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Study Objectives: Insomnia and sleep problems are common in pregnancy and have potentially negative impacts on both parental and infant health. This study examined the Sleeping for Two adaptation of cognitive behavioral therapy for insomnia (CBT-I) in pregnancy., Methods: A parallel (1:1) randomized controlled trial evaluated CBT-I ( n = 32) compared to a treatment as usual waitlist ( n = 32) among pregnant individuals from Alberta, Canada experiencing insomnia. Five weekly individual sessions of CBT-I pivoted from in-person delivery to telehealth due to the COVID-19 pandemic physical distancing regulations. Insomnia symptom severity (primary outcome), insomnia diagnosis by structured interview, self-reported sleep problems, as well as sleep parameters measured by diary and actigraphy were assessed pretreatment at 12-28 weeks gestation, 1-week posttreatment, and 6 months postpartum. Birth information (secondary outcomes) were collected via delivery record and parent report of infant sleep (exploratory outcome) was taken at 6 months postpartum., Results: Multilevel modeling using an intention-to-treat approach showed that CBT-I was associated with a decrease in insomnia symptoms and improved sleep quality across time compared to treatment as usual. The CBT-I group had fewer diagnoses of insomnia posttreatment, but the difference did not reach statistical significance until 6 months postpartum. Participants with worse sleep quality at baseline benefitted substantially more from CBT-I vs treatment as usual waitlist., Conclusions: CBT-I delivered in pregnancy can reduce symptoms of insomnia and improve sleep quality, which could in turn minimize risk of negative consequences for birthing parent and infant health., Clinical Trial Registration: Registry: ClinicalTrials.gov; Name: Sleeping for Two: RCT of CBT-Insomnia in Pregnancy; URL: https://www.clinicaltrials.gov/study/NCT03301727; Identifier: NCT03918057., Citation: MacKinnon AL, Silang K, Watts D, et al. Sleeping for Two: a randomized controlled trial of cognitive behavioral therapy for insomnia in pregnancy. J Clin Sleep Med . 2025;21(2):365-376., (© 2025 American Academy of Sleep Medicine.)

The COVID-19 pandemic has prompted extensive disruptions to the daily lives of children and adolescents worldwide, which has been associated with an increase in anxiety and depressive symptoms in youth. However, due to public health measures, in-person psychosocial care was initially reduced, causing barriers to mental health care access. This study investigated the feasibility, acceptability and preliminary effectiveness of iCOPE with COVID-19, a brief telemental health intervention for children and adolescents to address anxiety symptoms. Sessions were provided exclusively using videoconferencing technology. Feasibility and acceptability were measured with client satisfaction data. The main outcome measure for effectiveness was anxiety symptom severity measured using the Screen for Child Anxiety and Related Disorders (SCARED). Results indicated that the treatment was well accepted by participants. Significant reductions in anxiety were noted for social anxiety, and were observed to be trending towards a mean decrease for total anxiety. The findings suggest that this brief telemental health intervention focused on reducing anxiety related to COVID-19 is acceptable and feasible to children and adolescents. Future research using a large sample and with a longer follow-up period could inform whether symptom decreases are sustained over time., (© 2023 Association for Behavioral and Cognitive therapist behaviors Therapies.)

