Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Symphyseal morphology and jaw muscle recruitment levels during mastication in musteloid carnivorans.

In studies of mammalian mastication, a possible relationship has been proposed between bilateral recruitment of jaw adductor muscle force during unilateral chewing and the degree of fusion of the mandibular symphysis. Specifically, species that have unfused, mobile mandibular symphyses tend to utilize lower levels of jaw adductor force on the balancing (nonchewing) than the working (chewing) side of the head, when compared to related species with fused symphyses. Here, we compare jaw adductor recruitment levels in two species of musteloid carnivoran: the carnivorous ferret (unfused symphysis), and the frugivorous kinkajou (fused symphysis). During forceful chewing, we observe that ferrets recruit far more working-side muscle force than kinkajous, regardless of food toughness and that high working-to-balancing side ratios are the result of increased working-side force, often coupled with reduced balancing-side force. We propose that in carnivorans, high working-to-balancing side force ratios coupled with an unfused mandibular symphysis are necessary to rotate the hemimandible for precise unilateral occlusion of the carnassial teeth and to sustain laterally oriented force on the jaw to engage the carnassial teeth during shearing of tough foods. In contrast, the kinkajou's flattened cheekteeth permit less precise occlusion and require medially-oriented forces for grinding, thus, a fused symphysis is mechanically beneficial.

Effect of clinician information sessions on diagnostic testing for Chagas disease.

BACKGROUND: Chagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11). CONCLUSION/SIGNIFICANCE: In this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.

Alcohol drinking during early adolescence activates microglial cells and increases frontolimbic Interleukin-1 beta and Toll-like receptor 4 gene expression, with heightened sensitivity in male rats compared to females.

Adolescent drinking is risky because neural circuits in the frontal lobes are undergoing maturational processes important for cognitive function and behavioral control in adulthood. Previous studies have shown that myelinated axons in the medial prefrontal cortex (mPFC) are particularly sensitive to alcohol drinking, especially in males. Pro-inflammatory mediators like toll-like receptor 4 (TLR4) and interleukin-1 beta (IL1b) have been implicated in alcohol induced-inflammation and demyelination; thus, herein we test the hypothesis that voluntary alcohol drinking early in adolescence elicits a pro-inflammatory state that is more pronounced in the brain of males compared to females. Adolescent male and female Wistar rats self-administered sweetened alcohol or sweetened water from postnatal days 28-42 and separate sets of brains were processed for 1) immunolabeling for ionized calcium-binding adapter molecule 1 to analyze microglial cell morphology, or 2) qPCR analysis of gene expression of pro-inflammatory mediators. Binge drinking alcohol activated microglia in the mPFC and hippocampus of both males and females, suggesting that voluntary alcohol exposure initiates an inflammatory response. Il1b mRNA was upregulated in the mPFC of both sexes. Conversely, Tlr4 mRNA levels were elevated after drinking only in males, which could explain more robust effects of alcohol on myelin in this region in developing males compared to females. Il1b mRNA changes were not observed in the hippocampus, but alcohol elevated Tlr4 mRNA in both sexes, highlighting regional specificity in inflammatory responses to alcohol. Overall, these findings give insight into potential mechanisms by which low-to-moderate voluntary alcohol intake impacts the developing brain. This article is part of the special Issue on 'Vulnerabilities to Substance Abuse'.

Evaluating bony predictors of bite force across the order Carnivora.

In carnivorans, bite force is a critical and ecologically informative variable that has been correlated with multiple morphological, behavioral, and environmental attributes. Whereas in vivo measures of biting performance are difficult to obtain in many taxa-and impossible in extinct species-numerous osteological proxies exist for estimating masticatory muscle size and force. These proxies include both volumetric approximations of muscle dimensions and direct measurements of muscular attachment sites. In this study, we compare three cranial osteological techniques for estimating muscle size (including 2D-photographic and 3D-surface data approaches) against dissection-derived muscle weights and physiological cross-sectional area (PCSA) within the jaw adductor musculature of 40 carnivoran taxa spanning eight families, four orders of magnitude in body size, and the full dietary spectrum of the order. Our results indicate that 3D-approaches provide more accurate estimates of muscle size than do surfaces measured from 2D-lateral photographs. However, estimates of a muscle's maximum cross-sectional area are more closely correlated with muscle mass and PCSA than any estimates derived from muscle attachment areas. These findings highlight the importance of accounting for muscle thickness in osteological estimations of the masticatory musculature; as muscles become volumetrically larger, their larger cross-sectional area does not appear to be associated with a proportional increase in the attachment site area. Though volumetric approaches approximate muscle dimensions well across the order as a whole, caution should be exercised when applying any single method as a predictor across diverse phylogenies.

