Myeloperoxidase Staining Research Articles

Systemic capsaicin inhibits neuronal activation in the brainstem during postoperative ileus in the mouse

Neuronal inhibitory reflex mechanisms contribute to postoperative ileus after abdominal surgery. During this condition, sensory neurons in the brainstem are activated. We aimed to determine the contribution of capsaicin-sensitive afferents to central vagal sensitivity in mice during postoperative ileus. Under enflurane anesthesia, C57BL/6 mice were laparotomized and the small bowel was manipulated to induce ileus or was left untouched as a sham-treatment group. A subgroup of ileus animals was pre-treated with Capsaicin (1 microm/kg, i.p.) 48 h before small bowel manipulation. The animals were killed 24 h later and the brainstem was removed for Fos immunohistochemistry, which was quantified in the nucleus of the solitary tract (nTS). Spontaneous jejunal motility was recorded in vitro. Leukocyte infiltration in the intestinal muscularis was studied by myeloperoxidase staining as an index of postoperative inflammation. There were 30+/-9 Fos-positive neurons counted in the nTS after ileus and 6+/-2 in sham controls (Bregma -7.70 mm, P=0.01). A reduction to 8+/-3 was observed after Capsaicin pre-treatment in ileus animals (P<0.05). Peak amplitudes of spontaneous jejunal motility were 2+/-0.3 cmH2O during postoperative ileus, 3+/-0.6 cmH2O after ileus with capsaicin pre-treatment, and 10+/-2 cmH2O in control animals (N=6, both P<0.05). The number of leukocytes infiltrating the muscularis was 39+/-9/mm2 during ileus and 1.8+/-1/mm2 in controls (mean+/-SEM, P<0.01, N=6). After capsaicin, this number increased to 72+/-28/mm2 in ileus animals (P<0.05 vs control animals, N=7). The inhibition of capsaicin-sensitive vagal afferent pathways appears to boost rather than to attenuate the inflammatory response during postoperative ileus, while intestinal motility remained unchanged. This suggests a protective role of the capsaicin-sensitive afferent innervation for the inflammatory phase of postoperative ileus.

Hyperbaric oxygen therapy reduces neuroinflammation and expression of matrix metalloproteinase‐9 in the rat model of traumatic brain injury

The acute inflammatory response plays an important role in secondary brain damage after traumatic brain injury (TBI). Neutrophils provide the main source of matrix metalloproteinases (MMPs) which also play a deleterious role in TBI. Numerous preclinical studies have suggested that hyperbaric oxygen therapy (HBOT) may by beneficial in various noncerebral and cerebral inflammatory diseases. The goal of this study was to evaluate the effects of HBOT on inflammatory infiltration and the expression of MMPs in correlation with secondary cell death in the rat model of dynamic cortical deformation (DCD). Twenty animals underwent DCD with subsequent HBOT (2.8 ATA, two sessions of 45 min each); 10 animals: DCD and normobaric oxygenation (1 ATA), 10 animals: not treated after DCD. Cell death was evaluated by TUNEL. Neutrophils were revealed by myeloperoxidase staining. Immunohistochemical staining for MMP-2 and -9 and tissue inhibitors of MMP-1 (TIMP-1) and -2 was also performed and the results were quantitatively evaluated by image analysis. In the animals treated by HBOT, a significant decrease in the number of TUNEL-positive cells and neutrophilic inflammatory infiltration was seen in comparison with nontreated animals and those treated by normobaric oxygen. The expression of MMP-9 was also significantly lower in the treated group. Staining for MMP-2 and TIMP-2 did not change significantly. Our results demonstrate that HBOT decreased the extent of secondary cell death and reactive neuroinflammation in the TBI model. The decline of MMP-9 expression after HBOT may also contribute to protection of brain tissue in the perilesional area. Further research should be centred on the evaluation of long-term functional and morphological results of HBOT.

Granulocytic Sarcoma in Erythroleukemia (M6).