Background: Insomnia and sleep disturbances are common in pregnancy and have potentially significant consequences for both maternal and infant health. There is limited research examining the effectiveness of cognitive behavioral therapy for insomnia (CBT-I) during pregnancy. With increased distress and limited access to services during the COVID-19 pandemic, there is also an unprecedented need for telehealth delivery of treatment programs for pregnant women. The aims of this trial are to evaluate the impact of the Sleeping for Two adaptation of CBT-I in pregnancy (in-person or telehealth) versus treatment as usual (TAU) in reducing symptoms of insomnia (primary outcome), as well as increasing gestational length and reducing symptoms of depression (secondary outcomes)., Methods: A two-arm, single-blinded, parallel group randomized controlled trial (RCT) design with repeated measures will be used to evaluate the impact of CBT-I compared to TAU among a sample of 62 pregnant women, enrolled between 12 and 28 weeks of gestation, who self-identify as experiencing insomnia. Five weekly individual sessions of CBT-I will be delivered in person or via telehealth depending on physical distancing guidelines. Assessment of insomnia diagnosis by structured interview, self-reported insomnia symptom severity and sleep problems, and sleep quantity and quality as measured by a daily diary and actigraphy will occur at 12-28 weeks of pregnancy (T1), 1 week post-treatment (T2), and 6 months postpartum (T3)., Discussion: CBT-I delivered in pregnancy has the potential to reduce symptoms of insomnia and depression and could lead to reduced risk of preterm birth, all of which can minimize risk of negative maternal and child health and developmental consequences in the short (e.g., infant death) and long terms (e.g., developmental delays). This RCT builds on a successful open pilot trial conducted by our team and will provide further evaluation of a novel evidence-based treatment for pregnancy-related insomnia, which can be widely disseminated and used to treat individuals that are most in need of intervention. Findings will enhance understanding of pregnancy-related sleep problems, as well as means by which to improve the health and sleep of mothers and their children., Trial Registration: ClinicalTrials.gov NCT03918057. Registered on 17 April 2019., (© 2021. The Author(s).)

Objective: Cognitive-behavioral therapy for insomnia (CBT-I) is an effective insomnia treatment but has yet to be applied to adolescents with sleep disruption following concussion. This pilot study evaluated CBT-I to improve insomnia in adolescents with protracted concussion recovery., Setting: Tertiary pediatric hospital., Participants: Participants (N = 24) were 12 to 18 years old (M = 15.0, SD = 1.4), 15.1 weeks (SD = 9.2) postinjury, and presenting with sleep disruption and persistent postconcussion symptoms., Design: A single-blind, parallel-group randomized controlled trial (RCT) design comparing 6 weeks of CBT-I and a treatment-as-usual control group. Outcomes were measured before treatment, at treatment completion, and 4 weeks after completion., Main Measures: Primary outcome was Insomnia Severity Index. Secondary outcomes included Pittsburgh Sleep Quality Index, Dysfunctional Beliefs and Attitudes about Sleep Scale, 7-night sleep diary, PROMIS Depression, PROMIS Anxiety, and Health and Behavior Inventory., Results: Adolescents who received CBT-I demonstrated large and clinically significant improvements in insomnia ratings at posttreatment that were maintained at follow-up. They also reported improved sleep quality, fewer dysfunctional beliefs about sleep, better sleep efficiency, shorter sleep-onset latency, and longer sleep time compared with those with treatment as usual. There was also a modest reduction in postconcussion symptoms., Conclusion: In this pilot RCT, 6 weeks of CBT-I produced significant improvement in sleep in adolescents with persistent postconcussion symptoms. A larger trial is warranted.

The transition to parenthood has been identified as a significant relationship stressor. Many couples report declines in relationship satisfaction and difficulty with individual stress and co-parenting-problems that have been associated with both child temperament as well as emotional and behavioral problems. Several parenting and relationship interventions have been developed to buffer against these difficulties. In the current study, we report secondary analyses of a randomized controlled trial of brief (6-hr) interventions that focused on improving either relationship satisfaction or co-parenting, delivered during pregnancy and the early postpartum period. In this trial, 90 opposite-sex couples (180 participants), who were pregnant with their first child, and were assessed as being at high risk for declines in relationship satisfaction, were randomized to receive either (1) a relationship intervention, (2) a co-parenting intervention, or (3) an information control. At 12 months postpartum, couples who received either the relationship or co-parenting intervention rated their infants as having lower negative emotionality and as having fewer externalizing symptoms compared to the information-only control. Lower externalizing symptoms at 12 months were, in turn, associated with reduced externalizing symptoms at 24 months postpartum. Whereas, lower ratings of child negative emotionality at 12 months were associated with reduced internalizing symptoms at 24 months postpartum. These results indicate that brief relationship or co-parenting interventions delivered during the transition to parenthood have secondary benefits for child mental health., Competing Interests: The authors declare no conflicts of interest.

