Inappropriate prescribing in a large community-dwelling older population: a focus on prevalence and how it relates to patient and physician characteristics.

WHAT IS KNOWN AND OBJECTIVE: The prescription of potentially inappropriate medications (PIMs) for older adults is a well-known population health concern. Updated country-specific estimates of inappropriate prescribing in older adults using germane explicit criteria are needed to facilitate physician-tailored quality improvement strategies. Therefore, we sought to determine the prevalence of PIMs for older adults in Emilia-Romagna, Italy, using the updated Maio criteria. We also evaluated patient and general practitioner (GP) characteristics related to inappropriate prescribing. METHODS: Older adults (≥ 65) in 2012 were evaluated in a one-year retrospective study using administrative health care data. The 2011 Maio criteria includes 25 medications reimbursed by the Italian National Formulary, in the following categories in terms of severity: 16 medications that 'should always be avoided,' 3 that are 'rarely appropriate,' and 6 that have 'some indications although they are often misused.' To evaluate the extent of associations between patient and GP related characteristics, we used generalized estimating equations with an exchangeable covariance design to fit robust logistic regression models. RESULTS AND DISCUSSION: A total of 865,354 older adults were in the cohort and 28% had at least one PIM. Of the entire cohort, 8%, 10%, and 14% of individuals were prescribed at least one medication that 'should always be avoided,' is 'rarely appropriate,' and has 'some indications but are often misused,' respectively. Older patients (≥ 75) and females were more likely to be exposed to PIMs. 2,923 GPs were identified in the region, each having prescribed at least one PIM, of which older GPs (≥ 56), male GPs, and solo practice GPs were more likely to prescribe PIMs to their older patients. WHAT IS NEW AND CONCLUSION: The high prevalence of PIM exposure among older adults is a substantial issue in the region. Knowing how patient and GP characteristics relate to PIMs exposure may improve the design and targeting of initiatives for improving prescribing safety in this population.

Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice.

Cyclophosphamide (CTX) increases the antitumor effectiveness of adoptive immunotherapy in mice, and combined immunotherapy regimens are now used in some clinical trials. However, the mechanisms underlying the synergistic antitumor responses are still unclear. The purpose of this study was (a) to evaluate the antitumor response to CTX and adoptive immunotherapy in mice bearing four different syngeneic tumors (two responsive in vivo to CTX and two resistant); and (b) to define the mechanism(s) of the CTX-immunotherapy synergism. Tumor-bearing DBA/2 mice were treated with a single injection of CTX followed by an intravenous infusion of tumor-immune spleen cells. In all the four tumor models, a single CTX injection resulted in an impressive antitumor response to the subsequent injection of spleen cells from mice immunized with homologous tumor cells independently of the in vivo response to CTX alone. Detailed analysis of the antitumor mechanisms in mice transplanted with metastatic Friend leukemia cells revealed that (a) the effectiveness of this combined therapy was dependent neither on the CTX-induced reduction of tumor burden nor on CTX-induced inhibition of some putative tumor-induced suppressor cells; (b) the CTX/immune cells' regimen strongly protected the mice from subsequent injection of FLC, provided the animals were also preinoculated with inactivated homologous tumor together with the immune spleen cells; (c) CD4(+) T immune lymphocytes were the major cell type responsible for the antitumor activity; (d) the combined therapy was ineffective in mice treated with antiasialo-GM1 or anti-IFN-alpha/beta antibodies; (e) spleen and/ or bone marrow cells from CTX-treated mice produced soluble factors that assisted in proliferation of the spleen cells. Altogether, these results indicate that CTX acts via bystander effects, possibly through production of T cell growth factors occurring during the rebound events after drug administration, which may sustain the proliferation, survival, and activity of the transferred immune T lymphocytes. Thus, our findings indicate the need for reappraisal of the mechanisms underlying the synergistic effects of CTX and adoptive immunotherapy, and may provide new insights into the definition of new and more effective strategies with chemotherapy and adoptive immunotherapy for cancer patients.

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice.

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.

The fine tuning of metabolism, autophagy and differentiation during in vitro myogenesis.

Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation.

High frequency of antithyroid autoantibodies in pregnant women at increased risk of gestational diabetes mellitus.

