Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

Magainin 2-derived stapled peptides derived with the ability to deliver pDNA, mRNA, and siRNA into cells.

We have developed cell-penetrating stapled peptides based on the amphipathic antimicrobial peptide magainin 2 for intracellular delivery of nucleic acids such as pDNA, mRNA, and siRNA. Various types of stapled peptides with a cross-linked structure were synthesised in the hydrophobic region of the amphipathic structure, and their efficacy in intracellular delivery of pDNA was evaluated. The results showed that the stapled peptide st7-5 could deliver pDNA into cells. To improve the deliverability of st7-5, we further designed st7-5_R, in which the Lys residues were replaced by Arg residues. The peptide st7-5_R formed compact and stable complexes with pDNA and was able to efficiently transfer pDNA into the cell. In addition to pDNA, st7-5_R was also able to deliver mRNA and siRNA into the cell. Thus, st7-5_R is a novel peptide that can achieve efficient intracellular delivery of three different nucleic acids.

Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.

Structure-activity relationship study of amphipathic antimicrobial peptides using helix-destabilizing sarcosine.

Antimicrobial peptides (AMPs) are potential therapeutic agents against bacteria. We recently showed that a rationally designed AMP, termed Stripe, with an amphipathic distribution of native cationic and hydrophobic amino acids on its helical structure exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria with negligible hemolytic activity and cytotoxicity. In this study, the structure-activity relationship of Stripe was elucidated by designing a series of antimicrobial peptides whereby amino acid residues of Stripe were exchanged with helix-destabilizing sarcosine residues. Stripe 1-5 peptides with hydrophobic amino acids substituted with sarcosine were predominantly unstructured and showed no antimicrobial activity, except against Escherichia coli (E. coli) (DH5α) cells. The activity against E. coli (DH5α) cells and the helicity of Stripe 1-5 peptides decreased concomitantly as the number of sarcosine residue substitutions increased. Stripe 1-5 peptides showed no hemolytic activity or cytotoxicity. The results indicate that sarcosine substitutions provide an approach to study the structure-activity relationship of helical AMPs, and the helicity of Stripe is an important feature defining its activity.

Helical Antimicrobial Peptide Foldamers Containing Non-proteinogenic Amino Acids.

Antimicrobial peptides (AMPs) are potential novel therapeutic drugs against microbial infections. Most AMPs function by disrupting microbial membranes because of their amphipathic properties and ordered secondary structures. In this minireview, we describe recent efforts to develop helical AMP foldamers containing non-proteinogenic amino acids, such as α,α-disubstituted α-amino acids, ß-amino acids, γ-amino acids, side-chain stapling and N-alkyl glycines.

Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence.

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

Status of fracture risk assessment and osteoporosis treatment in Japanese patients with rheumatoid arthritis.

OBJECTIVES: This study aimed to investigate the prevalence of patients with rheumatoid arthritis (RA) at a high risk of major osteoporosis (OP)-related fractures and the status of OP-related medical treatment for these patients. METHODS: We enrolled 120 patients aged ≥40 years (average, 69.1 years) with RA. The Fracture Risk Assessment Tool (FRAX®) was used to evaluate the fracture risk. Of the 120 patients, the femoral neck bone mineral density (BMD) was evaluated in 102 patients, and their FRAX® scores were calculated alongside the BMD values. Patients observed to be at a high risk of a major OP-related fracture (10-year probability >20% or hip fracture risk >3%), according to FRAX®, were identified as those requiring OP treatment; medication ratio for OP (percentage of patients actually receiving medication among patients requiring OP treatment) was assessed. RESULTS: OP treatment was indicated in 75 (63%) patients; the medication ratio for OP was 49%. The use of biological disease-modifying anti-rheumatic drugs and corticosteroids showed a positive effect; however, the use of methotrexate showed a negative effect on the medication ratio. CONCLUSION: The number of potential patients requiring OP treatment is underestimated. All patients with RA should be assessed to determine their eligibility for OP treatment.

Rational Design of Helix-Stabilized Antimicrobial Peptide Foldamers Containing α,α-Disubstituted Amino Acids or Side-Chain Stapling.

Antimicrobial peptides (AMPs) are expected to be good candidate molecules for novel antimicrobial therapies. Most AMPs exert their antimicrobial activity through disruption of microbial membranes due to their amphipathic properties. Recently, the helical peptide 'Stripe' was reported by our group, a rationally designed amphipathic AMP focused on distribution of natural cationic and hydrophobic amino acid residues. In this study, a set of Stripe-based AMP foldamers was designed, synthesized and investigated that contain α,α-disubstituted amino acids or side-chain stapling to stabilize their helical structures. Our results showed that a peptide containing 2-aminoisobutyric acid (Aib) residues exhibited potent antimicrobial activity against both Gram-positive S.aureus (MIC value: 3.125 µM) and Gram-negative bacteria (including a multidrug-resistant strain, MDRP, MIC value: 1.56 µM), without significant hemolytic activity (>100 µM). Electrophysiological measurements revealed that this peptide formed stable pores in a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) bilayer but not in a dioleoylphosphocholine (DOPC) bilayer. The introduction of Aib residues into Stripe could be a promising way to increase the antimicrobial activity of AMP foldamers, and the peptide could represent a promising novel therapeutic candidate to treat multidrug-resistant bacterial infection.

