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Additional file 1: Appendix 1. Overview of the statements and panel scores for every Delphi round.

Our objectives were to determine (1) the effect of a single dose of an oral Ca bolus within 24 h after parturition on plasma Ca concentration, (2) the response of primiparous (PP) and multiparous (MP) cows to this supplementation strategy, and (3) differential responses based on plasma Ca at enrollment. For objective 1, cows from 1 commercial dairy in New York State were enrolled within 19 h after parturition (mean ± standard deviation = 8.3 ± 5.3 h) and randomized within parity group (first, second, and ≥third) to control [CON (n = 25); no placebo] or a single dose bolus treatment [BOL (n = 25); 3 oral Ca boluses supplying 54 to 64 g of Ca]. Plasma Ca was measured repeatedly between 1 and 24 h following treatment. For objectives 2 and 3, cows on 6 commercial farms in New York State were assigned to treatment as for objective 1 (CON, n = 1,973; BOL, n = 1,976). Herd records for health, reproduction, and Dairy Herd Improvement Association test day milk production were collected. Mixed effect multivariable models were developed using repeated measures ANOVA, Poisson regression, or proportional hazard models. Objective 2 analyses considered treatment with periparturient risk factors, whereas objective 3 analyses also considered Ca status. No difference was observed for plasma Ca between 1 and 24 h after treatment. Primiparous cows assigned to BOL calving at >712 d old had decreased risk of one or more health disorders [≤30 d in milk; risk ratio (RR) = 0.65, 95% confidence interval (CI) = 0.51 to 0.84] and those with body condition score >3.5 responded to BOL with increased milk production (CON = 31.7 ± 1.1, BOL = 35.1 ± 1.1 kg/d), as did those with days carried calf >277 (CON = 31.9 ± 1.0, BOL = 34.7 ± 1.0 kg/d). Reduced risk of one or more health disorders was observed in parity ≥3 (RR = 0.85, 95% CI = 0.81 to 0.89) and MP cows with body condition score >3.5 (retained placenta; RR = 0.70, 95% CI = 0.58 to 0.84) or that were lame (displaced abomasum; RR = 0.49, 95% CI = 0.32 to 0.75). Differential responses for PP cows by Ca status were minimal. For MP cows with low plasma Ca, BOL decreased risk of additional Ca treatment (≤1.8 mmol/L; RR = 0.57, 95% CI = 0.40 to 0.80) as well as risk of one or more health disorders (≤2.15 mmol/L; RR = 0.90, 95% CI = 0.85 to 0.95). Supplementation with a single oral dose of Ca could be targeted to periparturient risk groups for improved health. Calcium status did not differentiate responses of PP cows, but MP cows with low Ca at parturition had improved health status when supplemented.

The objective of this study was to evaluate the association of postpartum plasma Ca concentration with early-lactation disease outcomes, culling within 60 d in milk, pregnancy to first service, and milk production. A total of 1,453 cows from 5 commercial dairy farms in New York State were enrolled in a prospective cohort study from February to November 2015. Blood samples were collected within 12 h of parturition, and plasma was submitted to a diagnostic laboratory for total Ca measurement. Early-lactation disease, reproductive performance, and milk production from Dairy Herd Improvement Association (DHIA) test-day data were compiled from each farm's management software. Multivariable Poisson regression models were built to evaluate the association of plasma Ca with the risks of retained placenta (RP), metritis, displaced abomasum (DA), clinical mastitis, culling within 60 d in milk, and pregnancy to first service. Repeated-measures ANOVA were used to evaluate the association of Ca at parturition with milk production across the first 9 DHIA tests. Herd was considered a random effect in all models. Primiparous cows were modeled separately from multiparous cows if differential responses were observed. Calcium was not associated with the risk of RP, metritis, clinical mastitis, or pregnancy to first service in primiparous or multiparous cows. For multiparous cows only, higher Ca concentration tended to be associated with increased culling within the first 60 d in milk. Multiparous cows with Ca ≤1.85 mmol/L had an increased risk of being diagnosed with a DA compared with cows with Ca >1.85 mmol/L. For the milk production models, Ca was not associated with the amount of milk produced within the first 9 DHIA tests in primiparous cows; however, multiparous cows with Ca ≤1.95 mmol/L produced, on average, 1.1 kg more milk per day across the 9 DHIA tests than their multiparous counterparts with Ca >1.95 mmol/L. Our results indicate that plasma Ca concentration measured within 12 h of parturition is a poor predictor of early-lactation health outcomes. Reduced Ca concentration in the immediate postpartum period was associated with higher milk production in multiparous cows. From these results, we caution that studies attempting to categorize subclinical hypocalcemia based on a single sample in the immediate postpartum period could misclassify the disorder.

