Application of Octenidine into Nasal Vestibules Does Not Influence SARS-CoV-2 Detection via PCR or Antigen Test Methods.

The targeted or universal decolonization of patients through octenidine for nasal treatment and antiseptic body wash for 3 to 5 days prior elective surgery has been implemented in several surgical disciplines in order to significantly reduce surgical site infections (SSIs) caused by Staphylococcus aureus carriage. However, as most healthcare facilities also screen patients on admission for pilot infection, it is imperative that a prophylactic nasal decolonization procedure not yield a false negative SARS-CoV-2 status in otherwise positive patients. We assessed the effect of a commercially available octenidine-containing nasal gel on two different screening methods-antigen (Ag) detection based on colloidal gold immunochromatography and RT-PCR-in a prospective-type accuracy pilot study in asymptomatic SARS-CoV-2-positive inpatients. All patients still showed a positive test result after using the octenidine-containing nasal gel for about 3 days; therefore, its application did not influence SARS-CoV-2 screening, which is of high clinical relevance. Of note is that Ag detection was less sensitive, regardless of the presence of octenidine. From an infection prevention perspective, these results favor octenidine-based decolonization strategies, even during seasonal SARS-CoV-2 periods. As only asymptomatic patients are considered for elective interventions, screening programs based on RT-PCR technology should be preferred.

Blockade of adhesion molecule lymphocyte function-associated antigen-1 improves long-term heart allograft survival in mixed chimeras.

BACKGROUND: The mixed chimerism approach for intentional induction of donor-specific tolerance was shown to be successful in various models from mice to humans. For transplant patients, the approach would obviate the need for long-term immunosuppression and associated side effects; moreover, it would preclude the risk of late graft loss due to chronic rejection. Widespread clinical application is hindered by toxicities related to recipient pre-conditioning. Herein we aimed to investigate a clinically relevant protocol for tolerance induction to cardiac allografts, sparing CD40 blockade or T-cell depletion. METHODS: B6 mice were conditioned with non-myeloablative total body irradiation, fully mismatched BALB/c bone marrow cells, and short-term therapy, based on either anti- lymphocyte function-associated antigen-1 (anti-LFA-1) or anti-CD40L. Multilineage chimerism was followed by flow-cytometric analysis, tolerance was assessed with skin and heart allografts from fully or major histocompatibility complex-mismatched donors. Mechanisms of tolerance were investigated by analysis of donor-specific antibodies (DSAs), mixed lymphocyte reaction (MLR) assays, and deletion of donor-reactive T cells. RESULTS: We found that the combination of cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and rapamycin with LFA-1 blockade enhanced bone marrow engraftment and led to more efficient T-cell engraftment and subsequent tolerization. Although fully mismatched skin grafts were chronically rejected, primarily vascularized heart allografts survived indefinitely and without signs of chronic rejection, independent of minor antigen mismatches. CONCLUSIONS: We have demonstarted a robust protocol for the induction of tolerance for cardiac allografts in the absence of CD40 blockade. Our findings demonstrate the potential of a clinically relevant minimal conditioning protocol designed to induce lifelong immunologic tolerance toward cardiac allografts.

Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade.

Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been reported with distinct models and protocols. We therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2b) mice received nonmyeloablative total body irradiation (3 Gy), costimulation blockade (anti-CD40L mAb and CTLA4Ig), and 2 × 107 bone marrow cells (BMC) from either of three donor strains: Balb/c (H2d) (MHC plus multiple minor histocompatibility antigen (mHAg) mismatched), B10.D2 (H2d) or B10.A (H2a) (both MHC mismatched, but mHAg matched). Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 of Balb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (p < 0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (p < 0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities. .

In vitro effect of octenidine dihydrochloride against Trichomonas vaginalis.

Trichomoniasis is the most common non-viral sexually transmitted disease. It is associated with a wide spectrum of complications, including infertility and increased susceptibility to human immunodeficiency virus (HIV). A rising number of reports of Trichomonas vaginalis strains resistant to metronidazole has driven the search for new compounds. In the present study, the in vitro effects of the common antiseptic octenidine dihydrochloride against T. vaginalis were tested on metronidazole-resistant and -susceptible strains. Assays were performed under microaerophilic conditions in three different media containing varying concentrations of protein. It was shown that octenidine dihydrochloride is highly effective against T. vaginalis, with no difference between metronidazole-resistant and -susceptible strains. The 50% effective concentration (EC50) values ranged from 5.7 to 21.37µg/mL after 5min, from 6.48 to 10.82µg/mL after 15min and from 0.68 to 2.11µg/mL after 30min of treatment depending on the protein concentration of the test medium. Octenidine dihydrochloride, already approved in some countries for the treatment of bacterial and fungal vaginal infections, appears to be a promising alternative treatment for trichomoniasis, particularly in mixed vaginal infections or in cases caused by metronidazole-resistant strains.

Polyclonal Recipient nTregs Are Superior to Donor or Third-Party Tregs in the Induction of Transplantation Tolerance.

Induction of donor-specific tolerance is still considered as the "Holy Grail" in transplantation medicine. The mixed chimerism approach is virtually the only tolerance approach that was successfully translated into the clinical setting. We have previously reported successful induction of chimerism and tolerance using cell therapy with recipient T regulatory cells (Tregs) to avoid cytotoxic recipient treatment. Treg therapy is limited by the availability of cells as large-scale expansion is time-consuming and associated with the risk of contamination with effector cells. Using a costimulation-blockade based bone marrow (BM) transplantation (BMT) model with Treg therapy instead of cytoreductive recipient treatment we aimed to determine the most potent Treg population for clinical translation. Here we show that CD4(+)CD25(+) in vitro activated nTregs are superior to TGFß induced iTregs in promoting the induction of chimerism and tolerance. Therapy with nTregs (but not iTregs) led to multilineage chimerism and donor-specific tolerance in mice receiving as few as 0.5 × 10(6) cells. Moreover, we show that only recipient Tregs, but not donor or third-party Tregs, had a beneficial effect on BM engraftment at the tested doses. Thus, recipient-type nTregs significantly improve chimerism and tolerance and might be the most potent Treg population for translation into the clinical setting.

Hand hygiene--evaluation of three disinfectant hand sanitizers in a community setting.

Hand hygiene is acknowledged as the single most important measure to prevent nosocomial infections in the healthcare setting. Similarly, in non-clinical settings, hand hygiene is recognised as a key element in helping prevent the spread of infectious diseases. The aim of this study was to evaluate the efficacy of three different disinfectant hand sanitizers in reducing the burden of bacterial hand contamination in 60 healthy volunteers in a community setting, both before and after education about the correct use of hand sanitizers. The study is the first to evaluate the efficacy and ease of use of different formulations of hand rubs used by the general population. The products tested were: Sterillium (perfumed, liquid), desderman pure gel (odorless, gel) and Lavit (perfumed, spray). Sterillium and desderman are EN1500 (hygienic hand rub) certified products (available in pharmacy) and Lavit is non EN1500 certified and available in supermarkets. The two EN1500 certified products were found to be significantly superior in terms of reducing bacterial load. desderman pure gel, Sterillium and Lavit reduced the bacterial count to 6.4%, 8.2% and 28.0% respectively. After education in the correct use of each hand rub, the bacterial load was reduced even further, demonstrating the value of education in improving hand hygiene. Information about the testers' perceptions of the three sanitizers, together with their expectations of a hand sanitizer was obtained through a questionnaire. Efficacy, followed by skin compatibility were found to be the two most important attributes of a hand disinfectant in our target group.

Mandibular corrective osteotomy using novel locking compression plate 3.5/4.5/5.0 mm metaphyseal plates.

OBJECTIVE: To describe a technique and the outcome of using 3.5/4.5/5.0 Metaphyseal Locking Compression Plate for corrective osteotomy of mandibular brachygnathia. STUDY DESIGN: Clinical report. ANIMAL: Eight-month Thoroughbred horse. METHODS: Severe mandibular brachygnathia was surgically treated by corrective osteotomy and fixation with 2 LCP 3.5/4.5/5.0 Metaphyseal plates inserted using minimally invasive technique. RESULTS: Severe mandibular brachygnathia was treated successfully with minor complications and stable fixation after 3 months. Cosmetic outcome and owner satisfaction was excellent. CONCLUSIONS: Corrective osteotomy and fixation with LCP 3.5/4.5/5.0 Metaphyseal plates can be considered as a surgical technique for correction of severe mandibular brachygnathia in weanlings.

