Does low-intensity pulsed ultrasound stimulate maturation of tissue-engineered cartilage?

Traumatic events are a primary cause of local lesions of articular cartilage. Tissue engineered, cartilage-like structures represent an alternative to current treatment methods. The time necessary for tissue maturation and the mechanical quality of the regenerate at implantation are both critical factors for clinical success. Low-intensity pulsed ultrasound has proven to accelerate chondrogenesis in vitro. The goal of this study was to evaluate whether low-intensity pulsed ultrasound is capable of accelerating the process of cartilage maturation and increasing regenerate stability. Hyaline-like cartilage specimens were generated in vitro and subcutaneously implanted in the backs of nude mice. Twenty-eight animals received 20 min of low-intensity pulsed ultrasound treatment daily, and 28 animals received a sham treatment. Specimens were explanted after 1, 3, 6, and 12 weeks, mechanically tested with the use of an indentation test, histologically examined, and processed for RT-PCR. The Young's moduli significantly increased from 3 to 12 weeks, and at 6 weeks were comparable to those of native articular cartilage. In histological examination, specimens showed neocartilage formation. There was no significant difference between ultrasound-treated and sham-treated groups. The mechanical stability of the neocartilage specimens increased with treatment time and reached values of native cartilage after 6 weeks in vivo. Low-intensity pulsed-ultrasound stimulation showed no stimulatory effect on tissue maturation. In contrast, ultrasound-treated specimens showed a reduced Col 2 expression at 1 week and were significantly less stiff compared to native cartilage at 6 and 12 weeks. An acceleration of the maturation of tissue-engineered neocartilage in a clinical setting by means of low-intensity pulsed ultrasound therefore appears rather unrealistic.     

An ordered sequence of expression of human MHC class-II antigens during B-cell maturation?

Studies with monoclonal antibodies confirm that human MHC class-II antigens are encoded by at least three pairs of loci. Here Keith Guy and Veronica van Heyningen suggest that as B cells mature theproducts of these loci are expressed in the sequence SB → DR → DC antigens — a sequence which parallels the order of the genes on chromosome 6.     

Developmental changes in line bisection: a result of callosal maturation?

Normal adults tend to bisect horizontal lines to the left of the objective middle, especially when using the left hand. This bias has been attributed to the dominance of the right hemisphere in spatial attention. The authors investigated the effect of hand use and line position in visual line bisection in right-handed children and adults, classified into 4 different age groups: 10-12, 13-15, 18-21, and 24-53 years (N = 98). All 4 groups showed the characteristic leftward bias when using the left hand. When using the right hand, the youngest group showed a rightward bias, whereas the other 3 groups all showed a leftward bias. This suggests a shift from contralateral to right-hemispheric control during puberty and may reflect maturation of the corpus callosum.     

Is lithium essential for epididymal sperm maturation?

A wider biological role of ultratrace element lithium in the mammalian reproduction has been reported, however, presence of lithium in the epididymal luminal fluid (ELF) and its influence on sperm during maturation events in the epididymal regions are still unknown. A pilot study was carried out in Jamunapari buck which revealed that levels of lithium in the ELF diminished gradually and significantly (P < 0.01) from caput to cauda epididymis, concomitantly, a distinct increase (P < 0.01) in the spermatozoan motility, viability and hypo-osmotic reactive sperm were observed, except spermatozoan motility that was found absent in the caput epididymis. Therefore, we hypothesize that levels of lithium in the epididymal regions is one of the motility initiation and/or regulatory factor for epididymal sperm maturation essential for acquiring fertilizing competence of sperm cells, hence, lithium could also be considered as one of the biomarker of sperm maturation in any species.     

Assessment of mumps virus-specific antibodies by different serological assays: which test correlates best with mumps immunity?

