Single Immunization of Newborn Mice with Heterologous Type-II Collagen Induces Arthritic Disease

Collagen-induced arthritis (CIA) is a widely accepted model of autoimmune disease with significant similarities to rheumatoid arthritis in humans. CIA is provoked in susceptible strains upon immunization of adult mice with native type-II collagen in complete Freund's adjuvant (CFA). Neonatal exposure to antigen is supposed to result in T cell clone deletion and induction of tolerance. Here we report that the neonatal injection of bovine type-II collagen (bCII) to ICR (CD-2) mice triggers the development of autoimmune chronic joint inflammation. Compared with standard CIA significant joint swelling was not observed and anti-collagen antibodies were not detected if the second challenge with the antigen was not supplied. Histopathologic examination of the joints showed cell infiltration, synovial hyperplasia and at the later period bone destruction. Mice immunized as neonates expressed Ag-specific proliferative response and delayed type hypersensitivity (DTH) reaction to bCII.     

Single immunization of newborn mice with heterologous type-II collagen induces arthritic disease

Collagen-induced arthritis (CIA) is a widely accepted model of autoimmune disease with significant similarities to rheumatoid arthritis in humans. CIA is provoked in susceptible strains upon immunization of adult mice with native type-II collagen in complete Freund's adjuvant (CFA). Neonatal exposure to antigen is supposed to result in T cell clone deletion and induction of tolerance. Here we report that the neonatal injection of bovine type-II collagen (bCII) to ICR (CD-2) mice triggers the development of autoimmune chronic joint inflammation. Compared with standard CIA significant joint swelling was not observed and anti-collagen antibodies were not detected if the second challenge with the antigen was not supplied. Histopathologic examination of the joints showed cell infiltration, synovial hyperplasia and at the later period bone destruction. Mice immunized as neonates expressed Ag-specific proliferative response and delayed type hypersensitivity (DTH) reaction to bCII.     

TRAIL对CIA小鼠脾细胞表达Th1/Th2细胞因子的影响

 摘要：目的 观察肿瘤坏死因子相关凋亡诱导配体（Tumor Necrosis Factor Related Apoptosis Inducing Ligand , TRAIL）对胶原诱导型关节炎（Collagen-induced arthritis, CIA）小鼠的治疗效果,探讨TRAIL治疗类风湿性关节炎（Rheumatoid Arthritis, RA）的可能机制。方法 牛Ⅱ型胶原蛋白加弗氏完全佐剂免疫DBA/1J小鼠建立CIA模型,采用重组腺相关病毒AAV-TRAIL关节腔内注射进行治疗。CBA和流式细胞术检测Th1/Th2细胞因子的分泌。结果 AAV-TRAIL可改善CIA小鼠的病情,体外研究其机制发现TRAIL可抑制CIA小鼠的脾细胞分泌Th1型细胞因子,但对Th2型细胞因子没有作用。结论TRAIL对CIA小鼠的治疗作用可能和TRAIL抑制Th1型细胞因子分泌相关。     

Glycogen synthase kinase-3beta inhibition attenuates the degree of arthritis caused by type II collagen in the mouse

Recently, glycogen synthase kinase-3 (GSK-3) has being identified as an ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and plays an important role in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3beta inhibition on the degree of arthritis caused by type II collagen (CII) in the mouse (collagen-induced arthritis; CIA). Mice developed erosive hind paw arthritis when immunized with CII in an emulsion in complete Freund's adjuvant (CFA). The incidence of CIA was 100% by day 28 in the CII-challenged mice and the severity of CIA progressed over a 35-day period with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint margins. Treatment of mice with the GSK-3beta inhibitor TDZD-8 (1 mg/kg/day i.p.) starting at the onset of arthritis (day 25) ameliorated the clinical signs at days 26-35 and improved histological status in the joint and paw. Immunohistochemical analysis for nitrotyrosine, poly(ADP-ribose) (PAR), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) revealed a positive staining in inflamed joints from mice subjected to CIA. The degree of staining for nitrotyrosine, PAR, iNOS, and COX-2 was significantly reduced in CII-challenged mice treated with the GSK-3beta inhibitor. Plasma levels of tumor necrosis factor (TNF)-alpha and the joint tissue levels of macrophage inflammatory protein (MIP)-1alpha and MIP-2 were also significantly reduced by GSK-3beta inhibition. These data demonstrate that GSK-3beta inhibition exerts an anti-inflammatory effect during chronic inflammation and is able to ameliorate the tissue damage associated with CIA.     

