FasL^＋组织工程化猪软骨细胞的同种异体移植

目的:探讨表达FasL的猪组织工程化软骨细胞在同种异体内的生长状况和免疫排斥反应。方法:实验于2002-09/2004-03在上海交通大学医学院,上海市免疫学研究所进行。①分离制备猪软骨细胞。②制备FasL 软骨细胞,构建重组pGCEN-FasL反转录病毒载体,转染PA317细胞。经G418筛选获得分泌pGCEN-FasL病毒颗粒的PA317细胞克隆,选择高滴度的病毒液,感染猪软骨细胞。再经过G418筛选获得FasL 的软骨细胞克隆,扩增培养。③FACS检测FasL的表达,应用JAM试验测定FasL 的软骨细胞诱导Fas 的Jurkat细胞及活化的T细胞的凋亡率。同时制备转染pGCEN反转录病毒空载体的软骨细胞为对照。④取FasL 的软骨细胞与可注射性生物材料PluronicF127混合,注射于同种异体猪的腹壁皮下。在第4,5周取材,通过组织病理和免疫组织化学等方法,检测软骨细胞生长和免疫排斥反应。结果:①经转染的软骨细胞表面FasL的表达率为57%。②FasL 的软骨细胞具有明显诱导Fas 细胞和活化的同种异体T细胞的凋亡,最大的凋亡率分别为53.41%,30.38%(效/靶=10∶1),对照组分别为32.27%,13.16%(效/靶=10∶1)。③组织工程化猪软骨结节的结构与正常软骨组织基本一致,可见清晰的软骨凹陷和软骨膜,仅细胞排列较正常软骨略显混乱、不均匀现象。④免疫组织化学染色显示FasL 的组织工程化猪软骨结节的Ⅱ型胶原蛋白分布均匀,形状清楚,与正常软骨比较基本一致。⑤第5周的软骨细胞表面的FasL分子表达明显,周围的炎性细胞浸润相对较少。而对照组的软骨细胞周围可见到大量浸润的炎性细胞。结论:成功构建FasL 软骨细胞并有效表达,抑制免疫排斥反应,为建立同种异体软骨细胞移植的免疫耐受提供了实验依据。     

Recombinant immunoblot assay reaction patterns and hepatitis C virus RNA in blood donors and non-A, non-B hepatitis patients

To establish the value of the second-generation recombinant immunoblot assay (RIBA-2) and cDNA polymerase chain reaction (cDNA PCR) for confirmation of hepatitis C virus (HCV) infection, anti-HCV reaction patterns and the presence of HCV RNA were examined in 610 blood donors and 255 non-A, non-B hepatitis patients who were positive or indeterminate in RIBA-2. Of RIBA-2-positive donors (n = 191) and patients (n = 224), 75.4 and 89.7 percent, respectively, were HCV RNA positive. The most frequently observed anti-HCV recognition patterns in HCV RNA-positive donors and patients were c22, c33c, and c100 and/or 5- 1-1 (67.3%, 57.7%) and c22, c33c (24.8%, 29.3%). Among subjects with a RIBA-2-indeterminate result, HCV RNA was detected more often in patients (n = 31) than in donors (n = 419): 67.7 and 2.1 percent, respectively. In viremic persons with single-band reactivity in RIBA-2, this reactivity was always directed against either c22 or c33c. HCV RNA was detected by cDNA PCR in none of 162 persons with only anti-c100 and/or anti-5-1-1 reactivity. Therefore, RIBA-2 reactivity against c100 in combination with 5-1-1 should not be considered positive but indeterminate. In RIBA-2-indeterminate persons, HCV RNA detection is necessary for reliable confirmation of HCV infection.     

Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus

BACKGROUND: In a confirmatory laboratory, the second-generation recombinant immunoblot assay (RIBA-2) was replaced by the third- generation RIBA (RIBA-3) in March 1993. The aim of this validation study was to compare the sensitivity and specificity of RIBA-2 and RIBA- 3 in a routine setting, by using a validated hepatitis C virus (HCV) RNA polymerase chain reaction to establish plasma viremia. STUDY DESIGN AND METHODS: RIBA-2 testing was performed (March 1991-March 1993) in 593 HCV RNA-positive and 1498 HCV RNA-negative subjects. RIBA-3 testing was performed (March 1993-May 1994) in 220 HCV RNA-positive and 530 HCV RNA-negative subjects. All samples reacted for anti-HCV in enzyme- linked immunosorbent assay. RESULTS: In HCV RNA-positive individuals, the sensitivity of RIBA-3 was significantly higher than that of RIBA-2 (99.5% vs. 93.3%, p = 0.0005). This was not caused by inclusion of the NS5 antigen, but by a higher sensitivity of the antigens c33 and c100 (RIBA-2: 94.3% and 62.6%; RIBA-3: 99.5% and 88.6%). Replacement of the c22 and c100 recombinant proteins by synthetic peptides significantly reduced nonspecific reactivity against these antigens (p < 0.0001). Unfortunately, increased nonspecific reactivity against the modified c33 antigen and the new NS5 antigen canceled out this effect. Two-band reactivity occurred more often in nonviremic persons than in viremic persons (32.7% vs. 8.2%, p < 0.0001). Risk factors for HCV infection were less frequently observed in 11 blood donors with two-band reactivity than in 6 blood donors with other positive RIBA-3 patterns (18% vs. 83%, p = 0.03). CONCLUSION: The higher sensitivity of RIBA-3 significantly reduced the number of indeterminate test results in HCV RNA-positive persons. Confirmatory laboratories must be aware of the frequent occurrence of nonspecific, isolated reactivity and even nonspecific, two-band reactivity in anti-HCV enzyme-linked immunosorbent assay-reactive blood donors.     

Control of inflammatory pain by chemokine-mediated recruitment of opioid-containing polymorphonuclear cells

Opioid-containing leukocytes can counteract inflammatory hyperalgesia. Under stress or after local injection of corticotropin releasing factor (CRF), opioid peptides are released from leukocytes, bind to opioid receptors on peripheral sensory neurons and mediate antinociception. Since polymorphonuclear cells (PMN) are the predominant opioid-containing leukocyte subpopulation in early inflammation, we hypothesized that PMN and their recruitment by chemokines are important for peripheral opioid-mediated antinociception at this stage. Rats were intraplantarly injected with complete Freund's adjuvant (CFA). Using flow cytometry, immunohistochemistry, and ELISA, leukocyte subpopulations, chemokine receptor (CXCR2) expression on opioid-containing leukocytes and the CXCR2 ligands keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) were quantified. Paw pressure threshold (PPT) was determined before and after intraplantar and subcutaneous injection of CRF with or without naloxone. PMN depletion was achieved by intravenous injection of an antiserum. Chemokines were blocked by intraplantar injection of anti-MIP-2 and/or anti-KC antiserum. We found that at 2 h post CFA (i) intraplantar but not subcutaneous injection of CRF produced dose-dependent and naloxone-reversible antinociception (P<0.05, ANOVA). (ii) Opioid-containing leukocytes in the paw and CRF-induced antinociception were reduced after PMN depletion (P<0.05, t-test). (iii) Opioid-containing leukocytes mostly expressed CXCR2. MIP-2 and KC, but not CINC-2 were detectable in inflamed but not in noninflamed tissue (P<0.05, ANOVA). (iv) Combined but not single blockade of MIP-2 and KC reduced the number of opioid-containing leukocytes and peripheral opioid-mediated antinociception (P<0.05, t-test; P>0.05, ANOVA). In summary, in early inflammation peripheral opioid-mediated antinociception is critically dependent on PMN and their recruitment by CXCR2 chemokines.     

Polymorphonuclear motility: measurement by computer-linked image analysis

Most methods of measuring neutrophil motility provide information mainly about the performance of a small proportion of the fastest moving cells. Application of a computer-linked image analysis technique, using the "Quantimet," provides a convenient, automated method of measuring the motility of the whole cell population. This makes it possible to test whether changes in motility represent a homogeneous alteration affecting all cells or a change in the numbers or performance of a subset of cells. In this study the neutrophils from patients with uncomplicated rheumatoid arthritis were found to perform similarly to normals, while cells from patients with Felty's syndrome were markedly slower. This was an overall, homogeneous slowing of the whole cell population, not due to a loss of fast moving cells.     

