Normal Untreated Mice Research Articles

High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

870. Gene Transfer in the Sandhoff Murine Model Using Lentiviral Vectors

The Sleeping Beauty (SB) transposon system created at the University of Minnesota is one of the few non-viral gene therapy systems that are able to integrate genes into human chromosomes. This is especially relevant for treating genetic disorders that require sustained expression of the therapeutic transgene. The purpose of this study is to evaluate SB-mediated transposition as a tool for safe, efficient, stable, non-viral gene therapy in a model lysosomal disorder, mucopolysaccharidosis (MPS) type VII. Cis SB transposon plasmids were constructed to deliver both the T2 transposon and SB11 transposase on the same plasmid (Geurts et al., Mol. Ther. v. 8, p. 108-17, 2003). For MPS VII, we constructed a transposon carrying the human -glucuronidase (GUSB) cDNA driven by a strong ubiquitous CAGGS promoter, and with SB11 transcribed from the intermediate-strength ubiquitin C promoter. Expression levels of SB11 in murine liver were confirmed by Western analysis. MPS VII mice completely deficient in GUSB activity were hydrodynamically injected with 25 g of plasmids that either did, or did not, express SB11. Blood samples for plasma isolation were collected 24 h after treatment and biweekly thereafter. An injection was deemed technically successful by the observation of plasma GUSB levels >100-fold of wild type levels 24 h following treatment. Four weeks after treatment, all plasma GUSB activities were below the lowest level of detection (probably not due to immune response because Bethesda Assays did not indicate the presence of GUSB inhibitors). Mice were euthanized 3 months after treatment. At that time GUSB activity in liver homogenates from mice that received transposase was 10-fold higher compared to that in the no-transposase control group, which was equal to approximately 1% of the value for normal untreated mice. Transgene frequency (as estimated by GUSB histochemical staining of frozen liver sections) was below 0.1% in the former group, but virtually undetectable in mice receiving transposon without transposase. Toluidine blue staining of liver sections was used to evaluate whether the amount of enzyme expressed from the transposons was sufficient to reduce the number and size of pathologic lysosomal inclusion in livers. GUSB activity levels in the spleen, lung, and kidney were 1% wild type levels and also produced transient expression of exceedingly high levels of circulating GUSB enzyme. These studies define a pattern of biodistribution and transgene expression that may be therapeutic for other MPS diseases in addition to MPS VII.

Optic nerve damage in experimental mouse ocular hypertension.

To evaluate optic nerve damage in mice after laser-induced ocular hypertension. Ocular hypertension was induced unilaterally in 13 NIH Black Swiss mice by laser photocoagulation of the limbus. Over the following 12 weeks, intraocular pressure (IOP) was measured at regular intervals by the microneedle method. The optic nerves of these mice and of seven normal untreated mice were then processed conventionally for electron microscopy, and cross sections of the nerve 300 micro m posterior to the globe were collected. Low- and high-magnification images were collected systematically and masked before analysis. For each nerve, cross-sectional area was measured in low-magnification micrographs, and axon and glia numbers were counted in high-magnification micrographs. In normal untreated mice, the average number of axons was 59,597 +/- 3,112 (mean +/- SD). Variation among these measurements was 5.7% +/- 3.9%. After laser treatment, the duration of high IOP ranged from 2 to 12 weeks (6.2 +/- 3.6 weeks, mean +/- SD). The mean IOP in the treated eyes was 1.3 times greater than the mean IOP in the control eyes (P = 0.0012). The maximum IOP in the treated eyes was 1.6 times greater than that observed in the control eyes (P < 0.0001). The optic nerve cross-sectional area, mean axon density, and total number of axons in the treated eyes were significantly less than in the control eyes (28.5% +/- 23.4%, 57.8% +/- 37.8%, and 63.1% +/- 38.1%, respectively; P < 0.005 for each). The decrease in optic nerve cross-sectional area and the positive integral of elevated IOP and duration of IOP elevation correlated significantly with total axon loss (r(2) = 0.79, P < 0.0001 and r(2) = 0.36, P = 0.040, respectively). The number of astrocytes per cross section of optic nerve was significantly greater in the treated eyes than in the control eyes (P = 0.014). Laser-induced ocular hypertension in mouse eyes can induce optic nerve axon loss that correlates with the magnitude and duration of elevated IOP.

Acceptance of islet allografts in the liver of mice by blockade of an inducible costimulator.

