Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species.

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52-48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19-100.00%) and sensitivity was 52.94% (95% CI, 35.13-70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67-65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

SCROTAL THERMOGRAPHY IN THE EVALUATION OF SURGICAL OUTCOME AFTER VARICOCELE REPAIR: A CASE REPORT.

Scrotal thermography is a diagnostic method for varicocele. In short, there are five diagnostic thermographic criteria for varicocele, i.e., pattern of scrotal thermographic image indicative of varicocele, temperature at pampiniform plexus ≥34 C°, temperature difference between left and right pampiniform plexus ≥0.5 C°, enhancement of image during Valsalva maneuver, and temperature at pampiniform plexus ≥ temperature at ipsilateral thigh. Three or more positive signs are indicative of varicocele. The aim of this report is to present the use of digital thermography as a diagnostic method to evaluate the outcome of varicocele repair. We present a case of a student diagnosed with varicocele grade III, and assessed preoperatively and followed up postoperatively by scrotal thermography. According to thermographic indicators, our patient was positive for varicocele diagnosis before surgical treatment. Three months after varicocele repair, the patient did not show positive thermographic indicators of varicocele while physical examination and color Doppler ultrasound were equivocal. This case report suggests that infrared digital thermography of scrotum could be very valuable for monitoring patients in the period after surgery for varicocele, however, it should be confirmed in a larger number of patients.

Application of Supercritical Solvent Impregnation for Production of Zeolite Modified Starch-Chitosan Polymers with Antibacterial Properties.

In the present study, supercritical solvent impregnation (SSI) has been applied to incorporate thymol into bio-composite polymers as a potential active packaging material. Thymol, a natural component with a proven antimicrobial activity, was successfully impregnated into starch-chitosan (SC) and starch-chitosan-zeolite (SCZ) films using supercritical carbon dioxide (scCO2) as a solvent. Experiments were performed at 35 °C, pressures of 15.5 and 30 MPa, and an impregnation time in the range of 4-24 h. The highest impregnation yields of SC films with starch to chitosan mass ratios of 1:1 and 1:2 were 10.80% and 6.48%, respectively. The addition of natural zeolite (15-60%) significantly increased the loading capacity of films enabling thymol incorporation in a quantity of 16.7-27.3%. FTIR and SEM analyses were applied for the characterization of the films. Mechanical properties and water vapor permeability of films before and after the impregnation were tested as well. Thymol release kinetics in deionized water was followed and modeled by the Korsmeyer-Peppas and Weibull model. SCZ films with thymol loading of approximately 24% exhibited strong antibacterial activity against E. coli and methicillin-resistant Staphylococcus (S.) aureus (MRSA).

Association study of cytochrome P450 1A1*2A polymorphism with prostate cancer risk and aggressiveness in Croatians.

Cytochrome P450 1A1 (CYP1A1) is an enzyme participating in the bioactivation of various endogenous and environmental reactive compounds that can bind to DNA and thus induce cancerogenesis. Gene encoding the enzyme is expressed in the prostate tissue and is polymorphic. CYP1A1*2A gene polymorphism is associated with elevated enzyme activity and/or inducibility which can lead to accumulation of genotoxic compounds and consequently to cancerogenesis. We examined the association of this polymorphism with prostate cancer (PCa) risk and aggressiveness. The case-control study consisted of 120 PCa patients and 120 benign prostatic hyperplasia (BPH) controls, in Croatian population. Regarding aggressiveness, PCa patients were grouped according to the Gleason score (GS), tumor stage (T) and existence of distant metastasis (M). The polymorphism was analyzed using real-time polymerase chain reaction (PCR). We did not observe association of mutated allele with PCa risk, neither with PCa aggressiveness. Furthermore, frequency of polymorphic genotype was slightly higher in BPH group (16.6% vs. 14.2%, respectively) and also in less aggressive form of PCa (20.4% vs. 9.6% for GS < 7; 15.6% vs. 9.1% for T < 3; 16.7% vs. 10.0% for no distant metastasis). Comparing our findings with other published results, we can assume that the ethnicity influence the genotype distribution and thus may affect the etiology of PCa, even possibly in the way to cause an opposite effect among different ethnic groups. Given the small number of participants, results should be validated on the larger sample size.

Comparison between clinical significance of serum proinflammatory protein interleukin-6 and classic tumor markers total PSA, free PSA and free/total PSA prior to prostate biopsy.