BACKGROUND: Thyroid autoantibodies (ThyAb) and subclinical hypothyroidism occur more frequently in pregnant women with insulin-dependent diabetes mellitus than in healthy pregnant women. Few studies have investigated the presence of ThyAb in women with gestational diabetes mellitus (GDM), and no significant association between diabetes in pregnancy and thyroid function has been reported. OBJECTIVE: To assess the thyroid biochemical profile and estimate the prevalence of ThyAb in a group of pregnant women at increased risk of GDM due to family and personal risk factors, and to investigate the relationship between a positive family history of diabetes or thyroid diseases and the eventual presence of ThyAb during pregnancy. METHODS: Oral glucose tolerance, serum ThyAb and thyroid function were evaluated in 181 pregnant women with increased risk for GDM (study group). Seventeen healthy pregnant women without risk factors for GDM and with a normal glucose tolerance were recruited as controls. RESULTS: The women who developed GDM showed a mean free thyroxine concentration significantly lower than that observed in the healthy pregnant women and in those with impaired gestational glucose tolerance and normal glucose tolerance. Twenty-nine of the 181 women in the study group (16%) were ThyAb positive. However, the risk of being ThyAb positive during pregnancy was three times greater in the women with positive family history of both diabetes mellitus and thyroid disease than in those with no family history of these conditions. CONCLUSIONS: This study showed that women with increased risk of GDM, mostly those with family history of diabetes mellitus and thyroid disease, also have an increased risk of being ThyAb positive during pregnancy. It also highlighted the importance of evaluating thyroid function in pregnant women with impaired glucose tolerance, in view of their increased risk of subclinical hypothyroidism.

Detection of phosphatidylcholine-specific phospholipase C in NIH-3T3 fibroblasts and their H-ras transformants: NMR and immunochemical studies.

Although evidence supports constitutive activation of phosphatidylcholine specific phospholipase C (PC-plc) in rastransformed fibroblasts, no studies have been devoted to measure the basal activity levels of this enzyme, its molecular characteristics and subcellular localization. This paper reports for the first time measurements of the activity of different enzymes responsible for PC hydrolysis (PC-plc; phospholipases A2 (pla2) and A1 (pla1)) in homogenates of murine NIH-3T3 fibroblasts (3T3) and their transformants obtained by human H-ras transfection (3T3ras). To this end, 31P NMR analyses were carried out on total cell homogenates, incubated in the presence of mixed diheptanoylphosphatidylcholine: sphingomyelin (DHPC:SM) unilamellar vesicles (SLUV), in which DHPC acts as a suitable substrate for water-soluble lipolytic enzymes. The basal PC-plc activity levels (0.66 +/- 0.14 and 0.38 +/- 0.10 nmol/10(6) cells.hour in 3T3 and 3T3ras fibroblasts, respectively),were substantially higher (over 30-50x) than those reported in the literature for normal mammalian cells (dog heart myocytes). Moreover the PC-plc activity was about 15-30 times lower than the overall PC deacylation activity in both clones. The use of high titer polyclonal antibodies, raised in a rabbit against bacterial PC-plc, allowed identification of one cross-reactive mammalian PC-plc component (M(r) 66 kD) in cell lysates of both 3T3 and 3T3ras fibroblasts, and detection, by indirect immunofluorescence, of its subcellular localization. In control 3T3 fibroblasts (in the late log-phase of growth) the enzyme was exclusively located in the cytosol, while in H-ras transformed cells it was massively exposed on the external side of the membrane. This new finding strongly suggests that the oncogenic product p2Iras is able to induce (or mediate) translocation of PC-plc across the plasma membrane of ras transformed cells, with possible implications not only on cell biochemistry (enhancement of PC-plc activity, and consequent production of intra- and extracellular PCho and accumulation of neutral lipids) but also on cell-cell interaction mechanisms which facilitate tumour invasion and metastasis of oncogene-transformed cells.

[Tubercular Addison's disease with high titers of microsomal thyroid antibodies and reduced thyroid function reserve]. / Morbo di Addison tubercolare con elevato titolo di anticorpi antimicrosomiali tiroidei e ridotta riserva funzionale della tiroide.

This case reports deals with a male patient, white, aged 51, bricklayer, tobacco smoker. At 43 years old a tubercular epididimite was surgically treated with orchiectomy. Recently, the reports marked weakness, weight loss, headache, vertigo, hypotension. On admittance to the hospital, hyperpigmentation was also present. Previous therapy (cortisone acetate 40 mg/die), was suspended, clinical investigations showed impaired adrenal and thyroidal functions; antimicrosomal antibodies were also present. Therefore therapy with cortisone acetate (25 mg x 2/die, 9-alpha-fluorohydrocortisone 0.1 mg/die and L-thyroxine 100 mcg/die) was instituted with marked improvement of the patient's conditions. This case reports highlights the importance of proceeding with complete investigations on the system, even when dealing with a tubercular Addison's disease.

