Hiroyuki Usuda

Development, differentiation, and phenotypic heterogeneity of murine tissue macrophages.

In murine ontogeny, macrophage precursor cells develop in the yolk sac and fetal liver. Primitive macrophages also appear in the yolk sac, migrate to various tissues, and differentiate into several fetal macrophage populations. Because the development of the monocytic cell lineage is incomplete in the early stage of fetal hematopoiesis, primitive/fetal macrophages are considered to originate from granulocyte‐macrophage colony‐forming cells or earlier macrophage precursors, bypassing the early monocytic cell series. In adult mice rendered severely monocytopenic by administration of strontium‐89, resident macrophages are maintained by self‐renewal. In contrast, administration of liposome‐encapsulated dichloromethylene diphosphonate (clodronate) results in the elimination of various tissue macrophage populations. The repopulation of affected macrophages is dependent on the increase of precursors in the liver and spleen during the period of macrophage depletion. Such precursors reconstitute heterogeneous macrophage subpopulations. In mice homozygous for the osteopetrosis (op) mutation, the absence of macrophage colony‐stimulating factor (M‐CSF) activity results in a deficiency of monocytes and monocyte‐derived macrophages. However, immature macrophages are present in various tissues. Administration of M‐CSF to op/op mice induces the increased proliferative capacity and the morphological maturation of macrophages. However, the responses of individual tissue macrophage subpopulations to M‐CSF are different. These results indicate that macrophage development, differentiation, and proliferation are regulated by the tissue microenvironment including the in situ production of macrophage growth factors in both fetal and adult life.

Limited Resection for Noninvasive Bronchioloalveolar Carcinoma Diagnosed by Intraoperative Pathologic Examination

BACKGROUND The establishment of limited resection procedures for non-small cell lung cancer is expected. Many groups have suggested noninvasive bronchioloalveolar carcinoma (BAC) to be a potential indication for limited resection. METHODS We designed a prospective phase II study evaluating limited resection for noninvasive BAC diagnosed by intraoperative pathologic examination. From 1999 to 2007, limited resection was the procedure in 46 patients (16 men and 30 women; median age, 69 years; range, 49 to 83) who were diagnosed intraoperatively as having noninvasive BAC. The first end point was the predictive value of the intraoperative pathologic examination for noninvasive BAC diagnosis. The second end point was overall survival, disease-free survival, and cancer-specific survival, calculated using the Kaplan-Meier method. RESULTS We performed wedge resections for 44 patients and segmentectomy for 2 patients. Permanent pathologic examination revealed 3 patients had primary lung adenocarcinomas other than noninvasive BAC. The predictive value of intraoperative pathologic examination for noninvasive BAC diagnosis was 94%. During a median 51-month follow-up, there were only 2 cancer unrelated deaths. The 5-year overall survival rate and the disease-free survival rate were 93%, and the 5-year cancer-specific survival rate was 100%. CONCLUSIONS The results of our prospective phase II study indicate that limited resection, mainly by wedge resection, is a potentially curative surgical procedure and may be an acceptable alternative to lobectomy for patients with noninvasive BAC. Furthermore, an intraoperative pathologic diagnosis of noninvasive BAC is strongly predictive and allows for an intraoperative decision to perform a limited resection in these patients.

An In Vitro Coculture Model of Transmigrant Monocytes and Foam Cell Formation

Abstract —To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of foam cells in vitro, we have developed a 2-coculture system with rabbit aortic endothelial cells (AECs), aortic smooth muscle cells (SMCs), and a mixture of matrix proteins on polyethylene filters in chemotaxis chambers. AECs were seeded on a mixture of type I and IV collagen with or without various types of serum lipoproteins (method 1) or on matrix proteins secreted by SMCs (method 2). In these coculture systems, rabbit AECs can maintain a well-preserved monolayer for up to 2 weeks. When human CD14-positive monocytes were added to the upper medium of the system, with monocyte chemotactic protein-1 treatment ≈60% of the monocytes transmigrated within 24 hours and were retained for up to 7 days, whereas without MCP-1 treatment,

Plasma Cells and the Chronic Nonsuppurative Destructive Cholangitis of Primary Biliary Cirrhosis

