Effects of exogenous tumour necrosis factor-alpha on the secretory function of the bovine reproductive tract depend on tumour necrosis factor-alpha concentrations.

The aim of study was to correlate tumour necrosis factor-alpha (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F(2alpha), PGE(2) nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic--1 microg or luteotrophic--10 microg). In the peripheral blood, 1 microg TNF concentrations achieved within the range of 30-45 pg/ml, and 10 microg TNF provoked a sharp increase in achieved concentrations at a range of 250-450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF(2alpha) and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE(2) (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis, metritis).

Overexpression of connective tissue growth factor in podocytes worsens diabetic nephropathy in mice.

Connective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes.

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis.

Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-beta-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation.

Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A.

Sindbis virus can infect a broad range of insect and vertebrate cell types due to the widespread distribution of the cellular receptor for the virus. The development of Sindbis virus vectors that target specific cell types could have important implications for the design of gene therapy strategies. To achieve this goal we have designed and constructed Sindbis virus particles displaying the IgG-binding domain of protein A. The protein A-envelope chimeric Sindbis virus vector has minimal infectivities against baby hamster kidney and human cell lines. When used in conjunction with monoclonal antibodies that react with cell-surface antigens, however, the protein A-envelope chimeric virus was able to infect human cell lines with high efficiency. Infection rates were 90% or higher for human lymphoblastoid cells. A variety of cells could be targeted by changing the monoclonal antibody without generating a new recombinant virus.

Milky spots as the implantation site for malignant cells in peritoneal dissemination in mice.

We examined the site-specific implantation of cancer cells in peritoneal tissues after an i.p. inoculation of 10(5) P388 leukemia cells. Twenty-four h after the inoculation, the number of viable cancer cells infiltrating into specific tissue sites of the peritoneum was estimated by an i.p. transfer method. A descending order of tissue implantation with cancer cells was established as omentum > gonadal fat > mesenterium > posterior abdominal wall > stomach, liver, intestine, anterior abdominal wall, and lung. A significant correlation was established between the logarithm of the number of infiltrating cancer cells and the logarithm of the number of milky spots. Next, the omentum was examined microscopically after i.p. inoculation with P388 leukemia cells labeled with bromodeoxyuridine or B-16 PC melanoma, which were differentiated from the other cells by an immunocytological method using anti-bromodeoxyuridine antibody or by the melanin of the B-16 PC melanoma cells. These cancer cells were found microscopically to be infiltrating only the milky spots, whereas none were seen at the other sites. These results suggest that cancer cells seeded i.p. specifically infiltrate the milky spots in the early stage of peritoneal metastases.

Structural alterations of the superior temporal gyrus in schizophrenia: Detailed subregional differences.

BACKGROUND: Reduced gray matter volumes in the superior temporal gyrus (STG) have been reported in patients with schizophrenia. Such volumetric abnormalities might denote alterations in cortical thickness, surface area, local gyrification or all of these factors. The STG can be anatomically divided into five subregions using automatic parcellation in FreeSurfer: lateral aspect of the STG, anterior transverse temporal gyrus of Heschl gyrus (HG), planum polare (PP) of the STG, planum temporale (PT) of the STG and transverse temporal sulcus. METHODS: We acquired magnetic resonance imaging (MRI) 3T scans from 40 age- and sex-matched patients with schizophrenia and 40 healthy subjects, and the scans were automatically processed using FreeSurfer. General linear models were used to assess group differences in regional volumes and detailed thickness, surface area and local gyrification. RESULTS: As expected, patients with schizophrenia had significantly smaller bilateral STG volumes than healthy subjects. Of the five subregions in the STG, patients with schizophrenia showed significantly and marginally reduced volumes in the lateral aspect of the STG and PT of the STG bilaterally compared with healthy subjects. The volumetric alteration in bilateral lateral STG was derived from both the cortical thickness and surface area but not local gyrification. There was no significant laterality of the alteration in the lateral STG between patients and controls and no correlation among the structures and clinical characteristics. CONCLUSIONS: These findings suggest that of five anatomical subregions in the STG, the lateral STG is one of the most meaningful regions for brain pathophysiology in schizophrenia.

Caspase-dependent apoptosis by ectopic expression of E2F-4.

E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.

Chronologic changes in the clinicopathologic findings and survival of gastric cancer patients.