An inducible costimulator (ICOS) has been found to be a novel costimulator for T-cell activation, although its precise role in transplant immunobiology remains unclear. This study determined whether ICOS plays an essential role in rejection of intrahepatic islet allografts in streptozotocin-induced diabetic mice. Mononuclear cells in the liver of mice were isolated and examined by flow cytometry with respect to expression of ICOS in association with rejection, and the effects of in vivo treatment with an anti-ICOS antibody on survival of intrahepatic islet allografts were determined. RESULTSFlow cytometric analysis of mononuclear cells in the liver of normal untreated mice revealed that ICOS is expressed on CD4+CD3int natural killer T cells. The expression of ICOS was up-regulated on CD4+CD3bright T cells and expanded CD8 T cells in the liver in association with rejection. Posttransplant short-term administration of anti-ICOS antibody alone produced a significant prolongation of islet allograft survival. Administration of the antibody in conjunction with a subtherapeutic regimen of FK506 prevented rejection, leading to the acceptance of islet allografts. ICOS has an essential role in rejection of intrahepatic islet allografts and the blockade of ICOS interaction might be a novel approach for preventing islet allograft rejection.

COX-2 inhibition and retinal angiogenesis in a mouse model of retinopathy of prematurity.

The prostaglandin-cyclooxygenase (COX) pathway influences new blood vessel growth in a variety of tissues. This study was conducted to determine the cellular location of COX-2 in the retina and whether the inhibition of COX-2 would reduce retinal angiogenesis in a rodent model of retinopathy of prematurity (ROP). ROP was induced in C57BL/6 mice by exposing 7-day-old mice to 75% oxygen (hyperoxia) for 5 days followed by 5 days in room air (relative hypoxia and retinal angiogenesis). Normal mice were those with a normally developing retinal vasculature exposed to room air from birth until postnatal day (P)17. The COX-2 inhibitor, rofecoxib (15 mg/kg body weight intraperitoneally) was administered to normal and ROP mice from P12 to P17. Immunohistochemistry for COX-2 was performed on retinas from all groups by the avidin-biotin method. Histologic methods were used to count blood vessel profiles (BVPs) in the inner retina (inner limiting membrane, ganglion cell layer, and inner plexiform layer) with a masked approach. Intense COX-2 immunolabeling was specifically localized to ganglion cells and blood vessels of all mice retinas. In ROP mice, COX-2 immunolabeling was detected on blood vessels extending into the vitreous cavity. Quantitation of BVPs in the inner retina revealed an increase in untreated ROP mice compared with untreated normal mice (P < 0.001). Rofecoxib decreased BVPs by approximately 45% in normal mice and 37% in ROP mice. COX-2 is localized to sites associated with retinal blood vessels. The finding that the selective COX-2 inhibitor, rofecoxib, attenuated the retinal angiogenesis that accompanies ROP, and normal retinal development indicates that COX-2 plays an important role in blood vessel formation in the retina.

Expression of BDNF and TrkB in mouse taste buds after denervation and in circumvallate papillae during development.

BDNF (brain-derived neurotrophic factor) is a member of the neurotrophin family which affects the proliferation and survival of neurons. Using an immunocytochemical method, we examined the expression of BDNF and its receptor, TrkB, in the taste bud cells of the circumvallate papillae of normal mice and of mice after transection of the glossopharyngeal nerves. We additionally observed the expression of BDNF and TrkB in the developing circumvallate papillae of late prenatal and early postnatal mice. In normal untreated mice, BDNF was expressed in most of the taste bud cells; TrkB was detected in the plasma membrane of taste bud cells and in the nerve fibers. Double-labeling studies showed that BDNF and NCAM (neural cell adhesion molecule) or TrkB and NCAM colocalized in some of the taste bud cells, but that most taste bud cells were immunopositive for only BDNF or TrkB. NCAM-immunoreactive cells are known to be type-III cells, which have afferent synaptic contacts with the nerve terminals. Five days after denervation, the number of taste buds and nerve fibers markedly decreased; however, the remaining taste bud cells still expressed BDNF and TrkB. By 10 days after denervation, most of the taste buds had disappeared, and there were a few TrkB-immunoreactive nerve fibers in the connective tissue core. By 4 weeks after denervation, numerous TrkB-immunoreactive nerve fibers had invaded the papillae, and a few taste buds expressing BDNF and TrkB had regenerated. At E (embryonic day) 15 during development, the circumvallate papillae appeared, and then TrkB-immunoreactive nerve fibers entered the connective tissue core, and some of these fibers further invaded among the dorsal epithelial cells of the papillae. TrkB-immunoreactive oval-shaped cells were occasionally found in the dorsal epithelium. Such TrkB-immunoreactive nerve fibers and cells were also observed at E16-18. However, BDNF was not expressed in the papillae through the late prenatal days of E15 to E18. At P (postnatal day) 0, a cluster of BDNF-and TrkB-immunoreactive cells appeared in the dorsal epithelium of the papillae, and was presumed to be primitive taste buds. We conclude that TrkB-immunoreactive nerve fibers are necessary for papillary and taste bud formation during development and for the regeneration of taste buds after denervation. BDNF in the taste bud cells may act as a neurotrophic factor for innervating sensory neurons--through TrkB receptors of the axons of those neurons, and also may exert autocrine and paracrine trophic actions on neighboring taste bud cells by binding to their TrkB receptors.

