Directing HER4 mRNA expression towards the CYT2 isoform by antisense oligonucleotide decreases growth of breast cancer cells in vitro and in vivo.

BACKGROUND: The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4. METHODS: We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells. RESULTS: Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P<0.0001). In addition, the SSO treatment induced a decreased activity of Akt. We confirmed the SSO-dependent switching of the HER4 isoform CYT1 to CYT2 expression in a xenografted mouse tumour model driven by subcutaneously injected MCF7 cells. We hence demonstrated the feasibility of SSO-directed splice-switching activity in vivo. Furthermore, the SSO treatment efficiently decreased the growth of the xenografted tumour (P=0.0014). CONCLUSION: An SSO directing the splicing of HER4 towards the CYT2 isoform has an inhibitory effect of cancer cell growth in vitro and in vivo. These results may pave the way for the development of new anticancer drugs in HER4-deregulated cancers in humans.

Diabetes-induced changes in mannan-binding lectin levels and complement activation in a mouse model of type 1 diabetes.

Circulating mannan-binding lectin (MBL) levels are elevated in type 1 diabetes. Further, high MBL levels are associated with the development of diabetic nephropathy. In animals, a direct effect of MBL on diabetic kidney changes is observed. We hypothesized that MBL levels and detrimental complement activation increase as a consequence of diabetes. We measured plasma MBL before and 7 weeks after inducing diabetes by streptozotocin. Mice have two MBLs, MBL-A and MBL-C. Diabetes induction led to an increase in MBL-C concentration, whereas no change during the study was found in the control group. The increase in MBL-C was associated with the increasing plasma glucose levels. In accordance with the observed changes in circulating MBL levels, liver expression of Mbl2mRNA (encoding MBL-C) was increased in diabetes. Mbl1expression (encoding MBL-A) did not differ between diabetic and control animals. The estimated half-life of recombinant human MBL was significantly prolonged in mice with diabetes compared with control mice. Complement activation in plasma and glomeruli did not differ between groups. We demonstrate for the first time that MBL levels increase after induction of diabetes and in parallel with increasing plasma glucose. Our findings support the previous clinical observations of increased MBL in type 1 diabetes. This change may be explained by alternations in both MBL production and turnover.

Histological changes in chronic autoimmune SKG-arthritis evaluated by quantitative three-dimensional stereological estimators.

OBJECTIVES: To investigate the quantitative arthritic and bone erosive changes, including the number of osteoclasts and osteoclast precursors in the new SKG-model of inflammatory polyarthritis using three-dimensional (3D) stereological methods. METHODS: Arthritis was induced in female SKG-mice with Zymosan A. Quantitative histology was made in four control mice and four mice with arthritis euthanised after 6 and 12 weeks. The right hind paw was embedded undecalcified in methylmethacrylate and cut exhaustively generating vertical uniform random sections. A computer controlled microscope and stereological software was used for histological quantification. Total volumes were estimated according to the Cavalieri principle, total surfaces were estimated using the vertical sections design, and the number of osteoclasts was counted in a physical fractionator. RESULTS: The arthritis score increased during the 12-week period and was paralleled by an increase in the volume of inflammatory tissue (r=0.96, p<0.001). The number of osteoclasts on bone (r=0.77, p<0.05) and osteoclast-covered bone surface (r=0.62, p<0.05) increased resulting in a decrease in the volume of bone (r=-0.65, p<0.05). However, the number of osteoclast precursors declined between week 6 and 12 (p<0.05). Furthermore, the total cartilage surface (r=-0.74, p<0.05) and cartilage volume (r=-0.74, p<0.05) decreased during the 12 weeks of arthritis. CONCLUSIONS: In this study we demonstrated changes in 3D stereological parameters of inflammatory tissue, bone erosion, osteoclasts, and cartilage in mouse paws during the course of arthritis in the SKG mouse. This is the first time 3D quantitative histology has been applied in a mouse model of rheumatoid arthritis.

Welfare assessment in porcine biomedical research - suggestion for an operational tool.