There has been increased interest in the role of B cells in the pathogenesis of primary biliary cirrhosis (PBC). Although the vast majority of patients with this disease have anti‐mitochondrial antibodies, there is no correlation of anti‐mitochondrial antibody titer and/or presence with disease severity. Furthermore, in murine models of PBC, it has been suggested that depletion of B cells may exacerbate biliary pathology. To address this issue, we focused on a detailed phenotypic characterization of mononuclear cell infiltrates surrounding the intrahepatic bile ducts of patients with PBC, primary sclerosing cholangitis, autoimmune hepatitis, chronic hepatitis C, and graft‐versus‐host disease, including CD3, CD4, CD8, CD20, CD38, and immunoglobulin classes, as well as double immunohistochemical staining for CD38 and IgM. Interestingly, CD20 B lymphocytes, which are a precursor of plasma cells, were found in scattered locations or occasionally forming follicle‐like aggregations but were not noted at the proximal location of chronic nonsuppurative destructive cholangitis. In contrast, there was a unique and distinct coronal arrangement of CD38 cells around the intrahepatic ducts in PBC but not controls; the majority of such cells were considered plasma cells based on their expression of intracellular immunoglobulins, including IgM and IgG, but not IgA. Patients with PBC who manifest this unique coronal arrangement were those with significantly higher titers of anti‐mitochondrial antibodies. Conclusion: These data collectively suggest a role for plasma cells in the specific destruction of intrahepatic bile ducts in PBC and confirm the increasing interest in plasma cells and autoimmunity. (HEPATOLOGY 2012)

Carcinosarcoma of the urinary bladder: Expression of epithelial markers and different expression of heat shock proteins between epithelial and sarcomatous elements

A case of carcinosarcoma composed of both adenocarcinoma and saarcomataus elements in the non‐trigone region of the urinary bladder is presented. The epithelial element was a well to pooriy differentiated adenocarcinome with focal squamous metaplasia. The sarcomatous elements disclosed spindle cell sarcoma with focal epltheliold pattern and myxold change In the stroma, together with chondrosarcomatous and rhabdomyosarcomatous elements. By Immunohistochemical examination, not onty the carcinoma element but also the sarcomatous elements showed a positive lmmunoreaction for cytokeratin (CK), epithelial membrane antigen (EMA) and carcinoembryonic antigen. Some population of sarcomatous elements expressed smooth muscle actin and muscle specific actin (MSA) and a limited portion of epitheliold area showed a positive Immunoreaction for desmin, MSA and myoglobin, Indicating leiomyosarcomatous and rhabdomyosarcomatous differentiation, respectively. Unexpectedly, tumor cells In the chondrosarcomatws element revealed a simultaneous positvity of CK and EMA as well as S‐100 protein. Both epithelial and sarcomatous elements showed an Intensive positive Immunoreaction for p53 and heat shock protein (HSP) 70. However, HSP27 and HSPGO were detected in most epitheilal elements and only in a small number of tumor cells in the sarcomatous area. These findings indicate that sarcomatous elements, Including heterologous elements, may derive from epithelial elements with partial or complete loss of epithelial features, and different factors other than p53 and HSP7O may associate wtth the morphological alteration of carcinoma.

Complete remission of multiple hepatocellular carcinomas associated with hepatitis C virus-related, decompensated liver cirrhosis by oral administration of enteric-coated tegafur/uracil

We report a case of complete remission of multiple hepatocellular carcinomas after oral administration of enteric-coated tegafur/uracil. A 77-yr-old woman was diagnosed as having recurrent hepatocellular carcinoma associated with decompensated liver cirrhosis. We administered enteric-coated tegafur/uracil to this patient. After 1 month of oral administration, there was a decrease in tumor markers. An image analysis showed disappearance of hepatocellular carcinoma. No recurrence of the hepatocellular carcinoma was recognized for 18 months up to the time of the patients death, which was due to massive bleeding from a hemorragic rectal ulcer. At autopsy, the tumor lesion had necrotized. Oral administration of enteric-coated granules containing tegafur/uracil may provide an effective treatment for hepatocellular carcinoma.

Composite paraganglioma-ganglioneuroma of the urinary bladder.

Presented herein is the case of a 73‐year‐old man, complaining of dysuria, who had a composite paraganglioma–ganglioneuroma of the urinary bladder (CPGUB). At cystoscopy a submucosal tumor was found in the urinary bladder and resected after transurethral biopsy. The levels of serum catecholamine and 24 h urinary excretion of catecholamine and vanillylmandelic acid were elevated. Grossly, the resected tumor, measuring 4 × 3 × 2.5 cm, had a brownish cut surface with no necrosis and hemorrhage. Histologically, the tumor had alternating cellular and fibrous areas. The cellular areas consisted of polygonal cells, arranged in well‐defined nests (Zellballen) and positive for Grimelius staining, with abundant amphophilic to acidophilic cytoplasm, occasionally containing eosinophilic hyaline globules and brown pigments. Although the nuclei of several polygonal cells were bizarre, mitoses and vascular invasion were not found. Fibrous areas consisted of spindle cells, resembling Schwann cell, admixed with ganglionic cells. To the authors’ knowledge, only four cases of CPGUB have been reported in the English‐language literature. Detailed reported cases and the present case showed no malignant features, such as extra‐bladder infiltration and metastasis, and no recurrence in the short length of follow up. Accumulation of long‐term follow‐up cases may provide valuable prognostic information on this composite tumor.