昆明山海棠对CIA大鼠模型中HIF-1α表达的影响

目的:研究昆明山海棠(THH)对胶原诱导性关节炎(CIA)大鼠模型中低氧诱导因子1α(HIF-1α)表达的影响及其作用机制。方法:建立CIA大鼠模型,随机分组,治疗组分别采用高、中、低剂量THH每天灌胃1次,连续用药30d。动态观察关节炎指数(AI)及关节的病理改变,用RT-PCR及免疫组化染色法分别检测HIF-1αmRNA及其蛋白的表达。结果:THH可明显抑制CIA大鼠的足爪肿胀,明显减轻滑膜增殖及炎症反应,HIF-1α表达明显降低。结论:THH通过降低HIF-1α的表达,减轻CIA模型大鼠关节的炎症反应。     

The plasminogen activator/plasmin system is essential for development of the joint inflammatory phase of collagen type II-induced arthritis

The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA, we found that none of the 50 plasminogen-deficient littermates that were tested developed CIA within a 40-day period. Antibody generation after CII immunization as well as the binding of labeled anti-CII antibodies to the surface of cartilage were similar in wild-type and plasminogen-deficient mice. No sign of inflammation was seen when plasminogen-deficient mice were injected with a mixture of monoclonal antibodies against CII. However, after daily injections of human plasminogen, these mice developed arthritis within 5 days. Our finding that infiltration of inflammatory cells into the synovial joints was impaired in plasminogen-deficient mice suggests that uPA and plasminogen are important mediators of joint inflammation. Active plasmin is therefore essential for the induction of pathological inflammatory joint destruction in CIA.     

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL-10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra-articular injection with the IL-10-expressing virus (Ad5E1mIL-10) caused substantial over-expression of IL-10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL-10 was noted for a few days, being maximal at day 1. One intra-articular injection of Ad5E1mIL-10 in the knee joints of collagen type II (CII)-immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL-10 expression by Ad5E1mIL-10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL-10 over-expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall-induced arthritis model revealed that local IL-10 over-expression markedly suppressed the production of tumour necrosis factor-alpha (TNF-alpha) and IL-1alpha, but had no significant effect on IL-1beta and IL-12 production in the inflamed synovium. These data indicate that local over-expression of IL-10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down-regulation of TNF-alpha.     

合成环瓜氨酸化蛋白短肽的免疫原性和致关节炎性研究

目的:构建环瓜氨酸化蛋白短肽诱导性小鼠关节炎模型,探讨该短肽的免疫原性、致关节炎性。法:36 只 DBA/1 小鼠随机分为3 组,于第0 天、第21 天分别以域型胶原(C ,CIA)、环瓜氨酸化波形蛋白短肽(CCit-Vim,CCV-IA)和偶联血蓝蛋白(KLH)的环瓜氨酸化波形蛋白短肽(CCit-Vim+KLH,CCV+K-IA)皮下注射。ELISA 检测血清抗C 抗体、抗CCit-Vim 抗体、抗CCP 抗体及TNF,间接免疫荧光法(IIF)检测抗大鼠食管角蛋白抗体(AKA),对关节炎指数(AI)、足容积、踝关节病理学进行评价。结果:CCV+K-IA 小鼠关节炎发病率为25%(3/12),但关节炎出现时间晚,持续时间短,发病率及关节炎程度均低于CIA;CCV-IA 无关节炎发生。CCV+K-IA 产生抗CCit-Vim 抗体高于CIA(P = 0.031),产生抗CCP 抗体反而低于CIA(P =0.007)。CCV+K-IA、CCV-IA 产生的抗C域抗体水平均低于CIA(P<0.05)。CCV+K-IA 与CIA 的TNF 高于CCV-IA(P<0.05)。CCV+K-IA 的AKA 阳性率高于其余两组(50% vs CCV-IA 25%、CIA 16.67%)。CCV-IA 和CCV+K-IA 踝关节病理显示轻度滑膜增生,无滑膜血管翳形成及炎性细胞浸润。结论:偶联KLH 的CCit-Vim 短肽不仅具有强的免疫原性,而且具有致关节炎性;与C 比较,CCit-Vim+KLH 能诱导出更高的AKA 阳性率。     

