Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

AIMS: To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. METHODS: This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. RESULTS: Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessles. CONCLUSIONS: These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.     

Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

RPG1: an essential gene of Saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase

In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G₁ phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or α-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G₁ cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G₁ at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G₁ phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3. Received: 6 October 1997 / 8 November 1997     

Optic nerve atrophy and CT scanning: the target sign

Three patients are described who demonstrate the "target-sign" of optic nerve atrophy on CT scanning. This sign is produced by the centrally located atrophic optic nerve surrounded by a patulous perioptic subarachnoid space. An explanation for this phenomenon is proposed.     

Avoidance of “First-Pass” Elimination of Rectally Administered Propranolol in Relation to the Site of Absorption in Rats

The extent of first-pass elimination of racemic propranolol and dextropropranolol in doses of 0.25 or 0.50 mg was investigated in relation to the site of drug administration in the rectum of rats. The compounds were given orally, i.v., and rectally at distances of 2 and 1 cm from and directly at the anus by low volume zero-order 30 min infusion. Unchanged propranolol was determined in blood, and propranolol and three metabolites were measured in urine. The systemic availability of propranolol after oral administration was approximately 6 %. Rectal administration at 2 cm, at 1 cm and directly at the anus (0.2 cm) gave two, three and six times higher values, respectively. The more distal application site produced urinary metabolite profiles that were comparable to those observed after oral administration, while application directly at the anus was similar to i.v. dosing. In all experiments log-linear elimination phases with comparable elimination half-lives (range 12–18 min) were found, except with the 0.50 mg dose after i.v. and rectal administration close to the anus which showed a non-linear profile. The mean systemic availability after rectal administration of 0.25 mg dextro-propranolol close to the anus was 50 and 64 % as compared to a 0.25 and 0.125 mg i.v. dose, respectively. The rectal route may be used for propranolol to partially prevent hepatic first-pass metabolism. However, avoidance of presys-temic elimination is maximal only in the immediate vicinity of the anus as the venous blood supply of the upper part of the rectum of rats appears to be connected to the portal system and the lower part to the general circulation.     

Engineered NS1 for Sensitive,Specific Zika Virus Diagnosis from Patient Serology

Dengue virus (DENV) and Zika virus (ZIKV) belong to the Flaviviridae family of viruses spread by Aedes aegypti mosquitoes in tropical and subtropical areas. Accurate diagnostic tests to differentiate the 2 infections are necessary for patient management and disease control. Using characterized ZIKV and DENV patient plasma in a blind manner, we validated an ELISA and a rapid immunochromatographic test for ZIKV detection. We engineered the ZIKV nonstructural protein 1 (NS1) for sensitive serologic detection with low cross reactivity against dengue and developed monoclonal antibodies specific for the ZIKV NS1 antigen. As expected, the serologic assays performed better with convalescent than acute plasma samples; the sensitivity ranged from 71% to 88%, depending on the performance of individual tests (IgM/IgG/NS1). Although serologic tests were generally less sensitive with acute samples, our ZIKV NS1 antibodies were able to complement the serologic tests to achieve greater sensitivity for detecting early infections.     

Mitigating disparity in children with acute appendicitis: Impact of patient-driven protocols

PurposePrevious reports in the literature demonstrate racial and ethnic disparities for children diagnosed with acute appendicitis, with minorities experiencing worse outcomes. At our institution, we have developed an evidence based patient driven protocol for children following laparoscopic appendectomy. However, the influence of such protocol on mitigating racial and ethnic disparities in outcomes remains unknown. The purpose of our study is to assess the impact of our protocol by evaluating the influence of race and ethnicity on surgical outcomes among children treated for acute appendicitis.Material and methodsA retrospective review of prospectively collected data was conducted. Children undergoing a laparoscopic appendectomy at our freestanding children's hospital between December 2015 and July 2017 were included. Demographic data, post-operative length of stay, same day discharge rates and hospital readmission rates were abstracted from patient medical records. Patients were classified by their race and ethnic background. Comparative analysis was performed in STATA with a p value < .05 determined as significant.ResultsA total of 786 children were included, with the majority being either White (70%, n = 547), Black (8%, n = 62) or Hispanic (17%, n = 133); 569 patients (72%) were found to have non-perforated appendicitis. There was no statistically significant difference in the rates of same day discharge among White, Black or Hispanic children respectively (88% vs. 77% vs. 86%, p = .126). Of the 217 children with perforated appendicitis, Hispanic children had increased rates of perforation (41%, n = 55) compared to White and Black children respectively (23%, n = 128 and 29%, n = 18, p = .001). However, average post-operative length of stay were similar among White, Black and Hispanic children (96 h vs. 95 h vs. 98 h, p = .015). On multivariate analysis, the only significant risk factor for an elevated post-operative length of stay was the presence of a perforation.ConclusionOur evidence based patient driven protocol effectively mitigates racial and ethnic disparities found in children with acute appendicitis. Further prospective investigation into the role of such patient-driven protocols to mitigate healthcare disparities is warranted.Levels of EvidenceTherapeutic study; Level 3.     

Cardiopulmonary bypass

The purpose of cardiopulmonary bypass is to maintain perfusion and oxygenation of the vital organs in the absence of heart and lung function, usually to facilitate surgery on the heart, but occasionally in other situations. Although the intricacies of the modern extracorporeal circuit and the conduct of cardiopulmonary bypass are the domain of the clinical perfusion scientist (‘perfusionist’), safe surgery mandates a good understanding of some fundamentals by the anaesthetist and the surgeon. This review is aimed at the anaesthetist. First, we will systematically examine the main components of the extracorporeal circuit, travelling in the direction that blood travels, from the venous cannula to the arterial cannula. Then we will describe the process of preparing for bypass, ‘going on’, conducting a bypass run, and weaning and separation from bypass. It is crucial to have clear communication between the surgeon, perfusionist and anaesthetist. This can be difficult for the novice because a quite specific language has evolved in cardiac operating theatres to signal key events in the cardiopulmonary bypass sequence. As we go through this article, we will highlight commonly used terminology and expressions used.