[ANP in the cirrhotic patient. A clinical contribution]. / L'ANP nel paziente cirrotico. Contributo clinico.

Pathogenesis of ascites in patients affected by liver cirrhosis is still debated; humoral and haemodynamic factors can play a role. Plasmatic renin activity (PRA), plasmatic aldosterone (PA), atrial natriuretic peptide (ANP) plasma levels, blood Na, K, urea, urinary K and Na were evaluated in 14 patients affected by liver cirrhosis (11 males and 3 females, aged from 38 to 62 years), 8 of them with ascites. The results were compared with those obtained in a control group poised to age and sex to the experimental group. 4 out of 14 patients suffering from ascites unresponsive to medical treatment were submitted to peritoneal venous jugular shunt (PVGS) and blood samples for PRA, PA and ANP were withdrawn immediately before, 4, 8 hours following surgery. The patients affected by liver cirrhosis without ascites showed PRA and PA levels similar to those observed in the control group, while ANP plasma levels were significantly higher (50.6 + 9.6 vs. 39.7 + 9.5 Pg/ml) (p < 0.02). In patients with ascites ANP, PA and PRA levels were higher than those observed in non ascites patients (ANP = 147.8 + 97.3 vs. 50.6 + 9.6 pg/ml; PA = 20.6 + 2.7 vs 7.8 + 0.8 ng/dl; PRA = 4.48 + 0.5 vs 1.9 + 0.34 ng/ml/h). In patients submitted to PVGS, PA and PRA levels were reduced 4 and 8 hours following the surgery, while ANP levels showed significant increase. A natriuretic and diuretic response has been observed even in the absence of ANP plasma levels variations.(ABSTRACT TRUNCATED AT 250 WORDS)

[Behavior of serum angiotensin converting enzyme in hyperthyroidism correlated to that of TSH]. / Il comportamento del serum angiotensin converting enzyme nell'ipertiroidismo correlato a quello del TSH.

The study examined the relations between serum levels of angiotensin-converting enzyme and those of thyroid-stimulating hormone in a group of hyperthyroid patients, and the respective therapy. The study continued for 12 months, from the onset of disease until remission. From an analysis of the results it was seen that levels of SACE and TSH during the first 4 months were significantly different to those in normal subjects: levels of SACE were increased, whereas TSH levels had fallen. This difference gradually diminished over the course of the following 8 months, and SACE and TSH values returned to within normal limits. The increment of SACE may depend on the presence of damage to the vascular endothelium within and surrounding the thyroid, in addition to intense vasodilation caused by the hyperthyroid state. The correlation between increased SACE levels and the course of disease is further confirmed by the fact that levels returned to within normal values during thyrostatic therapy; this was also observed in the case of TSH.

[Syndrome X and surgical stress. A clinical case]. / Sindrome X e stress chirurgico. Caso clinico.

The syndrome X is a clinical disease characterised by anginous pain with the absence of significant and angiographically visible stenosis of the coronary tree. D. P. M., a 61-year-old woman suffering from biliary lithiasis, underwent cholecystectomy. During the immediate postoperative period, the patient showed difficulty in regaining consciousness and there were electrocardiographic signs of extensive anterior ischemia; prior to the operation only a 1st degree atrio-ventricular block and a positive history of occasional precordial pain had been reported. On the 2nd postoperative day the patient complained of violent retrosternal pain irradiated to the left shoulder. Given that the signs of ischemia had regressed, various instrumental tests were performed: echocardiogram, cycloergometric test, dipyridamole test, cold pressure test, Holter's dynamic ECG, all of which were within the normal; moreover, selective coronarography did not reveal significant stenosis of the coronary tree. The patient was therefore diagnosed as suffering from syndrome X. In the light of the present case, the authors conclusion may be summarised as follows: the diagnosis of syndrome X, which is by definition not easy, may sometimes become critical, as in the present case, since rapid intervention would have enabled prophylactic therapy to be performed to combat surgical stress.

Stable transfection of provirus of human immunodeficiency virus into a murine packaging cell line.

