Effect of modified alkaline supplementation on bone metabolic turnover in rats.

This study aims to determine the effects of a high protein diet and alkaline supplementation on bone metabolic turnover in rats. Eight-week-old male Sprague-Dawley rats were investigated by bone status, including bone mineral density (BMD) and biomechanical markers from blood and urine. Thirty rats were randomly divided into three groups and treated for 8 weeks as follows: baseline control group (n. 10, C), high-protein supplemented diet group (n. 10, chronic acidosis, CA group) and supplemented chronic acidosis (n.10, SCA). Diet-treated rats were fed an acidic high-protein diet and the supplementation consisted in a modified alkaline formula (Basenpulver, NaMed, Italy). At the end of the experimental period, the rats were sacrificed, blood samples were drawn and femur and tibia were removed for analysis of bone mineral density (BMD) by dual energy X-ray absorptiometry (DEXA). In the CA group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased 2.1-fold (p<0.05 vs normal diet controls) as well as kidney weight. However, serum Ca and P concentration, as well as urinary Dpd excretion were not significantly changed. Femural and tibial BMD was significantly decreased in the CA group (p<0.05), but alkaline supplementation prevented such phenomenon (p<0.05 vs CA). These results suggest that blood Ca and P concentrations in chronic acidosis condition during the 12-week supplementation might be maintained by hypercalciuria and hyperphosphaturia at the expenses of bone structure. However, modified alkaline supplementation is able to prevent such derangements.

Antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (IL-12) or other cytokines compared with exogenous IL-12.

BACKGROUND: Numerous animal model studies have examined the ability of genetically engineered tumor cells to release cytokines and to elicit an immune memory against the parental tumor. Often only a single cytokine is studied, and few comparative studies have been conducted. PURPOSE: We evaluated the antitumor efficacy of adenocarcinoma cells engineered to release interleukin (IL)-12 in a mouse model system. The efficacy of this cytokine was compared with that of other cytokines released by engineered adenocarcinoma cells and that of exogenous IL-12 administered both locally and intraperitoneally. METHODS: BALB/cAnCr mice were inoculated with syngeneic parental mammary adenocarcinoma (TSA) cells in quantities sufficient to lead to tumors in all inoculated mice. TSA cells engineered to release IL-12 (TSA-IL12) were also injected into normal and selectively immunosuppressed BALB/cAnCr mice. Tumor incidence, growth, and rejection patterns were evaluated by the measurement of neoplastic masses and by the study of the histologic and ultrastructural features of the tumor site. The effects of local or intraperitoneal administration of recombinant IL-12 (rIL-12) on tumor-bearing animals were also studied. RESULTS: Most mice rejected TSA-IL12 cells through a CD8-positive, T-lymphocyte-dependent reaction associated with macrophage infiltration, vessel damage, and necrosis. The systemic immunity of mice that had rejected TSA-IL12 cells to a subsequent challenge with parental TSA cells was less efficient than that elicited by TSA cells engineered to release IL-4 or IL-10 but equivalent to that elicited by TSA cells engineered to release IL-2, IL-7, and interferon alfa. Compared with TSA cells engineered to produce other cytokines, TSA-IL12 cells were the most efficient in curing mice with established TSA tumors; injection of 0.1 million proliferating cells contralaterally to the tumor growth area cured five of 15 mice bearing 1-day-old tumors; injection of the same dose of proliferating cells into the tumor growth area cured two of 20 tumor-bearing mice. However, two 5-day courses with a nontoxic dose (0.1 microgram) of rIL-12 given intraperitoneally cured a similar proportion of these animals (six of 20). Only two of 20 mice with 7-day-old TSA tumors were cured by vaccination with proliferating TSA-IL12 cells, whereas 24 of 30 mice with such tumors were cured by intraperitoneal administration of rIL-12. CONCLUSIONS: TSA cells engineered to release IL-12 are rejected by most mice; the ensuing immune memory for TSA parental cells, however, was less efficient than that elicited by proliferating TSA cells engineered to release other cytokines (e.g., IL-4, IL-10, and possibly interferon gamma). The immune reaction elicited by TSA-IL12 cells was the most efficient in curing mice with established TSA tumors; notably though, the same or a better cure rate was obtained with rIL-12 given intraperitoneally.

Ceramide inhibition of NF-kappaB activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response.

Protein kinase C (PKC) is known to activate NF-kappaB whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1, 2). In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF-7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC-mediated activation of NF-kappaB by reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCbeta2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC-DA) was resistant to ceramide. A kinase-inactive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC-DA prevented, at least partly, the ability of ceramide to inhibit activation of NF-kappaB. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophosphorylation sites.

Regulation of the chemokine system at the level of chemokine receptor expression and signaling activity.

