Youth Mental Health Outcomes up to Two Years After SARS-CoV-2 Infection Long-COVID or Long-Pandemic Syndrome: A Retrospective Cohort Study.

PURPOSE: Youth mental distress has substantially increased during the COVID-19 pandemic. However, it is unclear if mental symptoms are directly related to SARS-CoV-2 infection or to social restrictions. We aimed to investigate mental health outcomes in infected versus uninfected adolescents, for up to two years after an index polymerase chain reaction (PCR) test. METHODS: A retrospective cohort study, based on electronic health records from a large nationally representative Israeli health fund, among adolescents aged 12-17 years with a PCR test for SARS-CoV-2 between March 1, 2020 and March 1, 2021. Infected and uninfected individuals were matched by age, sex, test date, sector, and socioeconomic status. Cox regression was used to derive hazard ratios (HRs) for mental health outcomes within two years from PCR test for infected versus uninfected individuals, while accounting for pre-existing psychiatric history. External validation was performed on UK primary care data. RESULTS: Among 146,067 PCR-tested adolescents, 24,009 were positive and 22,354 were matched with negative adolescents. SARS-CoV-2 infection was significantly associated with reduced risks for dispensation of antidepressants (HR 0.74, 95% confidence interval [CI] 0.66-0.83), diagnoses of anxiety (HR 0.82, 95% CI 0.71-0.95), depression (HR 0.65, 95% CI 0.53-0.80), and stress (HR 0.80, 95% CI 0.69-0.92). Similar results were obtained in the validation dataset. DISCUSSION: This large, population-based study suggests that SARS-CoV-2 infection is not associated with elevated risk for mental distress in adolescents. Our findings highlight the importance of taking a holistic view on adolescents' mental health during the pandemic, with consideration of both SARS-CoV-2 infection and response measures.

Data-Driven Assessment of Adolescents' Mental Health During the COVID-19 Pandemic.

OBJECTIVE: Adolescents' mental health was severely compromised during the COVID-19 pandemic. Longitudinal real-world studies on changes in the mental health of adolescents during the later phase of the pandemic are limited. We aimed to quantify the effect of COVID-19 pandemic on adolescents' mental health outcomes based on electronic health records. METHOD: This was a retrospective cohort study using the computerized database of a 2.5 million members, state-mandated health organization in Israel. Rates of mental health diagnoses and psychiatric drug dispensations were measured among adolescents 12 to 17 years of age with and without pre-existing mental history, for the years 2017 to 2021. Relative risks were computed between the years, and interrupted time series (ITS) analyses evaluated changes in monthly incidence rates of psychiatric outcomes. RESULTS: The average population size was 218,146 in 2021. During the COVID-19 period, a 36% increase was observed in the incidence of depression (95% CI = 25-47), 31% in anxiety (95% CI = 23-39), 20% in stress (95% CI = 13-27), 50% in eating disorders (95% CI = 35-67), 25% in antidepressant use (95% CI = 25-33), and 28% in antipsychotic use (95% CI = 18-40). A decreased rate of 26% (95% CI = 0.80-0.88) was observed in ADHD diagnoses. The increase of the examined outcomes was most prominent among youth without psychiatric history, female youth, general secular Jewish population, youth with medium-high socioeconomic status, and those 14 to 15 years of age. ITS analysis confirmed a significantly higher growth in the incidence of psychiatric outcomes during the COVID-19 period, compared to those in previous years. CONCLUSION: This real-world study highlights the deterioration of adolescents' mental health during the COVID-19 pandemic and suggests that youth mental health should be considered during health policy decision making. DIVERSITY & INCLUSION STATEMENT: We worked to ensure sex and gender balance in the recruitment of human participants. We worked to ensure race, ethnic, and/or other types of diversity in the recruitment of human participants. We actively worked to promote sex and gender balance in our author group. The author list of this paper includes contributors from the location and/or community where the research was conducted who participated in the data collection, design, analysis, and/or interpretation of the work.

Long covid outcomes at one year after mild SARS-CoV-2 infection: nationwide cohort study.

OBJECTIVES: To determine the clinical sequelae of long covid for a year after infection in patients with mild disease and to evaluate its association with age, sex, SARS-CoV-2 variants, and vaccination status. DESIGN: Retrospective nationwide cohort study. SETTING: Electronic medical records from an Israeli nationwide healthcare organisation. POPULATION: 1 913 234 Maccabi Healthcare Services members of all ages who did a polymerase chain reaction test for SARS-CoV-2 between 1 March 2020 and 1 October 2021. MAIN OUTCOME MEASURES: Risk of an evidence based list of 70 reported long covid outcomes in unvaccinated patients infected with SARS-CoV-2 matched to uninfected people, adjusted for age and sex and stratified by SARS-CoV-2 variants, and risk in patients with a breakthrough SARS-CoV-2 infection compared with unvaccinated infected controls. Risks were compared using hazard ratios and risk differences per 10 000 patients measured during the early (30-180 days) and late (180-360 days) time periods after infection. RESULTS: Covid-19 infection was significantly associated with increased risks in early and late periods for anosmia and dysgeusia (hazard ratio 4.59 (95% confidence interval 3.63 to 5.80), risk difference 19.6 (95% confidence interval 16.9 to 22.4) in early period; 2.96 (2.29 to 3.82), 11.0 (8.5 to 13.6) in late period), cognitive impairment (1.85 (1.58 to 2.17), 12.8, (9.6 to 16.1); 1.69 (1.45 to 1.96), 13.3 (9.4 to 17.3)), dyspnoea (1.79 (1.68 to 1.90), 85.7 (76.9 to 94.5); 1.30 (1.22 to 1.38), 35.4 (26.3 to 44.6)), weakness (1.78 (1.69 to 1.88), 108.5, 98.4 to 118.6; 1.30 (1.22 to 1.37), 50.2 (39.4 to 61.1)), and palpitations (1.49 (1.35 to 1.64), 22.1 (16.8 to 27.4); 1.16 (1.05 to 1.27), 8.3 (2.4 to 14.1)) and with significant but lower excess risk for streptococcal tonsillitis and dizziness. Hair loss, chest pain, cough, myalgia, and respiratory disorders were significantly increased only during the early phase. Male and female patients showed minor differences, and children had fewer outcomes than adults during the early phase of covid-19, which mostly resolved in the late period. Findings remained consistent across SARS-CoV-2 variants. Vaccinated patients with a breakthrough SARS-CoV-2 infection had a lower risk for dyspnoea and similar risk for other outcomes compared with unvaccinated infected patients. CONCLUSIONS: This nationwide study suggests that patients with mild covid-19 are at risk for a small number of health outcomes, most of which are resolved within a year from diagnosis.

Estimating the effect of cesarean delivery on long-term childhood health across two countries.

Assessing the impact of cesarean delivery (CD) on long-term childhood outcomes is challenging as conducting a randomized controlled trial is rarely feasible and inferring it from observational data may be confounded. Utilizing data from electronic health records of 737,904 births, we defined and emulated a target trial to estimate the effect of CD on predefined long-term pediatric outcomes. Causal effects were estimated using pooled logistic regression and standardized survival curves, leveraging data breadth to account for potential confounders. Diverse sensitivity analyses were performed including replication of results in an external validation set from the UK including 625,044 births. Children born in CD had an increased risk to develop asthma (10-year risk differences (95% CI) 0.64% (0.31, 0.98)), an average treatment effect of 0.10 (0.07-0.12) on body mass index (BMI) z-scores at age 5 years old and 0.92 (0.68-1.14) on the number of respiratory infection events until 5 years of age. A positive 10-year risk difference was also observed for atopy (10-year risk differences (95% CI) 0.74% (-0.06, 1.52)) and allergy 0.47% (-0.32, 1.28)). Increased risk for these outcomes was also observed in the UK cohort. Our findings add to a growing body of evidence on the long-term effects of CD on pediatric morbidity, may assist in the decision to perform CD when not medically indicated and paves the way to future research on the mechanisms underlying these effects and intervention strategies targeting them.

Results of an early second PCR test performed on SARS-CoV-2 positive patients may support risk assessment for severe COVID-19.

Identifying patients at increased risk for severe COVID-19 is of high priority during the pandemic as it could affect clinical management and shape public health guidelines. In this study we assessed whether a second PCR test conducted 2-7 days after a SARS-CoV-2 positive test could identify patients at risk for severe illness. Analysis of a nationwide electronic health records data of 1683 SARS-CoV-2 positive individuals indicated that a second negative PCR test result was associated with lower risk for severe illness compared to a positive result. This association was seen across different age groups and clinical settings. More importantly, it was not limited to recovering patients but also observed in patients who still had evidence of COVID-19 as determined by a subsequent positive PCR test. Our study suggests that an early second PCR test may be used as a supportive risk-assessment tool to improve disease management and patient care.

Remote allergen exposure elicits eosinophil infiltration into allergen nonexposed mucosal organs and primes for allergic inflammation.

The natural history of allergic diseases suggests bidirectional and progressive relationships between allergic disorders of the skin, lung, and gut indicative of mucosal organ crosstalk. However, impacts of local allergic inflammation on the cellular landscape of remote mucosal organs along the skin:lung:gut axis are not yet known. Eosinophils are tissue-dwelling innate immune leukocytes associated with allergic diseases. Emerging data suggest heterogeneous phenotypes of tissue-dwelling eosinophils contribute to multifaceted roles that favor homeostasis or disease. This study investigated the impact of acute local allergen exposure on the frequency and phenotype of tissue eosinophils within remote mucosal organs. Our findings demonstrate allergen challenge to skin, lung, or gut elicited not only local eosinophilic inflammation, but also increased the number and frequency of eosinophils within remote, allergen nonexposed lung, and intestine. Remote allergen-elicited lung eosinophils exhibited an inflammatory phenotype and their presence associated with enhanced susceptibility to airway inflammation induced upon subsequent inhalation of a different allergen. These data demonstrate, for the first time, a direct effect of acute allergic inflammation on the phenotype and frequency of tissue eosinophils within antigen nonexposed remote mucosal tissues associated with remote organ priming for allergic inflammation.

CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules.

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.

Thy1+ NK [corrected] cells from vaccinia virus-primed mice confer protection against vaccinia virus challenge in the absence of adaptive lymphocytes.

While immunological memory has long been considered the province of T- and B-lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1(+) subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1(+) NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.

A safety and tolerability study of differently-charged nanoparticles for local pulmonary drug delivery.

Nanoparticle (NP) based drug delivery systems provide promising opportunities in the treatment of lung diseases. Here we examined the safety and tolerability of pulmonary delivered NPs consisting of PEG-PLA as a function of particle surface charge. The rationale for such a comparison should be attributed to the differential pulmonary toxicity of positively and negatively charged PEG-PLA NP. Thus, the local and systemic effects of pulmonary administered NPs were investigated following 5days of daily endotracheal instillation to BALB/c mice that were euthanized on the eighth or nineteenth day of the experiment. We collected bronchoalveolar lavages and studied hematological as well as histochemistry parameters. Notably, the cationic stearylamine based PEG-PLA NPs elicited increased local and systemic toxic effects both on the eighth and nineteenth day. In contrast, anionic NPs of similar size were much better tolerated with local inflammatory effects observed only on the eighth experimental day after pulmonary instillation. No systemic toxicity effect was observed although a moderate change was noted in the platelet count that was not considered to be of clinical significance. No pathological observations were detected in the internal organs following instillation of anionic NPs. Overall these observations suggest that anionic PEG-PLA NPs are useful pulmonary drug carriers that should be considered as a promising therapeutic drug delivery system.

Route of adenovirus-based HIV-1 vaccine delivery impacts the phenotype and trafficking of vaccine-elicited CD8+ T lymphocytes.

Candidate HIV-1 vaccine regimens utilizing intramuscularly (i.m.) administered recombinant adenovirus (rAd)-based vectors can induce potent mucosal cellular immunity. However, the degree to which mucosal rAd vaccine routing might alter the quality and anatomic distribution of vaccine-elicited CD8(+) T lymphocytes remains unclear. We show that the route of vaccination critically impacts not only the magnitude but also the phenotype and trafficking of antigen-specific CD8(+) T lymphocytes in mice. I.m. rAd immunization induced robust local transgene expression and elicited high-frequency, polyfunctional CD8(+) T lymphocytes that trafficked broadly to both systemic and mucosal compartments. In contrast, intranasal (i.n.) rAd immunization led to similarly robust local transgene expression but generated low-frequency, monofunctional CD8(+) T lymphocytes with restricted anatomic trafficking patterns. Respiratory rAd immunization elicited systemic and mucosal CD8(+) T lymphocytes with phenotypes and trafficking properties distinct from those elicited by i.m. or i.n. rAd immunization. Our findings indicate that the anatomic microenvironment of antigen expression critically impacts the phenotype and trafficking of antigen-specific CD8(+) T lymphocytes.

Efficient generation of mucosal and systemic antigen-specific CD8+ T-cell responses following pulmonary DNA immunization.

Although mucosal CD8(+) T-cell responses are important in combating mucosal infections, the generation of such immune responses by vaccination remains problematic. In the present study, we evaluated the ability of plasmid DNA to induce local and systemic antigen-specific CD8(+) T-cell responses after pulmonary administration. We show that the pulmonary delivery of plasmid DNA formulated with polyethyleneimine (PEI-DNA) induced robust systemic CD8(+) T-cell responses that were comparable in magnitude to those generated by intramuscular (i.m.) immunization. Most importantly, we observed that the pulmonary delivery of PEI-DNA elicited a 10-fold-greater antigen-specific CD8(+) T-cell response in lungs and draining lymph nodes of mice than that of i.m. immunization. The functional evaluation of these pulmonary CD8(+) T cells revealed that they produced type I cytokines, and pulmonary immunization with PEI-DNA induced lung-associated antigen-specific CD4(+) T cells that produced higher levels of interleukin-2 than those induced by i.m. immunization. Pulmonary PEI-DNA immunization also induced CD8(+) T-cell responses in the gut and vaginal mucosa. Finally, pulmonary, but not i.m., plasmid DNA vaccination protected mice from a lethal recombinant vaccinia virus challenge. These findings suggest that pulmonary PEI-DNA immunization might be a useful approach for immunizing against pulmonary pathogens and might also protect against infections initiated at other mucosal sites.

X4 human immunodeficiency virus type 1 gp120 down-modulates expression and immunogenicity of codelivered antigens.

In order to increase the immune breadth of human immunodeficiency virus (HIV) vaccines, strategies such as immunization with several HIV antigens or centralized immunogens have been examined. HIV-1 gp120 protein is a major immunogen of HIV and has been routinely considered for inclusion in both present and future AIDS vaccines. However, recent studies proposed that gp120 interferes with the generation of immune response to codelivered antigens. Here, we investigate whether coimmunization with plasmid-encoded gp120 alters the immune response to other coadministered plasmid encoded antigens such as luciferase or ovalbumin in a mouse model. We found that the presence of gp120 leads to a significant reduction in the expression level of the codelivered antigen in vivo. Antigen presentation by antigen-presenting cells was also reduced and resulted in the induction of weak antigen-specific cellular and humoral immune responses. Importantly, gp120-mediated immune interference was observed after administration of the plasmids at the same or at distinct locations. To characterize the region in gp120 mediating these effects, we used plasmid constructs encoding gp120 that lacks the V1V2 loops (DeltaV1V2) or the V3 loop (DeltaV3). After immunization, the DeltaV1V2, but not the DeltaV3 construct, was able to reduce antigen expression, antigen presentation, and subsequently the immunogenicity of the codelivered antigen. The V3 loop dependence of this phenomenon seems to be limited to V3 loops known to interact with the CXCR4 molecule but not with CCR5. Our study presents a novel mechanism by which HIV-1 gp120 interferes with the immune response against coadministered antigen in a polyvalent vaccine preparation.

A matter of timing: unsynchronized antigen expression and antigen presentation diminish secondary T cell responses.

Despite the low and short expression of secondary Ag, prime-boost immunizations using homologous or heterologous vectors are capable of amplifying memory CD8(+) T cells. This is mainly attributed to the rapid presentation of Ag by APCs and the high proliferative capacity of memory CD8(+) T cells. Nevertheless, certain viruses and vectors often require prolonged Ag presentation for optimal T cell priming, and the influence of such a prolonged presentation during secondary immune induction is not clear. To address this issue, we primed and boosted mice intradermally (i.d.) with plasmid DNA that was recently reported to require prolonged Ag presentation for maximal CD8(+) T cell priming. Although functional memory CD8(+) T cells were present in the mice after i.d. priming, the secondary CD8(+) T cell response elicited was limited and reached a similar level of that observed during priming. The initial levels of secondary Ag expressed in the boosted mice were sufficient to prime CD8(+) T cell response in naive hosts, suggesting that lower Ag load alone does not explain the limited secondary immune responses observed. Removal of the injection site 5 or 10 days after i.d. boosting immunization resulted in diminished Ag presentation and no expansion of memory CD8(+) T cells. In fact, Ag-presenting activity following boost occurred mainly two weeks postimmunization, a time when the Ag was no longer expressed in situ. These findings suggest that when the boosting vector triggers prolonged Ag presentation, the lack of synchronicity between Ag accessibility and Ag presentation limits secondary immune responses.

