Biophysical Profiling Research Articles

Systems serology for decoding infection and vaccine-induced antibody responses to HIV-1

Experimental and analytical advances have enabled systematic, high-resolution studies of humoral immune responses, and are beginning to define mechanisms of immunity to HIV. High-throughput, information-rich experimental and analytical methods, whether genomic, proteomic, or transcriptomic, have firmly established their value across a diversity of fields. Consideration of these tools as trawlers in 'fishing expeditions' has faded as 'data-driven discovery' has come to be valued as an irreplaceable means to develop fundamental understanding of biological systems. Collectively, studies of HIV-1 infection and vaccination including functional, biophysical, and biochemical humoral profiling approaches have provided insights into the phenotypic characteristics of individual and pools of antibodies. Relating these measures to clinical status, protection/efficacy outcomes, and cellular profiling data using machine learning has offered the possibility of identifying unanticipated mechanisms of action and gaining insights into fundamental immunological processes that might otherwise be difficult to decipher. Recent evidence establishes that systematic data collection and application of machine learning approaches can identify humoral immune correlates that are generalizable across distinct HIV-1 immunogens and vaccine regimens and translatable between model organisms and the clinic. These outcomes provide a strong rationale supporting the utility and further expansion of these approaches both in support of vaccine development and more broadly in defining mechanisms of immunity.

Biophysical and Pharmacological Profiling of Multiple Voltage-Gated Sodium Channel Subtypes on QPatch II

Voltage - gated sodium channels (VGSC) are responsible for the initiation and propagation of action potentials in excitable cells. VGSC have been identified as excellent drug targets for treatment of pain, epilepsy and to other neurological disorders. Early compounds, however, were developed using empirical approaches. The identification of the molecular identity of VGSC in combination with technological advances, such as the automated patch clamp technique, provide the basis for a rational design of subtype – selective compounds. To date, 9 functional mammalian isoforms (NaV1.1–1.9) have been described in the literature. The various subtypes differ in their expression pattern and exhibit distinct biophysical and pharmacological profiles. All have in common that they produce a transient inward current in response to membrane depolarization. During this process the VGSC transitions from a closed to and open into an inactivated state. Interestingly, inhibitor compounds often exhibit different pharmacological profiles dependent upon the ion channel conformational state. In the present study, the second generation QPatch (QPatch II; Sophion Bioscience) was used in combination with adaptive voltage protocols to investigate state-dependent inhibition of tetrodotoxin (TTX) and tetracaine on 8 different VGSC subtypes (NaV1.1-8). A first step was to determine the half inactivation potential V½(inactivation) for each individual cell. This value was then used during the next steps as preconditioning pulse. Such an adaptive protocol allowed to determine IC50 values for both the closed and the inactivated state and reduce heterogeneity of the cells. Both IC50 values and biophysical parameters of the different subtypes align well with literature values.

Artificial strain of human prions created in vitro

The molecular mechanism that determines under physiological conditions transmissibility of the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD) is unknown. We report the synthesis of new human prion from the recombinant human prion protein expressed in bacteria in reaction seeded with sCJD MM1 prions and cofactor, ganglioside GM1. These synthetic human prions were infectious to transgenic mice expressing non-glycosylated human prion protein, causing neurologic dysfunction after 459 and 224 days in the first and second passage, respectively. The neuropathology, replication potency, and biophysical profiling suggest that a novel, particularly neurotoxic human prion strain was created. Distinct biological and structural characteristics of our synthetic human prions suggest that subtle changes in the structural organization of critical domains, some linked to posttranslational modifications of the pathogenic prion protein (PrPSc), play a crucial role as a determinant of human prion infectivity, host range, and targetting of specific brain structures in mice models.

