Mutations in topoisomerase IIβ result in a B cell immunodeficiency.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization

Disrupting the Btk Pathway Suppresses COPD-Like Lung Alterations in Atherosclerosis Prone ApoE−/− Mice Following Regular Exposure to Cigarette Smoke

Chronic obstructive pulmonary disease (COPD) is associated with severe chronic inflammation that promotes irreversible tissue destruction. Moreover, the most broadly accepted cause of COPD is exposure to cigarette smoke. There is no effective cure and significantly, the mechanism behind the development and progression of this disease remains unknown. Our laboratory has demonstrated that Bruton’s tyrosine kinase (Btk) is a critical regulator of pro-inflammatory processes in the lungs and that Btk controls expression of matrix metalloproteinase-9 (MMP-9) in the alveolar compartment. For this study apolipoprotein E null (ApoE−/−) mice were exposed to SHS to facilitate study in a COPD/atherosclerosis comorbidity model. We applied two types of treatments, animals received either a pharmacological inhibitor of Btk or MMP-9 specific siRNA to minimize MMP-9 expression in endothelial cells or neutrophils. We have shown that these treatments had a protective effect in the lung. We have noted a decrease in alveolar changes related to SHS induced inflammation in treated animals. In summary, we are presenting a novel concept in the field of COPD, i.e., that Btk may be a new drug target for this disease. Moreover, cell specific targeting of MMP-9 may also benefit patients affected by this disease

Pauler Ákos és az ókortudomány

<p>(A) Oil Red O stained cross sections of aortic arch from ApoE^-/- mice after 13 weeks of WD + SHS, WD only, Chow + SHS, and Chow only. Wild type mice fed Chow with and without SHS exposure are shown as controls (WT + SHS, WT Air). Four to six ApoE^-/- mice per group were examined along with 3 WT mice per group. (B) Movat’s Pentachrome stained cross sections of aortic arch from ApoE^-/- mice after 13 weeks of WD + SHS, WD only, Chow + SHS, and Chow only. Three to four mice per group were examined.</p

Metalloproteinase-9 contributes to endothelial dysfunction in atherosclerosis via protease activated receptor-1

<div><p>The atherosclerotic process begins when vascular endothelial cells undergo pro-inflammatory changes such as aberrant activation to dysfunctional phenotypes and apoptosis, leading to loss of vascular integrity. Our laboratory has demonstrated that exposure of mice to second hand smoke triggers an increase in expression of metalloproteinase-9. Further, metalloproteinase-9 released by second hand smoke—activated leukocytes may propagate pro-atherogenic alterations in endothelial cells. We have shown that levels of metalloproteinase-9 were increased in the plasma from apolipoprotein E deficient (ApoE^-/-) mice exposed to second hand smoke relative to non-exposed controls. Moreover, we have collected data from two different, but complementary, treatments of second hand smoke exposed atherosclerotic mice. Animals received either cell specific metalloproteinase-9 directed siRNA to minimize metalloproteinase-9 expression in neutrophils and endothelial cells, or a pharmacological inhibitor of Bruton’s tyrosine kinase which indirectly limits metalloproteinase-9 production in neutrophils. These treatments reduced atherosclerotic changes in mice and improved overall vascular health. We also demonstrated that metalloproteinase-9 could activate endothelial cells and induce their apoptosis via cleavage of protease activated receptor-1. In summary, better understanding of metalloproteinase-9’s pathogenic capabilities as well as novel signaling pathways involved may lead to development of treatments which may provide additional benefits to atherosclerosis patients with a history of second hand smoke exposure.</p></div

Level of MMP-9 (total and endogenous active) in plasma of WD + SHS exposed ApoE^-/- mice.

