Generation of autoreactive CTL by tumour vaccines containing foreign T helper epitopes.

The aim of this study was to evaluate the effect of including a foreign T helper cell epitope in vaccines designed for generation of CTL against self-antigens and for inhibition of tumour growth. Two different vaccine designs were composed, a minimal epitope vaccine and a modified full length self-antigen, both based on OVA containing either a colinearily synthesized or an inserted Th-epitope, respectively. These vaccines were used for immunization of tolerant OVA transgenic mice (RIP-OVA(low)) and non-tolerant C57BL/6 mice. First, it was shown that transgenic mice were tolerant to OVA in the CD4 compartment. Secondly, only the vaccines containing the foreign Th-epitope and not the wild-type constructs were able to induce self-reactive CTL in the transgenic mice. Thirdly, these self-reactive CTL induced by the Th-epitope modified constructs also inhibited tumour growth in the OVA transgenic mice. Overall, these results demonstrate that inclusion of a foreign Th-epitope circumvents the tolerance in this OVA transgenic strain. In addition, these results show the importance of including strong T-cell help in cancer vaccines.

In situ depletion of CD4+ T cells in human skin by Zanolimumab.

CD4(+) T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4(+) T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, kappa) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4(+), but not CD8(+) CD3(+) T cells. The capacity of Zanolimumab to deplete the CD4(+) T cells in the skin may be of importance in diseases where CD4(+) T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.

Mendelian analysis of a metastasis-prone substrain of BALB/c nude mice using a subcutaneously inoculated human tumour.

Most nude mice do not allow the formation of metastases after heterotransplantation of human malignant tumours. Here we describe a substrain of BALB/c nude mice (BALB/c/AnNCr) that reproducibly allows some human cancers to metastasize. By Mendelian analysis of hybrids between this substrain and C57BL/6J +/+ mice we found that the ability to allow a human tumour (MDA-MB-435 BAG) to express its metastatic phenotype is determined by a recessively inheritable trait in the mouse host. We are presently working to identify the genetics responsible for development of metastases. The study also includes immunohistochemical and electron microscopic analysis of the test tumour, originally assumed to be a human mammary carcinoma, but shown to possess characteristics of a malignant melanoma (1). The ultimate aim of our ongoing study is to establish a substrain of nude mice that will allow metastasis in all recipients.

Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA radiation.

BACKGROUND: Apoptosis has been proposed to act as an important mechanism for eliminating keratinocytes that have been irreversibly damaged by ultraviolet (UV) irradiation. One way to induce apoptosis in keratinocytes is through activation of the cell surface receptor Fas (CD95), either with the ligand (FasL) or directly with UV radiation. OBJECTIVES: To investigate the regulation of Fas and FasL expression in human skin and the formation of apoptotic cells after in vivo exposure to UVB or long-wave UVA radiation. METHODS: Volunteers were irradiated with either 3 minimal erythema doses (MED) of UVB (n = 6) or 3 MED of long-wave UVA (n = 6) on buttock skin 12, 24 and 72 h before skin punch biopsies were taken. Expression of Fas and FasL was demonstrated by immunohistochemistry on cryostat sections. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labelling reaction. RESULTS: In five of six subjects, exposure to UVB radiation resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 24 and 72 h after irradiation. In all subjects, exposure to long-wave UVA resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 12 h after irradiation. In five of six subjects, exposure to UVB radiation resulted in temporarily decreased expression of FasL, but after 72 h the expression of FasL had returned to the preirradiation level. The expression of FasL on epidermal cells after exposure to long-wave UVA showed considerable variation. UVB irradiation was a stronger inducer of epidermal apoptosis than was UVA irradiation. The number of apoptotic epidermal cells did not correlate with expression of Fas or FasL. CONCLUSIONS: In human skin the expression of Fas on epidermal cells increases after in vivo exposure to UVB or long-wave UVA. Exposure to UVB causes a temporary decrease in the expression of FasL on epidermal cells.

Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response.

Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases. Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection. To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re-infected with P. aeruginosa 14 days after the initial infection. Singly-infected BALB/c mice, as well as non-infected mice, were used as controls. Decreased mortality and milder lung inflammation in re-infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly-infected mice. The improved outcome in re-infected mice correlated with changes in CD4 cell numbers. Surface expression of LFA-1 on pulmonary CD4 cells was increased in re-infected compared with singly-infected mice. Moreover, resistance to re-infection was paralleled by a shift towards a Th1-dominated response and increased IL-12 production. No significant increase in serum IgG was observed in the re-infected mice. In conclusion, these results indicate a protective role for a Th1-dominated response, independent of antibody production, in chronic P. aeruginosa lung infection in CF.

Urokinase plasminogen activator is localized in stromal cells in ductal breast cancer.

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade that facilitates cancer invasion through degradation of the extracellular matrix, and high levels of uPA in human breast cancer tissue correlate with poor prognosis. We previously found that, in ductal breast cancer, uPA mRNA is highly expressed by myofibroblasts surrounding invasively growing cancer cells. However, the localization of uPA protein has not been settled in the published literature. Because uPA is a secreted molecule, it could conceivably be localized differently from its mRNA. We have studied the localization of uPA immunoreactivity in detail. Twenty-five cases of invasive ductal carcinoma were analyzed with three different uPA antibody preparations, all of which gave an essentially identical stromal staining pattern. Using double immunofluorescence, we identified uPA immunoreactivity in myofibroblasts and macrophages in all cases examined. Additionally, in approximately half of the tumors, we saw uPA staining of endothelial cells. In 3 of the 25 cases, a small subpopulation of the cancer cells was uPA-positive. We conclude that uPA immunoreactivity is almost exclusively associated with stromal cells, which thus play a major role in generation of proteolytic activity in ductal breast cancer.

The metastasis-associated Mts1(S100A4) protein could act as an angiogenic factor.

The involvement of Mts1(S100A4), a small Ca(2+)-binding protein in tumor progression and metastasis had been demonstrated. However, the mechanism by which mts1(S100A4) promoted metastasis had not been identified. Here we demonstrated that Mts1(S100A4) had significant stimulatory effect on the angiogenesis. We detected high incidence of hemangiomas--benign tumors of vascular origin in aged transgenic mice ubiquitously expressing the mts1(S100A4) gene. Furthermore, the serum level of the Mts1(S100A4) protein increased with ageing. Tumors developed in Mts1-transgenic mice revealed an enhanced vascular density. We showed that an oligomeric, but not a dimeric form of the Mts1(S100A4) protein was capable of enhancing the endothelial cell motility in vitro and stimulate the corneal neovascularization in vivo. An oligomeric fraction of the protein was detected in the conditioned media as well as in human serum. The data obtained allowed us to conclude that mts1(S100A4) might induce tumor progression via stimulation of angiogenesis.

Occlusion versus air exposure on full-thickness biopsy wounds.

The benefits of moisture-retaining dressings on wound healing are well documented in experimental animal models but not in humans. To examine the effect of occlusion, the effects of three brands of synthetic occlusive dressings (Comfeel Plus, DuoDerm CGF, OpSite) were compared with air exposure in epithelial resurfacing and proliferation in acute, full-thickness skin wounds in humans. In 10 healthy males, four 4 mm standardised wounds were made with a sterile punch biopsy on each lower extremity. Epithelialisation of the wounds was assessed histologically and blindly postwounding on days 7 and 14. Wound margin epidermal proliferation was evaluated immunohistochemically with Ki67. Epithelial percentage coverage increased significantly (p = 0.007) with the occlusive dressings (62 +/- 6%, mean +/- SEM), compared with air exposure, (39 +/- 7%) on day 7 but not on day 14 (p = 0.500). Epidermal cell proliferation showed no significant intergroup difference on either day. Treatment with occlusive dressings increased early epithelial migration of acute full-thickness biopsy wounds compared with air exposure in healthy men.