The aim of the study was to clarify whether serum levels of proinflammatory cytokine interleukin-6 (IL-6) could be a useful marker in prostate diseases. Serum IL-6 was determined prior to prostate biopsy procedure in 82 patients with prostate adenocarcinoma (PCa), 25 patients with benign prostatic hyperplasia (BPH), 24 patients with high-grade prostatic intraepithelial neoplasia (PIN) and 17 patients with chronic prostatitis. Serum IL-6 levels were compared with total PSA (tPSA), free PSA (fPSA) and the free/total ratio (f/tPSA) serum levels. Statistically significant difference was not found in serum IL-6 levels among the four groups (p = 0.088). However, the patients with poorly differentiated PCa with Gleason score (GS) 4 + 3 = 7 and > 7 had significantly higher serum IL-6 levels than the patients with moderately differentiated PCa with GS 3 + 4 = 7 and < 7 (p = 0.007). The findings suggest that serum IL-6 level might be a potentially useful marker for poorly differentiated PCa.

Cornstarch aerogels with thymol, citronellol, carvacrol, and eugenol prepared by supercritical CO2- assisted techniques for potential biomedical applications.

This study focuses on the development of bioactive materials using environmentally friendly techniques, renewable, biocompatible, and biodegradable polysaccharide, as well as natural bioactive compounds (NBCs) found in plant extracts. First, cornstarch aerogels with a porosity of 86 % and a specific surface area of 225 m2/g were produced via supercritical CO2- assisted drying. Further, thymol, citronellol, carvacrol, and eugenol were incorporated into the aerogels by supercritical CO2- assisted impregnation, which allowed variation in loadings of NBCs (12.8-17.6 %). Interaction between cornstarch aerogels and NBCs determined impregnation rate, pore wall thickness (in the range 18-95 nm), liquid absorption capacity (from 265 to 569 %), dehydration mass loss, and release in phosphate-buffered saline. Controlled release of NBCs was maintained over a 3-day period. Moreover, impregnated aerogels showed a significant antioxidant effect with the highest value for DPPH radical inhibition of 25.5 % determined for the aerogels impregnated with eugenol. Notable antimicrobial activity against tested Gram-negative bacteria, Gram-positive bacteria, and fungi was also observed, being the highest for thymol-loaded aerogel with the diameter of the inhibition zones of up to 37.5 mm. This work shows a promising green approach for the production of bioactive two-component starch-based materials for potential applications in skin infection treatment.

Banana Lectin: A Novel Immunomodulatory Strategy for Mitigating Inflammatory Bowel Disease.

Compared to the general population, patients with inflammatory bowel disease (IBD) are less likely to be vaccinated, putting them at an increased risk of vaccine-preventable illnesses. This risk is further compounded by the immunosuppressive therapies commonly used in IBD management. Therefore, developing new treatments for IBD that maintain immune function is crucial, as successful management can lead to better vaccination outcomes and overall health for these patients. Here, we investigate the potential of recombinant banana lectin (rBanLec) as a supporting therapeutic measure to improve IBD control and possibly increase vaccination rates among IBD patients. By examining the therapeutic efficacy of rBanLec in a murine model of experimental colitis, we aim to lay the foundation for its application in improving vaccination outcomes. After inducing experimental colitis in C57BL/6 and BALB/c mice with 2,4,6-trinitrobenzene sulfonic acid, we treated animals orally with varying doses of rBanLec 0.1-10 µg/mL (0.01-1 µg/dose) during the course of the disease. We assessed the severity of colitis and rBanLec's modulation of the immune response compared to control groups. rBanLec administration resulted in an inverse dose-response reduction in colitis severity (less pronounced weight loss, less shortening of the colon) and an improved recovery profile, highlighting its therapeutic potential. Notably, rBanLec-treated mice exhibited significant modulation of the immune response, favoring anti-inflammatory pathways (primarily reduction in a local [TNFα]/[IL-10]) crucial for effective vaccination. Our findings suggest that rBanLec could mitigate the adverse effects of immunosuppressive therapy on vaccine responsiveness in IBD patients. By improving the underlying immune response, rBanLec may increase the efficacy of vaccinations, offering a dual benefit of disease management and prevention of vaccine-preventable illnesses. Further studies are required to translate these findings into clinical practice.

Green Processing of Neat Poly(lactic acid) Using Carbon Dioxide under Elevated Pressure for Preparation of Advanced Materials: A Review (2012-2022).

