Bone marrow-derived B cells preserve ventricular function after acute myocardial infarction.

OBJECTIVES: In view of evidence that mature cells play a role in modulating the stem cell niche and thereby stem cell potential and proliferation, we hypothesized that a mature bone marrow (BM) mononuclear cell (MNC) infusion subfraction may have particular potency in promoting hematopoietic or resident stem cell-induced cardiac repair post-infarction. BACKGROUND: Treatment of acute myocardial infarction (MI) with BM MNC infusion has shown promise for improving patient outcomes. However, clinical data are conflicting, and demonstrate modest improvements. BM MNCs consist of different subpopulations including stem cells, progenitors, and differentiated leukocytes. METHODS: Stem cells (c-kit+) and subsets of mature cells including myeloid lineage, B and T-cells were isolated from bone marrow harvested from isogeneic donor rats. Recipient rats had baseline echocardiography then coronary artery ligation; 1 x 10(6) cells (enriched subpopulations or combinations of subpopulations of BM MNC) or saline was injected into ischemic and ischemic border zones. Cell subpopulations were either injected fresh or after overnight culture. After 2 weeks, animals underwent follow-up echocardiography. Cardiac tissue was assayed for cardiomyocyte proliferation and apoptosis. RESULTS: Fractional ventricular diameter shortening was significantly improved compared with saline (38 +/- 3.2%) when B cells alone were injected fresh (44 +/- 3.0%, p = 0.035), or after overnight culture (51 +/- 2.9%, p < 0.001), or after culture with c-kit+ cells (44 +/- 2.4%, p = 0.062). B cells reduced apoptosis at 48 h after injection compared with control cells (5.7 +/- 1.2% vs. 12.6 +/- 2.0%, p = 0.005). CONCLUSIONS: Intramyocardial injection of B cells into early post-ischemic myocardium preserved cardiac function by cardiomyocyte salvage. Other BM MNC subtypes were either ineffective or suppressed cardioprotection conferred by an enriched B cell population.

Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity.

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.

Characterization of primitive marrow CD34+ cells that persist after a sublethal dose of total body irradiation.

Knowledge of the molecular events that occur during hematopoietic stem/progenitor cell (HSPC) development is vital to our understanding of blood cell production. To study the functional groups of genes characteristic of HSPCs we isolated a subpopulation of CD34+ bone marrow (BM) cells from nonhuman primates that persisted in vivo after a sublethal dose of total body irradiation (TBI). CD34+ cells isolated during the phase of maximal hematopoietic suppression show a transcriptional profile characteristic of metabolically inactive cells, with strong coordinate downregulation of a large number of genes required for protein production and processing. Consistent with this profile, these CD34+ cells were not able to generate hematopoietic colonies. Transcriptional profiling of these CD34+ cells in conjunction with a pathway analysis method reveals several classes of functionally related genes that are upregulated in comparison to the CD34+ cells obtained prior to TBI. These families included genes known to be associated with self-renewal and maintenance of HSPCs (including bone morphogenetic proteins), resistance to apoptosis (Bcl-2) as well as genes characteristic of a variety of nonhematopoietic tissues (gamma-aminobutyric acid/glycine receptor, complement receptor C1qRp). In contrast, during the period of hematopoietic recovery, the CD34+ cells expressed higher level of genes encoding factors regulating maturation and differentiation of HSPCs. Our data indicate that the primitive BM CD34+ cell population that persists after radiation possesses a transcriptional profile suggestive of pluripotency.

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide.

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.

BACKGROUND & AIMS: The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II. METHODS: Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1. CONCLUSIONS: After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.