Pulmonary delivery of DNA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA-PEI nanoparticles enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice.

During persistent infection and hypoxic-stress, Mycobacterium tuberculosis (Mtb) expresses a series of Mtb latency antigens. The aim of this study was to evaluate the immunogenicity of a DNA vaccine encoding the Mtb latency antigen Rv1733c and to explore the effect of pulmonary delivery and co-formulation with poly (d,l-lactide-co-glycolide) (PLGA)-polyethyleneimine (PEI) nanoparticles (np) on host immunity. Characterization studies indicated that PLGA-PEI np kept their nanometer size after concentration and were positively charged. The np were able to mature human dendritic cells and stimulated them to secrete IL-12 and TNF-alpha comparable to levels observed after lipopolysaccharide (LPS) stimulation. Mtb latency antigen Rv1733c DNA prime combined with Rv1733c protein boost enhanced T cell proliferation and IFN-gamma secretion in mice in response to Rv1733c and Mtb hypoxic lysate. Rv1733c DNA adsorbed to PLGA-PEI np and applied to the lungs increased T cell proliferation and IFN-gamma production more potently compared to the same vaccinations given intramuscularly. The strongest immunogenicity was obtained by pulmonary priming with np-adsorbed Rv1733c DNA followed by boosting with Rv1733c protein. These results confirm that PLGA-PEI np are an efficient DNA vaccine delivery system to enhance T cell responses through pulmonary delivery in a DNA prime/protein boost vaccine regimen.

Preparation and characterization of diphtheria toxoid-loaded elastic vesicles for transcutaneous immunization.

The objective was to develop surfactant-based vesicle formulations containing diphtheria toxoid (DT) for transcutaneous immunization. Formulation variables were molar ratio of the surfactants (sucrose-laurate ester, octaoxyethylene-laurate ester, and sodium bistridecyl sulfosuccinate), DT concentration, pH, and ionic strength. The formulations were characterized by visual inspection, dynamic light scattering and zeta potential measurements. DT loading efficiency and capacity of the vesicle formulations were studied by protein assay and enzyme-linked immunosorbent assay. DT-containing vesicle formulations were optimized for colloidal stability. The pH had a dramatic effect on DT-vesicle association: at pH 4.5 > 70% of the protein was associated with the vesicles, vs. < 20% at pH 5.0. By varying the ionic strength of the buffer, we revealed that hydrophobic interactions play an important role in the association. DT-vesicles formulated at pH 4.5 were stable and showed high association of DT with preserved antigenicity, and are therefore excellent candidates for future in vivo studies.

Pulmonary DNA vaccination: concepts, possibilities and perspectives.

Mucosal immunity establishes the first line of defence against pathogens entering the body via mucosal surfaces. Besides eliciting both local and systemic immunity, mucosal vaccination strategies that are non-invasive in nature may increase patient compliance and reduce the need for vaccine application by trained personnel. A relatively new concept is mucosal immunization using DNA vaccines. The advantages of DNA vaccines, such as the opportunity to combine the genetic information of various antigen epitopes and stimulatory cytokines, the enhanced stability and ease of production make this class of vaccines attractive and suitable for mucosal application. In contrast to the area of intranasal vaccination, only a few recent studies have focused on pulmonary immunization and the involvement of the pulmonary immune system in eliciting protective immune responses against inhaled pathogens. This review focuses on DNA vaccine delivery to the lung as a promising approach to prevent pulmonary-associated diseases caused by inhaled pathogens. Attractive immunological features of the lung as a site for immunization, the mechanisms of action of DNA vaccines and the pulmonary application of such vaccines using novel delivery systems will be discussed. We also examine pulmonary diseases prone to prevention or therapeutical intervention by application of DNA vaccines.

Non-invasive pulmonary aerosol delivery in mice by the endotracheal route.