Inhibition of Huntington synthesis by antisense oligodeoxynucleotides.

The Huntington disease gone encodes the protein huntington, which is widely expressed during embryonic development and in mature tissues. In order to elucidate the physiological function of huntington, which so far is unknown, we intend to study the effect of antisense down-regulated huntington expression. We have found an inhibiting effect of a phosphorothioated oligodeoxynucleotide (PS-ODN) added to the culture medium of embryonic teratocarcinoma cells (NT2) and postmitotic neurons (NT2N neurons) differentiated from the NT2 cells. Specific inhibition of expression of endogenous huntington was achieved in NT2N neurons in the concentration range of 1-5 microM PS-ODN, whereas no inhibition was obtained in NT2 cells. We describe in detail the selection of the target sequence for the antisense oligo and the uptake, intracellular distribution, and stability of the antisense PS-ODN in the two cell types. Antisense down-regulation of huntington in this model of human neurons represents a suitable approach to study its normal function.

CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene.

An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its growth in a T cell-competent host adapts to the T cell system through a selective elimination of highly immunogenic tumour cells that are susceptible to CD8+ T cell-mediated lysis.

Cystic vestibular schwannoma--clinical and experimental studies.

The cystic variant of vestibular schwannoma (VS) presents a therapeutic dilemma. Several studies have previously demonstrated that the surgical outcome in this tumour entity is less favourable than that of solid tumours of comparable size. The "wait and scan" policy has not been recommended for these tumours, as the cystic elements expand, causing displacement of the brainstem and compression of the 4th ventricle, resulting in hydrocephalus. The large tumour size at diagnosis and the cystic contents do not support the role of radiosurgery as a therapeutic option. We have previously published the surgical outcome of 23 cystic VS. The present study includes 44 patients (44 cystic tumours) in a series of 773 tumours (5.7%) who underwent surgery in the period 1976 to 1996. This paper presents the neuroradiological and histological features of the tumours, as well as the results of tumour specimen implantation and surgery in athymic nude mice. Therapeutic options are also discussed.

Histologic, cytologic, and bacteriologic examinations of experimentally induced Salmonella typhimurium infection in Lewis rats.

BACKGROUND AND PURPOSE: Histopathologic changes, cellular composition, and bacterial spreading were studied in rat spleen after experimentally induced infection with Salmonella typhimurium. METHODS: Lewis rats were inoculated intraperitoneally with 10(6) bacteria. Spleen weight, cell numbers, and cell surface markers were studied together with histopathologic changes, and expression of inducible nitric oxide synthase (iNOS). The spread of bacteria to blood, spleen, liver, mesenteric lymph nodes, lung, and kidney was studied at 12 hours, and 1, 3, 7, 14, and 28 days after inoculation. RESULTS: Experimentally induced infection caused an increase in spleen weight and leukocyte numbers, and a decrease in CD49d, on postinoculation days (PID) 3 through 7. Numerous granulomas were disseminated throughout the splenic red pulp also on PID 3 through 7. From PID 14 on, clearance of cellular exudate and regeneration of tissue structure were observed. Massive expression of iNOS was seen on PID 3. Bacterial growth was observed in liver and spleen from 12 hours to 14 days after inoculation. Bacteria were detected in blood on PID 3 and mesenteric lymph nodes were infected from PID 3 through 14. CONCLUSIONS: Salmonella typhimurium was rapidly taken up by the reticuloendothelial system. The infection induced weight increase and reversible changes in the spleen, peaking on PID 3 with granuloma formation and infiltration with macrophages. On PID 3, extensive production of iNOS within the granulomas was observed, suggesting initial killing of phagocytosed bacteria, followed by bacterial clearance and tissue regeneration. Cell surface marker expression on CD4+ T cells indicated no change in their numbers; however, there was a time-dependent change in expression of CD49d.

Effect of photodynamic therapy on a heterotransplanted human parotid tumor.

