Pneumococcal carriage and disease in adults in England 2011-2019: the importance of adults as a reservoir for pneumococcus in communities.

BACKGROUND: Pneumococcal carriage in children has been extensively studied, but carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is less understood. METHODS: Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by PCR. Pneumococcal carriage in adults 18-44 years was compared with carriage among PCV-vaccinated children 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and IPD data for England for the same ages for 2014-2019. Age-group specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for adults aged 65+ years. RESULTS: In total 98 isolates (97 carriers) were identified from 1,631 adults aged 18+ years (age and sex standardized carriage prevalence 6.4%), with only three identified solely by PCR. Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R=0.9). Serotypes 3, 37 and 8 represented a higher proportion of adult carriage than expected from direct low-level transmission from children to adults. The predicted carriage serotype distributions for 65+ years aligned more closely with the carriage serotype distribution for young adults than young children. CONCLUSIONS: The nasal wash technique is highly sensitive; additional benefit of PCR is limited. Comparison of carriage serotype distributions suggests some serotypes may be circulating preferentially within these specific young adults. Our data suggest that for some serotypes carried by adults 65+ years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered.

Motivation for physical activity and the moderating effect of cancer diagnosis: A nationally representative cross-sectional study.

The aim of this study was to investigate associations between types of motivation for physical activity and self-reported weekly aerobic moderate-to-vigorous physical activity (MVPA) in the 2012 and 2014 waves of the nationally representative Health Information National Trends Survey 4 (n = 7307). We further explored differential associations between MVPA and types of motivation for physical activity by cancer survivor status. We found that those who were more motivated by "getting enjoyment from exercise" reported 26.4% more MVPA (+49.8 min/week) than those who were less motivated by this factor, adjusting for covariates (p = 0.025). Conversely, those who were more motivated by "concern over the way you look" reported 22.1% less MVPA (-55.5 min/week) than those who were less motivated by this factor, adjusting for covariates (p = 0.002). We found no evidence for a relationship between motivation from either "pressure from others" or "feeling guilty when you skip exercising" and MVPA. We identified a significant interaction for "feeling guilty when you skip exercising" and cancer survivor status, adjusting for covariates (p = 0.034). Cancer survivors who reported being more motivated by "feeling guilty when you skip exercising" reported 36.2% less MVPA (-71.75 min/week) than those who were less motivated by this factor; there was no statistically reliable difference in those without a history of cancer. Findings are concordant with previous literature highlighting the primacy of enjoyment for physical activity adherence. There is a need for further inquiry into guilt-related motivation for physical activity among cancer survivors, as it may have a unique, negative impact in this population.

Manual therapy, exercise therapy, or both, in addition to usual care, for osteoarthritis of the hip or knee. 2: economic evaluation alongside a randomized controlled trial.

OBJECTIVE: To evaluate the cost effectiveness of manual physiotherapy, exercise physiotherapy, and a combination of these therapies for patients with osteoarthritis of the hip or knee. METHODS: 206 Adults who met the American College of Rheumatology criteria for hip or knee osteoarthritis were included in an economic evaluation from the perspectives of the New Zealand health system and society alongside a randomized controlled trial. Resource use was collected using the Osteoarthritis Costs and Consequences Questionnaire. Quality-adjusted life years (QALYs) were calculated using the Short Form 6D. Willingness-to-pay threshold values were based on one to three times New Zealand's gross domestic product (GDP) per capita of NZ$ 29,149 (in 2009). RESULTS: All three treatment programmes resulted in incremental QALY gains relative to usual care. From the perspective of the New Zealand health system, exercise therapy was the only treatment to result in an incremental cost utility ratio under one time GDP per capita at NZ$ 26,400 (-$34,081 to $103,899). From the societal perspective manual therapy was cost saving relative to usual care for most scenarios studied. Exercise therapy resulted in incremental cost utility ratios regarded as cost effective but was not cost saving. For most scenarios combined therapy was not as cost effective as the two therapies alone. CONCLUSIONS: In this study, exercise therapy and manual therapy were more cost effective than usual care at policy relevant values of willingness-to-pay from both the perspective of the health system and society. Trial registration number Australian New Zealand Clinical Trials Registry ACTRN12608000130369.

