Amelanotic malignant melanoma of unknown primary origin metastasizing to the bone marrow: a case report and review of the literature.

We herein describe the case of a 77-year-old Japanese man who presented with progressive thrombocytopenia. No lymphadenopathies, bone lesions, hepatosplenomegaly or masses within any internal organs were detectable. Bone marrow smears revealed diffuse infiltration of large atypical cells morphologically resembling mature lymphoid neoplasms. A flow cytometric analysis showed that the tumor cells strongly expressed CD56 without myeloid or lymphoid antigens, suggesting that they were non-hematologic in origin. Ultimately, amelanotic malignant melanoma of unknown primary origin was diagnosed based on positive immunostaining for S100 proteins, HMB-45 and Melan-A. This case illustrates the usefulness of flow cytometric analyses for making such diagnoses. We also review the available literature on similar cases.

Novel complementary peptides to target molecules.

We generated an evolutionary computer program that generates complementary peptide (C-pep) sequences, with the potential to interact with a target peptide, by comparing several physico-chemical parameters of each pair of the complementary peptides being analyzed. We generated C-peps to target several molecules. About 30% of synthesized C-peps interfered with the function of their targets. C5a stimulates generation of TNFα and other inflammatory cytokines. Inhibition of C5a should be effective against sepsis, which impairs the status of cancer-bearing patients. One of the inhibitory C-peps of C5a, termed AcPepA, was effective in Cynomolgus monkeys intravenously infused with a lethal dose of bacterial LPS (4 mg/kg) destined to die. The monkeys were rescued by intravenous administration of 2 mg/kg/h of AcPepA. The excellent therapeutic effect of AcPepA is likely to be due to restriction of high mobility group box 1 (HMGB1) surge induced by the effect of C5a on C5L2, which is the second C5a receptor, since the released HMGB1 has the capacity to stimulate TLR4 as an endogeneous ligand resulting in further activation of inflammatory cells to release inflammatory cytokines forming a positive feedback circuit of inflammation.

Resistance to chemotherapeutic agents and promotion of transforming activity mediated by embryonic stem cell-expressed Ras (ERas) signal in neuroblastoma cells.

Neuroblastoma is a common childhood tumor derived from neural crest precursor cells. In the present study, we investigated the expression and function of embryonic stem cell-expressed Ras (ERas), a novel Ras family protein previously reported as the specific expression gene in embryonic stem cells (ES cells), in neuroblastoma cell lines. Our results showed that the expressions of ERas were detected in neuroblastoma cell lines by RT-PCR and Western blotting. Therefore, we transfected a full length ERas expression vector into the neuroblastoma cell line SH-SY5Y, which has weak endogenous expression of ERas, and obtained clones with higher levels of expression. Overexpression of ERas did not increase the growth rate of the ERas transfectants but promoted their transforming activity. The ERas transfectants were more resistant to all the chemotherapy agents than the parental cell line. The ability of ERas to rescue cells from the toxic effect of chemotherapeutic agents was inhibited by the phosphatidylinositol 3'-kinase (PI3K) inhibitor PD294002. These results show that the ERas/PI3K pathway may provide resistance to chemotherapy and promote transforming activity in neuroblastoma.

Procarboxypeptidase R deficiency causes increased lethality in concanavalin A-induced hepatitis in female mice.

Carboxypeptidase R (CPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is an enzyme generated by proteolytic cleavage of its zymogen (proCPR). CPR removes the C-terminal arginine from inflammatory peptides such as C3a and C5a, bradykinin, enkephalin, and the thrombin-cleaved N-terminal fragment osteopontin (cleaved N-OPN). In the mouse model of concanavalin A (Con A)-induced immune-mediated fulminating hepatitis, cleaved N-OPN is one of the important peptides that induce the production of chemokines or cytokines. In the current study using proCPR deficient mice, we showed that injection of Con A into the mouse tail vein can induce a significantly higher lethality in proCPR-deficient female but not in male mice. Furthermore, a lack of CPR activity increased serum macrophage inflammatory protein-2 (MIP-2) and high-mobility group box 1 (HMGB1) levels after Con A injection. These in vivo findings suggest that CPR helps to protect against Con A-induced hepatitis.

TNF-alpha-induced aquaporin 9 in synoviocytes from patients with OA and RA.