To evaluate the effect of photodynamic therapy on human parotid tumors we used tumor specimens obtained from parotid surgery on a consecutive group of patients. The tumors were transplanted into a subcutaneous pocket of nude mice. The original human tumors were pleomorphic adenoma (four), adenolymphoma (one), acinic cell carcinoma (one), sarcoma (one) and low-grade adenocarcinoma (one). The most aggressive growth was seen in the low-grade adenocarcinoma. We re-implanted this tumor on ten mice bilaterally, and treated the tumors with photodynamic therapy (PDT), resulting in a mean depth of tumor necrosis of 5.4 mm (1-10 mm). In three cases we found vital tumor cells in the periphery of the tumor after treatment, with several new blood vessels in the surrounding tissue, indicating a great potential for neo-angiogenesis in this tumor. In order to evaluate the possible nerve damage subsequent to the photodynamic therapy, the ischiadic nerve in 24 lower limbs of nude mice were investigated. In one case only the macroscopical and histological investigation revealed signs of nerve damage. The current study demonstrates that the nude mice implantation model is excellent to investigate growth in both malignant and benign parotid tumors, and to test new therapeutic modalities. Photodynamic therapy seems to have a possible role in the future management of the malignant lesions of the parotid gland, in cases where radical surgery for some reason is not achievable.

Effects of Radix Angelicae sinensis and shuanghuanglian on a rat model of chronic Pseudomonas aeruginosa pneumonia.

OBJECTIVE: To study the effect of two kinds of Chinese herbal medicine, Radix angelicae sinensis (RAS) ([Chinese characters: see text]) and Shuanghuanglian (SHL) ([Chinese characters: see text]) on chronic Pseudomonas aeruginosa (PA) lung infection in a rat model mimicking cystic fibrosis (CF). METHODS: Rats were divided into RAS, SHL and control groups. All rats were challenged intratracheally with alginate embedded PA and the treatments with herbal medicine started on the same day of challenge. The drugs were administered subcutaneously once a day for ten days and the control group was treated with sterile saline. The rats were sacrificed two weeks after challenge. RESULTS: Significantly improved lung bacterial clearance (P < 0.05, P < 0.01) and milder macroscopic lung pathology (P < 0.005) were found in the two treated groups compared to the control group. In the SH treated group, the neutrophil percent in the peripheral blood leukocytes (P < 0.05), the anti-PA IgG level in serum (P < 0.05), the incidence of lung abcesses (P < 0.005) and the incidence of acute lung inflammation (P < 0.05) were significantly lower than in the control group. The RAS treatment reduced fever (P < 0.05), decreased the incidence of lung abcesses (P < 0.005) and lung mast cell number (P < 0.05), and lowered anti-PA IgG1 level in serum (P < 0.05) when compared to the control group. The anti-PA bacterial activity test in SHL was weakly positive whereas in RAS it was negative. CONCLUSION: The treatment with both herbal medicines could increase the resistance of the rats against PA lung infection and they therefore might be potential promising drugs for stimulation of the immnune system in CF patients with chronic PA lung infection.

Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis.

Neutrophil-borne heparin-binding protein (HBP) is a multifunctional protein involved in the progression of inflammation. HBP is stored in neutrophil granules and released upon stimulation of the cells in proximity to endothelial cells. HBP affects endothelial cells in multiple ways; however, the molecular and cellular mechanisms underlying the interaction of HBP with these cells are unknown. Affinity isolation and enzymatic degradation demonstrated that HBP released from human neutrophils binds to endothelial cell-surface proteoglycans, such as syndecans and glypican. Flow cytometry indicated that a significant fraction of proteoglycan-bound HBP is taken up by the endothelial cells, and we used radiolabeled HBP to determine the internalization rate of surface-bound HBP. Confocal and electron microscopy revealed that internalized HBP is targeted to perinuclear compartments of endothelial cells, where it colocalizes with mitochondria. Western blotting of isolated mitochondria from HBP-treated endothelial cells showed that HBP is present in 2 forms - 28 and 22 kDa. Internalized HBP markedly reduced growth factor deprivation-induced caspase-3 activation and protected endothelial cells from apoptosis, suggesting that uptake and intracellular routing of exogenous HBP to mitochondria contributes to the sustained viability of endothelial cells in the context of locally activated neutrophils.

Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3.

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.

Cancer invasion and tissue remodeling--cooperation of protease systems and cell types.

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non-neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI-1 and MMPs) can be expressed by either the non-neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non-neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family.

Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc-transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.

Early immune response in susceptible and resistant mice strains with chronic Pseudomonas aeruginosa lung infection determines the type of T-helper cell response.

Most cystic fibrosis (CF) patients become chronically infected with Pseudomonas aeruginosa in the lungs. The infection is characterized by a pronounced antibody response and a persistant inflammation dominated by polymorphonuclear neutrophils. Moreover a high antibody response correlates with a poor prognosis. We speculated that a change from this Th2-like response to a Th1-like response might decrease the lung inflammation and thus improve the prognosis in CF patients. To investigate this, we infected C3H/HeN and BALB/c mice intratracheally with P. aeruginosa. In addition, we studied the early immune response leading to different Th responses. Mortality was lower in the C3H/HeN mice (p<0.005), they cleared the bacteria faster (day 3 p<0.01, day 7 p<0.02), had a milder lung inflammation (day 7 p<0.01, day 14 p< or =0.0005) and had a Th1-like IgG subclass switch. At day 3, the C3H/HeN mice produced less NO and TNF-alpha, (p<0.01 and p<0.03) and had the lowest IL-10/IL-12 ratio (p< or =0.05). At day 7, the C3H/HeN mice had the highest IFN-gamma (p<0.02), and the lowest IL-4 (p<0.02) production in the lungs. In conclusion, these results show that the Th1-reacting C3H/HeN mice with chronic P. aeruginosa lung infection have a better disease outcome compared to the Th2-reacting BALB/c mice, indicating that a Th1 response might be beneficial in CF patients with chronic P. aeruginosa lung infection.

Serological diagnosis of experimental Enterococcus faecalis endocarditis.

A modified rat model of endocarditis with catheterization for 2 days was established in female Lewis rats using different inocula of Enterococcus faecalis (strain no. EF 19) in order to measure IgG antibodies in serum during the course of infection. Increasing the inocula intravenously resulted in an increase in the CFU/g vegetation and the CFU/g spleen, the ID50 being about 10 CFU/ml and the ID90 about 1x10(2) CFU/ml. The lowest bacterial inoculum infecting 100% of the rats was 3x10(3) CFU/ml, and for further investigations we used this inoculum size. Rats were sacrificed on day 2, 5, 7, 9, 11 and 28 after infection. The CFU/g vegetation and the CFU/g spleen increased until day 7 and then decreased. Serum samples were collected from 129 rats at different times after challenge. Three different ELISA systems were established to measure the IgG antibody responses: E. faecalis sonicate ELISA (a pool of four sonicates of strain no. EF 10, EF 11, EF 19 and EF 48), E. faecalis whole cell ELISA (strain no. EF 19) and E. faecalis purified cell wall ELISA (strain no. EF 19). An IgG antibody response was detected already on day 2, and except for a minor decrease on day 6/7 the antibody response continued to increase until day 14 (whole cell ELISA and sonicate ELISA) and day 21 (purified cell wall ELISA) when a plateau was reached. Significant increases in IgG antibody responses (p<0.05) were found between groups of rats from days 0-2, 2-8/9 and 8/9-14 in the E. faecalis whole cell and sonicate ELISAs and from days 0-2, 2-10/11 and 10/11-21 in the E. faecalis purified cell wall ELISA. In conclusion, we established a model of endocarditis in rats with catheterization for 2 days and were able to demonstrate an increase in IgG antibodies during the course of infection.