An Unusual Cause of Cranial Dural Thickening.

We describe an unusual cause of cranial dural thickening in an elderly female with a chronic meningeal inflammatory process. A 70-year-old ethnically Chinese, Singaporean female presented with a history of chronic daily headache with no other meningeal signs. Serial MRI brains showed progressive pachymeningeal and leptomeningeal enhancement in the left frontal region with underlying vasogenic oedema, similar appearances in the right frontal region to a lesser extent, and persistent inflammatory changes in her bilateral paranasal sinuses. Investigative work-up showed a chronically raised ESR with a normal CRP, negative ANCA, and a chronically raised serum IgA kappa paraprotein. Bone marrow trephine biopsy was suggestive of a low level plasma cell disorder. Olfactory cleft biopsy showed no evidence of IgG4-related disease or vasculitis and no significant plasma cell infiltrate. Histopathological examination from a meningeal biopsy revealed a diagnosis of an en-plaque meningioma (the WHO, 2016; Grade I) causing an unusual granulomatous reaction. We discuss the radiological and histological relations of this rare form of meningioma. Clinicians can consider en-plaque meningioma in the differential diagnosis of linear dural thickening and enhancement.

MOG-IgG-associated demyelination: focus on atypical features, brain histopathology and concomitant autoimmunity.

INTRODUCTION: Antibodies to myelin oligodendrocyte glycoprotein (MOG) have been demonstrated in patients with optic neuritis (ON), encephalitis and myelitis. OBJECTIVE: To describe the clinical and paraclinical features in patients with MOG-associated demyelination, focusing on unusual cases, brain biopsy and concomitant autoimmunity. METHODS: A single centre retrospective observational case series, analysing demographic, clinical, laboratory, histopathology and radiological data from MOG- positive patients. RESULTS: We identified 20 adults. The male/female ratio was 1.5. Mean age at onset was 31.6 years and mean disease duration was 7.5 years. The most frequent presentation was myelitis (45%), followed by ON (30%). One case had simultaneous myelitis and ON. Two patients had a cortical syndrome, 1 patient had an encephalopathic presentation and 1 cryptogenic focal epilepsy. Anti-neutrophil cytoplasmic antibodies (ANCA) were found in 3 cases, while 1 patient had an antibody to glutamic acid decarboxylase (GAD). Brain biopsy was performed in 2 patients. Relapsing course was identified in 60% of patients. We also discuss 3 cases with atypical features, brain histopathology and concomitant autoimmunity. CONCLUSION: MOG- associated demyelination represents a new disease entity. Unusual cases are reported, expanding the disease spectrum. Elucidating this further should be the focus of prospective studies.

Narrow band imaging and serology in the assessment of premalignant gastric pathology.

BACKGROUND: Patient outcomes in gastric adenocarcinoma are poor due to late diagnosis. Detecting and treating at the premalignant stage has the potential to improve this. Helicobacter pylori is also a strong risk factor for this disease. AIMS: Primary aims were to assess the diagnostic accuracy of magnified narrow band imaging (NBI-Z) endoscopy and serology in detecting normal mucosa, H. pylori gastritis and gastric atrophy. Secondary aims were to compare the diagnostic accuracies of two classification systems using both NBI-Z and white light endoscopy with magnification (WLE-Z) and evaluate the inter-observer agreement. METHODS: Patients were prospectively recruited. Images of gastric mucosa were stored with histology and serum for IgG H. pylori and Pepsinogen (PG) I/II ELISAs. Blinded expert endoscopists agreed on mucosal pattern. Mucosal images and serological markers were compared with histology. Kappa statistics determined inter-observer variability for randomly allocated images among four experts and four non-experts. RESULTS: 116 patients were prospectively recruited. Diagnostic accuracy of NBI-Z for determining normal gastric mucosa was 0.87(95%CI 0.82-0.92), H. pylori gastritis 0.65(95%CI 0.55-0.75) and gastric atrophy 0.88(95%CI 0.81-0.94). NBI-Z was superior to serology at detecting gastric atrophy: NBI-Z gastric atrophy 0.88(95%CI 0.81-0.94) vs PGI/II ratio < 3 0.74(95%CI 0.62-0.85) p<.0001. Overall NBI-Z was superior to WLE-Z in detecting disease using two validated classifications. Inter-observer agreement was 0.63(95%CI 0.51-0.73). CONCLUSIONS: NBI-Z accurately detects changes in the GI mucosa which currently depend on histology. NBI-Z is useful in the detection of precancerous conditions, potentially improving patient outcomes with early intervention to prevent gastric cancer.

