Macrophage deficiency in osteopetrotic (op/op) mice inhibits activation of satellite cells and prevents hypertrophy in single soleus fibers.

Effects of macrophage on the responses of soleus fiber size to hind limb unloading and reloading were studied in osteopetrotic homozygous (op/op) mice with inactivated mutation of macrophage colony-stimulating factor (M-CSF) gene and in wild-type (+/+) and heterozygous (+/op) mice. The basal levels of mitotically active and quiescent satellite cell (-46 and -39% vs. +/+, and -40 and -30% vs. +/op) and myonuclear number (-29% vs. +/+ and -28% vs. +/op) in fibers of op/op mice were significantly less than controls. Fiber length and sarcomere number in op/op were also less than +/+ (-22%) and +/op (-21%) mice. Similar trend was noted in fiber cross-sectional area (CSA, -15% vs. +/+, P = 0.06, and -14% vs. +/op, P = 0.07). The sizes of myonuclear domain, cytoplasmic volume per myonucleus, were identical in all types of mice. The CSA, length, and the whole number of sarcomeres, myonuclei, and mitotically active and quiescent satellite cells, as well as myonuclear domain, in single muscle fibers were decreased after 10 days of unloading in all types of mice, although all of these parameters in +/+ and +/op mice were increased toward the control values after 10 days of reloading. However, none of these levels in op/op mice were recovered. Data suggest that M-CSF and/or macrophages are important to activate satellite cells, which cause increase of myonuclear number during fiber hypertrophy. However, it is unclear why their responses to general growth and reloading after unloading are different.

Angiomyofibroblastoma of the vulva.

BACKGROUND: Angiomyofibroblastoma (AMF) is a rare benign mesenchymal neoplasm that arises in the pelviperial region. CASE: A patient presented with a painless mass in the right vulva. Under the preoperative diagnosis of Bartholin cyst, she underwent a simple tumor excision. Pathological examination revealed an AMF. Immunohistochemical examination showed that tumor cells were positive for estrogen receptor, progesterone receptor, vimentin, and CD34. She has been with no evidence of local recurrence for ten months after surgery. CONCLUSION: AMF of the vulva is a distinctive mesenchymal tumor that is curable with a simple excision.

Transition of low-grade to high-grade endometrial stromal sarcoma: a case report.

BACKGROUND: The transition of low-grade endometrial stromal sarcoma (ESS) to high-grade ESS remains a rare clinical event. CASE: A patient presented with abdominal pain and abnormal genital bleeding. She underwent a supracervical hysterectomy with bilateral salpingo-oophorectomy, omentectomy, and resection of peritoneal disseminated lesions. Pathological examination revealed low-grade ESS in the uterus and omentum. Immunohistochemical examination showed immunoreactivity for CD10 and Ki-67 (MIB1) in the uterus and omentum. However, estrogen receptor, progesterone receptor, alpha-SMA, desmin, h-caldesmon, and CAM5-2 were negative. P53 immunoreactivity was noted only in the omental lesion. Despite performing six courses of adjuvant chemotherapy, she recurred in the abdomen. She underwent ileostomy and resection of peritoneal disseminated lesions. Pathology showed high-grade ESS in the recurrent lesion of the ileum, which was characterized by severe cytologic atypia, high mitotic index, multifocal necrosis, increased Ki-67 index, and immunoreactivity for p53. CONCLUSION: Although rare, the transition of low-grade ESS to high-grade ESS may occur and suggests the worsening of the prognosis. Pathological examination and immunohistochemistry are useful for the diagnosis of the transition of low-grade ESS to high-grade ESS.

Effect of exercise training on the density of endothelial cells in the white adipose tissue of rats.

We examined the effects of a 9-week exercise training (TR) in Wistar male rats, beginning at 4 weeks of age, on the density of endothelial cells (ECs) in epididymal white adipose tissue (WAT) and the mRNA expression of angiogenic factors in adipose tissue stromal vascular fraction (SVF) cells. The number of ECs and mRNA expressions were assessed by lectin staining and real-time reverse transcriptase-polymerase chain reaction, respectively. Compared with control (CR) rats, TR rats gained weight more slowly and had significantly lower final weight of WAT due to the reduction in the size and the number of adipocytes. TR significantly increased the number of ECs per square millimeter and per adipocyte (1.37- and 1.23-fold, respectively) in WAT. This is probably because the number of adipocytes is fewer while the number of ECs is constant in the WAT of TR rats, because the regression line of TR rats for adipocyte number-dependent EC number was shifted toward the left without significant differences in the slopes between groups. TR also induced the upregulation of mRNA expression of vascular endothelial growth factor (Vegf)-A and Vegf-receptor-2 in SVF cells, thereby retaining a constant number of ECs in the WAT.

Increased growth of brown adipose tissue but its reduced thermogenic activity in creatine-depleted rats fed beta-guanidinopropionic acid.

To study the responses of thermogenic activity in brown adipose tissue (BAT) to creatine depletion, male Wistar rats were fed creatine analogue beta-guanidinopropionic acid (beta-GPA) for about 10 weeks. Compared to control rats, a marked decrease in the levels of high-energy phosphates, such as phosphocreatine and ATP, was noted in BAT of beta-GPA rats. Conversely, upward trends in other chemical components (DNA, glycogen, and total protein) in BAT as well as an increase in BAT mass were observed in beta-GPA rats, suggesting a tendency to hyperplasia of the BAT. The thermogenic activity (which was assessed by guanosine 5'-diphosphate binding to BAT mitochondria) in the mitochondria recovered from BAT of beta-GPA rats, however, was not increased in response to such changes but rather decreased. Moreover, uncoupling protein (UCP) content in the mitochondrial fraction of beta-GPA rats was significantly lower than that in control rats (the relative amounts were 77 +/- 6 and 100 +/- 4%, respectively). Nevertheless, surprisingly, the level of UCP mRNA was remarkably greater in beta-GPA rats than in control rats. These observations indicate that there is a discordance between BAT growth and activity in beta-GPA rats, thereby suggesting that a failure on and after UCP translation may be involved in the impairment of BAT thermogenic activity with creatine depletion. The impairment of BAT thermogenic activity, that is, UCP activity may indicate that uncoupling or heat production was inhibited in order to increase the ATP synthesis in BAT of beta-GPA rats in compensation for a reduction in the levels of high-energy phosphates (including ATP), with resultant hypothermia.

Interleukin-1-dependent mitogenic responses induced by protoscoleces of Echinococcus multilocularis in murine lymphocytes.

Mitogenic effects of protoscoleces (PSCs) of Echinococcus multilocularis on murine lymphocytes were studied. Spleen cells from normal BALB/c mice showed significant proliferative responses when cocultured with PSCs. Proliferative responses were observed in both the T and B cell populations. The PSCs also stimulated cells of the macrophage/monocyte lineage to secrete interleukin-1 (IL-1). Depletion of plastic- and Sephadex G-10-adherent cells from the spleen cell population significantly reduced the proliferative responses to PSCs and the low responsiveness was restored by addition of plastic-adherent cells to these cultures. Furthermore, addition of anti-IL-1 serum to the spleen cell cultures stimulated with PSCs completely suppressed the proliferative responses. These findings demonstrate that the mitogenic effect of PSC on lymphocytes depends on IL-1 secreted by cells of macrophage/monocyte lineage.

Age-associated increase of basal corticosterone levels decreases ED2high, NF-kappaBhigh activated macrophages.

The proportion of cells with a high density of ED2 (ED2high cells) in peritoneal cells from old rats was significantly lower than that from young rats. The expression of major histocompatibility complex class II (MHC class II) molecules, the antigen presentation, production of interleukin (IL)-1beta and IL-6, and nuclear factor-kappaB activity in ED2high cells were markedly higher than those in cells with a low density of ED2 (ED2low cells), although no significant difference was observed in the expression of MHC class II molecules and the antigen presentation between ED2high cells from young and old rats. Meanwhile, basal corticosterone concentration in serum and glucocorticoid (GC) receptor mRNA expression in peritoneal cells increased significantly in old rats. The proportion of ED2high cells was increased by adrenalectomy in young rats. Furthermore, nuclear translocation of GC receptor was observed in ED2low cells, whereas GC receptor was detected in cytoplasmic extracts from ED2high cells. These results suggest that the decrease in functional ED2high macrophages with age results in the age-associated decline of immune responses, which is regulated, in part, by the basal GC concentration.

