Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.     

Effectiveness and safety of repeated quadruple therapy in Helicobacter pylori infection after failure of second-line quadruple therapy: repeated quadruple therapy as a third-line therapy

Backgrounds: Quadruple therapy using a proton‐pump inhibitor, bismuth, metronidazole, and tetracycline is a standard second‐line therapy for Helicobacter pylori infection, achieving an eradication rate of about 80% in Korea. A standard third‐line therapy is not currently established, although various protocols have been proposed. We performed this study to evaluate the effectiveness of a retrial with quadruple therapy before starting a third‐line treatment with new drugs. Materials and Methods: In 80 of 746 patients treated with a second‐line quadruple therapy at the Korea University Ansan Hospital between January 2002 and September 2010, treatment for H. pylori had failed, and 45 of these patients were eligible for this study. Eradication of H. pylori was assessed by repeated endoscopy or by the ¹³C‐urea breath test at least 4 weeks after therapy. The patients with treatment failure were treated again with quadruple regimen for 2 weeks and reevaluated for treatment effectiveness and safety. Results: The eradication rate with second‐line quadruple therapy was 86.9%. Of the 80 patients who failed treatment for H. pylori with the initial second‐line quadruple therapy, 64 patients were treated again with the same regimen. Of the 45 retreated patients in this study, three patients were lost to follow‐up and two complied poorly with medication. The eradication rate in the 40 patients retreated was 75.0% at per‐protocol analysis. Seventeen patients experienced mild adverse events. Conclusions: A retrial of quadruple therapy before use of a third‐line therapy may be safe and effective for patients who fail to respond to second‐line quadruple therapy.     

短波对兔VX2肿瘤热疗效果实验研究

目的 探讨短波热疗对荷VX2肿瘤的抑制作用.方法 选择一级新西兰兔49只,随机分成热化组(RH)13只、热疗组(R)13只、化疗组(H)12只和空白对照组(K)11只.移植VX2肿瘤10天后,对RH组施加热疗和化疗,R组施加热疗,H组施加化疗,K组不作任何处理,监测肿瘤的变化.结果 RH组、R组、H组和K组平均瘤重为9.1 g、29.81 g、16.62 g和29.93 g.RH组瘤重极显著小于对照组(P＜0.01),并显著小于H组(P＜0.05).肿瘤平均大小为:7.81 cm3、26.28 cm3、11.66 cm3和30.94 cm3,RH组肿瘤平均体积比H组小,但无显著性差异.结论 短波热疗结合化疗对兔VX2肿瘤的抑制作用明显高于单纯化疗.单纯热疗未见对肿瘤有明显的抑制作用.     

Neoadjuvant BRAF‐targeted therapy in regionally advanced and oligometastatic melanoma

Current management of locoregional and oligometastatic melanoma is typically with surgery; however, some patients are unable to undergo resection due to location/size of their tumors and/or the anticipated morbidity of the surgery. While there are currently no established guidelines for neoadjuvant therapy in melanoma, neoadjuvant BRAF‐targeted therapy may make resection more feasible. A retrospective analysis was conducted of 23 patients with BRAFV600‐mutant, stage III/IV melanoma treated with BRAF‐targeted therapy prior to surgery, with no adjuvant treatment. Surgical specimens, preoperative imaging, and clinical outcomes were evaluated. Results: Ten of 23 patients (44%) attained a pathologic complete response (pCR), with no correlation between RECIST response based on preoperative imaging and pathologic response. After a median of 43‐month follow‐up, only 1 patient (10%) with a pCR recurred, while 8 of 13 (62%) patients without a pCR recurred. Patients with a pCR had significantly improved relapse‐free (RFS) and overall survival (OS) compared to patients with residual tumor. Neoadjuvant BRAF‐targeted therapy is associated with a high pCR rate in patients with stage III‐IV melanoma, which may correlate with improved RFS and OS.     