A retrospective analysis of case files registered over 4 years, revealed eight cases with delusion of parasitosis. The onset, clinical features and types of referral are discussed. This rare disorder which responds readily to pimozide should be studied in greater detail.

In 2004 is in het kader van het programma Resultaten Scoren de ‘Richtlijn Detox: verantwoord ontgiften door ambulante of intramurale detoxificatie’ opgesteld (Resultaten Scoren, 2004). De Richtlijn Detox biedt een basis voor het handelen van arts en verpleegkundige. IrisZorg

Objective: To analyse the relation between antioxidant vitamins A, E, and malondialdehyde (MDA) lipoperoxidation product plasma concentrations with incident dementia. Design: A nested case–control within the PAQUID (Personnes Agees QUID) cohort. Setting: The PAQUID population-based prospective cohort in southwestern France. Subjects: Among 626 subjects with blood collection at baseline, 46 developed a dementia during the follow-up and were considered to be cases. Each case was matched (on age and sex) to three controls. Results: Plasma vitamin E concentrations were lower among cases (mean value at 22.62 μmol/l (s.d.: 7.38) vs 24.99 (s.d.: 6.73 among controls). The same trend was observed for vitamin A concentrations, but the difference was not significant. On the contrary, MDA concentrations tended to be higher (mean value 1.35 μmol/l (s.d.: 0.53) vs 1.23 (s.d.: 0.44)) among cases. In logistic regression models, plasma values were split into tertiles. Adjusted for confounders, the risk of dementia was significantly increased in the lowest vitamin E tertile (≤21.0 μmol/l) (OR=3.12, P=0.033) compared to the highest one (≥25.5 μmol/l). The risk of Alzheimer's disease was also increased, with borderline significance (OR=3.06, P=0.053). Risks associated with vitamin A were nonsignificant. Similarly, there was a trend to an increased risk of dementia in the highest tertile of MDA (OR=1.67, P=0.31). Conclusions: These results suggest that subjects with low plasma vitamin E concentrations are at a higher risk of developing a dementia in subsequent years.

Summary Objective To explore demographic and clinical factors associated with radiographic severity of first metatarsophalangeal joint osteoarthritis (OA) (First MTPJ OA). Design Adults aged ≥50 years registered with four general practices were mailed a Health Survey. Responders reporting foot pain within the last 12 months were invited to undergo a clinical assessment and weight-bearing dorso-plantar and lateral radiographs of both feet. Radiographic first MTPJ OA in the most severely affected foot was graded into four categories using a validated atlas. Differences in selected demographic and clinical factors were explored across the four radiographic severity subgroups using analysis of variance (ANOVA) and ordinal regression. Results Clinical and radiographic data were available from 517 participants, categorised as having no (n = 105), mild (n = 228), moderate (n = 122) or severe (n = 62) first MTPJ OA. Increased radiographic severity was associated with older age and lower educational attainment. After adjusting for age, increased radiographic first MTPJ OA severity was significantly associated with an increased prevalence of dorsal hallux and first MTPJ pain, hallux valgus, first interphalangeal joint (IPJ) hyperextension, keratotic lesions on the dorsal aspect of the hallux and first MTPJ, decreased first MTPJ dorsiflexion, ankle/subtalar joint eversion and ankle joint dorsiflexion range of motion, and a trend towards a more pronated foot posture. Conclusions This cross-sectional study has identified several dose–response associations between radiographic severity of first MTPJ OA and a range of demographic and clinical factors. These findings highlight the progressive nature of first MTPJ OA and provide insights into the spectrum of presentation of the condition in clinical practice.