Insulin stimulates the expression of basic fibroblast growth factor in rat brown adipocyte primary culture.

The effect of insulin on the hyperplasia of brown adipose tissue (BAT) was investigated using the primary culture of rat brown adipocyte precursor cells (RBAC). Results showed insulin to significantly increase the number of RBAC, but not bovine capillary endothelial cells, in the presence of fetal bovine serum. Insulin also increased the expression of basic fibroblast growth factor (bFGF) mRNA and the related protein in the primary culture of RBAC. In addition, insulin enhanced the capillary growth in an in vitro angiogenesis model in which microvascular fragments and RBAC isolated from rat BAT were grown in coculture. The level of bFGF-related protein in the coculture was higher in the presence of insulin than in the absence of insulin. These findings suggest that insulin may play an important role in the proliferation as well as in the differentiation of brown adipocytes, with resulting hyperplasia of BAT (including the formation of new capillaries) through increased production of bFGF in brown adipocytes.

Glucocorticoid-mediated generation of suppressor macrophages with high density Fc gamma RII during acute cold stress.

Acute cold stress induces adherent cells with suppressor function, resulting in immunosuppression. Glucocorticoids (GC) are known as an inhibitor of the immune system and are increased by stress through stimulation of the hypothalamic-pituitary-adrenal axis. To elucidate the mechanisms underlying acute cold stress-induced immunosuppression, functions and surface phenotypes of murine peritoneal cells of the monocyte/macrophage lineage from acute cold-stressed mice (5 C for 3 or 24 h) in addition to the role of GC in the immunomodulation were investigated. Flow cytometric analysis revealed that the proportion of MAC-1+ cells with a high density of Fc gamma RII (Fc gamma RIIbright cells) was markedly increased in the peritoneal exudate cells from acute cold-stressed mice. These Fc gamma RIIbright cells were also stained with F4/80, a monoclonal antibody directed specifically against the mouse mature macrophages. The prominent suppressor activity for Concanavalin A (Con A) responses of control spleen cells was found in Fc gamma RIIbright cells, whereas MAC-1+ cells, with a low density of Fc gamma RII (Fc gamma RIIdull cells), from the stressed mice did not suppress the Con A responses. Fc gamma RIIbright cells from control mice also suppressed the Con A responses; the inhibitory effect was considerably less than that of cells from acute cold-stressed mice. As was anticipated, serum corticosterone levels were markedly increased in acute cold-stressed mice. In addition, expression of GC receptor messenger RNA was observed in Fc gamma RIIbright cells from these mice. The increase in Fc gamma RIIbright cells in peritoneal exudate cells caused by acute cold stress was inhibited by adrenalectomy or administration of a saturating amount of the GC antagonist RU 38486 (mifepristone). On the contrary, administration of the GC agonist, dexamethasone, markedly increased the proportion of Fc gamma RIIbright cells in peritoneal exudate cells of control mice. These results suggest that the generation of Fc gamma RIIbright suppressor cells of monocyte/macrophage lineage by acute cold stress was mediated to a greater or lesser degree by the action of GC through the GC receptor.

Identification of parathyroid hormone messenger ribonucleic acid in an apparently nonfunctioning parathyroid carcinoma transformed from a parathyroid carcinoma with hyperparathyroidism.

mRNA coding for pre-pro-PTH, a precursor of PTH, was sought in an apparently nonfunctioning parathyroid carcinoma that had transformed from one that was previously functioning. Total poly(A+) RNA was prepared by phenol-chloroform-isoamyl alcohol extraction and oligo-dT-cellulose affinity chromatography from the tumor tissue and bovine parathyroid glands. In the rabbit reticulocyte lysate cell-free translation system, total poly(A+) RNA from the tumor as well as that from bovine parathyroid glands directed the translation of a product which was specifically precipitated by an anti-PTH serum and which migrated at the same position as pre-pro-PTH on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicated the presence of mRNA coding for pre-pro-PTH (PTH mRNA) in an apparently nonfunctioning parathyroid carcinoma, suggesting that PTH synthesis is not always absent in parathyroid carcinomas which are not accompanied by hyperparathyroidism.

