Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

TIA-1 cytotoxic granule-associated RNA binding protein improves the prognostic performance of CD8 in mismatch repair-proficient colorectal cancer

Background

Evidence suggests a confounding effect of mismatch repair (MMR) status on immune response in colorectal cancer. The identification of innate and adaptive immune cells, that can complement the established prognostic effect of CD8 in MMR-proficient colorectal cancers patients, representing 85% of all cases, has not been performed.

Methodology/Principal Findings

Colorectal cancers from a test (n = 1197) and external validation (n = 209) cohort of MMR-proficient colorectal cancers were mounted onto single and multiple punch tissue microarrays. Immunohistochemical quantification (score 0-3) was performed for CD3, CD4, CD8, CD45RO, CD68, CD163, FoxP3, GranzymeB, iNOS, mast cell tryptase, MUM1, PD1 and TIA-1 tumor-infiltrating (TILs) reactive cells. Coexpression experiments on fresh colorectal cancer specimens using specific cell population markers were performed. In the test group, higher numbers of CD3+ (p<0.001), CD4+ (p = 0.029), CD8+ (p<0.001), CD45RO+ (p = 0.048), FoxP3+ (p<0.001), GranzymeB+ (p<0.001), iNOS+ (p = 0.035), MUM1+ (p = 0.014), PD1+ (p = 0.034) and TIA-1+ TILs (p<0.001) were linked to favourable outcome. Adjusting for age, gender, TNM stage and post-operative therapy, higher CD8+ (p<0.001; HR (95%CI): 0.66 (0.64-0.68)) and TIA-1+ (p<0.001; HR (95%CI): 0.56 (0.5-0.6)) were independent prognostic factors. Moreover, among patients with CD8+ infiltrates, TIA-1 further stratified 355 (35.6%) patients into prognostic subgroups (p<0.001; HR (95%CI): 0.89 (95%CI: 0.8-0.9)). Results were confirmed on the validation cohort (p = 0.006). TIA-1+ cells were mostly CD8+ (57%), but also stained for TCRγδ (22%), CD66b (13%) and only rarely for CD4+, macrophage and NK cell markers.

Conclusions

TIA-1 adds prognostic information to TNM stage and adjuvant therapy in MMR-proficient colorectal cancer patients. The prognostic effect of CD8+ TILs is confounded by the presence of TIA-1+ which translates into improved risk stratification for approximately 35% of all patients with MMR-proficient colorectal cancers.     

Antiproliferative effects of insulin-like growth factor-binding protein-3 in mesenchymal chondrogenic cell line RCJ3.1C5.18. relationship to differentiation stage

Chondrogenesis results from a complex equilibrium between chondrocyte proliferation and differentiation. Insulin-like growth factors (IGFs) have a crucial role in chondrogenesis, but their mechanisms of action are not well defined. IGF-binding protein-3 (IGFBP-3) is the major carrier for circulating IGFs in postnatal life, and has been shown to have IGF-independent effects on proliferation of several cancer cell lines. In this study, we have evaluated the IGF-independent and -dependent effects of IGFBP-3 on chondrocyte proliferation and the relationship of these effects with chondrocyte differentiation stage. We used the RCJ3.1C5.18 nontransformed mesenchymal chondrogenic cell line, which, over 2 weeks of culture, progresses through the differentiation pathway exhibited by chondrocytes in the growth plate. We demonstrated that IGFBP-3 inhibited, in a dose-dependent manner (1-30 nm), the proliferation of chondroprogenitors and early differentiated chondrocytes, stimulated by des-(1-3)-IGF-I and longR(3)-IGF-I (IGF-I analogs with reduced affinity for IGFBP-3), and by insulin and IGF-I. In terminally differentiated chondrocytes, IGFBP-3 retained the ability to inhibit cell proliferation stimulated by IGF-I, but had no effect on cell growth stimulated by insulin, or des-(1-3)-IGF-I or longR(3)IGF-I. By monolayer affinity cross-linking, we demonstrated a specific IGFBP-3-associated cell-membrane protein of approximately 20 kDa. We determined that IGFBP-3 has an antiproliferative effect on chondrocytes and, that this effect is related to the differentiation process. In chondroprogenitors and early differentiated chondrocytes, antiproliferative effect of IGFBP-3 is mainly IGF-independent, whereas, following terminal differentiation this effect is IGF-dependent.     

Steady-state kinetics of MgATP splitting by native myosin RLC-free subfragment 1

MgATP positively regulates the dimerisation reaction, resulting in an increase in the rate of MgATP splitting with increasing MgATP concentration. We investigated the stoichiometry of this dimerisation reaction and found that each subunit in the dimer bound one molecule of MgATP at the dimerisation site. We studied changes with temperature in the MgATPase activity of S1 in the dimeric form for temperatures of 18-25 degrees C. Between 18.0 and 21.2 degrees C, kcat increased steadily with temperature. Between 21.2 and 21.8 degrees C, there was a large decrease in kcat. A strong increase in kcat occurred at temperatures above 21.8 degrees C, corresponding to a new reversible conformation of S1, unable to dimerise. The steep decrease in kcat between 21.2 and 21.8 degrees C is due to a temperature transition in the monomer-dimer equilibrium.     