We have previously shown that medroxyprogesterone acetate (MPA), either alone or combined with conjugated equine estrogens (CEE), significantly decreased insulin sensitivity (SI), compared with both untreated controls and those treated with CEE alone. The purpose of this study was to determine the effects of estradiol (E2), with and without nomegestrol acetate (NA; a potent progestin that lacks androgenic activity), on SI and arterial antioxidant activity, as determined by F2-isoprostanes. Thirty-six adult female cynomolgus monkeys (Macaca fascicularis) were ovariectomized and fed a moderately atherogenic diet, with one of the following three treatments added to the diet, for 12 weeks: 1) no treatment (control); 2) E2; or 3) continuous combined E2 + NA (E2+NA). SI and glucose effectiveness were assessed by the frequently sampled i.v. glucose tolerance test using a third-phase insulin infusion after 10 weeks of treatment. Cholesterol content and F2-isoprostanes were measured in the thoracic aorta after 12 weeks of treatment. E2 treatment resulted in a significantly greater SI, compared with control or E2+NA-treated monkeys (10.03 +/- 0.91 vs. 6.35 and 6.49 x 10(-4) min(-1) microU(-1) mL; P < 0.05). In contrast to our studies of CEE and MPA, E2+NA treatment, though reducing the SI below that of the E2 group, did not reduce the SI below that of control monkeys. As expected, the short period of treatment resulted in no significant differences in aortic cholesterol content. There was no treatment effect on total F2-isoprostanes (representing F2-isoprostane formation caused primarily by autooxidation), suggesting minimal antioxidant activity. However, there was a treatment difference in the prostaglandin F2alpha (PGF2alpha) isomer (a prostaglandin (PG) isomer formed by both autooxidation of arachidonate and cyclooxygenase activity). PGF2alpha concentrations were 32% lower with E2 treatment, compared with controls, and 36% lower, compared with E2+NA treatment (0.48 +/- 0.08 vs. 0.71 +/- 0.12 and 0.75 +/- 0.06; P < 0.05), suggesting differences in PG synthesis between hormone treatments. In conclusion, NA, a progestin without androgenic activity, may still affect some cardiovascular risk factors differently than estrogen-only therapy. However, it seems to be less detrimental than MPA.

To determine whether leptin has insulin sensitizing effects in normal rodents, we measured plasma glucose and insulin concentrations in male Sprague-Dawley rats treated with leptin or vehicle by continuous sc infusion for 48 h. In additional experiments, we examined the acute effect of iv leptin upon insulin sensitivity under conditions of clamped glycemia. Subcutaneous leptin was administered at 10.0 and 1.0 μg/h. To avoid confounding effects of differences in food intake, both leptin- and vehicle-treated rats were fasted during the 48-h period of infusion. Infusion of leptin, 10 μg/h, significantly reduced both plasma glucose and insulin. Leptin, 1.0 μg/h, also decreased plasma glucose and insulin, although the effects on insulin did not achieve statistical significance. Leptin at either dose did not alter body weight or epididymal fat mass compared with vehicle treated controls. Leptin, 10 μg/h, decreased circulating insulin-like growth factor-1 levels. No differences in GLUT-4 content in either in brown or epididymal fat were observed as a result of leptin-treatment. Leptin, 10 μg/h, significantly decreased urine osmolality, increased water intake, and reduced renal potassium excretion compared with vehicle-infused rats. In additional rats, we measured the acute effect of iv leptin on insulin sensitivity determined as whole body glucose utilization during hyperinsulinemic glucose clamps performed at glucose targets of 60 and 90 mg/100 ml. Glucose utilization was increased by 29% during the last 135 min of glycemia clamped at 60 mg/100 ml (P < 0.05) and by 30% during the last 135 min of glycemia clamped at 90 mg/dl (P < 0.01) in rats infused with leptin compared with vehicle. In summary, leptin increased insulin sensitivity in normal rats both under fasting conditions and in the presence of hyperinsulinemia at clamped glucose. These effects did not appear dependent on altered body weight. Leptin also altered salt and water metabolism under fasting conditions resulting in increased water intake and more dilute urine.

