Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses.

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1ß, IL-6, IL-10, MCP-1, MIP-1α, MIP-1ß, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2-10 µm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.

Comparative Evaluation of Electronic Acupuncture Pen and 2% Lignocaine Gel as an Intraoral Topical Anesthetic Agent in Children.

Background The most exasperating aspect for pediatric patients in a dental setup is the fear and anxiety caused by injections, called "blenophobia". There are numerous local anesthetic agents available to reduce the needle prick pain. Taking into consideration the paradigm shift, there is always a possibility for alternate treatment options. This study aimed to evaluate and compare the effectiveness of transcutaneous electrical nerve stimulation (TENS) application through an electronic acupuncture pen (Meridian Energy Acupuncture Pen W-912 GENERIC) and 2% lignocaine gel as an intraoral topical anesthetic agent in children. Method Patients aged 6 to 12 years were eligible for inclusion. The topical anesthetic agents were administered to the patients in a bilateral split-mouth technique. In Group A, 2% topical anesthetic gel was administered on the first visit on one side of the mandible and topical anesthesia through the electronic acupuncture pen was administered on the next visit, on the opposite side of the mandible. The electronic acupuncture pen was applied on one side of the mandible on the first visit, and on the next appointment, 2% topical anesthetic gel on the other side was administered in Group B. Sound, eye, motor scale (SEM) and faces pain scale-Revised (FPS) were used as tools of evaluation after local anesthesia was administered.  Results  The comparison between electronic acupuncture pen and 2% lignocaine gel using the SEM scale shows a statistically insignificant difference (p-value = 0.082). Similarly, a comparison of FPS values between both groups indicates no significant difference (p-value = 0.582). However, results show a reduced pain perception in both groups. Conclusion Topical anesthetic agents are commonly used to reduce needle prick pain in children. TENS through the electronic acupuncture pen, a revisited aid in scientific research, has proved its efficacy as a topical pain reduction measure during dental treatment. This device overcomes the shortcomings of the anesthetic gels and also nullifies the chances of overdosage, hypersensitivity, and disagreeable taste. Thus, this tool can be used in dental practice for the management of pain in children.

Comparative Evaluation of Antibacterial Efficacy of Emblica Officinalis Lollipop Against Streptococcus Mutans Counts in Institutionalized Visually Impaired Children.

Background Among the 1.21 billion population of India, 26.8 million individuals have disabilities, and around five million are visually impaired. These children encounter problems related to oral health maintenance, thus further leading to dental problems. Even though Pediatric dentists treat visually impaired children for their oral problems, they emphasize mainly on the prevention of dental caries. Dental caries has a multifactorial etiology, and dentists are unable to find a complete solution for its prevention. However, reducing Streptococcus mutans has been seen to reduce the caries rate in the past. The use of the herbal product Emblica officinalis to reduce Streptococcus mutans levels has been documented. An affordable delivery system is required to use Emblica officinalis for its anticaries action. Thus, a unique delivery system of herbal sugar-free lollipops containing Emblica officinalis extract was made and can effectively deliver antimicrobial action in visually impaired children. Aim To evaluate the antibacterial efficacy of Emblica officinalis lollipop on Streptococcus mutans counts and pH levels in institutionalized visually impaired children. Method A total of 60 institutionalized visually impaired children (age: 4 to 14 years) were selected. The study consisted of two groups (experimental "Emblica officinalis lollipop" and control "placebo lollipop"), and the children were divided equally into both groups. Children from the study and control groups were subjected to the respective lollipops twice daily for seven days. Streptococcus mutans count and pH count were evaluated at baseline and after seven days post-intervention of the respective lollipop. Results The results showed that in both groups, Streptococcus mutans count was reduced post-intervention. However, the efficacy of the study group (Emblica officinalis lollipop) in inhibiting the Streptococcus mutans count was better than the control group (placebo lollipop). An increase in the pH level was seen post-intervention for both the study and control groups. And on the intergroup comparison, no statistical significance was found. Conclusion The use of Emblica officinalis lollipop is effective in inhibiting the Streptococcus mutans count when compared with the placebo lollipop. While marginal pH change was seen in both groups. Thus, the herbal modality most acceptable without any pharmaceutical concerns should be chosen. Emblica officinalis lollipops can be used in institutionalized visually impaired children to reduce the oral Streptococcus mutans count and maintain a healthy oral cavity.

Effectiveness of Two Different Behavior Modification Techniques for Anxiety Reduction in Children.

Background Dental anxiety has been a major concern for dentists while operating children. For a smooth, uneventful treatment, operators must incorporate various behavior management techniques in their practice. The incorporation of magic tricks as behavior management techniques has been used earlier by physicians and nurses to reduce pre-operative anxiety in hospitals. This study aimed to compare the impacts of magic tricks on reducing dental anxiety in children. Material and methods Patients aged four to 11 years were eligible for inclusion. The study comprised two groups of 15 children each. During the first visit, children weren't subjected to any behavior management. Behavior management aids (magic tricks and audiovisuals) were used during the second visit. Hemodynamic parameters along with an anxiety scale were used to assess anxiety in children before, during, and after treatment procedures. Venham's picture test and modified visual analog scale were also used to assess the anxiety. Results A reduction in anxiety was seen in both groups after behavior management was used. The hemodynamic parameters like blood pressure and pulse rate were seen to decrease during the second visit, while the oxygen saturation was seen to increase. Conclusion The study demonstrates that magic trick along with audiovisual aids was effective in controlling dental anxiety. Thus, magic tricks can be used in dental practice as a behavior management aid for children to facilitate cooperative behavior. Reducing a child's dental anxiety through various different magic trick aids could be a potential behavior management modality that needs further research.

Evaluation of antibacterial efficacy of Triphala toothwipes on oral Streptococcus mutans count in intellectually disabled children.

AIM: To evaluate antibacterial efficacy of Triphala toothwipes on oral Streptococcus mutans counts in intellectually disabled (ID) children. METHODS: Twenty-seven children with a mild ID were randomly divided into two groups: A - Triphala and B - placebo group. Toothwipes were given to caregivers of children belonging to the respective groups, and were instructed to use them 1 h after their meals, twice a day for 7 days. Simplified Plaque index was recorded and plaque samples were collected for microbiological examination at baseline, 48 h, and 7 days. The obtained data were tabulated and analyzed. RESULTS: Triphala group showed a statistically significant reduction of S. mutans after 48 h and 7 days. Both Triphala and placebo groups showed a statistically significant reduction in dental plaque after 48 h and 7 days. CONCLUSION: Triphala toothwipes are effective against the oral S. mutans compared with placebo toothwipes, while both the toothwipes are equally effective in reducing dental plaque. Thus, Triphala toothwipes can be used as an adjunct aid along with routine oral hygiene practices in individuals who lack psychomotor skills or are dependent on others to maintain oral hygiene, including infants, preschoolers, geriatric population, and children with special health care needs.

Comparison of impact of oral hygiene instructions given via sign language and validated customized oral health education skit video on oral hygiene status of children with hearing impairment.

INTRODUCTION: Children with special health-care needs have limitations in oral hygiene performance due to their potential motor, sensory, and intellectual disabilities and so are more prone to have compromised oral health. AIM: This study aimed to compare the impact of oral hygiene instructions given via sign language and a validated customized oral health education skit video on oral hygiene status of children with hearing impairment (CHI). SETTINGS AND DESIGN: Ethical clearance was obtained from the institutional ethical committee for research activities. The study was carried out across CHI schools of Wardha district, Maharashtra, India. METHODOLOGY: Sixty-eight CHI, within the age group of 6-13 years, were divided into two educational intervention groups: customized oral health educational video (Group A) and sign language (Group B). A structured questionnaire was designed to gather information about the routine oral hygiene practices via the Indian Sign Language. Baseline Gingival Index (GI)-S and Plaque Index-S indices were recorded. Based on the group assigned, oral hygiene instructions were given on a daily basis. Reassessment was done after 4 weeks. STATISTICAL ANALYSIS: Unpaired t-tests were performed (P < 0.05) to determine if significant differences exist between the two groups. RESULTS: Postintervention plaque scores between Group A and Group B were 0.12 ± 0.22 and 0.07 ± 0.22, respectively, and the difference between the two was statistically insignificant (P = 0.330). For GI, scores in Group A and Group B were 0.03 ± 0.12 and 0.04 ± 0.12, respectively, and the difference was statistically insignificant (P = 0.669). CONCLUSION: Both sign language and the validated customized video modeling have been proved to be positively influencing the oral hygiene status of CHI equivalently.

Evaluating Immunogenicity Risk Due to Host Cell Protein Impurities in Antibody-Based Biotherapeutics.

A potential risk factor for immunogenicity of a biotherapeutic is the low levels of host cell protein (HCP) impurities that remain in the product following the purification process. During process development, significant attention has been devoted to removing HCPs due to their potential safety risk. Samples from different purification steps of several monoclonal antibodies (mAbs) purified by one type of platform were evaluated for their residual Chinese Hamster Ovary (CHO) cell-derived HCP content. HCPs in both in-process (high levels of HCP) and highly purified (low levels of HCP) samples were identified and quantitated by proteomic analysis via mass spectrometry. The responses to HCPs were evaluated in an in vitro assay using PBMC from a population of healthy and disease state individuals. Results indicated that samples with up to 4000 ppm HCP content (levels 200 times greater than the drug substance) did not pose a higher immunogenicity risk than highly purified mAb samples. As an orthogonal method to predict immunogenicity risk, in silico algorithms that probe amino acid sequence for foreign epitope content were used to evaluate over 20 common HCPs (identified in the different mAb samples). Only a few HCPs were identified as high risk by the algorithms; however, the in vitro assay results indicated that the concentration of these HCPs from in-process biotherapeutic mAb samples was not sufficient to stimulate an immune response. This suggests that high levels of HCP in mAb biotherapeutics purified by this type of platform do not increase the potential risk of immunogenicity of these molecules. Insights from these studies can be applied to HCP control and risk assessment strategies.

Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

Physical characterization and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting.

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5-10 µm in size displayed elevated cytokine release profiles compared with other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared with controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by microflow imaging, transmission electron microscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in vitro PBMC studies to rank-order the immunogenic potential of various types of mAb particles are discussed.