人巨细胞病毒PPUL44蛋白单克隆抗体CH13抗原决定簇位点的确定

目的 :确定人巨细胞病毒 PPU L44蛋白单克隆抗体 CH13在 PPU L44上的抗原决定簇位置 ,探讨随机多肽文库在研究相互作用蛋白质的氨基酸序列方面的应用。方法 :用 CH13在一个随机多肽文库中筛选出能与CH13结合的克隆 ,测定上述克隆随机多肽的 DNA序列 ,用随机多肽的氨基酸同源序列与 PPUL44蛋白质的氨基酸序列比较。结果 :能与 CH13结合的随机多肽的氨基酸序列显示出高度的一致性 ;随机多肽同源序列NEGEAFGPDVSG与 PPUL44蛋白位于第 32 1～ 332的氨基酸序列高度同源 ;而随机多肽序列 FQIL VCVESVL R与 PPU L44位于第 181～ 184的氨基酸序列有 4个相邻的氨基酸一致。结论 :CH13的抗原决定簇位于 PPU L44蛋白第 32 1～ 332氨基酸序列附近 ;CH13与 PPU L44位于第 181～ 184氨基酸序列可能有一定反应。随机多肽文库在研究相互作用蛋白质的氨基酸序列方面具有广泛的应用前景。     

Lung oxidative stress as related to exercise and altitude. Lipid peroxidation evidence in exhaled breath condensate: a possible predictor of acute mountain sickness

Lung oxidative stress (OS) was explored in resting and in exercising subjects exposed to moderate and high altitude. Exhaled breath condensate (EBC) was collected under field conditions in male high-competition mountain bikers performing a maximal cycloergometric exercise at 670 m and at 2,160 m, as well as, in male soldiers climbing up to 6,125 m in Northern Chile. Malondialdehyde concentration [MDA] was measured by high-performance liquid chromatography in EBC and in serum samples. Hydrogen peroxide concentration [H₂O₂] was analysed in EBC according to the spectrophotometric FOX₂ assay. [MDA] in EBC of bikers did not change while exercising at 670 m, but increased from 30.0±8.0 to 50.0±11.0 nmol l⁻¹ (P<0.05) at 2,160 m. Concomitantly, [MDA] in serum and [H₂O₂] in EBC remained constant. On the other hand, in mountaineering soldiers, [H₂O₂] in EBC under resting conditions increased from 0.30±0.12 μmol l⁻¹ at 670 m to 1.14±0.29 μmol l⁻¹ immediately on return from the mountain. Three days later, [H₂O₂] in EBC (0.93 ±0.23 μmol l⁻¹) continued to be elevated (P<0.05). [MDA] in EBC increased from 71±16 nmol l⁻¹ at 670 m to 128±26 nmol l⁻¹ at 3,000 m (P<0.05). Changes of [H₂O₂] in EBC while ascending from 670 m up to 3,000 m inversely correlated with concomitant variations in HbO2 saturation (r=−0.48, P<0.05). AMS score evaluated at 5,000 m directly correlated with changes of [MDA] in EBC occurring while the subjects moved from 670 to 3,000 m (r=0.51, P<0.05). Lung OS may constitute a pathogenic factor in AMS.     

Detection of precursor Th cells in mesenteric lymph nodes after oral immunization with protein antigen and cholera toxin

We have characterized the earliest antigen-specific Th cells in murine mesenteric lymph nodes (MLN), following oral immunization with the hen egg lysozyme (HEL) as antigen and cholera toxin (CT) as adjuvant. We did this by analyzing in vitro proliferation and cytokine production in response to HEL by the MLN T cells. MLN cells taken 5 days after a single oral immunization with HEL and CT provided the earliest source of proliferating HEL-specific T cells. This proliferation was completely inhibited by anti-IL-2, but not inhibited by anti-IL-4 antibody. IL-2 protein was detected in culture supernatants but not IL- 4 using ELISA or bioassays. IL-4 mRNA was not found in responding cells using RT-PCR. Some of the day 5 MLN cultures produced IFN-gamma in response to HEL, but isolated T cells from the same MLN did not. Exogenous IL-4 alone did not stimulate day 5 MLN T cells, but IL-4 did synergize with HEL to induce a large proliferative response. The data indicate that the HEL-specific CD4 T cell pool in MLN 5 days after oral immunization is composed of undifferentiated precursor Th cells. These cells have the potential for IL-2 production and IL-4R expression upon re-stimulation in vitro.      

A novel human homologue of yeast nucleosome assembly protein, 65 kb centromeric to the p57KIP2 gene, is biallelically expressed in fetal and adult tissues

Three genes on 11p15.5 are known to undergo genomic imprinting. The gene for insulin-like growth factor II (IGF2) is normally expressed from the paternal allele, while H19 and p57KIP2, a cyclin-dependent kinase inhibitor, are expressed from the maternal allele. Five germline balanced chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS) have been mapped to 11p15.5 between p57KIP2 and IGF2, and all are derived from the maternal chromosome. By positional cloning from BWS breakpoints, we have isolated a gene 100 kb and 65 kb centromeric to the proximal end of this BWS breakpoint cluster and p57KIP2, respectively. This gene is homologous to yeast nucleosome assembly protein (NAP1) and to a human homologue of NAP1, and we designate it hNAP2 (human nucleosome assembly protein 2). hNAP2 diverges in its expression pattern from IGF2, H19, and p57KIP2, and it shows biallelic expression in all tissues tested. Thus, hNAP2 is functionally insulated from the imprinting domain of 11p15.      

Accuracy of a multidisciplinary team-led discussion in predicting postmastectomy radiotherapy

Introduction

Immediate breast reconstruction (IBR) is performed increasingly following mastectomy for breast cancer. The literature suggests higher reconstructive failure and poorer cosmesis in the subgroup of patients receiving postmastectomy radiotherapy (PMRT) following IBR. We set out to determine the accuracy of a multidisciplinary team (MDT) discussion in predicting PMRT.

Methods

Preoperative MDT discussions were recorded prospectively over a 12-month period (from February 2011) in a symptomatic breast unit. The estimated need for PMRT was stratified into ‘PMRT not required’, ‘PMRT possibly required’, ‘PMRT probably required’ and ‘PMRT required’ groups.

Results

Of 156 referrals included in the study, 76 patients (49%) underwent mastectomy: 61 simple mastectomy, 10 skin sparing mastectomy (SSM) and delayed-immediate breast reconstruction, 3 SSM and implant-based IBR, and 2 mastectomy IBR with an autologous flap. The IBR rate was therefore 19.7%. The proportion of patients who received PMRT was 14% (3/21) in the ‘PMRT not required’, 30% (7/23) in the ‘PMRT possibly required’, 65% (9/14) in the ‘PMRT probably required’ and 94% (17/18) in the ‘PMRT required’ groups. Assigning a linear numerical score (1–4) to these groups (higher score representing greater likelihood of receiving PMRT), the predicted need for PMRT correlated with the proportion of patients who ultimately received PMRT (linear regression r²=0.98, p=0.01).

Conclusions

This study has examined the factors influencing MDT discussions regarding IBR, demonstrating that the MDT is reasonably accurate at predicting need for PMRT. Whether such accuracy is clinically adequate and/or reproducible across units is debatable.     

Single incision glove port laparoscopic colorectal cancer resection

Introduction

The advantages of single port surgery remain controversial. This study was designed to evaluate the safety and feasibility of single incision glove port colon resections using a diathermy hook, reusable ports and standard laparoscopic straight instrumentation.

Methods

Between June 2012 and February 2014, 70 consecutive patients (30 women) underwent a colonic resection using a wound retractor and glove port. Forty patients underwent a right hemicolectomy through the umbilicus and thirty underwent attempted single port resection via an incision in the right rectus sheath (14 high anterior resection, 13 low anterior resection, 3 abdominoperineal resection).

Results

Sixty-two procedures (89%) were completed without conversion to open or multiport techniques. Four procedures had to be converted and additional ports were needed in four other patients. The postoperative mortality rate was 0%. Complications occurred in six patients (9%). Two cases were R1 while the remainder were R0 with a median nodal harvest of 20 (range: 9–48). The median length of hospital stay was 5 days (range: 3–25 days) (right hemicolectomy: 5 days (range: 3–12 days), left sided resection: 6 days (range: 4–25 days). At a median follow-up of 14 months, no port site hernias were observed.

Conclusions

Single incision glove port surgery is an appropriate technique for different colorectal cancer resections and has the advantage of being less expensive than surgery with commercial single incision ports.     

Serum erythropoietin and erythropoiesis in high- and low-fetal hemoglobin beta-thalassemia intermedia patients

Clinical data suggest that in beta-thalassemia-intermedia patients, higher levels of circulating fetal hemoglobin (HbF) are associated with greater disease severity at comparable degrees of anemia. We assessed the influence of the amount of circulating HbF on serum erythropoietin (s-Epo) levels and on serum transferrin receptor, a measure of erythropoiesis, in 30 beta-thalassemia-intermedia patients. Twenty-four showed more than 40% HbF (21 of whom with beta (0)-thalassemia) and 6 presented lower HbF levels (beta(+)-thalassemia). The two groups of patients did not differ in age (15.3 v 19 years, respectively) or degree of anemia (Hb = 8.8 g/dL in both groups). Log (s-Epo) was correlated inversely with Hb (r = -0.47; P < .01), and directly with HbF (r = .55; P < .001). Multivariate regression analysis showed that Hb and HbF were independently correlated with s-Epo levels. High-HbF patients had greater s-Epo values at the same Hb level than low-HbF patients. Considering that iron-deficiency anemia control patients represented the predicted physiologic response of s-Epo to anemia, the observed/predicted s-Epo ratio in low-HbF thalassemic patients was no different from controls, but was increased in the high-HbF group. High- HbF patients also showed an expansion of erythropoiesis as much as four to nine times the normal value at the same Hb level as low-HbF patients. We conclude that HbF exerts an independent regulatory effect on erythropoietin production and erythropoiesis that is detectable only when HbF levels exceed 40%.     

Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro

Two anti-nerve growth factor receptor (LNGFR or p75NGFR) antibodies, Me20.4 and Me8211, label stromal cells with dendritic features in fresh smears and in formalin-fixed, paraffin-embedded human bone marrow (BM). The LNGFR+ cells have an oval nucleus, a scanty cytoplasm with long dendrites that intermingle with the hematopoietic cells, line the abluminal side of sinus endothelial cells, and provide the scaffold for the hematopoietic marrow. At the electron microscopy level, the immunogold tag labels the body and the long branching dendrites of fibroblast-like cells with scanty cytoplasm containing mitochondria, endoplasmic reticulum, and dense bodies. The LNGFR+ cells are positive for alkaline phosphatase, reticulin, collagen III, vimentin, TE-7, and CD13 but negative for endothelial (vWF, CD34, Pal-E), neural (CD56, neurofilament) and leukocyte markers (CD45, CD68). The LNGFR+ stromal cells appear in the fetal BM before the hematopoietic activity begins, originate from the vessel adventitia, and radiate in the Bm cavity. Long-term BM culture (LTBMC) in vitro contain LNGFR+ stromal cells. We document the presence of RNA message for the low- (LNGFR) and the high- affinity NGF receptor (NTRK1) by using RT-PCR on fresh BM aspirate and on LTBMC. BM biopsies from patients with hematologic fibrogenic diseases and in cytokine-treated cancer patients are evaluated for LNGFR+ cells: the amount of stained cells is correlated with the traditional reticulin stain in cases of myelofibrosis, therapy-related myelodysplasia, leukemia, and detected an increase of stromal cells in cytokine-treated patients. The anti-LNGFR antibodies represent a specific membrane marker for the adventitial reticular cells (ARC) of the human marrow and allow precise evaluation and quantitation of this important BM microenvironment component in vivo and in vitro.     

Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment

Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.