The role of modified biophysical profile in high risk pregnancies and fetal outcome

Objectives: In a high risk population where chances of adverse outcome are relatively high and almost all the pregnancies are under strict monitoring, biophysical profiling further helps to identify adverse outcome and thus a basis for intervention. In this study, we aim to evaluate the role of modified biophysical profile in high risk pregnancies and fetal outcome. Methods: Total 125 high risk pregnancies and were monitored for modified biophysical profiling from GA 34 weeks onwards. AFI<8 and non-reactive NST were considered as abnormal BPP. Apgar <7 at 5 min, MSL, NNU admission and neonatal death were considered as adverse fetal outcomes. Chi-square test was used to compare the data. Results: Mean age was 24.32±4.37 (range 19-35) years. Mean age at enrolment was 35.23±1.78 weeks. A total of 41 (32.8%) patients had AFI<8. Non-reactive NST was seen in 52 (41.6%) patients. Overall abnormal biophysical profile (NR-NST/AFI<8) was seen in 62 (49.6%) patients. Incidence of meconium stained liquor, Apgar<7 at 5m, NNU admission and NNU expiry was 15.2%, 20.8%, 26.4% and 4.0% respectively. NST and overall BPP showed a statistically significant association with all the outcomes however, AFI failed to show a significant association with NNU expiry. For all the outcomes NST had higher sensitivity as compared to AFI. Combined BPP showed a higher sensitivity than either of two components. Conclusion: Modified BPP was found to be useful in identification of adverse fetal outcomes, thus highlighting its role in planning interventions to avert extreme events. Keywords:

High‐resolution definition of humoral immune response correlates of effective immunity against HIV

Defining correlates of immunity by comprehensively interrogating the extensive biological diversity in naturally or experimentally protected subjects may provide insights critical for guiding the development of effective vaccines and antibody‐based therapies. We report advances in a humoral immunoprofiling approach and its application to elucidate hallmarks of effective HIV‐1 viral control. Systematic serological analysis for a cohort of HIV‐infected subjects with varying viral control was conducted using both a high‐resolution, high‐throughput biophysical antibody profiling approach, providing unbiased dissection of the humoral response, along with functional antibody assays, characterizing antibody‐directed effector functions such as complement fixation and phagocytosis that are central to protective immunity. Profiles of subjects with varying viral control were computationally analyzed and modeled in order to deconvolute relationships among IgG Fab properties, Fc characteristics, and effector functions and to identify humoral correlates of potent antiviral antibody‐directed effector activity and effective viral suppression. The resulting models reveal multifaceted and coordinated contributions of polyclonal antibodies to diverse antiviral responses, and suggest key biophysical features predictive of viral control.

Linking High-Throughput Screens to Identify MoAs and Novel Inhibitors of Mycobacterium tuberculosis Dihydrofolate Reductase.

Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of action (MoA) and reveal actionable chemical matter. Here, we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning was employed to identify chemical features with predicted efficacy targets for a given phenotypic screen. We validate the approach on data from a set of 55 000 compounds in 24 historical internal antibacterial phenotypic screens and 636 bacterial targets screened in high-throughput biophysical binding assays. Models were built to reveal the relationships between phenotype, target, and chemotype, which recapitulated mechanisms for known antibacterials. We also prospectively identified novel inhibitors of dihydrofolate reductase with nanomolar antibacterial efficacy against Mycobacterium tuberculosis. Molecular modeling provided structural insight into target-ligand interactions underlying selective killing activity toward mycobacteria over human cells.

Use of Doppler velocimetry in diagnosis and prognosis of intrauterine growth restriction (IUGR): A Review.

Intrauterine growth restriction (IUGR) is a condition which has been difficult to assess at an early stage, resulting in the delivery of children who have poor genetic growth potential. Currently, IUGR classification is based upon the system of ultrasound biometry. Doppler velocimetry allows the measurement of hemodynamic flow of major fetal vessels, comparing the flow indices and patterns of normal and IUGR cases. In this review, the effectiveness of Doppler velocimetry in assessing blood flow in major vessels including the umbilical artery, ductus venosus, and middle cerebral artery was studied for both diagnostic and prognostic screening of IUGR. The umbilical artery is the most frequently studied vessel in Doppler velocimetry due to its accessibility and the strength of its associations with fetal outcomes. Abnormalities in the ductus venosus waveform can be indicative of increased resistance in the right atrium due to placental abnormalities. The middle cerebral artery is the most studied fetal cerebral artery and can detect cerebral blood flow and direction, which is why these three vessels were selected to be examined in this context. A potential mathematical model could be developed to incorporate these Doppler measurements which are indicative of IUGR, in order to reduce perinatal mortality. The purpose of the proposed algorithm is to integrate Doppler velocimetry with biophysical profiling in order to determine the optimal timing of delivery, thus reducing the risks of adverse perinatal outcomes.