2-Deoxyglucose enhances epileptic tolerance evoked by transient incomplete brain ischemia in mice

The aim of the study was to assess the influence of chronic treatment with a non-metabolisable glucose analogue, 2-deoxyglucose (2-DG) at a 150 mg/kg dose on long-term epileptic tolerance (ET) evoked by 30 min bilateral carotid artery clamping (BCCA) in mice. The effects of protein synthesis inhibition with cycloheximide (CHX), given in three daily doses of 2.5 mg/kg starting either 1 day before (peri-insult regimen) or 1 day after the priming insult (post-insult regimen), on ET development was also studied. Seizures were induced 14 days after BCCA with 3.5 mg/kg of bicuculline; this dose (CD 97) evokes convulsions in 97% of normal untreated mice. BCCA resulted in decreased mortality, prolonged latency to the onset of generalised convulsions and decreased overall seizure score. CHX given in the post-insult regimen did not influence, while the peri-insult regimen abolished, all signs of BCCA-evoked ET. 2-DG treatment of sham-operated animals resulted in a moderate but significant decrease in mortality rate and a tendency toward a lower seizure score. BCCA combined with 2-DG treatment resulted in a marked decrease in mortality rate, as well as reduction in all indicators of seizure susceptibility. CHX abolished the antiepileptic effects of BCCA alone, as well as BCCA combined with 2-DG, while it did not influence the 2-DG-related decrease in mortality. We conclude that the development of BCCA-induced epileptic tolerance, as well as unmasking antiepileptic effects of 2-DG by BCCA, is dependent on protein synthesis.

Treatment with sd/01 relative to chemotherapy in mice

The effectiveness of Filgrastim SD/01 (SD/01) has been demonstrated in the relief of neutropenia associated with cancer chemotherapy. The studies reported to date administered SD/01 primarily on the day after chemotherapy in a similar manner to the injection of conventional Filgrastim. It was not clear whether the same dosing paradigm would be optimum for an engineered form of Filgrastim with substantially altered pharmacokinetics. In order to examine the optimum timing of SD/01 relative to chemotherapy, mice were treated with 325 mg/kg cyclophosphamide (CY) on study day 0. They were also treated with a bolus dose of 1000 g/kg SD/01 on days −5, −4, −3, −2, −1, 1, 2, 3, 4 or 5. Blood was collected for complete blood counts on study days −1, +1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. Normal untreated mice have an ANC of 0.75 to 1.25 × 103/μL. Following CY treatment ANC fell to a nadir of .01 × 103/μL on day 4 then quickly rebounded to a high of 3.5 × 103/μL on day 8, returning to normal by day 10. Post-CY treatment with SD/01 on days 1, 2 or 3 improved the ANC nadir and brought counts back to baseline between days 4 and 5 (one day earlier). Delaying the SD/01 to days 4 and 5 after CY did not accelerate the return to baseline ANC. Pretreatment with SD/01 did not impact the depth or timing of the nadir or the day ANC passed the baseline count during recovery. Pre-treatment with SD/01 raised the ANC depending on the interval between treatment and CY exposure. Thus, 5 days pretreatment yielded an ANC of 14 × 103/μL, 4 days 11 × 103/μL, 3 days 8 × 103/μL, 2 days 3 × 103/μL, and 0.53 × 103/μL with 1 day pre-treatment. Pre-treatment with SD/01 (ie before chemo) is relatively ineffective compared to post-chemo treatment. Day 1 post-chemotherapy treatment appears to be the most effective timing for decreasing the severity and duration of neutropenia—delaying administration makes it less effective, and pre-treating does not appear to improve or worsen ANC recovery.