Introduction: Insomnia during pregnancy is prevalent, yet little is known about preferred treatments for the disorder. The current study investigated both pregnant women's and their partners' preferences for treatment of maternal insomnia, comparing the two most empirically supported therapies: cognitive-behavioral therapy for insomnia (CBT-I) and pharmacotherapy., Method: We recruited pregnant women and their partners ( N = 212) from a low-risk maternity clinic in Calgary, AB, Canada. Participants read expert validated treatment descriptions of pharmacotherapy and CBT-I and indicated their preference for treatment of maternal insomnia. Participants also completed the Insomnia Severity Index and Edinburgh Postnatal Depression Scale., Results: The majority of pregnant women (72.6%) and their partners (73.6%) selected CBT-I as their preferred choice for treatment of maternal insomnia, with a high concordance between partners. Correlates of maternal treatment choice included previous use of treatment and partner preference., Discussion: Findings suggest that pregnant women and their partners prefer CBT-I to pharmacotherapy as a treatment for maternal insomnia. Partners' preference may be an important consideration for women selecting treatment for insomnia. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Couple therapy reduces relational and individual distress and may affect utilization of other health services, particularly among higher service utilizers. Although average decreases in service utilization are predicted among recipients of couple therapy, low utilizers of services may appropriately increase use. The relationship between couple therapy and service utilization was examined among a sample of 179 U.S. military veterans who received treatment in Veterans Affairs (VA) specialty couple therapy clinics. Consistent with hypotheses, overall mental and physical health visits decreased from the 12 months preceding couple therapy to the 12 months following treatment. Moderator analyses showed that decreases were greatest among individuals who were rated by their therapist as having completed a full course of couple therapy, suggesting that change was attributable to intervention. Pretreatment service utilization also moderated observed change-higher utilizers' use of services decreased substantially, whereas lower utilizers' slightly increased. Cost analyses revealed that the estimated per person mean cost in our sample decreased by $930.33 in the year following compared to the year prior to couple therapy, as per 2008 VA cost data. As service utilization data were only available for one partner and only for 1 year posttherapy, the true magnitude of this effect may be underestimated. Our findings are relevant to policy makers as they demonstrate that couple therapy reduces average service utilization and associated costs and addresses calls for analyses of cost effectiveness of systemic interventions., (© 2016 Family Process Institute.)

Background: Clinically significant psychological distress in pregnancy is common, with epidemiological research suggesting that between 15 and 25 % of pregnant women experience elevated symptoms of stress, anxiety, and depression. Untreated psychological distress in pregnancy is associated with poor obstetrical outcomes, changes in maternal physiology, elevated incidence of child physical and psychological disorders, and is predictive of maternal postpartum mood disorders. Despite the wide-ranging impact of antenatal psychological distress on mothers and their children, there is a gap in our knowledge about the most effective treatments that are available for psychological distress experienced in pregnancy. Additionally, no trials have focused on potential physiological changes that may occur as a result of receiving mindfulness training in pregnancy. The proposed trial will determine the effectiveness of an 8-week modified Mindfulness-based Cognitive Therapy (MBCT) intervention delivered during pregnancy., Methods: A randomized controlled trial (RCT) design with repeated measures will be used to evaluate the effectiveness of MBCT to treat psychological distress in pregnancy. A sample of 60 consenting pregnant women aged 18 years and above will be enrolled and randomized to the experimental (MBCT) or control (treatment as usual) condition. Primary (e.g., symptoms of stress, depression, and anxiety), secondary (cortisol, blood pressure (BP), heart rate variability (HRV), and sleep) and other outcome data (e.g., psychological diagnoses) will be collected via a combination of laboratory visits and at-home assessments from both groups at baseline (T 1 ), immediately following the intervention (T 2 ), and at 3 months postpartum (T 3 ). Descriptive statistics will be used to describe sample characteristics. Data will be analyzed using an intention-to-treat approach. Hierarchical linear models will be used to test intervention effects on primary and secondary outcomes., Discussion: The trial is expected to improve knowledge about evidence-based treatments for psychological distress experienced in pregnancy and to evaluate the potential impact of mindfulness-based interventions on maternal physiology., Trial Registration: ClinicalTrials.gov: NCT02214732 , registered on 7 August 2014. Protocol Version 2.0., 5 September 2016.