Abstract 5371: PRMT5 inhibitors as novel treatment for cancers

Abstract Increased expression or dysregulation of protein arginine methyltransferase 5 (PRMT5) activity is associated with poor prognosis in many cancers. Through increased methylation of epigenetic and non-epigenetic targets, the aberrant activity of PRMT5 has been associated with many pro-tumourigenic cellular changes such as, increased levels of protein synthesis, dysregulation of cell cycle, cellular adaptation to hypoxic conditions, and suppression of normal cell death pathways. Genetic studies suggest that suppression of PRMT5 activity can reverse many of these pro-tumourigenic effects making PRMT5 an attractive drug discovery target. We screened a library of 350,000 lead-like compounds with a biochemical assay measuring the methylation of a histone H4 peptide by the recombinant human PRMT5/MEP50 complex. Biochemical and biophysical profiling of the inhibitory compounds indicated that several distinct binding modes were exhibited by the different chemical scaffolds. Inhibitors displayed competitive, noncompetitive or uncompetitive interactions with respect to S-adenosyl methionine and the peptide substrate. Medicinal chemistry developed several classes of potent, highly selective inhibitors of PRMT5 methyltransferase activity from the hit set. The optimised tool compound, CTx-034, is a potent inhibitor of PRMT5 methyl transferase activity (KD = 2 nM), which is highly selective (&amp;gt;100-fold) versus a panel of 18 methyltransferases (including 6 PRMT family members), 11 lysine demethylases, and 15 safety related targets (GPCRs, ion channels, enzymes). Treatment of cancer cell lines with CTx-034 reduces cellular levels of symmetrically dimethylated H4 Arginine 3 (H4R3me2s), in a dose dependent manner (IC50 = 4 nM) to levels undetectable by Western blot. Furthermore, within this chemical series the ability of compounds to reduce cellular levels of H4R3me2s closely correlates with PRMT5 inhibitory activity supporting PRMT5 as the cellular target of these compounds, and suggesting that PRMT5 is the major writer of this histone mark in many cancer cell lines. CTx-034 also inhibits the symmetric dimethylation of arginine on other histone and non-histone cellular substrates of PRMT5, including H3R2me2s and SmD1. Conversely, CTx-034 treatment does not reduce levels of H4R3 asymmetric dimethylation, a histone mark catalysed by PRMT1. Finally, CTx-034 has good oral bioavailability and pharmacokinetic properties in rodents and twice-daily dosing (10 - 100 mg/kg) over 10-14 days produces a dose dependent reduction of the H4R3me2s mark in bone marrow cells and peripheral white blood cells. This treatment is well tolerated by the mice, with no significant reduction in body weight or changes in haematological parameters observed. CTx-034 provides an excellent tool compound for cellular and in vivo proof of concept studies. Citation Format: Hendrik Falk, Richard C. Foitzik, Elizabeth Allan, Melanie deSilva, Hong Yang, Ylva E. Bozikis, Marica Nikac, Scott R. Walker, Michelle A. Camerino, Ben J. Morrow, Alexandra E. Stupple, Rachel Lagiakos, Jo-Anne Pinson, Romina Lessene, Wilhelmus JA Kersten, Danny G. Ganame, Ian P. Holmes, Gill E. Lunniss, Matthew Chung, Stefan J. Hermans, Michael W. Parker, Alison Thistlethwaite, Karen White, Susan A. Charman, Brendon J. Monahan, Patricia Pilling, Julian Grusovin, Thomas S. Peat, Stefan Sonderegger, Emma Toulmin, Stephen M. Jane, David J. Curtis, Paul A. Stupple, Ian P. Street. PRMT5 inhibitors as novel treatment for cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2015-5371

Fetal assessment methods for improving neonatal and maternal outcomes in preterm prelabour rupture of membranes.