PURPOSE: To determine the chronologic changes in the clinicopathologic features of gastric cancer patients. PATIENTS AND METHODS: The clinicopathologic findings of 1,795 patients with gastric cancer were examined retrospectively from hospital records obtained between 1969 and 1995. The patients were divided into three generations on the basis of chronologic order. The first generation included patients treated over the period 1969 to 1977; the second generation, 1978 to 1986; and the third generation, 1987 to 1995. RESULTS: The chronologic changes in the clinicopathologic findings for all gastric cancers included increases in the superficial type based on macroscopic appearance (P < .005), small-sized tumor (P < .025), superficial depth of invasion (P < .005), and earlier histologic stages (P < .005), in addition to a decrease in lymph node metastasis (P < .005). Overall, the postoperative survival rate has improved over time in gastric cancer patients, with 5-year survival rates of 36.0%, 53.3%, and 68.6% in the first, second, and third generations, respectively. In stages 1,2, and 3, the survival rate in the third generation was the highest of the three generations, whereas in stage 4, the survival rate did not differ between the three generations. Patients who underwent a D2 dissection showed a higher survival rate than those with D1 or D3 dissections, but there was no statistical difference in the survival of patients with D1, D2, and D3 dissections when stage 4 patients were excluded. CONCLUSION: The chronologic changes in gastric cancer patients over the past 27 years have included an increase in the incidence of earlier-staged gastric cancers, which has had a significant impact on the improved postoperative survival rate.

Soluble interleukin-6 (IL-6) receptor in the sera of pregnant women forms a complex with IL-6 and augments human chorionic gonadotropin production by normal human trophoblasts through binding to the IL-6 signal transducer.

To study the role of soluble interleukin-6 receptor (sIL-6R) during pregnancy, sIL-6R levels in the sera of pregnant women in the first, second, and third trimesters were determined and found to remain unchanged during pregnancy, but were significantly higher than those in nonpregnant women in the follicular, ovulatory, and luteal phases of the menstrual cycle (P < 0.001). IL-6 levels, however, in the sera of pregnant women at all trimesters showed no difference from those in nonpregnant women at any stage of the menstrual cycle. Recombinant sIL-6R (rsIL-6R) augmented hCG production by rIL-6-stimulated trophoblasts dose dependently, but failed to enhance hCG production by unstimulated trophoblasts. rIL-6- and rsIL-6R-induced hCG production was significantly blocked by anti-IL-6R antibody, PM1; antisignal transducing glycoprotein 130 (gp130) antibody, GPX7; or a tyrosine kinase inhibitor, genistein. Thus, sIL-6R in serum from pregnant women forms a complex with placental and decidual IL-6 in a manner similar to trophoblast membrane-bound IL-6R. These two discrete types of IL-6R and IL-6 complex might act cooperatively by binding to gp130 and subsequently evoking tyrosine kinase activity in the trophoblasts to produce hCG in vivo.

Leukemia inhibitory factor produced at the fetomaternal interface stimulates chorionic gonadotropin production: its possible implication during pregnancy, including implantation period.

We investigated the role of leukemia inhibitory factor (LIF) at the implantation site of human embryos. The first trimester decidual tissue produced higher levels of LIF than chorionic tissue, but the decidua produced much smaller amounts of interleukin-6 (IL-6) than the chorion in vitro, as determined by enzyme-linked immunosorbent assay. The reverse transcription-polymerase chain reaction and immunohistochemical analysis revealed the expression and localization, on the trophoblasts, of glycoprotein 130 (gp130), an IL-6 signal transducer receptor component shared by the cytokines such as LIF and IL-6. Trophoblasts stimulated by recombinant LIF (rLIF) produced CG titer at the amount similar to that induced by rIL-6. Recombinant LIF-induced CG production was significantly blocked by anti-gp130 antibody but not by anti-IL-6 receptor antibody, whereas rIL-6-induced CG was completely blocked by both antibodies. Recombinant LIF- and rIL-6-induced CG productions were both significantly blocked by genistein, a tyrosine kinase inhibitor, suggesting an involvement of tyrosine kinase in gp130-mediated CG production. Since CG is capable of stimulating trophoblast growth and differentiation as well as placental metabolism, LIF produced at the fetomaternal interface are considered to stimulate the trophoblasts to produce CG, which may contribute to the maintenance of the placental functions and embryonal growth.

Stability and localization of biotin-labeled murine monoclonal antibody to human gastric cancer in normal mice and nude mice-human tumor models.

We examined the tissue localization of biotin-labeled murine monoclonal antibody (MAb) S202 directed against the human scirrhous gastric carcinoma cell line MK-01 in normal and tumor-bearing mice after intravenous (IV) administration. The biotin-labeled MAb proved to be stable in vivo under normal conditions, antibody titer being 1:256 at 4 hr after IV injection. At 24 hr after injection, the tumor was stained by the avidin-biotin-peroxidase complex (ABC) method. Biotin-labeled MAb was found to be suitable for detection of the xenografted tumor of nude mice. This study provides new information concerning the dynamics of the distribution of biotin-labeled MAb in vivo.

Chorioamnionitis reduces placental endocrine functions: the role of bacterial lipopolysaccharide and superoxide anion.

