Acute Placental Infection Due to Klebsiella pneumoniae: Report of a Unique Case

A 40-year-old woman, gravida 9, with seven healthy children and a history of one abortion (p 7 + 1) , presented at 18 weeks of gestation with fever and malodorous vaginal discharge. Ultrasound revealed a macerated fetus. The placenta showed acute chorioamnionitis and acute villitis with microabscess formation. Blood and vaginal cultures both grew Klebsiella pneumoniae. This is the first reported case in English literature of Klebsiella pneumoniae causing suppurative placentitis leading to fetal demise

c-Abl binding and phosphorylation of geminin Y150 in G₂/M/early G₁ cells promotes overexpressed geminin oncogenicity in HME cells.

<p>(A) Immunoprecipitation of cycling, G₀/G₁, S, G₂/M or M/G₁ HME cells with anti-Cdt1, -c-Abl, -Sp1 (negative control) and -geminin antibodies. (B) c-Abl immunoprecipitated from S or G₂/M HME cells (upper panels) was used to <i>in vitro</i> phosphorylate GST-WT-, -Y98A-, -Y111A-, -Y150A-geminin or GST-survivin (negative control, lower panels). (C) c-Abl immunoprecipitated from G₂/M was used to <i>in vitro</i> phosphorylate GST-WT-geminin in the presence of the increasing concentrations of CKII inhibitor, TBB (left panels) or c-Abl inhibitor, imatinib (right panels). (D) The percentage of p-(S10)-H3⁺-cells in HME, uninduced or induced Gem9 following transfection of si-control or si-c-Abl (for 72 hr) or treatment with vehicle or 5 µM of imatinib (during the last 24 h). Data are represented as mean ± SD of triplicates done three separate times, where *  =  <i>p</i>≤<i>0.05</i> and **  =  <i>p</i>≤<i>0.001</i>. (E) Metaphase spread analysis of chromosome condensation in uninduced or induced Gem9 cells before or after transfection of sic-Abl or treatment with 5 µM of imatinib. (F) FACS analysis of uninduced or induced Gem9 cells transfection of sic-Abl or treatment with 5 µM of imatinib. Aneuploid cells are shown in the red circles and their percentage is in insets. R1 = G₀/G₁, R3/R4/R5 = early/mid/late S, R2 = G₂/M and R6 = >4N cells. Experiments were done three separate times in triplicates.</p

Stabilization of geminin protein by c-Abl phosphorylation and the expression of geminin and c-Abl in breast cancer cell lines.

<p>(A) The expression level of geminin in naïve HME, uninduced and induced Gem9 cells following c-Abl silencing or inactivation using imatinib or transfection of the dominant negative c-Abl (K290R). (B) The expression of c-Abl, geminin, p-CrkII in induced Gem9, MCF7 or MDA-MB-231 cells silenced from c-Abl or treated with imatinib. (C) The expression level of c-Abl, p-CrkII and geminin in induced Gem9 or MDA-MB-231 cells silenced of c-Abl or treated with nilotinib. (D) RT/PCR analysis of geminin mRNA in uninduced, induced Gem9 or MDA-MB-231 cells in the presence of vehicle or imatinib. (E) The expression of geminin in naïve HME or induced Gem9 cells in the presence of vehicle, imatinib or imatinib + MG132. (F) The expression of c-Abl and geminin mRNAs and proteins (inset) in several breast cancer cell lines. Note that Hs578T cells express high level of geminin mRNA, but no protein. (G) The re-expression of geminin in Hs578T cells reconstituted with WT and not kinase dead (KD) c-Abl (left). The re-expression of geminin in Hs578T cells reconstituted with WT or constitutively active (CA) c-Abl was blocked by imatinib (right). (H) The expression of endogenous (left) or overexpressed (right) geminin in MDA-MB-231 cells following treatment with the translational inhibitor cycloheximide (CHX) for 0-10 h with cells collected at 2 h intervals. (I) The expression of exogenous Myc tagged WT- (left), Y150A- (middle) or Y150E- (right)-geminin following no treatment (1^st lanes), 10 h of CHX (2^nd lanes), 10 h CHX followed by 24 h of complete serum (3^rd lanes) or 10 h CHX followed by 24 h of complete serum + 10 µM of imatinib (4^th lanes).</p

The expression of geminin and c-Abl in breast tumor samples.

