Chloroacetate Esterase Staining Research Articles

Porphyromonas gingivalis Strain W83 Infection Induces Liver Injury in Experimental Alcohol-Associated Liver Disease (ALD) in Mice

The liver plays a vital role in the defense against infections. Porphyromonas gingivalis (P. gingivalis), a dominant etiologic oral bacterium implicated in periodontal disease (PD), has been associated with various systemic diseases. This study aimed to investigate the influence of P. gingivalis on alcohol-associated liver diseases (ALD). Mice were fed a Lieber–DeCarli liquid diet containing 5% ethanol for 10 days after an initial adaptation period on a diet with lower ethanol content for 7 days. Two days before tissue sample collection, the mice were administered P. gingivalis strain W83 (Pg) through intraperitoneal injection (IP). Pair-fed mice with Pg infection (PF+Pg) exhibited an activated immune response to combat infections. However, alcohol-fed mice with Pg infection (AF+Pg) showed liver injury with noticeable abscess lesions and elevated serum alanine aminotransferase (ALT) levels. Additionally, these mice displayed liver infiltration of inflammatory monocytes and significant downregulation of proinflammatory cytokine gene expression levels; and AF+Pg mice also demonstrated increased intrahepatic neutrophil infiltration, as confirmed by chloroacetate esterase (CAE) staining, along with elevated gene expression levels of neutrophil cytosol factor 1 (Ncf1), neutrophilic inflammation driver lipocalin 2 (Lcn2), and complement component C5a receptor 1 (C5ar1), which are associated with neutrophilic inflammation. Interestingly, compared to PF+Pg mice, the livers of AF+Pg mice exhibited downregulation of gene expression levels of NADPH oxidase 2 (Cybb), the leukocyte adhesion molecule Cd18, and the Toll-like receptor adaptor Myd88. Consequently, impaired clearance of P. gingivalis and other bacteria in the liver, increased susceptibility to infections, and inflammation-associated hepatic necrotic cell death were observed in AF+Pg mice, which is likely to have facilitated immune cell infiltration and contributed to liver injury. Furthermore, in addition to the Srebf1/Fasn pathway induced by alcohol feeding, Pg infection also activated carbohydrate response element-binding protein (ChREBP) in AF+Pg mice. In summary, this study demonstrates that P. gingivalis infection, acting as a “second hit”, induces dysfunction of immune response and impairs the clearance of bacteria and infections in alcohol-sensitized livers. This process drives the development of liver injury.

Characterisation of tumour-immune phenotypes and PD-L1 positivity in squamous bladder cancer

AimsImmune checkpoint inhibitor (ICI) therapy has become a viable treatment strategy in bladder cancer. However, treatment responses vary, and improved biomarkers are needed. Crucially, the characteristics of immune cells remain understudied especially in squamous differentiated bladder cancer (sq-BLCA). Here, we quantitatively analysed the tumour-immune phenotypes of sq-BLCA and correlated them with PD-L1 expression and FGFR3 mutation status.Methods Tissue microarrays (TMA) of n = 68 non-schistosomiasis associated pure squamous cell carcinoma (SCC) and n = 46 mixed urothelial carcinoma with squamous differentiation (MIX) were subjected to immunohistochemistry for CD3, CD4, CD8, CD56, CD68, CD79A, CD163, Ki67, perforin and chloroacetate esterase staining. Quantitative image evaluation was performed via digital image analysis.ResultsImmune infiltration was generally higher in stroma than in tumour regions. B-cells (CD79A) were almost exclusively found in stromal areas (sTILs), T-lymphocytes and macrophages were also present in tumour cell areas (iTILs), while natural killer cells (CD56) were nearly missing in any area. Tumour-immune phenotype distribution differed depending on the immune cell subset, however, hot tumour-immune phenotypes (high density of immune cells in tumour areas) were frequently found for CD8 + T-cells (33%), especially perforin + lymphocytes (52.2%), and CD68 + macrophages (37.6%). Perforin + CD8 lymphocytes predicted improved overall survival in sq-BLCA while high PD-L1 expression (CPS ≥ 10) was significantly associated with higher CD3 + , CD8 + and CD163 + immune cell density and high Ki67 (density) of tumour cells. Furthermore, PD-L1 expression was positively associated with CD3 + /CD4 + , CD3 + /CD8 + and CD68 + /CD163 + hot tumour-immune phenotypes. FGFR3 mutation status was inversely associated with CD8 + , perforin + and CD79A + lymphocyte density.ConclusionsComputer-based image analysis is an efficient tool to analyse immune topographies in squamous bladder cancer. Hot tumour-immune phenotypes with strong PD-L1 expression might pose a promising subgroup for clinically successful ICI therapy in squamous bladder cancer and warrant further investigation.

A rare der(10)t(X;10)(p11.2;p11.2) in an elderly patient with therapy-related acute myelomonocytic leukemia.