Manual therapy, exercise therapy, or both, in addition to usual care, for osteoarthritis of the hip or knee: a randomized controlled trial. 1: clinical effectiveness.

OBJECTIVE: To evaluate the clinical effectiveness of manual physiotherapy and/or exercise physiotherapy in addition to usual care for patients with osteoarthritis (OA) of the hip or knee. DESIGN: In this 2 × 2 factorial randomized controlled trial, 206 adults (mean age 66 years) who met the American College of Rheumatology criteria for hip or knee OA were randomly allocated to receive manual physiotherapy (n = 54), multi-modal exercise physiotherapy (n = 51), combined exercise and manual physiotherapy (n = 50), or no trial physiotherapy (n = 51). The primary outcome was change in the Western Ontario and McMaster osteoarthritis index (WOMAC) after 1 year. Secondary outcomes included physical performance tests. Outcome assessors were blinded to group allocation. RESULTS: Of 206 participants recruited, 193 (93.2%) were retained at follow-up. Mean (SD) baseline WOMAC score was 100.8 (53.8) on a scale of 0-240. Intention to treat analysis showed adjusted reductions in WOMAC scores at 1 year compared with the usual care group of 28.5 (95% confidence interval (CI) 9.2-47.8) for usual care plus manual therapy, 16.4 (-3.2 to 35.9) for usual care plus exercise therapy, and 14.5 (-5.2 to 34.1) for usual care plus combined exercise therapy and manual therapy. There was an antagonistic interaction between exercise therapy and manual therapy (P = 0.027). Physical performance test outcomes favoured the exercise therapy group. CONCLUSIONS: Manual physiotherapy provided benefits over usual care, that were sustained to 1 year. Exercise physiotherapy also provided physical performance benefits over usual care. There was no added benefit from a combination of the two therapies. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry ACTRN12608000130369.

Growth of subcutaneous chicken testicular transplants.

Avian genetic resources have declined dramatically over the past half century, partly because the poultry community has been slow to adopt cryoconservation of avian germplasm. Techniques for gonadal cryopreservation and functional recovery have recently been developed but only some have been optimized. Chicks were castrated at 2 or 6 d and testicles were autotransplanted subcutaneously in one piece after disruption of the tunica membranes to optimize transplantation procedures without the complication of tissue rejection or immunosuppression. At 22 wk of age, the roosters were euthanized and growth of the testicular tissue was evaluated. Mortality with castration at 2 d was high but was much reduced with castration at 6 d. Transplantation of whole testicles subcutaneously on the back of chicks, without complete removal of the tunica membranes, yielded good growth of tissue with transplantation at 2 or 6 d of age. These results will contribute to the use of testicular cryopreservation and transplantation as an effective conservation strategy for avian germplasm. Further definition of the age of treatment will improve the overall efficiency.

Cryoconservation of avian gonads in Canada.

Avian genetic resources have declined dramatically over the past half century as the cost of maintaining populations has exceeded the perceived benefit of keeping them. Despite the early importance of poultry in the development of cryopreservation techniques, very little avian germplasm has been conserved. Cryopreservation and recovery of avian gonads preserve the W chromosome and overcome problems of freezing and recovering semen or conserving and manipulating embryonic cells, and the use of vitrification procedures for preserving gonads minimizes cellular damage. On the basis of research demonstrating the biological possibility of cryopreserving and transplanting avian gonads, 5,125 testicles and 2,667 ovaries from 10 populations of Japanese quail, 9 populations of chickens, and 1 population of Chilean tinamou were cryopreserved and sent to the Canadian Animal Genetic Resources program for long-term storage. These gonads represent 20 of the 33 distinct avian populations currently maintained at Canadian public institutions of agricultural research.

Chimeric plumage coloration produced by ovarian transplantation in chickens.

Ovaries from Rhode Island Red donors were transplanted orthotopically into White Leghorn recipients. At maturation, recipients were mated with Rhode Island Red roosters to test the origin of their ovaries, using plumage coloration as a marker. A chick with chimeric plumage coloration was produced, indicating mechanisms that produce follicles with both donor and recipient ovarian contents. This study suggests that ovarian transplantation could be useful for in vivo studies of cytological and molecular mechanisms involved in avian folliculogenesis.