Increased peroxidation of lipids in red blood cells (RBC) in patients with advanced chronic renal failure (CRF) reflects increased generation of reactive oxygen species (ROS), which may contribute to the metabolic damage induced by CRF and to its progression. We have evaluated parameters indicative of lipoperoxidation (LPO) of RBC at baseline in patients with CRF compared to controls, and the effects of a very low protein diet supplemented with amino and keto acids and vitamins A, C, E (VLPD) over a 6-month period. The presence of peroxidation damage in CRF patients before the administration VLPD was demonstrated by elevated levels of free malondialdehyde (MDA) (p.0003) and decreased levels of polyunsaturated fatty acids (PUFA), particularly C20:4 (p.001), C22:4 (p.0001) and C22:5 (p.0001) when compared to controls. Similarly, RBC vitamin E content was significantly decreased (p.0001) while enzymatic activities were unalterated. VLPD reduced erythrocyte LPO as suggested by (a) decreased levels of free and total RBC MDA (p.003 and p.03, respectively), (b) increased levels of PUFA, particularly C22:4 and C22:5 (p.003 and p.03, respectively), and (c) increased levels of vitamins A and E (p.001 and p.04, respectively) as compared to prediet results. Antioxidant enzyme activities were not modified. These results suggest that VLPD has a protective role against LPO of erythrocytes in patients with CRF.

Faces with threatening versus positive expressions are better remembered in visual working memory (WM) and are especially effective at capturing attention. We asked how the presence of a single threatening or happy face affects WM for concurrently viewed faces with neutral expressions. If threat captures attention and attention determines WM, then a WM performance cost for neutral faces should be evident. However, if threat boosts processing in an object-specific, noncompetitive manner, then no such costs should be produced. Participants viewed three neutral and one angry or happy face for 2 s. Face recognition was tested 1 s later. Although WM was better for singletons than nonsingletons and better for angry versus happy singletons, WM for neutral faces remained unaffected by either singleton. These results, combined with eye movement and response time analyses, argue against a selective attention account of threat-based benefits to WM and support object-specific enhancement via threat processing.

Rheumatoid factor is a commonly requested test in the investigation of joint problems. Although the test has been found to be both sensitive and specific for rheumatoid arthritis in patients attending Rheumatology Clinics, when the test is applied to general hospital or community populations it performs poorly. Thus the setting in which rheumatoid factor tests are requested and awareness of the test’s limitations are critical for appropriate use and interpretation of results. We studied 295 consecutive requests for testing at four centres. The majority were performed for the investigation of joint problems but only 6% of these were positive, suggesting unselective requesting. Half of the results were used to make diagnoses and influence management. However, significant numbers of clinicians felt that the test result had either excluded or confirmed a diagnosis of rheumatoid arthritis suggesting that results were being over-interpreted and that clinical decisions made on this basis may have been inappropriate. We conclude that in this setting rheumatoid factor testing is of limited value and generates misleading information.

Insulin-like growth factor-I (IGF-I) is a highly conserved 70-residue circulating peptide that mediates many of the systemic growth-promoting effects of GH. This laboratory has found previously that GH rapidly stimulates hepatic IGF-I transcription in hypophysectomized (hypox) rats by activating promoter 1, the major rat IGF-I gene promoter. In this study, the hormonal regulation of IGF-I expression through promoter 2, a minor promoter in most tissues but active in the liver, was investigated. Through use of a sensitive RNase protection assay, GH was shown to rapidly induce the accumulation of correctly initiated transcripts directed by this promoter in hepatic nuclei. Using in vitro DNase-I footprinting, six DNA-protein interactions were identified within promoter 2 with hepatic nuclear extracts from juvenile male hypox rats given a single ip injection of GH or saline 60 min before death. These DNA-protein-binding complexes also were investigated for specificity and for regulation by GH by gel mobility shift assays. All DNA-protein interactions were detected in hepatic nuclear protein extracts from hypox rats and did not change within 15-120 min after GH treatment. These results thus identify and characterize a series of constitutive nuclear protein-binding sites within the second rat IGF-I promoter that may be involved in mediating its transcriptional activity.