An 80-year-old male was referred to our hospital with dyspnea on effort. He had a history of diffuse large B-cell lymphoma 2 years ago, for which he was treated with chemotherapy, including rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisolone. His laboratory data included a white blood cell count of 1.9 × 109/L, a hemoglobin concentration of 10.5 g/dl, and a platelet count of 92 × 109/L. His serum lactate dehydrogenase level was 196 U/L. A bone marrow examination revealed a hypercellular bone marrow with a blast frequency of 23.4%. The blast cells were positive for both nonspecific esterase and naphthol AS-D chloroacetate esterase staining (Figure 1). A cytogenetic study of the patient's bone marrow cells demonstrated the following karyotype: 47,XY, +8, der(10)t(X;10)(p11.2;p11.2)[7]/46,XY[13] (Figure 2). A diagnosis of therapy-related acute myelomonocytic leukemia was made due to his history of chemotherapy-treated lymphoma. Although the patient received two courses of azacitidine monotherapy, it was not effective, and he died 3 months after being diagnosed with leukemia. To the best of our knowledge, only five cases of acute myeloid leukemia (AML) involving a translocation consisting of 10p11 and Xp11 breakpoints have been reported [1-5]. Of these, one case demonstrated the t(X;10)(p11;p11) translocation as a sole abnormality [3]. This translocation has never been reported in other solid tumors. Hence, t(X;10)(p11;p11) is considered to be a rare, but recurrent, cytogenetic aberration in AML. The reported cases have several things in common, that is, they all involved balanced translocations, most of them involved therapy-related leukemia, and all of the patients except one (a 50-year-old [5]) were children. Although our case also involved therapy-related leukemia, it was very rare, as it involved an 80-year-old patient who harbored an unbalanced der(10)t(X;10)(p11;p11) translocation. As for the association between t(X;10)(p11;p11) and the pathogenesis of AML, our case involved der(10)t(X;10)(p11;p11), but lacked der(X)t(X;10)(p11;p11) due to the unbalanced nature of the translocation. Therefore, der(10)t(X;10)(p11;p11), rather than der(X)t(X;10)(p11;p11), is proposed to play a crucial role in the leukemogenesis in t(X;10)(p11;p11)-harboring AML. The patient's consent was obtained for the publication of the case report. The authors declare no conflict of interest. Masahiro Manabe, Nao Tanizawa, Satoru Nannno, and Ki-Ryang Koh designed the study. Masahiro Manabe, Yuuji Hagiwara, and Reiko Asada analyzed the data. Masahiro Manabe and Ki-Ryang Koh collected the clinical data and specimens. Masahiro Manabe, Yuuji Hagiwara, and Reiko Asada wrote the manuscript. All of the authors have reviewed and approved the final manuscript. All data generated during this study are included in this manuscript.

Increased hepatic JNK activation by ethanol is mediated by transcriptional suppression of mitogen‐activated protein kinase phosphatase 1 (Mkp1): role of cAMP‐specific protein kinase A

Background Increased and sustained JNK activation has been shown to be detrimental for hepatocyte survival and liver injury in various diseases, including alcohol-associated liver disease (ALD). Among three JNK isoforms, JNK1 and 2 are expressed in hepatocytes and both play a role in mouse models of ALD. Mkp1 phosphatase has been identified as a regulator of JNK activation. A previous study showed that Mkp1 degradation plays a critical role in sustained JNK activation and hepatocyte death from acute ethanol exposure. However, the effect of chronic ethanol exposure on hepatocyte Mkp1 expression is not known. Methods Male C57B/6 mice were subjected to intragastric (iG) feeding of ethanol for 8 weeks with weekly ethanol binge (5g/kg) starting in the second week of iG feeding. Pair-fed control mice were fed an isocaloric high-fat liquid diet. Plasma ALT, AST and histologic grading were performed to evaluate steatosis, liver inflammation and injury. Liver immunohistochemistry was performed to evaluate apoptosis (TUNEL staining). Myeloperoxidase (MPO) activity assay and chloroacetate esterase (CAE) staining for neutrophil infiltration were also performed. Liver RNA sequencing was performed on an Illumina NextSeq 500. Data analysis was done using the Tuxedo suite pipeline. Validation studies were carried out by performing real time qPCR and Western blot. Primary mouse hepatocytes were exposed to 50 mM ethanol for 48 hours followed by TNF stimulation. Statistical analysis was performed using ANOVA. Results Histological examination of liver sections confirmed the development of severe alcohol-associated steatohepatitis in ethanol fed mice via significant micro- and macro-vesicular steatosis, polymorphonuclear leukocyte (PMN) infiltration, CAE and TUNEL-positive cells. ALT and AST levels were significantly elevated in ethanol-fed mice. JNK activation was evident by Western blot analysis of phosphorylated JNK levels. RNA Seq analysis showed significant changes in expression of several Mkps; however, only Mkp1 levels were significantly downregulated, which was further validated by real time qPCR. Importantly, ethanol exposure of primary mouse hepatocytes led to a significant decrease in Mkp1 mRNA expression which further declined following TNF stimulation. These changes in Mkp1 were accompanied by sustained JNK activation and cell death. Moreover, ethanol exposure and TNF stimulation markedly increased cAMP specific PDE4 expression resulting in reduction in hepatocyte cAMP levels. Significantly, PDE4 inhibition and PKA specific agonists prevented the suppressive effect of ethanol and TNF on Mkp1 and resultant activation of JNK and cell death. Conclusion Our results strongly implicate ethanol-induced decline in hepatic Mkp1 as a significant mechanism regulating JNK activation and hepatocyte death in ALD. Further, these data strongly suggest that the ethanol induced decrease in cAMP/PKA signaling plays a causal role in regulating the Mkp1/JNK axis in hepatocytes and development of liver injury.

Decitabine-Induced Early Platelet Response, a Predictor of Favorable Outcome during Hypomethylating Treatment of MDS, Is Associated with In Vivo Megakaryocytic Differentiation

Introduction Despite broad use of hypomethylating agents (HMAs) in MDS/AML treatment, the number of established outcome predictors is still very limited (Stomper and Lübbert, Sem Hematol 2019). One of them, the early, isolated and sometimes massive increase of platelets, is recurrently observed in patients with MDS treated with HMAs. It is not observed with non-HMA cytidine analogs such as low-dose cytarabine. In HMA-treated MDS patients, an early platelet increase is a predictor of overall and leukemia-free survival (van den Bosch et al., Leuk Res 2004; van der Helm et al., Br J Haematol 2011). HMAs induce cellular differentiation in vitro, by gene reactivation in the malignant cells. However, evidence for HMA-induced in vivo differentiation is still very limited. We hypothesized that the megakaryocytic cell lineage in MDS is a target for HMA-induced cellular maturation in vivo. Methods We systematically analyzed the bone marrow morphology of 34 higher-risk MDS patients (median age: 71.5 years, range 51-79) before and after 1 cycle of treatment with the HMA decitabine (DAC). All patients had been treated at a single center within 3 prospective clinical trials (Wijermans et al., Ann Hematol 2005; Lübbert et al., J Clin Oncol 2011). One treatment cycle consisted of 45 mg/m2 DAC per day (15 mg/m2 intravenously over 4 hours every 8 hours) for 3 consecutive days, repeated 6 weeks later. The early platelet response was evaluated after 1 cycle of DAC treatment. Based on the criteria of the International Working Group, an absolute increase in platelet count of 30x109/l or more compared to the pre-treatment count was defined as a platelet response. The histological analysis of the bone marrow specimens was performed by an experienced hematopathologist blinded to the treatment timepoints. Up to 200 megakaryocytes (MK) per sample were quantified at a magnification of 400 x using chloroacetate esterase staining. Results Thirteen of 34 patients (38%) showed a platelet response already after 1 cycle of DAC treatment, 21 (62%) did not. The median pre-treatment platelet count did not differ in patients with or without an early platelet response (median of 34x109/l in both groups, range 7-169 and 8-265, respectively). After 1 cycle of DAC treatment, patients with a platelet response had a median platelet count of 117x109/l (range 78-281), patients without this response had a median platelet count of 32 x109/l (range 4-155). Overall survival (OS) was measured from the time of early platelet response assessment after completion of 1 treatment cycle, i.e. after 6 weeks. The presence of a platelet response after 1 DAC cycle was associated with a longer OS compared to the absence of this early platelet response: median of 26.6 versus 14.0 months (p=0.04). Both pre- and post-treatment bone marrow biopsies of patients with an early platelet response showed higher numbers of MK, as well as significant differences in MK morphology compared to biopsies from patients without an early platelet response. Regarding MK numbers, increased MK density in specimens of patients with an early platelet response was observed both before (mean MK number per high power field 6.2 vs. 2.6, p=0.02) and after the application of DAC (mean MK number 10.4 vs. 3.1, p=0.01). Regarding MK maturation stage, more pre-treatment juvenile MK (on average 32.4% vs. 20.5% of all MK, p=0.03) and MK with typical myeloproliferative stigmata (present in 5/13 vs. 2/21 biopsies) were observed in patients with an early platelet response, compared to patients without this response. Regarding the induction of megakaryocytic maturation during this early treatment phase, more post-treatment "naked", mature MK nuclei indicative of active platelet shedding (on average 9.5% vs. 3.8% of all MK, p=0.01), were noted in patients with an early platelet response than in patients without an early platelet response. Conclusions This is, to the best of our knowledge, the first systematic hematopathological analysis of changes in quantitative and morphological MK features in bone marrow specimens of MDS patients during HMA treatment. DAC, which has in vitro differentiation-inducing effects on megakaryoblastic cells, induced maturation also of dysplastic MK in vivo in higher-risk MDS patients with an early platelet response to this HMA. The predictive value of an early platelet increase, a very easy-to-determine parameter, during this type of treatment is confirmed. Disclosures No relevant conflicts of interest to declare.

Histological Findings in Infected and Noninfected Second Stage Revision Knee Arthroplasties.

Tissues from a periprosthetic joint infection (PJI) of the knee contain a heavy neutrophil polymorph (NP) infiltrate (> 5 NPs per high-powered field [HPF] by Musculoskeletal Infection Society [MSIS] criteria). PJI of the knee can be treated by a two-stage procedure and our aim was to determine whether the MSIS histological criteria for PJI diagnosis are valid in a second-stage revision knee arthroplasty. Periprosthetic tissues from 45 second-stage revision knee cases were analyzed histologically by hematoxylin-eosin and chloroacetate esterase (CAE) staining for the identification of NPs. The number of NPs was determined semiquantitatively and results correlated with the microbiological and clinical findings. In 9 of the 45 cases, an organism was cultured in two or more samples, meeting MSIS microbiological criteria for a definite diagnosis of PJI; histologically, seven of these cases contained > 5 per NPs per HPF on average, with the remaining two cases containing 1 NP and 2 NPs per HPF. In noninfected second-stage revisions, NPs were not seen in 30 cases with 6 cases showing less than 1 NP per HPF on average. The sensitivity, specificity, accuracy, and positive and negative predictive values of MSIS histological criteria (> 5 NPs per HPF) to diagnose PJI were 78%, 100%, 96%, 100%, and 95%, respectively. MSIS histological criteria for the diagnosis of PJI are valid for most but not all infected second-stage revision knee arthroplasties. Correlation of histology with clinical, microbiology and other laboratory findings is required to establish a diagnosis of PJI in second-stage revision knee arthroplasties.

Role of Aldose Reductase in LPS‐inducible Intrahepatic Immune Infiltration and Inflammation

Aldose reductase (AR) is the rate‐limiting enzyme of the polyol pathway that converts glucose to sorbitol, which is further metabolized to fructose. AR has been widely investigated in pathogenesis of diabetic complications, cardiovascular disorders and cancer. Previous studies show that activation of AR is involved in glucose‐induced hepatitis in mice, hepatitis in rats and human autoimmune hepatitis. Recent findings demonstrate that AR is involved in inflammatory hepatic ischemia‐reperfusion injury. The gut‐liver axis is known to be involved in the development of both alcoholic and non‐alcoholic liver diseases. Gut permeability releases endotoxin (lipopolysaccharide, LPS) into circulation and then triggers inflammation. Inflammatory responses are among the most critical pathological processes associated with liver injury. This study investigated the role of AR in intrahepatic immune infiltration/activation and inflammation using mouse models and human specimens from patient with inflammatory liver disease.Wild type (WT, C57BL/6) and aldose reductase knockout mice (ARKO, AKR1B3−/−) were subjected to stimulation with or without LPS (2.0mg/kg) through intraperitoneal injection. Liver histology (H&E staining), intrahepatic immune cell markers (F4/80, chloroacetate esterase stain) and apoptosis (TUNEL) were examined. Intrahepatic immune cells were isolated from mouse livers and analyzed by flow cytometry using immunofluorescence antibodies: anti‐CD11b, anti‐F4/80, anti‐ly6G and anti‐ly6C. The expression levels of AR in hepatitis patient livers were evaluated by real‐time PCR.Proinflammatory cytokines (TNFα, IL‐1β, IL‐6) and chemokines (CXCL2 and MCP‐1/CCL2) were significantly upregulated in WT mice exposed to LPS. The genetic deletion of AR greatly attenuated LPS‐induced cytokines/chemokines. LPS resulted in the infiltration of circulating neutrophils and monocytes into the liver and increased CD11b+Kuffer subsets in WT mice. The lack of AR in ARKO mice was associated with decreased populations of neutrophils, monocytes and CD11b+ Kuffer cells. Specifically, administration of LPS increased neutrophils (F4/80−/CD11b+/Ly6G+/SSCint) by 5 fold in WT mice and AR deficiency reduced this to 3 fold; LPS elevated intrahepatic inflammatory monocytes (F4/80−/CD11b+ /ly6Chi) by 2 fold in WT mice and AR deficiency reduced this to 1 fold. LPS increased the CD11b+Kupffer cell subset (F4/80+CD11b+) by 2 fold in WT mice and AR deficiency decreased this to 1 fold. Hepatic AR expression was significantly upregulated by 2 fold in patients with inflammatory liver disease. Taken together, our data show that AR is a key mediator of hepatic inflammation in response to endotoxin in mice. The upregulation of hepatic AR in patients with hepatitis emphasize the relevance of AR in human liver diseases associated with inflammation. The data also suggest that AR may be a novel therapeutic target for inflammatory hepatic injury.Support or Funding InformationNIH‐NIEHS, NIH‐NIAAA, DOD, VA, and UofL‐IPIBSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute monocytic leukemia diagnosed by flow cytometry includes acute myeloid leukemias with weakly or faintly positive non-specific esterase staining.

A diagnosis of acute monocytic leukemia (AML-M5) based on α-naphthyl butyrate esterase (α-NB) staining has some problems, because AML-M5 leukemic cells often show weak or faint positivity on α-NB staining. In these situations, some cases of AML-M5 tend to be misdiagnosed as AML-M0. Therefore, we evaluated the significance of weak or faint α-NB staining in AML-M5 diagnosed by flow cytometry (FCM). Nineteen AML cases in which leukemic cells were negative for naphthol AS-D chloroacetate esterase staining were studied. For FCM, we defined leukemic cells as having a monocytic nature when more than 10% of the leukemic cells were positive for at least one of the following antigens: CD4, CD11c, CD14, and CD64. The monocytic nature determined by FCM was consistent with positive or weak positivity on α-NB staining. Five of 6 cases in which leukemic cells exhibited faint positivity for α-NB staining could be diagnosed as AML-M5 by FCM, while negative α-NB staining was consistent with a diagnosis of AML-M0. These results suggest that AML-M5 should be taken into consideration even when leukemic cells are faintly positive for α-NB staining.