In recent years, increasing interest in using the pig (Sus scrofa) for biomedical research has become evident. Today, the pig is considered an advantageous alternative animal model for various human diseases and conditions. However, even though a considerable amount of biomedical research has been done on pigs, hardly any studies include systematic welfare assessment. Still, it is essential to assess welfare of laboratory pigs, both domestic pig breeds and smaller purpose-bred breeds, as (1) scientific obligations entail responsibility to ensure and document a fair welfare standard for animals used for experimental purposes; and (2) the scientific outcome can be dependent upon the welfare state of the animals. In order to be able to quantify and control laboratory pig welfare, a practical tool is needed. The purpose of the present paper is to provide an overview of the current status of the extent of welfare assessment in pigs used in biomedical research and to suggest a welfare assessment standard for research facilities based on an exposition of ethological considerations relevant for the welfare of pigs in biomedical research. The tools for porcine welfare assessment presented suggest a method for monitoring the welfare status of individual laboratory pigs, intended to relieve the practical scoring of the welfare of individual pigs as well as the interpretation of the findings.

Interleukin-20 plays a critical role in maintenance and development of psoriasis in the human xenograft transplantation model.

BACKGROUND: Interleukin (IL)-20 is a recently discovered cytokine displaying increased levels in psoriatic lesions. Interestingly, IL-20 levels decrease with antipsoriatic treatment, correlating with clinical improvement. However, the role of IL-20 in the aetiology of psoriasis is unknown. OBJECTIVES: In this study, we investigate the effects both of blocking IL-20 signalling in psoriatic plaques and of adding IL-20 to nonlesional psoriasis skin. METHODS: We employed the human skin xenograft transplantation model in which psoriatic plaques and nonlesional keratome skin biopsies obtained from donors with moderate to severe plaque psoriasis were transplanted on to immuno-deficient mice. The transplanted mice were treated with anti-IL-20 antibodies or recombinant human IL-20. RESULTS: We demonstrate that blocking IL-20 signalling with anti-IL-20 antibodies induces psoriasis resolution and inhibits psoriasis induction. We also demonstrate that continuous IL-20 infusion, together with injection of additional nonactivated leucocytes, promotes induction of psoriasis in nonlesional skin from patients with psoriasis. CONCLUSIONS: The results suggest that IL-20 plays a critical role in the induction and maintenance of psoriasis, and IL-20 is suggested as a new possible specific target in psoriasis treatment.

Microglia and macrophages express tumor necrosis factor receptor p75 following middle cerebral artery occlusion in mice.

The proinflammatory and potential neurotoxic cytokine tumor necrosis factor (TNF) is produced by activated CNS resident microglia and infiltrating blood-borne macrophages in infarct and peri-infarct areas following induction of focal cerebral ischemia. Here, we investigated the expression of the TNF receptors, TNF-p55R and TNF-p75R, from 1 to 10 days following permanent occlusion of the middle cerebral artery in mice. Using quantitative polymerase chain reaction (PCR), we observed that the relative level of TNF-p55R mRNA was significantly increased at 1-2 days and TNF-p75R mRNA was significantly increased at 1-10 days following arterial occlusion, reaching peak values at 5 days, when microglial-macrophage CD11b mRNA expression was also increased. In comparison, the relative level of TNF mRNA was significantly increased from 1 to 5 days, with peak levels 1 day after arterial occlusion. In situ hybridization revealed mRNA expression of both receptors in predominantly microglial- and macrophage-like cells in the peri-infarct and subsequently in the infarct, and being most marked from 1 to 5 days. Using green fluorescent protein-bone marrow chimeric mice, we confirmed that TNF-p75R was expressed in resident microglia and blood-borne macrophages located in the peri-infarct and infarct 1 and 5 days after arterial occlusion, which was supported by Western blotting. The data show that increased expression of the TNF-p75 receptor following induction of focal cerebral ischemia in mice can be attributed to expression in activated microglial cells and blood-borne macrophages.

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.

Experience with mouse hepatitis virus sanitation in three transplantable murine tumour lines.

