Comparative evaluation of two different xenogenic acellular matrices on full-thickness skin wound healing.

OBJECTIVE: The purpose of the study was to compare the healing potential of bubaline small intestinal matrix (bSIM) and fish swim bladder matrix (FSBM) on full-thickness skin wounds in rabbits. METHOD: Four full-thickness skin wounds (each 20×20mm) were created on the dorsum of 18 rabbits that were divided into three groups based on treatment: untreated sham control (I), implanted with double layers of bSIM (II) and implanted with double layers of FSBM (III). Macroscopic, immunologic and histologic observations were made to evaluate wound healing. RESULTS: Gross healing progression in the bSIM and FSBM groups showed significantly (p<0.05) less wound contraction compared with the sham group. The IgG concentration in rabbit sera was significantly (p<0.05) lower in the FSBM group compared with the bSIM group by enzyme-linked immunosorbent assay. The stimulation index of peripheral blood lymphocytes was significantly (p<0.05) lower in the FSBM group compared with the bSIM group by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Implantation of FSBM resulted in improved re-epithelialisation, neovascularisation and fibroplasia. CONCLUSION: The FSBM is a more effective dermal substitute when compared with the bSIM for full-thickness skin wound repair in rabbit.

Nutritional deficiencies that may predispose to long COVID.

Multiple nutritional deficiencies (MND) confound studies designed to assess the role of a single nutrient in contributing to the initiation and progression of disease states. Despite the perception of many healthcare practitioners, up to 25% of Americans are deficient in five-or-more essential nutrients. Stress associated with the COVID-19 pandemic further increases the prevalence of deficiency states. Viral infections compete for crucial nutrients with immune cells. Viral replication and proliferation of immunocompetent cells critical to the host response require these essential nutrients, including zinc. Clinical studies have linked levels of more than 22 different dietary components to the likelihood of COVID-19 infection and the severity of the disease. People at higher risk of infection due to MND are also more likely to have long-term sequelae, known as Long COVID.

Reliable and efficient parameter estimation using approximate continuum limit descriptions of stochastic models.

Stochastic individual-based mathematical models are attractive for modelling biological phenomena because they naturally capture the stochasticity and variability that is often evident in biological data. Such models also allow us to track the motion of individuals within the population of interest. Unfortunately, capturing this microscopic detail means that simulation and parameter inference can become computationally expensive. One approach for overcoming this computational limitation is to coarse-grain the stochastic model to provide an approximate continuum model that can be solved using far less computational effort. However, coarse-grained continuum models can be biased or inaccurate, particularly for certain parameter regimes. In this work, we combine stochastic and continuum mathematical models in the context of lattice-based models of two-dimensional cell biology experiments by demonstrating how to simulate two commonly used experiments: cell proliferation assays and barrier assays. Our approach involves building a simple statistical model of the discrepancy between the expensive stochastic model and the associated computationally inexpensive coarse-grained continuum model. We form this statistical model based on a limited number of expensive stochastic model evaluations at design points sampled from a user-chosen distribution, corresponding to a computer experiment design problem. With straightforward design point selection schemes, we show that using the statistical model of the discrepancy in tandem with the computationally inexpensive continuum model allows us to carry out prediction and inference while correcting for biases and inaccuracies due to the continuum approximation. We demonstrate this approach by simulating a proliferation assay, where the continuum limit model is the well-known logistic ordinary differential equation, as well as a barrier assay where the continuum limit model is closely related to the well-known Fisher-KPP partial differential equation. We construct an approximate likelihood function for parameter inference, both with and without discrepancy correction terms. Using maximum likelihood estimation, we provide point estimates of the unknown parameters, and use the profile likelihood to characterise the uncertainty in these estimates and form approximate confidence intervals. For the range of inference problems considered, working with the continuum limit model alone leads to biased parameter estimation and confidence intervals with poor coverage. In contrast, incorporating correction terms arising from the statistical model of the model discrepancy allows us to recover the parameters accurately with minimal computational overhead. The main tradeoff is that the associated confidence intervals are typically broader, reflecting the additional uncertainty introduced by the approximation process. All algorithms required to replicate the results in this work are written in the open source Julia language and are available at GitHub.

Variable Abnormalities in T and B Cell Subsets in Ataxia Telangiectasia.