Lipid peroxidation is believed to be involved in malaria and plasma of malaria patients may be damaged by reactive oxygen soecies This study compares determinations of malondialdehyde ifiDA), vitamins A and E by high performance liquid chromatography, thiobarbituric acid reactive substances (TBA-RS) by fluorometric analysis and fatty acids by gas chromatography. During malarial infection, polyunsaturated fatty acids (PUFA), vitamins A and E decreased significantly when TBA-RS increased significantly. However, there was no significant change >n total or MDA bound to amino-groups. On the other hand, there was no correlation between TBA-RS production and PUFA decrease or between TBA-RS production and vitamin A or E consumption. These results support the concept that other compounds such as proteins and cholesterol may be involved in the formation of these TBA-RS.

In 14 patients undergoing haemodialysis, lipoperoxidation (LPO) processes were determined in plasma and red blood cells (RBC) before and after a dialysis session by determining (a) the direct substrate, polyunsaturated fatty acids (PUFA); (b) the end product of LPO, malondialdehyde (MDA); and (c) the hydrophobic antioxidant systems, vitamins A and E. In plasma before dialysis, linoleic and arachidonic acid, and the antioxidant vitamin E, were significantly lowered as compared to the healthy controls (p < 0.05). On the contrary, the free MDA level was enhanced (p < 0.05). These results were emphasized by a dialysis session. In RBC of these patients, no difference in linoleic acid, free MDA, or vitamin E level were observed before or after dialysis when compared to controls. However, only vitamin A was significantly higher in haemodialysis patients (before and after dialysis) and in renal failure patients (p < 0.05) than in the healthy control group. The present results suggest that increased RBC vitamin A may offer some degree of protection against oxidative stress in erythrocytes, but not in plasma where LPO is demonstrated.

The complete DNA sequence of the 1.7 kilobase pairs (kbp) 5' of the rat growth hormone gene (rGH) has been determined and analyzed for Z-DNA-forming potential. Regions of alternating purine-pyrimidine (APP) sequences located between -1047 and -986 [(GT)31], between -445 and -433 bp, and between -426 and -403 bp relative to the rGH RNA transcription initiation site were identified and shown to form Z-DNA in negatively supercoiled plasmids by two-dimensional gel electrophoresis. Free-energy calculations indicated that Z-DNA forms most readily in the proximal Z-DNA regions. Diethyl pyrocarbonate footprinting of physiologically supercoiled plasmid DNA confirmed the presence of Z-DNA from -444 to -404 bp spanning the two most proximal APP sequences and a short non-APP sequence in between. DNA sequence analysis also predicted a region of DNA curvature near this proximal Z-DNA region. Formation of Z-DNA in the distal Z-DNA region consisting of a (GT)31 repeat was constrained at physiological plasmid superhelical densities. This may be related to the presence of DNA sequences (-1584 to -1559) 512 bp upstream of (GT)31 that undergo cruciform formation and thereby utilize the available free energy. Removal of 580 bp containing the cruciform region resulted in Z-DNA formation within (GT)31, thus demonstrating that deletion mutations can exert topological changes at a distance within the rGH 5'-flanking region. Methylation of two specific cytosines in the rGH 5'-flanking DNA that have been associated with inhibition of rGH promoter activity had no effect on Z-DNA formation. No evidence for DNA secondary structure formation within the rGH second exon-intron or 3'-flanking region was observed. We conclude that the rGH 5'-flanking region undergoes secondary-structure formation at physiological superhelical densities, thus providing a potential mechanism(s) for modulating rGH activity.