Development of Amphipathic Antimicrobial Peptide Foldamers Based on Magainin 2 Sequence.

Magainin 2 (Mag 2), which is isolated from the skin of frogs, is a representative antimicrobial peptide (AMP), exerts its antimicrobial activity via microbial membrane disruption. It has been reported that both the amphipathicity and helical structure of Mag 2 play an important role in its antimicrobial activity. In this study, we revealed that the sequence of 17 amino acid residues in Mag 2 (peptide 7) is required to exert sufficient activity. We also designed a set of Mag 2 derivatives, based on enhancement of helicity and/or amphipathicity, by incorporation of α,α-disubstituted amino acid residues into the Mag 2 fragment, and evaluated their preferred secondary structures and their antimicrobial activities against both Gram-positive and Gram-negative bacteria. As a result, peptide 11 formed a stable helical structure in solution, and possessed potent antimicrobial activities against both Gram-positive and Gram-negative bacteria without significant cytotoxicity.

Rational design of novel amphipathic antimicrobial peptides focused on the distribution of cationic amino acid residues.

Antimicrobial peptides (AMPs) have garnered much attention as novel therapeutic agents against infectious diseases. They exhibit antimicrobial activity through microbial membrane disruption based on their amphipathic properties. In this study, we rationally designed and synthesized a series of novel AMPs Block, Stripe, and Random, and revealed that Stripe exhibits potent antimicrobial activity against Gram-positive and Gram-negative microbes. Moreover, we also demonstrated that Stripe disrupts both Gram-positive and Gram-negative mimetic bacterial membranes. Finally, we investigated the hemolytic activity and cytotoxicity in human blood cells and human cell lines, and found that Stripe exhibited neither. These data indicated that Stripe is a promising antimicrobial reagent that does not display significant cytotoxicity.

The prognostic value of positron emission tomography/computed tomography in rheumatoid arthritis patients with methotrexate-associated lymphoproliferative disorders.

Recently, methotrexate-associated lymphoproliferative disorders (MTX-LPDs) in rheumatoid arthritis (RA) have been found to commonly occur in association with iatrogenic immunodeficiency. Several factors have been reported to be related to the prognosis. We herein investigate the efficacy of 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) in predicting the prognosis of MTX-LPD. We performed a retrospective analysis of the clinical features, characteristics, and outcomes of 18 patients with MTX-LPDs who were treated from 2004 to 2015. All of the patients were diagnosed with MTX-LPD based on the histological examination of biopsy specimens. Spontaneous regression was detected after the cessation of MTX in 5 of 18 cases (28%). The maximum standardized uptake value (SUVmax) of the FDG uptake on PET/CT was significantly lower, and the maximum size of the LPD-associated tumor was significantly smaller among the patients who showed spontaneous regression (p = 0.01, p = 0.04, respectively). Both the SUVmax and the maximum tumor size were related to better overall survival (p = 0.02, p = 0.04, respectively). Thus, PET/CT can be used to predict spontaneous regression and the prognosis at the diagnosis of MTX/LPD. Cases that showed spontaneous regression never relapsed during the follow-up period, despite the usage of several anti-rheumatoid arthritis drugs, including biological agents. The early detection of LPDs and the early cessation of MTX are important for the management of RA patients. An evaluation by F-FDG-PET/CT can be useful for predicting spontaneous regression and the prognosis.

Clinical characteristics of methotrexate-associated lymphoproliferative disorders: relationship between absolute lymphocyte count recovery and spontaneous regression.

Several reports have shown that patients with rheumatoid arthritis (RA) are at increased risk of developing lymphoproliferative disorders (LPD). Methotrexate (MTX) has been recognized as a major cause of LPD. Sometimes spontaneous regression (SR) occurs after withdrawal of MTX. Recent studies suggest that the early recovery of the absolute lymphocyte count (ALC) after withdrawal of MTX is associated with the spontaneous regression of MTX-LPD. We retrospectively analyzed 26 patients with MTX-LPD to identify predictive factors for spontaneous regression. The spontaneous regression after withdrawal of MTX occurred in 13 of 26 (50%) cases. We assessed the ALC at the time of MTX cessation and 1 month after cessation in 23 evaluable cases. The spontaneous regression was observed in 3 of 11 in the ALC recovery group (27%) and in 8 of the 12 in the ALC non-recovery group (67%). Thus, we could not detect any relationship between the recovery of ALC after withdrawal of MTX and the spontaneous regression. The patients in the ALC recovery group had a poorer prognosis than those in the ALC non-recovery group (2-year overall survival: 65.6 vs. 100%, p = 0.05). According to these results, the recovery of the ALC might not be useful as a predictor of the spontaneous regression. Furthermore, the existence of extranodal sites and advanced-stage were associated with non-SR. It is suspected that MTX-LPD patients with high disease activity at the time of their diagnosis might have little hope of spontaneous regression. This result indicated the importance of the early detection of MTX-LPD.