Therapeutic Effect of Emodin on Collagen-Induced Arthritis in Mice

Emodin, an anthraquinone isolated from the Chinese herb Radix et Rhizoma Rhei, has been reported to have anti-inflammatory, antibacterial, and antitumor activities. However, the effect of emodin on collagen-induced arthritis (CIA) has not yet been investigated. The purpose of this study was to investigate whether emodin has a protective effect against collagen-induced arthritis in mice and its possible mechanisms. CIA was induced in mice by immunization with bovine type II collagen. The mice were treated with emodin (5, 10, and 20 mg/kg/day, i.g.) from days 21 to 42 after immunization. The clinical scores and hind paw swelling were evaluated. The expression of prostaglandin E₂ (PGE₂) and cyclooxygenase-2 (COX-2) in synovial tissues was determined. The levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in the plasma were measured by enzyme-linked immunosorbent assay. The results showed that emodin treatment significantly alleviated the severity of the disease, based on the reduced hind paw swelling and clinical scores, compared with untreated CIA mice. Comparing with untreated CIA mice, emodin treatment inhibited the levels of TNF-α and IL-6 in the plasma, PGE₂ production, and COX-2 protein expression in synovial tissues in a dose manner. In conclusion, our results suggest that anti-inflammatory effects of emodin against collagen-induced arthritis in mice may be due to its ability to inhibit pro-inflammatory mediators. Emodin may be a promising potential therapeutic reagent for arthritis treatment.     

Paeoniflorin suppresses inflammatory mediator production and regulates G protein-coupled signaling in fibroblast – like synoviocytes of collagen induced arthritic rats

OBJECTIVE: To investigate the effects of Paeoniflorin (Pae) on inflammatory mediators and G protein - coupled signaling in fibroblast - like synoviocytes (FLS) from collagen induced arthritic (CIA) rats. METHODS: SD rats were injected with type II collagen. Pae (25, 50, 100 mg. kg(-1)) was administered to CIA rats. The inflammation of CIA rats was evaluated by paw swelling, arthritis index and histopathology of joints. FLS were isolated and cultured. Interleukin (IL)-1 activity was measured by the (3)H-TdR - intake method Tumor necrosis factor alpha (TNF-alpha), prostaglandin E(2) (PGE(2)) and cAMP were measured by radioimmunoassay. Protein kinase A (PKA) was assessed by luminescent kinase assay. Gi was detected by Western blot. RESULTS: Inflammation in CIA rats was accompanied by hyperplastic synovium, pannus and cartilage erosion in joints. IL-1 activity and Gi expression increased, PGE(2) and TNF-alpha production were enhanced, but cAMP level and PKA activity decreased. Pae significantly suppressed the inflammatory response and inflammatory mediators (IL-1, TNF-alpha and PGE(2)) in vivo. Pae inhibited Gi expression and restored cAMP level and PKA activity in FLS of CIA rats in vivo and vitro. CONCLUSION: Inflammatory mediators and G protein - coupled signaling were associated with the pathogenesis of synovitis in CIA rats. Pae, as a new monomer, had anti-inflammatory effects on the animal model of CIA in rats, but also had regulatory effects on FLS from CIA rats in vitro. These results highlight Pae as a good candidate for therapeutic intervention in RA.     