Aggressive polyfibromatosis: A 10 year follow-up

Polyfibromatosis syndrome is a condition characterized by the occurrence of several cutaneous fibrotic conditions including Dupuytren's contracture and keloid formation. A 10 year follow-up of a patient with an aggressive type of polyfibromatosis associated with erosive arthropathy is presented. The underlying pathogenesis and management of this uncommon condition is discussed.     

Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria

The polymorphic frequency of the gene for beta s-globin involved in the generation of sickle trait and sickle cell anemia in the human population is caused by the enhanced resistance of sickle trait individuals to Plasmodium falciparum malaria, as supported by epidemiologic and in vitro studies. However, the mechanism for the protective effect of sickle hemoglobin in vivo has not been fully defined. The generation of transgenic mice expressing high levels of human beta s- and alpha-chains has allowed us to study this phenomenon in vivo in an experimental model. We infected the transgenic beta s mice with two species of rodent malaria and found a diminished and delayed increase in parasitemia as compared with controls. This is in contrast to our previous studies involving the introduction of a beta A transgene, which does not alter the infection. The use of this model allowed us to address the question of the mechanism of protection against malaria in mice expressing sickle hemoglobin. We find that splenectomy of transgenic mice completely reverses the protection against Plasmodium chabaudi adami infection. The results reported have shown a relationship between the presence of the beta s gene product and partial resistance to malaria in an experimental model in vivo and shows that the spleen plays an important role in this protection.     

Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4

A number of observations indicate that exposure to IL-4 is essential for the priming of Th2-type effector T cells and that exposure to IL-12 is essential for the priming of Th1-type effector T cells. However, the initial source of IL-4 in the early immune response has not been clearly identified. Dendritic cells (DC) are the most potent antigen- presenting cells (APC) in priming naive T cells. In this report, we show that DC exposed to IL-10 may play an important role in the priming of IL-4-secreting cells in the early immune response. DC isolated from splenic adherent cell cultures treated with rIL-10 (IL-10-DC) primed naive ovalbumin (OVA)-TCR transgenic T cells to secrete IL-4 upon re- stimulation with OVA and splenic APC. By contrast, DC isolated from rIL- 12, rIL-4 or control treated cultures induced almost exclusively Th1- type effector T cells. IL-4 secretion was detected in the primary cultures of IL-10-DC plus naive CD4+ T cells and the priming of IL-4- secreting T cells by IL-10-DC was dependent on endogenous IL-4 production in the priming culture since anti-IL-4 neutralizing antibody completely abrogated the priming of IL-4-secreting cells. Anti-B7-2 but not anti-B7-1 inhibited the ability of IL-10-DC to prime T cells to secrete IL-4. Furthermore, the ability of IL-10 DC to prime for IL-4- secreting T cells was closely related to the down-regulation of CD40 ligand-mediated IL-12 p70 production by DC in the primary cultures and was markedly reduced by adding exogenous IL-12 to the priming cultures. Thus, our findings indicate that early immunologic events that drive Th2 differentiation involve the effects of IL-10 on DC.      

Kundenverständnis in der Gesetzlichen Krankenversicherung

Background

Empirical and scientific studies prove the causality between a company’s sustainable value creation and its focus on the respective target customer. The most success is granted to those companies that establish a robust relationship with their customers on a broad analytical basis.

Results

This study aimed to establish a multidimensional, customer-related value management system that allows segmentation of the customer base of a compulsory health insurance fund according to strategic and action-oriented parameters. For the first time, a customized system was developed specifically for this branch.

Conclusions

Customer understanding is the basis for the development of strategic initiatives for communication, customer retention, and sales; for product portfolio management; and for risk management systems in the sense of broad, value-based management of medical care.