The chemokine system is highly influenced by the microenvironmental context. Regulation of the chemokine system occurs not only at the level of agonist production, but also at the level of chemokine receptor expression. This review provides examples of regulation of the system at the receptor level by modulation of receptor expression in canonical cellular targets (tuning of the system), and induction of novel receptors (shaping of the system), with particular attention to dendritic cells as a cellular model. Receptor signaling activity represents a further potential level of regulation of the system. Finally, chemokines can also influence the microenvironment by modulating gene expression in target cells.

[A clinical study of the use of a combination of glucomannan with lactulose in the constipation of pregnancy]. / Studio clinico dell'impiego di una associazione di glucomannano con lattulosio nella stipsi gravidica.

RATIONALE: Constipation is a problem frequently encountered during pregnancy as is excessive weight gain. Treatments of common use to control constipation are endowed with some drawbacks and they are not active in controlling weight increase. A preparation of lactulose and glucomannan in previous studies proved very effective and well tolerated in patients affected by stypsis and evidentiated also activity both in controlling excessive food intake and in correcting some metabolic imbalances regarding lipids and urea. MATERIAL AND METHODS: 50 pregnant females affected by constipation were treated with sachets containing a preparation of glucomannan (1.45 g) and lactulose (4.2 g) in a posology of 2 (1-4) sachets a day for 1-3 months. RESULTS: Treatment induced a return to normal frequency of weekly number of evacuations (4.9-5.8/week) and a parallel control of weight gain (within 20% of initial body weight). The latter finding seems to be related to hunger control induced by glucomannan at the gastric level which prevents an excessive food intake.

[Hydronephrosis in pregnancy. Ultrasonographic study]. / Idronefrosi della gravidanza. Studio ecografico.

Hydronephrosis is a common pathology during pregnancy and according to some authors affects between 80 and 90% of women in the third trimester. The right side is most frequently affected. Moreover, there appears to be no relation between parity and hydronephrosis or between previous infections of the urinary tract and hydronephrosis; however its presence, especially if severe, is associated with urinary infections with a frequency which is significantly greater than in patients with a urinary tract within normal limits. There are two pathogenetic hypotheses: one identifies the inhibitory effect of progesterone and prostaglandins on muscular tone and the peristalsis of smooth ureteral muscle, with consequent dilatation and anfractuosity of the excretory tract above the pelvic brim; the other is the mechanical hypothesis based on the possible compression of the ureter caused by the pregnant womb at the level of the pelvic brim at the intersection with the ilial and ovarian vessels. One hypothesis does not exclude the other, but on the contrary they complement each other and give added plausibility to a multifactorial pathogenesis. The study of hydronephrosis in pregnancy has made considerable progress since the introduction of echotomography in image diagnosis, a low-cost method which is easy to use and does not harm the product of conception. Echographs with 3.5 MHz probes were used to carry out a prospective study by examining the kidneys and excretory tract in 370 asymptomatic pregnant women during the first, second and third trimesters of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

[Selective episiotomy. Comparison of two suture technics]. / Episiotomia selettiva. Due tecniche di sutura a confronto.

AIMS: The study aimed to highlight any differences between the sequelae of episiorrhaphy performed using the Blair-Donatti and Guilmen-Pontonnier techniques. METHODS: A total of 202 pregnant women were recruited in the Obstetrics Ward of Codogno Civic Hospital in 1994 and 1995. Subjects were randomly divided into two groups matched for socioeconomic status, age and parity (group A: Guilmen-Pontonnier suture, group B: Blair-Donatti suture). The following parameters were evaluated: pain twenty-four hours, sixty-six hours and one, two and three months after labour, pain during sexual relations, the onset of infection, hematoma, dehiscence of the wound, and lastly the cosmetic results. RESULTS: No significant differences were found between the parameters examined in both groups, barring the improved cosmetic result in group A.

Autophagy in term normal human placentas.

Autophagy is an inducible catabolic process that responds to environment and is essential for cell survival during stress, starvation and hypoxia. Its function in the human placenta it is not yet understood. We collected 14 placentas: 7 at vaginal delivery and 7 at elective caesarean section after uneventful term pregnancies. The presence of autophagy was assessed in different placental areas by immunoblotting, immunohistochemistry and electron microscopy. We found that autophagy is significantly higher in placentas obtained from cesarean section than in those from vaginal delivery. Moreover there is a significant inverse relationship between autophagy and umbilical arterial glucose concentration.

Inhibition of human breast cancer cell growth and enzymatic activity by a fermented nutraceutical: an in vitro and in vivo study .