In this report we present in detail a non-invasive pulmonary application method that can be a useful tool in studying drug and vaccine delivery to the lower airways. In this method the formulation is sprayed directly into the lungs of mice via the endotracheal route using a MicroSprayer aerolizer. Mean droplet size produced was 8 microm, appropriate for deposition in the large airways. Endotracheal application of suspension of fluorescent nanospheres, 200 nm in size, by this method resulted in nanoparticle deposition in the smaller airways (bronchi and bronchioles). Mice showed full recovery one day after administration of 50 microl of formulation. Furthermore, no mortality was observed as a result of the technique. We conclude that this endotracheal application is a useful tool for studying pulmonary drug delivery in mice. The technique is especially useful for the pulmonary application of vaccines, since it enables multiple administrations without a need for analgesics.

Cationic submicron emulsions for pulmonary DNA immunization.

Pulmonary immunization against inhaled pathogens such as Mycobacterium tuberculosis would induce local and systemic immune responses and protect from entry and dissemination of the pathogen. The aim of this study was to evaluate cationic submicron emulsion as a potential carrier for DNA vaccines to the lung. DNA loaded emulsions were 128-152 nm in size and retained positive zeta potential above +40 mV during 3 months of storage. Loading efficiency was above 99%, DNA was protected from DNase I degradation up to 60 min and was stable in presence of 75% fetal calf serum (FCS). The plasmid DNA was detected in the endo-lysosomal compartment of the human bronchial cell line, Calu-3, 6 h after application. No cytotoxic effect on these cells was observed. Human dendritic cells were matured in presence of DNA loaded emulsion, although to a lesser extent than DNA solution indicating slower release and lower exposure to unmethylated CpG sequences. These results indicate that cationic submicron emulsions are potential DNA vaccine carriers to the lung since they are able to transfect pulmonary epithelial cells, which possibly induce cross priming of antigen presenting cells and directly activate dendritic cells, resulting in stimulation of antigen specific T-cells.

PLGA-PEI nanoparticles for gene delivery to pulmonary epithelium.

Pulmonary gene delivery is thought to play an important role in treating genetically related diseases and may induce immunity towards pathogens entering the body via the airways. In this study we prepared poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles bearing polyethyleneimine (PEI) on their surface and characterized them for their potential in serving as non-viral gene carriers to the pulmonary epithelium. Particles that were synthesized at different PLGA-PEI ratios and loaded with DNA in several PEI-DNA ratios, exhibited narrow size distribution in all formulations, with mean particle sizes ranging between 207 and 231 nm. Zeta potential was strongly positive (above 30 mV) for all the PEI-DNA ratios examined and the loading efficiency exceeded 99% for all formulations. Internalization of the DNA-loaded PLGA-PEI nanoparticles was studied in the human airway submucosal epithelial cell line, Calu-3, and DNA was detected in the endo-lysosomal compartment 6 h after particles were applied. Cytotoxicity of these nanoparticles was dependent on the PEI-DNA ratio and best cell viability was achieved by PEI-DNA ratios 1:1 and 0.5:1. These findings demonstrate that PLGA-PEI nanoparticles are a potential new delivery system to carry genes to the lung epithelium.

Pulmonary delivery of chitosan-DNA nanoparticles enhances the immunogenicity of a DNA vaccine encoding HLA-A*0201-restricted T-cell epitopes of Mycobacterium tuberculosis.

In this study, we used an HLA-A2 transgenic mouse model to investigate the effects of pulmonary delivery of a new DNA plasmid encoding eight HLA-A*0201-restricted T-cell epitopes from Mycobacterium tuberculosis formulated in chitosan nanoparticles. It was shown that the chitosan-DNA formulation was able to induce the maturation of dendritic cells (DCs) while chitosan solution alone could not, indicating the DNA was released from the particles and able to stimulate DCs. Pulmonary administration of the DNA plasmid incorporated in chitosan nanoparticles was shown to induce increased levels of IFN-gamma secretion compared to pulmonary delivery of plasmid in solution or the more frequently used intramuscular immunization route. These results indicate that pulmonary delivery of DNA vaccines against tuberculosis may provide an advantageous delivery route compared to intramuscular immunization, and that increased immunogenicity can be achieved by delivery of this DNA encapsulated in chitosan nanoparticles.