Comparative efficacy and acceptability of psychotherapies for depression in children and adolescents: A systematic review and network meta‐analysis

Previous meta-analyses of psychotherapies for child and adolescent depression were limited because of the small number of trials with direct comparisons between two treatments. A network meta-analysis, a novel approach that integrates direct and indirect evidence from randomized controlled studies, was undertaken to investigate the comparative efficacy and acceptability of psychotherapies for depression in children and adolescents. Systematic searches resulted in 52 studies (total N=3805) of nine psychotherapies and four control conditions. We assessed the efficacy at post-treatment and at follow-up, as well as the acceptability (all-cause discontinuation) of psychotherapies and control conditions. At post-treatment, only interpersonal therapy (IPT) and cognitive-behavioral therapy (CBT) were significantly more effective than most control conditions (standardized mean differences, SMDs ranged from −0.47 to −0.96). Also, IPT and CBT were more beneficial than play therapy. Only psychodynamic therapy and play therapy were not significantly superior to waitlist. At follow-up, IPT and CBT were significantly more effective than most control conditions (SMDs ranged from −0.26 to −1.05), although only IPT retained this superiority at both short-term and long-term follow-up. In addition, IPT and CBT were more beneficial than problem-solving therapy. Waitlist was significantly inferior to other control conditions. With regard to acceptability, IPT and problem-solving therapy had significantly fewer all-cause discontinuations than cognitive therapy and CBT (ORs ranged from 0.06 to 0.33). These data suggest that IPT and CBT should be considered as the best available psychotherapies for depression in children and adolescents. However, several alternative psychotherapies are understudied in this age group. Waitlist may inflate the effect of psychotherapies, so that psychological placebo or treatment-as-usual may be preferable as a control condition in psychotherapy trials.     

New Insights into the Circadian Rhythm of Acute Myocardial Infarction in Subgroups

The aim of this study was to determine the existence of the circadian rhythm (CR) in the onset of acute myocardial infarction (AMI) in different patient subgroups. Information was collected about 41,244 infarctions from the database of the ARIAM (Analysis of Delay in AMI) Spanish multicenter study. CR in AMI were explored in subgroups of cases categorized by age, gender, previous ischemic heart disease (PIHD), outcome in coronary care unit, infarction electrocardiograph (ECG) characteristics (Q wave or non‐Q wave), and location of AMI. Cases were classified according to these variables in the different subgroups. To verify the presence of CR, a simple test of equality of time series based on the multiple‐sinusoid (24, 12, and 8 h periods) cosinor analysis was developed. For the groups as a whole, the time of pain onset as an indicator of the AMI occurrence showed a CR (p<0.0001), with a morning peak at 10:10 h. All the analyzed subgroups also showed CR. Comparison between subgroups showed significant differences in the PIHD (p<0.01) and infarction ECG characteristics (p<0.01) groups. The CR of the subgroup with Q‐wave infarction differed from that of non‐Q wave subgroup (p<0.01) when the patients had PIHD (23% in Q wave infarction vs. 39.2% in non‐Q wave). AMI onset followed a CR pattern, which is also observed in all analyzed subgroups. Differences in the CR according to the Q/non‐Q wave infarction characteristics could be determined by PIHD. The cosinor model fit with three components (24, 12, and 8 h periods) showed a higher sensitivity than the single 24 h period analysis.     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

Finite element modelling of radial shock wave therapy for chronic plantar fasciitis

Therapeutic use of high-amplitude pressure waves, or shock wave therapy (SWT), is emerging as a popular method for treating musculoskeletal disorders. However, the mechanism(s) through which this technique promotes healing are unclear. Finite element models of a shock wave source and the foot were constructed to gain a better understanding of the mechanical stimuli that SWT produces in the context of plantar fasciitis treatment. The model of the shock wave source was based on the geometry of an actual radial shock wave device, in which pressure waves are generated through the collision of two metallic objects: a projectile and an applicator. The foot model was based on the geometry reconstructed from magnetic resonance images of a volunteer and it comprised bones, cartilage, soft tissue, plantar fascia, and Achilles tendon. Dynamic simulations were conducted of a single and of two successive shock wave pulses administered to the foot. The collision between the projectile and the applicator resulted in a stress wave in the applicator. This wave was transmitted into the soft tissue in the form of compression–rarefaction pressure waves with an amplitude of the order of several MPa. The negative pressure at the plantar fascia reached values of over 1.5 MPa, which could be sufficient to generate cavitation in the tissue. The results also show that multiple shock wave pulses may have a cumulative effect in terms of strain energy accumulation in the foot.     