In order to generate HIV (murine leukemia virus (MuLV)) pseudotypes, HIV genome was transfected into the ecotropic murine packaging cell line (GP+E86) and four of the nine transfected clones were extensively characterized. One clone (801), harbouring a full copy of integrated HIV sequences, exhibited a detectable level of intracellular HIV p24 antigen expression. Northern blot analysis revealed that clone 801 expressed all three classes of HIV mRNAs. Multispliced 2 kb mRNAs were detected in another clone (8.14). Two other clones (1.31 and 1.32) also exhibited a complete HIV provirus, but did not show any viral expression, as evaluated by Northern blot analysis or HIV p24 ELISA. Reverse transcription-polymerase chain reaction (RT-PCR) experiments revealed the presence of full length genomic RNA in four transfected clones, which were extensively characterized. A co-cultivation of clone 801 with human CD4' cells resulted in syncytia formation. By electron microscopy, mature HIV particles were observed after co-cultivation of uninfected C8166 cells with 801 cells. These results demonstrated that the murine clone was stably transfected with the complete HIV genome and was capable of shuttling infectious HIV to human cells. Clone 801 was co-cultivated with murine NIH-3T3 fibroblasts. In several experiments, HIV infection of NIH-3T3 cells was revealed by PCR technique. Thus, 801 cells appear to produce low levels of HIV (MuLV) pseudotypes capable of transferring the HIV genome into mouse cells.

Thyroid autoimmunity in pregnant women at risk for GDM.

Presence of antithyroid autoantibodies (ThyAb) during pregnancy is strictly related to the risk of developing post partum thyroiditis (PPT) and this risk is increased in IDDM pregnant women. Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance of variable severity that begins, or is first diagnosed, during pregnancy. GDM is considered a risk factor for both type 1 and type 2 diabetes and various non-organ specific autoantibodies have been found to be associated with GDM, although there is little information on the association of GDM with thyroid autoimmunity. In this study oral glucose tolerance and prevalence of ThyAb were evaluated in a group of 41 pregnant women at increased risk of developing GDM and in a healthy control group. Our results showed that 22% of GDM risk group had impaired glucose gestational tolerance (IGGT) or GDM at the time of oral glucose tolerance test (OGTT). Moreover, ThyAb prevalence found in the women at increased risk of GDM (14.6%) was similar to that observed in healthy pregnant controls (12.5%). Nevertheless ThyAb frequency was higher in those GDM risk women with family history of diabetes (30.7%).

[Cigarette smoking and hormonal changes in the course of the menstrual cycle]. / Fumo di sigaretta e modificazioni ormonali nel corso del ciclo.

ACTH, cortisol and PRL plasma levels were determined on day 7, 14, and 21 of the menstrual cycle in 20 females with hypertrichosis of whom 10 were smokers and 10 non-smokers. Significantly raised levels of all hormones considered were found in smokers throughout the cycle. These findings suggest a possible relationship between objective symptoms (hypertrichosis) and nicotine, mediated by complex changes in the endocrine pattern and presumably also due to the action of nicotine on the central nervous system.

Carbohydrate-recognition and angiogenesis.

Angiogenesis is required for the continual growth of the tumor and provides a gateway for cells to escape the confines of the primary tumor. Angiogenic stimulus triggers a cascade of functional responses leading to local basement membrane dissolution, endothelial cell migration, proliferation and microvessel morphogenesis. In this commentary, we review the significance of carbohydrate-binding proteins involved in angiogenesis. The importance of carbohydrate-recognition processes to angiogenesis stems from the observation that angiogenic factors like fibroblast growth factor family and vascular endothelial growth factors bind initially to the extracellular matrix proteoglycans before binding to their cognate receptors, and some of the adhesion molecules bind to glycoconjugates present on the surface of the endothelial cells. The possible significance of these interactions will be discussed.

[Effect of smoking on the hypophyseo-adrenal axis]. / Effetto del fumo sull'asse ipofisi-surrene.

Plasma concentrations of adrenocorticotrophin (ACTH) and cortisol were measured during insulin-induced hypoglycemia and lysin-8-vasopressin (LVP) test in 60 healthy subjects, non-smokers and habitually smokers of 10 or more cigarettes/24 hours. A marked and statistically significant rise of both hormones was found in non-smoker subjects, whereas smokers showed poor and not significant modifications. These results suggest that continuous chronic inhalation of nicotine may act as a powerful stimulus on the hypothalamo-hypophyseal structures that control the hypothalamic CRF and/or ACTH production and release. Central nervous mechanisms of hormonal regulation may become less sensitive and efficient when an acute rise of ACTH is required, as during stimulating tests. Our investigation confirms that cigarette smoking is heavily responsible of endocrine disorders, in particular of hypophyseal-adrenal axis.

