Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory

Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae

Abstract Tissue-resident memory CD4 T ($T_{RM}$) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against re-infection. However, it is not clear how they are maintained in respiratory tissues. Here we demonstrate that B. pertussis-specific CD4 $T_{RM}$ cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 $T_{RM}$ cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 $T_{RM}$ cells was suppressed by anti-IL-12p40, but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1$\beta$ or IL-18. Intranasal immunization of mice with a whole cell pertussis vaccine induced respiratory CD4 $T_{RM}$ cells that were re-activated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate CD4 $T_{RM}$ cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen

A Pertussis Outer Membrane Vesicle-Based Vaccine Induces Lung-Resident Memory CD4 T Cells and Protection Against Bordetella pertussis, Including Pertactin Deficient Strains

Pertussis is a respiratory infectious disease that has been resurged during the last decades. The change from the traditional multi-antigen whole-cell pertussis (wP) vaccines to acellular pertussis (aP) vaccines that consist of a few antigens formulated with alum, appears to be a key factor in the resurgence of pertussis in many countries. Though current aP vaccines have helped to reduce the morbidity and mortality associated with pertussis, they do not provide durable immunity or adequate protection against the disease caused by the current circulating strains of Bordetella pertussis, which have evolved in the face of the selection pressure induced by the vaccines. Based on the hypothesis that a new vaccine containing multiple antigens could overcome deficiencies in the current aP vaccines, we have designed and characterized a vaccine candidate based on outer membrane vesicle (OMVs). Here we show that the OMVs vaccine, but not an aP vaccine, protected mice against lung infection with a circulating pertactin (PRN)-deficient isolate. Using isogenic bacteria that in principle only differ in PRN expression, we found that deficiency in PRN appears to be largely responsible for the failure of the aP vaccine to protect against this circulating clinical isolates. Regarding the durability of induced immunity, we have already reported that the OMV vaccine is able to induce long-lasting immune responses that effectively prevent infection with B. pertussis. Consistent with this, here we found that CD4 T cells with a tissue-resident memory (TRM) cell phenotype (CD44+CD62LlowCD69+ and/or CD103+) accumulated in the lungs of mice 14 days after immunization with 2 doses of the OMVs vaccine. CD4 TRM cells, which have previously been shown to play a critical role sustained protective immunity against B. pertussis, were also detected in mice immunized with wP vaccine, but not in the animals immunized with a commercial aP vaccine. The CD4 TRM cells secreted IFN-γ and IL-17 and were significantly expanded through local proliferation following respiratory challenge of mice with B. pertussis. Our findings that the OMVs vaccine induce respiratory CD4 TRM cells may explain the ability of this vaccine to induce long-term protection and is therefore an ideal candidate for a third generation vaccine against B. pertussis

Novel gene therapeutic approaches to prolong corneal allograft survival

Cornea transplantation (penetrating keratoplasty) is the most frequent form of allograft transplantation in humans with a 90 % success rate in the first 2 years, however, the rate of survival decreases over time and drops to approximately 62% after 10 years. The predictions for \u27high risk\u27 graft survival are even poorer. Despite advances in microsurgery and immunosuppressive treatment protocols a significant number of corneal grafts still undergo immune-mediated allograft rejection. Therefore, alternative approaches are needed to prevent corneal transplant rejection such as genetic modification of donor corneas. Gene therapy has been widely applied in preclinical studies to manipulate the corneal graft rejection process. The main goal of corneal gene therapy is to achieve long-term allograft survival without the need for other treatments or only restricted to short periods of time thereby reducing undesired side effects. Herein, two distinctive strategies in a preclinical model of rat cornea transplantation to prevent allograft rejection were investigated using lentiviral (LV) vectors as vehicles for therapeutic gene delivery and overexpression in the corneal allograft. The first part of this work was based on known anti-apoptotic properties of nerve growth factor (NGF). As previously reported, adenovirus (Ad)-mediated NGF overexpression reduces apoptosis in corneal endothelial cells and prolongs allograft survival in an in vivo rat model, however, the mechanisms were not fully understood and the use of adenoviral vectors raised concern. The aim of this part of the project was to develop an anti-apoptotic strategy using alternative, low immunogenic LV vectors expressing NGF (LV.NGF) for rat cornea transplantation and investigation of the underlying mechanisms of cytoprotection. It was shown that LV.NGF transduced corneal endothelial cells secreted biologically active NGF. In addition, supernatants from LV.NGF transduced immortalized human corneal endothelial cells (HCEC) significantly reduced thapsigargin (TG)-induced apoptosis in control PC12 cells, however, no protective effect of either recombinant NGF or supernatant containing NGF could be observed in HCEC. Subsequently, a protocol for LV-mediated transduction of ex vivo cultured corneas was established. However, in contrast to previous results showing that Ad.NGF gene transfer in cultured corneas resulted in significant prolongation of allograft survival, donor corneas transduced with LV.NGF did not show any protective effect in vivo. Different expression levels of the therapeutic gene after Ad- or LV gene transfer may account for these results. The second part of this project was to investigate the role of LV-mediated overexpression of Programmed Death Ligand-1 (PD-L1) on rat corneal allograft survival. In contrast to LV.NGF treatment, allogeneic LV.PD-L1 transduced corneas showed a high percentage (83%) of graft survival. This striking result was associated with a reduction of natural killer T (NKT) cell and cytotoxic CD8+ T cell infiltration to the graft as measured by flow cytometry. Graft opacity, as an indicator of cell infiltration, was present but was significantly reduced in PD-L1 transduced corneas compared to control or EGFP expressing corneas. Herein, the data provide evidence that therapeutic applications of LV-based cornea gene therapy might be a valuable clinical approach for treatment of corneal endothelium failure and/or graft rejection

Gene therapy approaches to prevent corneal graft rejection: where do we stand?

U radu su prezentirani rezultati ergonomske prosudbe svih relevantnih čimbenika iz radnog i(li) prometnog okoliša koji utječu na izvedbu i sigurnost vozača cestovnih vozila kompilacijom i komparacijom spoznaja iz recentne znanstvene i stručne literature. Između ostalih čimbenika iz radnog i(li) prometnog okoliša, analizirao se utjecaj antropometrijske prilagođenosti vozača gabaritama upravljačnice, utjecaj čimbenika promjene ITM s navršenom dobi, a zbog dokazanog utjecaja istoga na fizičko radno opterećenje vozača. Također su se analizirale mjere za poboljšanje odvijanja javnog gradskog putničkog prometa, s ciljem unaprijeđenja sigurnosti i izvedbe vozača cestovnog vozila u urbanim sredinama. Sažeto su se analizirali čimbenici umora vozača. Istražilo se kako se primjenom iRAP metodologije i statističkom obradom čimbenika prometnih nesreća može pojednostaviti selekcija lokacija prometnih nesreća u urbanim sredinama za koje umor može biti potencijalni uzrok. Odredile su se smjernice s ciljem poboljšanja sigurnosti cestovnog prometa i izvedbe vozača u urbanim sredinama.This graduate thesis presents the results of an ergonomic assessment of all relevant factors from the working and/or traffic environment affecting the performance and safety of road vehicle drivers by compiling and comparing the findings from recent scientific and professional literature. Among other factors from the working and traffic environment, the impact of driver’s anthropometric adjustment to the cab dimensions, as well as the influence of changes in the BMI (Body Mass Index) with age was studied, due to the proven impact of the mentioned on the driver’s physical workload. Also, measures for improving the performance of public city transport were investigated in order to improve the safety and performance of road vehicle drivers in urban environment. The factors of driver’s fatigue were briefly examined. The research also included the study of how by using the iRAP methodology and the statistical processing of factors of traffic accidents, the selection of the traffic accidents’ locations in urban environments, for which the fatigue can be a potential cause, could be simplified. In order to improve road safety and driver’s performance in urban environments the guidelines were set