Characterization of idiotopes on MOPC 315 IgA using monoclonal antiidiotypic antibodies

The isologous antiidiotypic response in BALB/c mice to immunization with the DNP-binding IgA myeloma protein, MOPC 315, alters the expression of the anti-DNP antibody repertoire and confers immunity against MOPC 315 myeloma tumors. In order to characterize the idiotopes on MOPC 315 IgA which elicit this response we have isolated four monoclonal antiidiotypic antibodies (AIA), D10 (IgG_2a), A2(IgG₁), G3 (IgG_2b) and F1 (IgG_2a), produced by splenocytes of BALB/c mice immunized with MOPC 315 IgA in three independent fusion experiments. These AIA react with MOPC 315 IgA. reassociated H³¹⁵ L³¹⁵ and F³¹⁵_V but not with free H³¹⁵, L³¹⁵, V³¹⁵_H or V³¹⁵₂. In addition the AIA do not react with the closely related DNP-binding IgA myeloma protein, MOPC 460, suggesting that they are directed against private idiotopes on MOPC 315 IgA. These idiotopes can be divided into two groups. Group I, defined by D10, A2 and G3 consists of two overlapping idiotopes, one of which is related to the hapten-binding site. The two idiotopes are formed by an interaction of amino acids in H³¹⁵ and L³¹⁵. Group II defined by F1 consists of one idiotope which is related to the hapten-binding site. This idiotope is comprised of an aminoacid sequence on H³¹⁵ which requires an interaction with either L³¹⁵ or L⁴⁶⁰ for expression. A2 and G3 react identically with the same idiotope but were derived from two independent fusion experiments. This indicates an identity of AIA clonotypes among individual mice and suggests that the isologous AIA response to MOPC 315 IgA is restricted.     

The Effect of Aspirin on Recurrent Fetal Loss in Experimental Antiphospholipid Syndrome

PURPOSE: To evaluate the effect of aspirin treatment upon fetal loss in mice with experimental antiphospholipid syndrome (APLS). MATERIALS AND METHODS: Experimental APLS was induced in pregnant mice by passive transfer of mouse monoclonal anticardiolipin antibody. The mice were treated with high (100μg/d) or low (10μg/d) does of aspirin, using vitaminC(100μg/d or 10μg/d)as a control. The mice were assessed for the presence of lupus anticoagulants (prolonged aPTT), thrombocytopenia, degree of fetal resorption rate and mean embryo and placental weights. RESULTS: The mice with APLS had a higher fetal resorption rate(45.7± 12.2% vs 2.5 ± 0.4%, P<0.001), reduced placenta mean weight(104 ± 8 mgvs 169 ±7mg, P<0.001), prolonged aPTT (94± 14sec vs 39±4sec, P<0.001), and reduced mean platelet count(597± 186 ± 10³/mm³vs 847±51 ± 10³/mm³,P<0.001). The groupof mice with APLS, who were treated with low-dose aspirin, had a lower resorption rate (11.1 ±9.3% vs 45.7±12.2%, P<0.001), a higher placenta mean weight (178 ± 8 mg vs 104 ± 8 mg, P<0.001), a higher mean embryo weight (1042 ± 134 mg vs 721±91 mg, P<0.001), and a lower aPTT (58±15 sec vs 94±14 sec, P, <0.001). Micewho were treated with high-dose aspirin also had a lower resorption rate, although not as much as in the low-dose aspirin group (34.2 ± 12.7% vs 45.7 ± 12.2%, P<0.001). CONCLUSION: Aspirin, especially in low dose, has a protective effect against obstetrical complications associated with experimental APLS.     

Induction of immunoglobulin-producing human peripheral blood lymphocytes in serum-free medium

Induction of immunoglobulin-secreting cells from human peripheral blood lymphocytes in a serum-free culture medium was studied. Albumin, transferrin, insulin and fibronectin can replace serum entirely for support of pokeweed mitogen (PWM)-stimulated B lymphocytes, measured by a reverse hemolytic plaque assay using protein A-coated red cells. In this serum-free system, growth and maturation to IgM and IgG secretion occur at the same or higher efficiency as in conventional serum-containing medium, with maximum numbers of plaque-forming cells on day 6 at optimal dose of PWM, 0.5 ～ 5 μg/ml. This system can be used to avoid the interference from undefined serum components.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Osteoprotegerin reduces the loss of periarticular bone mass in primary and secondary spongiosa but does not influence inflammation in rat antigen-induced arthritis