[Changes in the levels of ACTH and cortisol after passive exposure to cigarette smoke in smokers and non-smokers]. / Modificazioni dei livelli di ACTH e cortisolo dopo esposizione passiva al fumo di sigaretta in fumatori e non fumatori.

Plasma ACTH and cortisol levels were studied in smokers and non smokers, (exposed or not to smoke of the environment), after passive exposure to cigarette smoking. Non smokers, usually not exposed to smoke, show a rise in both hormones, whereas smokers and non smokers commonly exposed to smoke don't show any change in ACTH and cortisol levels. These data suggest that nicotine acts as an acute stimulus on the hypophysis-adrenal axis even passively inhaled.

[Oral administration of nicotine and plasma levels of ACTH and cortisol in smokers and non-smokers]. / Somministrazione orale di nicotina e livelli plasmatici di ACTH e di cortisolo in fumatori e non fumatori.

Plasma ACTH and cortisol levels after oral administration of nicotine (chewing gum containing nicotine 2 mg) in short and long time (10 and 45 min) were studied in smokers and non smokers. Non smokers after short time administration showed significant rise in ACTH and cortisol. No modifications were seen in the other groups of subjects. These data confirm that nicotine stimulates hypophysis-adrenal axis in non smokers and that this effect is connected with nicotine contents of cigarettes rather than other volatile substances.

[ACTH and cortisol after cigarette smoke exposure during the dexamethasone suppression test in smokers and non-smokers]. / Studio dell'ACTH e del cortisolo dopo fumo di sigaretta, nel corso della prova di inibizione con desametasone, in fumatori e non fumatori.

In this research the effect of nicotine, (smoke of cigarette), was studied in smokers and non smokers during dexamethasone inhibitory test (1 mg h 23.00). ACTH and cortisol plasma levels, physiologically suppressed at 08.00, increased, after 30 min from smoking, only in group of non smokers. These data suggest that nicotine, in non smokers, could induce a maximum stimulus on diencephalic structures, so to overcome the inhibition of dexamethasone.

[Modification of left ventricular filling after acute administration of nifedipine in hypertensive patients. Doppler echocardiographic study]. / Modificazioni del riempimento ventricolare sinistro dopo somministrazione acuta di nifedipina in pazienti ipertesi. Studio eco-Doppler.

This study was undertaken to determine the acute effects of nifedipine on left ventricular filling, examined by Doppler echocardiography in patients with arterial hypertension and normal left ventricular systolic function. Twenty-seven patients with mild to moderate systemic hypertension, and 18 normotensive subjects were studied. Pulsed Doppler, M-mode and B-mode measurements were performed before and 30 min after sublingual nifedipine administration (10 mg). Peak E, peak A, area A, area E, ratio area A/area E, E deceleration and E acceleration time were measured. Before administering nifedipine, some of these diastolic filling parameters were significantly different in the hypertensive patients as compared with normal subjects. After nifedipine, in patients with arterial hypertension, peak E and area E increased significantly (p less than 0.01), while ratio peak A/E and ratio area A/area E showed a significant decrease (p less than 0.001). No significant changes were observed in normal subjects. The result of this study demonstrates that the acute administration of nifedipine modifies transmitral flow velocity pattern in hypertensive patients, suggesting that the left ventricular filling abnormalities are in part dynamic and reversible.

Site-specific anti-c-erb A antibodies recognizing native thyroid hormone receptors: their use to detect the expression and localization of alpha and beta c-erb A proteins in rat liver.

The cell-specific expression and tissue distribution of c-erbA proteins alpha and beta is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the alpha or beta form of thyroid hormone receptors. The cDNAs coding for h c-erbA beta 1, alpha 1 and alpha 2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-beta 62-81 immunoprecipitated only the beta 1 receptor. The antiserum anti-alpha 144-162 determined precipitation of both alpha 1 and alpha 2 proteins but not of the beta 1 receptor. Anti-alpha 2 431-451 produced a selective precipitation of alpha 2, and had no effect on alpha 1 or beta 1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-beta 62-81 and anti-alpha 144-162 caused a partial shift of the labeled T3-receptor complex to a higher molecular form, while the antibody directed against c-erbA alpha 2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.