 R1 domain. Surprisingly, NAB proteins bearing some of these mutations increased Egr1 activity dramatically. The mechanism underlying the unexpected behavior of these mutants was elucidated by the discovery that NAB conserved domain 1 (NCD1) not only binds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repression by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. To examine NAB repression of a native Egr target gene, we show that NAB2 represses Egr2/Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repression is reversed by coexpression of dominant negative NAB2. Because of their specific ability to alleviate NAB repression of Egr target genes, the dominant negative NAB mutants will be useful in elucidating the mechanism and function of NAB corepressors.

The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex.

Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus.

Targeted disruption of the murine tissue factor gene results in embryonic lethality.

Tissue factor (TF) is an integral membrane glycoprotein that is believed to be the physiologic initiator of the blood coagulation cascade. Disruption of the mouse tissue factor gene leads to embryonic lethality between days E9.5-E11.5 of gestation. On E9.5, TF(-/-) embryos appear indistinguishable from their TF(+/+) and TF(+/-) littermates. By E10.5, TF(-/-) embryos are severely growth retarded, appear nearly bloodless, and are in most cases dead. Initial observations suggest that TF(-/-) embryos are dying of circulatory failure. Approximately 15% of the TF(-/-) embryos survive beyond E10.5, but none complete gestation. Heterozygotes appear normal and free of bleeding complications.

Gated blood pool SPECT and phase analysis to assess simulated Wolff-Parkinson-White syndrome in the baboon.

This study assesses the diagnostic potential of a tomographic technique with phase analysis to detect premature electroventricular contraction patterns simulated by pacing in the baboon. The data of gated SPECT were analysed by backprojection of the Fourier coefficients, followed by angulation and integration to thick slices of the entire ventricular mass yielding separate ventricular contraction patterns in three perpendicular views. Electrodes were implanted in each baboon: at the sinoatrial node; posterior, left ventricular; anterior left ventricular; on the left and the right lateral ventricular walls. The atrium was stimulated throughout at a fixed rate. Subsequent ventricular stimuli followed during the QRS complex, such to invoke the appearance of pre-excitation QRS morphology. The first points of activation (FPA) from this algorithm were correctly detected for the RV, for the anterior and posterior sites, although the latter two manifested first points in the RV. LV pacing manifested also as a FPA in the RV, but was followed by a true subsequent point in the LV.

Cardiac function and Fourier phase data from simulated Wolff-Parkinson-White syndrome in a baboon model.

The diagnostic value of Fourier phase analysis and planar scintigraphy in Wolff-Parkinson-White (WPW) syndrome has been suspect. This study investigates phase analytical data from planar radionuclide ventriculography of six baboons with simulated WPW syndrome by means of implanted electrodes. An electrode in the atrium controlled the heart rate and a subsequent stimulation was delivered by electrodes placed at different sites on the ventricles, delayed to cause the characteristic delta wave of the WPW syndrome. Sensitivity for accurately-localizing variously-situated first points of activation (FPAs) from the Fourier phase images was found highest for premature right ventricular (RV) activation, and for atrioventricular (AV) delays around 125 msec. Other sites were subject to artifacts. Changes in cardiac function, phase delay, and histogram parameters were not statistically meaningful.

Closure of infected median sternotomy wound with pectoralis major muscle flaps. A report of 3 cases.

Three patients who developed mediastinal sepsis and sternal dehiscence after coronary artery bypass grafting were treated by closure of the wound using bilateral pectoralis major muscle flaps. Excellent cosmetic and functional results were obtained and morbidity was reduced.

Pneumonectomy for severe inflammatory lung disease. Results in 64 consecutive cases.

Severe inflammatory lung disease resulting in severe unilateral pulmonary pathology necessitating pneumonectomy is still encountered in third world populations. A retrospective study of the last 64 patients undergoing pneumonectomy was performed. The underlying lung pathology was: destroyed lung due to tuberculosis in 33 patients; severe bronchiectasis in 25; necrotizing pneumonia in 4; lung abscess in 1 and hypoplastic lung in 1 patient. The perioperative management of these patients is outlined. Perioperative complications included respiratory failure in 4, secondary haemorrhage in 2 and post-pneumonectomy empyema in 5 patients. There were 2 mortalities (3.1%), both due to contralateral spillage with fulminant respiratory failure. Excellent results were achieved in 89% of the patients.