SF-6D and EQ-5D result in widely divergent incremental cost-effectiveness ratios in a clinical trial of older women: implications for health policy decisions.

SUMMARY: Using two instruments (SF-6D and EQ-5D) to estimate quality adjusted life years (QALYs), we conducted an economic evaluation of a 12-month randomized controlled trial with a 12-month follow-up study in older women to evaluate the value for money of two doses of resistance training compared with balance and tone classes. We found that the incremental QALYs estimated from the SF-6D were two- to threefold greater than those estimated from the EQ-5D. INTRODUCTION: Decision makers must continually choose between existing and new interventions. Hence, economic evaluations are increasingly prevalent. The impact of quality-adjusted life year (QALY) estimates using different instruments on the incremental cost-effectiveness ratios (ICERs) is not well understood in older adults. Thus, we compared ICERs, in older women, estimated by the EuroQol-5D (EQ-5D) and the Short Form-6D (SF-6D) to discuss implications on decision making. METHODS: Using both the EQ-5D and the SF-6D, we compared the incremental cost per QALY gained in a randomized controlled trial of resistance training in 155 community-dwelling women aged 65 to 75 years. The 12-month randomized controlled trial included a subsequent 12-month follow-up. Our focus, the follow-up study, included 123 of the 155 participants from the Brain Power study; 98 took part in the economic evaluation (twice-weekly balance and tone exercises, n = 28; once-weekly resistance training, n = 35; twice-weekly resistance training, n = 35). Our primary outcome measure was the incremental cost per QALY gained of once- or twice-weekly resistance training compared with balance and tone exercises. RESULTS: At cessation of the follow-up study, the incremental QALY was -0.051 (EQ-5D) and -0.144 (SF-6D) for the once-weekly resistance training group and -0.081 (EQ-5D) and -0.127 (SF-6D) for the twice-weekly resistance training group compared with balance and tone classes. CONCLUSION: The incremental QALYs estimated from the SF-6D were two- to threefold greater than those estimated from the EQ-5D. Given the large magnitude of difference, the choice of preference-based utility instrument may substantially impact health care decisions.

Production of donor-derived offspring after ovarian transplantation between Muscovy (Cairina moschata) and Pekin (Anas platyrhynchos) ducks.

Transplantation of ovarian tissue between different breeds of chickens or Japanese quail can result in normal egg production and give rise to donor-derived offspring with a high frequency. The feasibility of interspecific ovarian transplantation was evaluated by transplanting ovarian tissue from Muscovy ducks to Pekin ducks just after hatch, with subsequent treatment of the recipients with an immunosuppressant. Among the 8 mature Pekin ducks that had received ovarian tissue from Muscovy ducklings, 2 produced fertile eggs when mated with Muscovy drakes, and one of these produced only Muscovy ducks, which were derived from the transplanted Muscovy ovary. The other produced mule ducks that were hybrids of the host Pekin duck and the Muscovy drake. Further development of interspecific transplantation of ovaries may provide a simple method for the conservation and propagation of endangered birds.

Guidelines for conducting and reporting economic evaluation of fall prevention strategies.

UNLABELLED: Falls in older people result in substantial health burden. Compelling evidence indicates that falls can be prevented. We developed comprehensive guidelines for economic evaluations of fall prevention interventions to facilitate publication of high-quality economic evaluations of the effective strategies and aid decision making. INTRODUCTION: The importance of economics applied to falls and fall prevention in older people has largely been overlooked. The use of different methodologies to assess the costs and health benefits of the interventions and their comparators and the inconsistent reporting in the studies limits the usefulness of these economic evaluations for decision making. We developed guidelines to encourage and facilitate completion of high-quality economic evaluations of effective fall prevention strategies. METHODS: We used a generic checklist for economic evaluations as a platform to develop comprehensive guidelines for conducting and reporting economic evaluations of fall prevention strategies. We considered the many challenges involved, particularly in identifying, measuring, and valuing the relevant cost items. RESULTS: We recommend researchers include cost outcomes and report incremental cost-effectiveness ratios in terms of falls prevented and quality adjusted life years in all clinical trials of fall prevention interventions. Studies should include the following cost categories: (1) implementing the intervention, (2) delivering the comparator group intervention, (3) total health care costs, (4) costs of fall-related health care resource use, and (5) personal and informal carer opportunity costs. CONCLUSIONS: This paper provides a timely benchmark to promote comparability and consistency for conducting and reporting economic evaluations of fall prevention strategies.

Economic evaluation of dose-response resistance training in older women: a cost-effectiveness and cost-utility analysis.

SUMMARY: We estimated the incremental cost-effectiveness of a once-weekly or twice-weekly resistance training intervention compared with balance and tone classes in terms of falls prevented and quality-adjusted life years (QALYs) gained. Both resistance training interventions were more likely to save health care resource money and offer better health outcomes for falls prevention than balance and tone classes. INTRODUCTION: This study aims to estimate the incremental cost-effectiveness and cost-utility of a once-weekly or twice-weekly resistance training intervention compared with twice-weekly balance and tone classes in terms of falls prevented and QALYs gained. METHODS: Economic evaluation was conducted concurrently with a three-arm randomized controlled trial including 155 community-dwelling women aged 65 to 75 years, Mini Mental State Examination ≥24, and visual acuity 20/40 or better. Participants received the once-weekly resistance training (n = 54), the twice-weekly resistance training (n = 51) or the twice-weekly balance and tone (the comparator) classes (n = 50) for 1 year. Measurements included the number of falls for each participant, healthcare resource utilization, and associated costs over 9 months; health status was assessed using the EQ-5D and SF-6D to calculate QALYs. RESULTS: Based on the point estimates from our base case analysis, we found that both once- and twice-weekly resistance training groups were less costly (p < 0.05) and more effective than twice-weekly balance and tone classes. The incremental QALYs assessed using the SF-6D were 0.003 for both the once- and twice-weekly resistance training groups, compared with the twice-weekly balance and tone classes. The incremental QALYs assessed using the EQ-5D were 0.084 for the once-weekly and 0.179 for the twice-weekly resistance training groups, respectively, compared with the twice-weekly balance and tone classes. CONCLUSIONS: An individually tailored resistance training intervention delivered once or twice weekly provided better value for money for falls prevention than balance and tone classes.

International comparison of cost of falls in older adults living in the community: a systematic review.

SUMMARY: Our objective was to determine international estimates of the economic burden of falls in older people living in the community. Our systematic review emphasized the need for a consensus on methodology for cost of falls studies to enable more accurate comparisons and subgroup-specific estimates among different countries. INTRODUCTION: The purpose of this study was to determine international estimates of the economic burden of falls in older people living in the community. METHODS: This is a systematic review of peer-reviewed journal articles reporting estimates for the cost of falls in people aged > or =60 years living in the community. We searched for papers published between 1945 and December 2008 in MEDLINE, PUBMED, EMBASE, CINAHL, Cochrane Collaboration, and NHS EED databases that identified cost of falls in older adults. We extracted the cost of falls in the reported currency and converted them to US dollars at 2008 prices, cost items measured, perspective, time horizon, and sensitivity analysis. We assessed the quality of the studies using a selection of questions from Drummond's checklist. RESULTS: Seventeen studies met our inclusion criteria. Studies varied with respect to viewpoint of the analysis, definition of falls, identification of important and relevant cost items, and time horizon. Only two studies reported a sensitivity analysis and only four studies identified the viewpoint of their economic analysis. In the USA, non-fatal and fatal falls cost US $23.3 billion (2008 prices) annually and US $1.6 billion in the UK. CONCLUSIONS: The economic cost of falls is likely greater than policy makers appreciate. The mean cost of falls was dependent on the denominator used and ranged from US $3,476 per faller to US $10,749 per injurious fall and US $26,483 per fall requiring hospitalization. A consensus on methodology for cost of falls studies would enable more accurate comparisons and subgroup-specific estimates among different countries.

Does a home-based strength and balance programme in people aged > or =80 years provide the best value for money to prevent falls? A systematic review of economic evaluations of falls prevention interventions.