This review provides a concise overview of up-to-date developments in the processing of neat poly(lactic acid) (PLA), improvement in its properties, and preparation of advanced materials using a green medium (CO2 under elevated pressure). Pressurized CO2 in the dense and supercritical state is a superior alternative medium to organic solvents, as it is easily available, fully recyclable, has easily tunable properties, and can be completely removed from the final material without post-processing steps. This review summarizes the state of the art on PLA drying, impregnation, foaming, and particle generation by the employment of dense and supercritical CO2 for the development of new materials. An analysis of the effect of processing methods on the final material properties was focused on neat PLA and PLA with an addition of natural bioactive components. It was demonstrated that CO2-assisted processes enable the control of PLA properties, reduce operating times, and require less energy compared to conventional ones. The described environmentally friendly processing techniques and the versatility of PLA were employed for the preparation of foams, aerogels, scaffolds, microparticles, and nanoparticles, as well as bioactive materials. These PLA-based materials can find application in tissue engineering, drug delivery, active food packaging, compostable packaging, wastewater treatment, or thermal insulation, among others.

Predictive Value of Red Blood Cell Distribution Width, Mean Platelet Volume and Platelet Distribution Width in Children with Acute Appendicitis.

BACKGROUND AND OBJECTIVES: Acute appendicitis in pediatric patients is one of the most common surgical emergencies, but the early diagnosis still remains challenging. The aim of this study was to determine the predictive value of Red blood cell distribution width (RDW), Mean platelet volume (MPV) and Platelet distribution width (PDW) in children with acute appendicitis. MATERIALS AND METHODS: This study was a retrospective assessment of laboratory findings (RDW, MPV, PDW) of patients who underwent surgical treatment for acute appendicitis from January 2019 to December 2020. RESULT: During this period, 223 appendectomies were performed at our Institute. In 107 (43%) cases appendicitis was uncomplicated, while in 116 (46.6%) it was complicated. WBC and RDW/MPV ratio were significant parameters for the diagnosis of acute appendicitis with cut-off values of 12.86 (susceptibility: 66.3%; specificity: 73.2%) and 1.64 (susceptibility: 59.8%; specificity: 71.9%), respectively. WBC and RDW/RBC ratio were independent variables for the diagnosis of complicated appendicitis. The cut-off values were 15.05 for WBC (sensitivity: 60.5%; specificity: 70.7%) and 2.5 for RDW/RBC ratio (sensitivity: 72%; specificity: 52.8%). CONCLUSIONS: WBC is an important predictor of appendicitis and complicated appendicitis. RDW, MPV and PDW alone have no diagnostic value in pediatric acute appendicitis or predicting the degree of appendix inflammation. However, the RDW/MPV ratio can be an important predictor of appendix inflammation, with higher values in patients with more severe appendix inflammation. RDW/RBC ratio may be an important predictor of complicated appendicitis.

Recombinant Bet v 1-BanLec chimera modulates functional characteristics of peritoneal murine macrophages by promoting IL-10 secretion.

Allergen-specific immunotherapy (AIT) is a desensitizing treatment for allergic diseases that corrects the underlined pathological immune response to innocuous protein antigens, called allergens. Recombinant allergens employed in the AIT allowed the production of well-defined formulations that possessed consistent quality but were often less efficient than natural allergen extracts. Combining recombinant allergens with an adjuvant or immunomodulatory agent could improve AIT efficacy. This study aimed to perform structural and functional characterization of newly designed recombinant chimera composed of the Bet v 1, the major birch pollen allergen, and Banana Lectin (BanLec), TLR2, and CD14 binding protein, for the application in AIT. rBet v 1-BanLec chimera was designed in silico and expressed as a soluble fraction in Escherichia coli. Purified rBet v 1-BanLec (33.4 kDa) retained BanLec-associated biological activity of carbohydrate-binding and preserved IgE reactive epitopes of Bet v 1. The chimera revealed secondary structures with predominant ß sheets. The immunomodulatory capacity of rBet v 1-BanLec tested on macrophages showed changes in myeloperoxidase activity, reduced NO production, and significant alterations in the production of cytokines when compared to both rBanLec and rBet v 1. Comparing to rBet v 1, rBet v 1-BanLec was demonstrated to be more efficient promoter of IL-10 production as well as weaker inducer of NO production and secretion of pro-inflammatory cytokines TNFα, and IL-6. The ability of rBet v 1-BanLec to promote IL-10 in together with the preserved 3D structure of Bet v 1 part implies that the construct might exert a beneficial effect in the allergen-specific immunotherapy.

Cross-Reactive Effects of Vaccines: Heterologous Immunity between Tetanus and Chlamydia.