BACKGROUND: Ataxia-telangiectasia (AT) is a rare genetic condition, caused by biallelic deleterious variants in the ATM gene, and has variable immunological abnormalities. This study aimed to examine immunologic parameters reflecting cell development, activation, proliferation, and class switch recombination (CSR) and determine their relationship to the clinical phenotype in AT patients. METHODS: In this study, 40 patients with a confirmed diagnosis of AT from the Iranian immunodeficiency registry center and 28 age-sex matched healthy controls were enrolled. We compared peripheral B and T cell subsets and T cell proliferation response to CD3/CD28 stimulation in AT patients with and without CSR defects using flow cytometry. RESULTS: A significant decrease in naïve, transitional, switched memory, and IgM only memory B cells, along with a sharp increase in the marginal zone-like and CD21low B cells was observed in the patients. We also found CD4+ and CD8+ naïve, central memory, and terminally differentiated effector memory CD4+ (TEMRA) T cells were decreased. CD4+ and CD8+ effector memory, CD8+ TEMRA, and CD4+ regulatory T cells were significantly elevated in our patients. CD4+ T cell proliferation was markedly impaired compared to the healthy controls. Moreover, immunological investigations of 15 AT patients with CSR defect revealed a significant reduction in the marginal zone, switched memory, and more intense defects in IgM only memory B cells, CD4+ naïve and central memory T cells. CONCLUSION: The present study revealed that patients with AT have a broad spectrum of cellular and humoral deficiencies. Therefore, a detailed evaluation of T and B cell subsets increases understanding of the disease in patients and the risk of infection.

Artemisia dracunculus L. modulates the immune system in a multiple sclerosis mouse model.

Background: Multiple sclerosis along with its animal model, experimental autoimmune encephalomyelitis (EAE), are chronic inflammatory and degenerative diseases of the central nervous system (CNS). Due to the unknown cause of the disease, the most common treatments of MS are targeted for the reduction of inflammation and the repairment of CNS tissue damage, especially myelin restoration. Due to the immune protective nature of herbs, it may be useful to evaluate the impact of herbs in the diet regimen of MS patients along with their immune-mediated effects. The purpose of this study was to investigate the effect of an aqueous extract of Artemisia dracunculus (Tarragon) on the treatment of EAE in C57BL/6 mice.Methods: In this experimental study, mice were divided into the following control, untreated EAE, and A. dracunculus treated EAE groups. EAE was induced by myelin oligodendrocyte glycoprotein (MOG35-55) in female C57BL/6 mice. The symptoms of the disease and the weight of the mice were recorded daily. On day 33 after EAE induction, the mice were sacrificed and the specimens were collected. Cell proliferation and cytokine release (TGF-ß, IL-17 and IL-23) from mice cultured spleen cells was measured by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and ELISA respectively.Results: Administration of the extract of A. dracunculus mitigated EAE symptoms (P < 0.05). Furthermore, there was a reduction in the levels of inflammatory cytokines including IL-17 (P = 0.009) and IL-23 (P = 0.012) and confirmed increased serum antioxidant levels in A. dracunculus treated EAE mice (P = 0.008).Conclusions: These observations indicate that A. dracunculus extracts could reduce inflammatory cytokines and attenuate certain signs of EAE, suggesting the potential of a useful adjuvant therapy for MS.

An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation.

BACKGROUND: In the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells. However, these assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. However, this assay has not been established using primary chicken cells to evaluate T cell proliferation by flow cytometry. RESULTS: Here, we established an assay to evaluate the proliferation of primary chicken splenocytes based on the incorporation of a thymidine analog (EdU) and a click reaction with a fluorescent azide, detected by a flow cytometer. We also established a protocol that combines EdU incorporation and immunostaining to detect CD4+ and CD8+ proliferating T cells. By inducing cell proliferation with increasing concentrations of a mitogen (Concanavalin A), we observed a linear increase in EdU positive cells, indicating that our protocol does not present any deficiency in the quantity and quality of reagents that were used to perform the click reaction. CONCLUSIONS: In summary, we established a reliable protocol to evaluate the proliferation of CD4+ and CD8+ chicken T cells by flow cytometry. Moreover, as this is an in-house protocol, the cost per sample using this protocol is low, allowing its implementation in laboratories that process a large number of samples.

Safety of measles, mumps, and rubella vaccine in egg allergy: in vivo and in vitro management.

BACKGROUND: Egg allergy is the second most prevalent form of food allergy in childhood. In spite of the evidence accumulated, inoculating egg allergy children with attenuated vaccines grown on chick embryo cell cultures, such as the measles, mumps, and rubella (MMR) vaccine, is regarded (erroneously) as potentially dangerous or even anaphylactogenic, by many. An issue perceived as particularly conflicting also by Health Professionals. CASE PRESENTATION: A 15-year-old boy, with a history of severe egg allergy in early infancy, who was still sensitized to egg allergens, including baked egg, had never received MMR vaccination, in fear of possible anaphylaxis, in spite of the fact that this vaccination is mandatory in the first year of life, in Italy. Because of that, he was not allowed to attend school, longer, and was referred to us in order to assess the potential risk of MMR vaccination. Upon thorough allergologic workup, sensitization to MMR vaccine components was excluded by an in vivo approach, consisting in skin prick tests, intradermal tests, and subcutaneous injection test, corroborated by vaccine-specific B-lymphocyte proliferation assay, ex vivo. T-cell proliferation in response to MMR vaccine was also excluded. Eventually, the boy was inoculated with MMR vaccine and was readmitted to school. CONCLUSIONS: The diagnostic strategy adopted appears feasible and easy-to-perform and may be adopted in controversial cases (as the one reported), characterized by previous severe allergic reactions to egg. The B-lymphocyte proliferation assay we developed may represent a useful and reliable tool not only in research but also in clinical practice.

Formulation and evaluation of letrozole-loaded spray dried liposomes with PEs for topical application.

Letrozole (LET), an aromatase inhibitor widely used as a first-line drug for the estrogen-dependent breast cancer treatment in postmenopausal women. In this study, an attempt has been made to develop LET topical drug delivery which would be a more efficient system to treat elevated blood levels of estrogen found in breast cancer patients. The technique involves, encapsulation of the LET in phospholipids using spray dryer. The LET spray-dried powder (LT-SDP) powder was tested by Fourier transform infrared, X-RD, and differential scanning calorimetry. These studies confirm the entrapment efficiency (EE) of the system. The LT-SDP in the form dispersion was further evaluated. The confocal laser scanning microscopy (CLSM) showed spherical vesicles, the particle size, polydispersity index, and the EE was found to be 284.0 nm, 0.247, and 59.08%, respectively. LT-SDP dispersion was added into a cream base with peppermint and olive oil as natural penetration enhancers. Optimized formulation showed superior skin targeting in in vitro and in vivo studies. Cell proliferation assay and flow cytometry was carried out using human cancer cell line of breast MDA-MB-231 which showed superior anti-proliferative action and enhanced apoptosis activity of LT-SDP cream (43.9%) in comparison. The CLSM micrograph, skin irritation, and histopathology studies showed the penetration ability and inertness of the LT-SDP cream, respectively. In vivo bioavailability studies showed an almost four-fold increase in the plasma concentration (11.3 versus 4.2) while the mean residence time (81.11 versus 64.42 h) and half-life (51.01 versus 39.36 h) were reasonably higher than plain LET cream.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

Inferring parameters for a lattice-free model of cell migration and proliferation using experimental data.

Collective cell spreading takes place in spatially continuous environments, yet it is often modelled using discrete lattice-based approaches. Here, we use data from a series of cell proliferation assays, with a prostate cancer cell line, to calibrate a spatially continuous individual based model (IBM) of collective cell migration and proliferation. The IBM explicitly accounts for crowding effects by modifying the rate of movement, direction of movement, and the rate of proliferation by accounting for pair-wise interactions. Taking a Bayesian approach we estimate the free parameters in the IBM using rejection sampling on three separate, independent experimental data sets. Since the posterior distributions for each experiment are similar, we perform simulations with parameters sampled from a new posterior distribution generated by combining the three data sets. To explore the predictive power of the calibrated IBM, we forecast the evolution of a fourth experimental data set. Overall, we show how to calibrate a lattice-free IBM to experimental data, and our work highlights the importance of interactions between individuals. Despite great care taken to distribute cells as uniformly as possible experimentally, we find evidence of significant spatial clustering over short distances, suggesting that standard mean-field models could be inappropriate.

LTCC Packaged Ring Oscillator Based Sensor for Evaluation of Cell Proliferation.