A 37 year old white South African woman presented with a two week history of fevers, rigors, and watery diarrhoea up to six times a day. She had a complex past medical history, developing insulin dependent diabetes mellitus aged 2 years and hypothyroidism aged 11 years. At 13 years she was diagnosed with idiopathic thrombocytopenic purpura (ITP) and two years later underwent splenectomy, with a partial response. At 25 years, she developed haemolytic anaemia. Initial treatment with azathioprine was withdrawn owing to thrombocytopenia and she was subsequently treated for three years with oral cyclophosphamide. On moving to the United Kingdom, treatment was changed to a combination of danazol, hydroxychloroquine, and 10–30 mg oral prednisolone a day. At the age of 33 she was diagnosed with primary antibody deficiency on the basis of recurrent pneumonia, autoimmune disease, borderline low and falling total IgG, low IgG2 subclass, and failure to produce a specific IgG response to tetanus and pneumovax immunisations. Intravenous immunoglobulin replacement treatment was started In the six years before the above presentation she had also described intermittent watery diarrhoea, controlled with loperamide and on several occasions remitted when the steroid dose for ITP was increased. Stool culture and parasitology, abdominal ultrasound scan, gastroscopy and duodenal biopsy, flexible sigmoidoscopy, barium meal and follow through, and barium enema were all normal. Biopsies of the rectum and sigmoid colon showed a chronic inflammatory cell infiltrate compatible with immunodeficiency. Other problems included hypoadrenalism attributed to either prolonged steroid use or autoimmune disease, and …

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.

Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling. Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT.

Differentiation of developing T cells into the type 1 (IFN-gamma-producing) or type 2 (IL-4-producing) subsets is a central theme of immune regulation. The balance of IL-4 and IL-12 present during T cell activation has been considered the major influence on type 1 versus type 2 development. Here we show that CD4 T cells can become biased towards type 1 or type 2 phenotypes during their initial activation in the absence of IL-4 or IL-12. This type of regulation is dependent on the balance of MAPkinase, protein kinase C, and calcineurin signaling after TCR engagement. Later maturation of Th1 or Th2 effectors is dependent on IL-12 or IL-4. However Tc1 CD8 effector development is independent of IL-12, and Tc2 cell generation requires both appropriate TCR signals and IL-4 early in effector development. Using an altered peptide ligand to stimulate TCR transgenic T cells, we show that altered signaling regulates the numbers of CD8 cells capable of developing into Tc2 effectors, and also their responsiveness to IL-4. Together, the results support a two-stage model of differentiation in which intermediate cells biased towards the type 1 or type 2 pathways after activation, are subsequently matured in response to IL-12 or IL-4, respectively.

Chronic granulocytic leukaemia (CGL) is a rare disease. For most patients the only curative treatment (an allogeneic stem cell transplant) is not available. Survival varies between a few months to many years from diagnosis, and an accurate prediction of the duration of survival could help patients and clinicians make informed decisions about the many treatment options. In 1984, the Sokal score was introduced to stratify patients into risk groups. Recently, a new prognostic scoring system was proposed by Hasford and co-workers for interferon treated patients. We have analysed survival on an unselected population based cohort of patients using both the Hasford and the Sokal scores. In the group overall, neither score was predictive of survival, but in younger patients (< 60 years) treated with interferon, the Hasford score was highly predictive of survival, dividing patients into groups with a five year survival of 77% (45 patients) v 33% (six patients) v 14% (31 patients) (p = 0.01).

