Eosinophil chemotactic lymphokine produced by spleen cells of Schistosoma japonicum-infected mice

Eosinophil chemotactic factor (ECF) was detected in the cell-free supernatant of the culture of spleen cells obtained from Schistosoma japonicum-infected mice. ECF production by spleen cells was dependent on both the amount of soluble egg antigen (SEA) added and the number of cells in the culture. When sufficient amount of SEA was added to the culture, ECF production was detectable by 6 h after incubation, reached a plateau by 24 h and then rapidly decreased. ECF was produced by nonadherent, Thy 1.2-positive cells, indicating that it is a lymphokine. After Sephadex G-75 gel chromatography, estimated molecular weight of this ECF was 15,000-20,000 daltons. When eosinophils were preincubated with this ECF lymphokine, their chemotactic reactivity to S. japonicum egg-derived ECF was significantly enhanced, although this pretreatment caused neither the deactivation of chemotactic reactivity to homologous ECF nor the enhancement of the random migration of eosinophils. Thus, this ECF lymphokine seems to be important not only as a chemoattractant but also as an activator of eosinophilotaxis around deposited eggs in schistosomiasis japonica.     

Diurnal dynamics of cytokine spectrum produced by immunocompetent cells of intact mice

The production of cytokines by immunocompetent cells of intact mice was studied in various phases of the diurnal cycle. The ratio of produced cytokines was shown to differ in various phases of the diurnal cycle. The correlation between spontaneous production of various cytokines disappeared at 20:00, which reflects the development of physiological immunosuppression due to evening increase in physical activity of mice and corresponding changes in neuroendocrine status. Our results illustrate the existence of diurnal variations in intercellular interactions in the immune system. These data should be taken into account in the study of the mechanisms for cytokine immunoregulation and development of new schemes for cytokine immunocorrection. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 10, pp. 448–451, October, 2007     

Mucin is produced by clara cells in the proximal airways of antigen-challenged mice

Airway mucus hypersecretion is a prominent feature of many obstructive lung diseases. We thus determined the ontogeny and exocytic phenotype of mouse airway mucous cells. In naive mice, ciliated (approximately 40%) and nonciliated (approximately 60%) epithelial cells line the airways, and > 95% of the nonciliated cells are Clara cells that contain Clara cell secretory protein (CCSP). Mucous cells comprise < 5% of the nonciliated cells. After sensitization and a single aerosol antigen challenge, alcian blue-periodic acid Schiff's positive mucous cell numbers increase dramatically, appearing 6 h after challenge (21% of nonciliated/nonbasal cells), peaking from Days 1-7 (99%), and persisting at Day 28 (65%). Throughout the induction and resolution of mucous metaplasia, ciliated and Clara cell numbers identified immunohistochemically change only slightly. Intracellular mucin content peaks at Day 7, and mucin expression is limited specifically to a Clara cell subset in airway generations 2-4 that continue to express CCSP. Functionally, Clara cells are secretory cells that express the regulated exocytic marker Rab3D and, in antigen-challenged mice, rapidly secrete mucin in response to inhaled ATP in a dose-dependent manner. Thus, Clara cells show great plasticity in structure and secretory products, yet have molecular and functional continuity in their identity as specialized apical secretory cells.     

Molecular weight and other characteristics of mycobacterial growth inhibitory factor produced by spleen cells obtained from mice immunized with viable attenuated mycobacterial cells.

Exposure of mycobacterial growth inhibitory factor (MycoIF) to trypsin, chymotrypsin, or neuraminidase decrease its ability to produce intracellular inhibition of mycobacterial growth within macrophages, suggesting that MycoIF was a glycoprotein. MycoIF was unaffected by deoxyribonuclease or ribonuclease. Supernatant fluids from antigenically stimulated H37Ra-immunized mouse spleen cells exposed to puromycin were unable to produce significant intracellular inhibition. This indicated that the presence of MycoIF activity in supernatant fluids required protein synthesis. The filtration of MycoIF-containing supernatant fluids on Sephadex G-150 demonstrated that significant MycoIF activity appeared only in those fractions which eluted on the downward side of the serum albumin peak. Based on protein standards filtered through the Sephadex gel, the molecular weight of MycoIF was calculated to be between 20,000 and 35,000. These calculations assumed that MycoIF is a globular protein. Attempts to purify MycoIF by anion exchange chromatography (diethylaminoethylcellulose) was not successful.     