Granulocytic Sarcomas are extramedullary tumors composed of immature myeloid cells, these tumors can be seen in myeloid malignancies such as chronic myelogenous leukemia, myelodysplastic syndrome and acute myelogenous leukemia. They can be seen at the time of diagnosis of the myeloid malignancy, prior to diagnosis, or after treatment as a first sign of relapse. We report here the case of a young woman who presented with a 6 month history of a slowly enlarging nasal mass. Biopsies of the lesion had been inconclusive until a CBC revealed abnormal blasts in the peripheral blood. Subsequently bone marrow biopsy revealed more than 59% erythroid precursors with a preponderance of immature erythroblasts and 33% myeloblast consistent with AML (M6). Reanalysis of the nasal biopsy demonstrated positive staining for CD68, Myeloperoxidase, Lysozyme, and CD117, but negative for CD34, suggestive of granulocytic sarcoma. The patient received induction chemotherapy with idarubicin and cytarabine and achieved bone marrow remission along with complete resolution of the nasal mass. To our knowledge this is the first report of a granulocytic sarcoma in association with AML M6.

TEL-AML1 Transgenic Zebrafish Model of Acute Lymphoblastic Leukemia (ALL).

TEL-AML1 (ETV6-RUNX1) fusion is the product of the t(12;21) chromosomal translocation, the most common chromosomal rearrangement in childhood cancer. The translocation fuses two highly conserved transcription factors, TEL and AML1 that have essential roles in hematopoiesis. Genetic studies of identical twins with concordant leukemia, the detection of leukemia-specific fusion genes in neonatal blood spots, and the existence of multiple leukemic subclones at ALL diagnosis point to prenatal origin of the fusion and long latency before leukemia development. Additionally, a study in growth factor-dependent cell lines and transgenic mice, and several mouse transplant models suggest that TEL-AML1 is insufficient by itself for leukemic transformation. To determine whether TEL-AML1 has selective transforming impact on a particular stem cell lineage, we established stable zebrafish transgenic lines expressing the TEL-AML1 fusion protein both constitutively, and within the lymphoid progenitors. In these lines, either the zebrafish β-actin (ZBA) promoter or the xenopus elongation factor-1 (XEF1) promoter drives TEL-AML1 expression constitutively, while the zebrafish RAG-2 promoter (RAG2) selectively drives TEL-AML1 expression in committed lymphocyte precursors, but not in earlier multilineage hematopoietic precursors. TEL-AML1 expression, alone or fused to EGFP, was detected at 24-hour post fertilization (hpf) with fluorescent microscopy or RT-PCR and confirmed with western blot analysis. In-Situ Hybridization and confocal microscopy revealed that transgenic zebrafish maintained TEL-AML1 expression throughout adulthood. The expression of TEL-AML1 was associated with accumulation of immature hematopoietic progenitor cells, mostly in the kidney and spleen of several transgenic zebrafish lines, within a few weeks of development, indicating an expansion of the progenitor cell population. To date, a small number (5 out of 391 transgenic fish; 1.3%) of founders and progeny of zebrafish transgenic for ZBA- or XEF1-TEL-AML1 fusion developed an infiltrating lymphoid neoplasm, most likely acute lymphoblastic leukemia with a latency of 8–12 month. The lymphoblasts were negative for myeloperoxidase and PAS staining, with abundant expression of both EGFP in lymphoblasts from EGFP-TEL-AML1 line, and pre-B-cell leukemia transcription factor-1 (PBX1; also called cALLa) indicating that Leukemias originated in a TEL-AML1 expressing cell(s), and mimic the human CD10-positive precursor-B cell ALL. None of 350 wild-type, 125 control EGFP fish or 353 RAG2-TEL-AML1 transgenic zebrafish had any leukemia or hematopoietic changes. Therefore, the expression of TEL-AML1 fusion in our ZBA- and XEF1-TEL-AML1 transgenic lines generates a premalignant state, which appears to require additional genetic events for acquisition of the leukemic phenotype. The fact that none of the RAG2 zebrafish expressing TEL-AML1 in committed lymphoid progenitors developed leukemia or progenitor expansion indicates that the leukemic stem cell associated with TEL-AML1 leukemia is an immature progenitor earlier than the committed lymphoid progenitors where RAG2 expression is detected. The TEL-AML1 transgenic zebrafish lines provide a basis for large scale and targeted mutagenesis screens aimed at identifying mutations and cooperating events that are required for TEL-AML1 induced leukemias.

Peculiar Morphological, Cytochemical, Biochemical and Immunophenotyping Features in One Case of Acute Myeloid Leukaemia at the Onset.