Diagnosis and treatment of adult acute myeloid leukemia with NPM-MLF1 fusion gene and literatures review

Objective To evaluate the clinical features, treatment and prognosis of acute myeloid leukemia (AML) with nucleophosmin (NPM)-myelodysplasia/myeloid leukemia factor (MLF)1 fusion gene. Methods On 16 February 2016, a case of AML patient with NPM-MLF1 fusion gene who was admitted to Changhai Hospital Affiliated to Second Military Medical University was included in this study. When the patient was admitted, bone marrow morphology, cytochemical staining and immunophenotype analysis of leukemia blasts, cytogenetics and molecular genetic analysis were conducted and diagnosis was made base on those test results. On 17 February 2016, the patient recieved two courses of daunorubicin+ arabinosylcytosin (DA) 3+ 7 induction therapy. Subsequently, the patient recieved one course of fludarabine, cytarabine and granulocyte colony-stimulating factor (FLAG) regimen and 5 courses of high-dose of cytarabine. In the meantime, bone marrow morphology and minimal residual disease (MRD) were conducted during the chemotherapy. On 6 April 2017, the patient received bone marrow morphology, immunophenotype analysis of leukemia blasts and molecular genetic analysis on her visit. The clinical features, diagnosis and treatment of this patient were analyzed retrospectively, and the related literatures were reviewed. Results ①When the patient was admitted, bone marrow morphology revealed hyper-cellular marrow with 48.0% leukemia blasts. Cytochemical staining showed that peroxidase (POX) staining in some leukemia blasts were positive, and naphthol AS-D chloroacetate esterase (CE) staining in a few leukemia blasts were positive, and non-specific estertase (NSE) staining in some leukemia blasts were positive. The results of immunophenotype analysis revealed the ratio of the blasts cell was about 39.9%, and the ratio of immature cell was about 13.6%. R banding karyotype analysis marked with 46, XX, t(3; 5)(q25; q34)[3]/46, XX[7]. Molecular genetic analysis showed NPM/MLF1 fusion gene was positive. According to above, the diagnosis of patient was AML-subtype M4 with NPM-MLF1 fusion gene. ②After the patient was treated with the first course of induction therapy, and the reexamined bone marrow morphology post therapy showed partial remission (PR). The patient received complete remission (CR) after the second course of induction therapy. After the patient received consolidation therapy, and bone marrow morphology showed the patient obtained sustained CR status. On the patient′s last visit, bone marrow morphology revealed a total of 25.0% leukemia blasts, consisting of 21.0% blasts and 4.0% immature cell, suggesting AML recurrenced. Immunophenotyping of leukemia blasts showed approximately 28.5% of the early myeloid cells in bone marrow which was consistent with AML recurrence. Molecular genetic analysis showed that the MPM-MLF1 fusion gene was positive (66.1%). During the treatment, bone marrow MRD decreased, then increased. The patient went back to local hospital for treatment after AML relapsed. Conclusions AML-subtype M4 patient with NPM-MLF1 fusion gene can achieve CR after chemotherapy, but prone to early recurrence, and NPM-MLF1 fusion gene may be a poor prognostic factor for AML. Key words: Leukemia, myeloid, acute; Drug therapy; NPM-MLF1; t (3;5)

Quantification of neutrophil polymorphs in infected and noninfected second-stage revision hip arthroplasties.

In the diagnosis of periprosthetic joint infection (PJI), a heavy neutrophil polymorph (NP) infiltrate (>5 per high-power field [HPF] by MSIS criteria) is characteristically seen in periprosthetic tissues. PJI is commonly treated by a two-stage procedure with surgical clearance of infected tissues followed by intensive antibiotic treatment before re-implantation; tissues are sampled at the time of second stage but whether MSIS histological criteria can be used to diagnose the presence or absence of infection in second stage samples has not been established. Periprosthetic tissues from 31 cases of second-stage revision hip arthroplasty (including 3 cases fulfilling the microbiological MSIS criteria for PJI), were analysed histologically after haematoxylin-eosin and chloroacetate esterase (CAE) staining. The extent of the NP infiltrate was determined semiquantitatively and correlated with the microbiological diagnosis. CAE staining facilitated identification of NPs in arthroplasty tissues and showed that in those cases where an organism was cultured in 2 or more samples, meeting the MSIS microbiological criteria for definite diagnosis of PJI, there was a heavy polymorph infiltrate (>5 NP per HPF on average). It was noted that isolated or scattered NPs were seen in 42.8% of periprosthetic tissues from noninfected second-stage revisions. The MSIS histological criteria which support a diagnosis of PJI in specimens from a primary revision hip arthroplasty (i.e. >5 NPs per HPF on average) are also valid for the assessment of a second-stage specimens. NPs can be seen in samples of periprosthetic tissue from uninfected second-stage revisions, indicating that strict histological criteria should be used in evaluating their significance in this context.

