A quantitative recipe for engineering protein polymer nanoparticles.

Protein polymers can assemble switchable nanostructures with emerging applications as biomaterials and nanomedicines. For example, above a critical micelle temperature (CMT) some elastin-like polypeptide (ELP) diblock copolymers assemble spherical nanoparticles, which may modulate cellular internalization and in vivo biodistribution. To achieve engineering-level control over their properties, this report explores a comprehensive library of ELP monoblock and diblock polymers. For the first time, we report that a surprisingly high core molecular weight is required for stable nanoparticle formation; furthermore, nanoparticle size depends on polymer molecular weight. A mathematical model was developed to characterize four ELP monoblock libraries and to predict the phase behavior of corresponding diblock copolymers. The CMT was almost entirely dependent on the hydrophobic core ELP, while the bulk phase transition temperature (Tt,bulk ) depends predominantly on the hydrophilic block. Nanoparticle assembly was accompanied by a conversion in secondary structure of the hydrophobic block from random coil and beta-sheets to type-2 ß turns. For the first time, this report enables the rational design of ELP protein polymer nanoparticles with physico-chemico properties that will be suitable for biological applications.

Kinetic quantification of protein polymer nanoparticles using non-invasive imaging.

Protein polymers are repetitive amino acid sequences that can assemble monodisperse nanoparticles with potential applications as cancer nanomedicines. Of the currently available molecular imaging methods, positron emission tomography (PET) is the most sensitive and quantitative; therefore, this work explores microPET imaging to track protein polymer nanoparticles over several days. To achieve reliable imaging, the polypeptides were modified by site-specific conjugation using a heterobifunctional sarcophagine chelator, AmBaSar, which was subsequently complexed with (64)Cu. AmBaSar/(64)Cu was selected because it can label particles in vivo over periods of days, which is consistent with the timescales required to follow long-circulating nanotherapeutics. Using an orthotopic model of breast cancer, we observed four elastin-like polypeptides (ELPs)-based protein polymers of varying molecular weight, amino acid sequence, and nanostructure. To analyze this data, we developed a six-compartment image-driven pharmacokinetic model capable of describing their distribution within individual subjects. Surprisingly, the assembly of an ELP block copolymer (78 kD) into nanoparticles (R(h) = 37.5 nm) minimally influences pharmacokinetics or tumor accumulation compared to a free ELP of similar length (74 kD). Instead, ELP molecular weight is the most important factor controlling the fate of these polymers, whereby long ELPs (74 kD) have a heart activity half-life of 8.7 hours and short ELPs (37 kD) have a half-life of 2.1 hours. These results suggest that ELP-based protein polymers may be a viable platform for the development of multifunctional therapeutic nanoparticles that can be imaged using clinical PET scanners.

Biomolecular engineering of intracellular switches in eukaryotes.

Tools to selectively and reversibly control gene expression are useful to study and model cellular functions. When optimized, these cellular switches can turn a protein's function "on" and "off" based on cues designated by the researcher. These cues include small molecules, drugs, hormones, and even temperature variations. Here we review three distinct areas in gene expression that are commonly targeted when designing cellular switches. Transcriptional switches target gene expression at the level of mRNA polymerization, with examples including the tetracycline gene induction system as well as nuclear receptors. Translational switches target the process of turning the mRNA signal into protein, with examples including riboswitches and RNA interference. Post-translational switches control how proteins interact with one another to attenuate or relay signals. Examples of post-translational modification include dimerization and intein splicing. In general, the delay times between switch and effect decreases from transcription to translation to post-translation; furthermore, the fastest switches may offer the most elegant opportunities to influence and study cell behavior. We discuss the pros and cons of these strategies, which directly influence their usefulness to study and implement drug targeting at the tissue and cellular level.

Clinician-patient communication: evidence-based recommendations to guide practice in cancer.

GOALS OF WORK: To develop recommendations for effective communication between cancer health care providers and patients based on a systematic review of methods of clinician-patient communication that may affect patient outcomes associated with distress at critical points in the course of cancer care. MATERIALS AND METHODS: A systematic review of the literature was conducted, and evidence-based recommendations were formulated to guide clinician-patient communication in cancer care. A formal external review was conducted to validate the relevance of these recommendations. MAIN RESULTS: Recommendations for communication in cancer care are presented, based on guidelines from the Australian National Breast Cancer Centre and the Australian National Cancer Control Initiative,an updated systematic review of the research evidence, and a consensus by the Clinician-Patient Communications Working Panel of the Program in Evidence-Based Care of Cancer Care Ontario. The recommendations were sent to 110 Ontario practitioners for external review: 33 responded (30% response rate). Most of these respondents (87%) agreed with the draft recommendations and approved of their use as a practice guideline (90%). A condensed version of the recommendations, including 10 key points, was also created. CONCLUSIONS: There is evidence to support general clinician-patient communication approaches, although the preferences of cancer patients regarding such communication exhibit individual and cultural variability. Recommendations are provided, based on evidence, the consensus of an expert panel, and feedback from a survey of external practitioners. Evidence evaluating the role of decision aids and strategies to facilitate better communication is inconsistent, although such tools may be of value for some patients.

A framework for the organization and delivery of systemic treatment.

BACKGROUND: Increasing systemic treatment and shortages of oncology professionals in Canada require innovative approaches to the safe and effective delivery of intravenous (IV) cancer treatment. We conducted a systematic review of the clinical and scientific literature, and an environmental scan of models in Canada, the United Kingdom, Australia, and New Zealand. We then developed a framework for the organization and delivery of IV systemic treatment. METHODS: The systematic review covered the medline, embase, cinahl, and HealthStar databases. The environmental scan retrieved published and unpublished sources, coupled with a free key word search using the Google search engine. The Systemic Treatment Working Group reviewed the evidence and developed a draft framework using evidence-based analysis, existing recommendations from various jurisdictions, and expert opinion based on experience and consensus. The draft was assessed by Ontario stakeholders and reviewed and approved by Cancer Care Ontario. RESULTS: The poor quantity and quality of the evidence necessitated a consensus-derived model. That model comprises four levels of care determined by a regional systemic treatment program and three integrated structures (integrated cancer programs, affiliate institutions, and satellite institutions), each with a defined scope of practice and a specific organizational framework. INTERPRETATION: New models of care are urgently required beyond large centres, particularly in geographically remote or rural areas. Despite limited applicable evidence, the development and successful implementation of this framework is intended to create sustainable, accessible, quality care and to measurably improve patient outcomes.

Treatment of depression in cancer patients.

QUESTION: What is the efficacy of pharmacologic and non-pharmacologic treatments for major depression and other depressive disorders in cancer populations? PERSPECTIVES: Depression occurs at an increased rate in medically ill populations, including patients with cancer. In the general population, depression has been shown to be responsive to structured forms of psychotherapy and to pharmacologic interventions. The Supportive Care Guidelines Group conducted a systematic review of the evidence for the effectiveness of those therapies in patients with depression and cancer and developed the present clinical practice guideline based on that review and on expert consensus. OUTCOMES: Outcomes of interest included symptomatic response to treatment, discontinuation rate of treatment, adverse effects, and quality of life. METHODOLOGY: Clinical recommendations were developed by the Supportive Care Guidelines Group based on a systematic review of the published literature through June 2005, feedback obtained from Ontario health care providers on the draft recommendations, the Report Approval Panel (rap) of Cancer Care Ontario's Program in Evidence-Based Care, and expert consensus. RESULTS: The systematic review of the literature included eleven trials (seven of pharmacologic agents and four of non-pharmacologic interventions). Feedback received from 44 responding health care providers and the rap on the draft recommendations was addressed and documented in the guideline. Among providers, 82% agreed with the draft recommendations as stated, 68% agreed that the report should be approved as a practice guideline, and 73% indicated that they would be likely to use the guideline in their own practice. PRACTICE GUIDELINE: These recommendations apply to adult cancer patients with a diagnosis of major depression or other non-bipolar depressive disorders. They do not address the treatment of non-syndromal depressive symptoms, for which specific antidepressant treatment is not usually indicated. The guideline is intended both for oncology health professionals and for mental health professionals engaged in the treatment of cancer patients. Expert consensus was central to the development of the guideline recommendations because of limited evidence in cancer patients. RECOMMENDATIONS: Treatment of pain and other reversible physical symptoms should be instituted before or with initiation of specific antidepressant treatment. Antidepressant medications should be considered for the treatment of moderate-to-severe major depression in cancer patients. Current evidence does not support the relative superiority of one pharmacologic treatment over another, nor the superiority of pharmacologic treatment over psychosocial interventions. The choice of an antidepressant should be informed by individual medication and patient factors: the side effect profiles of the medication, tolerability of treatment (including the potential for interaction with other current medications), response to prior treatment, and patient preference. Cancer patients diagnosed with major depression may benefit from a combined modality approach that includes both psychosocial and pharmacologic interventions. Psychosocial treatment approaches that may be of value include those that provide information and support and those that address any combination of emotional, cognitive, and behavioural factors. QUALIFYING STATEMENTS: Referral to a mental health specialist is appropriate when the diagnosis of depression is unclear, when the syndrome is severe, when patients do not respond to treatment, or when other complicating factors that may affect the choice of treatment are present. Although care has been taken in the preparation of the information contained in this guideline, any person seeking to apply or to consult the guideline is expected to use independent medical judgment in the context of individual clinical circumstances or to seek out the supervision of a qualified clinician.

Second-line or subsequent systemic therapy for recurrent or progressive non-small cell lung cancer: a systematic review and practice guideline.

PURPOSE: This clinical practice guideline, based on a systematic review, evaluates second-line or subsequent therapy for patients with recurrent or progressive non-small cell lung cancer. METHODS: Relevant randomized trials and meta-analyses were identified through a systematic search of the literature. External feedback was obtained from practitioners in Ontario, and the guideline was approved by the provincial Lung Cancer Disease Site Group. RESULTS: Twenty-four randomized trials met the eligibility criteria. Two phase III trials demonstrated a significant benefit in overall survival and quality of life (QOL) for single-agent docetaxel. A pooled analysis comparing docetaxel administered weekly versus three-weekly found similar survival between the schedules and a non-significant reduction in febrile neutropenia for the weekly regimen. One phase III trial found that single-agent pemetrexed provided similar survival and QOL, compared to docetaxel. Another phase III trial demonstrated that oral topotecan was non-inferior to docetaxel for one-year survival rate, although QOL significantly favored docetaxel over topotecan. Docetaxel-based and other combination chemotherapy regimens have not been shown to be superior to single-agent docetaxel. One phase III trial revealed a statistically significant survival and QOL benefit for erlotinib over placebo for patients who were not eligible for further chemotherapy. Modest tumor response rates and symptom control have been demonstrated for gefitinib; however, a statistically significant survival benefit has not been established for gefitinib over placebo. CONCLUSION: Second-line or subsequent therapy with single-agent docetaxel, pemetrexed, or erlotinib offers patients a significant survival and QOL advantage.

A comparison of monocyclic and bicyclic phospholanes as acyl-transfer catalysts.

The synthesis and evaluation of chiral phosphines 11, 15a, 19a, 24a, and 28a as nucleophilic catalysts for anhydride activation and kinetic resolution of alcohols is described. The relative reactivity follows the order 11a > 11b > 15a > 1 in the monocyclic series, and 24a > 19a > 28ain the bicyclic series, with an overall rate advantage of ca. 2 orders of magnitude for the bicyclic phospholanes over the monocyclic analogues. The increased reactivity of the bicyclic phospholanes for the acylation of alcohols is attributed to conformational effects and ground-state destabilization in a highly associative mechanism. Kinetic resolution data demonstrate promising enantioselectivities for 24a.

Kinetic resolution of allylic alcohols using a chiral phosphine catalyst.

[reaction: see text] The kinetic resolution of racemic allylic alcohols 3, 6, and 12--17 has been explored using the PBO catalyst 7 for activation of isobutyric anhydride. Trisubstituted allylic alcohols (12--15; 17) are the best substrates and react with an enantioselectivity of s = 32--82 at -40 degrees C.

The hypertension trials.

ESTABLISHED ANTIHYPERTENSIVE TREATMENT: Previous studies have clearly demonstrated the benefits of antihypertensive therapy, particularly in reducing the incidence of stroke. However, there is still concern that the full potential for reversing coronary heart disease in the hypertensive patient has not been realized by treatment with established agents, the diuretics and the beta-blockers. TRIALS ON NEWER AGENTS: A question that still remains to be answered is whether treatment regimens based on alternative drugs, including calcium channel blocking agents and angiotensin converting enzyme inhibitors, will confer an advantage over older drugs. This question is at last being addressed in long-term morbidity and mortality trials with these newer agents.

Calcium channel blockers in the dock: innocent or guilty?

Several studies have recently been published which have raised doubts over the long-term safety of calcium channel blockers (CCB). These have included retrospective case control studies in hypertension and meta-analyses of small scale studies in unstable angina and myocardial infarction (MI). Most of the reports were primarily concerned with the use of the short acting dihydropyridine nifedipine. Despite wide media coverage of these reports, the results are by no means irrefutable and, because of the nature of the studies themselves, are open to several criticisms. Calcium channel blockers are currently being evaluated in large-scale, prospective, randomised controlled studies, but results are unlikely to be available within the next few years. Meanwhile, the consensus view seems to be that short acting dihydropyridines should in general be avoided and have no place in the management of unstable angina and post MI. In the setting of stable angina, long acting dihydropyridines should generally be used in conjunction with a beta-blocker. In hypertensive patients, long acting dihydropyridines may be used as alternative antihypertensive agents in patients in whom the first line agents (diuretics and beta-blockers) are poorly tolerated, contra-indicated or ineffective.

Autosomal recessive juvenile cataract in Hutterites.

Autosomal recessive inheritance of juvenile cataract is described amongst several related sibships of Lehrerleut Hutterites. The main features of the cataract include onset between three and seven years of age; rapid progression to maturity within one to three months; normal intelligence; no systemic associations, and no urinary reducing substances and normal erythrocyte galactokinase activity. Genetic analysis demonstrates the close relationship between parents of affected sibships with a coefficient of inbreeding of affected sibships of 0.0512. Estimates of heterozygote frequency within Lehrerleut Hutterites at 0.128 indicate that if current inbreeding practice continues additional cases can be expected.

The requirements for transferrin-dependent adherence of human granulocytes to pollen grains.

Human granulocyte/pollen binding protein (GPBP), previously identified as serum transferrin, promoted prolonged firm adherence of neutrophils to Timothy grass pollen. Some characteristics of this adherence reaction are reported. GPBP-induced binding was time-, temperature- and concentration-dependent. Maximal adherence was observed by 2 h and was only slightly decreased at 18 h. The optimal temperature for adherence was 37 degrees C. Concentrations of GPBP as low as 1.25 microgram/ml gave significantly greater binding than the albumin or lactoferrin control. Eosinophils, monocytes and lymphocytes did not appear to participate in GPBP-induced pollen binding reactions at concentrations up to 300 micrograms/ml. In the presence of GPBP, neutrophils adhered to a range of grass, weed and tree pollens. These included timothy, meadow, false oat, rye, giant and short ragweed, plantain, silver birch and ash. GPBP did not facilitate the adherence of granulocytes to inert particles of similar size such as Sephadex beads and agarose. The adherence was Mg++- but not Ca++-dependent and was not inhibited by a monoclonal antibody to the transferrin receptor (OKT9). Transferrin/GPBP did not bind to either neutrophils or pollen grains. A purified commercial transferrin reacted in all respects like GPBP in these pollen binding studies. These observations indicate that GPBP/transferrin-induced adherence of granulocytes to pollen grains is a hitherto unrecognized property of transferrin which appears unrelated to iron transport or the conventional transferrin receptor.

Enhancement of leukocyte cytotoxicity after exercise-induced asthma.

We have previously shown that there were elevations of neutrophil chemotactic activity (NCA) and increases in the percentages of neutrophil and monocyte complement rosettes after exercise-induced asthma (EIA). These observations suggested that leukocyte activation may occur after EIA, possibly as a result of the release of mast-cell-associated mediators. In the present study, we have attempted to establish whether neutrophils and monocytes are functionally altered after EIA as assessed by changes in their cytotoxic capacity. Cytotoxicity was assessed by a direct visual killing assay using opsonized (complement-coated) schistosomula of Schistosoma mansoni as target organisms. Neutrophils and mononuclear cells obtained from 8 patients after exercise-induced asthma (EIA+ve) had increased cytotoxicity for opsonized schistosomula for as long as 60 min after exercise. These changes were preceded by elevations in the concentrations of serum high molecular weight NCA (which were maximal at 10 min after exercise). In asthmatic patients who did not develop exercise-induced asthma (EIA-ve), no significant increases in neutrophil or mononuclear cell killing of schistosomula, or serum NCA concentrations, were observed. There was a highly significant correlation (p less than 0.001) between the reduction in FEV1 and the increases in neutrophil cytotoxicity. In 5 EIA+ve patients, administration of disodium cromoglycate (cromolyn) prior to the exercise task inhibited both the enhancement in neutrophil and mononuclear cell cytotoxicity, as well as the elevations in circulating NCA and the reductions in FEV1.(ABSTRACT TRUNCATED AT 250 WORDS)

Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter.