Human gingival fibroblasts (hGFs) play a major role in the maintenance and repair of gingival connective tissue. The mitogen insulin with IGFs etc. synergizes in facilitating wound repair. Although curcumin (CUR) and insulin regulate apoptosis, their impact as a combination on hGF in wound repair remains unknown. Our study consists of: 1) analysis of insulin-mediated mitogenesis on CUR-treated hGF cells, and 2) development of an in vitro model of wound healing.
Materials and Methods:
Apoptotic rate in CUR-treated hGF cells with and without insulin was observed by AnnexinV/PI staining, nuclear morphological analysis, FACS and DNA fragmentation studies. Using hGF confluent cultures, wounds were mechanically created in vitro and incubated with the ligands for 48 h in 0.2% fetal bovine serum DMEM.
CUR alone showed dose-dependent (1–50 μM) effects on hGF. Insulin (1 μg/ml) supplementation substantially enhanced cell survival through up-regulation of mitogenesis/anti-apoptotic elements.
The in vitro model for gingival wound healing establishes that insulin significantly enhanced wound filling faster than CUR-treated hGF cells over 48 h. This reinforces the pivotal role of insulin in supporting CUR-mediated wound repair. The findings have significant bearing in metabolic dysfunctions, e.g. diabetes, atherosclerosis, etc., especially under Indian situations.
Curcumin; fibroblast; gingival healing; insulin; regeneration
Polyphenols as “sensitizers” together with cytotoxic drugs as “inducers” cooperate to trigger apoptosis in various cancer cells. Hence, their combination having similar mode of mechanism may be a novel approach to enhance the efficacy of inducers. Additionally, this will also enable to achieve the physiological concentrations facilitating significant increase in the activity at concentrations which the compound can individually provide. Here we propose that polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC, antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in polyphenols alone. We conclude that differential sensitization of HBCCs with low dose polyphenol augments apoptotic efficacy of CC. This may offer a novel approach to achieve enhanced action of CC with concomitant reduction of side effects enabling improved management of hormone-dependent breast cancer.
Nanomedicines have enormous potential to improve the precision of cancer therapy, yet our ability to efficiently home these materials to regions of disease in vivo remains very limited. Inspired by the ability for communication to improve targeting in biological systems, such inflammatory cell recruitment to sites of disease, we construct systems where synthetic biological and nanotechnological components communicate to amplify disease targeting in vivo. These systems are composed of ‘Signalling’ modules (nanoparticles or engineered proteins) that target tumours and then locally active the coagulation cascade to broadcast tumour location to clot-targeted ‘Receiving’ nanoparticles in circulation that carry a diagnostic or therapeutic cargo, thereby amplifying their delivery. We show that communicating nanoparticle systems can be composed from multiple types of Signalling and Receiving modules, can transmit information via multiple molecular pathways in coagulation, can operate autonomously, and can target over 40-fold higher doses of chemotherapeutics to tumours than non-communicating controls.
The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 107 plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 108 p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 109 p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.
Background & objectives:
Reflux oesophagitis (RE), is one of the most prevalent chronic gastrointestinal disorders commonly referred to as gastroesophageal reflux disease (GERD) and requires long term therapy. The present study was designed to investigate the protective effects of Panax quinquefolium
(PQ), administered with variable doses, on experimentally induced reflux oesophagitis (RE) in rats.
Forty two female Sprague-Dawley (180-220 g) rats were randomly divided to receive standardized root powder of PQ (50-200mg/kg, po), standard anti-reflux (omeprazole, 5 mg/kg, ip) and anti-oxidant (α-tocopherol, 16 mg/kg, po). After 45 min drug pretreatment, RE was produced in rats by simultaneous ligation of the pyloric end and forestomach. Several parameters, including macroscopic lesion index, glutathione system, lipid peroxidation (LPO) and tissue myeloperoxidase (MPO) activity were measured. Alterations in ICAM-1, CINC-2 and MCP-1 gene expression were examined through reverse transcriptase polymerase chain reaction (RT-PCR).
PQ significantly attenuated the severity of the macroscopic signs of RE-induced tissue damage, replenished the depleted GSH level and reduced the RE-associated LPO levels dose dependently. In contrast, omeprazole though effectively improved the mucosal damage, it failed to bring significant attenuation of RE-associated changes in LPO, GSH level and MPO activity. α-Tocopherol significantly ameliorated RE-induced tissue injury and improved LPO level and GSH/GSSG ratio but failed to counteract RE-induced MPO activity. PQ at dose of 100 mg/kg significantly downregulated ICAM-1 and CINC-2 expression whereas it showed no effect over MCP-1 expression.
Interpretation & conclusions:
The present data indicate that PQ protects against RE-induced oesophageal damage via a mechanism that inhibits the influx of inflammatory cell to oesophagus and a consequence excessive oxidative load, opening the avenue to its promising protective role in patients with gastroesophageal reflux disease (GERD).
Acid reflux; anti-inflammatory; oesophagitis; oxidative stress; Panax quinquefolium
Ayurveda is getting its due recognition as a rationale system of medicine worldwide despite the fact that medical and scientific fraternity of the globe has very strong opposite opinion regarding safety and efficacy of Ayurvedic medicines. Meanwhile, provisions of Intellectual Property Rights under World Intellectual Property Organization (WIPO) and Patents have attracted many individuals and organizations to explore possibilities of commercial benefits with Ayurvedic traditional knowledge. Although rules are not favoring to grant a patent on prior published knowledge, biopiracy managed grant of Patent on knowledge of Ayurvedic medicinal plants which has been successfully checked with references of data base of Traditional Knowledge Digital Library (TKDL). Current provisions of the Patent law of India are obstructive in nature for getting patent on Ayurvedic medicines. If we have to invite researchers from basic science to ensure quality, safety and efficacy of Ayurvedic medicines, there is an urgent need to amend laws of patent with pragmatic promotional policies. This will encourage more patents on numerous pharmaceutical, nutraceutical and cosmaceutical products based on Ayurveda. As every action of today's world is based on economic criteria so why stakeholders of Ayurveda should be deprived of it. New inventions would drive acceptance of Ayurveda as a global system of medicine.
Ayurvedic pharmaceuticals; cosmaceuticals; IPR; nutraceuticals; product patent; TKDL
Amongst the mandates of United Nations, health of mankind is the thrust area of UN through World Health Organization (WHO). Planning and execution of policies for mainstreaming of traditional medicines (TRM) of respective countries along with conventional system of medicine (allopathy), first in the country of origin followed by the international arena, is the priority agenda of operations of WHO. Within Indian context, WHO accorded prime focus to Ayurveda in its activities related to TRM.Sponsorship and encouragement of studies substantiating parameters of standardization, safety and efficacy of herbal medicines of Ayurveda are under chief consideration of WHO. In this review, several guidelines of WHO are summarized. Department of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homoeopathy (AYUSH), Central Council of Research in Ayurveda and Siddha and numerous other collaborative centers of WHO in India are assigned with several Appraisal Project Work (APW) and Direct Financial Cooperation (DFC) projects that will strengthen Ayurveda as evidence-based medicine for its global acceptance. Implementation of pharmacovigilance program in Ayurveda, publication of documents for rational use and initiatives to prepare consumer guidelines for appropriate use of Ayurvedic medicines are some other contributions of WHO toward advancement of Ayurveda at national as well as global level. Here, we suggest further exploration, interaction and interpretation of traditional knowledge in the light of contemporary core sciences and biomedical sciences that can pave the way for accreditation of Ayurveda worldwide as an established system of medicine.
Ayurveda; efficacy; standardization; safety; traditional medicine
The emergence of a highly pathogenic H5N1 influenza virus in Hong Kong in 1997 and the subsequent appearance of other H5N1 strains and their spread to several countries in south-east Asia, Africa, the Middle East, and Europe has evoked fear of a global influenza pandemic. Vaccines offer the best hope to combat the threat of an influenza pandemic. However, the global demand for a pandemic vaccine cannot be fulfilled by the current egg-based vaccine manufacturing strategies, thus creating a need to explore alternative technologies for vaccine production and delivery. Several egg-independent vaccine approaches such as cell culture-derived whole virus or subvirion vaccines, recombinant protein-based vaccines, virus-like particle (VLP) vaccines, DNA vaccines and viral vector-based vaccines are currently being investigated and appear promising both in preclinical and clinical studies. The present review will highlight the various egg-independent alternative vaccine approaches for pandemic influenza.
H5N1 influeza; cell-derived vaccine; egg-independent; pandemic influenza; viral vector
Inflammatory responses to implanted biomedical devices elicit a foreign body fibrotic reaction that limits device integration and performance in various biomedical applications. We examined chronic inflammatory responses to microgel conformal coatings consisting of thin films of poly(N-isopropylacrylamide) hydrogel microparticles cross-linked with poly(ethylene glycol) diacrylate deposited on poly(ethylene terephthalate) (PET). Unmodified and microgel-coated PET disks were implanted subcutaneously in rats for 4 weeks and explants were analyzed by histology and immunohistochemistry. Microgel coatings reduced chronic inflammation and resulted in a more mature/organized fibrous capsule. Microgel-coated samples exhibited 22% thinner fibrous capsules that contained 40% fewer cells compared to unmodified PET disks. Furthermore, microgel-coated samples contained significantly higher levels of macrophages (40%) than unmodified PET controls. These results demonstrate that microgel coatings reduce chronic inflammation to implanted biomaterials.
foreign body response; macrophage; hydrogel; polyethylene terephthalate; fibrous capsule
Sandhana kalpana (biomedical fermented formulations) are one of the best dosage forms of Ayurveda in practice since thousands of years. In order to prepare these medicaments, certain sets of conditions are prearranged, which lead to fermentation. Thus, products bequeath with self-generated ethyl alcohol, which potentiate these preparations (Asava–Arishta), pharmaceutically and therapeutically. Commonly, medicinal and commercial components of these formulations are prompting many researchers to contribute in manufacturing, quality control, safety, and efficacy of these formulations. To cope up with this, literature related to Asava–Arishta has been surveyed from the Vedic period to recent publications of Government of India, ie, Ayurvedic Formulary of India, and presented briefly here. In this review paper, we have discussed pioneering facts such as nature and amount of carbohydrate, type of containers, optimum temperature, variety and relevance of initiator of fermentation, manufacturing, regulatory rules, and business aspects of Asava-Arishta. After going through this basic information, any academician or researcher may show a way to further strengthen this dosage form.
Asava; Arishta; ethyl alcohol; fermentation; quality control; Sandhana kalpana
Histone methylation plays an important role in regulating chromatin-mediated gene control and epigenetic-based memory systems that direct cell fate. Enzymes termed histone demethylases directly remove the methyl marks from histones, thus contributing to a dynamically regulated histone methylated genome, however the biological functions of these newly identified enzymes remains unclear. The JMJD2A-D family belongs to the JmjC domain-containing family of histone demethylases (JHDMs). Here, we report the cloning and functional characterization of the Drosophila HDM gene Dmel\Kdm4A that is a homolog of the human JMJD2 family. We show that homologs for three human JHDM families, JHDM1, JHDM2 and JMJD2 are present in Drosophila and that are each expressed during the Drosophila lifecycle. Disruption of Dmel\Kdm4A results in a reduction of the male lifespan and a male-specific wing extension/twitching phenotype that occurs in response to other males, and is reminiscent of an inter-male courtship phenotype involving the courtship song. Remarkably, certain genes associated with each of these phenotypes are significantly downregulated in response to Dmel\Kdm4A loss, most notably the longevity associated Hsp22 gene and the male sex-determination fruitless gene. Our results have implications for the role of the epigenetic regulator Dmel\Kdm4A in the control of genes involved in lifespan and male-specific sex-determination in the fly.
The unprecedented global spread of highly pathogenic avian H5N1 influenza viruses within the past ten years and their extreme lethality to poultry and humans has underscored their potential to cause an influenza pandemic. Combating the threat of an impending H5N1 influenza pandemic will require a combination of pharmaceutical and nonpharmaceutical intervention strategies. The emergence of the H1N1 pandemic in 2009 emphasised the unpredictable nature of a pandemic influenza. Undoubtedly, vaccines offer the most viable means to combat a pandemic threat. Current egg-based influenza vaccine manufacturing strategies are unlikely to be able to cater to the huge, rapid global demand because of the anticipated scarcity of embryonated eggs in an avian influenza pandemic and other factors associated with the vaccine production process. Therefore, alternative, egg-independent vaccine manufacturing strategies should be evaluated to supplement the traditional egg-derived influenza vaccine manufacturing. Furthermore, evaluation of dose-sparing strategies that offer protection with a reduced antigen dose will be critical for pandemic influenza preparedness. Development of new antiviral therapeutics and other, nonpharmaceutical intervention strategies will further supplement pandemic preparedness. This review highlights the current status of egg-dependent and egg-independent strategies against an avian influenza pandemic.
The present study was undertaken to evaluate the anti-ulcer and antioxidant activities of the ethanol extract of Lagenaria breviflora (EELB) whole fruit in laboratory rats.
The anti-ulcer property of the ethanolic extract of the whole fruit of Lagenaria breviflora (LB) was assessed using the cold-restraint stress-induced (CRU) gastric ulcer, pyloric ligation-induced (PL) gastric ulcer, aspirin-induced (ASP) gastric ulcer and alcohol-induced (AL) gastric ulcer models. The scavenging activity of the LB extract was examined with 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), Nitric oxide, Hydroxyl radical and Superoxide anion scavenging models.
EELB (50, 100, 150 and 200 mg/kg, b.w.) protected against the CRU gastric ulcer dose dependently. Similarly, 150 mg/kg b.w. of the LB extract protected against the PL gastric ulcer, ASP gastric ulcer and AL gastric ulcer and was comparable to omeprazole (10 mg/kg b.w.) or Suscralfate (500 mg/kg b.w.), respectively. The in vitro antioxidant activity of LB was demonstrated by its ability to quench free radicals generated by nitric oxide and superoxide anion with a concomitant scavenging potential against DPPH-induced radical formation.
Taken together, the study showed that the whole fruit extract possess potent anti-ulcer and antioxidant activities.
Anti-ulcer; antioxidant; free radical scavenger
Ayurvedic dosage forms are very exclusive in its pharmaceutics and therapeutics. Sneha Kalpana is a group of products of medicated taila and ghee, these drugs are treating very wide range of diseases among patients of all age groups. Liposomal system of drug delivery is a new invention in conventional system of medicine. This system is also covering a high degree of objective of therapeutics at different targets successfully. Probably, here is very distinctive similarity between these two on account of their aqueous and oleaginous origin. Most likely, these are two faces of same coin. A brief survey of literature is done here to explore possibilities of further investigation in benefit of mankind by applying wisdom of both fields together. In fact, this is a review paper based on certain hypothesis which may be established or rejected factually by further researches.
Aqueous; liposomes; oleaginous; Sneha Kalpana; therapeutics
Pharmacovigilance is a corrective process originating in pharmaco-epidemiology. The 1997 Erice Declaration, presented at the World Health Organisation, became the basis on which the concept was implemented internationally for conventional systems of medicine. The increasing international acceptance of Ayurveda, led regulators to implement a similar program for Ayurveda, particularly as some medical professionals, scientists and members of the public reported adverse reactions after taking Ayurvedic formulations. The World Health Organisation therefore persuaded the Department of AYUSH, Ministry of Health and Family Welfare, Government of India, to implement a pharmacovigilance program for Ayurveda, as a means to ensuring the safety and efficacy of Ayurvedic medicines. After a year of due diligence, the pharmacovigilance program was launched nationally on 29 September 2008. Since that time, Ayurveda, Siddha and Unani medicines have been monitored according to the provisions of a protocol prepared by the National Pharmacovigilance Resource Centre, IPGTRA, Jamnagar, and approved by Department of AYUSH. The program was reviewed, first, on 21st January 2009 by the National Pharmaco-vigilance Consultative Committee for ASU drugs (NPCC-ASU), and again, on 15 Feburary, 2010, when an evaluation meeting effectively rubber stamped the program. Among the outcomes of these meetings were several suggestions of measures to improve the program’s efficiency. Recent developments include the constitution of pharmacovigilance centers at all Ayurveda Teaching institutes and research centers.
Adverse drug reaction; Awareness; Ayurvedic medicine; Pharmacovigilance; Safety
In the present research paper, the work done on pharmaceutical study of Lauha Bhasma conducted in the Department of Rasa Shastra under the postgraduate research programme is being presented. The pharmaceutical processing of Lauha Bhasma was performed by following samanya shodhana, vishesha shodhana and marana of Lauha. Under the process of marana, three specific pharmaceutical techniques were followed, viz. bhanupaka, sthalipaka and putapaka. During the putapaka process, an electric muffle furnace (EMF) was used. The temperature of puta was studied in two batches, viz. in Batch I, a temperature of 800°C was maintained whereas in Batch II, a temperature of 600°C was maintained. The purpose behind selecting two temperatures was to validate the process of marana of Lauha and to determine an ideal temperature for the preparation of Lauha Bhasma in EMF. It is found that after 20 puta at a temperature of 600°C, the Lauha Bhasma was prepared properly. The entire characteristic of Lauha Bhasma, like “pakwa jambu phala varna,” varitar, etc. was attained at 600°. At a temperature of 800°C, the process could not be carried out smoothly. The pellets turned very hard and brassy yellow in color. The desired color was attained only after decreasing the temperature in further puta.
Ayurveda; electric muffle furnace; Lauha; marana; puta; shodhana
The one of the oldest system of medicine, Ayurveda is momentous in audience of worldwide on virtue of its holistic approach of life. Formulations of Ayurveda consist of substances of herbal, mineral/metal and animal origin which are processed pharmaceutical to have therapeutic effects. This is attribute of processes of Shodhan (purification/potentiation), Bhavana (impregation /levigation) and Marana (incineration/calcinations) of Rasa Shastra which acclimatize these toxic industrial matter to a effective remedies known as herbo mineral formulations (Rasaoushadhies) of Ayurveda. In recent past there is prevalence of some doubt on safety and efficacy of these medicines. In this review paper we tried to justify application of these medicines as these are time tested and showed wonderful clinical adaptability. We also attempted to establish new facts of figures of core science in explanation of these medicines.
Shodhan ’a-MaranaBhasma; Nanotechnology; Metallopharmaceuticals
Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocytes/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-α, IL-1β, MCP-1) in response to materials implanted acutely in the murine intraperitoneal space. These microgel coatings can be applied to biomedical implants as a protective coating to attenuate biofouling, leukocyte adhesion and activation, and adverse host responses for biomedical and biotechnological applications.
cell adhesion; cytokine; foreign body response; hydrogel; macrophage; polyethylene terephthalate
Dopaminergic neurodegeneration during Parkinson disease (PD) involves several pathways including proteasome inhibition, α-synuclein (α-syn) aggregation, mitochondrial dysfunction, and glutathione (GSH) depletion. We have utilized a systems biology approach and built a dynamic model to understand and link the various events related to PD pathophysiology. We have corroborated the modeling data by examining the effects of α-syn expression in the absence and presence of proteasome inhibition on GSH metabolism in dopaminergic neuronal cultures. We report here that the expression of the mutant A53T form of α-syn is neurotoxic and causes GSH depletion in cells after proteasome inhibition, compared to wild-type α-syn-expressing cells and vector control. Modeling data predicted that GSH depletion in these cells was due to ATP loss associated with mitochondrial dysfunction. ATP depletion elicited by combined A53T expression and proteasome inhibition results in decreased de novo synthesis of GSH via the rate-limiting enzyme γ-glutamyl cysteine ligase. Based on these data and other recent reports, we propose a novel dynamic model to explain how the presence of mutated α-syn protein or proteasome inhibition may individually impact on mitochondrial function and in combination result in alterations in GSH metabolism via enhanced mitochondrial dysfunction.
Parkinson's disease; Neurodegeneration; α-Synuclein; Protein aggregation; Proteasome inhibition; Mitochondrial dysfunction; Glutathione; Systems biology; Dynamic model; In silico; Free radicals
Non-synonymous single nucleotide polymorphisms (SNPs) within vital DNA repair genes may cause reduction of activity leaving the genome unrepaired resulting in genomic instability and cancer.
Materials and methods
The present endeavour involved study on the association of the SNP rs13181 (Lys751Gln/A18911C) in the Nucleotide Excision Repair (NER) pathway gene ERCC2 (excision repair cross-complementing rodent repair deficiency, complementation group 2) with the risks of Squamous Cell Carcinomas of the Head and Neck (SCCHN) and Breast cancer using a case-control based association study among 685 (400 controls and 285 SCCHN-affected cases) and 395 (227 normal healthy female controls and 168 breast cancer cases) ethnically-matched samples, respectively from north India using Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism (PCR-RFLP) analysis.
Results showed significant association of rs13181 homozygous mutant (CC) [Odds Ratio (OR) 4.412, 95% Confidence Interval (CI) 2.413 to 8.068], heterozygous (AC) (OR 2.086, 95% CI 1.246 to 3.492) and combined mutant (AC + CC) (OR 2.672, 95% CI 1.647 to 4.334) genotypes with predisposition to Breast cancer. Statistically significant increase in SCCHN risk was also associated with the mutant genotypes of rs13181 (ERCC2), viz. homozygous mutant (CC) (OR 1.680, 95% CI 1.014 to 2.784), heterozygous (AC) (OR 1.531, 95% CI 1.092 to 2.149) and combined mutant (AC + CC) (OR 1.560, 95% CI 1.128 to 2.158) genotypes.
The results of this case-control study indicate that the polymorphism rs13181 might be a risk factor for predisposition towards SCCHN and breast cancer among north Indian subpopulations.
Because of high prevalence of adenovirus (Ad) infections in humans, it is believed that preexisting Ad-neutralizing antibodies (‘vector immunity’) may negatively impact the immune response to vaccine antigens when delivered by human Ad (HAd) vectors. In order to evaluate whether bovine adenovirus subtype 3 (BAd3), a nonhuman Ad vector can effectively elude high levels of preexisting vector immunity, naïve or HAd serotype 5 (HAd)-primed mice were immunized with BAd-H5HA [BAd3 vector expressing the hemagglutinin (HA) gene from H5N1 influenza virus]. Even in the presence of very high levels of HAd-specific neutralizing antibody, no significant reductions in HA-specific humoral and cell-mediated immune responses were observed in HAd-primed mice immunized with BAd-H5HA. In naïve mice immunized with HAd-H5HA (HAd5 vector expressing H5N1 HA) and boosted with BAd-H5HA, the humoral responses elicited were significantly higher (P<0.01) than with either with either HAd-H5HA or BAd-H5HA alone, while the CMI responses were comparable in the groups. This finding underlines the importance of a heterologous prime-boost approach for achieving an enhanced immune response. The immunization of naïve or HAd-primed mice with BAd-H5HA bestowed full protection from morbidity and mortality following a potentially lethal challenge with A/Hong Kong/483/97. These results demonstrate the importance of BAd vectors as an alternate or supplement to HAd vectors for influenza pandemic preparedness.
Development of effective and immunogenic vaccines against highly pathogenic avian influenza H5N1 viruses with the potential to cause a pandemic is a public health priority. The global demand for a vaccine cannot be met in the event of an influenza pandemic because of the limited capacity to manufacture egg-derived vaccines as well as potential problems with the availability of embryonated eggs. Thus, there is an urgent need to develop alternative, egg-independent vaccines. We developed an adenoviral vector-based vaccine that contains hemagglutinin proteinfrom clade 1 and clade 2 viruses, as well as conserved nucleoprotein, to broaden the vaccine coverage against H5N1 viruses.
Pandemic Influenza; Vaccine; Adenoviral vector; Avian influenza virus
Nanoparticles possessing multiple functionalities provide synthetic handles for varied surface chemistries, making them useful for a range of applications such as biotargeting and drug delivery. However, the combination of interfering functionalities on the same particle is often challenging. We have employed a synthetic scheme involving chemical protection/deprotection to combine interfering functional groups on the same hydrogel nanoparticle. The synthesis of amine-containing poly(N-isopropylacrylamide) nanogels was carried out via free radical precipitation polymerization by incorporating a Fmoc-protected amine PEG macromonomer. The Fmoc group was then removed to obtain free amines, which were shown to be available for conjugation. We further explored pNIPAm-co-acrylic acid nanogels with a protected amine-PEG, yielding zwitterionic particles. With careful attention to the order of the chemoligation and deprotection steps, these interfering functional groups can be forced to behave in a pseudo-orthogonal fashion, allowing for multiple chemoligation steps that employ both the amine and carboxylic acid groups.
Nanogels; Chemoligation; Orthogonal functionality; Bioconjugation
Small interfering RNAs (siRNAs) mediate cleavage of specific, complementary mRNA sequences and thus regulate gene expression. Not surprisingly, their use for treatment of diseases that are rooted in aberrant gene expression, such as cancer, has become a paradigm that has gained wide interest. Here, we report the development of dendrimer-conjugated magnetofluorescent nanoworms that we call “dendriworms” as a modular platform for siRNA delivery in vivo. This platform maximizes endosomal escape to robustly produce protein target knockdown in vivo, and is tolerated well in mouse brain. We demonstrate that siRNA-carrying dendriworms can be readily internalized by cells and enable endosomal escape across a wide range of loading doses, whereas dendrimers or nanoworms alone are inefficient. Further, we show that dendriworms carrying siRNA against the epidermal growth factor receptor (EGFR) reduce protein levels of EGFR in human glioblastoma cells by 70−80%, 2.5-fold more efficiently than commercial cationic lipids. Dendriworms were well-tolerated after 7-days of convection-enhanced delivery to the mouse brain and in an EGFR-driven transgenic model of glioblastoma, anti- EGFR dendriworms led to specific and significant suppression of EGFR expression. Collectively, these data establish dendriworms as a multimodal platform that enables fluorescent tracking of siRNA delivery in vivo, cellular entry, endosomal escape, and knockdown of target proteins.
dendrimer; siRNA; nanoparticle; EGFR; glioblastoma
RNA interference (RNAi) is a cellular process whereby the silencing of a particular gene is mediated by short RNAs (siRNAs). Although siRNAs have great therapeutic potential, cellular delivery has been a challenge. Nanoparticle-siRNA conjugates have emerged as potential delivery vehicles; however, reports describing the effects of nanoparticle conjugation on RISC incorporation and subsequent gene silencing have been mixed. In this report, we have systematically evaluated the effect of siRNA coupling strategies using a model nanoparticle system with varying conjugation schemes. We show that the accessibility of the siRNA linked to the nanoparticle and the lability of the cross-linker are critical for efficient gene knockdown.