The influence of diet on masticatory motor patterns in musteloid carnivorans: An analysis of jaw adductor activity in ferrets (Mustela putorius furo) and kinkajous (Potos flavus).

Broad similarities in the timing of jaw adductor activity driving jaw movements across distantly related and morphologically disparate species have led to the hypothesis that mammalian masticatory motor patterns are conserved. However, some quantitative analyses also suggest that masticatory motor patterns have evolved in concert with dietary and/or morphological specialization. Here, we assess this relationship in two closely related carnivoran species with divergent diets and morphology: carnivorous ferrets and frugivorous kinkajous. Using electromyography to characterize jaw adductor activity during rhythmic mastication, we test the hypotheses that (1) carnivoran masticatory motor patterns differ from those of non-carnivorans based on previously published data, and (2) differences between ferret and kinkajou motor patterns are associated with dietary and morphological differences. We find that both species exhibit highly synchronous jaw adductor activity that is likely typical of most carnivorans. Kinkajous differ from ferrets, however, in having a balancing-side zygomaticomandibularis that is active later than all other adductors. The significance of these different masticatory motor patterns may relate to morphological differences in the dentition of ferrets and kinkajous. Whereas ferret cheek teeth have vertical occlusal surfaces that limit jaw closing to a primarily dorsally directed movement, kinkajous have relatively flat occlusal surfaces that allow more transverse movement, which may be essential for processing fruits. Our results suggest that some aspects of masticatory motor patterns are highly conserved yet some components are modified in concert with functional and morphological evolution of the masticatory apparatus.

Mobile Phone and Social Media Use of Homeless Youth in Denver, Colorado.

The purpose of this study was to investigate homeless youth mobile phone and social media use, to plan health promotion efforts. Nearly half (46.7%) of runaway/homeless youth in this sample (n = 181) owned a mobile phone and a majority of those devices were smart phones. Ownership did not vary significantly by shelter location, though regular use of Facebook was more prevalent among those in housing programs or camping, than those living on the streets. Over 90% of youth in the sample reported using Facebook. Such media use might facilitate parent, family, and health provider communications with homeless youth.

Nutrition Considerations in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a serious degenerative muscular disease affecting males. Diagnosis usually occurs in childhood and is confirmed through genetic testing and/or muscle biopsy. Accompanying the disease are several nutrition-related concerns: growth, body composition, energy and protein requirements, constipation, swallowing difficulties, bone health, and complementary medicine. This review article addresses the nutrition aspects of DMD.

Low calpain-9 is associated with adverse disease-specific survival following endocrine therapy in breast cancer.

BACKGROUND: The calpains are intracellular cysteine proteases that function in a variety of important cellular functions, including signalling, motility, apoptosis and survival. In breast cancer high calpain-1 and calpain-2 expression has been associated with adverse clinical outcome. Calpain-9 was thought to be exclusively expressed in the digestive tract; however recent studies have shown that this protein is also expressed in breast tissue. METHODS: We investigated the expression of calpain-9 in a large cohort of early stage breast cancer patients (n = 783) using immunohistochemistry on a tissue microarray. Patients had long-term follow-up information available for analysis. RESULTS: Low expression of calpain-9 was associated with patients over 40 years of age (P = 0.025), smaller tumour size (P = 0.001), lower tumour stage (P = 0.009), a more favourable Nottingham Prognostic Index value (P = 0.002) and positive oestrogen receptor status (P = 0.014). Calpain-9 expression was not associated with survival in the total patient cohort, however low calpain-9 expression was associated with adverse survival in patients who received endocrine therapy (P = 0.033), which remained significant in multivariate Cox regression analysis accounting for potential confounding factors (hazard ratio (HR) = 0.56, 95% confidence interval (95% CI) = 0.36-0.89, P = 0.013). Low calpain-9 expression was also associated with adverse survival in patients with an intermediate Nottingham Prognostic Index value (P = 0.009), and remained so in multivariate analysis (HR = 0.54, 95% CI = 0.36-0.82, P = 0.003). CONCLUSIONS: This study suggests that calpain-9 may play a role in breast cancer and that low expression is associated with poorer patient clinical outcome following endocrine therapy. Validation studies are warranted as determining expression of calpain-9 may provide important prognostic information.