The Diagnosis of Infection in Metal-on-Metal Hip Arthroplasties

BackgroundDistinction of aseptic from septic hip arthroplasty failure can be challenging. Some studies report an increased incidence of septic failure with metal-on-metal (MoM) hip arthroplasties. The Musculoskeletal Infection Society (MSIS) have formulated criteria to facilitate the diagnosis of periprosthetic joint infection (PJI). In this study, we determined the prevalence and histologic features of septic MoM hip failure. MethodsOverall, 104 cases of failed MoM hip arthroplasty, classified as septic or aseptic by MSIS microbiological criteria, were analyzed. The overall prevalence of septic failure was determined and the nature of the causative organisms noted. The extent of the neutrophil polymorph (NP) infiltrate in periprosthetic tissue in all cases was analyzed by hematoxylin-eosin and chloroacetate esterase staining. ResultsThe prevalence of septic MoM hip arthroplasty failure was 6.7%. Infective organisms were coagulase-negative Staphylococcus in 4 cases; Staphylococcus aureus, Streptococcus, and Propionibacterium species were isolated in the remaining cases. Chloroacetate esterase staining facilitated identification of NPs. All cases of PJI contained more than 5 NPs per high-power field (HPF) on average. Four cases of aseptic MoM implant failure contained scanty or scattered NPs (less than 5 per HPF on average). ConclusionThe prevalence of PJI as a cause of MoM hip arthroplasty failure was relatively high compared to other hip bearing combinations; however, the organisms responsible were similar. Histologically, a minority of aseptic MoM implant failures contained some NPs, but the MSIS criteria for the histologic diagnosis of PJI (>5 NPs/HPF) correctly identified all microbiologically confirmed cases of septic failure.

The role of 12/15‐Lipoxygenase in a Mouse Model of Alcoholic Liver Disease

Introduction/Aim12/15‐Lipoxygenase (12/15‐LOX) catalyzes the oxidation of free and esterified polyunsaturated fatty acids, predominantly, linoleic and arachidonic, generating the whole spectrum of bioactive lipid mediators. This enzyme is involved in the regulation of various homeostatic processes as well as in the pathogenesis of multiple diseases. Recently published studies from our group and others have shown that experimental and clinical alcoholic liver disease was associated with elevated oxidized linoleic acid metabolites (OXLAMs), specifically 9‐ and 13‐hydroxy‐octadecadienoic acids (9‐ and 13‐HODEs). The aim of the present study was to examine the role of 12/15‐LOX in an animal model of ALD.Materials and MethodsEight‐week‐old male 12/15‐LOX knockout mice (12/15‐LOX−/−), and their genetically unaltered wild type (WT) counterparts were fed Lieber–DeCarli control (isocaloric maltose‐dextrin) or ethanol (5% w/v) liquid diets ad libitum for 10 days plus a single binge ethanol administration (5 g/kg) by gavage, whereas mice in control groups were gavaged with isocaloric maltose dextrin. Liver histopathological changes were estimated by hematoxylin and eosin staining. Liver triglyceride accumulation was assessed by biochemical assay and Oil Red O staining. Liver injury was evaluated by plasma ALT and AST activity. Liver inflammation was determined by hepatic proinflammatory gene expression. Chloroacetate esterase staining was used to evaluate necroinflammatory changes. TUNEL staining was used to determine hepatic apoptotic cell death. Plasma OXLAMs were analyzed on LC‐MS and GCxGC/TOF‐MS platforms.ResultsChronic‐binge alcohol administration resulted in a marked increase in plasma OXLAM levels, specifically 9‐HODE and 13‐HODE, in parallel with up‐regulation of hepatic 12/15‐LOX in WT animals. These effects were associated with hepatic steatosis, inflammation and injury. 12/15‐LOX genetic depletion did not reduced hepatic steatosis caused by EtOH, but did ameliorate hepatic injury as assessed by ALT (231.2+32.8 U/L in WT vs 148.4+48.6 U/L in 12/15‐LOX−/−) and AST (279.1+36.7 U/L in WT vs 177.1+28.3 U/L in 12/15‐LO−/−) levels. 12/15‐LOX deficiency did not affect hepatic apoptosis; while neutrophil infiltration was significantly reduced in 12/15‐LOX deficient mice compared to WT animals. 12/15‐LOX deficiency prevented EtOH‐induced pro‐inflammatory cytokine and chemokine expression, including Tnf‐α and Mcp1 mRNA levels.Conclusion12/15 LOX plays an important role in the pathogenesis of experimental ALD, in part, via increased production of OXLAMs. 12/15‐LOX and OXLAMs may represent novel therapeutic targets in ALD managementSupport or Funding InformationStudy supported by the NIH and Veterans Administration.

Use of chloroacetate esterase staining for the histological diagnosis of prosthetic joint infection.