Vaccines can have heterologous effects on the immune system, i.e., effects other than triggering an immune response against the disease targeted by the vaccine. We investigated whether monoclonal antibodies (mAbs) specific for tetanus could cross-react with Chlamydia and confer heterologous protection against chlamydial infection. The capability of two tetanus-specific mAbs, namely mAb26 and mAb51, to prevent chlamydial infection has been assessed: (i) in vitro, by performing a neutralization assay using human conjunctival epithelial (HCjE) cells infected with Chlamydia trachomatis serovar B, and (ii) in vivo, by using a guinea pig model of Chlamydiacaviae-induced inclusion conjunctivitis. The mAb26 has been superior in comparison with mAb51 in the prevention of chlamydial infection in HCjE cells. The mAb26 has conferred ≈40% inhibition of the infection, compared to less than 5% inhibition in the presence of the mAb51. In vivo, mAb26 significantly diminished ocular pathology intensity in guinea pigs infected with C. caviae compared to either the mAb51-treated or sham-treated guinea pigs. Our data provide insights that tetanus immunization generates antibodies which induce heterologous chlamydial immunity and promote protection beyond the intended target pathogen.

Cooperative binding of anti-tetanus toxin monoclonal antibodies: Implications for designing an efficient biclonal preparation to prevent tetanus toxin intoxication.

Oligoclonal combinations of several monoclonal antibodies (MAbs) are being considered for the treatment of various infectious pathologies. These combinations are less sensitive to antigen structural changes than individual MAbs; at the same time, their characteristics can be more efficiently controlled than those of polyclonal antibodies. The main goal of this study was to evaluate the binding characteristics of six biclonal equimolar preparations (BEP) of tetanus toxin (TeNT)-specific MAbs and to investigate how the MAb combination influences the BEPs' protective capacity. We show that a combination of TeNT-specific MAbs, which not only bind TeNT but also exert positive cooperative effects, results in a BEP with superior binding characteristics and protective capacity, when compared with the individual component MAbs. Furthermore, we show that a MAb with only partial protective capacity but positive effects on the binding of the other BEP component can be used as a valuable constituent of the BEP.

Modulation of functional characteristics of resident and thioglycollate-elicited peritoneal murine macrophages by a recombinant banana lectin.

We demonstrated that a recombinant banana lectin (rBanLec), which structural characteristics and physiological impacts highly resemble those reported for its natural counterparts, binds murine peritoneal macrophages and specifically modulates their functional characteristics. By using rBanLec in concentrations ranging from 1 µg to 10 µg to stimulate resident (RMs) and thioglycollate-elicited (TGMs) peritoneal macrophages from BALB/c and C57BL/6 mice, we have shown that effects of rBanLec stimulation depend on its concentration but also on the functional status of macrophages and their genetic background. rBanLec, in a positive dose-dependent manner, promotes the proliferation of TGMs from both BALB/c and C57BL/6 mice, while its mitogenic influence on RMs is significantly lower (BALB/c mice) or not detectable (C57BL/6 mice). In all peritoneal macrophages, irrespective of their type and genetic background, rBanLec, in a positive dose dependent manner, enhances the secretion of IL-10. rBanLec stimulation of RMs from both BALB/c and C57BL/6 resulted in a positive dose-dependent promotion of proinflammatory phenotype (enhancement of NO production and IL-12 and TNFα secretion, reduction of arginase activity). Positive dose-dependent skewing toward proinflammatory phenotype was also observed in TGMs from C57BL/6 mice. However, the enhancement of rBanLec stimulation promotes skewing of TGMs from BALB/c mice towards anti-inflammatory profile (reduction of NO production and IL-12 secretion, enhancement of arginase activity and TGFß and IL-4 secretion). Moreover, we established that rBanLec binds oligosaccharide structures of TLR2 and CD14 and that blocking of signaling via these receptors significantly impairs the production of TNFα and NO in BALB/c macrophages. Since the outcome of rBanLec stimulation depends on rBanLec concentration as well as on the functional characteristics of its target cells and their genetic background, further studies are needed to investigate its effects under physiological and specific pathological conditions.

The effect of infectious dose on humoral and cellular immune responses in Chlamydophila caviae primary ocular infection.