Human breast cancer cell lines MCF-7 (ER-positive) and Hs578T (ER-negative) were cultured and one lot incubated for 48 h with 5-50 mug/ml of a fermented phytocompound (MK: Manda-Koso, Innoshima, Japan). In vitro, it appeared a dose-dependent decrease of cell viability (5-57%) in MK group in both cell lines (P < 0.001, plateau: 30 microg/ml), decreased beta-galactosidase activity, enhanced apoptosis, and inversely increased Bax/Bcl2 ratio (P < 0.01) with an upregulation of p53 (P < 0.05). In the in vivo model, Balb-c mice were inoculated with tumor cells and the treatment group was fed with 20 mg of MK. Tumor weight in MK-fed group was time-course reduced by 22% to 51% at 2 and 4 weeks, respectively (P < 0.05) with increased survival (P < 0.05). Tumour tissue of MK-fed mice showed a downregulated Bcl-2 with increased Bax/Bcl-2 ratio, reduced PCNA, and activated caspase 3. Although more studies are ongoing to foster the clinical applicability of MK integrated within a rational chemopreventive and therapeutic strategy, a p53-mediated mechanism is likely to play a relevant role, besides its reported antioxidant capacity, NK cell activity enhancement, cancer-cytostatic activity properties.

Phenotypic and morphological characterization of neuroblastoma cells constitutively expressing B-myb.

B-myb gene is expressed in neuroblastoma cells and down-regulated during differentiation. We used B-myb-transfected LAN-5 cells, which constitutively express high level of B-myb, to detect changes at phenotypic and morphological levels in basal and differentiation conditions. Our results demonstrate that the overexpression of B-myb markedly affects the cytoskeletal composition, the pattern of neurotransmitter enzymes and the extracellular matrix expression. In general, B-myb transfected neuroblastoma cells show a broad potentiality without a direction toward a specific neuroectodermal differentiation pathway. On the other hand, we confirm inhibition of the neuronal differentiation upon retinoic acid (RA) treatment of B-myb transfected cells. Furthermore, the ultrastructural analyses are supportive of a change in the metabolism in B-myb transfected cell treated with RA. Our data suggest that B-myb expression is compatible with an early phase of differentiation of neuroectodermal cells, but must be down-regulated for the completion of the differentiative programme.

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.

Local and systemic antitumor response after combined therapy of mouse metastatic tumors with tumor cells expressing IFN-alpha and HSVtk: perspectives for the generation of cancer vaccines.

In this study, we have evaluated the local versus systemic antitumor response in tumor-bearing mice subjected to a combined therapeutic regimen based on the injection of genetically modified Friend erythroleukemia cells (FLC) producing IFN-alpha and expressing the HSVtk (tk) gene, and we have investigated the host immune mechanisms involved in tumor rejection and development of antitumor immunity. Repeated subcutaneous (s.c.) injections of IFNtk-expressing tumor cells, followed by GCV administration, were effective in counteracting the growth of both contralateral parental tumors as well as visceral metastases, whereas similar treatments with control tk cells (i.e. nonproducing IFN) were ineffective. Morphologic analyses of the homolateral and contralateral tumor tissues and in vivo immunosuppression experiments with specific monoclonal antibodies revealed that both CD4+ and CD8+ T lymphocytes played essential roles in the generation of a definite antitumor response after the combined therapeutic regimen. We have also compared the effectiveness of irradiated versus viable tumor vaccines co-expressing the two genes in the FLC model and in the poorly immunogenic metastasizing TS/A adenocarcinoma tumor system. Repeated injections of high doses of irradiated IFN-alpha-tk-expressing tumor cells followed by GCV administration resulted in the cure of the majority of mice bearing established metastatic tumors, while repeated inoculations of the same number of viable tumor vaccines were much less effective. We conclude that; (1) IFN-alpha is an essential cofactor in the generation of a systemic antitumor immunity following the prodrug-induced tumor cell killing; (2) vaccines co-expressing an autotoxic gene and a cytokine gene may represent promising new tools for the treatment of some cancer patients.

Extracellular matrix remodelling in a murine mammary adenocarcinoma transfected with the interferon-alpha 1 gene.

The rejection of interferon alpha 1 gene-transfected mammary adenocarcinoma cells (TSA-IFN alpha) injected into syngeneic BALB/c mice was accompanied by an unusual stromal reaction and marked CD8-positive T-lymphocyte involvement. To investigate the biological background of this reaction, the possibility was evaluated that an interaction between TSA-IFN alpha and stromal cells might remodel the extracellular matrix (EM). When fibroblasts were co-cultured with TSA-IFN alpha or treated with exogenous IFN alpha, there was no change in their replication rate or collagen synthesis. By contrast, their fibronectin (FN) production and release were increased, resulting in enhanced fibroblast chemotaxis. These findings were mirrored by increased FN staining in the peritumoural and tumoural areas in vivo. IFN alpha thus determines increased FN production and hence massive local recruitment and activation of fibroblasts, with a modification of the EM. The several activities of IFN alpha should thus be considered prior to its employment in clinical trials.