Malignant progression of B16 melanoma cells induced in vitro by growth factors produced by highly malignant cells

Four mouse B16 melanoma subclones (G3.15, G3.5, G3.12 and G3.26) exhibit progressively greater growth capacity in vitro and in vivo. Previously, non-metastatic G3.15 cells were sequentially converted, in monolayer cultures, to the moderately-metastatic G3.5 cells, and then to a highly-metastatic G3.5^* phenotype. Both conversions were induced by hypoxia followed by confluence, and also occurred in tumors. G3.5^* cells were comparable with, yet distinguishable from, G3.12 cells in being growth-autonomous in culture. In this study, the presumption that rapidly-growing G3.26 cells represented the ultimate progression step in this clonal system was examined. Both G3.12 and G3.5^* cells converted in vitro to the G3.26 phenotype during growth in serum-free medium conditioned by G3.26 cell growth. By selective filtration of conditioned medium and characterization of the stability of growth- and conversion-promoting activities, three distinct activities were found to promote a two-step G3.12 to G3.26 phenotype conversion: (1) a < 10 kDa filtrate stimulated slight attachment and proliferation of G3.12 cells, effects that were reversible, partly attributable to accumulated lactate, and fully mimicked by medium acidification to pH 6.5; (2) medium acidification, together with a heat- and acid-stable but partially trypsin-sensitive > 10 kDa activity, induced G3.12 G3.5^* conversion that resulted in acquisition of growth autonomy; and (3) a heat-, acid- and trypsin-sensitive > l0 kDa activity induced G3.5^* G3.26 conversion, characterized by anchorage-independent growth in soft agar, and potent lung colonization following intravenous injection. Phenotype analysis of G3.12 tumors and lung metastases revealed that G3.5^*-like cells were regularly present in tumors and metastases, whereas G3.26-like cells occurred almost exclusively in large lung metastases. While G3.12 cells might convert to G3.5^* cells in order to disseminate, G3.26 cells are apparently not involved in metastatic spread but probably account for the rapid growth of established metastases.     

Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice

T cell-derived cytokines are important in the development of an effective immune response, but when dysregulated they can promote disease. Here we identify a four-helix bundle cytokine we have called interleukin 31 (IL-31), which is preferentially produced by T helper type 2 cells. IL-31 signals through a receptor composed of IL-31 receptor A and oncostatin M receptor. Expression of IL-31 receptor A and oncostatin M receptor mRNA was induced in activated monocytes, whereas epithelial cells expressed both mRNAs constitutively. Transgenic mice overexpressing IL-31 developed severe pruritis, alopecia and skin lesions. Furthermore, IL-31 receptor expression was increased in diseased tissues derived from an animal model of airway hypersensitivity. These data indicate that IL-31 may be involved in promoting the dermatitis and epithelial responses that characterize allergic and non-allergic diseases.     

Differing effects on superior cervical ganglia in neonatal mice produced by antisera to nerve growth factors from mice and snakes.

Rabbit or horse antisera to Nerve Growth Factors from mouse salivary glands and the venoms of five snakes were injected into neonatal mice. The mice were killed 10 days later and the superior cervical ganglia removed, weighed and examined histologically. Treatment with antisera to the snake Nerve Growth Factors had no effect on ganglion weight, maximum neuronal density or mean neurone diameter. This was true even for the one antiserum that in vitro showed a weak cross-reactivity with the mouse antigen. In contrast, treatment with the antiserum to mouse Nerve Growth Factor produced a partial destruction of the superior cervical ganglia (immunosympathectomy). The weights of the ganglia in animals treated for the first 5 days post partum with increasing volumes (a total of 0.5 ml) of antiserum to the mouse factor fell by some 80% the total neurone number by approx 50% the maximum neurone density by 40% and the mean neurone dia. by 17%. The effect was found to be dose dependent.It is considered that caution is required in extrapolating results from in vitro studies on Nerve Growth Factor to the situation obtaining in vivo and that the inability of snake antisera to produce immunosympathectomy in neonatal mice may result from differences in the antigenic determinants of the mouse and snake Nerve Growth Factors. The marked effect of antiserum to the mouse salivary gland factor in neonatal mice reported by earlier workers has been confirmed. No single explanation, however, can be given for the differences in the reduction in neurone numbers found in the present and previous studies. Furthermore, it is concluded that neither the previous nor the present studies afford evidence that the antiserum directly causes neuronal death.     