On July 28th 2005, a 53-year old woman was admitted to our Hematology Dpt. with fever (37.5 C), fatigue, marked gingiva hypertrophy, mild hepatomegaly and absence of lymphoadenopaty and splenomegaly. Laboratory tests showed high leukocyte count of 182 x109/L, moderately increased platelet count (438 x109/L), normocytic anemia (10,9 g/dl, MCV 84 fl). Biochemical parameters, including lysozyme, were in the normal range except for high level of lactate dehydrogenase (2524 UI/l). Peripheral blood (PB) smear showed the presence of 94% of blasts, without evident granules. At the cytochemical myeloperoxidase staining blast cells revealed the presence of single or multiple Auer rods, while cytoplasmic granules were positive to the α-naphtyl butyrate esterase. In the bone marrow (BM) aspirate blasts were 80% showing same morphological and cytochemical features as in peripheral blood, while the granulocytic maturating series was 14%. No evidence of morphologically identifiable monocytic series in PB neither in BM. Megakariocytes were slightly increased and there was trilineage dysplasia. Blast immunophenotyping in PB and in BM was as follow: CD45, CD33, CD13, CD7 and HLA-Dr positive, 36% MPO positive and 48% co-express CD11c an CD117. Moreover blasts were CD14, CD19 and CD56 negative. FAB diagnosis was AML-M4. Interphase Fluorescence in situ Hybridization (FISH) for detection of AML1/ETO and bcr/abl gene rearrangements was negative. After treatment with 2 g hydroxyurea and leucapheresis, the WBC count was still high (140 x109/L) while after 1g of aracytin, WBC count decreased to 31000/mm3. Patient is presently enrolled into the GIMEMA protocol AML-12.This case shows at the onset following abnormalities:- an increased platelet count is a rare evenience in an AML at the onset;- negativity for lysozyme;- absence of CD14;- presence of Auer rods in quite all the blast population, which is a very rare evenience associated with monocytic subtypes of acute myeloblastic leukemia (AML);- atypical homogenous cytochemical pattern of blasts, showing Auer rods MPO positive and granules ANBE positive;- absence of any morphological differentiation of monocytic series. S4479

Multilineage Involvement of Light Microscopic Myeloperoxidase-Negative Acute Leukemia

The classification of acute leukemia has traditionally been based on a combination of morphology and cytochemical staining data, including myeloperoxidase (MPO) reaction; however, a recent World Health Organization (WHO) classification entails use of cytogenetic and molecular findings in addition to the classic morphological and immunophenotypic analyses. Nevertheless, there have been rare cases in which blastic cells show multilineage phenotypes. These cases may be classified as acute leukemia of ambiguous lineage in the recent WHO classification. We report the case of a 49-year-old man with acute leukemia with multilineage phenotypes. Morphological findings led to a diagnosis of acute myeloid leukemia M2 by the French-American-British classification, but at light microscopy the results of MPO staining were negative for blast cells. In contrast, results of reverse transcription polymerase chain reaction and fluorescence-activated cell sorter analyses were positive for expression of MPO messenger RNA and protein. The blast cells expressed CD4, CD19, CD22, CD33, CD38, CD79a, and HLA-DR and showed rearrangement of the immunoglobulin heavy chain and TCR-3 genes. Results of immunoelectron microscopic analysis of the blast cells were positive for MPO, CD19, CD33, CD34, CD38 and glycophorin A but not for platelet peroxidase. According to these results, the blast cells had at least 4 lineage phenotypes. We concluded that the multiparameter analyses conducted in this case, including immunological and ultrastructural assays, were important in arriving at the appropriate diagnosis of acute leukemia of ambiguous lineage in the new WHO classification.

The Application of Molecular Analyses for Primary Granulocytic Sarcoma with a Specific Chromosomal Translocation

Primary granulocytic sarcoma (GS) is a rare disease defined by the absence of antecedent or concomitant leukemic cells in the bone marrow and the peripheral blood. Immunohistochemical staining for myeloperoxidase is necessary for a definite diagnosis. Otherwise, primary GS is often misdiagnosed as a malignant lymphoma or other malignancies. Primary GS is well known to frequently develop into acute myeloid leukemia (AML). Here we describe a 28-year-old woman with primary GS manifesting as an epidural tumor in the sacral region accompanied by meningeal dissemination. Fluorescence in situ hybridization analysis detected the AML1/MTG8 fusion gene in neoplastic cells obtained from her cerebrospinal fluid specimen and the epidural mass. The AML1/MTG8 fusion gene transcript was also detected by a nested reverse transcriptase-polymerase chain reaction analysis of mononuclear cells from the bone marrow, although leukemic cells were not recognized in a microscopical examination of the patient's bone marrow. Systemic chemotherapy with high-dose cytarabine followed by local radiotherapy was performed, and the patient clinically achieved a complete response. These molecular analyses provide a precise method of diagnosis, especially with respect to the French-American-British AML classification, according to the characteristic karyotypic alterations, and a patient consequently can quickly be given appropriate systemic chemotherapy as induction therapy.