Fetal assessment following preterm prelabour rupture of membranes (PPROM) may result in earlier delivery due to earlier detection of fetal compromise. However, early delivery may not always be in the fetal or maternal interest, and the effectiveness of different fetal assessment methods in improving neonatal and maternal outcomes is uncertain. To study the effectiveness of fetal assessment methods for improving neonatal and maternal outcomes in PPROM. Examples of fetal assessment methods that would be eligible for inclusion in this review include fetal cardiotocography, fetal movement counting and Doppler ultrasound. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (30 June 2014) and reference lists of retrieved studies. Randomised controlled trials comparing any fetal assessment methods, or comparing one fetal assessment method to no assessment. Two review authors independently assessed trials for inclusion into the review. The same two review authors independently assessed trial quality and independently extracted data. Data were checked for accuracy. We included three studies involving 275 women (data reported for 271) with PPROM at up to 34 weeks' gestation. All three studies were conducted in the United States. Each study investigated different methods of fetal assessment. One study compared weekly endovaginal ultrasound scans with no assessment (n = 93), one compared amniocentesis with no assessment (n = 47), and one compared daily nonstress testing with daily modified biophysical profiling (n = 135). We were unable to perform a meta-analysis, but were able to report data from individual studies.There was no convincing evidence of increased risk of neonatal death in the group receiving endovaginal ultrasound scans compared with the group receiving no assessment (risk ratio (RR) 7.30, 95% confidence interval (CI) 0.39 to 137.54; one study, 92 women), or in the group receiving amniocentesis compared with the group receiving no amniocentesis (RR 1.00, 95% CI 0.07 to 15.00; one study, 44 women). For both these interventions, we inferred that there were no fetal deaths in the intervention or control groups. The study comparing daily nonstress testing with daily modified biophysical profiling did not report fetal or neonatal death. Primary outcomes of maternal death and serious maternal morbidity were not reported in any study. Overall, there were few statistically significant differences in outcomes between the comparisons.The overall quality of evidence is poor, because participant blinding was not possible for any study. There is insufficient evidence on the benefits and harms of fetal assessment methods for improving neonatal and maternal outcomes in women with PPROM to draw firm conclusions. The overall quality of evidence that does exist is poor.Further high-quality randomised controlled trials are required to guide clinical practice.

Disruption of Myelin Leads to Ectopic Expression of KV1.1 Channels with Abnormal Conductivity of Optic Nerve Axons in a Cuprizone-Induced Model of Demyelination

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K+ channels (KV1.1 and 1.2 α subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of KV1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or KV1.1-subunit selective (dendrotoxin K) blockers of K+ currents suggest enhanced KV1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K+ currents mediated by recombinant channels comprised of different KV1.1 and 1.2 stoichiometries revealed that the enrichment of KV1 channels KV1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the patho-physiology of MS and related disorders.

Definition and management of fetal growth restriction: a survey of contemporary attitudes

ObjectiveTo evaluate opinions among Irish obstetricians and obstetric trainees regarding the optimal definition, assessment and management of pregnancies affected by intrauterine growth restriction (IUGR). Study designAn anonymous, structured, web-based survey that comprised 14 questions was sent to 200 obstetricians and obstetric trainees in Ireland. ResultsOf the 113 participants (57% response rate), the majority (50%) were consultants, with over 10 years’ clinical experience (46%), who worked in large maternity units (58%) with neonatal units providing care for preterm IUGR fetuses (94%). Eighty-three clinicians (74%) agreed that an estimated fetal weight (EFW) below the 10th centile constitutes small-for-gestational age (SGA). The majority (n=93; 82%) would deliver the SGA fetus between 37+0 and 39+6 weeks gestation. In total, the survey yielded 30 different IUGR definitions; the top three definitions were (i) an EFW below the 5th centile (n=18; 16%), (ii) an EFW below the 10th centile with oligohydramnios and abnormal umbilical artery (UA) Doppler (n=16; 14%), and (iii) an EFW below the 10th centile (n=12; 11%). In the evaluation of the preterm IUGR fetus with abnormal UA Doppler, the assessment of amniotic fluid volume, middle cerebral artery, ductus venosus, cardiotocograph (CTG) and biophysical profiling was performed in 74%, 60%, 60%, 54% and 52% respectively. The majority of clinicians applied three or more assessment modalities and 60% referred to a maternal-fetal medicine (MFM) subspecialist. Interestingly, even among MFM subspecialists there was no common consistent management approach. Most doctors (81%) would deliver the IUGR fetus for CTG abnormalities but MFM subspecialists more commonly deliver on the basis of absent end-diastolic flow in the UA alone (37% vs. 10%; p=0.006). Two-thirds of doctors (n=74) would implement customised growth charts if they became available for their population and over 80% thought that a national guideline on IUGR would be beneficial. ConclusionThe results of this survey confirm the inconsistencies surrounding the clinical management of IUGR pregnancies and highlight the need for standardisation of terminology and antenatal surveillance, implementation of fetal weight customisation and national guidance for Ireland.