Following infection, the balance between protective immunity and immunopathology often depends on the initial infectious load. Several studies have investigated the effect of infectious dose; however, the mechanism by which infectious dose affects disease outcomes and the development of a protective immune response is not known. The aim of this study was to investigate how the infectious dose modulates the local and systemic humoral and the cellular immune responses during primary ocular chlamydial infection in the guinea pig animal model. Guinea pigs were infected by ocular instillation of a Chlamydophila caviae-containing eye solution in the conjunctival sac in three different doses: 1×102, 1×104, and 1×106 inclusion forming units (IFUs). Ocular pathology, chlamydial clearance, local and systemic C. caviae-specific humoral and cellular immune responses were assessed. All inocula of C. caviae significantly enhanced the local production of C. caviae-specific IgA in tears, but only guinea pigs infected with the higher doses showed significant changes in C. caviae-specific IgA levels in vaginal washes and serum. On complete resolution of infection, the low dose of C. caviae did not alter the ratio of CD4+ and CD8+ cells within guinea pigs' submandibular lymph node (SMLN) lymphocytes while the higher doses increased the percentages of CD4+ and CD8+ cells within the SMLN lymphocytes. A significant negative correlation between pathology intensity and the percentage of CD4+ and CD8+ cells within SMLN lymphocyte pool at selected time points post-infection was recorded for both 1×104, and 1×106 IFU infected guinea pigs. The relevance of the observed dose-dependent differences on the immune response should be further investigated in repeated ocular chlamydial infections.

A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C.

Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC- and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development.

Infectious dose and repeated infections are key factors influencing immune response characteristics in guinea pig ocular chlamydial infection.

The aim of this study was to determine whether infectious dose of Chlamydia caviae after repeated infections influences the immunological responses and subsequent clearance of pathogen at the ocular surface of guinea pigs. Animals were infected three times via the conjunctiva at six- and twelve-week intervals by applying either 1 × 10(4) or 1 × 10(6) inclusion-forming units (IFUs) of C. caviae. Ocular pathology, infection course, C. caviae-specific serum IgG levels and their capacity to bind and neutralize infection ex vivo were assessed. Animals infected with 1 × 10(4) IFUs had completely diminished ocular infection and pathology after the 2nd infection with increased levels of C. caviae-specific serum IgG and their effective capacity to bind and neutralize C. caviae. Only partial protection was observed in animals infected with 1 × 10(6) IFUs after the 2nd and 3rd infections. Our findings show that full protection was observed in animals repeatedly infected with the lower dose. The lower dose appeared not to compromise the host immune system, thereby enabling fast clearance of the pathogen and the establishment of competent neutralizing antibodies.

Key protection factors against tetanus: Anti-tetanus toxin antibody affinity and its ability to prevent tetanus toxin - ganglioside interaction.

Antibodies capable to neutralize tetanus toxin (TeNT) are key factors in protection against tetanus disease. Although antibody-based therapeutics for treatment of tetanus exist on the market its production is tedious. Hence, the tetanus-specific antibodies preparation that could be easily produced in large scale in vitro would be beneficial. Monoclonal antibodies (MAbs) are considered for a long time as a reagent of choice, but the core drawback is how to select a MAb that would be safe in providing efficacious protection. In this study we have investigated the parameters crucial for a single MAb to be assigned as protective. Eight murine MAbs were characterized in vitro for their reactivity toward TeNT and assessed in vivo for protectiveness against TeNT intoxication. Correlation of in vitro and in vivo data has revealed that in vitro selection of MAb that is protective in vivo could be performed by a combination of two assays: the measurement of MAb affinity toward TeNT taking Ka 1 × 10(8) M(-1) as a threshold level, and the evaluation of its capability to prevent TeNT-ganglioside interaction. Single MAb could be taken into consideration as a potential therapeutic only if it has a capacity to completely inhibits TeNT-ganglioside complex formation.

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers.

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.

Biodiesel synthesis at high pressure and temperature: analysis of energy consumption on industrial scale.

Analysis of several different schemes for industrial FAME production at higher pressure and temperature (catalytic or non-catalytic synthesis) was realized with the aim to find the best route to reduce the energy consumption (EC) and to improve the life cycle energy efficiency. Obtained results indicated that the EC (MJ/kg FAME) mainly depends on degree of conversion of triglycerides being almost 25% smaller if degree of conversion increase from 97 mass% to complete conversion. Further significant decrease of EC might be obtained at subcritical conditions but only after substantial decrease of methanol to oil molar ratio (from 42 to 15) which requires use of appropriate catalyst. On account of that, the kinetics of heterogeneous catalyzed methanolysis of triglycerides was analyzed using data published in literature (CaO) as well as own experimental data (K(2)CO(3)/Al-O-Si) with a goal to obtain reliable kinetic rate constant which might be used for process simulation. This study shows that if heterogeneous process of biodiesel synthesis is realized at subcritical conditions then further decrease of EC is possible.