Therapeutic effect of interferon-gamma gene transfer in experimental visceral leishmaniasis.

Interferon (IFN)-gamma, in both natural endogenous form and administered as exogenous protein, induces control over visceral Leishmania donovani in experimentally infected BALB/c mice. To further characterize the therapeutic role of IFN-gamma in host defense against intracellular L. donovani, the efficacy of IFN-gamma delivered by gene transfer was tested. One week after infection, normal and IFN-gamma gene-disrupted (GKO) BALB/c mice were injected with an IFN-gamma gene-bearing mammalian expression plasmid (pIFN). Plasmid-specific IFN-gamma transcripts were detected in liver and spleen. Whereas liver parasite burdens more than doubled in untreated and mock-treated normal and GKO mice during the subsequent 2 weeks, animals injected with pIFN had controlled visceral infection and reduced parasite burden. These results indicate that, in infected tissues, IFN-gamma delivered by gene transfer enhances control over disseminated intracellular infection.

Catabolism of lipid-free recombinant apolipoprotein serum amyloid A by mouse macrophages in vitro results in removal of the amyloid fibril-forming amino terminus.

Serum amyloid A fibrils are formed when the normally rapid catabolism of the acute-phase reactant apolipoprotein serum amyloid A (apoSAA) is incomplete; thus amyloidosis may be viewed as a condition of dysregulated proteolysis. There is evidence that apoSAA is dissociated from plasma high-density lipoprotein (HDL) prior to fibril formation. The objective of this study was to investigate degradation of lipid-free apoSAA by tissue macrophages derived from amyloid-susceptible CBA/J mice in vitro. Peritoneal macrophages derived from untreated (normal) mice converted apoSAA (12 kDa) to a single 4 kDa C-terminal peptide while splenic macrophages converted apoSAA to 10, 7 and 4 kDa C-terminal peptides and a 4 kDa peptide that lacked the C- and N-terminal regions. Similar patterns of proteolysis occurred when peritoneal and splenic macrophages from amyloidotic CBA/J mice were used. Conditioned medium prepared from peritoneal, but not splenic macrophages, degraded apoSAA. Specific sites of cleavage indicated activity of cathepsin G- and elastase-like neutral proteases. The data indicate that lipid-free apoSAA can be degraded by secreted or cell-associated neutral proteases that are generated by macrophages to yield peptides that lack fibrillogenic potential.

ALLEVIATION OF GRAFT VERSUS HOST DISEASE BY INHIBITION OF TNF?? PRODUCTION USING IMMUNOSUPPRESSIVE ALLOTRAP PEPTIDES

126 Immunomodulatory peptides derived from HLA (Allotrap peptides) have been studied extensively both in vitro and in vivo models of immunosuppression. These peptides bind to heme oxygenase-1 (HO-1) and modulate both protein and activity levels. In vivo inhibition of HO-1 activity leads to upregulated protein expression and activity. Immunosuppressive activity of peptides correlated with modulation of HO-1 activity. One of the end products of HO activity, CO, is known to activate guanylate cyclase which is responsible for synthesis and release of cGMP, a well-known second messenger. cGMP, among other properties, is directly implicated in the synthesis and release of the pro-inflammatory cytokine, TNFα. In this study, we analyzed whether Allotrap peptides could influence TNFα production by activated macrophages. RAW264.7 cells (a mouse macrophage cell line) were activated with LPS in the presence of peptides for 24h and TNFα levels in culture medium were measured. Compared to unstimulated controls, LPS caused significant TNFα release. Addition of immunomodulatory peptides resulted in a dose dependent inhibition(up to 80%) of TNFα production (IC50=20 µM). Negative control peptides had no effects. In vivo, the effect of peptide therapy was tested in a B6 to B6D2F1 GVHD model, where TNFα production has been shown to correlate with disease development. Injection of immunomodulatory peptides to normal untreated mice caused an increase in HO-1 protein expression, and an increase in HO-1 enzyme activity in spleen cells. To test the immunomodulatory activity of HO-1 upregulation, normal B6 mice were treated with peptide. Three days later, unfractionated B6 spleen cells from peptide-treated or mannitol-treated (control) mice were injected into B6D2F1 mice. Severity of GVHD was determined by animal survival. Pre-treatment of B6 mice with 5 or 10 mg/kg Allotrap peptide had little effect. However, in recipients receiving spleen cells from animals treated with 20 mg/kg peptide a significant delay in the onset of GVHD was observed (median survival time was 45 days versus 22 days in mannitol-treated controls, p=0.02). These results demonstrate that HO-1 modulating peptides inhibited TNFα production in vitro and diminished induction of GVHD in a parent into F1 mouse model.