Modulation of the Lung Inflammatory Response to Serotype 8 Pneumococcal Infection by a Human Immunoglobulin M Monoclonal Antibody to Serotype 8 Capsular Polysaccharide

The human monoclonal antibody to serotype 8 pneumococcal capsular polysaccharide D11 [immunoglobulin M(kappa)] protects wild-type and complement component 4 knockout (C4 KO) mice against lethal intratracheal challenge with serotype 8 pneumococcus, but it does not promote polymorphonuclear leukocyte (PMN)-mediated pneumococcal killing in vitro. In this study, we investigated the effect of D11 on the blood and lung bacterial burdens and the serum and lung expression of inflammatory chemokines and cytokines in an intratracheal challenge model with serotype 8 pneumococcus in C4 KO mice. Pneumococcus was not detected in the blood of D11-treated mice, whereas control mice had high-grade bacteremia with >10(7) CFU. Control mice had a >5-log increase in lung CFU and D11-treated mice manifested a nearly 3-log increase in lung CFU compared to the original inoculum 24 h after infection. Serum and lung levels of soluble macrophage inflammatory protein 2 (MIP-2) and interleulin-6 (IL-6) as measured by an enzyme-linked immunosorbent assay were lower in D11-treated mice than in control mice 24 h after infection. Real-time PCR was performed to examine lung mRNA chemokine and cytokine expression. The results showed that D11-treated mice had significantly less gamma interferon, MIP-2, IL-12, monocyte chemoattractant protein 1/JE, and tumor necrosis factor alpha expression than control mice 24 h after infection. Histopathology and immunohistochemical staining of lung tissues revealed that D11-treated mice had less inflammation, fewer PMNs, and less myeloperoxidase staining than control mice 24 h after infection. These findings suggest that the efficacy of certain serotype-specific antibodies against pneumococcal pneumonia could be associated with modulation of the lung inflammatory response and a reduction in host damage.

CD62 Blockade with P-Selectin Glycoprotein Ligand-Immunoglobulin Fusion Protein Reduces Ischemia-Reperfusion Injury After Rat Intestinal Transplantation

Intestinal transplantation (ITx) is severely limited by ischemia-reperfusion (I/R) injury. This study investigates I/R injury and ameliorates its consequences by using a recombinant protein targeted against selectins (recombinant P-selectin glycoprotein ligand-immunoglobulin [rPSGL-Ig]). An isogeneic model of ITx was undertaken with control animals (no therapy) and treatment animals (rPSGL-Ig). Survival was assessed. Separate groups underwent an analysis examining tissue at multiple time points after I/R injury including histopathology; myeloperoxidase staining; immunostaining for CD3 and ED2; polymerase chain reaction analysis of interleukin (IL)-8/cytokine-inducible neutrophil chemoattractant, IL1beta, IL-6, interferon-gamma, IL-2, IL-4, and IL10; and western blots for hemoxygenase-1, BCL-2, and BCL-xl. Standard statistical analysis was undertaken. Treatment with rPSGL-Ig resulted in significantly improved survival after ITx. Analysis demonstrated diminished injury on histopathology and reduced tissue infiltration of neutrophils and lymphocytes. Significant differences in the cytokine profile after ITx were seen between the two groups including the production of inflammatory cytokines at 24 hr and the Th1 and Th2 cytokines at 2 and 4 hr. Last, treatment resulted in increased production of hemoxygenase, BCL-2, and BCL-xl. The results of this investigation of I/R injury after ITx revealed that rPSGL-Ig treatment led to marked improvement in outcome. The mechanism of action seems to involve the blockade of neutrophil and lymphocyte infiltration leading to a decreased inflammatory response possibly driven by Th2 cytokines. The results not only lend insight into the mechanisms behind I/R injury after ITx but also demonstrate a potential therapeutic modality to ameliorate its consequences.

Mast Cell Tryptase and Microphthalmia Transcription Factor Effectively Discriminate Between Cutaneous Mast Cell Disorders and Leukemia Cutis: A Comparative Cytochemical and Immunohistochemical Study

Cutaneous mast cell disorders are uncommon but a subset of these disorders, especially mastocytoma, can histologically mimic leukemia cutis. Our objective was to employ a panel of cytochemical and immunohistolochemical markers to determine which markers would be most useful in distinguishing these entities. We studied 17 cases of cutaneous mast cell disorders (UP, TMEP, and mastocytoma) and 12 cases of leukemia cutis (myeloid, myelomonocytic and monocytic) with toluidine blue, Giemsa, pinacyanol erythrocyanate (PE), mast cell tryptase, microphthalmia transcription factor (MITF), CD117, myeloperoxidase, CD43 and CD68. We found that PE stained the majority of cases of mast cell disorders (11/15) but did not stain leukemia cutis. Tryptase and MITF stained all cases of mast cell disorders but not leukemia cutis. Although CD117 showed strong staining in all cases of mast cell disorders, it also stained up to 25% of cases of leukemia cutis (2/8). Staining for myeloperoxidase was specific to leukemia cutis but detected only leukemias with myeloid differentiation (6/12). Our results show that mast cell tryptase and microphthalmia transcription factor are equally effective in distinguishing mast cell disorders from leukemia cutis. Interestingly, pinacyanol erythrocyanate appears to be specific for mast cell disease, as no staining was seen in leukemia cutis.

Microarray Analysis of the Short Isoform of Ikaros Overexpressed in Patients with Hematologic Malignancies.

We analyzed expression of the transcription factor Ikaros in human hematologic malignancies and found overexpression of the short isoform of Ikaros, Ik-6, in patients with blast crisis of chronic myeloid leukemia and pre-B cell acute lymphoblastic leukemia. We have made several stable transfectants which overexpress Ik-6 and analyzed the consequence of Ik-6 overexpression.First, overexpression of Ik-6 in FDH-1, which was established from a patient with early pre-B acute lymphoblastic leukemia, and Ramos conferred resistance to dexamethasone- and anti-IgM-induced apoptosis, respectively. Upon treatment with dexamethasone, FDH-1 cells demonstrated massive apoptosis measured by staining with Annexin V. In contrast to mock transfectants, Ik-6 transfectants showed less marked apoptosis (mock 86.4% vs Ik-6 34.4–73.6%, p=0.01). We also used Ramos as a model of anti-IgM-induced apoptosis, and flow cytometric analysis was performed with Annexin-V staining. In contrast to mock transfectants, Ik-6 transfectants showed less pronounced apoptosis (mock 40.4% vs Ik-6 23.1–27.4%, p=0.03). Next, overexpression of Ik-6 in Nalm6 lead to impaired cell growth and cell cycle progression. Cell growth and cell cycle were analyzed and compared with mock transfectants and Ik-6 transfectants using MTT assay and DNA content analysis with PI staining, respectively. MTT assay demonstrated that, after three days of growth, Ik-6 transfectants grew more slowly than mock transfectants (Ik-6/mock ratio 0.62–0.67; p=0.01). DNA content analysis showed that, after three days of growth, more Ik-6 transfectants underwent G0–G1 arrest than mock transfectants (Ik-6/mock ratio 1.15–1.21; p=0.01), and less Ik-6 transfectants entered into S phase than mock transfectants (Ik-6/mock ratio 0.84–0.90; p=0.05).Finally, overexpression of Ik-6 promoted all-trans retinoic acid (ATRA)-induced granulocytic differentiation, and, on the other hand, inhibited vitamin D3 (VD3)-induced monocytic differentiation of HL-60. Upon induction of granulocytic differentiation by ATRA, mock transfectants showed 74.5% positivity of myeloperoxidase (MPO) staining. In contrast, Ik-6 transfectants showed 51.5–56.8% of positivity of MPO staining (p=0.04). NBT reduction assay demonstrated 43.8% positivity with mock transfectants and 62.3–67.3% positivity with Ik-6 transfectants (p=0.001). Upon induction of monocytic differentiation by VD3, mock transfectants showed 51.0% positivity of nonspecific esterase (NSE) staining. In contrast, Ik-6 transfectants showed 26.8–36.0% of positivity of NSE staining (p=0.03).To understand the underlying mechanisms, we report the results of microarray analysis of Ik-6 transfectants.

Heme oxygenase-1 expression in human lungs with cystic fibrosis and cytoprotective effects against Pseudomonas aeruginosa in vitro.

Inflammation and oxidative stress play important roles in cystic fibrosis (CF) lung disease. Inflammatory/oxidant-mediated induction of heme oxygenase-1 (HO-1) is believed to be a cytoprotective response. This study examined HO-1 expression in lung samples from patients with CF using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. In addition, we evaluated myeloperoxidase staining as a marker of acute inflammation and potentially an increase in oxidant stress and Prussian blue and ferritin staining to assess iron status of the lung. Macrophage HO-1 staining was increased in diseased lungs as compared with normal control subjects and correlated with myeloperoxidase staining. Quantitative reverse transcription-polymerase chain reaction further supported an increase in HO-1 expression in CF lung disease. Although iron staining was minimal, ferritin staining was increased in diseased lungs in concert with HO-1 staining. To determine whether HO-1 induction was cytoprotective, we evaluated a CF airway epithelial cell line, IB3.1, in response to Pseudomonas aeruginosa-induced injury/apoptosis in cells overexpressing HO-1 by either transient or stable transfection of pcDNA3.1/HO-1 construct. Overexpression of HO-1 resulted in protection against P. aeruginosa-induced injury/apoptosis. This suggests that the induction of HO-1 in patients with CF is a cytoprotective event and that augmenting its expression is a potential therapy against bacterial injury.

Heme-oxygenase-1 expression correlates with severity of acute cellular rejection in lung transplantation.

Heme-oxygenase-1 (HO-1) has been shown to play an important role in oxidative stress, and recent studies indicate that it is a graft survival protein in cardiac and liver transplant models. Our laboratory previously found HO-1 to be increased in human lung allografts with acute cellular rejection (ACR) and in active obliterative bronchiolitis. To better understand the role of HO-1 in ACR we studied the relationship between HO-1 expression and ACR in a rodent model of lung transplantation. Orthotopic left lung transplantation was performed from Lewis (donor) to Sprague-Dawley (recipient) rats, and ACR (Grade A0 to A4) was evaluated at days 3, 5, and 7. HO-1 expression was assessed by immunohistochemistry and Western analysis, and compared with the degree of ACR. Myeloperoxidase staining was evaluated as an indirect measure of oxidant stress. Donors and recipients were also treated with either an inhibitor of HO activity, tin protoporphyrin or an inducer, cobalt protoporphyrin, and the severity of ACR was compared with that in untreated allografts. HO-1 expression was elevated in transplanted versus native lungs or isografts, and the degree of elevation was closely correlated with ACR grade (p < 0.001). Similarly, myeloperoxidase expression increased with time and severity of ACR. Administration of the metalloporphyrins, tin protoporphyrin and cobalt protoporphyrin, produced no significant difference in the degree of ACR, but did alter the severity of ischemia-reperfusion injury. Similar to what occurs in human lung transplantation, HO-1 expression is increased in a rodent lung transplant model of ACR and correlates with the severity of rejection. Altering its expression does not appear to affect the degree of ACR.

HMG-CoA reductase inhibitor, atorvastatin, promotes sensorimotor recovery, suppressing acute inflammatory reaction after experimental intracerebral hemorrhage.

Statins have neuroprotective effects on ischemic stroke. They modify the endothelial function, increase blood flow, and inhibit thrombus formation, which are independent of lipid-lowering effects. However, whether statins have a protective effect toward hemorrhagic stroke is yet unknown. To test this possibility, we attempted to determine the effect of atorvastatin on experimental intracerebral hemorrhage (ICH). ICH was induced using stereotaxic infusion of collagenase into the left basal ganglia in adult rats. Atorvastatin (1 mg/kg or 10 mg/kg) or phosphate-buffered saline was administered for 2 weeks. To monitor the sensorimotor deficits, limb placing and Rotorod tests were performed. Hematoma volume, brain water content, and hemispheric atrophy were analyzed. Immunohistochemical staining for myeloperoxidase (MPO), microglia (OX42), inducible nitric oxide synthase (iNOS), or endothelial nitric oxide synthase (eNOS) was performed. Perihematomal cell death was determined by TUNEL staining. The atorvastatin-treated ICH group showed better performance on Rotorod and limb placing tests when compared with the vehicle-treated group (P<0.01). The hematoma volumes between groups were not different, but the brain water content and hemispheric atrophy were reduced in the atorvastatin-treated ICH group. Atorvastatin reduced TUNEL-positive cells, iNOS expression, and MPO-positive or OX42-positive cells in the perihematomal regions in a dose-dependent manner, whereas it increased eNOS expression. The present study shows that atorvastatin reduces the perihematomal cell death via antiinflammation, which is associated with sensorimotor recovery after experimental ICH.

The Psuedo-Chediak–Higashi Anomaly: An Unusual Staining Pattern in an Indian Child with Acute Myeloid Leukemia

The authors describe the psuedo-Chediak–Higashi anomaly in a 12-year-old boy with acute myeloid leukemia (AML-M2). There were large purple granules in the blasts, promyelocytes, and myelocytes. Instead of the previously described patterns, the authors observed a unique rim pattern staining of the granules in both the May–Grunwald–Giemsa and the myeloperoxidase stains. Moreover, many of the granules had central vacuoles with strong myeloperoxidase positivity at the periphery. The bone marrow had a much higher positivity for these mega-granules as compared to the peripheral blood. On remission, these granules were no longer seen. To the best of the authors' knowledge, this pattern of staining has not been previously reported in the literature.

Myeloperoxidase-positive inflammatory cells participate in bile duct damage in primary biliary cirrhosis through nitric oxide-mediated reactions

Previous studies have suggested that increased nitric oxide (NO)-mediated products are found in the livers of subjects with primary biliary cirrhosis (PBC), but the mechanisms involved remain enigmatic. We took advantage of immunohistochemistry and several unique monoclonal antibodies to study inflammatory cells responsible for the generation of NO, the enzymes responsible for NO production, the expression of 3-nitrotyrosine, and the presence of CD68 + and/or myeloperoxidase (MPO) + cells. We examined a total of 113 liver specimens, including 64 with PBC, 19 with primary sclerosing cholangitis (PSC), 6 with non-A, non-B hepatitis, 6 with alcoholic liver disease, 4 with cryptogenic cirrhosis, 4 with biliary atresia, and 10 normal subjects. Twenty-two percent of PBC had elevated expression of 3-nitrotyrosine in their bile duct epithelial cells (BECs) ( P = .0316). Furthermore, the BECs in PBC also demonstrated apoptotic changes. MPO-positive inflammatory cells were also noted adjacent to the basement membrane. In contrast, the liver of normal subjects showed few apoptotic changes in the bile ducts, with no evidence of MPO staining in the portal area. Furthermore, sections from livers of subjects with stage I or stage II PBC demonstrated significantly increased inflammatory cell infiltration ( P = .0064) and elevated 3-nitrotyrosine expression in BECs ( P = .0246) compared with stage III and IV. The presence of 3-nitrotyrosine was closely associated with infiltrating CD68- and/or MPO-positive cells. There was also a stage-associated difference in the presence of bile duct infiltrating cells and 3-nitrotyrosine in PBC with an increase dominant in early stage disease. In conclusion, NO and reactive oxygen species, collectively determined as 3-nitrotyrosine, are associated with bile duct destruction in PBC and are particularly prevalent in early stage disease. (H epatology 2003;38:1018-1025.)

Myeloperoxidase–Positive Inflammatory Cells Participate in Bile Duct Damage in Primary Biliary Cirrhosis Through Nitric Oxide–Mediated Reactions

Previous studies have suggested that increased nitric oxide (NO)–mediated products are found in the livers of subjects with primary biliary cirrhosis (PBC), but the mechanisms involved remain enigmatic. We took advantage of immunohistochemistry and several unique monoclonal antibodies to study inflammatory cells responsible for the generation of NO, the enzymes responsible for NO production, the expression of 3–nitrotyrosine, and the presence of CD68+ and/or myeloperoxidase (MPO)+ cells. We examined a total of 113 liver specimens, including 64 with PBC, 19 with primary sclerosing cholangitis (PSC), 6 with non–A, non–B hepatitis, 6 with alcoholic liver disease, 4 with cryptogenic cirrhosis, 4 with biliary atresia, and 10 normal subjects. Twenty–two percent of PBC had elevated expression of 3–nitrotyrosine in their bile duct epithelial cells (BECs) (P = .0316). Furthermore, the BECs in PBC also demonstrated apoptotic changes. MPO–positive inflammatory cells were also noted adjacent to the basement membrane. In contrast, the liver of normal subjects showed few apoptotic changes in the bile ducts, with no evidence of MPO staining in the portal area. Furthermore, sections from livers of subjects with stage I or stage II PBC demonstrated significantly increased inflammatory cell infiltration (P = .0064) and elevated 3–nitrotyrosine expression in BECs (P = .0246) compared with stage III and IV. The presence of 3–nitrotyrosine was closely associated with infiltrating CD68– and/or MPO–positive cells. There was also a stage–associated difference in the presence of bile duct infiltrating cells and 3–nitrotyrosine in PBC with an increase dominant in early stage disease. In conclusion, NO and reactive oxygen species, collectively determined as 3–nitrotyrosine, are associated with bile duct destruction in PBC and are particularly prevalent in early stage disease.