Long term outcome after surgical correction of mandibular brachygnathia with unilateral type 1 external skeletal fixation.

OBJECTIVE: To describe complications and long-term outcome after surgical correction of severe overbite in 7 horses using corrective osteotomy and a Type I external fixator. STUDY DESIGN: Case series. ANIMALS: Horses (n = 7). METHODS: Seven horses with severe mandibular brachygnathia were treated by corrective osteotomy and a Type I external fixator. Data on surgical technique, complications, long-term outcome and owner satisfaction were recorded. RESULTS: Severe mandibular brachygnathia was corrected successfully in all horses. Short term follow-up revealed a relatively high morbidity due to several complications such as surgical site infection, sequestrum formation and instability due to early pin loosening. Long-term over all owner satisfaction was very high. CONCLUSIONS: Corrective osteotomy and fixation with an external fixator is an effective surgical technique for correction of severe mandibular brachygnathia and offers good results in a long-term perspective.

Engraftment of retrovirally transduced Bet v 1-GFP expressing bone marrow cells leads to allergen-specific tolerance.

Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5. Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells. Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays. Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays. These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients.

While costimulation blockade-based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor-reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 10(7) T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti-CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short-term treatment of sensitized BMT recipients with rapamycin or anti-LFA-1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long-term mixed chimerism and donor-specific tolerance. Thus, rapamycin and anti-LFA-1 mAb are effective in overcoming the potent barrier that donor-reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.

Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning.

In order to develop minimally toxic bone marrow transplantation (BMT) protocols suitable for use in a wider range of indications, it is important to identify ways to enhance BM engraftment at a given level of recipient conditioning. CXCL12/stromal cell-derived factor-1α plays a crucial physiological role in homing of hematopoietic stem cells to BM. It is regulated by the ectopeptidase dipeptidyl peptidase IV (DPPIV; DPP4) known as CD26, which cleaves dipeptides from the N-terminus of polypeptide chains. Blocking DPPIV enzymatic activity had a beneficial effect on hematopoietic stem cell engraftment in various but very specific experimental settings. Here we investigated whether inhibition of DPPIV enzymatic activity through Diprotin A or sitagliptin (Januvia) improves BM engraftment in nonmyeloablative murine models of syngeneic (i.e., CD45-congenic) and allogeneic (i.e., Balb/c to B6) BMT (1 Gy total body irradiation, 10-15 × 10(6) unseparated BM cells/mouse). Neither Diprotin A administered in vivo at the time of BMT and/or used for in vitro pretreatment of BM nor sitagliptin administered in vivo had a detectable effect on the level of multilineage chimerism (follow-up >20 weeks). Similarly, sitagliptin did not enhance chimerism after allogeneic BMT, even though DPPIV enzymatic activity measured in serum was profoundly inhibited (>98% inhibition at peak exposure). Our results provide evidence that DPPIV inhibition via Diprotin A or sitagliptin does not improve engraftment of unseparated BM in a nonmyeloablative BMT setting.

Therapeutic efficacy of polyclonal tregs does not require rapamycin in a low-dose irradiation bone marrow transplantation model.

BACKGROUND: Mixed chimerism is an effective strategy for the induction of transplantation tolerance but the toxicity of recipient conditioning makes current bone marrow (BM) transplantation (BMT) protocols unsuitable for widespread clinical application. Therapies promoting BM engraftment under minimal conditioning would facilitate translation of this concept to the clinic. Recently, we have shown that regulatory T cell (Treg) therapy has potent engraftment-enhancing effects in an irradiation-free noncytotoxic BMT protocol, but only if it is combined with rapamycin treatment. METHODS: Here, we investigated whether polyclonal Treg therapy is effective in promoting chimerism and tolerance in an otherwise unsuccessful BMT protocol using low-dose total body irradiation (1 Gy) and costimulation blockade and determined whether Tregs do so on their own without rapamycin. RESULTS: The application of polyclonal FoxP3-transduced recipient Tregs led to durable multilineage chimerism and donor-specific skin graft tolerance whereas recipients receiving costimulation blockade alone or green flourescent protein (GFP)-transduced cells failed to develop chimerism. Infused Tregs had a limited life span as indicated by polymerase chain reaction analysis but rather contribute to de novo induction of subsequent Treg generations. Deletion of donor-reactive T cells was observed but progressed more slowly over time compared with recipients of a nonmyeloablative BMT protocol using 3 Gy total body irradiation. CONCLUSIONS: In conclusion, Treg therapy promotes BM engraftment on its own in a low-dose irradiation BMT protocol, leading to chimerism and tolerance maintained through deletional and nondeletional mechanisms.