ALLEVIATION OF GRAFT VERSUS HOST DISEASE BY INHIBITION OF TNFα PRODUCTION USING IMMUNOSUPPRESSIVE ALLOTRAP PEPTIDES

126 Immunomodulatory peptides derived from HLA (Allotrap peptides) have been studied extensively both in in vitro and in vivo models of immunosuppression. These peptides bind to heme oxygenase-1 (HO-1) and modulate both protein and activity levels. In vivo inhibition of HO-1 activity leads to upregulated protein expression and activity. Immunosuppressive activity of peptides correlated with modulation of HO-1 activity. One of the end products of HO activity, CO, is known to activate guanylate cyclase which is responsible for synthesis and release of cGMP, a well-known second messenger. cGMP, among other properties, is directly implicated in the synthesis and release of the pro-inflammatory cytokine, TNFα. In this study, we analyzed whether Allotrap peptides could influence TNFα production by activated macrophages. RAW264.7 cells (a mouse macrophage cell line) were activated with LPS in the presence of peptides for 24h and TNFα levels in culture medium were measured. Compared to unstimulated controls, LPS caused significant TNFα release. Addition of immunomodulatory peptides resulted in a dose dependent inhibition (up to 80%) of TNFα production (IC50=20υM). Negative control peptides had no effects. In vivo, the effect of peptide therapy was tested in a B6 to B6D2F1 GVHD model, where TNFα production has been shown to correlate with disease development. Injection of immunomodulatory peptides to normal untreated mice caused an increase in HO-1 protein expression, and an increase in HO-1 enzyme activity in spleen cells. To test the immunomodulatory activity of HO-1 upregulation, normal B6 mice were treated with peptide. Three days later, unfractionated B6 spleen cells from peptide-treated or mannitol-treated (control) mice were injected into B6D2F1 mice. Severity of GVHD was determined by animal survival. Pre-treatment of B6 mice with 5 or 10 mg/kg Allotrap peptide had little effect. However, in recipients receiving spleen cells from animals treated with 20 mg/kg peptide a significant delay in the onset of GVHD was observed(median survival time was 45 days versus 22 days in mannitol-treated controls, p= 0.02). These results demonstrate that HO-1 modulating peptides inhibited TNFα production in vitro and diminished induction of GVHD in a parent into F1 mouse model.

Oral administration of a Kampo (Japanese herbal) medicine Juzen-taiho-to inhibits liver metastasis of colon 26-L5 carcinoma cells.

We have investigated the inhibitory effect of oral administration of Juzen‐taiho‐to, a Kampo Japanese herbal medicine, on liver metastasis by the inoculation of a liver‐metastatic variant (L5) of murine colon 26 carcinoma cells into the portal vein. Oral administration of Juzen‐taiho‐to for 7 days before tumor inoculation resulted in dose‐dependent inhibition of liver tumor colonies and significant enhancement of survival rate as compared with the untreated control, without side effects. We also found that liver metastasis of L5 cells was enhanced in BALB/c mice pretreated with anti‐asialo GM1 serum or 2‐chloroadenosine, and in BALB/c nu/nu mice, compared to normal mice. This indicates that NK cells, macrophages, and T‐cells play important roles in the prevention of metastasis of tumor cells. Juzen‐taiho‐to significantly inhibited the experimental liver metastasis of colon 26‐L5 cells in mice pretreated with anti‐asialo GM1 serum and untreated normal mice, whereas it did not inhibit metastasis in 2‐chloroadenosine‐pretreated mice or T‐cell‐deficient nude mice. Oral administration of Juzen‐taiho‐to activated peritoneal exudate macrophages (PEM) to become cytostatic against the tumor cells. These results show that oral administration of Juzen‐taiho‐to inhibited liver metastasis of colon 26‐L5 cells, possibly through a mechanism mediated by the activation of macrophages and/or T‐cells in the host immune system. Thus, Juzen‐taiho‐to may be efficacious for the prevention of cancer metastasis.

Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation.

Selective accumulation of eosinophils and activated CD4+ cells is now considered a central event in the pathogenesis of asthma, and this process is thought to be mediated by a number of cytokines including tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the Type 2 cytokines interleukin-4 (IL-4) and IL-5. To carry out a detailed time-course analysis of cellular changes in the bronchoalveolar lavage fluid (BAL), peripheral blood (PB), and bone marrow (BM), and of changes in the aforementioned cytokines in BAL and serum, Balb/c mice were sensitized by intraperitoneal injection with ovalbumin (OVA) adsorbed to aluminum hydroxide on two occasions 5 days apart, and were subjected to an OVA aerosol challenge 12 days after the second sensitization. This resulted in an airways inflammatory response characterized by early transient neutrophilia, marked eosinophilia, and, to a lesser extent, lymphocytosis in the BAL. Inflammatory events were first observed 3 h and 24 h after antigen challenge in the lung tissue and BAL, respectively, and lasted for 21 days. In the BM, we detected a 1.5- and 5-fold increase in the total number of cells and eosinophils, respectively, 4 days after the second sensitization. This was followed by a decrease, although BM eosinophilia remained clearly present at the time of antigen challenge. A second eosinopoietic event was observed in the BM shortly after challenge and reached a peak at day 3. BM cellularity returned to normal at day 21 after challenge. Serum OVA-specific IgE was first detected 3 days following the second sensitization (150 ng/ml). IgE levels then decreased but remained at the 75 ng/ml range at the time of the aerosol challenge. During the sensitization period, TNF-alpha (approximately 25 pg/ml), IL-4 (approximately 40 pg/ml), and IL-5 (approximately 250 pg/ml) were detected in serum, but not in the BAL fluid (BALF) and returned to background levels at the time of the antigen challenge. After antigen challenge, TNF-alpha, IL-4, IL-5, and GM-CSF were detected in serum. Peak levels were observed at 3 h (approximately 40 pg/ml), 3 h (approximately 120 pg/ml), 12 h (approximately 350 pg/ml), and 3 h (approximately 10 pg/ml), respectively, and returned to background levels 24 h after challenge. In the BALF, we detected peak levels of TNF-alpha, IL-4, IL-5, and GM-CSF at 6 h (approximately 250 pg/ml), 24 h (approximately 140 pg/ml), 24 h (350 pg/ml), and 3 h (approximately 10 pg/ml), respectively, with a return to background levels 5 days after challenge. No IL-10 could be detected at any time point during sensitization or after challenge in either serum or BAL. We also detected approximately 40 pg/ml of interferon-gamma (IFN-gamma) in the serum of normal untreated mice. Serum IFN-gamma levels fluctuated during sensitization and after challenge, but never exceeded those observed in untreated mice. Thus, the cytokine profile observed in this experimental model of allergic inflammation is characterized by IL-4 and IL-5 dominance, with an apparently minor TNF-alpha and GM-CSF contribution and relatively low or undetectable levels of IFN-gamma and IL-10.

Stimulation and inhibition of proliferation in the small intestinal crypts of the mouse after in vivo administration of growth factors.

The effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), insulin-like growth factor (IGF) I and II, acidic fibroblast growth factor (FGF), tumour necrosis factor alpha (TNF alpha), macrophage inhibitory protein 1 alpha (MIP-1 alpha) (LD78), and TGF beta-1 on cell proliferation in the crypts of the small intestine of mice were investigated. Various doses and dosing regimens were tested. Three in vivo assays were developed, in each case involving detailed cell positional analysis of methyl tritiated thymidine labelling and mitotic activity. These allowed deductions to be made about the regions of the crypt and hence regions of the proliferative hierarchy (stem cells versus dividing transit cells) that are affected by treatment with growth factors. The assays involved: (1) normal untreated mice (an assay most likely to be effective for detecting inhibitors); (2) mice shortly after whole body irradiation when compensatory proliferation has been endogenously triggered (another assay for inhibitory factors, possibly ones associated specifically with the regenerative process); and (3) mice at late times (96 hours) after irradiation in the regression phase after a proliferative overshoot (an assay designed to detect stimulators). Little effect was seen after treatment with acidic FGF, TNF alpha, or MIP-1 alpha but EGF, IGF-I and II, and TGF alpha can all be seen to exert some stimulatory effects on labelling or mitosis. EGF and IGF-I stimulate both unirradiated mice and 96 hour recipients, while TGF alpha had a greater effect on the 96 hour animals. In all cases, multiple doses were used. TGF beta-1 was an effective inhibitor of proliferation in unirradiated and early regenerating (18 hour) animals. EGF was the most effective of the stimulators, raising the levels of proliferation at all positions in the crypt, but particularly in the upper crypt. IGF-I also exerted its effect predominantly in the upper crypt, while TGF alpha raised proliferation at all cell positions. TGF beta-1 tended to have its strongest inhibitory effects in the lower (stem cell) regions of the crypt.

Effect of combined administration of a synthetic low-toxicity lipid A derivative, DT-5461a, and indomethacin in various experimental tumor models of colon 26 carcinoma in mice.

We investigated the antitumor effects of a synthetic lipid A derivative, DT-5461a, in combination with indomethacin in three experimental tumor models (peritoneal carcinomatosis, liver tumor, and lung tumor models) of transplanted colon 26 carcinoma in mice. This carcinoma produces the immunosuppressive prostaglandin E2 (PGE2). Intravenous administration of DT-5461a alone resulted in little or no prolongation of survival time [increase in life span (ILS): -2%-22%]. When indomethacin was given in drinking water a slight or moderate increase in survival time was seen (ILS: 4%-45%). In contrast, the combination of DT-5461a and indomethacin induced an additive increase in life span (ILS: 16% to more than 193%). The strongest antitumor effect of this combined therapy was seen in the peritoneal carcinomatosis model; in this model, plasma PGE2 concentrations were considerably higher than in normal mice, and concentrations were further but transiently increased by DT-5461a administration. Following oral indomethacin administration, these elevated PGE2 concentrations were reduced to the level in untreated normal mice. Furthermore, intratumoral tumor necrosis factor (TNF) activity in the group receiving the combined therapy was significantly higher than that in the DT-5461a-treated group. No TNF production was induced by the administration of indomethacin alone. These results suggest that the antitumor effect of DT-5461a can be enhanced by combination with indomethacin, and that the inhibition of PGE2 production may have a role in this antitumor effect.

Colony-stimulating factor-1 injections improve but do not cure skeletal sclerosis in osteopetrotic ( op) mice

The osteopetrotic ( op) mutation in mice is characterized by general skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Contrary to previous reports, we have noted persistent metaphyseal sclerosis in op mice treated with CSF-1 at doses above physiological concentrations of circulating CSF-1. We pursued this observation by quantitating osteoclasts and macrophages in the first 500 μm (area A) and the subsequent 1000 μm (area B) in the proximal tibial metaphysis using tartrate-resistant acid phosphatase and F4/80 as cell markers. In untreated normal mice, osteoclasts and macrophages were found in areas A (9.1 and 13.8 cells/1000 μm 2) and B (4.1 and 9.4 cells/1000 μm 2), respectively. In untreated mutants, osteoclasts and macrophages as percentages of normal were, respectively, 0% and 2% (area A) and 30% and 13% (area B). After CSF-1 treatment (0.15, 0.3, 0.5, and 1.0 × 10 6 U/day) for 28 days, marrow cavity size and numbers of osteoclasts and macrophages increased significantly in area B. However, area A remained sclerotic, with few macrophages (3% to 20%), and although osteoclast numbers were normal, their distribution was not, being absent in subepiphyseal sites. High CSF-1 gene expression occurs at bone modeling sites, co-localizes with osteoblasts, and temporally correlates with their differentiation. The failure of physiological concentrations of circulating CSF-1 to restore osteoclasts and macrophages and to cure sclerosis in subepiphyseal areas (A) of op/op mutants suggests that this factor must be presented locally to certain site-specific osteoclast and macrophage progenitors by osteoblasts and/or endothelial cells. Furthermore, the presence of osteoclasts in some skeletal sites in untreated op mice demonstrates that the development of some osteoclasts is independent of CSF-1.

