Flow-Based Fmoc-SPPS Preparation and SAR Study of Cathelicidin-PY Reveals Selective Antimicrobial Activity.

Antimicrobial peptides (AMPs) hold promise as novel therapeutics in the fight against multi-drug-resistant pathogens. Cathelicidin-PY (NH2-RKCNFLCKLKEKLRTVITSHIDKVLRPQG-COOH) is a 29-residue disulfide-cyclised antimicrobial peptide secreted as an innate host defence mechanism by the frog Paa yunnanensis (PY) and reported to possess broad-spectrum antibacterial and antifungal properties, exhibiting low cytotoxic and low hemolytic activity. Herein, we detail the total synthesis of cathelicidin-PY using an entirely on-resin synthesis, including assembly of the linear sequence by rapid flow Fmoc-SPPS and iodine-mediated disulfide bridge formation. By optimising a synthetic strategy to prepare cathelicidin-PY, this strategy was subsequently adapted to prepare a bicyclic head-to-tail cyclised derivative of cathelicidin-PY. The structure-activity relationship (SAR) of cathelicidin-PY with respect to the N-terminally positioned disulfide was further probed by preparing an alanine-substituted linear analogue and a series of lactam-bridged peptidomimetics implementing side chain to side chain cyclisation. The analogues were investigated for antimicrobial activity, secondary structure by circular dichroism (CD), and stability in human serum. Surprisingly, the disulfide bridge emerged as non-essential to antimicrobial activity and secondary structure but was amenable to synthetic modification. Furthermore, the synthetic AMP and multiple analogues demonstrated selective activity towards Gram-negative pathogen E. coli in physiologically relevant concentrations of divalent cations.

A Prospective Analysis of the SVS WIfI Classification System to Stratify Immediate and 1-Year Patient Outcomes.

The purpose of this study was to prospectively enroll patients that presented to the emergency department with a lower extremity infection, stratify risk and record outcomes. Risk stratification was performed based on the Society of Vascular Surgery Wound, foot Infection, and Ischemia (WIfI) classification system. This study aimed to establish the efficacy and validity of this classification in predicting patient outcomes during immediate hospitalization and throughout a 1 year follow up. A total of 152 patients were enrolled in the study and of these, 116 met the inclusion criteria and had at least 1 year of follow up for analysis. Each patient was assigned a WIfI score based on wound, ischemia, and foot infection severity according to the classification guidelines. Patient demographics as well as all podiatric and vascular procedures were recorded. The major end points of the study were rates of proximal amputation, time to wound healing, surgical procedures, surgical dehiscence, readmission rates, and mortality. A difference in rates of healing (p = .04), surgical dehiscence (p < .01), and 1 year mortality (p = .01) with increasing WIfI stage as well as across the individual component scores was noted. This analysis further supports the application of the WIfI classification system early during patient care to stratify risk and identify the need for early intervention and a multispecialty team approach to potentially improve outcomes in the severe multicomorbid patient.

Recent Progress in the Synthesis of Homogeneous Erythropoietin (EPO) Glycoforms.

Erythropoietin (EPO) has been regarded as a therapeutic glycoprotein for the clinical treatment of anaemia since its approval by the Food and Drug Administration (FDA) in 1989. Commercial production of the 165-residue glycoprotein is by recombinant protein expression using mammalian cell lines that renders a complex mixture of glycoforms that have an identical amino acid sequence but variations in the structures of the pendant glycans. This heterogeneous nature of human recombinant EPO restricts structural and bioactivity studies in medicinal chemistry. Consequently, chemical synthesis provides an elegant approach for the preparation of complex homogeneous glycoproteins from a readily accessible pool of amino acids and sugars. In addition, the combination of chemical and biosynthesis enables robust and large-scale production of homogeneous EPO. The scope of this minireview is to summarise the recent advances in the chemical and semisyntheses of homogeneous EPO glycoforms, highlighting the versatile approaches to the preparation and structural manipulations of the carbohydrate chains incorporated into synthetic EPO glycoproteins.