OBJECTIVES: To investigate the value for money of strategies to prevent falls in older adults living in the community. DESIGN: Systematic review of peer reviewed journal articles reporting an economic evaluation of a falls prevention intervention as part of a randomised controlled trial or a controlled trial, or using an analytical model. MEDLINE, PUBMED, EMBASE and NHS EED databases were searched to identify cost-effectiveness, cost-utility and cost-benefit studies from 1945 through July 2008. MAIN OUTCOME MEASURES: The primary outcome measure was incremental cost-effectiveness, cost-utility and cost-benefit ratios in the reported currency and in pounds sterling at 2008 prices. The quality of the studies was assessed using two instruments: (1) an economic evaluation checklist developed by Drummond and colleagues and (2) the Quality of Health Economic Studies instrument. RESULTS: Nine studies meeting our inclusion criteria included eight cost-effectiveness analyses, one cost-utility and one cost-benefit analysis. Three effective falls prevention strategies were cost saving in a subgroup of PARTICIPANTS: (1) an individually customised multifactorial programme in those with four or more of the eight targeted fall risk factors, (2) the home-based Otago Exercise Programme in people > or =80 years and (3) a home safety programme in the subgroup with a previous fall. These three findings were from six studies that scored > or =75% on the Quality of Health Economic Studies instrument. CONCLUSIONS: Best value for money came from effective single factor interventions such as the Otago Exercise Programme which was cost saving in adults 80 years and older. This programme has broad applicability thus warranting warrants health policy decision-makers' close scrutiny.

Studies of the protein synthetic activity of lysates from HeLa cells incubated in hypertonic medium.

When HeLa S-3 cells are incubated with medium made hypertonic by adding NaCl, protein synthesis is inhibited. When the cells are returned to normal conditions protein synthesis is restored. To study the molecular mechanism of this regulation of protein synthesis, lysates were prepared from HeLa S-3 cells grown in minimum essential medium (normal, N); from cells which were incubated with additional (100 mM) NaCl (hypertonic, H); and from cells which were treated similarly in hypertonic medium and then restored to isotonic conditions (hypertonic-isotonic, H-I). Lysates of H cells exhibited reduced endogenous protein synthesis. Studies with mixed lysates from H and N cells implicated that the H lysate did not contain a soluble, non-labile macromolecule (greater than 10 000 daltons) with an inhibitory activity upon the protein synthesis. Analysis by Edman reaction of H lysates showed a reduced incorporation of [35S]methionine into N-terminal suggesting that the initiation step of protein synthesis was affected. However, sucrose gradietn analysis indicated that lysates of H cells were still able to form 80-S initiation complexes with [35S] methionyl-tRNAIMet. The block in initiation was not complete. The lesion could not be reversed by adding post-ribosomal supernatant or a ribosomal salt wash from N cells to ribosomes from the H cells. The data show that the ribosomal fraction is primarily involved in the inhibition.

Association between activity at onset of symptoms and outcome of acute myocardial infarction.

OBJECTIVES: This study sought to compare the clinical features and outcome of a first myocardial infarction with onset of symptoms during or within 30 min of exercise, at rest and in bed. BACKGROUND: It is not known whether activity at onset influences outcome of acute myocardial infarction. METHODS: Information collected using a standard questionnaire was used to relate activity at the onset of symptoms to in-hospital outcome in 2,468 consecutive patients admitted to a coronary care unit with a first myocardial infarction between 1975 and 1993. RESULTS: Patients with exercise-related onset were more likely to be younger and male. Those with onset in bed were more likely to be older and have a history of stable or unstable angina. Compared with patients whose symptoms began at rest, those with exercise-related onset had a lower in-hospital mortality rate after adjusting for age, gender and year of admission (odds ratio [OR] 0.60, 95% confidence interval [CI] 0.40 to 0.89), and patients with onset in bed had a higher mortality rate (OR 1.38, 95% CI 1.03 to 1.85). The incidence of cardiac failure requiring diuretic therapy was also lower for exercise-related onset (OR 0.83, 95% CI 0.67 to 1.04) and higher when onset was in bed (OR 1.36, 95% CI 1.11 to 1.66). CONCLUSIONS: There is an association between activity at onset and outcome of acute myocardial infarction. Differences in pathophysiology or in the population at risk could explain this observation.