Objective: To assess the effect of osteoprotegerin (OPG) on joint swelling, synovial inflammation and cartilage destruction, periarticular and axial bone volume, and bone turnover in rat antigen-induced arthritis (AIA). Design: Rats were treated with OPG (3 mg/kg/day) at regular intervals from day 1 to day 20 of AIA. Disease activity was evaluated by measurement of joint swelling as well as, joint inflammation and destruction by histology. Bone volume and cellular turnover parameters of secondary spongiosa of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. Periarticular bone volume of the primary spongiosa at the right tibia head was measured by linear scanning. The findings were compared with those of PBS-treated AIA and healthy animals. Result: OPG treatment did not reduce joint swelling or histological signs of inflammation. Cartilage destruction was reduced. However, this effect did not reach statistical significance . In the secondary spongiosa OPG treatment reduced the loss of periarticular bone volume. However, the latter did not reach the level of healthy controls. OPG treatment significantly reduced parameters of bone formation and bone resorption. In the primary spongiosa, OPG-treatment led to a higher amount of mineralized tissue and a greater number of trabeculae compared to PBS-treated animals with AIA or healthy controls. In the axial skeleton, OPG treatment reduced bone formation and bone resorption parameters compared to healthy animals. This treatment had no influence on bone volume. Conclusions: In periarticular bone of AIA rats, OPG treatment reduced the loss of bone volume and decreased the bone turnover, thus preventing periarticular bone destruction. OPG treatment had no influence on inflammatory process or on cartilage destruction. Received 2 June 2005; returned for revision 26 July 2005; returned for final revision 9 August 2005; accepted by M. Parnham 24 September 2005 Presented in part at the 66. Annual Meeting of the American College of Rheumatology, New Orleans, U.S.A., October 2002, and at the 25. Annual Meeting of the American Society of Bone and Mineral Research, Minneapolis, USA, September 2003 Supported by grants from the Thuringian Ministry of Science, Research and Art (B307-01025, B378-01017), the Interdisciplinary Center for Clinical Research (IZKF) Jena, and the Deutsche Forschungsgemeinschaft (Br 1372/5-1) Osteoprotegerin was generously provided by Amgen (Thousand Oaks, CA, USA). Drs. Neumann and Oelzner contributed equally to this work.     

The renin–angiotensin system in kidney development: role of COX‐2 and adrenal steroids

Recent data from studies in rodents with targeted gene disruption and pharmacological antagonists have shown that the renin–angiotensin–aldosterone system (RAAS) and cyclooxygenase type‐2 (COX‐2) are necessary for late stages of kidney development. The present review summarizes data on the developmental changes of RAAS and COX‐2 and the pathways by which they are activated; their possible interplay and the mechanisms by which they affect kidney development. Intrarenal and circulating renin and angiotensin II (ANG II) are stimulated at birth in most mammals. In rats, renin and ANG II stay significantly elevated in the suckling period while aldosterone stabilizes at an adult level. COX‐2 is stimulated in thick ascending limb of Henle's loop in the suckling period at a time when urine concentrating ability is not developed. Data suggest that this induction is mediated by combined low plasma glucocorticoid concentration and by a low NaCl intake. Studies with selective inhibitors of COX‐2 and COX‐2 null mice show that COX‐2 activity stimulates renin secretion from JG‐cells during postnatal kidney development and that lack of COX‐2 activity leads to pathological change in cortical architecture and eventually to renal failure. In the postnatal period, ANG II initiates and maintains pelvic and ureteric contractions necessary for urine flow. Lack of ANG II in the neonatal period is thought to cause injury by a chronic increase of renal pelvic pressure. Aldosterone is crucial for survival and growth in the neonatal period through its effects on sodium reabsorption and the intrarenal sensitivity to aldosterone is increased in the postnatal period. Final maturation of the kidney occurs through an intimate interplay between a low dietary sodium intake and a non‐responsive HPA‐axis which stimulates cortical COX‐2 activity. COX‐2 supports increased activity of the RAAS and may contribute to a low concentrating ability.     