Cell-mediated immune response in human hepatic echinococcosis. Analysis of antigen-induced lymphoproliferation and NK activity

In vitro cellular immune responsiveness was studied in 25 patients undergoing surgery for hepatic hydatid disease and in 22 matched healthy controls. Proliferation of peripheral blood mononuclear cells (PBMC) induced by phytohaemagglutinin (PHA) was not statistically different in the two groups, while proliferation induced by antigenic preparations obtained from the human commensal microorganism Candida albicans was depressed in patients as compared to healthy subjects. Confirming previous data, antigen specific proliferative response to hydatid cyst fluids was greatly enhanced in patients as compared to controls (P less than 0.01). On the other hand, natural killer (NK) activity was significantly reduced (P less than 0.005). Both impairment of NK activity and lymphoproliferation induced by commensal microorganisms suggest that patients following the parasitic infection present a condition of relevant hyporesponsiveness in cell-mediated defence.     

Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems

Summary Monoclonal antibody production kinetics in batch systems is linearly related to the integral of the viable cells curve. This favors the secretion theory over that of release by dead cells. Regression of the production curve over the integral of the viable cells curve in different media can also give information on the nutritional requirements of the cells for growth and production.     

Feasibility of a large prospective study in general practice: an Italian experience. Gruppo di Studio sulla Pressione Arteriosa nell'Anziano.

A study of blood pressure control in elderly outpatients was carried out with the participation of 444 Italian general practitioners. Of 4096 patients aged 65 years or over who were considered for recruitment, 3959 (96.7%) fulfilled all the criteria of admission and were followed up for 12 months. The findings regarding one of the aims of the study--that is, to assess the feasibility of a large scale trial in general practice--are reported. Most (87%) of the doctors completed the study. Their adherence to the protocol was highly satisfactory, leading to an acceptable quality of work. Patients'' compliance was also good; 98.6% (3898) of the patients who had fulfilled the admission criteria agreed to participate in the study, and only 4% (158) dropped out. Both of these observations support the feasibility of carrying out prospective studies in general practice. The creation of networks of general practitioners who are prepared to carry out research in their practices would allow treatment and preventive measures to be studied simply and at low cost in the appropriate setting.     

Opossum adrenal medulla: I. Postnatal development and normal anatomy

The anatomy and histology of the adrenal gland in the adult opossum were found to be typical for mammals. The development of the adrenal medulla was also found to follow the typical mammalian pattern. Primitive sympathetic cells were found in both intra- and extra-adrenal locations in the newborn at a time when chromaffin precursor cells were migrating to the adrenal anlage. Pheochromoblasts first appeared within the forming medulla where at a later stage chromaffin cells could be observed forming columns of cells between adjacent sinusoids. Unlike in other mammals, much of this development takes place postnatally when the neonate is in the mother's marsupium. The value of the developing opossum adrenal medulla as an experimental model is stressed, since a significant amount of development takes place in an environment that is accessible to experimental manipulation.     

Suppression of a polyclonal B-cell response by supernatants from the murine autologous mixed lymphocyte reaction

Previously, we have demonstrated that supernatants from autologous mixed lymphocyte (AMLR) cultures contain helper factors which can mediate the development of a cytotoxic T-cell response to hapten modified self. In the current study, the effect of AMLR supernatants on the humoral response was explored. BALB/C splenic non-T cells produced a large polyclonal antibody response to lipopolysaccharide (LPS), as measured in a Protein A SRBC plaque assay. Surprisingly, syngeneic AMLR supernatants suppressed the LPS-induced generation of plaque-forming cells. The presence of T cells in the stimulated cultures did not affect suppressor activity. The decreased response was not the result of a shift in kinetics, as maximal activity was observed on Day 4, whether or not AMLR supernatants were added. The AMLR culture supernatants were most effective in suppressing the plaque-forming cell response when added at the initiation of culture. AMLR supernatants added after 24 hr of culture resulted in only about 50% of maximum suppression. Supernatants added at 48 or 72 hr had no effect. Interferon-gamma (IFN-gamma) has been detected in AMLR culture supernatant and has been reported to suppress the development of plaque-forming cells in response to LPS. However, it is unlikely the suppressive activity observed in these studies is due to IFN-gamma. Dialysis of the AMLR culture supernatant against a pH 2 buffer for 24 hr or incubation at 70, 80, or 90 degrees C for 10 min, treatments that inactivate IFN-gamma, enhanced suppression. These results suggest that in addition to cytotoxic-T-cell helper factors, the cellular interactions in the AMLR induces the production of a stable mediator(s) which is able to directly suppress B cells at an early stage of their development into plasma cells.