Poor sleep and low social support have each been associated with mortality and morbidity from chronic illness, and a small body of research suggests that the two interact to influence systemic inflammation whereby good social relationships may buffer the relationship between poor sleep and increased inflammation. The current study investigated interactions between sleep and social support in the prediction of inflammation in a clinical population (prehypertensive and hypertensive individuals) at high risk for the development of cardiovascular disease. Using a standardized subjective measure of sleep quality, we found that social support moderated the association between sleep and circulating levels of both IL-6 and CRP, such that poor sleep appeared to confer a risk of increased inflammation only in those participants who also reported low social support. In women, the same relationship was observed for TNF-α. These results extend previous findings into a clinical population and also demonstrate that sleep quality and social support interact in the prediction of two previously uninvestigated clinically relevant inflammatory markers (CRP and TNF-α). High levels of perceived social support may compensate for the negative health impact of poor sleep quality and vice versa., (© 2015 Society for Psychophysiological Research.)

Background: Among the most serious sequelae to an initial episode of Major Depressive Disorder (MDD) during adolescence is the significant increase in the probability of recurrence. This study reports on an integrated CBT/IPT program, provided in a group format, that was developed to decrease the rate of MDD recurrence in emerging adults., Methods: Participants were 89 young adults who were not depressed at study entry but had experienced MDD during adolescence. Participants were assigned to a CBT/IPT prevention program or to an assessment only control condition and were followed through the first 2 years of college., Results: Risk for MDD recurrence was reduced more than 50% for the prevention program participants compared to assessment only controls. The intervention also conferred beneficial effects on academic performance for those students who completed the majority of the group sessions., Limitations: The study included a self-selected sample of emerging adults who were aware of their history of depression. Due to the small sample size, it will be important to evaluate similar interventions in adequately-powered trials to determine if this is a replicable finding., Conclusions: With 51% of the assessment only participants experiencing a MDD recurrence during the first 2 years of college, these findings support the need for programs designed to prevent MDD recurrence in young adults. The current program, based on IPT and CBT principles, appears to reduce the rate of MDD recurrence among previously depressed emerging adults., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Previous studies of couple therapy have conceptualized change as a gradual process. However, there is growing evidence that, for many clients, the majority of gains in other treatment modalities are often achieved between just 2 sessions. Isolating the frequency, nature, and predictors of these sudden gains (SGs) in couple therapy can add to a growing understanding of how and why couple therapy works. In a sample of 67 couples receiving treatment-as-usual couple therapy in two Veterans Affairs hospitals, 25% of individuals experienced a SG in relationship satisfaction. The magnitude of these SGs were large (d=1.62) and fully explained the total pre-post change for individuals who experienced them. Individuals with SGs showed significantly greater satisfaction gains during therapy; however SGs were not related to relationship satisfaction or relationship status at 18-month follow-up. SGs were predicted by the content of the previous session, putative change mechanisms of communication, intimacy, and behavior, as well as the partner's SGs during the same period. Results suggest that SGs are an important component of change during couple therapy for some individuals, challenging the assumption of continuous change in previous studies. In addition, predictors of SGs were generally consistent with theoretical and empirical examinations of mechanisms of change in couple therapy., (Copyright © 2011. Published by Elsevier Ltd.)

Major depressive disorder (MDD) is characterized by a high risk of recurrence, especially among individuals whose initial episode occurs during adolescence. Identifying predictors of recurrence of MDD among young samples is therefore of paramount clinical importance. Survival analytic models were used to evaluate the effects of dysfunctional cognitions and Axis II personality pathology on MDD recurrence in a sample of 130 previously, but not currently, depressed young adults. Participants were initially assessed for depression, dysfunctional attitudes, and personality pathology during their first semester in college and then reevaluated via the Longitudinal Interval Follow-up Evaluation interview every 6 months for 18 months. Baseline level of depressive symptoms significantly (HR-1.07, p = .002) predicted recurrence of MDD. In the survival analyses with baseline level of depression serving as a current mood state covariate, overall personality pathology (HR-1.04, p < .05), but not cognitive distortions, uniquely predicted MDD recurrence. In similar analyses, none of the specific DSM-IV personality disorder cluster scores uniquely predicted recurrence. We discussed the theoretical, empirical, and clinical implications of these findings, and we noted the limitations of the study.