Psychometric properties of an automated telephone-based PHQ-9.

OBJECTIVE: This study aims to evaluate the psychometric properties of an automated version of the Patient Health Questionnaire-9 (PHQ-9) to further facilitate its use in primary care. We automated the PHQ-9 using a computer telephony modality (interactive voice response system) called telephone-linked communication (TLC). SUBJECTS AND METHODS: Eighty subjects were divided into four depression categories: none, mild, moderate, and severe. The automated questionnaire, TLC-PHQ-9, was administered to all subjects five times over a 3-month period, at intervals of 0 (T1), 1, 3, 4, and 4 (T5) weeks, respectively. The Inventory of Depressive Symptomatology (IDS) was administered (paper-and-pencil) at T1 and T5. We examined (1) reliability, (2) validity, (3) sensitivity and specificity, and (4) sensitivity to change. RESULTS: (1) Test-retest reliability showed substantial agreement between T1 and T2, with a weighted kappa of 0.76 (95% confidence interval [CI] 0.67-0.85). Cronbach's coefficient alpha values ranged from 0.913 to 0.918 for each TLC-PHQ-9 assessment. (2) The weighted kappa of 0.78 (95% CI 0.70-0.87) for T1 and 0.73 (95% CI 0.63-0.83) for T5 showed strong agreement between TLC-PHQ-9 and IDS in all depression categories. (3) TLC-PHQ-9 demonstrated good sensitivity (82.4%) and very good specificity (90.7%) for moderate-plus depression and poorer sensitivity (54.2%) but very good specificity (97.8%) for severe-plus depression. (4) The weighted kappa of 0.53 (95% CI 0.35-0.70) indicated moderate agreement between TLC-PHQ-9 and IDS. CONCLUSIONS: An automated telephony administration of the PHQ-9 appears to be a valid and reliable tool for monitoring depression symptoms and has strong fidelity across patients.

Expression of the calpain system is associated with poor clinical outcome in gastro-oesophageal adenocarcinomas.

BACKGROUND: Surgery is critical in the management of gastro-oesophageal cancer, and the addition of neo-adjuvant chemotherapy has proved to be of benefit. The calpain system has been implicated in tumour progression and response to various anti-cancer therapies, and therefore expression of the system was determined in this tumour type. METHODS: Two cohorts of gastro-oesophageal adenocarcinomas were investigated for calpain-1, calpain-2, calpain-9 and calpastatin expression using conventional immunohistochemistry. 88 patients who received neo-adjuvant chemotherapy and 140 patients who received surgery alone were investigated using a tissue microarray approach. RESULTS: Calpain-1, calpain-2 and calpastatin expression was associated with adverse cancer-specific survival in the neo-adjuvant cohort (P = 0.004, P = 0.001 and P = 0.012 respectively); which remained significant in multivariate analysis (Hazard ratio (HR) = 0.337; 95% confidence interval (CI) = 0.140-0.81; P = 0.015, HR = 0.375; 95% CI = 0.165-0.858; P = 0.020 and HR = 0.481; 95% CI = 0.257-0.900; P = 0.022 respectively). Calpain-1 and calpastatin expression was also associated with adverse cancer specific survival in the primary surgery cohort (P = 0.001 and P = 0.013 respectively); which remained significant in multivariate analysis (HR = 0.309; 95% CI = 0.159-0.601; P = 0.001 and HR = 0.418; 95% CI = 0.205-0.850; P = 0.016 respectively). Calpain-9 expression was not associated with cancer-specific survival in the neo-adjuvant and primary surgery cohorts. CONCLUSION: Determining the expression levels of calpain-1, calpain-2 and calpastatin may provide clinically relevant prognostic information for gastro-oesophageal adenocarcinomas; these findings warrant further studies in larger cohorts of patients.