<p>(A) The normalized expression of geminin and c-Abl mRNA in normal (n = 5), luminal A (n = 7), luminal B (n = 9), Her2⁺ (n = 11) and TN/BL (n = 7) tumor samples. (B) Box plot of gene expression for combined gene set of geminin and c-Abl across cell lines grouped into clinical subtypes; triple negative breast cancer (TNBC, red), HER2-positive (HER2, yellow), and ER-positive (ER⁺, blue) based on annotation data from (38). (C) Number of total, geminin-positive and c-Abl-positive tumors detected using immunohistochemistry on normal/cancer adjacent (n = 66), DCIS (n = 180), invasive (n = 100) and metastatic (n = 165) breast tumors. (D) Representative immunohistochemical staining images of geminin (1 and 3) or -c-Abl (2 and 4) on invasive breast tumor samples. Scale bar  =  50 µm. (E) Number of geminin-positive or -negative in Her2⁺ (n = 32) or TN/BL (n = 72) showing cytoplasmic (Cyt), nuclear (Nuc), or both (Cyt + Nuc) c-Abl expression. (F) The level of geminin and c-Abl in the nuclear or cytoplasmic fractions of the indicated cell lines.</p

c-Abl binding and phosphorylation of geminin Y150 in G₂/M/early G₁ cells promotes overexpressed geminin oncogenicity in HME cells.

<p>(A) Immunoprecipitation of cycling, G₀/G₁, S, G₂/M or M/G₁ HME cells with anti-Cdt1, -c-Abl, -Sp1 (negative control) and -geminin antibodies. (B) c-Abl immunoprecipitated from S or G₂/M HME cells (upper panels) was used to <i>in vitro</i> phosphorylate GST-WT-, -Y98A-, -Y111A-, -Y150A-geminin or GST-survivin (negative control, lower panels). (C) c-Abl immunoprecipitated from G₂/M was used to <i>in vitro</i> phosphorylate GST-WT-geminin in the presence of the increasing concentrations of CKII inhibitor, TBB (left panels) or c-Abl inhibitor, imatinib (right panels). (D) The percentage of p-(S10)-H3⁺-cells in HME, uninduced or induced Gem9 following transfection of si-control or si-c-Abl (for 72 hr) or treatment with vehicle or 5 µM of imatinib (during the last 24 h). Data are represented as mean ± SD of triplicates done three separate times, where *  =  <i>p</i>≤<i>0.05</i> and **  =  <i>p</i>≤<i>0.001</i>. (E) Metaphase spread analysis of chromosome condensation in uninduced or induced Gem9 cells before or after transfection of sic-Abl or treatment with 5 µM of imatinib. (F) FACS analysis of uninduced or induced Gem9 cells transfection of sic-Abl or treatment with 5 µM of imatinib. Aneuploid cells are shown in the red circles and their percentage is in insets. R1 = G₀/G₁, R3/R4/R5 = early/mid/late S, R2 = G₂/M and R6 = >4N cells. Experiments were done three separate times in triplicates.</p

Death of geminin overexpressing cells specifically in the absence of c-Abl.

<p>(A) Representative bright field and fluorescence images showing naïve HME, uninduced and induced Gem9 cells transfected with sh-control or shc-Abl and grown in the presence or absence of doxycycline for 4 days. Scale bar  =  400 µm. (<b>B</b>) Representative bright-filed and fluorescence images of Gem 9 cells expressing sh-control or shc-Abl and grown in the presence or absence of doxycycline for 0 or 6 days. Scale bar  =  400 µm. (C) Quantitative analysis of the data in (A) and (B). (D) Phase contrast images showing colony formed in soft agar using naïve HME, uninduced and induced Gem9 cultures before or after treatment with 10 µM of imatinib (upper) Quantitative analysis of the soft agar experiment described in (lower). Data are represented as mean ± SD from triplicates done 3 separate times. ***  =  <i>p</i><<i>0.001</i>.</p