Effect of millimeter wave irradiation on tumor metastasis

One of the major side effects of chemotherapy in cancer treatment is that it can enhance tumor metastasis due to suppression of natural killer (NK) cell activity. The present study was undertaken to examine whether millimeter electromagnetic waves (MMWs) irradiation (42.2 GHz) can inhibit tumor metastasis enhanced by cyclophosphamide (CPA), an anticancer drug. MMWs were produced with a Russian-made YAV-1 generator. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm(2), respectively. Tumor metastasis was evaluated in C57BL/6 mice, an experimental murine model commonly used for metastatic melanoma. The animals were divided into 5 groups, 10 animals per group. The first group was not given any treatment. The second group was irradiated on the nasal area with MMWs for 30 min. The third group served as a sham control for group 2. The fourth group was given CPA (150 mg/kg body weight, ip) before irradiation. The fifth group served as a sham control for group 4. On day 2, all animals were injected, through a tail vein, with B16F10 melanoma cells, a tumor cell line syngeneic to C57BL/6 mice. Tumor colonies in lungs were counted 2 weeks following inoculation. CPA caused a marked enhancement in tumor metastases (fivefold), which was significantly reduced when CPA-treated animals were irradiated with MMWs. Millimeter waves also increased NK cell activity suppressed by CPA, suggesting that a reduction in tumor metastasis by MMWs is mediated through activation of NK cells.     

Shockwaves prevent from heart failure after acute myocardial ischaemia via RNA/protein complexes

Shock wave treatment (SWT) was shown to induce regeneration of ischaemic myocardium via Toll‐like receptor 3 (TLR3). The antimicrobial peptide LL37 gets released by mechanical stress and is known to form complexes with nucleic acids thus activating Toll‐like receptors. We suggested that SWT in the acute setting prevents from the development of heart failure via RNA/protein release. Myocardial infarction in mice was induced followed by subsequent SWT. Heart function was assessed 4 weeks later via transthoracic echocardiography and pressure–volume measurements. Human umbilical vein endothelial cells (HUVECs) were treated with SWT in the presence of RNase and proteinase and analysed for proliferation, tube formation and LL37 expression. RNA release and uptake after SWT was evaluated. We found significantly improved cardiac function after SWT. SWT resulted in significantly higher numbers of capillaries and arterioles and less left ventricular fibrosis. Supernatants of treated cells activated TLR3 reporter cells. Analysis of the supernatant revealed increased RNA levels. The effect could not be abolished by pre‐treatment of the supernatant with RNase, but only by a sequential digestion with proteinase and RNase hinting strongly towards the involvement of RNA/protein complexes. Indeed, LL37 expression as well as cellular RNA uptake were significantly increased after SWT. We show for the first time that SWT prevents from left ventricular remodelling and cardiac dysfunction via RNA/protein complex release and subsequent induction of angiogenesis. It might therefore develop a potent regenerative treatment alternative for ischaemic heart disease.     

Effect of Weak Sinusoidal Magnetic Field on Germination and Yield of Cotton (Gossypium spp.)

This paper presents the results of a preliminary study on the effects of sinusoidal magnetic fields on percentage germination, growth, and yield of cotton (Gossypium species). The paper covers a brief account of the experimental setup, protocol, and the essential parameters employed. The study was carried out using var.spch‐1 cotton seeds having three different germination capacities (Type 1—31%, Type 2—49%, Type 3—64%). The seeds were subjected to magnetic fields with nine different frequencies and intensities as follows: A. 1 Hz, 100 nT; B. 1 Hz, 1500 nT; C. 1 Hz, 4000 nT; D. 10 Hz, 100 nT; E. 10 Hz, 1500 nT; F. 10 Hz, 4000 nT; G. 100 Hz, 100 nT; H. 100 Hz, 1500 nT; I. 100 Hz, 4000 nT. In Type 3 cotton seeds exposed to 10 Hz, 100 nT fields, the yield was 75.3% higher than that of control, and in Type 2 cotton seeds exposed to 100 Hz, 1500 nT fields, the yield was 85.2% higher than that of control.

The experiment leads to two major clear‐cut findings:

1. Pre‐sowing exposure of seeds to extremely low frequency (ELF) magnetic fields catalyzes certain varieties of cotton to yield as much as double the crop output yielded by unexposed controls.

2. The yield is strongly frequency dependent, i.e., the cotton varieties show outstanding responses only at specific frequencies.