Alterations of superoxide dismutase iso-enzyme activity, content, and mRNA expression with aging in rat skeletal muscle.

The alterations of superoxide dismutase iso-enzyme (Cu,Zn-SOD and Mn-SOD) activities, contents, and mRNA expressions with aging were studied in rat soleus muscle (SO) and extensor digitorum longus muscle (EDL). The activity and content of Cu,Zn-SOD in both muscles were significantly higher in old rats (24 months old) than in young rats (4 months old), whereas those of Mn-SOD showed no difference between young and old rats. After normalization to citrate synthase (CS) activity, however Mn-SOD/CS ratio in SO also showed the age-related increase. Moreover, the activities of other major antioxidant enzymes, glutathione peroxidase (GPX) and catalase (CAT), indicated age-related increases only in SO. As for the expressions of mRNAs for SOD iso-enzymes, that of Cu,Zn-SOD in either muscle showed no significant change with aging, unlike its activity and content, although that of Mn-SOD was decreased with aging only in EDL. Thus, aging appeared to raise the level of antioxidant enzyme system in rat skeletal muscle. However, the resistance of Cu,Zn-SOD and Mn-SOD to oxidative stress accompanied by aging was different, the former being obviously greater than the latter. Such changes also differed in muscle fiber type suggesting that fast-twitch fibers are more susceptible to age-related oxidative stress than slow-twitch fibers.

Swimming training improves brown-adipose-tissue activity in young and old mice.

The impairment of brown adipose tissue (BAT) thermogenic activity with aging has been well documented. The current study investigated the effect of swimming training on BAT activity in 2-month-old (young) and 26-month-old (old) male mice. The trained mice underwent a 6-week swimming program (1 h/day, 5 days/week) in water at 35-36 degrees C. Compared with young sedentary mice, the BAT-to-body mass ratio was markedly smaller in old sedentary mice, accompanied by the decreased amount of protein, whereas there was no significant difference in uncoupling protein (UCP) content, UCP mRNA expression, or guanosine 5'-diphosphate (GDP) binding (an index of UCP activity) between young and old mice. Meanwhile, the swimming training definitely increased BAT mass and its protein content in both the young and old mice, suggesting hypertrophy and hyperplasia. In addition, after the swimming training, the amounts of protein, UCP antigen, and GDP binding in the mitochondria recovered from BAT of both mice increased significantly as compared with the respective sedentary groups, while the expression of UCP mRNA did not vary substantially. These findings suggest that, irrespective of age, swimming training enhances the thermogenic activity and capacity in BAT of mice.

Effect of age on brown adipose tissue activity in the obese (ob/ob) mouse.

Brown adipose tissue (BAT), a highly thermogenic tissue in young animals, is relatively atrophied and thermogenetically quiescent (e.g. as measured by colonic temperature) in mice that are obese or old. The aim of the current study was to investigate the effect of aging (3.1 (young) versus 14.6 (old) months old) on BAT activity in lean and obese (ob/ob) mice. In young but not in old mice, BAT mass in terms of weight per unit body weight was significantly lower in obese mice than in lean mice. A significant increase in BAT mass of obese mice with age was noted in terms of weight or weight per unit body weight, probably because of a tendency to become white adipose tissue and the deposit of fat, accompanied by the lowest levels of total protein, guanosine 5'-diphosphate binding, and uncoupling protein (UCP) antigen in the mitochondria of BAT, as well as the lowest colonic temperature among the groups examined. Unlike old lean animals, the old obese (ob/ob) animals did not increase but rather decreased the expression of mRNA for UCP in the mitochondria of BAT. These findings suggest that a marked decrease in BAT thermogenic capacity and activity is noted in old obese mice, probably due to synergism of aging and obesity.