A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic (LTCC) module with a flip chip bonding technique. A microcontroller operates the chip, while the whole measurement system was controlled by PC. The developed biosensor was applied for measurement of the proliferation stage of adherent cells where the sensor response depends on the ratio between healthy, viable and multiplying cells, which adhere onto the chip surface, and necrotic or apoptotic cells, which detach from the chip surface. This change in cellular adhesion caused a change in the effective permittivity in the vicinity of the sensor element, which was sensed as a change in oscillation frequency of the ring oscillator. The sensor was tested with human lung epithelial cells (BEAS-2B) during cell addition, proliferation and migration, and finally detachment induced by trypsin protease treatment. The difference in sensor response with and without cells was measured as a frequency shift in the scale of 1.1 MHz from the base frequency of 57.2 MHz. Moreover, the number of cells in the sensor vicinity was directly proportional to the frequency shift.

De-immunized and Functional Therapeutic (DeFT) versions of a long lasting recombinant alpha interferon for antiviral therapy.

Interferon α (IFN-α) exerts potent antiviral, immunomodulatory, and antiproliferative activity and have proven clinical utility in chronic hepatitis B and C virus infections. However, repeated IFN-α administration induces neutralizing antibodies (NAb) against the therapeutic in a significant number of patients. Associations between IFN-α immunogenicity and loss of efficacy have been described. So as to improve the in vivo biological efficacy of IFN-α, a long lasting hyperglycosylated protein (4N-IFN) derived from IFN-α2b wild type (WT-IFN) was developed. However, in silico analysis performed using established in silico methods revealed that 4N-IFN had more T cell epitopes than WT-IFN. In order to develop a safer and more efficient IFN therapy, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of 4N-IFN. Using the OptiMatrix in silico tool in ISPRI, the 4N-IFN sequence was modified to reduce HLA binding potential of specific T cell epitopes. Following verification of predictions by HLA binding assays, eight modifications were selected and integrated in three variants: 4N-IFN(VAR1), (VAR2) and (VAR3). Two of the three variants (VAR1 and VAR3) retained anti-viral function and demonstrated reduced T-cell immunogenicity in terms of T-cell proliferation and Th1 and Th2 cytokine levels, when compared to controls (commercial NG-IFN (non-glycosylated), PEG-IFN, WT-IFN and 4N-IFN). It was previously demonstrated that N-glycosylation improved IFN-α pharmacokinetic properties. Here, we further reduce immunogenicity as measured in vitro using T cell assays and cytokine profiling by modifying the T cell epitope content of a protein (de-immunizing). Taking into consideration the present results and previously reported immunogenicity data for commercial IFN-α2b variants, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be promising candidates for improved IFN-α therapy of HCV and HBV.

A Bayesian Computational Approach to Explore the Optimal Duration of a Cell Proliferation Assay.

Cell proliferation assays are routinely used to explore how a low-density monolayer of cells grows with time. For a typical cell line with a doubling time of 12 h (or longer), a standard cell proliferation assay conducted over 24 h provides excellent information about the low-density exponential growth rate, but limited information about crowding effects that occur at higher densities. To explore how we can best detect and quantify crowding effects, we present a suite of in silico proliferation assays where cells proliferate according to a generalised logistic growth model. Using approximate Bayesian computation we show that data from a standard cell proliferation assay cannot reliably distinguish between classical logistic growth and more general non-logistic growth models. We then explore, and quantify, the trade-off between increasing the duration of the experiment and the associated decrease in uncertainty in the crowding mechanism.

Immunomodulatory activities of isolated compounds from the root-bark of Cussonia arborea.