Mandibular corpus bone strains during mastication in goats (Capra hircus): a comparison of ingestive and rumination chewing.

OBJECTIVE: To compare the mechanical loading environment of the jaw in goats during ingestive and rumination chewing. DESIGN: Rosette strain gauges were attached to the external surface of the mandibular corpus in five goats to record bone strains during the mastication of hay and rumination. RESULTS: Strain magnitudes and maximum physiological strain rates during the mastication of hay are significantly higher than during rumination chewing on the working and balancing sides. Principal strain ratios and orientations are similar between the two chewing behaviours. Loading and chewing cycle duration are all longer during rumination chewing, whereas chew duty factor and variances in load and chewing cycle durations are higher during ingestive chewing. For most of the variables, differences in strain magnitudes or durations are similar at all three gauge sites, suggesting that rumination and ingestive chewing do not differentially influence bone at the three gauge sites. CONCLUSIONS: Despite lower strain magnitudes, the repetitive nature of rumination chewing makes it an important component of the mechanical loading environment of the selenodont artiodactyl jaw. However, similarities in principal strain orientations and ratios indicate that rumination chewing need not be considered as a unique loading behaviour influencing the biomechanics of the selenodont artiodactyl jaw. Differences in loading and chewing cycle durations during rumination and ingestion demonstrate flexibility in adult chewing frequencies. Finally, although the low within-sequence variability in chewing cycle durations supports the hypothesis that mammalian mastication is energetically efficient, chewing during rumination may not be more efficient than during ingestion.

A comparative study of incisor procumbency and mandibular morphology in vampire bats.

The three species of vampire bats (Phyllostomidae: Desmodontinae), Desmodus rotundus, Diaemus youngi, and Diphylla ecaudata, are the only mammals that obtain all nutrition from vertebrate blood (sanguinivory). Because of the unique challenges of this dietary niche, vampire bats possess a suite of behavioral, physiological, and morphological specializations. Morphological specializations include a dentition characterized by small, bladelike, non-occlusive cheek teeth, large canines, and extremely large, procumbent, sickle-shaped upper central incisors. The tips of these incisors rest in cuplike pits in the mandible behind the lower incisors (mandibular pits). Here, we use microCT scanning and high-resolution radiography to describe the morphology of the mandible and anterior dentition in vampire bats, focusing on the relationship between symphyseal fusion, mandibular pit size, incisor size, and procumbency. In Desmodus and Diaemus, highly procumbent upper incisors are associated with relatively small mandibular pits, an unfused mandibular symphysis with substantial bony interdigitations linking the dentaries, and a diastema between the lower central incisors that helps to facilitate the lapping of blood from a wound. In Diphylla, less procumbent upper incisors are associated with relatively large mandibular pits, a completely fused mandibular symphysis, and a continuous lower toothrow lacking a central diastema. We hypothesize that symphyseal morphology and the presence or absence of the diastema are associated with the angle of upper incisor procumbency and mandibular pit development, and that spatial constraints influence the morphology of the symphysis. Finally, this morphological variation suggests that Diphylla utilizes a different feeding strategy as compared to Desmodus and Diaemus, possibly resulting from the functional demands of specialization on avian, rather than mammalian, blood.

Acute effects of walking on inflammatory and cardiovascular risk in sedentary post-menopausal women.

Biochemical markers of inflammation are emerging as new predictors of risk of cardiovascular disease (CVD) and may alter acutely with exercise. Few studies have been conducted on the effects of walking on these markers or whether different walking intensities elicit varied effects. As there is growing interest in modifiable lifestyle factors such as walking to reduce CVD risk, these inflammatory responses warrant investigation. The aim of this study was to compare the effects of walking at 50% versus 70% of predicted maximal heart rate on C-reactive protein (CRP), plasma fibrinogen, and triglycerides in sedentary post-menopausal women. Twelve post-menopausal women (mean age 58 years, s +/-6; stature 1.62 m, s+/-0.06; body mass 66.8 kg, s +/-6.2) completed two 30-min treadmill walks in a randomized cross-over design. Fasted blood samples were taken (for the determination of plasma fibrinogen, CRP, and lipids) before, immediately after, and 1 and 24 h after exercise. Triglyceride concentrations decreased from pre-exercise to 24 h post exercise at both walking intensities (time x group interaction, P < 0.05). No significant effects were observed for plasma fibrinogen, CRP, total cholesterol, low-density or high-density lipoprotein cholesterol (time x group interaction, P > 0.05). The results of this study suggest that fasting plasma triglycerides are decreased on the morning after 30 min of brisk walking at either 50% or 70% of maximal heart rate (moderate and vigorous intensity).

Raloxifene attenuation of 5-FU/methotrexate cytotoxicity in human breast cancer cells: the importance of sequence in combination chemotherapy.

BACKGROUND: The selective estrogen receptor modulator raloxifene (RAL), used to treat and prevent osteoporosis, is under investigation for its use in the treatment and prevention of breast cancer. RAL in combination with the antimetabolites methotrexate (MTX) and 5-fluorouracil (5-FU) has not been extensively studied. Because RAL and the antimetabolites target different phases of the cell cycle and exhibit different mechanisms of action and clinical toxicity, the effects of sequence of administration on the growth inhibition of MCF-7 human breast cancer cells were investigated. MATERIALS AND METHODS: MCF-7 human breast cancer cells were exposed to vehicle alone, 10 microM MTX, 1 microM 5-FU, 10 microM RAL, 10 microM RAL 24 hours prior to 1 microM 5-FU followed 2 hours later by 10 microM MTX, and 1 microM 5-FU 2 hours prior to 10 microM MTX followed 24 hours later by 10 microM RAL. The cells were evaluated for viability and proliferation. The retinoblastoma (Rb) protein, a cell cycle regulator which when phosphorylated allows the progression of cells from G1- to S-phase, was used as a marker to determine the effects of early RAL and late RAL on cellular progression at the molecular level. RESULTS: Early RAL administration exhibited a cell viability of 66.83 +/- 6.17% of the control. However, late RAL administration exhibited cell viability 39.40 +/- 17.03% of the control. Late RAL was a more cytotoxic combination than RAL alone or early RAL. These findings from manual cell counting were also supported by cell flow cytometric analysis and Western blot data. CONCLUSION: Late RAL in combination with 5-FU and MTX, due to greater cytotoxicity, is a more desirable combination to treat breast cancer than RAL alone.

Sequence- and time-dependent antagonism between raloxifene and methotrexate in human breast cancer cells.

The selective estrogen receptor modulator (SERM) and an agent for the prevention of osteoporosis in postmenopausal women, raloxifene (Ral), decreased high-dose methotrexate (MTX) cytotoxicity in MCF-7 breast cancer cells. When Ral is given at least 24 hours prior to MTX, the resultant interaction is antagonistic. However, when breast cancer cells are exposed to Ral 24 hours after MTX, the interaction between Ral and MTX is not antagonistic. The proliferation of cells exposed to 10 microM Ral and 10 microM MTX alone or in combination with Ral 24 hours prior to MTX was in had the following order: MTX > Ral 24 hours prior to MTX > Ral. MTX administration 24 hours prior to Ral had the following inhibitory effects on the growth of cells: MTX 24 hours prior to Ral > or = MTX > Ral 24 hours prior to MTX > Ral > control (no drug exposure). To determine if the antagonistic interaction between Ral and MTX was a function of sequence and time, cells were exposed to Ral 24 hours and 36 hours prior to MTX exposure. The percentages of control rates were 43.48 +/- 3.90% and 54.43 +/- 2.93%, respectively, from a 24 hours and 36 hours exposure of Ral prior to MTX. The growth rates after 24 h and 36 h exposures to MTX alone were 30.30 +/- 0.61% and 33.11 +/- 2.27% of control rates, respectively. These studies suggest that: (a) the interactions between Ral and MTX are sequence-dependent; (b) Ral antagonizes the effect of MTX when Ral administration precedes MTX; and (c) Ral antagonism to MTX is a function of time.