The use of NaOCl in combination with CHX produces cytotoxic product

Objectives

The combination of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) yields a “precipitate potentially toxic” (PPT). The aim of this study was to evaluate the tissue response to implanted polyethylene tubes filled with PPT-soaked fibrin sponge.

Methods

Forty rats received four polyethylene tubes each; each tube was filled with fibrin sponge soaked by 2.5 % NaOCl, 2.0 % CHX, PPT (2.5 % NaOCl plus 2.0 % CHX), or not soaked (control). The observation time points were 7, 15, 30, 60, and 90 days. At each time point, eight animals were killed, and the tubes and surrounding tissues were removed, fixed, and prepared for light microscopic analysis by performing glycol methacrylate embedding, serial cutting into 3-μm sections, and hematoxylin–eosin staining. Qualitative and quantitative evaluations of the reactions were performed. Results were statistically analyzed by Kruskal–Wallis test (p?<?0.05).

Results

All chemical solutions caused moderate reactions at 7 days. On day 30, PPT group was more cytotoxic than the control group and the CHX group (p?<?0.05). On days 15 and 60, PPT group was more cytotoxic than the control group (p?<?0.05). On day 90, there was no statistically significant difference between the different groups.

Conclusion

PPT is more cytotoxic than NaOCl and CHX alone, particularly in the short term.

Clinical significance

Protocols which suggest the use of CHX and NaOCl must be revised because this mixture produces cytotoxic product.     

Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation

Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (T_FH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the T_FH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.Following infection, T-cell receptor (TCR) interactions with foreign peptide/MHC (pMHC) drive the rapid clonal expansion and differentiation of T cells into distinct effector subsets specialized against different classes of microbes. An early bifurcation in CD4 T-cell responses results in the generation of T helper (Th)1 effectors, which regulate innate cell microbicidal function and follicular helper T (T_FH) cells, which migrate to B-cell follicles to regulate germinal center (GC) responses and antimicrobial antibody production (1). After pathogen is cleared, T cells undergo a contraction phase during which the majority of effectors die by apoptosis, leaving behind a population of long-lived memory cells to provide protection upon subsequent reinfection. The decision to differentiate into Th1 and T_FH lineages appears to occur very early in the immune response (2, 3). Initial T-cell priming by dendritic cells (DCs) is sufficient to induce fate-committed Th1 and T_FH cells as early as 3 d after infection, whereas maintenance and further expansion of the T_FH compartment depends on T-cell interactions with B cells (2). Similarly, memory T-cell differentiation occurs very early after infection and is critically dependent on B-cell interactions for optimal priming (4, 5). Importantly, CD4 T-cell differentiation is coupled to division, and unlike CD8 T-cell differentiation, requires constant antigen recognition (6, 7).Although the strength of TCR–pMHC interactions has been shown to directly modulate T-cell expansion and clonal dominance within the Th cell compartment (8, 9), how this influences CD4 T-cell fate is not well understood. Cumulative TCR signaling can be influenced by both antigen affinity and antigen dose (10). In terms of proliferation, higher antigen dose can compensate for lower antigen affinity to some extent, but several reports have shown independent effects on T-cell responses both in vitro and in vivo (10–12). These data indicate that antigen affinity and antigen dose may promote qualitatively distinct TCR signals. Recently, modulation of the overall TCR signal by varying either TCR affinity or antigen dose was shown to influence the pattern of effector T-cell differentiation, with higher affinity ligands or higher antigen dose promoting T_FH generation (13–15). However, another study examining high and low avidity CD4 T-cell responses during viral infection found significant differences in Th1 but not T_FH generation (16). Sustained TCR–pMHC interactions have also been shown to promote memory T-cell differentiation, which is associated with increased TCR avidity (17, 18). These studies, however, have focused on the development of the Th1 memory compartment, which is phenotypically and functionally distinct from the T_FH memory compartment (19, 20). Thus, although strong TCR signals resulting from high antigen affinity or high antigen dose can clearly affect the extent and quality of T-cell differentiation, whether or not T cells can discriminate these signals, and how this contributes to T-cell differentiation during infection, has not been determined.To address this question, we infected mice with varying concentrations of Listeria expressing either high or low affinity antigens for the TCR. By normalizing the degree of proliferation induced by high and low affinity antigens we were able to discern distinct influences of antigen affinity and antigen dose on Th cell differentiation. We observed a strong positive correlation between antigen affinity and Th1 differentiation that occurs early and is dose independent. Importantly, high antigen dose does not compensate for the low efficiency of Th1 differentiation induced by low affinity antigen. In contrast, early T_FH effector generation was observed after priming with high, intermediate, and low affinity antigen, but was not maintained at later time points under conditions of low antigen dose. In addition, we found that T cells activated by either high or low affinity antigen are equally capable of memory T-cell differentiation. Surprisingly, memory T cells generated by either low antigen affinity or low antigen dose maintained their biased effector lineages following recall activation with high affinity antigen. These data indicate that differential strength of stimulation during primary T-cell activation can imprint unique and long lasting T-cell differentiation programs.     