Immunohistochemistry of cyclin D3 in pulmonary carcinomas

Cyclin D3, a cell cycle regulator, is encoded in the 6q21 chromosome region. Abnormalities of this gene and its protein product have not been found in normal tissues or in malignancies from human subjects. The expression of cyclin D3 was studied immunohistochemically in archival formalin-fixed, paraffin-embedded specimens from normal organs obtained from three autopsy cases and 237 human primary pulmonary carcinomas. In normal organs, nuclear positivity for cyclin D3 was observed in reactive type-2 pneumocytes, islets of Langerhans, lymphocytes from lymph nodes, superficial cells of transitional epithelium, epithelium of oesophagus, stomach, small intestine and gallbladder, endothelium, smooth muscles, and brain. Proliferating cells such as lymphocytes in the germinal centres and non-proliferating cells such as neurons both demonstrated cyclin D3 immunoreactivity. Cyclin D3 showed obvious nuclear immunoreactivity in 168 pulmonary carcinomas (71%). The proportion of tumour cells that were cyclin D3-positive ranged from 1% to 73% (median, 16%). There was no relationship between cyclin D3 immunoreactivity and histological typing, tumour differentiation, or pathological TNM staging. In pulmonary carcinomas, distinct expression of the cyclin D3 protein is unlikely to be implicated in tumorigenesis, because of its expression in only a small fraction of cancer cells. It may relate to cancer progression. The distribution of cyclin D3 reactivity in the normal tissues suggests that cyclin D3 affects other processes than cell cycle regulation in a lineage-specific manner.

Histopathological and cytological examination of autopsy cases with multiple organ dysfunction syndromes

To evaluate the role of small scavenger receptor A (SRA)‐positive (SRA+) cells and large SRA+ cells in the peripheral blood (PB) in the pathophysiological mechanisms underlying multiple organ dysfunction syndromes (MODS), 24 autopsy cases with MODS were examined. In addition to histopathological and immunohistochemical examination, cytokine levels of cardiac blood, the SRA index (number of small SRA+ cells in 10 high power fields, upper limit <30), and appearance of large SRA+ cells in PB were examined. The SRA index exceeded 30 in all cases. Large SRA+ cells and tumor necrosis factor (TNF)‐α+ cells were detected in PB in all cases. Macrophage colony stimulating factor (M‐CSF), interleukin (IL)‐6 and IL‐8 levels exceeded the normal level in all cases, and 18 and 15 cases had TNF‐α and IL‐1β levels above the normal threshold, respectively. Lung injury and necrosis of cardiac myocyte were observed in all cases. Neutrophils and platelets accumulated in the capillaries of injured organs and endothelial cells were extensively injured. From these results, large SRA+ cells differentiated from monocytes in PB were considered to play an important role in the development of MODS, and necrosis of cardiac myocytes together with lung injury might be the leading cause of death in MODS patients.

Increase of scavenger receptor A‐positive monocytes in patients with acute coronary syndromes

To evaluate the utility of the scavenger receptor A (SRA) index (no. SRA+ monocytes observed in 10 high‐power fields of peripheral blood (PB) smear samples, normal upper limit <30) as the indication of disrupted, fissured, or eroded plaque, 225 patients with acute myocardial infarction (AMI), 79 with unstable angina (UA) and 91 with stable angina (SA) were examined. Thrombus was gathered from 95 of 205 sequential AMI patients (46.3%), and classified into platelets, mixed, and two kinds of residual mural thrombus (RMT). RMT was observed in 56 of 169 (33.1%) AMI patients with SRA index ≥30 at hospitalization. The SRA index of 82.4% of AMI, and 75.9% of UA, and 70.3% of SA patients was ≥30 at hospitalization. For 36 AMI patients who initially had an SRA index of <30 at hospitalization, it exceeded 30 within 2 days, and the SRA index rapidly increased in most AMI patients after hospitalization. SRA+ monocytes were considered to differentiate from SRA– monocytes in PB. An abnormally high SRA index is considered to be a useful indication of disrupted or fissured or eroded plaque.