Specific association of retroviral envelope protein, p15E, with human cell surfaces.

Experiments were carried out to analyze the binding sites on human cells for highly purified retroviral protein p15E isolated from Feline Leukemia Virus, Rickard Strain. Binding of 125I-labeled p15E was tested with surfaces of human peripheral blood lymphocytes and 3 cell lines, Raji, MOLT-4, and U-937. 125I-labeled p15E showed specific binding to human peripheral blood lymphocytes. In addition, all of the cell lines tested showed binding of 125I-labeled p15E. Using U-937 cells, we characterized the interaction between p15E and the surface of these cells, and showed that the binding was specific by the following 3 different sets of evidence: (i) in equilibrium binding experiments, 18,000 binding sites with a dissociation constant of 2 x 10(-9) M were present on U-937 cells; (ii) trypsin or N-glycanase treatment decreased the binding sites of 125I-labeled p15E; and (iii) by affinity chromatography using p15E or BSA Sepharose columns, the isolated membranes of 125I-labeled U-937 cells previously treated with Triton X-100 showed a significantly higher binding to the p15E column than to the BSA column.

Hemorrhagic colitis with unusual colonoscopy features, complicated with chronic graft-versus-host disease after allogeneic bone marrow transplantation.

A 32-year-old man was admitted after bone marrow transplantation because of hematochezia. He had history of chronic graft-versus-host disease (GVHD) of the skin and the liver, and cytomegaloviral pneumonia. Barium enema and colonoscopy showed multiple colon ulcers in the ascending and transverse colon. This feature is very rare in chronic GVHD and resembles the feature in autoimmune disease such as periarteritis nodosa. Thus, this ulceration is thought to be caused by vasculitis due to an autoimmune reaction in chronic GVHD.

Epstein-Barr virus-negative high grade B cell lymphoma of donor origin developing 19 months after unrelated allogeneic bone marrow transplantation.

A 22-year-old man, in first complete remission of acute myelogenous leukemia, developed a high grade B cell lymphoma 19 months after an allogeneic bone marrow transplant (allo-BMT) from an HLA-identical unrelated donor. Biopsy of a cervical lymph node revealed a lymphoma that was negative for Epstein-Barr virus-encoded small nuclear RNAs (EBERs) in situ hybridization. Genotypic analyses identified the lymphoma to be of donor origin, and there was no evidence of the Epstein-Barr virus (EBV) DNA in the lymphoma by Southern blot analysis. The lymphoma went into complete remission, following four courses of combination chemotherapy, but relapsed after a month and the patient died of congestive heart failure. The patient was thought to be persistently immunosuppressed 11 months after cessation of immunosuppressants, and the lymphoma was thought to be induced by one or more factors other than EBV.

Amino acid sequence of acylphosphatase from rabbit skeletal muscle.

The complete amino acid sequence of acylphosphatase from rabbit skeletal muscle has been elucidated by automatic Edman degradation of peptides obtained from staphylococcal protease and trypsin digestions. The enzyme consisted of a single polypeptide chain of 98 amino acid residues, lacking only histidine. Its amino (N)-terminus was blocked by an acetyl group. The presented sequence of rabbit muscle enzyme was compared with those of equine and porcine muscle enzymes. There were four unique replacements, i.e., Arg-4, Asp-28, Arg-31, and Glu-56 in the sequences of both equine and porcine muscle enzymes were replaced by Gly, Gly, Lys, and Asp, respectively, in that of rabbit muscle enzyme. Extensive structural homology was observed among the three enzymes.

Antigenic determinants of acylphosphatase from porcine skeletal muscle.