Rapid optimization and prototyping for therapeutic antibody-like molecules

Multispecific antibody-like molecules have the potential to advance the standard-of-care in many human diseases. The design of therapeutic molecules in this class, however, has proven to be difficult and, despite significant successes in preclinical research, only one trivalent antibody, catumaxomab, has demonstrated clinical utility. The challenge originates from the complexity of the design space where multiple parameters such as affinity, avidity, effector functions, and pharmaceutical properties need to be engineered in concurrent fashion to achieve the desired therapeutic efficacy. Here, we present a rapid prototyping approach that allows us to successfully optimize these parameters within one campaign cycle that includes modular design, yeast display of structure focused antibody libraries and high throughput biophysical profiling. We delineate this approach by presenting a design case study of MM-141, a tetravalent bispecific antibody targeting two compensatory signaling growth factor receptors: insulin-like growth factor 1 receptor (IGF-1R) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3). A MM-141 proof-of-concept (POC) parent molecule did not meet initial design criteria due to modest bioactivity and poor stability properties. Using a combination of yeast display, structured-guided antibody design and library-scale thermal challenge assay, we discovered a diverse set of stable and active anti-IGF-1R and anti-ErbB3 single-chain variable fragments (scFvs). These optimized modules were reformatted to create a diverse set of full-length tetravalent bispecific antibodies. These re-engineered molecules achieved complete blockade of growth factor induced pro-survival signaling, were stable in serum, and had adequate activity and pharmaceutical properties for clinical development. We believe this approach can be readily applied to the optimization of other classes of bispecific or even multispecific antibody-like molecules.

Renal dialysis, diabetes and iugr: delivery and peripartum care

A woman with Type I diabetes presented at 6+0/40 with a history of severe retinopathy, hypertension and nephropathy. A previous combined renal and pancreatic transplant had failed necessitating haemodialysis five...

Beckwith–Wiedemann syndrome with placental chorangioma due to H19‐differentially methylated region hypermethylation: A case report

Beckwith-Wiedemann syndrome (BWS) is a common overgrowth syndrome that involves abdominal wall defects, macroglossia, and gigantism. It is sometimes complicated by placental tumor and polyhydramnios. We report a case of BWS, prenatally diagnosed with ultrasonography. A large and well-circumscribed tumor also existed on the fetal surface of the placenta, which was histologically diagnosed as chorangioma after delivery. Polyhydramnios was obvious and the fetal heart enlarged progressively during pregnancy. Because the biophysical profiling score dropped to 4 points at 33 weeks of gestation, we carried out cesarean section. By epigenetic analysis, H19-differentially methylated region hypermethylation was observed in the placental tumor, normal placental tissue, and cord blood mononuclear cells. This is the first report of BWS with placental tumor due to H19-differentially methylated region hypermethylation.

Biophysical profiling of tumor cell lines.

Despite significant differences in genetic profiles, cancer cells share common phenotypic properties, including membrane-associated changes that facilitate invasion and metastasis. The Corning Epic® optical biosensor was used to monitor dynamic mass rearrangements within and proximal to the cell membrane in tumor cell lines derived from cancers of the colon, bone, cervix, lung and breast. Data was collected in real time and required no exogenously added signaling moiety (signal-free technology). Cell lines displayed unique profiles over the time-courses: the time-courses all displayed initial signal increases to maximal values, but the rate of increase to those maxima and the value of those maxima were distinct for each cell line. The rate of decline following the maxima also differed among cell lines. There were correlations between the signal maxima and the observed metastatic behavior of the cells in xenograft experiments; for most cell types the cells that were more highly metastatic in mice had lower time-course maxima values, however the reverse was seen in breast cancer cells. The unique profiles of these cell lines and the correlation of at least one profile characteristic with metastatic behavior demonstrate the potential utility of biophysical tumor cell profiling in the study of cancer biology.