The evolution of diabetes in the male leptin receptor-deficient (fa/fa) Zucker diabetic fatty (ZDF) rat is associated with disruption of normal islet architecture, beta-cell degranulation, and increased beta-cell death. It is unknown whether these changes precede or develop as a result of the increasing plasma glucose, or whether the increased beta-cell death can be prevented. Early intervention with thiazolidinediones prevents disruption of the islet architecture. To determine the specific effects of rosiglitazone (RSG) on beta-cell mass dynamics, male fa/fa (obese) and +/fa or +/+ (lean) rats age 6 weeks were fed either chow (control group [CN]) or chow mixed with rosiglitazone (RSG group) at a dosage of 10 micromol. kg(-1) body wt.day(-1). Rats were killed after 0, 2, 4, 6, or 10 weeks of treatment (at age 6, 8, 10, 12, or 16 weeks). Plasma glucose increased from 8.9 +/- 0.4 mmol/l at 0 weeks to 34.2 +/- 1.8 mmol/l (P = 0.0001) at 6 weeks of treatment in obese CN rats and fell from 8.0 +/- 0.3 to 6.3 +/- 0.4 mmol/l in obese RSG rats (P = 0.02). beta-cell mass fell by 51% from 2 to 6 weeks of treatment (ages 8-12 weeks) in obese CN rats (6.9 +/- 0.9 to 3.4 +/- 0.5 mg; P0.05), whereas beta-cell mass was unchanged in obese RSG rats. At 10 weeks of treatment (age 16 weeks), beta-cell mass in obese CN rats was only 56% of that of obese RSG rats (4.4 +/- 0.4 vs. 7.8 +/- 0.3 mg, respectively; P = 0.0001). The beta-cell replication rate fell from a baseline value of 0.95 +/- 0.12% in lean rats and 0.94 +/- 0.07% in obese rats (at 0 weeks) to approximately 0.3-0.5% in all groups by 6 weeks of treatment (age 12 weeks). After 10 weeks of treatment, beta-cell replication was higher in obese RSG rats than in CN rats (0.59 +/- 0.14 vs. 0.28 +/- 0.05%, respectively; P0.02). Application of our mass balance model of beta-cell turnover indicated that net beta-cell death was fivefold higher in obese CN rats as compared with RSG rats after 6 weeks of treatment (age 12 weeks). The increase in beta-cell death in obese CN rats during the 6-week observation period was well correlated with the increase in plasma glucose (r2 = 0.90, P0.0001). These results suggest that the development of hyperglycemia in ZDF rats is concomitant with increasing net beta-cell death. beta-cell proliferation compensates for the increased beta-cell loss at a time when plasma glucose is moderately elevated, but compensation ultimately fails and the plasma glucose levels increase beyond approximately 20 mmol/l. Treatment with rosiglitazone, previously shown to reduce insulin resistance, prevents the loss of beta-cell mass in obese ZDF rats by maintaining beta-cell proliferation and preventing increased net beta-cell death.

We review evidence from experiments conducted in our laboratory on retrograde amnesia in rats with damage to the hippocampal formation. In a new experiment reported here, we show that N-methyl-D-aspartate (NMDA)-induced hippocampal damage produced retrograde amnesia for both hidden platform and two-choice visible platform discriminations in the Morris water task. For both problems there was a significant trend for longer training-surgery intervals to be associated with worse retention performance. Little support is offered by our work for the concept that there is a process involving hippocampal-dependent consolidation of memories in extrahippocampal permanent storage sites. Long-term memory consolidation may take place within the hippocampus. The hippocampus may be involved permanently in storage and/or retrieval of a variety of relational and nonrelational memories if it was intact at the time of learning, even involving information which is definitely not affected in anterograde amnesia after hippocampal damage.

We have identified a novel form of the full-length E2F-3 protein that we term E2F-3B. In contrast to full-length E2F-3, which is expressed only at the G1/S boundary, E2F-3B is detected throughout the cell cycle with peak levels in GO where it is associated with Rb. Transfection and in vitro translation experiments demonstrate that a protein identical to E2F-3B in size and iso-electric point is produced from the E2F-3 mRNA via the use of an alternative translational start site. This alternative initiation codon was mapped by mutagenesis to codon 102, an ACG codon. Mutation of the ACG codon at position 102 abolished E2F-3B expression, whereas the conversion of ACG 102 to a consensus ATG led to the expression of a protein indistinguishable from E2F-3B. Given these results, E2F-3B is missing 101 N-terminal amino acids relative to full-length E2F-3. This region includes a moderately conserved sequence of unknown function that is present only in the growth-promoting E2F family members, including E2F-1, 2 and full-length E2F-3. These observations make E2F-3B the first example of an E2F gene giving rise to two different protein species and also suggest that E2F-3 and E2F-3B may have opposing roles in cell cycle control.