A heavy neutrophil polymorph infiltrate [>5 per high-power field (HPF) after examination of at least 5 HPF by Musculoskeletal Infection Society (MSIS) criteria] is characteristically seen in peri-implant tissues of infected prosthetic hip and knee joints. We determined whether chloroacetate esterase (CAE) staining facilitated the identification of neutrophil polymorphs in peri-implant tissues in cases of hip and knee arthroplasty infection and reassessed MSIS criteria in the light of our findings. Frozen and paraffin sections of peri-prosthetic tissues of 76 cases of failed hip and knee arthroplasties classified as septic or aseptic loosening microbiologically were analysed histologically by both haematoxylin-eosin and CAE staining. The extent of the neutrophil polymorph infiltrate was determined semiquantitatively and correlated with the microbiological and clinical diagnosis. CAE staining facilitated identification of neutrophil polymorphs in arthroplasty tissues. All cases of aseptic loosening contained fewer than two neutrophil polymorphs per HPF. CAE staining showed that in some cases of septic loosening, fewer than five neutrophil polymorphs per HPF (on average) are present in peri-prosthetic tissues. The histological criterion of more than two neutrophil polymorphs per HPF showed increased sensitivity and accuracy for the diagnosis of septic loosening. CAE is a useful stain that facilitates the identification of neutrophil polymorphs in both frozen and paraffin sections of peri-implant tissues. CAE staining shows that some microbiologically confirmed cases of septic loosening contain relatively few neutrophil polymorphs, indicating that the MSIS histological criterion of more than five neutrophil polymorphs per HPF is too high an index figure for the diagnosis of all cases of hip and knee arthroplasty infection.

Alkaline phosphatase is a useful cytochemical marker for the diagnosis of acute myelomonocytic and monocytic leukemia in the dog.

Immunophenotyping has replaced cytochemical staining as the preferred technique for classifying acute leukemia. However, some acute myeloid leukemias (AML) lack lineage-associated markers. In our experience, alkaline phosphatase (ALP) is expressed in immature canine monocytes. We hypothesized that ALP is a useful marker for monocytic AML. The objective was to compare ALP expression in neoplastic cells from dogs with lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoid leukemia (ALL), and AML. Alkaline phosphatase results were retrieved from medical records of dogs with acute leukemia. Smears from dogs with lymphoma or leukemia were also prospectively stained for ALP activity. CLL was based on persistent lymphocytosis (10×10(9) /L) and acute leukemia on ≥20% blasts in blood or bone marrow. ALL was classified based on positive phenotyping for T- or B-lymphocyte antigens, and AML on positive phenotyping for CD11b, CD11c or CD14, or cytochemical staining for chloroacetate esterase, Sudan Black B, or myeloperoxidase. There was no ALP activity in all 49 lymphomas and 7 CLLs. Weak ALP activity was seen in 31% of 14 ALL (all T-ALL). ALP activity was seen in all 20 AML (P<.001 vs ALL) with strong activity in 64% (vs 25% ALL) in most neoplastic cells (median 75% vs 9% ALL, P=.020). Of AML, 80% were CD34+ (vs 39% ALL, P=.027) and 100% were MHCII- (vs 43% ALL, P=.002). ALP activity may be useful for AML confirmation in dogs, particularly if neoplastic cells only express CD34+ on immunophenotyping.

Diabetes increases pancreatitis induced systemic inflammation but has little effect on inflammation and cell death in the lung.

Acute pancreatitis (AP) can lead to a systemic inflammatory response that often results in acute lung injury and single or multiple organ failure. In a previous study we demonstrated that diabetes aggravates the local pathophysiological process during AP. In this study we explore, if diabetes also increases pancreatitis induced systemic inflammation and causes lung injury. Acute pancreatitis was induced in untreated and streptozotocin-treated diabetic mice by injection of cerulein. Systemic inflammation was studied by IL-6 ELISA in blood plasma and white blood cell count. Lung inflammation and lung injury were quantified by chloroacetate esterase staining, evaluation of the alveolar cellularity index and cleaved caspase-3 immunohistochemistry. In normoglycaemic mice AP increased the IL-6 concentration in plasma and caused lymphocytopenia. Diabetes significantly increased the IL-6 concentration in plasma and further reduced the number of lymphocytes during AP, whereas diabetes had little effect on these parameters in the absence of pancreatitis. However, diabetes only marginally increased lung inflammation and did not lead to cell death of the lung epithelium during AP. We conclude that diabetes increases parameters of systemic inflammation during AP, but that this increase is insufficient to cause lung injury.

Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food-induced anaphylaxis

Sensitization to food antigen can occur through cutaneous exposure. We sought to test the hypothesis that epicutaneous sensitization with food antigen predisposes to IgE-mediated anaphylaxis on oral allergen challenge. BALB/c mice were epicutaneously sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin over 7 weeks or orally immunized with OVA and cholera toxin (CT) weekly for 8 weeks and then orally challenged with OVA. Body temperature was monitored, and serum mouse mast cell protease 1 levels were determined after challenge. Tissue mast cell (MC) counts were examined by using chloroacetate esterase staining. Levels of serum OVA-specific IgE and IgG(1) antibodies and cytokines in supernatants of OVA-stimulated splenocytes were measured by means of ELISA. Serum IL-4 levels were measured by using an in vivo cytokine capture assay. Epicutaneously sensitized mice exhibited expansion of connective tissue MCs in the jejunum, increased serum IL-4 levels, and systemic anaphylaxis after oral challenge, as evidenced by decreased body temperature and increased serum mouse mast cell protease 1 levels. Intestinal MC expansion and anaphylaxis were IgE dependent because they did not occur in epicutaneously sensitized IgE(-/-) mice. Mice orally immunized with OVA plus CT did not have increased serum IL-4 levels, expanded intestinal MCs, or anaphylaxis after oral challenge, despite OVA-specific IgE levels and splenocyte cytokine production in response to OVA stimulation, which were comparable with those of epicutaneously sensitized mice. Epicutaneously sensitized mice, but not mice orally immunized with antigen plus CT, have expansion of intestinal MCs and IgE-mediated anaphylaxis after single oral antigen challenge. IgE is necessary but not sufficient for food anaphylaxis, and MC expansion in the gut can play an important role in the development of anaphylaxis.

