T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

Tunable PEGylation of branch-type PEI/DNA polyplexes with a compromise of low cytotoxicity and high transgene expression: in vitro and in vivo gene delivery.

Although PEGylated polyplexes for gene delivery are widespread, there is a need for an in-depth investigation of the role of the PEGylation degree on the delivery efficiency of the systems. For this, a low-toxicity series of polymers for gene delivery were designed via Michael addition of poly(ethylene glycol)methyl ether methacrylate (PEGMA) onto branched polyethylenimine PEI. The goal was to finely tune the PEGylation degree in order to determine the system offering the best compromise between low cytotoxicity and high transfection efficiency under both in vitro and in vivo conditions. From dynamic light scattering tests, zeta potential measurements and gel retardation assay, it was found that nanoparticle assembly of PEI-g-PEGMA and DNA exhibited stable complex formation when the PEGylation degree was below 2.9%. In addition, complexes formed from polymers with a PEGylation degree of at least 1.67% (from PEI-g-PEGMA-6 to PEI-g-PEGMA-18) all showed very low hemolysis activity. Transfection efficiencies of the prepared complexes were determined using the pEGFP-C3 vector and ß-galactosidase. Complexes made of PEI-g-PEGMA-6 and PEI-g-PEGMA-10 at a polymer nitrogen/DNA phosphorus weight ratio (Wn/Wp) of 5 led to the best transfection efficiencies. Moreover, PEGylation ensured low cytotoxicity of the complexes in particular at high Wn/Wp ratios. In vivo tests in a mouse model confirmed the in vitro results obtained for PEI-g-PEGMA-6-based complexes, at all Wn/Wp ratios tested, but also showed that a high PEGylation degree (5.2% for PEI-g-PEGMA-18), though inefficient in vitro could still lead to successful delivery in vivo, due to a prolonged contact time between the complex and the cells, and to the change in the biological environment. Overall, provided a fine tuning of the grafting density of PEGMA onto PEI and the polymer nitrogen/DNA phosphorus weight ratio, our results prove that PEI-g-PEGMA polymers constitute an efficient platform for successful in vitro and in vivo gene delivery, and ensure low cytotoxicity and prolonged cell viability.

Predicting children's behaviour during dental treatment under oral sedation.

AIMS: The primary aim of this study was to assess whether parents' own anxiety and their perception of their child's dental fear and child's general fear can predict preoperatively their child's behaviour during dental treatment under oral sedation. The secondary aim was to assess whether the child's age, gender and ASA classification grade are associated with a child's behaviour under oral sedation. STUDY DESIGN: Cross-sectional prospective study. METHODS: The Corah's Dental Anxiety Scale (DAS), Children's Fear Survey Schedule Dental-Subscale (CFSS-DS) and Children's Fear Survey Schedule Short-Form (CFSS-SF) questionnaires were completed by parents of children undergoing dental treatment with oral midazolam. Behaviour was rated by a single clinician using the overall behaviour section of the Houpt-Scale and scores dichotomised into acceptable or unacceptable behaviour. Data were analysed using χ (2), t test and logistic regression analysis. RESULTS: In total 404 children (215 girls, 53 %) were included, with the mean age of 4.57 years, SD = 1.9. Behaviour was scored as acceptable in 336 (83 %) and unacceptable in 68 (17 %) children. STATISTICS: The level of a child's dental fear, as perceived by their parent, was significantly associated with the behaviour outcome (p = 0.001). Logistic regression analysis revealed that if the parentally perceived child's dental fear (CFSS-DS) rating was high, the odds of the child exhibiting unacceptable behaviour under oral sedation was two times greater than if their parents scored them a low dental fear rating (OR 2.27, 95 % CI 1.33-3.88, p = 0.003). CONCLUSIONS: CFSS-DS may be used preoperatively to help predict behaviour outcome when children are treated under oral sedation and facilitate treatment planning.

Macrophage migratory inhibitory factor (MIF) expression in acute graft-versus-host disease (GVHD) in allogeneic hemopoietic stem cell transplant recipients.