Chorioamnionitis has been shown to be one of the most important factors in inducing preterm delivery. The present study was undertaken to examine the effects of chorioamnionitis on placental endocrine functions. Preterm placentas with histologic chorioamnionitis produced smaller amounts of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) than those without chorioamnionitis (P < 0.001). To examine the mechanism involved in the suppression of placental endocrine functions induced by chorioamnionitis, we initially confirmed the expression of lipopolysaccharide (LPS) receptor, i.e. the CD14 molecule, on trophoblasts by Northern blot analysis and immunohistochemistry. We then stimulated purified trophoblasts with LPS, which is the major agent which induces inflammatory responses in the host via the LPS receptor. The trophoblasts stimulated with LPS produced reduced amounts of hCG, hPL, and progesterone in a time- and dose-dependent fashion in spite of the induced manganese-superoxide dismutase (SOD) synthesis. Stimulation of trophoblasts with hypoxanthine and xanthine oxidase resulted in suppressed hCG production, while the simultaneous addition of SOD into the culture medium reversed the suppression of hCG production. LPS in the placenta with chorioamnionitis might directly stimulate trophoblasts through the LPS receptor (CD14), thus reducing placental endocrine functions. Superoxide anions which exogenously act on trophoblasts might be generated by simultaneous stimulation of neutrophils and monocytes at the feto-maternal interface by LPS, and additively reduce placental endocrine functions.

C4d and C4bp deposition along the glomerular capillary walls in a patient with preeclampsia.

Complement (C) 4d and cofactor C4b binding protein (C4bp) are detected in the glomerular capillary walls of a patient with preeclampsia. A 32-year-old nullipara had proteinuria of 1.2 g/d and edema at the 33rd week of pregnancy. Gradually the urinary protein excretion increased, reaching 5.1 g/d at the 37th week. The patient also showed hypertension at this stage. After normal mature delivery, the level of the urinary protein excretion remained at 3 to 4 g/d. Renal biopsy performed by means of light and electron microscopy, 15 days after delivery, showed almost normal glomeruli and modest subendothelial widening. Immunohistochemistry indicated that immunoglobulin (Ig) A, IgG, C1q, C3c, and C4c were not deposited in the glomeruli, whereas weakly positive IgM and fibrin-related antigen (FRA) were observed. Conversely, C4d, C3d, and C4bp were strongly deposited. Protein S (PS) also was observed, with a similar distribution pattern to that of C4bp. Immunoelectron microscopy showed the deposition of C4d along the capillary walls and of C4bp in the subendothelium. These findings suggest that the C4 activation process as well as the regulation process of C system and of the inflammatory coagulation axis by C4bp and PS may play an important role in the pathophysiology of preeclampsia, so-called glomerular capillary endotheliosis (GCE).

Intraglomerular metastasis from pancreatic cancer.

Few case reports have shown the presence of metastatic tumor cells in renal glomeruli. We report one case with intraglomerular metastasis proved at renal biopsy. A 60-year-old man suffered from weight loss and fever of unknown origin. Urinalysis revealed proteinuria with cellular and granular casts. Because vasculitis was suspected, renal biopsy was performed. Presence of tumor cells occupying the glomerular capillary lumina was shown by means of light microscopy and electron microscopy. Laboratory findings revealed elevated leukocyte count (28.9 x 10(3)/mm(3)), serum granulocyte colony-stimulating factor (G-CSF) (77 pg/mL), and serum CA 19-9 (21,885 U/mL). The patient soon developed disseminated intravascular coagulation and died. Autopsy findings revealed pancreatic cancer showing positive staining for G-CSF and CA 19-9. Tumor cells in the glomerular capillary lumina showed positive staining for CA 19-9 and proliferating cell nuclear antigen (PCNA). These results suggest that the pancreatic tumor cells producing G-CSF were entrapped in the glomerular capillary lumina where they proliferated. This may have been the first step in renal metastasis.

Production of interleukin-6 by normal human trophoblast.

The placenta plays a number of important roles during pregnancy, some of which might be mediated by cytokines with multiple activities such as IL-6. Using an IL-6-dependent cell line, MH6o.BSF2, we showed that the placenta released IL-6 into the culture supernatant. Analysis of single-cell suspensions of placental cells determined the major source of IL-6 to be trophoblast. Using a mouse monoclonal antibody specific for IL-6 (alpha BSF2-I66), immuno-histochemical analysis of placental specimens demonstrated the localization of IL-6 only in the trophoblast layer. Additional immunocytochemical studies with single-cell suspensions of trophoblasts demonstrated the preferential presence of IL-6 molecules in syncytiotrophoblasts rather than cytotrophoblasts. The evidence that a high titer of IL-6 is produced spontaneously by syncytiotrophoblasts indicates that IL-6 may play immunological roles in fetomaternal interactions by means of IL-6-driven multiple immunoregulatory activities.

Mechanical strength of calcium phosphate cement in vivo and in vitro.