Transmission of viral infection by tumour lines or other biological materials may have confounding effects on research. Many research organizations require screening for viral agents of all cell lines, tumours, sera and other biologicals before implantation or inoculation into animal models. Screening for viral contamination is done by the mouse antibody production (MAP) test, by cell culture, or alternatively by direct detection of the viral agents by polymerase chain reaction (PCR). The description of procedures for sanitation of infected cell lines or tumours is sparse. The present report describes the procedures used for sanitation of three transplantable murine tumour lines, which were transplanted in vivo in a mouse hepatitis virus (MHV)-infected colony of mice at the Department of Experimental Clinical Oncology (DECO). The tumours were frozen and serially transplanted three times in a quarantine colony of syngenic mice. Serological examination of the mice transplanted with tumours as well as their cage mates in the quarantine colony did not detect any antibodies against MHV. After repeated serial transplantation in seronegative animals, tumour material was frozen and thawed tumours were later used for transplantation into the newly established virus-free colony of mice at DECO. PCR-based detection of MHV did not reveal any contamination of the tumour examined by this technique, indicating that this murine tumour apparently did not transmit MHV or that MHV was eliminated from the tissue so fast after the infection that it could not be transmitted by the tumour tissue. It is concluded that MHV infection of mice with transplantable murine tumours does not necessarily cause the tumours to be contaminated.

Aged human bone marrow stromal cells maintaining bone forming capacity in vivo evaluated using an improved method of visualization.

Age-related decreased osteoblast function is a well-known but poorly understood phenomenon. Previous studies that examined the effects of donor age on osteoblast functions employed in vitro assays that may not reflect the true osteoblast capacity for bone formation. Thus, we have developed an in vivo assay for quantifying the bone forming capacity (BFC) and we compared the BFC of osteoblastic cells obtained from young and old donors. Osteoblasts were obtained from human bone marrow stromal cell cultures and implanted subcutaneously in immuno-deficient mice (NOD/LtSz- Prkdc(scid)). After 8 weeks, the implants were removed and embedded un-decalcified in methyl methacrylate (MMA). Sections were stained histochemically with Goldner's Trichrome stain and immuno-histochemically using human-specific antibodies against known osteogenic markers. Implanted human marrow stromal cells (hMSC) were able to form bone in vivo. The donor origin of bone was verified using several human-specific antibodies. Dose-response experiments demonstrated that 5 x 10(5) hMSC per implant gave the maximal bone formation after 8 weeks. No difference in BFC was observed between cells obtained from young (24-30 years old; mean age 27 +/- 2 years, n = 5) and old (71-81 years old; mean age 75 +/- 4 years, n = 5) donors. Our study demonstrates that the capacity of hMSC to form bone in vivo is maintained with age and suggests that the observed senescence-associated decrease in bone formation is due to a defect in the bone microenvironment, the nature of which remains to be determined.

Growth hormone receptor antagonist administration inhibits growth of human colorectal carcinoma in nude mice.

Increasing evidence has accumulated in support of the hypothesis that growth hormone (GH) and insulin-like growth factors (IGFs) play a role in carcinogenesis. In order to test this hypothesis, female nude mice were xenografted with two different human colorectal cancer cell lines (COLO 205 and HT-29) and randomized to receive placebo or a GH receptor antagonist (GHRA) (B2036-PEG) every second day for 16 days. The tumour volume was measured in each animal throughout the study and by the end of the experiment the tumour weights were recorded. After 16 days of therapy in nude mice with the COLO 205 colorectal cancer, GHRA treatment caused a 39% reduction in tumour volume (p < 0.02) and a 44% reduction in tumour weight (p < 0.01). GHRA treatment equally reduced circulating IGF-I and IGFBP-3 levels, while apoptosis was increased in the treatment group. Expression of IGF-I, IGF-II and the corresponding receptors in COLO 205 tumours was also decreased by the treatment. GHRA had no effect on the growth of the HT-29 colorectal cancer despite pronounced reduction in serum IGF-I. The present study thereby demonstrates a central role for the GH/IGF system in the pathogenesis of some colorectal cancers and suggests that specific GHR blockade may present a new concept in the treatment of colorectal cancer.

A murine vascular endothelial growth factor antibody inhibits in vivo growth of human Caki-I renal adenocarcinoma.

A number of studies have provided indirect evidence that angiogenesis is involved in tumour growth and metastasis formation of renal cell carcinoma (RCC). Nude mice bearing xenografts of human Caki-I RCC were treated i.p. for 3 weeks with a murine monoclonal antibody against vascular endothelial growth factor, VEGF (VEGF-ab). Tumour growth and mRNA expression of human and murine VEGF and murine VE-Cadherin in the tumours were measured. After 3 weeks of therapy, the tumour volume in the control nude mice was 548 +/- 98 mm3 compared to the tumours in the nude mice treated with VEGF-ab (122 +/- 24 mm3, p < 0.01). Treatment with VEGF-ab significantly reduced mRNA expression of murine VEGF-120 and murine VE-Cadherin (p < 0.05 and p < 0.01, respectively). The mRNA expression of human VEGF (hVEGF165, hVEGF189) and murine VEGF (mVEGF164) was unchanged due to antibody treatment. The mean percentage of apoptotic cells in tumours harvested from antibody-treated animals was significantly lower than in tumours from the control-treated animals (p < 0.02). These findings demonstrate for the first time that VEGF-ab significantly inhibit the growth of Caki-1 RCC in vivo.

Influence of surgery and endotoxin-induced sepsis combined on natural killer cell activity, oxidative burst of granulocytes and antigen presentation capability of monocytes.

BACKGROUND: Cell mediated immunity is affected in the course of sepsis and following surgical stress. The natural killer (NK) cells, the granulocytes and the monocytes constitute the immediate unspecific cell mediated immunity. We therefore investigated the effect of surgery- and endotoxin-induced sepsis on NK cells, granulocytes and monocytes in a two-hit model. METHODS: Three groups of 40 mice. Each group was divided into four groups of 10 mice. All the animals were anesthetized and subjected to either: laparotomy; treatment with Escherichia coli endotoxin i.p.; laparotomy followed 20 min later by endotoxin i.p.; or left untreated as a control group. In the first 40 mice the NK cell activity in the spleen and number of NK cells in the liver were measured, in the second the oxidative burst of granulocytes, and in the third the antigen presentation capacity of monocytes. RESULTS: Endotoxin stimulated the NK cell activity and up-regulated the antigen presentation capability on monocytes. In contrast, surgical stress reduced the NK cell activity, the number of NK cells and down-regulated the antigen presentation capability on monocytes. After surgery, followed by administration of endotoxin, the oxidative burst of granulocytes was stimulated while antigen presentation capability on monocytes was down-regulated. Endotoxin prevented or reverted the postoperative suppression of NK cell activity. CONCLUSION: Our two-hit model shows that some cell types of the unspecific immune system exhibit an excessive inflammatory response (NK cells, granulocytes) while specific functions of other cell types (monocytes) are simultaneously diminished. This diversity makes a potential therapeutic immunomodulation very complex as some cell types would need to be down-regulated while others need to be stimulated.

Effect of hemodiafiltration and sepsis on chemotaxis of granulocytes and the release of IL-8 and IL-10.

BACKGROUND: Extracorporeal circulation, such as cardiopulmonary bypass and hemodialysis, has been associated with an activation of the immune system. Continuous veno venous hemodiafiltration (CVVHD) is used in critically ill septic patients. During CVVHD, cytokines are excreted in ultrafiltrate. When the membranes, used in CVVHD, are incubated with leukocytes in vitro a slight production of cytokines is observed. Due to the underlying disease it is difficult to investigate the effect of CVVHD in septic patients. We therefore studied the separate effect of CVVHD on the chemotaxis of granulocytes, the proliferation of lymphocytes and the release of IL-8 and IL-10 in healthy pigs compared to an endotoxin and a control group. METHODS: Thirty-one pigs were anesthetized and mechanically ventilated. CVVHD was performed in 10 pigs. Eleven pigs received an infusion of Escherichia coli endotoxin 30 microg/kg, and 10 pigs served as a control group. The chemotaxis of granulocytes was measured in an assay chamber, and the cytokines IL-8 and IL-10 with an enzyme-linked immunosorbent assay. The adhesion molecules CD18 and CD62 on lymphocytes were measured using monoclonal antibodies, and the lymphocyte proliferation was measured without stimulation and in response to mitogens. RESULTS: CVVHD was accompanied by lymphocytopenia and increased spontaneous lymphoproliferative response, but no change in adhesion molecules on lymphocytes or cytokine levels in plasma, and no decrease in the chemotaxis of granulocytes. Following endotoxin we observed a pronounced lymphocytopenia and an increased secretion of IL-8 and IL-10, a decrease in the expression of CD18 on lymphocytes and in the stimulated lymphocyte proliferation and in the chemotaxis of granulocytes. CONCLUSION: CVVHD does not, in contrast to endotoxin-induced sepsis, influence chemotaxis of granulocytes, the production of IL-8 and IL-10 or the proliferation of lymphocytes.

Tail-vein injection of mannan-binding lectin DNA leads to high expression levels of multimeric protein in liver.

The human plasma protein mannan-binding lectin (MBL) is an essential part of the innate immune defense system. Low levels of MBL are associated with recurrent infections and other clinically significant signs of a compromised immune defense. Previous studies have addressed the possibility of reconstitution therapy by the use of recombinant or plasma-derived protein. Natural MBL is a multimeric protein, which consists of up to 18 identical polypeptide chains. Synthesis by in vitro methods of MBL with the proper multimeric structure is difficult. We here report that mice obtain MBL levels comparable to those found in normal human plasma when injected with an MBL expression construct as naked plasmid DNA contained in a large volume of physiologic salt solution. The expression was confined to the liver and high MBL expression levels were obtained with less than 5% of the liver cells transfected. The multimeric structure of the MBL found in plasma of injected mice was similar to that of natural MBL. Thus, liver expression following injection of naked DNA is an alternative to reconstitution therapy with a protein having a complex quaternary structure.

LDL receptor-GFP fusion proteins: new tools for the characterisation of disease-causing mutations in the LDL receptor gene.

The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of an LDL receptor mutation (W556S) found in FH patients and characterised as transport defective. In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-localisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum. Wild type (WT) and W556S LDL receptor GFP fusion proteins were expressed in mouse liver by means of hydrodynamic delivery of naked DNA. Two days after injection liver samples were analysed for GFP fluorescence. The WT LDL receptor GFP protein was located on the cell surface whereas the W556S LDL receptor GFP protein was retained in intracellular compartments. Thus, the GFP-tagged LDL receptor protein allows both detailed time lapse analysis and evaluations in animals for the physiological modelling of mutations. This method should be generally applicable in functional testing of gene products for aberrant processing.

Characterization of murine dendritic cells derived from adherent blood mononuclear cells in vitro.

The therapeutic potential of dendritic cells loaded with tumour antigens for the induction of effective immune responses against cancer is currently being tested in numerous clinical trials. In most cases, the dendritic cells are generated in vitro from peripheral blood monocytes. Many aspects of dendritic cell-based vaccination have not yet been examined in detail, and homologous mouse model systems may prove very valuable for optimizing clinical procedures. In the murine system, however, dendritic cells are usually isolated from either lymphoid tissues or bone marrow cultures. To date, murine monocyte-derived dendritic cells have been described only sporadically. Here, we describe a culture system for the generation of murine dendritic cells from adherent peripheral blood mononuclear cells by culturing in the presence of granulocyte-macrophage colony stimulating factor and interleukin-4. After 7 days of culture the nonadherent cells were harvested from the cultures. Most of these cells exhibited well-accepted characteristics of mature dendritic cells (e.g. veiled appearance, high expression of major histocompatibility complex class II and CD86) and stimulated vigorous proliferation of allogeneic T cells in a primary mixed leucocyte reaction following stimulation with bacterial lipopolysaccharide. Interestingly, staining the cells for expression of the putative antigen-uptake receptor DEC-205 revealed a distinct bimodal distribution.

The effect of CVVHD and endotoxin on the oxidative burst, adhesion molecules and distribution in tissues of granulocytes.

OBJECTIVE: Extracorporeal circulation, such as cardiopulmonary bypass and haemodialysis, has been associated with an activation of the immune system, especially the granulocytes. Continuous veno-venous haemodiafiltration (CVVHD) is used in critically ill septic patients. During CVVHD cytokines are excreted in the ultrafiltrate. But when the membranes used in CVVHD are cultured with granulocytes, the granulocytes are slightly activated. This effect is potentiated by endotoxin. We therefore, in vivo, compared the effect on granulocyte activation of CVVHD with an endotoxin group and a control group. METHODS: Thirty-one pigs were anaesthetized and mechanically ventilated. In ten pigs CVVHD was performed. Eleven pigs received an infusion of Escherichia coli endotoxin 30 mu/kg(-1) and ten pigs served as a control group. The adhesion molecules CD18 and CD62L were measured using monoclonal antibodies. The oxidative burst activity was assayed as superoxide dismutase-inhibitory reduction of cytochrome c. The number of granulocytes in peripheral blood and in the lungs and liver were counted. RESULTS: The infusion of endotoxin was followed by granulocytopenia, reduced oxidative burst activity, increased expression of CD18 and decreased expression of CD62L on granulocytes. Accumulation of granulocytes in liver and lung tissue was also noted in this group. CVVHD was only associated with a non-significant decrease in CD62L expression on granulocytes. It did not affect any of the other measured immunological parameters. CONCLUSION: In contrast to endotoxin-induced sepsis, the granulocytes were not activated during CVVHD.

The effect of surgical stress and endotoxin-induced sepsis on the NK-cell activity, distribution and pulmonary clearance of YAC-1 and melanoma cells.

Following surgery the activity of natural killer (NK) cells is decreased in the blood. It is possible that sepsis with release of endotoxin will further decrease the NK-cell activity. The purpose of the present study was to investigate the NK-cell cytotoxicity, the clearance in the lungs of YAC-1 and melanoma cells, as well as the distribution of NK-cells in the liver, following abdominal surgery and intraperitoneally (i.p.) administered endotoxin. Ten mice in each group were allocated to abdominal surgery, i.p. endotoxin or anaesthesia alone. Following abdominal surgery, the cytotoxicity of NK-cells isolated from the spleen was decreased and 4 h after injection the clearance of YAC-1 cells from the lungs was only 79.5+/-6.1% compared to 99.5+/-0.3% in the control group. The number of NK-cells in the liver was also significantly reduced following abdominal surgery. In contrast, i.p. endotoxin increased the activity of NK-cells by 28.5% compared to 11.8% in the control group and 8.1% in the surgery group, lowered the number of melanoma metastases in extrapulmonary organs and significantly increased the number of NK-cells in the liver. Following abdominal surgery, activity of NK-cells, pulmonary clearance and number of NK-cells in the liver were decreased. The number of NK-cells in the liver correlated with the NK-cell activity throughout the study. The increased NK-cell cytotoxicity and the increased number of NK-cells in the liver following i.p. administered endotoxin might initially be an appropriate measure against intra-abdominal infection.

The main regulatory region in the murine PSP gene is a parotid gland enhancer.

The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between -8.0 and -6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between -8.0 to -3.1 kb. This region was shown to activate a heterologus SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.

Murine mercury-induced autoimmunity: the role of T-helper cells.

Genetically mercury-susceptible (H-2s) mice in which the nude (athymic) mutation had been introduced, and euthymic (H-2s) mice treated with anti-CD4 monoclonal antibodies were used to determine the importance of T-helper (CD4+) cells for induction of autoimmunity by mercury, and to study the possibility of using anti-CD4 MAb for treatment of manifest autoimmunity. SJL/N and (A.SW x SJL-nu) F1 x SJL-nu BC (H-2s) mice homozygous for the nude mutation (nu/nu) were treated with 10 mg HgCl2/litre drinking water for 6 weeks. These mice developed neither the antinucleolar antibodies (ANoA) nor the systemic immune-complex (IC) deposits seen in mercury-treated littermates heterozygous for the nude mutation (nu/+). The nu/nu mice showed a significant and substantial reduction of splenocytes with pan-T-(CD3+), T-helper-(CD4+) and T-cytotoxic/suppressor (CD8+) markers, which was accompanied by a severe reduction of the proliferative response to Concanavalin A. Euthymic SJL/N mice given an initial intravenous (i.v.) injection of 100 micrograms anti-CD4 MAb (clone GK 1.5, rat IgG2b), followed by 6 weeks treatment with 100 micrograms anti-CD4 MAb intraperitoneally (i.p.) every third day in combination with 10 mg HgCl2/litre drinking water, did not develop ANoA or systemic IC-deposits. These features were seen in controls i.p. injected with rat IgG2b and given HgCl2 in the drinking water. The anti-CD4 MAb-treated mice showed very few CD4+ splenocytes, but a significant increase of CD8+ cells and severely impaired T-cell function. The possibility of treating longstanding autoimmune conditions with anti-CD4 MAb was examined by giving euthymic SJL mice HgCl2 for 3 months, followed by a mercury-free interval of 3 months and finally 7 weekly injections of 1 mg anti-CD4 MAb. This therapy caused a severe reduction of CD4+ cells, but there was no decline in the ANoA titre. In conclusion, induction of systemic autoimmunity by mercury was strictly dependent on T cells, specifically T-helper (CD4+) cells, and mercury-induced ANoA persisted for a long time after stopping mercury treatment. At this late stage, the autoimmune condition was no longer amenable for anti-CD4 MAb therapy.