Objective: In spite of high levels of antidepressant nonadherence frequently observed among depressed samples, relatively little research has investigated psychosocial predictors of adherence. Fostering greater collaboration in depression treatment to increase adherence has been advocated, but this strategy has not been adequately studied. The aim of this study was to examine the interaction of provider collaboration and patient reactance in the prediction of antidepressant adherence during the acute treatment phase., Method: Fifty outpatients diagnosed with major depressive disorder beginning antidepressant treatment within psychiatry clinics of the VA San Diego Healthcare System comprised the study sample. Patients were administered questionnaires following their medication evaluations to measure predictor variables. Antidepressant adherence was assessed via brief telephone interviews 3, 6, 9, and 12 weeks after treatment initiation. The roles of provider collaboration, patient reactance, and their interaction in adherence were examined using multiple regression analyses., Results: The interaction between provider collaboration and patient reactance accounted for 18.3% of the variance in 3-week adherence (P<.01). Among more reactant patients, greater levels of collaboration predicted better adherence, whereas among patients lower in reactance less collaboration predicted better adherence. No relationships were observed beyond the initial 3 weeks of treatment., Conclusions: This study demonstrates that interpersonal process variables are important in influencing antidepressant adherence and challenges the advocacy of more collaboration in antidepressant treatment as a "blanket strategy." Establishing a more collaborative set with reactant patients may ensure greater early treatment adherence, a critical period during which antidepressants have typically not yet taken effect., ((c) 2008 Wiley-Liss, Inc.)

Objective: To propose a paradigm shift in the role of the pharmacist by encouraging pharmacists' involvement in the achievement and maintenance of healthy lifestyles in their patients, thus reducing the need for long-term medications., Data Sources: Peer-reviewed medical literature, contemporary lay literature, news releases from health organizations/associations, United States health standards, and professional experiences of the authors., Summary: Our current paradigm for the management of chronic disease is malaligned. The medications we use are helpful for many patients with chronic disorders, but they are not curative and leave many individuals with unwanted adverse effects that are often treated with additional medication. The more effective approach to managing many patients with chronic diseases induced through unhealthy lifestyles would be to eat properly, exercise to maintain ideal body weight, and abstain from harmful substances. Managing the root causes of chronic disease would eliminate most of the unhealthy consequences and perhaps reduce the need for long-term medication. Pharmacists have the knowledge and capacity to assist in this endeavor., Conclusion: Influencing patients to achieve ideal body weight through proper diet and exercise, encouraging people to stay well, and then helping them to keep well are roles for the pharmacist. If pharmacists made wellness and disease prevention part of their practices, they would have greater impact on improving patients' overall health. Without treating the primary cause of a condition and only treating the symptoms of unhealthy lifestyles, we are in many ways "spitting into the wind."

Basic fibroblast growth factor (FGF-2) is an important stimulator of angiogenesis that has been implicated in neoplastic progression. Attempts to neutralize or modulate FGF-2 have met with some success in controlling neovascularity and tumor growth. In the present study, two peptides: one corresponding to the heparin binding domain and the other to the receptor binding domain of FGF-2, exerted dose-dependent inhibition of FGF-2-stimulated human umbilical vein endothelial cell proliferation (IC(50)=70 and 20 microg/ml, respectively). The identification of these functional regions suggested that targeting these domains might be an approach for the modulation of FGF-2 function. To investigate this possibility, we vaccinated mice with either the heparin binding domain peptide or the receptor binding domain peptide of FGF-2 in a liposome/adjuvant format, and analyzed the effect of vaccination on FGF-2-driven angiogenesis, tumor development and immune status. Mice vaccinated with the heparin binding domain peptide generated a specific antibody response to FGF-2, blocked neovascularization in a gelfoam sponge model of angiogenesis, and inhibited experimental metastasis by >90% in two tumor models: the B16BL6 melanoma and the Lewis lung carcinoma. These effects were not observed in mice treated with the receptor binding domain peptide conjugated to liposomes or liposomes lacking conjugated peptide. These data suggest that a heparin binding domain peptide of FGF-2, when presented to a host in a liposomal adjuvant formulation, can ultimately lead to inhibition of angiogenesis and tumor growth.

A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported. rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography. Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification. Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis. On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9). The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials. Methods development, process synthesis, and process scale-up data are presented and discussed., (Copyright 2000 Academic Press.)