Two different kinds of calcium phosphate cement were developed for implant fixation: cement A comprised of alpha-tricalcium phosphate (alpha-TCP) 95% and dicalcium phosphate dihydrate (DCPD) 5%, and cement B comprised of alpha-tricalcium phosphate 90% and dicalcium phosphate dihydrate 10%. The compression strength and pullout force of the new materials were tested both in vitro and in vivo. Microscopic observations were performed on the interface between bone and cement. Cement A showed a greater mechanical strength than cement B. The results suggest the clinical possibility of this calcium phosphate cement, which could be used as a material for enhancing implant fixation.

Comparative histocompatibility testing of seven calcium phosphate ceramics.

Five hydroxyapatite and two tricalcium phosphate ceramics were implanted in rat femora for one to six months. They were compared with each other in regard to biocompatibility and bone induction. After one month bone had grown directly on to the surface of all implants. After six months the on-growth of bone had increased with the exception of two dense HA materials. In these materials some bio-degradation had occurred with subsequent formation of a wide macrophage interlayer. Bio-degradation and biocompatibility of calcium phosphate ceramics seem to depend not only on density but also on other factors. Induction of bone could not be observed in soft tissue environment.

Analysis of immunoregulatory activity of a choriocarcinoma-derived factor: specific suppression of proliferative process of cell-mediated immune responses including LAK cell generation.

The immunosuppressive activity of a JEG-3 choriocarcinoma-derived factor in human IL-2-dependent T cell responses has been studied, together with its effect on IL-2-independent T cell responses induced by 10 nM TPA. The factor completely suppressed the IL-2-independent proliferative responses of T cells but failed to suppress antigenic expression of activation-associated CD 25 molecules. Further studies examined the effect of the factor on LAK cell generation induced by rIL-2. Recombinant IL-2-induced LAK cell proliferation was observed on Day 4 and Day 5, but not on Day 3. As the factor suppressed the responses of LAK cell proliferation, we tested whether it blocked the generation of Day 3, Day 4 and Day 5 LAK cells. The addition of the factor failed to suppress the generation of Day 3 LAK cells, while it partially suppressed the lytic activity of Day 4 LAK cells and completely suppressed that of Day 5 LAK cells. The data suggest the presence of a heterogeneous pattern for LAK cell generation; one without proliferation, but the other requiring proliferation, to acquire killer activity. Taken together with the evidence that the factor failed to suppress NK activity, the choriocarcinoma-derived factor suppressed only the proliferative events of immunocompetent cells, but inhibited neither their activation nor the differentiation events. This immunosuppressive factor might be involved in the prevention of host-mediated rejection of choriocarcinoma cells or maternal rejection of the fetus.

Cytokine production in chorioamnionitis.

Lymphohematopoietic cytokines play a significant role in many biological mechanisms including a number of reproductive processes such as ovulation, implantation, placentation, cervical dilation and parturition. Recent experiments have suggested that cytokines play a crucial role in the mechanisms of preterm labor and delivery, which are the leading causes of perinatal morbidity and mortality. Growing evidence suggests that infection is deeply concerned in the pathogenesis of preterm labor and delivery. Chorioamnionitis, a subset of intrauterine infection, has been identified in 20-33% of women with preterm delivery, and the inflammatory and related cytokines, interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), showed substantial increases in the amniotic fluid at women with intrauterine infection. Although the precise mechanism for chorioamnionitis-driven preterm labor mediated via cytokines is still unknown, both IL-1 and TNF-alpha along with IL-6 enhance prostaglandin production by human amnion cells, chorionic cells and decidual cells. Analysis of the regulatory sequences in the 5' upstream regions of receptor gene for human oxytocin, a potent uterotonic agent, suggests a close relationship between preterm labor and inflammatory cytokines through induction at the oxytocin receptor. Prompt identification of the patients with intra-amniotic infection may be useful in clinical practice. At present, the measurement of IL-8 in maternal serum or the measurement of IL-6 in cervical secretion may be helpful as a non-invasive screening for chorioamnionitis.

A rapid immunohistologic method for determining the limit of cancer invasion at the surgical margin of scirrhous carcinoma of the stomach during surgery.

The authors used the monoclonal antibody (MAb) S202 as an adjunct for detection of scirrhous gastric carcinoma cells in cytomorphologic preparations of the resected stomach. Twenty-six samples of scirrhous gastric cancer were stained by the avidin-biotin complex technique on fixed frozen sections. MAb S202 was strongly reactive on all sections. In 12 cases, samples obtained from surgical margins were examined by a rapid immunostaining method. In one case, conventional cytologic findings were equivocal, whereas by the rapid immunostaining method single cells were stained darkly, indicating malignancy. Thus, immunohistochemical analysis using MAb S202 should be carried out to determine the limit of invasive carcinoma at the time of surgery.