Mumps is one of the vaccine-preventable childhood diseases and it has not yet been eradicated in Germany. This raises the question as to whether the available mumps vaccines are effective enough to prevent mumps and which antibody test system allows the authentic assigning of mumps-specific immunity. In an attempt to answer this question, we analysed 227 sera samples from medical students of the University Hospital Frankfurt/Main, Germany, using different test systems: indirect immune fluorescence, neutralisation assay, routine ELISA and newly developed immunoassays, which contain the mumps nucleoprotein and the wild-type strain Enders ATCC VR106, respectively. Mumps vaccination coverage of the screened collective amounted to 75.1%, which differs notably from the detected mumps-specific seropositivity rates in the literature (range 53.3% to 82.4%). In contrast, a small group of unvaccinated students had much higher seropositivity rates. Of course, assigned vaccination coverage and calculated seropositivity rates are not effective enough to interrupt the transmission of the mumps virus. The often-occurring mumps outbreaks, some in highly vaccinated populations, may not always demonstrate vaccine failure. The investigation of newly developed test systems and the occurrence of different mumps virus genotypes should also be considered.     

Final steps of natural killer cell maturation: a model for type 1-type 2 differentiation?

Analysis of cytokine and differentiation antigen expression in human natural killer (NK) cells revealed that interleukin 13 (IL-13) and interferon gamma (IFN-gamma) are produced at sequential stages during irreversible IL-12-induced differentiation. In human NK cell clones, polyclonal CD3-CD161+CD56- cells and peripheral lymphocytes, IL-4 induced the proliferation of both IL-13+ NK and T cells, whereas IL-12 allowed a proliferation-independent accumulation of IFN-gamma+ cells. These data disproved the NK1-NK2 hypothesis and challenge the current T helper 1 (TH1)-TH2 paradigm. We propose that the cytokine environment regulates a type 2-->0-->1 developmental progression, with IL-12 needed for terminal differentiation and IL-4 delaying this process, rather than a type 1 versus type 2 decision of a type 0 cell.     

The precursor frequency of alloreactive CTLs is proportional to the number of T cell epitope specificities

The aim of this study is to find the experimental evidence that the precursor frequency of alloreactive CTLs is proportional to the number of the T-cell epitope specificities. The number of T-cell epitope specificities was manipulated by pulsing different number of HLA-A2 restricted peptide(s) onto the T2 cells, which acted as stimulating cells to elicit allo-reaction by co-culturing with peripheral blood lymphocytes (PBLs) of HLA-A2 negative individual. Ten HLA-A2 restricted peptides (all were normal cell components) were synthesized, and cell peptide extract was prepared by frozen and thawed.T2 cells loaded with different number of peptide(s) were co-cultured with PBLs of an HLA-A2 negative individual; the latter were stained with PKH67 in advance. Then the proliferation was monitored with flow cytometry, and the precursor frequency of the effector cells was analyzed by the ModFit Software. After 6 d of culture, no proliferation was observed in the bulk culture of PBL alone, and obvious proliferation took place when PBLs of the HLA-A2 negative were co-cultured with T2 cells loaded with or without loading peptide(s). The precursor frequency of the alloreactive CTLs was 0.052 819 for co-culture with T2 cells loaded without peptide; however it was 0.030 429 for T2 cells with EBV/ LMP2A and 0. 030 528 for T2 cells loaded with a single autogeneic peptide, and increased up to 0.144 942 for T2 cells loaded with 10 autogeneic peptides; the precursor frequency was 0.203 649 when co-cultured with T2 cells loaded with miscellaneous peptides extracted from the cytoplasm of T2 cells. This study reveals that the precursor frequency of alloreactive CTLs is proportional to the number of T-cell epitope specificities, and independent of the density of the allogeneic HLA ClassⅠmolecule. Our findings support the hypothesis that the alloreactive T cell populations comprise miscellaneous T cell clones; each is specific to corresponding pMHC. The novel constellation of peptides presented by allogeneic MHC molecules makes thousands of different epitopes, which account for the exceptional high precursor frequency of alloreactive T cells.     