Endostatin, a potent endogenous inhibitor of angiogenesis, inhibits the growth of primary tumors without induction of acquired drug resistance in mice. We report that a soluble recombinant human (rh) Endostatin produced with characteristics of the native Endostatin, effectively inhibited the growth of primary tumors and pulmonary metastases in a dose-dependent manner. We also show that deletion of two of the four zinc ligands of rhEndostatin did not affect this potent tumor inhibiton. The growth of established Lewis lung primary tumors implanted into mice was inhibited (80-90%) upon systemic treatment with 50 mg/kg/12 h of rhEndostatin. Using the B16-BL6 murine experimental pulmonary metastases model, rhEndostatin administered at 1.5 mg/kg/day or 4.5 mg/kg/day beginning 3- or 11-days post tumor cell injection, respectively, resulted in an approximate 80% inhibition of tumor growth. At effective anti-tumor doses of 1.5 and 50 mg/kg, pharmacokinetic modeling in mice showed (a) the protein was 100% bioavailable, (b) the AUC ranged from 16 to 700 ng ml/h and (c) the Cmax ranged from 161 to 4582 ng/ml. At the highest dose tested (300 mg/kg), delivered as a single bolus, no drug-related toxicity was observed in a Cynomolgus monkey infused with rhEndostatin. No toxicity was observed even at AUC and Cmax values that were 1.3- to 56-fold higher than those observed in mice with tumors that were potently inhibited. Our production system yields a well characterized, soluble and potent rhEndostatin at quantities sufficient for human use. The preclinical studies described herein are an important first step toward the assessment of Endostatin in the clinic.

Endogenous murine angiostatin, identified as an internal fragment of plasminogen, blocks neovascularization and growth of experimental primary and metastatic tumors in vivo. A recombinant protein comprising kringles 1-4 of human plasminogen (amino acids 93-470) expressed in Pichia pastoris had physical properties (molecular size, binding to lysine, reactivity with antibody to kringles 1-3) that mimicked native angiostatin. This recombinant Angiostatin protein inhibited the proliferation of bovine capillary endothelial cells in vitro. Systemic administration of recombinant Angiostatin protein at doses of 1.5 mg/kg suppressed the growth of Lewis lung carcinoma-low metastatic phenotype metastases in C57BL/6 mice by greater than 90%; administration of the recombinant protein at doses of 100 mg/kg also suppressed the growth of primary Lewis lung carcinoma-low metastatic phenotype tumors. These findings demonstrate unambiguously that the antiangiogenic and antitumor activity of endogenous angiostatin resides within kringles 1-4 of plasminogen.

An analytical immunoblotting procedure and a serological enzyme-linked immunosorbent assay (ELISA) for the characterization of antibodies to cholesterol are described. Hydrophobic membranes consisting of polyvinylidene fluoride (PVDF) are used to immobilize cholesterol for immunodetection by anti-sterol antibodies. To determine whether antibodies to cholesterol were induced after immunization with liposomal cholesterol, we separated total lipid extracts of very-low density lipoproteins by thin layer chromatography (TLC) on silica gel plates and transferred the separated lipid classes to PVDF membranes using isopropanol to facilitate passive diffusion. Lipid transfer was confirmed by exposure of membranes to iodine vapors or by staining of cholesterol with filipin complex. Serum from immunized mice reacted with cholesterol, whereas pre-immune serum or serum from mice injected with control liposomes did not bind. To determine the amount of anti-cholesterol activity in serum, we coated microtiter plates consisting of PVDF membrane wells with cholesterol. The PVDF membrane-based ELISA was found to be more reproducible and four-fold more sensitive than the conventional ELISA on polystyrene plates. These techniques may be useful in the analysis of anti-sterol antibodies and antibodies to other hydrophobic antigens.