Immunotherapy of experimental arthritis. Analysis of the articular cartilage of mice suppressed for collagen-induced arthritis by a T-cell hybridoma.

The present study elucidates the suppression of collagen-induced arthritis (CIA) by T-suppressor cells through the analysis of the joints and articular cartilage of mice suppressed for CIA by a T-cell hybridoma. T-cell hybridomas (T101N and T104B1) were derived from the somatic cell fusion of splenic and thymic cells of mice suppressed for CIA and the AKR BW 5147 thymoma cell line. CIA mice administered 1 X 10(5) T101N hybridoma cells intravenously were observed to have reduced hind paw pathology scores as well as reduced edema, compared with CIA mice or CIA mice administered 1 X 10(5) cells of a control T-cell hybridoma, T104B1. The hind paw articular cartilage of joints from mice with CIA administered T101N cells resembled normal joint architecture in histologic staining and alignment of articular cartilage surfaces. The histopathology observed in joints of mice administered T104B1 hybridoma cells resembled that of CIA mice with large pannus formation, fibrous bridging of the joint, soft-tissue metaplasia, and joint disorganization. The data indicate that specific T-cell hybridoma cell lines can modulate the joint histopathology observed in CIA to resemble the joint architecture of noninflammatory joints.     

小鼠对雨蛙肽诱发急性胰腺炎易感性的品系差异

目的: 小鼠是常用于胰腺炎研究的模式动物。然而不同品系的小鼠似乎对胰腺炎有着不同的易感性,但缺乏系统的对比研究。我们因此比较了C57BL/6J、BALB/c和ICR 小鼠对雨蛙肽诱导的急性胰腺炎易感性的差异。方法: 2月龄C57BL/6J、BALB/c和ICR雌鼠各12只,随机分为实验组(n=6)和对照组(n=6)。实验组予以腹腔注射雨蛙肽(50 μg/kg),每小时1次,共注射7次;对照组同法注射等量生理盐水。分别于第1次注射后的0、3、6、9、12、24 h取小鼠血浆,并于24 h处死小鼠取其胰腺组织。测定各组小鼠的血浆淀粉酶和脂肪酶活性;观察其胰腺组织的病理形态学改变程度及炎症因子表达水平。结果: 雨蛙肽诱导胰腺炎后,BALB/c和ICR小鼠的血浆淀粉酶和脂肪酶水平较C57BL/6J小鼠升高更为显著;C57BL/6J小鼠与其它2种品系的小鼠相比,其胰腺病理形态学改变程度较轻,胰腺组织炎症因子表达水平也较低。结论: BALB/c和ICR小鼠对雨蛙肽诱导的急性胰腺炎的敏感性比C57BL/6J小鼠高。这一结果对急性胰腺炎研究中小鼠品系的合理选择具有指导意义。     

通痹灵总生物碱抑制关节破坏的细胞免疫作用机制

目的:证实通痹灵总生物碱抑制胶原诱导型关节炎(CIA)大鼠关节炎症和关节破坏的药效,揭示其调节细胞免疫的作用机制。方法:牛II型胶原混合弗氏不完全佐剂于SPF级Wistar大鼠尾根部注射,足肿后随机分为造模加生理盐水组,甲氨蝶呤(MTX)治疗组和通痹灵总生物碱(以马钱子碱和士的宁为主的生物碱,TBL总碱)治疗组,容积法评价后肢肿胀度;给药后35d,乳腺钼靶机大鼠全身摄片,计分法评价各组大鼠X光片四肢96块骨侵蚀和100个关节间隙变化;处死动物,取左前肢和右后肢近端第3足趾关节苏木素-伊红(HE)染色,评价中性粒、淋巴细胞、浆细胞浸润、滑膜细胞增生的情况;MTT法检测TBL总碱对Jurkat细胞增殖的影响;Western blot检测TBL总碱对Jurkat细胞ERK1/2磷酸化的影响。结果:造模成功后,MTX和TBL总碱给药35d都可明显抑制大鼠关节肿胀和关节破坏,与造模加生理盐水组有统计学差异(P<0.01);TBL总碱可明显抑制CIA淋巴细胞、浆细胞的浸润和滑膜增生(P<0.05),MTX可抑制滑膜增生。TBL总碱可显著抑制T细胞增殖以及ERK1/2的磷酸化。结论:TBL总碱具有抑制关节免疫炎症和关节破坏的作用,其机制可能是通过干预T细胞MAPK信号转导,抑制T细胞的活化和增殖来实现的,为TBL总碱治疗RA提供了实验依据。     

p38 MAP Kinase inhibitor]

FR167653 is a potent inhibitor of p38 MAP Kinase and inhibits TNF-alpha and IL-1beta production in inflammatory cells. In this study we investigated the effect of FR167653 on CIA. CIA rats were subcutaneously injected with FR167653 (32 mg/kg/day) starting on the day of the booster injection and after the onset of arthritis in the prophylactic and therapeutic treatment groups, respectively. The hind paw swelling, radiolographic and histologic scores, and osteoclast number were evaluated. Serum and tissue cytokine levels were assessed by ELISA. Flow cytometric analysis of T-lymphocytes from bone marrow was also performed. The effect of FR167653 on in vitro osteoclast formation induced by sRANKL and TNF-alpha was examined. Hind paw swelling occurred in CIA rats but not in the prophylactic treatment group. Therapeutic treatment also significantly reduced the paw swelling. The mean radiographic, histologic score, and osteoclast number of the treatment group were significantly lower than those of CIA rats without treatment. FR167653 treatment reduced serum TNF-alpha and IL-1beta levels, ankle IL-1beta concentration, and CD4-CD8a+ T-cell population in bone marrow. Furthermore, FR167653 inhibited the osteoclast-like cell differentiation induced by both sRANKL and TNF-alpha in vitro. FR167653 prevented the onset of arthritis in a prophylactic treatment model and suppresses the progression of joint destruction in a therapeutic treatment model, suggesting that p38 MAP Kinase is a potential therapeutic target for rheumatoid arthritis.     

Experimental Leishmania (L.) amazonensis leishmaniasis: characterization and immunogenicity of subcellular fractions

A technique developed in Trypanosoma cruzi biochemical studies was successfully used to fractionate Leishmania (Leishmania) amazonensis promastigotes. Ultrastructural analyses revealed a membrane fraction (MF) associated to subpellicular microtubules, a ribosomal-rich microsomal fraction (MicF), and a flagellar fraction (FF) free of associated membrane. All fractions proved to be immunogenic through delayed type hypersensitivity reaction assays. Therefore, a protocol was designed to test whether these fractions could elicit a protective response in mice infected by L. (L), amazonensis. The protocol consisted of a BCG injection (as cellular immunity inducer), followed by cyclophosphamide (once its cytotoxic effect is over, this immunosupressor can increase the number of circulating leukocytes), then an injection with one of the fractions followed by a challenge. When compared to infected control animals, mice injected with any of the fractions presented a smaller footpad swelling, especially those injected with MicF or FF. Macroscopically, immunized mice under modulation by BCG presented no swelling. Histopathological studies performed on day 120 revealed fewer amastigotes and more intense inflammation in lesions of MicF and FF injected mice. Animals injected with MF presented an intermediate pattern. Parasite quantification corroborated these results. The results show that all fractions are potent immunostimulators, but MicF and FF have the strongest protective ability.     