Varying effects of different β-glucans on the maturation of porcine monocyte-derived dendritic cells

β-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, β-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of β-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 μg/ml) of five commercial β-glucan preparations, differing in structure and source, on porcine monocyte-derived dendritic cells (MoDCs). Although all β-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another β-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlan-stimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1β (IL-1β), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when β-glucans are used as dietary supplement.     

Frontal brain maturation and the stability of children’s shyness

Recent evidence suggests that relative to nonshy children, shy children exhibit a lower overall frontal EEG alpha/delta ratio (ADR) during middle childhood, possibly reflecting relatively less frontal brain maturation at this age. We examined this same ADR measure in relation to the stability of observed shyness and parent-reported child social anxiety measured across two laboratory visits separated by approximately 1 year during late childhood in 51 children (33% female, age range 10–16 years). We found that the overall frontal ADR score was significantly lower among children with high, stable observed shyness and parent-reported child social anxiety compared to children in the low, stable class. Findings provide convergent evidence suggesting that the stability of shyness in late childhood may be linked to relatively less overall frontal brain maturation at this age. We speculate on the adaptive function of delaying frontal brain maturation in the origins and maintenance of children's shyness.     

Low serum promotes maturation of excitation–contraction coupling in myotubes

Patch-clamping and the simultaneous fluorescence measurement of cytoplasmic Ca2+ ([Ca2+]i) were used to analyze the effect of serum on the functional features of excitation-contraction (E-C) coupling in mouse skeletal myotubes. In high-serum-treated (10%) myotubes, depolarization elicited Ca2+ release which continued for tens of milliseconds following the end of the pulse, after which [Ca2+]i decayed slowly. In low-serum-treated (0.5%) myotubes, the Ca2+ transient caused by depolarization had an increased rate of rise and peak amplitude, and [Ca2+]i began to decay rapidly upon repolarization. When a depolarizing pulse (0.5-1.0 s) was applied to low-serum-treated myotubes during a Ca2+ transient induced by 5-10 mM caffeine, repolarization usually caused the caffeine transient to terminate rapidly (RISC; repolarization-induced stop of caffeine-induced Ca2+ release). The RISC was less prominent in high-serum-treated myotubes. These results suggest that low serum promotes the maturation of myotubes so that Ca(2+)-release and Ca(2+)-removal activities are accelerated. Additionally, the essential features of the communication between the voltage sensor and the Ca(2+)-release channel are shared by myotubes and adult muscle fibers.     

A Modified Peptide Stimulation Method for Efficient Amplification of Cytomegalovirus （CMV）-Specific CTLs

CMV-specific immunity is essential for control of human cytomegalovirus （HCMV） infection. Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus （CMV） remains a significant cause of morbidity and mortality. Adoptive transfer of CMV-specific CD^8＋ T cell clones has been shown to reduce the rate of viral reactivation; however, the ex vivo production of cells for adoptive transfer is labor intensive and expensive. We report here a modified peptide stimulation method using CMV-specific epitope peptides to stimulate PBMCs for generation of CMV-specific CTLs. This method permits efficient amplification of CMV-specific CTLs and provides a large number of cells for FACS analysis from a single blood sample. Significantly, it achieves high frequencies of tetramer staining of CD^8＋ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. Thus, this approach expands and selects HLArestricted CMV-pp65-reactive T-cell lines of high specificity for potential adoptive immunotherapy.     

Naïve CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation

In defense of the host, the immune system must often raise an effective cytotoxic T lymphocyte (CTL) response from a small number of clonal precursors. The degree to which activation stimuli regulate the expansion and differentiation of na?ve CTLs, however, remains unknown. Using an engineered antigen-presenting cell (APC) system that allows control over antigenic stimulation, we studied the signaling duration requirements for priming and clonal expansion of na?ve CTLs. We found that na?ve CTLs become committed after as little as 2 h of exposure to APCs and that their subsequent division and differentiation can occur without the need for further antigenic stimulation of the daughter cells, whether priming is in vitro or in vivo. These data show that after a brief interaction with stimulatory APCs, na?ve CTLs initiate a program for their autonomous clonal expansion and development into functional effectors.     

治疗用合成肽乙肝疫苗对CTLs数量的影响

目的 研究治疗用合成肽乙型肝炎疫苗对慢性乙型肝炎（CHB）患者HBV特异性细胞毒性T淋巴细胞（CTLs）数量的影响,分析CTLs的数量与病毒载量（HBV DNA）的相关性.方法 15例HLA-A2阳性CHB患者随机分为三组：单用疫苗组、对照组和联合用药组（每组5例）.利用MHCI肽五聚体技术结合流式细胞分析技术,检测各组患者治疗前、给药期间及停药24周后患者外周血单个核细胞（PBMCs）中CTLs数量的变化,同时研究CTLs数量和病毒载量的关系.利用LightCycler 实时荧光定量PCR法对HBV DNA进行定量.结果 ①治疗前,三组患者五聚体阳性率（0.022%～0.028%）差异无统计学意义（P＞0.05）;②五聚体阳性率在治疗期间和随访时,单用疫苗组和联合用药组均明显高于对照组（P＜0.05）,单用疫苗组和联合用药组差异无统计学意义（P＞0.05）;③三组患者CTLs数量与病毒载量成负相关（P＜0.05）.结论 治疗用合成肽乙型肝炎疫苗能在一定程度上增加CTLs的数量,抑制HBV的复制.     

RLR-mediated production of interferon-β by a human dendritic cell subset and its role in virus-specific immunity

Cytosolic RIG-I-like helicases (RLR) are PRRs involved in type I IFN production and antiviral immunity. This study focuses to the comparison of the expression, function, and signaling cascades associated to RLR in the previously identified CD14(-)DC-SIGN(+)PPARγ(low)CD1a(+) and CD14(low)DC-SIGN(+)PPARγ(high)CD1a(-) human moDC subsets. Our results revealed that the expression of RLR genes and proteins as well as the activity of the coupled signaling pathways are significantly higher in the CD1a(+) subset than in its phenotypically and functionally distinct counterpart. Specific activation of RLR in moDCs by poly(I:C) or influenza virus was shown to induce the secretion of IFN-β via IRF3, whereas induction of proinflammatory cytokine responses were predominantly controlled by TLR3. The requirement of RLR-mediated signaling in CD1a(+) moDCs for priming na?ve CD8(+) T lymphocytes and inducing influenza virus-specific cellular immune responses was confirmed by RIG-I/MDA5 silencing, which abrogated these functions. Our results demonstrate the subset-specific activation of RLR and the underlying mechanisms behind its cytokine secretion profile and identify CD1a(+) moDCs as an inflammatory subset with specialized functional activities. We also provide evidence that this migratory DC subset can be detected in human tonsil and reactive LNs.     

DeFries–Fulker Analysis of Twin Data with Skewed Distributions: Cautions and Recommendations from a Study of Children’s Use of Verb Inflections

DeFries-Fulker (DF) analysis is an adaptation of multiple regression that is used to estimate heritability of extreme scores (h²‰‰_g) on a dimension. Probands are identified as scoring below a cutoff that defines impairment, and one then uses regression to predict the scores of co-twins from the proband scores and a term that denotes the genetic relationship between twins (1.0 for MZ and 0.5 for DZ twins). This paper reports illustrative data and simulations for the situation where the dimensional variable shows substantial negative skew. Two types of simulation were conducted: in the first, an underlying polygenic liability dimension was normally distributed: skewing was introduced by transforming or truncating the liability distribution. In the second set of simulations, skewing arose because an infrequent defective gene impaired scores. In both sets of simulations DF analysis was robust in the face of severe skewing of the data. DF analysis can provide two pointers to major gene effects on extreme scores on a trait with a skewed distribution: first, group heritability estimates will be higher for the original skewed data than for normalised data; second, estimates of h²‰‰_g will increase as the cutoff to identify probands is made more stringent. Both these features were seen in data from a test of verb inflections given to174 6-year-old twin pairs, suggesting that a single major gene may be implicated in causing impaired grammatical development.