Polyarteritis nodosa with uterine involvement.

Polyarteritis nodosa (PAN) is characterized by multisystem necrotizing vasculitis, primarily affecting small-to-medium-sized muscular arteries, and it is typically found in middle-aged men. PAN is rarely found in the female genital tract (including the uterus), and imaging of the uterus with PAN has not previously been reported. Reported is a case of a 78-year-old patient with uterus enlargement who was diagnosed with PAN through clinical findings and images. Computed tomography and magnetic resonance imaging findings of a uterus affected by PAN are presented and reviewed, and potential characteristic findings of the uterine with PAN are discussed.

Simvastatin inhibits production of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity. Evidence suggests that statins modulate immune functions and may have a beneficial effect on patients with RA. We investigated whether simvastatin could inhibit the expression of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing joint replacement therapy. METHODS: RA FLS were cultured with or without 0.05-10 microM simvastatin for 12 h. Cytokine mRNA expression and secretion levels were detected using real-time PCR and ELISA, respectively. Cell proliferation of FLS induced by TNF-alpha was determined by MTT assay. RESULTS: Real-time PCR analysis revealed that the levels of IL-6 and IL-8 mRNA expressed by FLS were reduced by simvastatin in a dose-dependent manner. Levels of IL-6 and IL-8 in FLS culture supernatants were decreased by simvastatin in a time-dependent and dose-dependent manner. MTT assay revealed that simvastatin could inhibit proliferation of FLS induced by TNF-alpha. These effects of simvastatin on IL-6 and IL-8 production and cell proliferation were reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not with farnesyl-pyrophosphate. CONCLUSION: Our results suggest that the beneficial effect of simvastatin in RA patients may involve inhibition of IL-6 and IL-8 production, as well as reduction of cell proliferation.

Leflunomide induced acute interstitial pneumonia.

We describe a 54-year-old woman with rheumatoid arthritis (RA), who developed acute respiratory failure 2 weeks after cessation of 6-week treatment with leflunomide. We diagnosed interstitial pneumonia, probably induced by leflunomide because acute respiratory failure was preceded by elevated serum liver enzyme concentration and hypertension. She showed dramatic improvement with prednisolone and cholestyramine. Prompt treatment may improve the prognosis. In Japan, leflunomide has been implicated as a possible cause to initiate or exacerbate interstitial pneumonia in patients with RA according to postmarketing surveillance. Clinicians should exclude pulmonary disease prior to initiating leflunomide treatment in patients with RA on the basis of a thorough history and physical examination, and chest radiograph.

[A case of Behçet's disease with esophageal ulcers complicated with systemic sclerosis, chronic hepatitis C, and pancytopenia].

A 62-year-old man was admitted to our hospital because of retrosternal burning pain and high fever in May, 2002. In 1995 chronic hepatitis C was diagnosed. Five years before admission he had been suffering from recurrent oral aphthous ulcers and genital ulcers. Distal scleroderma developed and the diagnosis of systemic sclerosis was made by skin biopsy in 1999. Prednisolone therapy, 5-30 mg/day, had been administered since then. In May 2000, he was referred to our department, and diagnosis of incomplete-type Behçet's disease was made because he had erythema nodosa, oral aphthous ulcers and genital ulcers. Asymptomatic mild pancytopenia was also found. In November 2000, gastrofiberscopy revealed that he had esophageal and gastric ulcers resistant to regular treatment and was diagnosed as entero-Behçet's disease, a subtype of the disease. The activity of esophageal and gastric ulcers was resistant to the low dose glucocorticoid and more than a moderate dose (30 mg/day) of prednisolone was necessary to reduce the activity. His gastrointestinal symptoms fluctuated with low dose prednisolone. Gastrofiberscopy on admission revealed that he had four shallow active oval ulcers in the middle-lower esophagus and distinct blind-fistula in the lower esophagus. Prednisolone were increased to 30 mg/day for his active entero-Behçet's disease, however, his burning retrosternal pain remained. He died on the 81st hospital day due to severe pneumonia. This is a rare case of Behçet's disease complicated with esophageal ulcers, systemic sclerosis, chronic hepatitis C, and pancytopenia. Of interest is the mechanism of coincidence of these diseases from the pathological point of view.

Renal complications in a patient with A-to-G mutation of mitochondrial DNA at the 3243 position of leucine tRNA.

A 27-year-old woman with short stature, sensorineural deafness, and renal dysfunction was hospitalized for evaluation. The serum lactate and pyruvate concentrations were elevated. The analysis of her mitochondrial DNA revealed an A-to-G mutation of the tRNA(Leu (UUR)) gene at the 3243 position. Renal biopsy revealed many sclerotic glomeruli, advanced tubulointerstitial changes, and numerous swollen mitochondria of the tubular cells. It was concluded that the patient's mitochondrial gene mutation was etiologically related to her nephropathy. The clinicopathologic features of this patient, as contrasted to the previous reports, suggested that renal involvement due to this mitochondrial gene mutation can be heterogeneous.