We reasoned that de novo oxidative damage, as a result of increased protein glycosylation, could participate in the mechanisms whereby diabetic erythrocytes acquire membrane abnormalities. To examine this hypothesis, the extent of erythrocyte membrane protein glycosylation and the oxidative status of spectrin, the major component of the erythrocyte membrane skeleton, were examined. Labeling erythrocyte membranes with [3H]borohydride, which labels glucose residues bound to proteins, revealed that several proteins were heavily glycosylated compared with nondiabetic erythrocyte membranes. In particular, the proteins beta-spectrin, ankyrin, and protein 4.2 were the most glycosylated. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis of diabetic erythrocyte membranes did not reveal any quantitative or qualitative abnormalities in spectrin or other membrane proteins, examination of spectrin oxidative status by amino acid analysis and with cis-dichlorodiammineplatinum(II) (cDDP), a chemical probe specific for protein methionine and cysteine residues, demonstrated that the diabetic spectrin was oxidatively damaged: spectrin from diabetic subjects contained 35% less methionine (P less than 0.002), 15% less histidine (P less than 0.006), and a twofold increase in cysteic acid (P less than 0.001) compared with normal spectrin. Diabetic spectrin bound 32% less cDDP than normal spectrin (P less than 0.001); the lowest cDDP binding was observed with spectrin from insulin-dependent diabetic subjects. The extent of cDDP binding to diabetic spectrin correlated moderately and inversely with glycosylated hemoglobin (GHb) levels (n = 12, r = -0.727). Erythrocyte deformability, measured by ektacytometry, was decreased between 5 and 23% of control measurements (average of approximately 10%) in 21 of 32 diabetic subjects surveyed.(ABSTRACT TRUNCATED AT 250 WORDS)

An efficient method for the synthesis and purification of a variety of iodinated phospholipid analogues is described. 1-Acyl-2-[[[3-(3-[125I]iodo-4-hydroxyphenyl)- propionyl]amino]caproyl]phosphatidylcholine (125I-PC) was prepared by alkylation of 1-acyl-2-(aminocaproyl)phosphatidylcholine with monoiodinated Bolton-Hunter reagent. 125I-Labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine were produced from 125I-PC by phospholipase D catalyzed base exchange in the presence of ethanol-amine or L-serine. All of these lipid analogues transferred readily from donor vesicles into recipient membranes. When an excess of acceptor vesicles was mixed with a population of donor vesicles containing the iodinated analogues, approximately 50% of the 125I-labeled lipids transferred to the acceptor vesicle population. In addition, under appropriate incubation conditions, these lipids were observed to transfer from vesicles to mammalian cells. Autoradiographic analysis of 125I-labeled lipids extracted from the cells after incubation with vesicles at 2 degrees C for 60 min revealed that a large proportion of the 125I-labeled phosphatidic acid was metabolized to 125I-labeled diglyceride and 125I-labeled phosphatidylcholine, whereas no metabolism of exogenously supplied 125I-labeled phosphatidylethanolamine or 125I-labeled phosphatidylcholine could be detected.

We have previously investigated the interaction of macrophages with red blood cells (RBC) displaying phosphatidylserine (PS) in their surface membranes after the transfer of the fluorescent lipid analog 1-acyl-2-[(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl] phosphatidylserine to the RBC (Tanaka, Y., and Schroit, A. J. (1983) J. Biol. Chem. 258, 11335-11343). This derivative, which is rapidly transferred to the RBC at 37 degrees C, results in the efficient binding and phagocytosis of the RBC by autologous macrophages. In the present study, we show that 51Cr-labeled RBC containing [(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminododecanoyl]phosphatidylserine (NBD-PS) are rapidly cleared from the peripheral circulation of syngeneic mice and accumulate in the liver and spleen. Fluorescence microscopy of Kupffer cells and splenic macrophages isolated from the liver and spleens of these animals revealed phagocytosed fluorescent RBC, suggesting the clearance was probably due to endocytosis of the RBC. The accumulation of these RBC in the spleen was dramatic, with approximately 30% of the injected cells localizing in this organ within 60 min. In contrast, the intravenous injection of RBC containing similar amounts of NBD-phosphatidylcholine or NBD-phosphatidylglycerol did not result in clearance which differed significantly from control (untreated) RBC populations. The observed clearance of NBD-PS-containing RBC was much different than the clearance of opsonized RBC which preferentially localized in the liver. These findings show that PS in RBC can serve as a signal for triggering their in vivo recognition and concomitant elimination from the circulation and suggest that the exposure of endogenous PS in the outer leaflet of RBC which occurs in certain pathological conditions could trigger their removal from the circulation.