Induction of relaxin messenger RNA expression in response to prolactin receptor activation requires protein kinase C delta signaling.

The ability of PRL or rat placental lactogen (rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized rat granulosa cell culture model. PRL receptor activation induced relaxin mRNA expression in a concentration- and time-dependent manner. High concentrations of PRL receptor agonist, equivalent to those of the second half of pregnancy in rats, were required to elicit relaxin mRNA expression. A 40-fold induction of relaxin mRNA was observed in cells treated 24 h with 1 microg/ml of rPL-1. Estrogen enhanced relaxin expression induced by PRL but did not affect relaxin expression on its own. PRL/rPL-1 induction of relaxin expression was independent of the extracellular regulated kinase (ERK) members of the mitogen-activated protein kinase (MAPK) pathway, based on the inability of the ERK kinase inhibitor PD98059 to block induction of relaxin expression. PRL/rPL-1 induction of relaxin expression required protein kinase C (PKC) delta, based on the ability of the preferential PKC delta inhibitor rottlerin to abolish induction of relaxin expression. Direct activation of PKC by phorbol myristate acetate, however, was not sufficient to promote induction of relaxin mRNA expression. Stats (signal transducers and activators of transcription) 3 and 5 DNA binding activities were induced by PRL/rPL-1 treatment of luteinized granulosa cells but only Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 treatment of luteinized granulosa cells resulted in increased phosphorylation on tyrosine-705 and serine-727 of Stat 3, and these responses were reduced and blocked, respectively, by rottlerin. Tyrosine and serine phosphorylations of Stat 3 in the corpus luteum were also increased in the second half of pregnancy when PL levels are highest. Stat 3, but not Stat 1 or 5, coimmunoprecipitated with luteal PKC delta during pregnancy; Stat 3 transiently coimmunoprecipitated with PKC delta from luteinized granulosa cells in response to PRL receptor activation; and Stat 3/PKC delta complex formation required PKC delta kinase activity. Taken together, these results show that PKC delta is obligatory for PRL/rPL-1-dependent relaxin expression, that PKC delta complexes with Stat 3 in response to PRL receptor activation, and that PKC delta is involved in the regulation of Stat 3 phosphorylation downstream of the PRL receptor. These results demonstrate that PRL/rPL-1 promotes relaxin expression in luteal cells and that this event is mediated, at least in part, via PKC delta.

Two forms of rat placental lactogen revealed by radioimmunoassay.

A radioimmunoassay (RIA) has been developed to the species of lactogenic hormone (rPL) present in late pregnant rat placenta. Partially purified rPL calibrated against oPRL in the rat liver receptor assay (RRA) was used as standard. None of the PL's, PRL's, and GH's from other species cross-reacted in the RIA when tested at 1000 ng/ml. Rat serum PL levels remained stable in samples stored at -70C but not at -20C. In previous studies, using RRA, two peaks of rPL were found in rat serum, one at Day 11-13 and one at Day 20. By RIA, however, only the 20,000 dalton species of rPL present in late pregnant serum was immunoreactive. The RIA did not detect the larger, 40-50,000 dalton molecular species of rPL predominant in mild-pregnant serum, thus revealing the presence of two possibly unrelated forms of rPL. Serum rPL concentrations at Day 21 of pregnancy increased exponentially as the number of fetuses increased until a maximum of nine fetus.

Secretion of placental lactogen, growth hormone, and prolactin in late pregnant rats.

The secretory patterns of placental lactogen (PL), GH, and PRL were determined in conscious unrestrained late pregnant rats by measurement of the plasma concentrations of each hormone by RIA. Blood samples were collected over a 4-h period at 15-min intervals on days 18 and 19 of gestation. From days 18-19, GH levels increased 2-fold, no change was observed in PRL levels, while PL concentrations increased slightly. There was an ultradian rhythm present in GH and PRL secretion, with a frequency of two to three secretory spikes per 4-h collection period. The variable concentrations of PL in the peripheral circulation were suggestive of an episodic or ultradian secretory pattern. PL levels increased 100% or more within 30 min (i.e. from 485 to 1132 ng/ml) or decreased as much as 65% within 60 min (i.e. from 792 to 247 ng/ml). The relative magnitude of these changes were similar to, but not coincident or correlated with, those for GH.

Rat placental lactogen-I variant (rPL-Iv), product of an unique gene, is biologically different from rPL-I.

The rat placenta expresses two rat placental lactogen-I (rPL-I) proteins: the normal rPL-I in the first half of pregnancy and a variant (rPL-Iv) in the second half of pregnancy. They are 70% identical at the amino acid level but arise from different cell types: rPL-I from giant cells and rPL-Iv from cytotrophoblasts. To assess whether rPL-Iv originates from alternative splicing of the rPL-I gene or is the product of a separate gene, genomic clones of rPL-I and rPL-Iv were isolated from a lambda EMBL-3 rat genomic library. Restriction enzyme analysis of the 14-kilobase full-length genomic clones of rPL-I and rPL-Iv indicated that the two genes are distinct. To assess the biological activity of the variant protein relative to other members of the rat PL/PRL/GH family, two expression systems were chosen to obtain the purified recombinant protein: 1) a secreted form of rPL-Iv was obtained from Chinese hamster ovary (CHO) cells transfected with rPL-Iv-complementary DNA; and 2) a rPL-Iv fusion protein (Bac-rPL-Iv) was obtained from Spodoptera frugiperda (Sf9) insect cells that had been infected with a recombinant baculovirus generated from rPL-Iv complementary DNA. An antibody was generated to the purified Bac-rPL-Iv fusion protein and used for affinity chromatography to purify recombinant rPL-Iv from the CHO cell media. The mitogenic activity of rPL-Iv was monitored in the Nb2 lymphoma cell bioassay. The relative potency of rPL-Iv compared with other members of the PL/PRL/GH family follows: ovine PRL 100, rPL-I 200, rPL-II 160, rPRL 40, CHO-rPL-Iv 0.7, and Bac rPL-Iv 0.4. Iodinated CHO-rPL-Iv showed minimal binding to Nb2 lymphoma cells, but at a 500-fold protein concentration rPL-I was able to displace [125I]rPL-I from the lymphoma cell PRL receptor. Using recombinant CHO-derived rPL-Iv as standard and antisera against the Bac-rPL-Iv fusion protein, a RIA was developed for rPL-Iv. In pregnant rats rPL-Iv appeared in the serum at day 14, rising to a peak of 2080 +/- 440 ng/ml at day 18, followed by a slight decline. These values reflect the levels of messenger RNA for rPL-Iv in rat placenta noted previously. In summary, rPL-Iv arises from a gene different from rPL-I and the rPL-I protein displays minimal binding and mitogenic activity in the Nb2 lymphoma cells.

Molecular cloning and expression of rat placental lactogen-Iv, a variant of rPL-I present in late pregnant rat placenta.

Late pregnant rat placenta was found to contain a messenger RNA (mRNA) that encodes an additional member of the prolactin-growth hormone family. This polypeptide was detected by hybridization of complementary DNA (cDNA) for rPL-I to a 1 kilobase mRNA transcript in late pregnant rat placenta. A cDNA clone for the new polypeptide was isolated from a phage lambda-gt10 library containing cDNA synthesized from day 18 rat placental mRNA. Sequencing of the day 18 cDNA clone revealed that it was most closely related structurally to rPL-I and at the amino acid terminal was identical to the rPL-I variant (rPL-Iv). The sequence of rPL-Iv (223 amino acids) is 85% homologous to rPL-I (230 amino acid). There are 71 base pair changes (85% nucleotide homology) between rPL-Iv and rPL-I which are not confined to any specific region of the molecule and which result in 36 amino acid changes. In vitro translation of rPL-Iv mRNA produced by transcription of the cDNA template yielded a 26 kilodalton polypeptide, the size of the expected precursor protein. In situ hybridization studies indicated that mRNA for rPL-Iv was present primarily in cytotrophoblasts of the basal zone as early as day 15 of gestation with some hybridization in a few giant cells as well.