Graft-versus-host disease (GVHD) is a major complication after hemopoietic stem cell transplantation (HSCT), but its pathogenesis remains uncertain. Macrophage migratory inhibitory factor (MIF) is an important mediator in the allo-immune reaction during renal transplantation, yet its role in hemopoietic stem cell transplantation (HSCT) remains unexplored. This study investigated the potential role of MIF in acute graft-versus-host disease (aGVHD) following allogeneic HSCT. Forty-six randomly selected patients undergoing autologous or allogeneic HSCT were studied. Immunohistochemistry and in situ hybridization were performed to examine tissue MIF mRNA and protein expression on skin and colonic biopsy specimens. The associated T cell and macrophage activation was also studied by immunohistochemical studies. A semi-quantitative method was used to assess MIF staining, as well as T cell and macrophage staining. Serial blood samples were analyzed by ELISA for serum MIF levels. Immunohistochemistry and in situ hybridization performed in 15 skin and 19 colonic biopsies from 17 patients who developed moderate to severe aGVHD showed a significant increase in MIF mRNA and protein expression compared with normal controls (seven skin and five colonic biopsies). MIF was localized within the epidermis and the vascular area of skin, but diffusely expressed in the entire thickness of colon. Macrophage and T lymphocyte infiltration was confined to areas of strong MIF expression. Serial analysis by ELISA showed that only patients who developed aGVHD (n = 19) exhibited an increase (two- to three-fold) in serum MIF during HSCT, but not in the allogeneic HSCT recipients without aGVHD (n = 7) or those who received autologous HSCT (n = 8). In 14 out of 19 patients, serum MIF peaked before the onset of aGVHD. Local and systemic up-regulation of MIF expression is associated with the occurrence of acute GVHD. Its pathogenetic role remains to be further determined.

A permanent record of antibiotic susceptibility results (disk method) using a Xerox photocopying machine.

A simple way to produce permanent antibiotic disk susceptibility and radioimmunodiffusion assay records with the aid of a photocopy machine and a flat mirror is described. Advantages and limitations of the procedure are discussed.

Actin-positive spindle cell lymphoma.

We report an unusual case of malignant lymphoma, large noncleaved B-cell type, exhibiting sarcomatoid and myxoid patterns and strong positive staining for muscle-specific actin. Despite vigorous chemotherapy, the 64-year-old male patient, who had lymphoma involving his right inguinal area, retroperitoneum, and anterior chest wall at the time of presentation, died 3 months later.

Liver transplantation in children: the experience of Queen Mary Hospital, Hong Kong.

Seven living-related liver transplants (LRLT) and two reduced-size liver transplants (RSLT) were performed on eight children who suffered from end-stage liver disease, having previously undergone one to three abdominal operations. Their ages at initial transplantation ranged from 8 months to 11 years (mean 35 months, median 12 months). Excluding the two older children aged 7 and 11 years, respectively, the rest of the children weighed 6 to 9.5 kg (mean 7.3 kg) at the time of the initial transplantation. Seven left lateral segments (S2 + 3) and two left lobes (S2 + 3 + 4) were used; of these the smallest graft had a graft-to-recipient body weight ratio of 0.9%. The volunteer living donors were four mothers, two fathers and one sister who were selected after medical and psychiatric evaluations, and their suitability was confirmed by hematological, biochemical, and radiological criteria. During a follow-up period of 3 to 30 months, all eight children are alive and well with normal liver function, one of them having undergone a retransplant LRLT because of hepatitis of undetermined etiology following a RSLT 1.5 years earlier. All seven donors had an uneventful postoperative course and were discharged on day 4 to 7 postoperatively. They have all resumed normal day-to-day activities. There were no complications in the donor group. A variety of complications occurred in the recipients, all of which were overcome. Operating microscope was used to perform all the arterial anastomoses using microvascular techniques. This method has proven to be a major factor in preventing arterial thrombosis even with the smallest of arterial anastomosis where a 1.5-mm diameter recipient artery was anastomosed to a 2.5-mm diameter donor hepatic artery.

Postoperative spindle-cell nodule of urinary bladder with unusual intracytoplasmic inclusions.

Postoperative spindle-cell nodule of the urinary bladder (or in the absence of previous surgical procedure, inflammatory pseudotumor) is a reactive process of unknown etiology mimicking a sarcoma. Intracytoplasmic inclusion bodies were noted inside the atypical spindle cells on Diff-Quik stained smears in a recent case of this entity. Although fluorescence was noted in the inclusion bodies with monoclonal antibodies against the major outer membrane protein of the Chlamydia species, the exact nature of these inclusion bodies remains unknown.

Simple BASIC program for calculating the cervicovaginal FNP and for estimating the sample size of the number of cervicovaginal smears to be rescreened.

OBJECTIVE: To guide cytotechnologists and pathologists in calculating the false negative proportion, or rate, and the number of Papanicolaou smears to be reevaluated for a meaningful assessment of screening performance, a computer program written in BASIC was prepared, based on several recent publications in the field of cytopathology. RESULTS: A complete program listing and sample runs to help users be cognizant of the necessary inputs to run the program. The output from the program gives the results of the various calculations. CONCLUSION: Since the tedious manual calculations are handled by the computer program, it is more likely for those involved in the interpretation of Papanicolaou smears to follow the approaches suggested by experts in these two areas.

Cancer pain management: a recent experience by anaesthesiologists in a teaching hospital in Hong Kong.

More than 60% of cancer patients suffers from unbearable pain, especially towards their terminal stages. Anaesthesiologists are involved in cancer pain management because of their expertise in analgesic pharmacology and neurolytic procedures. This manuscript reported on the experience of treating cancer pain in Chinese patient in Hong Kong with reference to current literature in other parts of the world. One hundred and sixty two Chinese patients were referred from other specialists to the Department of Anaesthesiology, Queen Mary Hospital for further management because of their cancer pain control were considered difficult. Upon referral, the mean visual analogue scale of pain (VAS) was 5.8 +/- 2.7. The pain caused insomnia (66.7%) and appetite loss (45%) as well. By far most (80%) patients' pain were successively controlled with oral systemic analgesics. These were prescribed in form of a combination of NSAID (72.2%), potent opioids (76.5%) and co-analgesics (21.6%). In our series, the mean oral morphine (MS Continus) requirements was 96.0 +/- 68.3 mg on discharge. Frequent nausea and constipation persisted in 16.0% and 8.0% respectively despite active treatment with anti-emetics and laxatives. Twenty eight neurolytic blocks was performed in 22 (13.6%) patients. Good pain relief was achieved in 78.6%. Overall speaking most patients (90.7%) were able to achieve adequate analgesia before death.

The development of hypercoagulability state, as measured by thrombelastography, associated with intraoperative surgical blood loss.

Thrombelastographic evidence of hypercoagulability, including shortening of r-time (P < 0.01); shortening of k-time (P < 0.01); and widening of trace angle (P < 0.01) were observed in a group of 21 Chinese surgical patients when (a) the amount of blood loss was at an estimated 10% of total blood volume and (b) the amount of blood loss was at an estimated 15% of blood volume. The amount of blood loss was documented by haemoglobin measurements. No evidence of hypercoagulability was observed at around one hour into the operation in the absence of bleeding. We conclude that a mild to moderate degree of surgical blood loss with haemodilution is associated with the development of hypercoagulability as measured by thrombelastography. Further studies looking at the thrombebolic outcome in such groups of patients is warranted. It is also suggested that caution should be exercised in the use of intraoperative isovolaemic haemodilution until the phenomenon is further investigated.

Photostatic copying of immunoelectrophoretic patterns with the use of color print filters.

Immunoelectrophoretograms can be replicated inexpensively, rapidly, and to scale by the use of color print filters on a photostatic copy machine. By this method additional copies are readily available for patient charts, permanent record, and any other use.

A BASIC data management program for clinical laboratory audits.

Presented in this article is a simple computer program written in BASIC for audit purposes. Although it is developed with a DEC PDP-11 microcomputer, the program can be modified for other microcomputers. Shorter and less versatile than most commercially available data management programs, it is suitable for the type of audit functions likely to be encountered in clinical laboratories.

Cystadenoma of the pancreas. An ultrastructural study.

A case of cystadenoma of the pancreas was studied at the light (LM) and the ultrastructural (EM) levels. The observation made under LM that the flattened epithelial cells lining the cystic spaces were similar to the centroacinar cells of a normal pancreas was supported by EM findings. Numerous glycogen granules were demonstrated in the cells, a feature characteristic of fetal but not adult centroacinar cells. A few epithelial cells contained occasional membrane bound dense granules of unknown nature. This observation and the finding of normally appearing islets of Langerhans raise the possibility that this may be an example of a mixed insular-duct cystadenoma. However, if the islets of Langerhans were considered entrapped structures, this tumor should be labelled as a simple serous cystadenoma.

Change in vascular cAMP and cGMP contents in portal hypertensive rats.

The purpose of this study was to investigate the possible changes of cyclic nucleotide contents in portal hypertensive rats. Portal hypertension was induced by partial portal vein ligation (PVL) in Sprague-Dawley rats. Sham-operated rats served as controls. Hemodynamic and cyclic nucleotide measurements were performed at 14 days after surgery. The portal venous pressure was significantly higher, while systemic arterial pressure and heart rate were lower in PVL rats than those in controls. Basal cAMP (PVL, 10.91 +/- 0.98, vs. sham, 8.08 +/- 0.81 pmol/mg protein) and cGMP (PVL, 0.91 +/- 0.12, vs. sham, 0.59 +/- 0.05 pmol/mg protein) contents in the tail artery were significantly higher in PVL rats. Isobutyryl methylxanthine (10(-5) M), a nonspecific phosphodiesterase inhibitor, exerted similarly stimulating effects on the tissue cGMP (PVL, 158 +/- 10, vs. sham, 178 +/- 20%) and cGMP (295 +/- 28 vs. 316 +/- 71%) levels in both PVL and control rats; so did forskolin (10(-6) M) on the cAMP (184 +/- 20 vs. 197 +/- 66%) content in both groups. Our results showed that the arterial cAMP and cGMP contents were higher in PVL rats, which may contribute to the reduction of peripheral resistance in portal hypertension.