A simple method for obtaining cultures enriched for Type II pneumonocytes from fetal rabbit

Summary A differential plating method permitted preparation of cultures significantly enriched for Type II pneumocytes. These cells were maintained in a differentiated state for at least 12 d, identifiable morphologically (by presence of osmiophilic lamellar inclusion bodies) and bio-chemically (by demonstration of synthesis of phosphatidyl choline and production of disaturated lecithin).     

阿仑膦酸钠对兔破骨细胞功能的影响

用骨陷窝形成分析法观察阿仑膦酸钠对体外培养破骨细胞功能的影响，探讨可能的作用机制。在建立兔破骨细胞培养方法的基础上，用不同浓度的阿仑膦酸钠分别与骨片或成骨细胞提前作用，然后再将骨片或成骨细胞分别与破骨细胞共同培养。结果发现当阿仑膦酸钠（10^-11,10^-9,10^-7mol/L)与骨片预处理后，破骨细胞在骨片上形成的骨吸收陷窝数目减少，其抑制率分别为19．5％（P＜0．05）、49．0％（P＜0．01）和74．5％（P＜0．01）。用阿仑膦酸钠（10^-9,10^-7,10^-5mol/L)预处理成骨细胞后，仅在高浓度（10^-5mol/L）时可见破骨细胞骨吸收陷窝的数目明显减少，其抑制率为62．8％（P＜0．01）。结果表明阿仑膦酸钠能直接或通过成骨细胞间接抑制破骨细胞的骨吸收活性。     

Interleukin 7 stimulates growth of fetal thymic precursors of cytolytic cells: induction of effector function by interleukin 2 and inhibition by interleukin 4

Interleukin 7 (IL-7) and interleukin 2 (IL-2) stimulate the outgrowth of distinct populations of thymocytes in lobe submersion cultures (LSC) established with day 12-14 murine fetal thymus. Analysis of the expression of cell surface markers in previous studies showed that IL-7 favors the expansion of a more immature population. In the experiments reported here, populations grown in IL-7 and IL-2 were found to differ functionally as assessed by the expression of cytolytic activity. Whereas cells derived from IL-2-supplemented LSC were highly cytolytic for a broad panel of targets, cells that emerged in IL-7-supplemented cultures exhibited little or no such activity, even in the presence of facilitating lectin. However, there were cells with cytolytic potential present in IL-7-grown populations, as demonstrated by the abrupt appearance of effector function 3 days after their exposure to IL-2. Limiting dilution analysis showed that the absolute number of cells in the cytolytic lineage in fetal thymic lobes increased during culture in LSC. Interestingly, identical increases occurred in IL-7-supplemented and IL-2-supplemented LSC, despite the fact that only the latter population exhibited appreciable lytic activity. Collectively, these results suggest that IL-7 stimulates outgrowth of cell populations which contain functionally inactive cytolytic precursors whose activity is inducible by IL-2. In contrast to IL-2, IL-4 failed to stimulate the appearance of a lytic population from IL-7-supplemented LSC. Furthermore, IL-4 interfered with cell proliferation and acquisition of lytic activity normally induced by IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-free fetal DNA and intact fetal cells in maternal blood circulation: implications for first and second trimester non-invasive prenatal diagnosis

Both intact fetal cells as well as cell-free fetal DNA are present in the maternal circulation and can be recovered for non-invasive prenatal genetic diagnosis. Although methods for enrichment and isolation of rare intact fetal cells have been challenging, diagnosis of fetal chromosomal aneuploidy including trisomy 21 in first- and second-trimester pregnancies has been achieved with a 50-75% detection rate. Similarly, cell-free fetal DNA can be reliably recovered from maternal plasma and assessed by quantitative PCR to detect fetal trisomy 21 and paternally derived single gene mutations. Real-time PCR assays are robust in detecting low-level fetal DNA concentrations, with sensitivity of approximately 95-100% and specificity near 100%. Comparing intact fetal cell versus cell-free fetal DNA methods for non-invasive prenatal screening for fetal chromosomal aneuploidy reveals that the latter is at least four times more sensitive. These preliminary results do not support a relationship between frequency of intact fetal cells and concentration of cell-free fetal DNA. The above results imply that the concentration of fetal DNA in maternal plasma may not be dependent on circulating intact fetal cells but rather be a product of growth and cellular turnover during embryonic or fetal development.