Basement membrane and matrix metalloproteinases in monocrotaline-induced liver injury.

Monocrotaline (MCT) is a pyrrolizidine alkaloid that causes liver injury in animals. In rats, injury is characterized by sinusoidal endothelial cell (SEC) damage and centrilobular parenchymal cell necrosis. Loss of endothelium is a possible outcome of the action of matrix metalloproteinases (MMPs), specifically MMP-9 from neutrophils and SECs and MMP-2 from SECs, on basement membrane collagen. Accordingly, the dynamics of MMPs in MCT-induced SEC damage were studied. Rats were treated with MCT (300 mg/kg, ip), and livers were collected at 8, 12, and 18 h. Immunofluorescence analysis of frozen sections of livers from MCT-treated rats revealed a progressive reduction in basement membrane heparan sulfate proteoglycan and collagen IV. A time-dependent increase in total type IV collagenase activity and MMP-9 content occurred in the livers of MCT-treated rats, as measured by fluorescent collagenase activity assay and gelatin zymography, respectively. Progressive neutrophil accumulation and activation in the liver after MCT treatment were demonstrated by an increased activity of myeloperoxidase and pronounced staining for hypochlorite-modified proteins generated via the myeloperoxidase-hydrogen peroxide-halide system. However, neutrophil depletion did not protect against MCT-induced SEC injury. Treatment of NP-26 cells, a sinusoidal endothelial cell line, with MCT resulted in dose-dependent release of MMP-9 from the cells. The results demonstrate the degradation of basement membrane components with a concurrent increase in the amount and activity of MMP-9, likely originating from sinusoidal endothelial cells, neutrophils, and probably other cell types. This suggests the possibility of a role for MMPs in the SEC detachment and loss that occurs during MCT hepatotoxicity.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats.

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 +/- 70 vs 1451 +/- 235 x 10(2) neutrophils/mg; P<0.01) and in the kidneys (526 +/- 89 vs 852 +/- 73 10(2) neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Neuroprotective effect of urinary trypsin inhibitor against focal cerebral ischemia-reperfusion injury in rats.

Acute inflammatory reactions cause neuronal damage in cerebral ischemia-reperfusion. Urinary trypsin inhibitor (UTI), a serine protease inhibitor, is cytoprotective against ischemia-reperfusion injury in the liver, intestine, kidney, heart, and lung through its antiinflammatory activity. Neuroprotective action of UTI on transient global cerebral ischemia has been documented. This is the first study to determine whether UTI is neuroprotective against transient focal cerebral ischemia. Adult male Wistar rats were randomly assigned to the following treatment groups: 0.9% saline (control, n = 9); 100,000 U/kg UTI (n = 9); and 300,000 U/kg UTI (n = 9). Treatments were performed intravenously 10 min before right middle cerebral artery occlusion for 2 h and subsequent reperfusion. Ninety-six hours after the onset of reperfusion, the motor neurologic deficit and the cerebral infarct size were evaluated. Furthermore, immunohistochemical staining for myeloperoxidase and nitrotyrosine to count infiltrating neutrophils and nitrated cells, respectively, was performed on the brain sections. Infarct volume in the 300,000 U/kg UTI group was smaller than in the 100,000 U/kg UTI and saline control groups (P < 0.05). Treatment with 300,000 U/kg UTI showed a trend to improve neurologic outcome but did not reach statistical significance (P = 0.0693). The significant decrease in neutrophil infiltration was observed in the ischemic hemisphere treated with 300,000 U/kg UTI compared with saline control (P < 0.05). Nitrotyrosine deposition in the ischemic hemisphere was significantly reduced in the 300,000 U/kg UTI group compared with saline control and 100,000 U/kg UTI groups (P < 0.05). Intravenous pretreatment with 300,000 U/kg UTI reduces focal ischemia-reperfusion injury in the rat brain, potentially opening a novel therapeutic avenue for the treatment of cerebral ischemia.