Laparoscopic repair of perforated peptic ulcer: single-center results

Background

Perforated peptic ulcer (PPU), the most common indication for emergency gastric surgery, is associated with high morbidity and mortality rates. Outcomes might be improved by performing this procedure laparoscopically, but no consensus exists on whether the benefits of laparoscopic repair (LR) of PPU outweigh the disadvantages.

Methods

From January 2002 to December 2012, 111 patients underwent surgery for perforated ulcer. A “laparoscopy-first” policy was attempted and then applied for 56 patients. The exclusion criteria for LR ruled out patients who had shock at admission, severe cardiorespiratory comorbidities, or a history of supramesocolic surgery. The aim of this study was a retrospective analysis of the 56 patients treated laparoscopically.

Results

The patient distribution was 30 men and 26 women, who had a mean age of 59 years (range 19–95 years). The mean ulcer size was 10 mm, and the Mannheim peritonitis index (MPI) was 21. LR was performed for 39 (69.6 %) of the 56 patients and included peritoneal lavage, suturing of the perforation, and omental patching. Conversion to laparotomy was necessary in 17 cases (30.4 %). The “conversion group” showed significant differences in ulcer size (larger ulcers: 1.9 vs 0.7 mm; p < 0.01), ulcer-site topography (higher incidence of posterior ulcers: 5 vs 0; p < 0.01), and MPI score (higher score: 24 vs 20; p < 0.05). The LR group had a mean operating time of 86 min (range 50–125 min), an in-hospital morbidity rate of 7.6 %, a mortality rate of 2.5 %, and a mean hospital stay of 6.7 days (range 5–12 days). None of these patients required reintervention.

Conclusions

The results showed that LR for PPU is feasible with acceptable mortality and morbidity rates. Skill in laparoscopic abdominal emergencies is required. Perforations 1.5 cm or larger, posterior duodenal ulcers, and an MPI higher than 25 should be considered the main risk factors for conversion.     

Computerunterstütztes „ring sizing“ für die Anuloplastie

Background

Current methods to determine the size of an annuloplasty ring for mitral valve reconstruction are semi-quantitative and dependent on the subjective assessment by the surgeon which requires a high degree of professional experience.

Aim

Computer-assisted ring sizing supports the surgeon with a quantitative measurement tool for an objective ring selection.

Material and methods

A fast modeling method for the annulus of the mitral valve was developed, which is easy to integrate into the clinical workflow. It enables precise measurement and preoperative deformation analysis of annuloplasty rings. Additionally, ring implants can be created based on the patient’s individual anatomy.

Results

Computer-assisted modeling and measurement tools enable an objective and reproducible ring selection. The possibility of manufacturing patient-specific designed rings can lead to a novel patient-centered treatment.     

Growth of children with end-stage renal disease undergoing daily hemodialysis

Background

The aim of this report is to describe the effect of daily hemodialysis on the growth of children with end-stage renal disease (ESRD).

Methods

We performed a prospective, observational study on 24 children with ESRD undergoing daily hemodialysis (DHD). The control group comprised 26 children on concurrent conventional hemodialysis (CHD), and the follow-up for both groups was 9.3?±?3.0 months. No patient received growth hormone (GH) therapy.

Results

At the onset of the study, the height-for-age Z-score was ?2.12?±?1.54 in the CHD group and ?2.84?±?2.27 in the DHD group (p?=?0.313). Assuming an increase of 0.5 standard deviation scores (SDS) of the height-for-age parameter as an improvement of growth, there were 33 % of patients in the DHD group and 8 % in the CHD group (p?=?0.035). The cumulative probability of gain in height for age at 12 months was 40 % in the DHD group versus 15 % in the CHD group (p?=?0.047). Also, 98 % of patients in the DHD group had an adequate total caloric intake, whereas 38 % in the CHD group reached this goal (p?<?0.001). No patient left the study due to intensification of the dialysis modality.

Conclusions

Our data show that the DHD favored a 0.5 SDS height gain in a third of patients without GH treatment. Dialysis intensification was not a cause for treatment dropouts, and DHD should be considered as a treatment for selected cases, especially small children.     

Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy

Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qβ-derived virus-like particles (Qβ–Fel d1). Qβ–Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcγRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcγRIIb. In addition, treatment with Qβ–Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qβ–Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qβ–Fel d1 may be a safe and effective treatment for cat allergy.Allergic reactions are associated with several hypersensitivity diseases including asthma, rhinoconjunctivitis, contact dermatitis, urticaria, anaphylaxis, and insect, drug, and food allergy. These diseases can affect all age groups and have reached epidemic proportions worldwide with increasing incidence over the last decades (Holgate, 1999). The most common forms of allergies, such as pollen, house dust, or animal dander allergies, are dependent on type 2 T cell responses (Georas et al., 2005), leading to the generation of IL-4 and IgE. IgE antibodies have a short half-life in serum but are stable if bound to Fcε receptors on circulating basophils and, in particular, tissue mast cells (Vieira and Rajewsky, 1988). Cross-linking of the IgE–FcεI receptor complex on these cells by allergen leads to degranulation within seconds, liberating a variety of preformed inflammatory mediators. The clinical effects attended by such allergic reactions vary according to the site of basophil and mast cell activation. Although inhalation or ingestion of allergens activates mucosal mast cells, i.v. or s.c. antigen entry activates circulating basophils and connective tissue mast cells.Most current therapies for the treatment of allergies block mast cell effector molecules (e.g., histamines) or nonspecifically suppress immune responses (e.g., steroids). Although effective, these treatments fail to affect the immunological conditions causing the allergies. In addition, different vaccination or desensitization strategies have been investigated, including the usage of allergen-derived peptides (Francis and Larché, 2005), recombinant hypoallergenic derivates (Niederberger et al., 2004; Saarne et al., 2005), oligonucleotides with CpG motives (Hessel et al., 2005), or allergen conjugated to carbohydrate-based particles (Andersson et al., 2004). However, only a few disease-modifying therapies for allergy have been approved so far. These immunotherapies consist of either multiple s.c. injections of increasing doses of allergen or multiple sublingual or oral administration of the allergen, resulting in long-term desensitization (Till et al., 2004). Although these allergen-specific immunotherapies have shown successes (Durham and Till, 1998), the procedures are time consuming and require 1–3 yr of regular treatments (Hedlin et al., 1986, 1991). Moreover, this therapy bears a high risk for anaphylactic reactions, especially after administration of higher allergen doses (Cox and Coulter, 1997; Bousquet et al., 1998).The mechanism by which this specific immunotherapy affects allergies is poorly understood. The reduction in allergic symptoms by this treatment has been hypothesized to be at least partly mediated by a shift from Th2 toward a Th1 response or the induction of regulatory T (T reg) cells (Akdis et al., 2005). Alternatively, and not mutually exclusive, the balance between allergen-specific IgE and IgG antibodies may regulate mast cell and basophil activity. In fact, Hulett et al. (1993) identified an FcR, with a binding site for IgG, which regulates high affinity IgE receptor–mediated mast cell activation (Daëron et al., 1995b). It is well established that the FcγRIIb (FcγIIb receptor), expressed on mouse and human basophils and mast cells (Bischoff, 2007), can down-regulate FcεRI signaling by cross-linking of the FcεRI–IgE and FcγRIIb–IgG-Ag complex (Daëron et al., 1995a; Katz, 2002; Bruhns et al., 2005; Kraft and Kinet, 2007; Nimmerjahn and Ravetch, 2008). Indeed, a fusion molecule between the cat allergen Fel d1 and the constant part of IgG1 has recently been shown to inhibit allergic symptoms by cross-linking of FcγRIIb with FcεRI in a mouse model of asthma (Zhu et al., 2005; Terada et al., 2006). In addition to signaling through FcγRIIb, allergen-specific IgG antibodies may sequester allergens and hence prevent their binding to IgE–FcεRI complexes. Moreover, it has been shown that the ratio of allergen-specific IgE and IgG antibodies may affect presentation of allergen-derived epitopes to T cells (Wachholz and Durham, 2004). Thus, allergen-specific antibodies may have multiple ways to modulate allergic responses.We have previously shown that antigens displayed in a repetitive fashion on virus-like particles (VLPs) derived from the coat protein of the bacteriophage Qβ are highly immunogenic in mice (Jegerlehner et al., 2002a,b; Lechner et al., 2002; Spohn et al., 2005) and humans (Maurer et al., 2005; Kündig et al., 2006; Ambühl et al., 2007; Tissot et al., 2008). Because bacterial host RNA is incorporated into the VLPs during self-assembly inside bacteria, Qβ-VLPs provide Toll-like receptor ligands, which induce strong IgG2a/c-dominated antibody responses (Forsbach et al., 2007, 2008; Jegerlehner et al., 2007). In the present study, the major cat allergen Fel d1 was coupled in an oriented fashion to Qβ-VLPs, resulting in a highly repetitive form of the allergen. This vaccine was strongly immunogenic in mice, yielding high and long-lasting antigen-specific serum IgG titers after only a single immunization. Vaccination with Qβ–Fel d1 resulted in strongly reduced immediate type I allergic responses in a mouse model of mast cell degranulation, vascular leakage, and anaphylaxis and completely abolished IgE B cell memory responses upon antigen recall. Strikingly, coupling of Fel d1 to Qβ-VLPs essentially abrogated its ability to induce allergic responses in mice or the degranulation of human basophils derived from allergic individuals. Thus, displaying Fel d1 on VLPs enhanced its immunogenicity and therapeutic efficacy while strongly reducing its reactogenicity.     