     

Guided Internet‐based vs. face‐to‐face cognitive behavior therapy for psychiatric and somatic disorders: a systematic review and meta‐analysis

Internet-delivered cognitive behavior therapy (ICBT) has been tested in many research trials, but to a lesser extent directly compared to face-to-face delivered cognitive behavior therapy (CBT). We conducted a systematic review and meta-analysis of trials in which guided ICBT was directly compared to face-to-face CBT. Studies on psychiatric and somatic conditions were included. Systematic searches resulted in 13 studies (total N=1053) that met all criteria and were included in the review. There were three studies on social anxiety disorder, three on panic disorder, two on depressive symptoms, two on body dissatisfaction, one on tinnitus, one on male sexual dysfunction, and one on spider phobia. Face-to-face CBT was either in the individual format (n=6) or in the group format (n=7). We also assessed quality and risk of bias. Results showed a pooled effect size (Hedges'' g) at post-treatment of −0.01 (95% CI: −0.13 to 0.12), indicating that guided ICBT and face-to-face treatment produce equivalent overall effects. Study quality did not affect outcomes. While the overall results indicate equivalence, there are still few studies for each psychiatric and somatic condition and many conditions for which guided ICBT has not been compared to face-to-face treatment. Thus, more research is needed to establish equivalence of the two treatment formats.     

Electromagnetic millimeter wave induced hypoalgesia: frequency dependence and involvement of endogenous opioids

Millimeter wave treatment (MMWT) is based on the systemic biological effects that develop following local skin exposure to low power electromagnetic waves in the millimeter range. In the present set of experiments, the hypoalgesic effect of this treatment was analyzed in mice. The murine nose area was exposed to MMW of "therapeutic" frequencies: 42.25, 53.57, and 61.22 GHz. MMWT-induced hypoalgesia was shown to be frequency dependent in two experimental models: (1) the cold water tail-flick test (chronic non-neuropathic pain), and (2) the wire surface test (chronic neuropathic pain following unilateral constriction injury to the sciatic nerve). Maximum hypoalgesic effect was obtained when the frequency was 61.22 GHz. Other exposure parameters were: incident power density = 13.3 mW/cm(2), duration of each exposure = 15 min. Involvement of delta and kappa endogenous opioids in the MMWT-induced hypoalgesia was demonstrated using selective blockers of delta- and kappa-opioid receptors and the direct ELISA measurement of endogenous opioids in CNS tissue. Possible mechanisms of the effect and the perspectives of the clinical application of MMWT are discussed.     

Prevalence of drug-resistant human immunodeficiency virus type 1 in therapy-naive patients and usefulness of genotype testing

In the present study, we performed genotypic drug-resistance testing in 116 therapy-naive human immunodeficiency virus type 1 (HIV-1)-infected patients between 1999 and 2002 at Nagoya National Hospital, Japan. The prevalence of drug-resistant HIV-1 with one or more major mutations significantly increased from 5.3% (4/75) in 1999-2001 to 17.1% (7/41) in 2002 (P=0.05), suggesting the spread of drug-resistant HIV-1. We identified a patient who possessed a protease (PR) inhibitor-resistant HIV-1 with a major mutation consisting of L90M before the initiation of therapy. The patient was administered zidovudine, lamivudine, and efavirenz as highly active antiretroviral therapy (HAART), as PR inhibitors were excluded based on the result of the drug-resistance testing. The treatment succeeded in strongly suppressing the proliferation of drug-resistant HIV-1 and concomitantly increased CD4 cell counts. Thus, we conclude that drug-resistance testing prior to the initiation of therapy is important for therapy-naive patients to devise the optimum therapy regimen for each individual.     

Detection of recombinant growth hormone by evanescent cascaded waveguide coupler on silica‐on‐silicon

An evanescent wave based biosensor is developed on the silica‐on‐silicon (SOS) with a cascaded waveguide coupler for the detection of recombinant growth hormone. So far, U ‐bends and tapered waveguides are demonstrated for increasing the penetration depth and enhancing sensitivity of the evanescent wave sensor. In this work, a monolithically integrated sensor platform containing a cascaded waveguide coupler with optical power splitters and combiners designed with S ‐bends and tapper waveguides is demonstrated for an enhanced detection of recombinant growth hormone. In the cascaded waveguide coupler, a large surface area to bind the antibody with increased penetration depth of evanescent wave to excite the tagged‐rbST is obtained by splitting the waveguide into multiple paths using Y splitters designed with s ‐bends and subsequently combining them back to a single waveguide through tapered waveguide and combiners. Hence a highly sensitive fluoroimmunoassay sensor is realized. Using the 2D FDTD (Finite‐difference time‐domain method) simulation of waveguide with a point source in Rsoft FullWAVE, the fluorescence coupling efficiency of straight and bend section of waveguide is analyzed. The sensor is demonstrated for the detection of fluorescently‐tagged recombinant growth hormone with the detection limit as low as 25 ng/ml. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)