The ontogeny of the somatotropin/insulin-like growth factor system was examined in well-fed pigs under basal conditions and during a short-term challenge of porcine ST (pST). The study was conducted with two replicates of eight castrate male pigs from 3.8 kg BW (10 d of age) to 92 kg BW (129 d of age). Pigs were reared individually with ad libitum access to milk replacer through 23 d of age. Thereafter, pigs were fed a corn, soybean meal, and dry skim milk diet formulated to exceed nutrient requirements by approximately 30%. Pigs were randomly assigned to receive daily i.m. injections of either 0 (buffer) or 120 microg/kg BW of pST for a duration of 4 d starting at 10, 19, 33, 43, 63, 83, and 125 d of age. Blood was collected via jugular venipuncture on d 0 and 4 of the challenge. Circulating levels of IGF-I were not dramatically affected by age, but levels of IGF-II were low from 10 to 19 d of age and then increased through later stages of growth. Circulating concentrations of IGF binding protein (BP)-3 increased with age (P.05), but levels of IGFBP-2, a 30-kDa IGFBP, and IGFBP-4 were unchanged (P.10). The pST challenge reduced plasma urea nitrogen at all ages, but the magnitude of the response was less in younger pigs compared with the maximum response in pigs greater than 30 kg BW (63 d of age). The IGF-I response to the pST challenge also increased from approximately 30% in young pigs to a threefold increase in older pigs. Regardless of age, concentrations of IGF-II were minimally affected by the pST challenge. Circulating levels of IGFBP-3 increased and IGFBP-2 levels decreased in response to the pST challenge, and the magnitude increased with age. The high nutritional status of pigs in the early phases of growth diminished the postnatal changes in IGF-I and IGFBP-2, but not IGF-II or IGFBP-3. Overall, data demonstrate a developmental regulation of the ST/IGF system, with pST challenges altering circulating concentrations of IGF-I, IGFBP-3, and IGFBP-2 coincident with changes in amino acid metabolism.

Epidemiological reports demonstrate an association between increased human morbidity and mortality with exposure to air pollution particulate matter (PM). Metal-catalyzed oxidative stress has been postulated to contribute to lung injury in response to PM exposure. We studied the effects of residual oil fly ash (ROFA), a component of ambient air PM, on the formation of lung carbonyls that are indicators of lipid peroxidation. Rats were instilled intratracheally with ROFA (62.5-1000 micrograms) and underwent lung lavage. Lavage fluid carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and measured by high performance liquid chromatography with UV detection. Dose-dependent increases in a peak that eluted with the same retention time as the acetaldehyde (CH3CHO) derivative was observed in rats treated with ROFA 15 min after instillation (up to 25-fold greater than saline treated controls). The identification of CH3CHO was confirmed using gas chromatography-mass spectroscopy. ROFA-induced increases in other lavage fluid carbonyls were not seen. Increased CH3CHO in lavage fluid was observed as late as 8 h later. No increase in CH3CHO was observed in plasma from ROFA-treated rats. An increased formation of CH3CHO was observed in a human airway epithelial cell line incubated with ROFA suggesting a pulmonary source of CH3CHO production. Instillation of solutions of metals (iron, vanadium, nickel) contained in ROFA, or instillation of another ROFA-type particle containing primarily iron, also induced a specific increase in CH3CHO. These data support the hypothesis that metals were involved in the increased CH3CHO formation. Thus metals on PM may mediate lung responses through induction of lipid peroxidation and carbonyl formation.

Lipoperoxidation final products represented by the TBARS (substances reacting with the Thiobarbituric acid), inflammatory reaction proteins and sera tocopherol have been studied in homozygous forms as well as in heterozygous forms of sickle cell diseases. The significant increase of TBARS (P0.001) measured by spectrofluorimetry, the considerable decrease of the sera alpha gamma tocopherol, measured by HPLC (P0.005) in all sickle cell patients, especially in crisis homozygous form, reinforce our previous study (22, 23, 24). The absence of links between the TBARS and the tocopherols (fig. 1) suggests that other defence mechanisms occur without vitamin E. The collapse of haptoglobinemia in homozygous sickle cell patients associated with the fall of hemoglobinemia indicates a severe tissue and intravascular hemolysis as a consequence of LPO. Furthermore, the simultaneous decrease of cholesterolemia seems to indicate important lipoperoxide activity detected in sickle cell patients.