<p>(A) Total MMP-9 in plasma at 7 weeks and endogenous active MMP-9 at 13 weeks exposure. Values are shown as mean ± STD, † p<0.1, ‡ p<0.05. For 7 weeks groups n = 5, for 13 weeks groups n = 6. (B) Comparison of endogenous active MMP-9 in plasma after 7 and 13 weeks exposure. Values are shown as mean ± STD, * p<0.01. For 7 weeks groups n = 5, for 13 weeks groups n = 6. (C) Gelatin zymography of pooled plasma taken after 5 to 20 weeks exposure, stained gel is shown along with inversion image and corresponding densitometry analysis of the most prominent band (indicated with arrow). For pooling plasma n = 4 at 5 weeks, n = 6 at 13 weeks, and n = 5 for remaining time points.</p

Histological analysis of aortic arch cross sections at the initial branch of the innominate artery from ApoE^-/- mice following four week treatments.

<p>(A) Representative Oil Red O stained sections of aortic arch show atherosclerotic lesions found at the initial branch of the innominate artery of control and treated mice; 4–8 animals per group were analyzed. Neutral lipids stained red. Movat’s Pentachrome and Picro-Sirius red stained sections of aortic arch are also shown; 3–5 mice per group were analyzed. (B) Representative Picro-Sirius red stained sections of aortic arch show atherosclerotic lesions found at the innominate artery of control and treated mice when viewed under normal (right) and polarized (left, center) light; 3–5 animals were evaluated per group.</p

Histological analysis of aortic arch cross sections at the lesser curvature from ApoE^-/- mice following four week treatments.

<p>(A) Representative Oil Red O stained sections of aortic arch show atherosclerotic lesions found at the lesser curvature of the aortic arch in control and treated mice; 5–8 animals were analyzed per group. Neutral lipids stained red. Movat’s Pentachrome and Picro-Sirius red stained sections are also shown. Four control animals, 5 BTK inhibitor, and 5 MECA:siMMP-9 treated animals were evaluated. (B) Representative Picro-Sirius red stained sections of aortic arches show atherosclerotic lesions found at the lesser curvature of control and treated mice viewed under polarized light. Four control animals, 5 BTK inhibitor, and 5 MECA:siMMP-9 treated animals were evaluated.</p

Changes in plasma levels of chemokines (KC, MCP-1, CRP and ICAM-1).

<p>Changes in plasma levels of chemokines (KC and monocyte chemotactic protein-1 [MCP-1]), C-reactive protein (CRP), and adhesion molecules (intercellular adhesion molecule, ICAM-1) at 13 weeks post WD and / or SHS exposure. Values are shown as mean ± STD, * p<0.01, ‡ p<0.05, † p<0.1. For WD + SHS group n = 5, Chow + SHS and WD Only groups n = 6.</p

Histological analysis of aortic arch cross sections from ApoE^-/- mice following two week treatments.

<p>(A) Representative sections of aortic arch show atherosclerotic lesions found at the initial branch of the innominate artery of control and treated mice, 5 mice per group were analyzed. Movat’s Pentachrome and Picro-Sirius red stained sections are also shown. (B) Representative Picro-Sirius red stained sections of aortic arches show atherosclerotic lesions found at the innominate artery branch of control and treated mice when viewed under polarized light. (C) Representative sections of aortic arches show atherosclerotic lesions found at the lesser curvature of control and treated mice. Movat’s Pentachrome and Picro-Sirius red stained sections are also shown; 4–5 animals per group were evaluated. (D) Representative Picro-Sirius red stained sections of aortic arches show atherosclerotic lesions found at the lesser curvature in control and treated mice when viewed under normal (right) and polarized (left, center) light; 4–5 animals per group were evaluated.</p

Picro-Sirius red stained cross sections of aortic arch viewed under polarized light.

<p>Aortic arch sections were examined from ApoE^-/- mice after 13 weeks of WD + SHS, WD only, Chow + SHS, and Chow only. Wild type mice fed rodent chow with and without smoke exposure are shown as controls (WT + SHS, WT Air). For ApoE^-/- mice 5–6 mice group were examined, 3 WT mice per group were examined.</p