Analysis of the quantitative precipitin reaction of acylphosphatase from porcine skeletal muscle with rabbit antiserum indicated the presence of at least two antigenic determinants on the porcine enzyme molecule. Immunological cross-reactivities of acylphosphatases from equine and rabbit skeletal muscles were examined. In double immunodiffusion with the antiserum, the precipitin lines of the porcine and equine enzymes completely fused, while the rabbit enzyme gave no precipitin line. The reaction between the 125I-labeled porcine enzyme and its antibody was inhibited to the same extent by the porcine and equine enzymes, but not by the rabbit enzyme. The three enzymes were similar in net charge and molecular weight on polyacrylamide gel electrophoreses. No conformational difference among the three enzymes was observed in their circular dichroism spectra. The amino acid composition of the rabbit enzyme differed from those of the porcine and equine enzymes in the contents of Glu, Gly, Lys, and Arg. Differences in the sequence of the rabbit enzyme from that of the porcine enzyme were investigated by comparison of the peptide maps of the tryptic peptides of the two enzymes. Four peptides of the rabbit enzyme were located at different positions from those of the porcine enzyme. Three of the four peptides from both enzymes were sequenced and all the tryptic peptides of both enzymes were characterized by amino acid analysis. The tryptic peptides of rabbit enzyme were tentatively aligned on the basis of their amino acid compositions and sequence homologies, compared with the corresponding peptides of the porcine enzyme. Among five amino acid residues of the porcine enzyme, Arg-4, Asp-28, Arg-31, Glu-56, and Ile-68, which are replaced in the rabbit enzyme, Arg-4 and Asp-28 are considered to be included in the antigenic determinants.

Amino acid sequence of acylphosphatase from porcine skeletal muscle.

The amino acid sequence of acylphosphatase from porcine skeletal muscle was determined. It consists of 98 amino acid residues with N-acetylserine at the amino (N)-terminus: Ac-Ser-Thr-Ala-Arg-Pro-Leu-Lys-Ser-Val-Asp-Tyr-Glu-Val-Phe-Gly -Arg-Val-Gln-Gly-Val-Cys-Phe-Arg-Met-Tyr-Thr-Glu-Asp-Glu-Ala-Arg-Lys-Ile -Gly-Val-Val-Gly-Trp-Val-Lys-Asn-Thr-Ser-Lys-Gly-Thr-Val-Thr-Gly-Gln -Val-Gln-Gly-Pro-Glu-Glu-Lys-Val-Asn-Ser-Met-Lys-Ser-Trp-Leu-Ser-Lys -Ile-Gly-Ser-Pro-Ser-Ser-Arg-Ile-Asp-Arg-Thr-Asn-Phe-Ser-Asn-Glu-Lys- Thr-Ile-Ser-Lys-Leu-Glu-Tyr-Ser-Asn-Phe-Ser-Ile-Arg-Tyr-OH. This sequence has three substitutions of amino acid residues, i.e., Thr/Ala, Ile/Val, and Ile/Val at positions 26, 68, and 96, respectively, from that of horse muscle acylphosphatase, formerly the only mammalian acylphosphatase with known sequence.

Acute cold stress induces suppressor macrophages in mice.

To elucidate mechanisms underlying acute cold stress-induced immunosuppression, functions of murine peritoneal cells of monocyte/ macrophage lineage from acute cold-stressed mice (exposed to 5 degrees C for 24 h) were investigated. Proliferative responses of spleen cells from control mice (reared at 25 degrees C) stimulated with concanavalin A (ConA) were significantly suppressed by adding peritoneal exudate cells from mice immediately after acute cold stress. The proportion of adherent cells was markedly increased in the peritoneal exudate cells from acute cold-stressed mice. These adherent cells from acute cold-stressed mice were shown to be the cells responsible for the suppressor activity for ConA responses of control spleen cells. Nonadherent cells did not suppress the ConA responses. The adherent cells in peritoneal exudate cells from control mice also suppressed the ConA responses, the inhibitory effect being considerably lower than that from acute cold-stressed mice. Addition of a nitric oxide synthase substrate analogue, NG-monomethyl-L-arginine, to the mixed cell cultures of normal spleen cells and adherent cells from acute cold-stressed mice inhibited nitric oxide release and completely abolished the suppressive effect of the adherent cells, suggesting that reactive nitrogen oxide released from the activated macrophages is apparently involved in the downregulation of proliferative responses of T cells. Thus the present findings suggest that acute cold stress induces macrophages with suppressor function and that this may contribute to the immune-suppressive state seen in spleen cells from acute cold-stressed mice.