Biophysical Profiling of Tumor Cell Lines

Despite significant differences in genetic profiles, cancer cells share common phenotypic properties, including membrane-associated changes that facilitate invasion and metastasis. The Corning Epic® optical biosensor was used to monitor dynamic mass rearrangements within and proximal to the cell membrane in tumor cell lines derived from cancers of the colon, bone, cervix, lung and breast. Data was collected in real time and required no exogenously added signaling moiety (signal-free technology). Cell lines displayed unique profiles over the time-courses: the time-courses all displayed initial signal increases to maximal values, but the rate of increase to those maxima and the value of those maxima were distinct for each cell line. The rate of decline following the maxima also differed among cell lines. There were correlations between the signal maxima and the observed metastatic behavior of the cells in xenograft experiments; for most cell types the cells that were more highly metastatic in mice had lower time-course maxima values, however the reverse was seen in breast cancer cells. The unique profiles of these cell lines and the correlation of at least one profile characteristic with metastatic behavior demonstrate the potential utility of biophysical tumor cell profiling in the study of cancer biology.

Identifying placental dysfunction in women with reduced fetal movements can be used to predict patients at increased risk of pregnancy complications

Maternal perception of fetal movements has historically been used to indicate fetal wellbeing, and has been used with varying success in recent years to identify those pregnancies at increased risk of stillbirth, and other placental pathologies. We present a hypothesis that links reduced fetal movements (RFM) to fetal growth restriction (FGR) and stillbirth through placental dysfunction, and suggests the possibility that this can allow development of a reliable method to identify those women experiencing RFM who are at increased risk of adverse outcome. Reduced fetal movement is thought to represent fetal compensation in a chronic hypoxic environment due to inadequacies in the placental supply of oxygen and nutrients. Placental analysis in FGR and in stillbirth has revealed a number of structural abnormalities and an imbalance in cell turnover, and in terms of function, FGR is also associated with reduced nutrient transport. Both FGR and stillbirth are linked to changes in maternal levels of placental hormones. However, no such studies have been performed in samples from pregnancies affected by RFM. Currently, there are no formal guidelines to direct the management of such women, although it is recommended they undergo measurement of symphysis-fundal height and cardiotocography, and possibly Doppler ultrasound and biophysical profiling. Novel tests could involve the measurement of placental-derived hormones in maternal serum. To address this hypothesis, macroscopic and microscopic analysis of placental samples from both normal pregnancies and those affected by RFM is needed to detect any changes in structure. Placental function could be evaluated by levels of placental hormones in maternal blood. If placental dysfunction can be linked to RFM, and a robust method of identifying those women with placental insufficiency can be developed; screening patients with RFM could lead to a reduction in perinatal morbidity and mortality.

Managing the Calf at Calving Time

Perinatal mortality rates are increasing internationally, particularly in Holstein-Friesian primiparae. The prevalence of perinatal mortality in US dairy herds is currently 8%. This article outlines our current knowledge of bovine perinatal pathophysiology and presents practical guidelines on management of the calf at calving time. Biophysical profiling of the newborn calf, without sophisticated equipment, is the first step in diagnosing at-risk perinates. Resuscitative techniques adaptable for both the producer and the veterinarian are detailed. In addition, management of prolonged recumbence, hypothermia, failure to suck, umbilical antisepsis and calf movement after calving are discussed. Knowledge gaps constraining future progress towards better newborn calf management are highlighted. Finally, current topics in perinatal mortality are presented.

Inpatient versus Outpatient Management of Monoamniotic Twins and Outcomes

A recent literature review holds that approximately one in 10 cases of monoamniotic twinning ends with perinatal death. An entangled umbilical cord may be fatal and is not a predictable complication. This multicenter retrospective records review was undertaken to learn whether outcomes might be improved by hospitalization when monoamniotic twins are at 24 to 28 weeks gestation and close fetal surveillance is maintained until delivery at 32 to 34 weeks. Review of a recent 10-year period at 4 centers totaling approximately 15,000 deliveries annually yielded 23 sets of monoamniotic twins, 11 managed as inpatients and 12 given outpatient care. Women in the inpatient group had continuous external fetal monitoring and underwent ultrasound (to monitor fetal growth) and Doppler flow studies at 4-week intervals. Outpatient management included nonstress testing or biophysical profiling at least 3 times a week. There were no fetal deaths in the inpatient group, but 3 such deaths occurred with outpatient management. Inpatients had gestational ages of 30 to 36 weeks when delivered. There were 7 elective operative deliveries in the inpatient group, and 4 sections were done because of a nonreassuring fetal heart tracing. Eleven infants had respiratory distress syndrome; 3, intraventricular hemorrhage; 3, anomalies; and one, sepsis. Two infants were apparently free of complications. In the outpatient group, gestational ages at delivery ranged from 27 to 34 weeks. Three cesarean sections were done electively and 6 others because of a nonreassuring fetal heart tracing. There were 2 cases each of preterm labor and intrauterine fetal demise and one each of severe preeclampsia, increased liver enzymes, and abruption. Sixteen infants in this group had respiratory distress syndrome, 4 had sepsis, and one each had intraventricular hemorrhage and atrial septal defect. Three infants each had patent ductus arteriosis and retinopathy of prematurity, whereas 4 were free of complications. It is not likely that a randomized trial comparing inpatient with outpatient management of monoamniotic twin pregnancies will prove feasible. Pending such a study, the investigators propose that inpatient management be considered.