Receptor Activity Modifying Proteins Have Limited Effects on the Class B G Protein-Coupled Receptor Calcitonin Receptor-Like Receptor Stalk.

The calcitonin receptor-like receptor (CLR) is a class B G protein-coupled receptor (GPCR) that forms the basis of three pharmacologically distinct receptors, the calcitonin gene-related peptide (CGRP) receptor, and two adrenomedullin (AM) receptors. These three receptors are created by CLR interacting with three receptor activity-modifying proteins (RAMPs). Class B GPCRs have an N-terminal extracellular domain (ECD) and transmembrane bundle that are both important for binding endogenous ligands. These two domains are joined together by a stretch of amino acids that is referred to as the "stalk". Studies of other class B GPCRs suggest that the stalk may act as hinge, allowing the ECD to adopt multiple conformations. It is unclear what the role of the stalk is within CLR and whether RAMPs can influence its function. Therefore, this study investigated the role of this region using an alanine scan. Effects of mutations were measured with all three RAMPs through cell surface expression, cAMP production and, in select cases, radioligand binding and total cell expression assays. Most mutants did not affect expression or cAMP signaling. CLR C127A, N140A, F142A, and L144A impaired cell surface expression with all three RAMPs. T125A decreased the potency of all peptides at all receptors. N128A, V135A, and L139A showed ligand-dependent effects. While the stalk appears to play a role in CLR function, the effect of RAMPs on this region seems limited, in contrast to their effects on the structure of CLR in other receptor regions.

Synthesis of truncated analogues of preptin-(1­16), and investigation of their ability to stimulate osteoblast proliferation.

Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the ß-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(1­16), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(1­16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(1­16) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (1­8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (1­8) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.

A murine model of mild traumatic brain injury exhibiting cognitive and motor deficits.

BACKGROUND: Mild traumatic brain injury (TBI) is a serious public health concern affecting more than 1.7 million people in the United States annually. Mild TBI is difficult to diagnose and is clinically associated with impaired motor coordination and cognition. METHODS: We subjected mice to a mild TBI (mTBI-1 or mTBI-2) induced by a weight drop model. We assessed brain injury histologically and biochemically, the latter by serum neuron-specific enolase and glial fibrillary acidic protein. Systemic and brain inflammation were measured by cytokine array. We determined blood-brain barrier integrity by cerebral vascular leakage of micromolecular and macromolecular fluorescent molecules. We evaluated mice using a rotarod device and novel object recognition to measure motor coordination and cognition, respectively. RESULTS: Mice undergoing mTBI-1 or mTBI-2 had significant deficits in motor coordination and cognition for several days after injury compared with controls. Furthermore, both mTBI-1 and mTBI-2 caused micromolecular leakage in the blood-brain barrier, whereas only mTBI-2 caused macromolecular leakage. Serum neuron-specific enolase and glial fibrillary acidic protein were elevated acutely and corresponded to the degree of injury, but returned to baseline within 24 h. Serum cytokines interleukin-6 and keratinocyte-derived chemokine were significantly increased within 90 min of TBI. Interleukin-6 levels correlated with the degree of injury. CONCLUSIONS: The current study provides a reproducible model of mild TBI in mice that exhibits pathologic features of mild TBI in humans. Furthermore, our data suggest that serum cytokines, such as IL-6, may be effective biomarkers for severity of head injury.

Glycolipid-peptide conjugate vaccines elicit CD8+ T-cell responses and prevent breast cancer metastasis.

