Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Gene expression changes in long-term culture of T-cell clones: genomic effects of chronic antigenic stress in aging and immunosenescence

The adaptive immune response requires waves of T-cell clonal expansion on contact with altered self and contraction after elimination of antigen. In the case of persisting antigen, as occurs for example in cytomegalovirus or Epstein-Barr virus infection, this critical process can become dysregulated and responding T-cells enter into a dysfunctional senescent state. Longitudinal studies suggest that the presence of increased numbers of such T-cells is a poor prognostic factor for survival in the very elderly. Understanding the nature of the defects in these T-cells might facilitate intervention to improve immunity in the elderly. The process of clonal expansion under chronic antigenic stress can be modelled in vitro using continuously cultured T-cells. Here, we have used cDNA array technology to investigate differences in gene expression in a set of five different T-cell clones at early, middle and late passage in culture. Differentially expressed genes were confirmed by real-time polymerase chain reaction, and relationships between these assessed using Ingenuity Systems evidence-based association analysis. Several genes and chemokines related to induction of apoptosis and signal transduction pathways regulated by transforming growth factor beta (TGFbeta), epidermal growth factor (EGF), fos and beta-catenin were altered in late compared to early passage cells. These pathways and affected genes may play a significant role in driving the cellular senescent phenotype and warrant further investigation as potential biomarkers of aging and senescence. These genes may additionally provide targets for intervention.     

粉叶小檗愈伤组织单细胞克隆

以粉叶小檗愈伤组织为材荆,用B5液体培养基进行悬浮培养建立悬浮细胞糸。经3～4次继代培养即可得到悬浮的单细胞。悬浮细胞通过细胞平板克隆(一般B5培养基平板克隆、优化培养基平板克隆和条件培养基平板克隆),经5代连续继代培养观察和薄板层析-分光光度法分析,发现用优化培养基进行平板克隆植板率最高,且克隆最易成功,并且还筛选到一株小檗碱产率高且稳定的克隆CV-57,其平均生手速率为每天14．412mg／L,为原始株系的1．91倍,平均小檗碱含量为干重的2．17％,是原始株系的2．26倍。     

长期肾移植受者外周血B淋巴细胞亚群分布特征

摘要 目的：观察移植肾功能稳定的长期受者（>10年）外周血B细胞亚群分布特征及其相关因素。方法：54名肾移植受者接受流式细胞仪检查,测算外周血总B细胞、未转化记忆B细胞、转化记忆B细胞、双阴性B细胞（CD19⁺CD27^-IgD^-）比例及数量（个/微升）。患者均服用包括环孢霉素的免疫抑制治疗。术后时间16.33±5.98年,GFR：91.63±11.28 mL/min/1.73 m²。结果：1长期肾移植患者外周血B细胞中幼稚B细胞最多（37.92% ± 22.06%）,未转化记忆B细胞最少(16.23% ± 11.10%)。B细胞亚群数量与白细胞总数、中性粒细胞比例等相关。2 以上述条件为控制因素行相关分析,转化记忆B细胞比例和GFR相关（r=-0.279,P=0.045）,双阴性B细胞数量和环孢霉素浓度相关（r=-0.300,P=0.029）。线性回归显示双阴性B细胞数目与环孢霉素浓度相关（R²=0.123,P=0.049）。3按GFR将患者分为肾功能减退组（GFR<90 mL/min/1.73 m²,n=19）和肾功能正常组（GFR≥90 mL/min/1.73 m²,n=35）。前者转化记忆B细胞比例显著升高（23.61% ± 10.96% vs.17.48%±8.91%,P=0.030）。按环孢霉素谷浓度将患者分为低浓度组（<64 mmol/L,n=28）和高浓度组（≥64 mmol/L,n=26）,前者双阴性B细胞数量显著升高（13.74±10.70 vs. 8.14±6.72/μL,P=0.027）。转化记忆B细胞比例与GFR分组相关（r=-0.326,P=0.018）,双阴性B细胞数量和环孢霉素浓度分组相关（r=-0.350,P=0.01）。结论：移植肾功能稳定的长期存活受者（>10年）外周血幼稚B细胞较多。转化记忆B细胞增多与移植肾功能减退相关,增多的双阴性B细胞和低孢霉素浓度治疗相关。     

Lung fibroblast clones from normal and fibrotic subjects differ in hyaluronan and decorin production and rate of proliferation

Development of fibrosis involves an increase in the deposition of connective tissue components including collagens, fibronectin and proteoglycans. One hypothesis to account for matrix deposition in fibrosis is that fibroblast with differing matrix producing capacity are involved in the fibrotic process. To test this hypothesis, primary fibroblast cultures and clones derived from these primary lines were established from the lung tissue of control patients and patients with pulmonary fibrosis. The primary lines and derived clones were studied in relation to their capacity to proliferate and to produce proteoglycans and hyaluronan. Primary fibroblast cultures and clones from normal subjects and patients with lung fibrosis differed considerably, with up to 13-fold difference, in both hyaluronan and proteoglycan production. The major proteoglycan produced was decorin in both controls and cultures from fibrotic patients, while cultures from patients with lung fibrosis had a higher expression of mRNA for both collagen and decorin. Clones derived from a primary line from a fibrotic patient secreted 3-fold greater amounts of decorin than those from a control subject. Furthermore, a negative correlation between proliferation and synthesis of decorin was noted. We suggest that different fibroblast clones accumulate in the lung, and that specific cell populations of high decorin producing fibroblasts may exist which are crucial in the pathogenesis of fibrosis.     

Resting heart rate,heart rate variability and functional decline in old age

Background:

Heart rate and heart rate variability, markers of cardiac autonomic function, have been linked with cardiovascular disease. We investigated whether heart rate and heart rate variability are associated with functional status in older adults, independent of cardiovascular disease.

Methods:

We obtained data from the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER). A total of 5042 participants were included in the present study, and mean follow-up was 3.2 years. Heart rate and heart rate variability were derived from baseline 10-second electrocardiograms. Heart rate variability was defined as the standard deviation of normal-to-normal RR intervals (SDNN). Functional status in basic (ADL) and instrumental (IADL) activities of daily living was measured using Barthel and Lawton scales, at baseline and during follow-up.

Results:

The mean age of the study population was 75.3 years. At baseline, higher heart rate was associated with worse ADL and IADL, and lower SDNN was related to worse IADL (all p values < 0.05). Participants in the highest tertile of heart rate (range 71–117 beats/min) had a 1.79-fold (95% confidence interval [CI] 1.45–2.22) and 1.35-fold (95% CI 1.12–1.63) higher risk of decline in ADL and IADL, respectively (p for trend < 0.001 and 0.001, respectively). Participants in the lowest tertile of SDNN (range 1.70–13.30 ms) had 1.21-fold (95% CI 1.00–1.46) and 1.25-fold (95% CI 1.05–1.48) higher risk of decline in ADL and IADL, respectively (both p for trends < 0.05). All associations were independent of sex, medications, cardiovascular risk factors and comorbidities.

Interpretation:

Higher resting heart rate and lower heart rate variability were associated with worse functional status and with higher risk of future functional decline in older adults, independent of cardiovascular disease. This study provides insight into the role of cardiac autonomic function in the development of functional decline.Elevated heart rate and reduced heart rate variability — the beat-to-beat variation in heart rate intervals — both reflect an altered balance of the autonomic nervous system tone characterized by increased sympathetic and/or decreased parasympathetic activity.^1–3 Sympathetic overactivity has been linked to a procoagulant state and also to risk factors for atherosclerosis, including metabolic syndrome, obesity and subclinical inflammation.^2–4 Moreover, increased heart rate is related to atherosclerosis, not only as an epiphenomenon of sympathetic overactivity, but also through hemodynamic mechanisms, such as high pulsatile shear stress, which leads to endothelial dysfunction.⁵Atherosclerosis has been linked to increased risk of functional decline in older people via cardiovascular events.⁶ As the world population is aging, the burden of functional disability is expected to increase.⁶ It has been hypothesized that heart rate and heart rate variability are markers of frailty, an increased vulnerability to stressors and functional decline.⁷ However, the direct link between these 2 parameters and risk of functional decline has not been fully established, and it is uncertain whether this association is independent of cardiovascular comorbidities.In this study, we examined whether heart rate and heart rate variability were cross-sectionally and longitudinally associated with functional status in older adults at high risk of cardiovascular disease, independent of cardiovascular risk factors and comorbidities.     

Lymphocyte antigens in Norwegian goats: serological and genetic studies

Altogether, 292 goat alloantisera were screened for antilymphocyte reactivity in a two-step dye exclusion microcytotoxicity test. Fifteen different lymphocyte antigen specificities were characterized by cluster analysis and absorption studies. The specificities were designated N1-N15 (N for Norwegian). Lymphocytes from 247 Norwegian dairy goats were tested. Each animal displayed from none to four of the characterized specificities. Lysostrip testing and family studies indicated that the specificities N1-N14 were coded for by multiple alleles belonging to at least two closely linked loci. It is suggested that these loci are part of the caprine major histocompatibility complex. Family studies gave strong evidence that the specificity N15 was not coded for by genes located in the same region as the other 14 specificities. Absorption studies showed that this specificity was located on both lymphocytes and erythrocytes.     

Lymphocyte maintenance during healthy aging requires no substantial alterations in cellular turnover

In healthy humans, lymphocyte populations are maintained at a relatively constant size throughout life, reflecting a balance between lymphocyte production and loss. Given the profound immunological changes that occur during healthy aging, including a significant decline in T‐cell production by the thymus, lymphocyte maintenance in the elderly is generally thought to require homeostatic alterations in lymphocyte dynamics. Surprisingly, using in vivo ²H₂O labeling, we find similar dynamics of most lymphocyte subsets between young adult and elderly healthy individuals. As the contribution of thymic output to T‐cell production is only minor from young adulthood onward, compensatory increases in peripheral T‐cell division rates are not required to maintain the T‐cell pool, despite a tenfold decline in thymic output. These fundamental insights will aid the interpretation of further research into aging and clinical conditions related to disturbed lymphocyte dynamics.     

人类诺如病毒反向遗传体系的构建与验证

为验证已报道的能复制人类诺如病毒(human norovirus,HuNoV)GⅡ.3 U201感染性克隆的复制能力,进一步在HuNoV临床病毒株中寻找更优序列,构建能高效复制的感染性克隆,本研究合成含有T7启动子和EF-1α启动子的HuNoV GⅡ.3 U201全长序列,构建质粒pU201和pEF-1α-U201及对...     

Isolation and characterization of diploid clones from adult and newborn rat liver cell lines

Summary A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and a pseudodiploid clone derived from adult rat liver cell line were positive for α-fetoprotein. This work was supported by a grant for cancer research from the Japanese Ministry of Education.     

Bone loss (osteopenia) in old male mice results from diminished activity and availability of TGF-β

One of the universal characteristics of the long bones and spines of middle-age and older mammals is a loss in bone mass (osteopenia). In humans, if this bone loss is severe enough, it results in osteoporosis, a skeletal disorder characterized by a markedly increased incidence of fractures with sequelae that may include pain, loss of mobility, and in the event of hip fracture, even death within a relatively few months of injury. An important contributing factor to the development of osteopororsis appears to be a diminution in the number and activity of osteoblasts responsible for synthesizing new bone matrix. The findings in the present and other similar studies suggest that this reduction in osteoblast number and activity is due to an age-related diminution in the size and osteogenic potential of the bone marrow osteoblast progenitor cell (OPC or CFU-f) compartment. We previously postulated that these regressive changes in the OPC/CFU-f compartment occurred in old animals because of a reduction in the amount and/or activity of TGF-β1, an autocrine growth factor important in the promotion of OPC/CFU-f proliferation and differentiation. In support of this hypothesis, we now report that (1) the osteogenic capacity of the bone marrow of 24-month-old BALB/c mice, as assessed in vivo, is markedly reduced relative to that of 3–4-month-old animals, (2) that the matrix of the long bones of old mice contains significantly less TGF-β than that of young mice, (3) that OPC's/CFU-f's isolated from old mice produce less TGF-β in vitro than those recovered from young mice, and (4) that OPC's/CFU-f's from old mice express significantly more TGF-β receptor (Types I, II, and III) than those of young animals and that such cells are more responsive in vitro to exogenous recombinant TGF-β1. We also find that colony number and proliferative activity of OPC's/CFU-f's of young mice and old mice, respectively, are significantly reduced when incubated in the presence of neutralizing TGF-β1 antibody. Collectively, these data are consistent with the hypothesis that in old male mice the reduction in the synthesis and, perhaps, availability from the bone matrix of TGF-β1 contributes to a diminution in the size and development potential of the bone marrow osteoprogenitor pool. J. Cell. Biochem. 70:478–488. © 1998 Wiley-Liss, Inc.     

Lymphocyte norepinephrine and epinephrine as indices of long-term changes in sympathoadrenal activity in human subjects

Researchers propose that long-term changes in sympathoadrenal activity during space flight can be evaluated by lymphocyte norepinephrine measurements in the early post-flight period. The metabolism of thrombocyte and lymphocyte catecholamines is explored to support the hypothesis.     

Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission spectrofluorometry

A method is described for monitoring intracellular ionized calcium (Ca²⁺) and determining kinetic and thermodynamic parameters of Ca⁺-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca²⁺ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca^2– that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca²⁺ with a rate constant of 0.031 ± 0.003 sec^–1, maximal rate of 23 ± 7 nM/sec, dissociation constant of 366 ± 63 nM, Hill coefficient of 2.3 ± 0.4, Q₁₀ of 2.58 ± 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca²⁺-extrusion system of lymphocytes and may be applicable to other blood cell types.Abbreviations DMSO dimethyl sulfoxide - HEPES [N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid],sodium salt - Indo-1/AM acetoxymethyl ester of indo-1 - IP₃ inositol 1,4,5-triphosphate - IP₄ inositol 1,3,4,5-tetrakisphosphate - RPMI Roswell Park Memorial Institute — 1640 culture medium - TPEN tetrakis-[2-pyridylmethyl]-ethylenediamine     

High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.

Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.     

cDNA捕捉法获得水稻杂种不育基因座位Sc区域的编码序列

摘要：cDNA捕获法足一种以表达为基础的基因分离技术,直接用目的区域的基因DNA捕捉该区域编码的cDNA,快速从大的基因组区域分离表达序列。本研究用一个水稻杂种不育基因座位Sc附近的大片段TAC基因组片段来捕捉该区域在水稻穗部表达的cDNA,共获得了6条不同的cDNA。将这些cDNA克隆进行测序分析,获得了该区域在水稻部表达的部分基因,其中1个是籼稻特有的基因。这些cDNA片段可成为新的标记用于目的基因的精细定位和候选基因序列分析。