Hypothesis: selective phosphodiesterase-5 inhibition improves outcome in preeclampsia

The pathogenesis of preeclampsia stems from aberrant changes at the placental interface. The trophoblastic endovascular invasion of tonic spiral arteries that converts them to passive conduits falters. Uteroplacental insufficiency and fetoplacental hypoxemia result. Secondary maternal oxidative stress and an excessive inflammatory response to pregnancy generate the clinical syndrome of preeclampsia. Current treatment focuses on preventing seizures, controlling hypertension, preserving renal function and delivering the baby. We propose that the pathophysiological changes induced by preeclampsia in the placenta parallel those caused by persistent hypoxemia in the lungs at high altitude or with chronic obstructive pulmonary disease. Unrelenting pulmonary hypoxic vasoconstriction induces pulmonary hypertension and cor pulmonale. Inhalation of nitric oxide and phosphodiesterase-5 inhibitors opposes pulmonary hypoxic vasoconstriction, alleviates pulmonary hypertension and improves systemic oxygenation. Notably nitric oxide donor therapy also counters hypoxemic fetoplacental vasoconstriction, a biological response analogous to pulmonary hypoxic vasoconstriction. Fetal oxygenation and nutrition improve. Placental upstream resistance to umbilical arterial blood flow decreases. Fetal right ventricular impedance falls. Heart failure (cor placentale) is avoided. Emergency preterm delivery can be postponed. Other than low dose aspirin and antioxidants vitamins C and E no available therapy specifically targets the underlying disease profile. We hypothesize that, like nitric oxide donation, pharmacological inhibition of placental phosphodiesterase-5 will also protect the fetus but for a longer time. Biological availability of guanosine 3 ′5 ′-cyclic monophosphate is boosted due to slowed hydrolysis. Adenosine 3 ′5 ′-cyclic monphosphate levels increase in parallel. Cyclic nucleotide accumulation dilates intact tonic spiral arteries and counters hypoxemic fetoplacental vasoconstriction. Intervillous and intravillous perfusion pick up. Maternal to fetal placental circulatory matching improves. Enhanced placental oxygen uptake alleviates hypoxemic fetal stress. Appropriate fetal nutrition resumes. Cor placentale and severe intrauterine growth restriction are averted. Increased maternal cyclic nucleotide concentrations promote systemic vasodilatation so that blood pressures fall. Preemption of oxidative stress initiated by “consumptive” oxidation of nitric oxide stabilizes the vascular endothelium and corrects coagulopathy. Anti-inflammatory and immunosuppressant adenosine 3 ′5 ′-cyclic monphosphate offsets the extreme gestational inflammatory response. Cellular injury and multi-organ damage are prevented. One tablet a day of the new long acting phosphodiesterase-5 inhibitor, tadalafil (half life of 17.5 h) theoretically should allow a preterm pregnancy affected by preeclampsia to continue safely. Selective monitoring of vital organ functions guards against life-threatening maternal complications. Regular biophysical profiling warns the obstetrician of impending fetal compromise. Fetal growth and vital organ maturation can continue. As a result workloads imposed upon neonatal intensivists will lighten. Parental anxiety and concern will be allayed. The cost of treating preeclamptic mothers and their extremely low birth weight infants will decrease. Money saved by midwifery services in poorer states can be used to pay for better prenatal care. Severe preeclampsia/eclampsia will be less common. Maternal and perinatal morbidity and mortality will be reduced. Because the human immunodeficiency virus often infects individuals at a workforce eligible age, the global acquired immunodeficiency syndrome pandemic has already brought many nations to the brink of economic ruin. Potentially productive lives saved for the future will help restore them fiscally.