Objectives: Metastasis is the principal cause of breast cancer mortality. Vaccines targeting breast cancer antigens have yet to demonstrate clinical efficacy, and there remains an unmet need for safe and effective treatment to reduce the risk of metastasis, particularly for people with triple-negative breast cancer (TNBC). Certain glycolipids can act as vaccine adjuvants by specifically stimulating natural killer T (NKT) cells to provide a universal form of T-cell help. Methods: We designed and made a series of conjugate vaccines comprising a prodrug of the NKT cell-activating glycolipid α-galactosylceramide covalently linked to tumor-expressed peptides, and assessed these using E0771- and 4T1-based breast cancer models in vivo. We employed peptides from the model antigen ovalbumin and from clinically relevant breast cancer antigens HER2 and NY-ESO-1. Results: Glycolipid-peptide conjugate vaccines that activate NKT cells led to antigen-presenting cell activation, induced inflammatory cytokines, and, compared with peptide alone or admixed peptide and α-galactosylceramide, specifically enhanced CD8+ T-cell responses against tumor-associated peptides. Primary tumor growth was delayed by vaccination in all tumor models. Using 4T1-based cell lines expressing HER2 or NY-ESO-1, a single administration of the relevant conjugate vaccine prevented tumor colonisation of the lung following intravenous inoculation of tumor cells or spontaneous metastasis from breast, respectively. Conclusion: Glycolipid-peptide conjugate vaccines that activate NKT cells prevent lung metastasis in breast cancer models and warrant investigation as adjuvant therapies for high-risk breast cancer.

Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors.

Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP1 production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM15-52 G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.

Clinical features and treatment outcomes of angioimmunoblastic T-cell lymphoma.

The objective of this retrospective study was to investigate clinical features and treatment outcomes in patients with angioimmunoblastic T-cell lymphoma (AITL), data of which were collected over a 15-year period. Sixty-five patients diagnosed with AITL were included in the study. About half of the patients (46.2%) presented with poor performance status (ECOG > or = 2); 72.3% of patients belonged to high intermediate or high-risk of IPI and same proportion belonged to Class 2 of PIT (Prognostic index for PTCL-U), and most patients (95.4%) were diagnosed at an advanced stage. At diagnosis, 27 patients (41.5%) presented with malignant pleural effusion, and 22 patients (33.8%) had skin involvement. Most of the initial chemotherapy regimens were anthracycline-based (88.2%). Overall response rate to initial chemotherapy was 86.2% (64.7% of complete response, 21.5% of partial response). The median progression-free survival and overall survival of all patients was 7.1 months (95% CI, 2.8 - 11.4) and 15.1 months (95% CI, 6.7 - 23.5), respectively. Age, performance status, and PIT scores were predictive prognostic factors for survival. In conclusion, although AITLs showed a good response to the initial chemotherapy, their response durations were short; therefore, chemotherapy for AITL should be modified or intensified as in high-dose chemotherapy.

Accuracy of noninvasive hemoglobin monitoring in patients at risk for hemorrhage.

BACKGROUND: Monitoring for acute blood loss is critical in surgical patients, and delays in identifying hemorrhage can result in poor outcomes. The current standard of care for monitoring patients at risk for bleeding is serial measurement of hemoglobin (Hgb) by standard laboratory complete blood count (CBC). Point-of-care testing (i.e., iSTAT) can be a rapid method of evaluating Hgb, and spectrophotometry-based devices (i.e., Radical-7) offer the advantages of being continuous and noninvasive. We sought to evaluate the accuracy of Radical-7 and iSTAT in measuring Hgb and assessing for blood loss when compared with the criterion standard CBC. METHODS: Adult patients at risk for hemorrhage admitted to the surgical intensive care unit of a tertiary referral, Level I trauma center were eligible for this study. Serial CBC Hgb measurements were drawn as clinically indicated. The Radical-7 device was placed on the patient for noninvasive Hgb measurements (SpHb), and at each CBC measurement, concurrent iSTAT Hgb measurements were obtained. Bland-Altman analysis was used to compare the three methods of measuring Hgb with accuracy defined as measurements within 1.0-g/dL CBC Hgb. Concordance measurements were also performed to compare trends between values. RESULTS: Eighty-eight patients were enrolled and underwent 572 CBC measurements. Bland-Altman analysis of SpHb versus CBC resulted in an estimated bias of 1.49 g/dL, with 95% limits of agreement of -2.2 g/dL to 5.0 g/dL. iSTAT versus CBC resulted in an estimated bias of -0.63 g/dL, with 95% limits of agreement of -3.4 g/dL to 2.2 g/dL. Changes in SpHb had concordance with CBC Hgb 60% of the time, compared with 76% for iSTAT versus CBC CONCLUSION: Radical-7 SpHb was inaccurate when compared with CBC Hgb levels, and serial SpHb achieved concordance with CBC Hgb 60% of the time. As such, the clinical utility of Radical-7 as a rapid, noninvasive predictor of acute hemorrhage may be limited. LEVEL OF EVIDENCE: Diagnostic study, level II; care management, level III.

Interleukin 6 mediates neuroinflammation and motor coordination deficits after mild traumatic brain injury and brief hypoxia in mice.

Traumatic brain injury (TBI) is a leading cause of mortality and disability. Acute postinjury insults after TBI, such as hypoxia, contribute to secondary brain injury and worse clinical outcomes. The functional and neuroinflammatory effects of brief episodes of hypoxia experienced following TBI have not been evaluated. Our previous studies have identified interleukin 6 (IL-6) as a potential mediator of mild TBI-induced pathology. In the present study, we sought to determine the effects of brief hypoxia on mild TBI and whether IL-6 played a role in the neuroinflammatory and functional deficits after injury. A murine model of mild TBI was induced by a weight drop (500 g from 1.5 cm). After injury, mice were exposed to immediate hypoxia (FIO2 = 15.1%) or normoxia (FIO2 = 21%) for 30 min. Serum and brain samples were analyzed for inflammatory cytokines 24 h after TBI. Neuron-specific enolase was measured as a serum biomarker of brain injury. Evaluation of motor coordination was performed for 5 days after TBI using a rotarod device. In some animals, anti-IL-6 was administered following TBI and hypoxia to neutralize systemic IL-6. Mice undergoing TBI had significant increases in brain injury. Exposure to brief hypoxia after TBI resulted in a more than 5-fold increase in serum neuron-specific enolase. This increase was associated with increases in serum and brain cytokine expression, suggesting that brief hypoxia exacerbates systemic and brain inflammation. Neutralization of IL-6 suppressed postinjury neuroinflammation and neuronal injury. In addition, TBI and hypoxia induced significant motor coordination deficits that were completely abrogated by IL-6 blockade. Exposure to hypoxia after TBI induces neuroinflammation and brain injury. These changes can be mitigated by neutralization of systemic IL-6. Interleukin 6 blockade also corrected the TBI-induced deficit in motor coordination. These data suggest that systemic IL-6 modulates the degree of neuroinflammation and contributes to reduced motor coordination after mild TBI.

A highly conserved tryptophan in the N-terminal variable domain regulates disulfide bond formation and oligomeric assembly of adiponectin.

Adiponectin is a collagenous adipokine with direct anti-diabetic and anti-atherogenic properties. It can assume an ensemble of oligomeric states, e.g. trimers, hexamers and octadecamers, each being involved in distinct signaling pathways relevant to adiponectin's diverse biological function in metabolism, immunity, inflammation and cellular homeostasis. Assembly of the active variants principally the octadecameric high molecular weight form is achieved via the tightly controlled oxidation of cysteine 39 located in the adiponectin hyper-variable domain (AHD, residues 18-44) between the signal sequence and the collagen-like domain. We show that mutation of a highly conserved tryptophan (W42A) in the AHD profoundly affects assembly by trapping full-length adiponectin in the oxidized trimeric or hexameric states with a concomitant major reduction in the high molecular weight form. Our biophysical measurements on synthesized analogues of the AHD suggests that the aberrant oligomer distribution can be explained based on the fact that the proximity of W42 to C39 causes a reduction in the rate of C39 oxidation, an effect that to our knowledge has not been documented before. At the biological level, the perturbed oligomer distribution of full-length mutant adiponectin leads to a major reduction in the AMP-activated protein kinase activation in endothelial cells and liver tissues.

Radioimmunotherapy with (131)I-rituximab for patients with relapsed/refractory B-cell non-Hodgkin's lymphoma (NHL).

AIM: To evaluate the efficacy and safety of radioimmunotherapy (RIT) with radioiodinated human/murine chimeric anti-CD20 monoclonal antibody rituximab ((131)I-rituximab) for treating Korean patients with relapsed or refractory B-cell non-Hodgkin's lymphomas (NHL). METHODS: All patients received unlabeled rituximab 70 mg immediately prior to the administration of a therapeutic dose (median dose: 7.3 GBq) of (131)I-rituximab. The tumor response was evaluated 1 month later by contrast enhanced (18) F-fludeoxyglucose positron emission tomography-computed tomography. RESULTS: Between May 2004 and October 2006, 24 patients received single treatment with (131)I-rituximab. The overall response rate (ORR) was 29%; 46% (three complete responses, two partial responses (PR) for patients with low grade B-cell NHL (LGL) and 9% (one PR) for patients with diffuse large B-cell lymphoma (DLBCL). After a median follow-up of 55 months, the median progression-free survival (PFS) for all the patients was 2.2 months. The median overall survival (OS) was 11.3 months. There were statistically significant differences between the LGL and the DLBCL for the median PFS (4.5 months vs 1.3 months, respectively, P = 0.0007) and the median OS (30.3 months vs 6.5 months, respectively, P = 0.0295). Grades 3-4 thrombocytopenia and neutropenia occurred in 33% (8/24) and 21% (5/24) of the patients, respectively. CONCLUSION: RIT with (131)I-rituximab seems to be effective and tolerable for patients with refractory LGL, although this treatment had modest activity in patients with refractory DLBCL. Further studies are warranted to determine the efficacy of (131)I-rituximab for treating the patients with DLBCL.

Prognostic factors for classifying extranodal NK/T cell lymphoma, nasal type, as lymphoid neoplasia.

This study evaluated the applicability of prognostic factors commonly used for diagnosis of classical lymphoma outcomes to extranodal NK/T cell lymphoma, nasal type (NTCL). Clinical features and their associations with lactate dehydrogenase (LDH) were evaluated in 70 patients. RLDH was defined as the ratio of LDH to the upper normal limit. RLDH was associated with stage (I-II vs. III-IV), lymph node involvement (LNI), and International Prognostic Index score (<2 vs. > or =2). Poor performance status and advanced stage were common in patients with local tumor invasiveness (LTI). LDH level, classified into three levels (low, high, and very high) was associated with survival (P < 0.001). In multivariate analysis, the predictive values of LDH level, B symptom, performance status, and stage remained significant whereas those of LTI and LNI did not. Scoring was performed by weighting each factor with 0.5 or 1.0 according to its hazard ratio. Scores were classified into four groups. Groups with high scores were associated with unfavorable outcomes (P < 0.001). Current study suggests that prognostic factors for NHL may be useful to predict the outcome of NTCL but the model should take LDH level and the prognostic weight of each factor into account.

Prognostic factor analysis and proposed prognostic model for conventional treatment of high-grade primary gastric lymphoma.

OBJECTIVES: We conducted a clinical risk factors analysis to define a prognostic model for high-grade primary gastric lymphoma (HG-PGL). METHODS AND RESULTS: The median event-free survival and overall survival of 214 HG-PGL patients were 54 and 104.5 months, respectively, after a median follow-up duration of 60 months. According to the prognostic factor analysis, survival, advanced age, male gender, higher LDH levels and the presence of ascites were identified as independent prognostic factors for HG-PGL. We identified four groups at different risk: group 1, no adverse effect; group 2, one factor; group 3, two factors; group 4, three or four factors. The new prognostic model showed excellent prognostic capacity to differentiate subgroups according to their risk stratification. CONCLUSIONS: The proposed new prognostic model for HG-PGL demonstrated a balanced distribution of patients into four groups with good prognostic capacity.