Studies on type II collagen induced arthritis in mice

A consistent and reproducible polyarthritis was induced in mice by immunizing them with type II collagen in Complete Freunds adjuvant (CFA) and Bacillus Calmette-Guerin (BCG) vaccine. Several inbred strains of mice were investigated for the ability to develop collagen induced arthritis (CIA). DBA/1 mice (H-2q) produced the highest incidence and the most severe arthritis of all the strains examined. Viable BCG vaccine was essential for the induction of a reproducible disease in this strain. The effects of some anti-inflammatory and anti-rheumatic compounds were examined on the developing and established lesions of CIA. These effects were determined by assessing the paw inflammation using a subjective scoring system and measuring foot weight. Furthermore, levels of serum amyloid P component (SAP) were also determined.Benoxaprofen, cyclophosphamide, indomethacin and prednisolone inhibited the paw inflammation in the developing disease whilst the anti-rheumatic compounds auranofin and D-penicillamine exacerbated the paw inflammation. Cyclophosphamide and prednisolone inhibited the established lesions but only prednisolone prevented the development of further lesions in the established disease. The SAP levels in the prednisolone treated group were also reduced. Auranofin treatment exacerbated the inflammation of both the established and the developing lesions in the same animal. D-penicillamine was inactive in the established disease.     

Oestrogen exhibits type II collagen protective effects and attenuates collagen-induced arthritis in rats

As anti-inflammatory treatments used in rheumatoid arthritis, such as glucocorticoids, often result in secondary detrimental effects on bone health, the objective of this study was to investigate the effects of oestrogen therapy (ET) on the development and activity of collagen-induced arthritis (CIA) in rats, with a focus on assessment of chondroprotective effects using biomarkers of type II collagen degradation. Forty female Lewis rats were allocated into four intervention groups: (i) control + vehicle; (ii) CIA + vehicle; (iii) CIA + ET; and (iv) CIA + prednisolone. During the 28-day intervention period we monitored body weight, time-point of disease onset, incidence of manifest disease and paw volume. Levels of the type II collagen degradation marker (CTX-II) were measured in serum. At euthanasia, hind paws were isolated, extracted for proteins and measured for the concentration of CTX-II. Matrix metalloproteinase (MMP) activity was evaluated using gelatinase zymography. Oestrogen treatment delayed the time-point of disease onset and reduced the incidence and degree of manifest immunoarthritis significantly, assessed by macroscopic evaluation of hind paw inflammation and paw volume. Measures of serum or tissue levels of CTX-II showed significantly reduced type II collagen degradation elicited by oestrogen treatment. In alignment, a decreased activity of MMP-2 and MMP-9 was found in the paw protein extracts. We have demonstrated that the anti-inflammatory effect of ET is linked to chondroprotective effects in an animal model of systemic immunoarthritis. As ET has positive rather than negative effects on bone health in contrast to prednisolone, these observations may be important for potential combination therapy.     

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-α in the induction of endothelin system genes

Objective

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice.

Treatment

CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100?mg/kg) once a day, starting from the day when arthritis was clinically detectable.

Methods

CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student’s t test.

Results

Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1β, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes.

Conclusion

These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.     

Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice

BALB/c male mice were mated with either Swiss-Webster or MF1 females to produce first generation cross-bred offspring. Hybridoma cell lines, from the fusion of P3-NS1-Ag4/1 myeloma cells with spleen cells sensitised to the porcine coronavirus causing transmissible gastroenteritis, were injected intraperitoneally into these mice to produce ascitic fluid containing monoclonal antibodies. Mice of 11 weeks of age weighing between 26 and 34 g were used. The volume of ascites produced by mice injected with four of the five hybrid cell lines tested was greater in the cross-bred offspring than in the BALB/c parent. The fifth cell line gave comparable volumes in the MF1 cross-breed and BALB/c parent but a lesser volume in the Swiss-Webster cross-breed. The antibody titres of the ascites as determined by virus neutralisation, radioimmune and indirect immune fluorescence assays, did not differ significantly between mouse types. The ability to use all offspring from a litter of cross-bred mice, irrespective of sex, and the increased volume of ascitic fluid formed in each mouse, permits fewer animals to be used for the production of ascites in these strains, thereby offering considerable economic and ethical advantages over the use of BALB/c mice.