OBJECTIVES: To determine whether aquaporins (AQPs) are expressed in the synovial tissues of patients with OA and RA, and to examine the patterns of expression in patients with and without hydrarthrosis. METHODS: AQPs were detected in synovial tissue samples from patients with OA and RA using RT-PCR and immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from patients with OA and RA were cultured and stimulated with TNF-alpha. The expression of AQPs in FLSs was examined using RT-PCR and western blot analyses and the function of aquaglyceroporins was examined by a glycerol uptake assay. RESULTS: AQP1, -3 and -9 mRNAs were expressed in synovial tissues from patients with OA and RA. AQP1, -3 and -9 proteins were also detected by immunohistochemistry. AQP9 mRNA was expressed more strongly in the synovial tissues of OA patients with hydrarthrosis than those without. AQP9 mRNA and protein expression were strongly induced with TNF-alpha treatment in FLSs, whereas the expression of AQP1 and -3 mRNAs was not induced with TNF-alpha treatment. AQP9 as an aquaglyceroporin was induced by TNF-alpha. CONCLUSIONS: AQP9 mRNA was detected in synovial tissues from OA and RA patients with hydrarthrosis. AQP9 expression was strongly induced in FLSs with TNF-alpha. Although the functions of AQP1, -3 and -9 in synovial tissues remain to be elucidated, it suggested that AQP9 might be related to the pathogenesis of hydrarthrosis and inflammatory synovitis.

Role of Rho/Rho-kinase and NO/cGMP signaling pathways in vascular function prior to atherosclerosis.

AIM: Atherosclerosis is a cardiovascular disease; however, there is little information on signal transduction for vascular function in the early stage of atherosclerosis. In this work, we investigated the role of Rho/Rho-kinase and nitrogen oxide (NO)/cyclic GMP (cGMP) signaling pathways in the aorta prior to atherosclerosis. METHODS: Tension, the expression of RhoA protein, Rho-kinase activity and the cGMP level were measured using endothelium-intact or -denuded aorta prepared from apolipoprotein E-deficient (apoE-KO) and C57BL/6 wild-type (WT) mice at 2 months of age. RESULTS: Phenylephrine (PE) induced less maximal contraction in the endothelium-denuded aorta from apoE-KO than from WT mice. A Rho-kinase inhibitor (Y-27632) reduced more effectively the contraction of apoE-KO than WT mice, but their RhoA proteins and Rho-kinase activities were not so different. Acetylcholine caused larger relaxation of the PE-stimulated, endothelium-intact aorta in apoE-KO due to endothelial NO release than WT mice. The basal cGMP level in the endothelium-intact aorta of apoE-KO mice was higher than that of WT. CONCLUSIONS: Smooth muscle contraction via alpha(1)-adrenergic receptor shows higher dependency on Rho-kinase activity, suggesting down-regulation of the mechanism different from Rho/Rho kinase signaling in the aorta prior to atherosclerosis. Endothelium-dependent relaxation is also intensified through the NO/cGMP pathway.

Proximal-type epithelioid sarcoma of the perineum: calling attention of urologists.

A subcutaneous mass in the perineum of a middle-aged man was excised and pathologically diagnosed at first as 'undifferentiated carcinoma' of unknown origin, which recurred 2 years later without any metastasis. Further histological evaluation ultimately established a correct diagnosis of 'proximal-type epithelioid sarcoma', a variant of rare epithelioid sarcoma. This type of tumor may confuse pathologists because its histological characteristics resemble undifferentiated carcinoma or malignant rhabdoid tumor. Frequent immunoreactivity of CD34, in addition to expression of keratins, epithelial membrane antigen and vimentin, provides strong support for the diagnosis of this rare neoplasm. Urologists should be aware that this sarcoma commonly occurs in the genital regions.

Interleukin-1beta induces the expression of aquaporin-4 through a nuclear factor-kappaB pathway in rat astrocytes.

Interleukin (IL)-1beta is known to play a role in the formation of brain edema after various types of injury. Aquaporin (AQP)4 is also reported to be involved in the progression of brain edema. We tested the hypothesis that AQP4 is induced in response to IL-1beta. We found that expression of AQP4 mRNA and protein was significantly up-regulated by IL-1beta in cultured rat astrocytes, and that intracerebroventricular administration of IL-1beta increased the expression of AQP4 protein in rat brain. The effects of IL-1beta on induction of AQP4 were concentration and time dependent. The effects of IL-1beta on AQP4 were mediated through IL-1beta receptors because they were abolished by co-incubation with IL-1 receptor antagonist. It appeared that IL-1beta increased the level of AQP4 mRNA without involvement of de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, did not inhibit the effects of IL-1beta. Inhibition of the nuclear factor-kappaB (NF-kappaB) pathway blocked the induction of AQP4 by IL-1beta in a concentration-dependent manner. These findings show that IL-1beta induces expression of AQP4 through a NF-kappaB pathway without involvement of de novo protein synthesis in rat astrocytes.

Aberrant MHC class II expression in mouse joints leads to arthritis with extraarticular manifestations similar to rheumatoid arthritis.

Genetic susceptibility to rheumatoid arthritis (RA) is associated with certain MHC class II molecules. To clarify the role of these determinants in RA, we generated the D1CC transgenic mouse that expressed genes involved in antigen processing and presentation by the MHC class II pathway in joints. The class II transactivator, which was transcribed from the rat collagen type II promoter and enhancer, directed the expression of these genes. In D1CC mice congenic for the H-2(q) (DBA/1) background, small amounts of bovine collagen type II in adjuvant induced reproducibly an inflammatory arthritis resembling RA. Importantly, these stimuli had no effect in DBA/1 mice. Eighty-nine percent of D1CC mice developed chronic disease with joint swelling, redness, and heat in association with synovial proliferation as well as pannus formation and mononuclear infiltration of synovial membranes. Granulomatous lesions resembling rheumatoid nodules and interstitial pneumonitis also were observed. As in patients with RA, anticyclic citrullinated peptide antibodies were detected during the inflammatory stage. Finally, joints in D1CC mice displayed juxtaarticular demineralization, severe joint space narrowing, and erosions, which led to ankylosis, but without the appearance of osteophytes. Thus, aberrant expression of MHC class II in joints facilitates the development of severe erosive inflammatory polyarthritis, which is very similar to RA.

Usefulness of D2-40 immunohistochemistry for differentiation between kaposiform hemangioendothelioma and tufted angioma.

BACKGROUND: Recent investigations have demonstrated the utility of the monoclonal antibody D2-40 as a marker for lymphatic endothelium. D2-40 can be used on formalin-fixed and paraffin-embedded materials. Our objective was to elucidate, using D2-40 immunohistochemistry, the differences among capillary hemangiomas, and especially between kaposiform hemangioendothelioma (KHE) and tufted angioma (TA). We studied four cases of KHE, nine cases of TA, and 31 cases of other vascular tumors. Antibodies against CD31, CD34, factor VIII-related antigen, and GLUT1 were also applied. RESULTS: In KHE, D2-40 was markedly reactive for three and partially for one of four cases in the peripheral area of Kaposi's sarcoma-like proliferative capillaries and negative in the surrounding dilated vessels. In TA, D2-40 was partially positive in the surrounding dilated vessels and negative in cannonball-like proliferative capillaries. CONCLUSIONS: Our results suggest that D2-40 is a useful antibody for immunohistochemical discrimination between KHE and TA. In addition, the difference of immunostaining pattern of D2-40 is limited to the peripheral area of capillary proliferation and surrounding dilated vessels; therefore, it is suggested that KHE and TA may reflect different stages in the evolution of a single entity. Namely, they may originate from stem cells possessing the characteristics of both lymphatic and blood vessel endothelial lineages.

Acute peritonitis and salpingitis associated with streptococcal toxic shock syndrome caused by Lancefield group G alpha-haemolytic Streptococcus dysgalactiae subsp. equisimilis.

The authors treated a patient for what appears to be the first reported occurrence of acute peritonitis and salpingitis associated with streptococcal toxic shock syndrome (STSS). This was caused by Lancefield group G alpha-haemolytic Streptococcus dysgalactiae subsp. equisimilis TKCH2004-001. The isolate showed M protein type stc36 and carried the spegg gene. To the best of the authors' knowledge, the present report represents the first case of STSS complicating acute peritonitis and salpingitis caused by Lancefield group G alpha-haemolytic S. dysgalactiae subsp. equisimilis.

Expression of myelencephalon-specific protease after cryogenic lesioning of the rat parietal cortex.

The gene for myelencephalon-specific protease (MSP) is a member of the kallikrein gene family and in rats is expressed mainly in the central nervous system. Its function and alteration in brain injury have not yet been clarified. We examined the expression of MSP after cryogenic injury (CI) using in situ hybridization, immunohistochemistry, and Western blotting. Analysis of MSP mRNA by in situ hybridization revealed a higher level of expression around the cryogenic area than on the contralateral side at 2-7 days after CI, with peak expression occurring 7 days after CI. Immunohistochemical analysis demonstrated expression of MSP protein at 1 day after CI, in the same region in which MSP mRNA was observed, with peak expression again at 7 days after CI, in the area around the lesion. Double immunohistochemical labeling revealed that MSP was expressed mainly in oligodendrocytes. These results suggest that expression of MSP may be related to the turnover of myelin-associated proteins and extracellular matrix proteins after CI. The regulation of active MSP may be important in the physiological or pathological changes involved in remyelination or demyelination.

Hepatitis induced by an IgM monoclonal antibody against procarboxypeptidase R.

Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.

Expression of glia maturation factor beta after cryogenic brain injury.

Glia maturation factor beta (GMFB) was identified as a growth and differentiation factor acting on neurons as well as glia. We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB-immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury. These findings may provide new insight into GMFB function in pathological conditions following brain injury.

Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in lipopolysaccharide-primed mice.

Carboxypeptidase R (CPR) is a heat-labile enzyme found in serum in addition to stable carboxypeptidase N. CPR cleaves the C-terminal basic amino acids, arginine and lysine, from inflammatory peptides such as complement C3a and C5a, bradykinin, and enkephalin. This enzyme is generated from procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor, following cleavage by proteolytic enzymes such as thrombin, plasmin, and trypsin. We generated proCPR-deficient mice by knocking out exons 4 and 5 of the proCPR gene, which are regarded as essential for CPR function. At LPS challenge, there was virtually no difference in lethality among proCPR(+/+), proCPR(+/-), and proCPR(-/-) mice. However, challenge with cobra venom factor, which can activate and deplete almost all complement in vivo, induced a lethal effect on proCPR(-/-) mice following LPS sensitization which up-regulates C5a receptor expression. In contrast, proCPR(+/+) and proCPR(+/-) mice were able to tolerate the cobra venom factor challenge with the limited dose (30 U). Although carboxypeptidase N plays a role in inactivation of inflammatory peptides in vivo, CPR may also be important in the regulation of hyperinflammation.

Expression of myelencephalon-specific protease in transient middle cerebral artery occlusion model of rat brain.

Myelencephalon-specific protease (MSP) is one of the serine proteases and is expressed in the central nervous system of rats. Its function and alternation in brain injury have not yet been clarified. In this study, we investigated the expression of MSP after transient middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. In situ localization of MSP mRNA demonstrated a higher level in the corpus callosum and around the ischemic area from 12 h to 14 days after MCA reperfusion, with the peak of expression coming 3 days after reperfusion in both regions. Immunohistochemically, the expression of protein was found 1 day after reperfusion in the same brain region that was observed for mRNA. The peak was 7 days after reperfusion in both regions. Micro-autoradiography, immunostaining and double immunohistochemical labeling revealed the expression of MSP to be located mainly in the oligodendrocytes. The present results indicate that MSP may be related to the turnover of the myelin-associated proteins and the extracellular matrix proteins after transient MCAO. The activation of MSP may play a role in remodeling processes such as neurite outgrowth and remyelination.

Chondrosarcoma of the distal phalanx of the second toe: a case report.

Although chondrosarcoma is a relatively common malignant bone lesion, these lesions rarely affect the foot. This article presents the case of a 32-year-old woman with a chondrosarcoma in the distal phalanx of the right second toe. Radiography and computed tomography showed cortical disruption and soft-tissue expansion. Histologically, the tumor was classified as grade 2 chondrosarcoma according to the American Joint Committee of Cancer pathologic staging system. Amputation at the distal half of the second proximal phalanx was performed to obtain clear surgical margins. A literature review and discussion of salient diagnostic and treatment issues is included.

Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain.

We recently reported that fibroblast growth factor 1 (FGF-1) upregulates apolipoprotein E (apoE) synthesis and its secretion as high density lipoprotein (HDL) in cultured astrocytes potentially by an autocrine or paracrine mechanism [Biochim. Biopys. Acta 1589 (2002) 261]. In order to examine pathophysiological relevance of this reaction, we studied association of the production of FGF-1 and apoE in the post-injury mouse brain. After the spot-injury of the brain by liquid nitrogen, the surface size of the wound shrunk more rapidly in the C57BL/6 wild-type mice than the apoE-knock out C57BL/6 mice. Immunohistochemical analysis of the lesions revealed that production of FGF-1 was identified in the reactive astrocytes by the day 2 after the injury in both types of mouse, prior to the production of apoE confirmed by the day 4 in the wild-type. These findings were consistent with our in-vitro observations and hypothesis that FGF-1 upregulates apoE synthesis and subsequently HDL production in the reactive astrocytes by an autocrine or paracrine manner. FGF-1 thus would exert its effect after the CNS damage through apoE secretion.

Reactive angioendotheliomatosis of the intestine.

We present a case of reactive angioendotheliomatosis (RAE) of the colon, featuring intravascular proliferation of endothelial cells with histologic resemblance to glomeruloid hemangioma. A 19-year-old Japanese male with an anal fistula was diagnosed endoscopically with Crohn's disease. Six months later, he was hospitalized for fever and abdominal pain. Emergency resection of ileocecum and splenic flexure of the colon was undertaken to control massive intestinal hemorrhage, and in all parts of the resected colon, foci of many small vessels with intravascular proliferation of endothelial cells were noted throughout the layers. Moreover, solid proliferation of endothelial cells was seen in the submucosa at the base of open ulcers. Two small granulomas, compatible with Crohn's disease, were also evident in the muscle layer of the terminal ileum. No other hemangiomas or hemangioma-like structures were observed with CT scans, and the vascular lesions were histologically diagnosed as RAE. The pathogenesis of this disorder is unknown, and most cases occur in skin with systemic disease. The present case might thus be a first case of RAE of the intestine without cutaneous involvement. Whether there is a relation with coexistent enteritis suggestive of Crohn's disease needs to be clarified.