Vitamin D Increases Cellular Turnover and Functionally Restores the Skeletal Muscle after Crush Injury in Rats

Insufficient skeletal muscle regeneration after injury often impedes the healing process and is accompanied by functional deficiencies or pain. The aim of our study was to provide evidence that vitamin D improves muscle healing after muscle injury. Therefore, we used male rats and induced an injury of the soleus muscle. After crush injury, animals received either 8.3 mg/kg (332,000 IU/kg) body weight vitamin D or vehicle solution, s.c. After assessment of muscle force at days 1, 4, 14, and 42 after injury, sampling of muscle tissue served for analysis of proliferation, apoptosis, satellite cells, and prolyl-4-hydroxylase-β expression. Vitamin D application caused a significant increase in cell proliferation and a significant inhibition of apoptosis at day 4 after injury compared to control animals. The numbers of satellite cells were not influenced by the vitamin D application, but there was an increase in prolyl-4-hydroxylase-β expression, indicative of increased extracellular matrix proteins. This cellular turnover resulted in a faster recovery of contraction forces at day 42 in the vitamin D group. Current data support the hypothesis that vitamin D promotes the regenerative process in injured muscle. Thus, vitamin D treatment may represent a promising therapy to optimize recovery after injury.

In vivo fluorescence imaging of apoptosis during foreign body response

Quantification of apoptotic tissues during inflammatory processes induced by biomaterials is challenging in vivo. Here we present a non-invasive method using a fluorescence imaging system which facilitates intermittent snap shots of the current state of local apoptotic tissue. For this purpose, apoptotic cells around two different subcutaneously implanted materials (titanium discs and copper-coated titanium discs) in hairless but immunocompetent mice were quantified after 4, 8 and 23 days of implantation. For validation, the results of fluorescence signals were compared to the histology of the inflammatory tissue using apoptotic-specific TUNEL-, macrophage-specific F4/80-, neutrophile-specific NIMP-R14- and chloroacetate esterase-staining. We could demonstrate that the fluorescence signals were well suited to quantify the extent of apoptosis in vivo and this is a good indication for the biocompatibility of biomaterials. This study shows that non-invasive monitoring of tissue processes following the implantation of biomaterials is possible in vivo and may help to reduce the number of animals in studies addressing biocompatibility.

Granulocytic Sarcoma of the Male Breast in Acute Myeloblastic Leukemia with Concurrent Deletion of 5q and Trisomy 8

We describe a unique case of Granulocytic Sarcoma (GS) in a male, who presented to us with a painless right breast mass without any prior history of Leukemia. GS is an extramedullary tumor of myeloproliferative precursors and may involve multiple sites of the body, but involvement of male breast is extremely rare. In the absence of clinical history or hematological abnormality, GS may be misdiagnosed, depending on the degree of myeloid differentiation present within the tumor. Often it is misdiagnosed as lymphoma. Diagnosis is made by finding eosinophilic myelocytes, myeloperoxidase, chloroacetate esterase staining, and lysozyme immunostain. Chemotherapy regimens similar to acute myeloid leukemia are recommended to treat GS. Recognition of this rare entity is important because early, aggressive chemotherapy can induce regression of the tumor and improve patient longevity.

Stable Form of JAB1 Enhances Proliferation and Maintenance of Hematopoietic Progenitors

Overexpression of JAB1 is observed in a variety of human cancers, but how JAB1 is involved in tumor development remained to be investigated. Here we analyzed mice with modified Jab1 expression. Mice ectopically expressing a more stable form of JAB1 protein under the control of a constitutive promoter were rescued from the embryonic lethality caused by the Jab1(-/-) allele and developed a myeloproliferative disorder in a gene dosage-dependent manner. Hematopoietic cells from the bone marrow of Jab1 transgenic mice had a significantly larger stem cell population and exhibited higher and transplantable proliferative potential. In contrast, Jab1(+/-) mice, which express approximately 70% as much JAB1 protein as their wild-type littermates, showed inefficient hematopoiesis. Expression of the tumor suppressor p16(INK4a) was inversely correlated with that of JAB1, and the oncoprotein SMYD3, a newly identified JAB1 interactor, suppressed transcription of p16 in cooperation with JAB1. Thus, the expression and function of JAB1 are critical for the proliferation and maintenance of hematopoietic progenitors.