Myeloproliferation in long‐term plasmacytoma‐regressor mice

MOPC-315 plasmacytoma-bearing BALB/c mice were treated with high doses of melphalan, causing a permanent and complete regression of the tumor. In the present study we analyzed plasmacytoma-regressor mice (PRM) 3-6 months after plasmacytoma regression. A second group of otherwise untreated normal mice was treated with melphalan (M--control group). A third group of mice remained untreated and served as an age- and sex-matched control group. PRM were cachectic and had an increased mortality rate compared to the M and the C control groups. Histopathological examination indicated that the spleen of PRM showed pronounced abnormalities, primarily in the red pulp. These abnormalities consisted of extramedullary hematopoiesis and myeloid-granulocytic hyperplasia. Spleens of M mice showed similar abnormalities but to a much lesser extent. Flow cytometric analysis of cellular surface markers of PRM splenocytes indicated a high number of large MAC-I- and GR-I-positive cells compared to splenocytes of M or C controls. These large cells also expressed Fc tau receptors (Fc tau RII), stained positively with non-specific esterase and adhered to plastic dishes; a certain percentage expressed MAC-2 and MAC-3 antigens. A quantitative suppression of CD4+ T cells and of B cells was also shown. Circulating levels of TNF were higher in PRM than in M or C mice. The capacity of splenocytes from PRM to secrete factors that stimulated CFU-GM colony formation in soft agar by bone-marrow cells from normal mice was significantly up-regulated compared to that of splenocytes from M or C mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of basic fibroblast growth factor in the healing stage of mouse gastric ulcer.

The aim of this study was to clarify the involvement of basic fibroblast growth factor (bFGF) in gastric ulcer healing. For this purpose, light and electron microscopic immunohistochemical studies for bFGF were performed using an experimental gastric ulcer model of mice. Ulceration was induced by the application of acetic anhydride to the serosal surface of the body of the stomach. Stomach tissues were investigated of mice at 5 days and 3 weeks respectively after treatment and also of untreated normal mice. Five days after treatment an ulcer was seen in the stomach of the experimental mice. Immunohistochemistry revealed that bFGF was localized in fibroblasts in the ulcer bed. The growth factor was distributed throughout the cytoplasm excluding organelles involved in the usual secretory system, such as rough endoplasmic reticulum, Golgi apparatus and secretory vacuoles. bFGF was also detected in the nucleus. Three weeks after treatment the surface of the ulcer lesion was completely covered by regenerated epithelium. The stomach tissues were immunohistochemically negative for bFGF both inside and outside the scar region; untreated normal stomach tissues were also negative for bFGF. These results suggest that the growth factor plays important roles in gastric ulcer healing.

Characterization of the exogenous interleukin-2 requirements for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers.

We have shown previously that thymocytes from MOPC-315-tumor-bearing mice treated with low-dose melphalan (L-phenylalanine mustard) (L-PAM TuB mice) are superior to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated normal mice or normal mice treated with low-dose melphalan in their ability to generate an antitumor cytotoxic response following 5-day in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2) [Mokyr MB, Bartik MM, Ahn M-C (1989) Cancer Res 49; 870]. Here we characterize the rIL-2 requirements for the generation of enhanced antitumor cytotoxicity by L-PAM TuB thymocytes relative to normal thymocytes upon in vitro stimulation with MOPC-315 tumor cells. Specifically, we show that delaying the addition of a low concentration of rIL-2 to 5-day in vitro stimulation cultures of thymocytes resulted in a progressive decline in the generation of antitumor cytotoxicity by both normal and L-PAM TuB thymocytes. However, even when rIL-2 was added on day 2 after culture initiation, thymocytes from L-PAM TuB mice generated a more potent antitumor cytotoxicity than did thymocytes from normal mice. In addition, when rIL-2 was added at the time of culture initiation, replacement of the conditioned medium with fresh medium lacking rIL-2 on day 3 of the 5-day in vitro stimulation culture period eliminated the ability of normal thymocytes, and reduced (but did not eliminate) the ability of L-PAM TuB thymocytes, to generate a significant level of antitumor cytotoxicity. A low concentration of fresh rIL-2 was sufficient to restore completely the generation of antitumor cytotoxicity by normal or L-PAM TuB thymocytes when added to the stimulation cultures immediately after the removal of the rIL-2-containing conditioned medium. The same low concentration of rIL-2 was also sufficient for restoring the generation of antitumor cytotoxicity by cultures of L-PAM TuB thymocytes, but not normal thymocytes, from which the rIL-2-containing medium was removed 1 day earlier. At the same time, conditioned medium from stimulation cultures of L-PAM TuB thymocytes was not superior to conditioned medium from stimulation cultures of normal thymocytes in supporting the generation of antitumor cytotoxicity by either normal or L-PAM TuB thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)