The role of natural killer T cells in costimulation blockade-based mixed chimerism.

Distinct lymphocyte populations have been identified that either promote or impede the establishment of chimerism and tolerance through allogeneic bone marrow transplantation (BMT). Natural killer T (NKT) cells have pleiotropic regulatory properties capable of either augmenting or downmodulating various immune responses. We investigated in this study whether NKT cells affect outcome in mixed chimerism models employing fully mismatched nonmyeloablative BMT with costimulation blockade (CB). The absence of NKT cells had no detectable effect on chimerism or skin graft tolerance after conditioning with 3Gy total body irradiation (TBI), and a limited positive effect with 1Gy TBI. Stimulation of NKT cells with alpha-galactosylceramide (alpha-gal) at the time of BMT prevented chimerism and tolerance. Activation of recipient (as opposed to donor) NKT cells was necessary and sufficient for the alpha-gal effect. The detrimental effect of NKT activation was also observed in the absence of T cells after conditioning with in vivo T-cell depletion (TCD). NKT cells triggered rejection of BM via NK cells as chimerism and tolerance were not abrogated when NKT cells were stimulated in the absence of both NK cells and T cells. Thus, activation of NKT cells at the time of BMT overcomes the effects of CB, inhibiting the establishment of chimerism and tolerance.

Hurdles to the induction of tolerogenic mixed chimerism.

To date, organ transplant patients have to deal with the numerous side effects of life-long dependence on immunosuppressive drugs, whereas at the same time these drugs fail to prevent chronic rejection in many cases. Finding ways to establish donor-specific immunological tolerance thus remains one of the major goals in transplantation medicine. Tolerance through mixed chimerism can be achieved in rodents and in humans by the transplantation of hematopoietic stem cells. Widespread clinical application of this tolerance approach is, however, prevented by the toxicities of current bone marrow transplantation protocols in humans. Cytotoxic recipient conditioning and the hazard of graft-versus-host disease are unacceptable risks for organ transplant recipients. However, considerable progress has been made toward nontoxic conditioning regimens in animal studies. Translation of these findings into large animal models and the clinical setting is expected to be an important step toward broad clinical application of the mixed chimerism approach in organ transplantation.

Glutamate-evoked alterations of glial and neuronal cell morphology in the guinea pig retina.

Neuronal activity is accompanied by transmembranous ion fluxes that cause cell volume changes. In whole mounts of the guinea pig retina, application of glutamate resulted in fast swelling of neuronal cell bodies in the ganglion cell layer (GCL) and the inner nuclear layer (INL) (by approximately 40%) and a concomitant decrease of the thickness of glial cell processes in the inner plexiform layer (IPL) (by approximately 40%) that was accompanied by an elongation of the glial cells, by a thickening of the whole retinal tissue, and by a shrinkage of the extracellular space (by approximately 18%). The half-maximal effect of glutamate was observed at approximately 250 mum, after approximately 4 min. The swelling was caused predominantly by AMPA-kainate receptor-mediated influx of Na+ into retinal neurons. Similar but transient morphological alterations were induced by high K+ and dopamine, which caused release of endogenous glutamate and subsequent activation of AMPA-kainate receptors. Apparently, retinal glutamatergic transmission is accompanied by neuronal cell swelling that causes compensatory morphological alterations of glial cells. The effect of dopamine was elicitable only during light adaptation but not in the dark, and glutamate and high K+ induced strong ereffects in the dark than in the light. This suggests that not only the endogenous release of dopamine but also the responsiveness of glutamatergic neurons to dopamine is regulated by light-dark adaptation. Similar morphological alterations (neuronal swelling and decreased glial process thickness) were observed in whole mounts isolated immediately after experimental retinal ischemia, suggesting an involvement of AMPA-kainate receptor activation in putative neurotoxic cell swelling in the postischemic retina.

Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura.

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 auto-antibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity.