A sensitive bioassay for chicken interleukin-18 based on the inducible release of preformed interferon-gamma

Conventional tests for measuring the biological activity of chicken interleukin (IL)-18 require primary chicken spleen cells. We now describe a sensitive bioassay that is based on interleukin-18-induced release of interferon (IFN)-gamma by a permanent chicken cell line. In B19-2D8 cells, cytoplasmically stored interferon-gamma is quickly secreted in response to interleukin-18 exposure.     

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.     

Dopamine in the orbitofrontal cortex regulates operant responding under a progressive ratio of reinforcement in rats

Prefrontocortical dopamine (DA) plays an essential role in the regulation of cognitive functions and behavior. The orbitofrontal cortex (OFC) receives a dopaminergic projection from the ventral tegmental area and is particularly important for goal-directed appetitive behaviors and for the neural representation of reward value. We here examined the effects of DA receptor blockers locally infused into the OFC, on instrumental behavior under a progressive schedule of reinforcement. After continuous reinforcement training (lever pressing for casein pellets) rats received bilateral intra-OFC-infusions of the DA D1-receptor antagonist SCH23390 (3 μg/0.5 μl), the DA D2-receptor antagonist sulpiride (3 μg/0.5 μl), or phosphate buffered saline through chronically indwelling cannulae. Immediately after infusion they were tested under a time-constrained progressive ratio schedule of reinforcement (3, 6, 9, 12, … lever presses for 1 casein pellet within 180 s). Both SCH23390 and sulpiride led to a significant reduction of the break point (cessation to respond to the increasing criterion of instrumental effort) compared to vehicle infusions. A food preference test revealed no drug effects on the amount of consumed pellets and on the preference of casein pellets over laboratory chow. Leftward shifts of the break point in progressive ratio tasks indicate a disturbance of the mechanisms that translate motivation into appetitive behavior under conditions of increasing instrumental effort. Therefore, our data indicate that orbitofrontal dopamine is necessary for reward-related instrumental behavior.     

In vivo labeling with1251-CRH of human ACTH-producing pituitary adenomas heterotransplanted to nude mice

Tissue from 23 pituitary adenomas causing Cushing’s disease was implanted subcutaneously into 159 NuNu/NMRi mice, resected after 21 or 35 days, and evaluated histologically and immunohistochemically. After 21 days, 74.3% of the grafts survived, 59% having less than 30% necrotic adenoma cells. After 35 days, 45% of the adenoma fragments survived, 37% having less than 30% necrotic adenoma cells. The preservation of the grafts was essentially dependent on the grade of vascularization accomplished by migration of the host’s capillaries. As assessed by adrenal weight and histologically, biological activity of the transplants could not be detected. Histologically, the grafts maintained the features of their primary tumors, and adrenocorticotropic hormone (ACTH) could be visualized immunohistologically.Seventeen mice with subsequently proved preserved adenoma tissue received an intravenous injection of 12.5 μCi¹²⁵l-corticotropin-releasing hormone (CRH) and light microscopy-autoradiography was performed. Specific labeling, as verified by positive and negative controls, was exhibited by 1 1 of 15 transplants originating from 3 highly differentiated ACTH cell adenomas. Four did not label clearly positive. Two grafts of an undifferentiated mucoid cell pituitary adenoma did not show any labeling.The nude mouse model is a useful tool for the study of ACTH-producing pituitary adenomas in vivo. Highly differentiated ACTH cell adenomas can be labeled with radioactive CRH in vivo.     

Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology

Although hemangioma is referred as to the most common tumor in infancy, the underlying pathogenetic events and the biologic origin of this benign vascular neoplasm have remained obscure. By using immunohistochemistry on frozen sections of infantile hemangiomas, we show here that proliferating endothelial cells abundantly expressed alpha(v)beta(3) but lacked beta(4) integrins. Instead, regressing and involuting infantile hemangiomas due to treatment with IFN-alpha showed positive staining of beta(4) integrin, which might point to the angiogenic significance of beta(4) integrin in infantile hemangiomas. Moreover, immunofluorescence analysis revealed the existence of HLA-DR(+), mostly CD68(+) and partly DC-SIGN/CD209(+) cells with dendritic cell morphology in the intimate vicinity of hemangiomatous vessels. Such cells were also detected in the dermal microvascular unit in normal skin. The coupled occurrence of vascular structures and perivascular cells that were stained positive with markers of monocyte or macrophage or dendritic cells might suggest that the development of infantile hemangioma is a result of vasculogenesis, that is, the formation of primitive blood vessels from angioblasts, rather than of angiogenesis, that is, the sprouting of capillaries from preexisting vessels.     

Rapid diagnosis of extrapulmonary tuberculosis by PCR: impact of sample preparation and DNA extraction

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.     

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by anti-tumor T cells

Unfractionated spleen cells taken from tumor-bearing mice 2weeks after tumor implantation contained tumor-primed T cellswhich produced cytokines including IL-2 and IFN- when culturedin vitro. With progressive tumor growth this initial lymphokine-producingcapacity decreased. Here, we investigated the ability of IL-12to (I) restore suppressed IFN- production, (II) cause tumorregression and (II) induce anti-tumor protective immunity. Additionof rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearingmice resulted in a striking enhancement in the production ofIFN- compared with cultures of these cells in the absence ofrIL-12 or of normal spleen cells in the presence of rIL-12.Five I.p. injections of rIL-12 into mice bearing s.c. tumorsinduced complete tumor regression. This was found when rIL-12was given at early (1–2 weeks), intermediate (4–5weeks) or even late (7 weeks) stages of tumor growth. Furthermore,IL-12-treated mice which rejected the primary tumor exhibitedcomplete resistance to a rechallenge with the same tumor butdid not reject a second syngenetic tumor. Immunohistochemicalanalyses following IL-12 treatment revealed that CD4⁺ and CD8⁺T cells infiltrate the tumor. More importantly, IFN- mRNA expressionwas observed in fresh tumor masses from tumor-bearing mice receivingIL-12 treatment The importance of IFN- was further demonstratedby the observation that the systemic administration of anti-IFN-mAb prior to IL-12 treatment completely abrogated the anti-tumoreffect of IL-12. Thus, these results indicate that administrationof modest levels of rIL-12 to tumor-bearing mice results intumor regression through mechanisms involving reversal of suppressedIFN- production by anti-tumor T cells and the establishmentof a tumor-specific protective immune response.     

Recombinant human granulocyte-macrophage colony-stimulating factor induces secretion of autoinhibitory monokines by U-937 cells

Colony-stimulating factors are required for survival proliferation, differentiation and functional activation of granulocytes, macrophages and their precursor cells. In the present report, however, we demonstrate antiproliferative activity of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) on monoblast cell line U-937 and provide evidence for the involvement of tumor necrosis factor alpha TNF-alpha and interleukin 1 beta (IL 1 beta) in its growth inhibitory action. GM-CSF (but not granulocyte CSF, G-CSF or macrophage CSF, M-CSF) suppressed DNA synthesis and self renewal of U-937 cells. Similarly, medium conditioned by U-937 cells in response to GM-CSF (GM-CSF U-937-CM) was able to reduce clonogenicity and [3H]thymidine uptake by U-937 cells. Since neutralization of GM-CSF present in GM-CSF U-937-CM by monoclonal antibody to GM-CSF did not abrogate the autoinhibitory activity present in GM-CSF U-937-CM, we considered the possibility that other soluble molecules are released by U-937 cells upon GM-CSF stimulation. Neutralization by antibodies to IL 1 beta and TNF-alpha suggested that both monokines could be the antiproliferative principle operating in GM-CSF U-937-CM. Moreover, employing IL 1 beta-specific enzyme-linked immunosorbent assay, TNF-alpha specific radioimmunoassay, Northern analysis using a cloned TNF-alpha-specific cDNA and an oligonucleotide probe for IL 1 beta, we demonstrate GM-CSF-inducible IL 1 beta and TNF-alpha gene expression by U-937 cells at the mRNA and protein level. Although M-CSF expression was induced under similar conditions, M-CSF failed to inhibit growth of U-937 cells.