Frequency of MART-1/MelanA and gp100/PMel17-specific T cells in tumor metastases and cultured tumor-infiltrating lymphocytes

Melanoma differentiation antigens, such as MART-1/MelanA and gp100/PMel17, frequently are observed as targets of tumor infiltrating lymphocytes (TIL) originated from HLA-A*0201-expressing patients with melanoma. Furthermore, particular clinical relevance was attributed to gp100/pMel17 based on the impression that the adoptive transfer of gp100-recognizing TIL was associated with clinical responses in a small group of patients. However, the actual frequency of specific T cells for these melanoma differentiation antigens has never been directly enumerated in ex vivo or in vitro expanded TIL cultures. Here, we enumerated melanoma differentiation antigen-specific T-cell precursor frequency in TIL using tetrameric HLA/epitope complexes, functionally characterizing their responsiveness to cognate epitope by cytokine release assay. T-cell precursor frequencies were enumerated in 11 fresh-tumor preparations and 17 TIL adoptively transferred into patients bearing HLA-A*0201. MART-1 or gp100-specific T cells could be detected respectively in 5 and 2 of the 11 fresh preparations and in 5 and 2 of the 17 adoptively transferred TIL. With one exception, melanoma differentiation antigen-specific T-cell precursor frequency in fresh material and TIL ranged between 5,000 to 21,000/10(6) CD8+ T cells. T-cell precursor frequency was not significantly higher in TIL whose administration was associated with clinical response. These data provide direct enumeration of MART-1/MelanA and gp100/pMel17 reactivity ex vivo and in vitro in the context of HLA-A*0201.