α-Difluoromethylornithine inhibits liver metastasis produced by intrasplenic injection of human tumor cells into nude mice

The purpose of these studies was to examine metastatic potentials of a human colon tumor xenograft (T6) and three different human tumor cell lines (LS174T, HT29 and A549) using the intrasplenic-nude mouse model system (ISMS model system). A further objective was to study the activity of-difluoromethylornithine (DFMO) against primary and metastatic growth of the xenograft and the three cell lines. DFMO is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting step in polyamine biosynthesis. Tumor burdens in the liver of nude mice were observed 6 weeks after the intrasplenic injection with LS174T and 12–14 weeks after intrasplenic injections with T6, HT29 and A549. Most of the mice developed primary tumor growth in the spleens. DFMO showed significant activity against liver metastases but had little or no activity against primary tumor growth in the spleens of the ISMS model and against s.c. growth of the xenograft. The studies demonstrated that the ISMS model system is an excellent system for studying metastatic behavior of human tumors and for studying the antimetastatic activity of experimental drugs.     

Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice

Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.     

Similar transforming growth factors (TGFs) produced by cells transformed by different isolates of feline sarcoma virus

Fisher rat embryo cells transformed by each of three independent isolates of feline sarcoma virus (FeSV) are shown to release transforming growth factors (TGFs) into cell culture medium. These acid- and heat-stable peptides compete for binding to, and stimulate phosphorylation of, EGF membrane receptors and promote anchorage-independent cell growth. Cells transformed by the Gardner and Snyder-Theilen strains of FeSV produce high titers of TGF (60-200 ng eq EGF/liter) while cells transformed by McDonough FeSV produce TGF at only low levels (<10 ng eq EGF/liter). Growth factors produced by cells transformed by each of the three FeSV isolates functionally and biochemically resemble each other, mouse sarcoma growth factor (SGF), and TGFs produced by human tumor cells.     

Differing effects on the pre- and paravertebral sympathetic ganglia of adult mice produced by antisera to nerve growth factors from mice and snakes

Antiserum to nerve growth factor isolated from mouse salivary glands causes, in adult mice, a reduction of approximately 50% in weight of the superior cervical ganglia. Prevertebral ganglia are less sensitive than paravertebral ganglia. The reduction in weight of the superior cervical ganglia results from a reduction in size of the principal sympathetic neurones (mean diameter falling by approximately 30%) rather than from a reduction in both cell size and cell numbers. If, as seems to be the case in neonatal mice, the antiserum acts by neutralizing endogenous nerve growth factor, no effect on adult neurone numbers would be expected. The origin of the reduction in neurone size is not understood.Antisera to nerve growth factor isolated from snake venoms do not affect the superior cervical ganglia of adult mice. It is suggested that chemical differences between the snake venom and mouse salivary gland nerve growth factors might lead to differences in the antisera produced from these respective antigens.     

Monokines produced by macrophages stimulate the growth of osteoblasts

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-1s, and TNFs, EGF, IGF-I and -II, TGF-beta, beta 2 microglobulin, G-CSF, CSF-1 and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-beta. HEP II, but not HEP I or TGF-beta, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.     

Polyclonal B-cell-activating factors produced by spleen cells of mice stimulated with a cell homogenate of Trypanosoma gambiense.

The effect of injection of a cell homogenate of Trypanosoma gambiense into mice on the production of soluble factors responsible for the induction of polyclonal B-cell activation (PBA) by their spleen cells was examined. PBA was induced by injection of the cell homogenate and was also detected in mice treated with either the serum or the spleen cells of mice treated with the cell homogenate. PBA-inducing activity became detectable in the serum and spleen cells as early as 12 h after injection of the cell homogenate, reached a peak on day 2, and then decreased. This activity was also detected in the culture medium of spleen cells obtained 2 days after injection of the cell homogenate. For determination of the type of spleen cells producing the PBA-inducing factor, the day 2 spleen cells were fractionated on the basis of differences in their adhesive properties. Spleen cells in the effluents from a Sephadex G-10 column (T and B cells) and a nylon wool column (T cells) and those adhering to a plastic flask (macrophages) all produced the PBA-inducing factor. The production of PBA-inducing factor by whole spleen cells and by cells adherent to the plastic flask was not affected by treatment with anti-Thy-1.2 antibody and complement. These data suggest that soluble factors derived from macrophages and T cells could contribute to the induction of PBA. The PBA-inducing activity in the conditioned medium was completely inactivated by treatment at pH 2.0, heating at 56 degrees C for 30 min, or exposure to 0.1% sodium dodecyl sulfate. The results are discussed in relation to cytokines that could affect B-cell activation.     

Syndecan in carcinomas produced from transformed epithelial cells in nude mice.

Expression of syndecan, a cell surface proteoglycan, was studied in carcinomas induced by implanting chemically transformed keratinocytes and mammary epithelial cells into nude mice. By immunohistochemistry and in situ hybridization, syndecan was localized in keratinizing cells within moderate- to well-differentiated squamous cell carcinomas in a pattern resembling that of normal epidermis, whereas almost total loss of expression was detected in poorly-differentiated areas within these tumors. In anaplastic spindle cell carcinomas, syndecan expression was barely detectable. In biphasic tumors, induced by mammary epithelial cells, and consisting of cysts overlaying an adenocarcinoma, syndecan was unquely localized to differentiated epithelial structures such as secretory epithelial lining of the cysts as well as aberrant glands and ducts within the carcinoma. Based on the expression pattern of syndecan in the tumors studied, we conclude that the expression of this developmentally regulated molecule is associated with epithelial differentiation also during neoplastic growth.     

Idd-linked genetic regulation of TACIhigh expressing B cells in NOD mice

In NOD mice, B cells play a key role in the initiation of type 1 diabetes pathogenesis. We have identified a novel NOD-specific B cell-related trait, i.e. the increased percentage of TACI(high)-expressing splenic B cells, by comparing NOD mice with non-autoimmune C57BL/6 mice. Using athymic NOD mice, we determined that this trait was T cell independent. We mapped the loci contributing to the increased proportion of TACI(high) expressing splenic B cells and found that the control of TACI expression was strongly linked to chromosome 1, in a region which includes the insulin-dependent diabetes (Idd) 5 loci. Moreover, another locus potentially involved was detected in the vicinity of Idd22 on chromosome 8. Interestingly, when analyzing age-dependent contribution to the obtained LOD scores we observed that the linkage to chromosome 8 was explained solely by mice > or =61 days of age, suggesting a temporal genetic regulation of TACI expression. In addition, analysis of genetic interaction between chromosome 1 and chromosome 8 indicated that the two loci acted in an additive fashion. Our findings corroborate the notion that B cell deviations contribute to type 1 diabetes development, and suggest a temporal regulation of TACI(high) expression, possibly influenced by the ongoing autoimmune process.     

Acquisition of steady-state operant behavior in long-living Ames Dwarf mice

Ames dwarf mice have a Prop-1 mutation that has been identified with increased levels of IGF-I in the central nervous system, upregulation of neuroprotective systems, and increased lifespan. To elucidate the behavioral effects of the Prop-1 mutation, 8 Ames dwarf and 7 normal mice (all of whom were 8 months of age or younger) were compared on a differential-reinforcement-of-low-rate-of-responding schedule of reinforcement and a matching-to-sample task. On both tasks, nosepokes were reinforced with access to a saccharin solution. Comparisons were based on several measures of behavioral efficiency: pause durations, intertrial intervals, and numbers of responses. Ames dwarf mice were generally less efficient than normal mice. One possible cause of this outcome is that relatively young Ames dwarf mice show less cognitive development than age-matched normal mice.