CONTEXT: Cussonia arborea Hochst. ex A. Rich (Araliaceae) is a folk medicine used to treat various diseases. However, there is no report of the root phytochemistry. OBJECTIVE: This study isolates and identifies the immunomodulatory compounds from root-bark of C. arborea. MATERIALS AND METHODS: The methanol extract (18 g) was subjected to repeated column chromatography resulting in isolation of five compounds (1-5). Structure determination was achieved by analysis of their 1 D and 2 D NMR, and mass spectroscopy. The compounds (100-1.0 µg/mL) were examined immunomodulatory for effect on production of reactive oxygen species (ROS) from whole blood phagocytes and on proliferation of T-cells. The compounds cytotoxicity (100-1.0 µg/mL) was evaluated on NIH-3T3 normal fibroblast cells. RESULTS: Three pentacyclic triterpenoids [3, 23-dihydroxy-12-oleanen-28-oic acid (1), 3ß-hydroxylolean-12-en-28-oic (2) and 23-hydoxy-oxo-urs-12-en-28-oic acid (5)], two phytosterols: [stigmasterol (3)] and [3-O-ß-d-glucopyranosyl stigmasterol (4)] were all isolated from the methanol soluble extract. All the tested compounds (1-4) were found to be nontoxic on NIH-3T3 cells. Compound 1 and 2 moderately inhibited the production of ROS (IC50 = 24.4 ± 4.3 and 37.5 ± 0.1 µg/mL, respectively) whereas compound 2 exhibited the highest inhibitory effect (IC50 = 12.6 ± 0.4 µg/mL) on proliferation of phytoheamagglutinin (PHA) activated T-cells. CONCLUSIONS: The isolated compounds (1-5) are reported for the first time from this species. In addition, compound 2 with suppressive potential on production of intracellular ROS and proliferation of T-cells could be of immense value in control of autoimmune diseases as well as in immune compromised patients.

A comparative analysis of the structural, functional and biological differences between Mouse and Human Nerve Growth Factor.

NGF is the prototype member of the neurotrophin family of proteins that promote the survival and growth of selected neurons in the central and peripheral nervous systems. As for all neurotrophins, NGF is translated as a pre-pro-protein. Over the years, NGF and proNGF of either human or mouse origin, given their high degree of homology, have been exploited for numerous applications in biomedical sciences. The mouse NGF has been considered the golden-standard for bioactivity. Indeed, due to evolutionary relatedness to human NGF and to its ready availability and by assuming identical properties to its human counterpart, the mouse NGF, isolated and purified from sub-maxillary glands, has been tested not only in laboratory practice and in preclinical models, but it has also been evaluated in several human clinical trials. Aiming to validate this assumption, widely believed, we performed a comparative study of the biochemical and biophysical properties of the mouse and human counterparts of NGF and proNGF. The mature and the precursor proteins of either species strikingly differ in their biophysical profiles and, when tested for ligand binding to their receptors, in their in vitro biological activities. We provide a structural rationale that accounts for their different functional behaviors. Despite being highly conserved during evolution, NGF and proNGF of mouse and human origins show distinct properties and therefore special care must be taken in performing experiments with cross-species systems in the laboratory practice, in developing immunoassays, in clinical trials and in pharmacological treatments.

Identification of coeliac disease triggering glutenin peptides in adults.

OBJECTIVE: Coeliac disease affects approximately 1% of Northern American and European populations. It is caused by an inappropriate immune response to dietary gluten. Gluten comprises of two major protein fractions: gliadins and glutenins. Glutenins have recently been found to be toxic to coeliac individuals. Proliferation assays suggest in some but not all paediatric coeliac individuals there may be immunological stimulation with high molecular weight (HMW) glutenins. Less evidence pertains to low molecular weight (LMW) glutenins. The aim is to assess adaptive, T-cell driven, and innate immune response in adult coeliac individuals towards HMW glutenin peptide, glut04, and LMW glutenin peptide, glt156. MATERIALS AND METHODS: Coeliac patients were recruited attending endoscopy for routine monitoring. Adaptive immune response towards glut04 and glt156 was measured by proliferation assays and measurement of interferon-γ secretion in 28 T-cell lines. The innate immune response was assessed by measurement of enterocyte cell height (ECH) in coeliac small intestinal biopsies following overnight incubation in organ culture chambers in a further nine individuals. RESULTS: There were 3/28 and 2/28 positive proliferation results using gluten-sensitive T-cells with glut04 and glt156, respectively. All coeliac biopsies tested in organ culture chambers demonstrated clear reduction in ECH with peptic-tryptic digest of whole industrial gluten, glut04 and glt156 when compared to negative control ovalbumin (p < 0.005). Three individuals had both T-cell and organ culture study data. Their proliferation assays showed no stimulation of the T-cells. CONCLUSIONS: This study demonstrates glutenin epitopes glut04 and glt156, while minor T-cell epitopes, are important in their ability to trigger the innate immune response.

An in vitro method to test the safety and efficacy of low-level laser therapy (LLLT) in the healing of a canine skin model.

BACKGROUND: Low-level laser therapy (LLLT) has been used clinically as a treatment modality for a variety of medical conditions including wound-healing processes. It is an attractive and emerging method to enhance wound healing and improve clinical outcomes both in human and veterinary medicine. Despite the fact that the use of LLLT continues to gain in popularity, there is no universally accepted theory that defends all its cellular effects and beneficial biological processes in tissue repair. The present study was designed to evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK) in an in vitro wound healing model. RESULTS: Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. Fifteen independent replicates were performed for each assay. Canine epidermal keratinocyte cells exposed to LLLT with 0.1, 0.2, and 1.2 J/cm(2) migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < 0.0001) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm(2). Irradiation with 10 J/cm(2) was characterized by decreased cellular migration and proliferation. These results revealed that LLLT has a measurable, dose-dependent effect on two different aspects of keratinocyte biology in vitro. CONCLUSION: In this in vitro wound-healing model, LLLT increased cellular migration and proliferation at doses of 0.1, 0.2, and 1.2 J/cm(2) while exposure to 10 J/cm(2) decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior.

Design and production of a multiepitope construct derived from hepatitis E virus capsid protein.

The aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against Hepatitis E virus (HEV). Initially, conserved and antigenic helper T-lymphocyte (HTL) epitopes in the HEV capsid protein were predicted by in silico analysis. Subsequently, a multiepitope comprising four HTL epitopes with high-affinity binding to the HLA molecules was designed, and repeated four times as high density multiepitope construct. This construct was synthesized and cloned into pET-30a (+) vector. Then, it was transformed and expressed in Escherichia coli BL21 cells. The high density multiepitope protein was purified by Ni-NTA agarose and concentrated using Amicon filters. Finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. The results showed that the high density multiepitope construct was successfully expressed and purified. SDS-PAGE and Western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kDa. Approximately 1 mg of the purified protein was obtained from each liter of the culture media. Moreover, the purified multiepitope protein was capable of induction of proliferation responses, IFN-γ ELISPOT responses and IFN-γ and IL-12 cytokines production in a significant level in peripheral blood mononuclear cells (PBMCs) isolated from HEV-recovered individuals compared to the control group. In conclusion, the newly produced multiepitope protein can induce significant T helper type 1 responses in vitro, and can be considered as a novel strategy for the development of HEV vaccines in the future.

Comparative histologic and immunologic evaluation of 1,4-butanediol diglycidyl ether crosslinked versus noncrosslinked acellular swim bladder matrix for healing of full-thickness skin wounds in rabbits.

BACKGROUND: Collagen-rich extracellular matrix from land-based mammalian tissues is increasingly used in regenerative medicine. However, its uses are associated with risk of disease transfer and may carry an ethnocultural stigma. In the present study, collagen-rich acellular swim bladder matrix (ASBM) from Rohu fish was prepared using sodium deoxycholate and crosslinked with 1,4-butanediol diglycidyl ether (BDDGE). Wound healing potential of ASBM and ASBM-BDDGE was compared in full-thickness skin wounds in rabbits. MATERIALS AND METHODS: Four full-thickness skin wounds (20 × 20 mm(2) each) were created on the dorsum of 18 rabbits and randomly divided into three equal groups. Wounds were left open, repaired with ASBM and ASBM-BDDGE in groups sham (I), ASBM (II), and ASBM-BDDGE (III), respectively. Planimetry, contracture, immunologic, and histologic observations were carried out to evaluate wound healing. RESULTS: Significantly (P < 0.05) lesser wound contraction was observed in ASBM (II) and ASBM-BDDGE (III) groups compared with sham (I) group. Total immunoglobulin G response in rabbit sera was decreased significantly (P < 0.05) in the ASBM-BDDGE (III) group compared with ASBM (II) group by enzyme-linked immunosorbent assay. Stimulation index of peripheral blood lymphocytes was decreased significantly (P < 0.05) in the ASBM-BDDGE (III) group compared with ASBM (II) group by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Histologically, improved epithelialization, neovascularization, fibroplasia, and best arranged collagen fibers were observed in ASBM (II) and ASBM-BDDGE (III) groups as early as on postimplantation day 21. CONCLUSIONS: Findings of this study indicate that BDDGE crosslinked ASBM derived from Rohu fish has potential for the clinical applications. Furthermore, it is expected that their clinical applications will not be limited by ethnocultural stigma.

Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay.

The golden standard for functional evaluation of immunodeficiencies is the incorporation of [(3)H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [(3)H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA+SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.