α-Integrin expression and function modulates presentation of cell surface calreticulin.

Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with α-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a ß1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated α-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.

The Crohn's disease-associated polymorphism in ATG16L1 (rs2241880) reduces SHIP gene expression and activity in human subjects.

Crohn's disease (CD) is a polygenic immune-mediated disease characterized by gastrointestinal inflammation. Mice deficient in the hematopoietic-restricted SH2 domain-containing inositolpolyphosphate 5'-phosphatase (SHIP) develop spontaneous CD-like ileal inflammation. Intriguingly, SHIP mRNA is not upregulated in biopsies from patients with ileal CD despite immune cell infiltration, but SHIP's role in human CD remains unknown. We analyzed SHIP mRNA expression and activity in biopsies and peripheral blood mononuclear cells (PBMCs) from control and treatment-naive subjects with ileal CD, and demonstrated that SHIP mRNA and activity were lower in hematopoietic cells in ileal biopsies and PBMCs from subjects with CD. In all tissues from our patient cohort and in PBMCs from a second healthy control cohort, subjects homozygous for the autophagy-related 16-like protein (ATG16L1) CD-associated gene variant (rs2241880), had low SHIP mRNA expression and activity. SHIP protein expression increased during autophagy and SHIP upregulation was dependent on ATG16L1 and/or autophagy, as well as the ATG16L1 CD-associated gene variant. Finally, homozygosity for the ATG16L1 risk variant and low SHIP mRNA expression is inversely related to increased (LPS+ATP)-induced IL-1ß production by PBMCs in our cohorts and was regulated by increased transcription of ILIB. These data suggest a novel mechanism by which the ATG16L1 CD-associated gene variant may predispose people to develop intestinal inflammation.

The role of SHIP1 in macrophage programming and activation.

The SHIP1 (SH2-containing inositol-5'-phosphatase 1) acts as a negative regulator of proliferation, survival and end cell activation in haemopoietic cells. It does so, at least in part, by translocating to membranes after extracellular stimulation and hydrolysing the phosphoinositide 3-kinase-generated second messenger, PtdIns(3,4,5)P(3) to PtdIns(3,4)P(2). SHIP1(-/-) mice have, as a result, an increased number of neutrophils and monocyte/macrophages because their progenitors display enhanced survival and proliferation. These mice also suffer from osteoporosis because of an increased number of hyperactive osteoclasts and a significant neutrophil infiltration of the lungs. Interestingly, SHIP1(-/-) mice do not display endotoxin tolerance and we have found that lipopolysaccharide-induced endotoxin tolerance is contingent on up-regulating SHIP1, through the production of autocrine-acting transforming growth factor-beta, in bone-marrow-derived macrophages and mast cells. Intriguingly, unlike bone-marrow-derived macrophages, SHIP1(-/-) peritoneal and alveolar macrophages produce 10-fold less NO than wild-type macrophages because these in vivo-generated macrophages have very high arginase I levels and this enzyme competes with inducible nitric oxide synthase for the substrate L-arginine. It is probable that, in the face of chronically increased PtdIns(3,4,5)P(3) levels in their myeloid progenitors, SHIP1(-/-) mice display a skewed development away from M1 (killer) macrophages (which have high inducible nitric oxide synthase levels and produce NO to kill microorganisms and tumour cells), towards M2 (healing) macrophages (which have high arginase levels and produce ornithine to promote host-cell growth and collagen formation). This skewing probably occurs to avoid septic shock and suggests that the phosphoinositide 3-kinase pathway plays a critical role in programming macrophages.

The role of SHIP in cytokine-induced signaling.

The phosphatidylinositol (PI)-3 kinase (PI3K) pathway plays a central role in regulating many biological processes via the generation of the key second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P3). This membrane-associated phospholipid, which is rapidly, albeit transiently, synthesized from PI-4,5-P2 by PI3K in response to a diverse array of extracellular stimuli, attracts pleckstrin homology (PH) domain-containing proteins to membranes to mediate its many effects. To ensure that the activation of this pathway is appropriately suppressed/terminated, the ubiquitously expressed tumor suppressor PTEN hydrolyzes PI-3,4,5-P3 back to PI-4,5-P2 while the 145-kDa hemopoietic-restricted SH2-containing inositol 5'- phosphatase, SHIP (also known as SHIP1), the 104-kDa stem cell-restricted SHIP (sSHIP) and the more widely expressed 150-kDa SHIP2 hydrolyze PI-3,4,5-P3 to PI-3,4-P2. In this review we will concentrate on the properties of the three SHIPs, with special emphasis being placed on the role that SHIP plays in cytokine-induced signaling.

1alpha,25-Dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase.

We investigated the basis for the induction of monocyte antimycobacterial activity by 1alpha,25-dihydroxyvitamin D(3) (D(3)). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonucleotides to the p110 subunit of PI 3-Kalpha. Cells infected with M. tuberculosis alone or incubated with D(3) alone produced little or undetectable amounts of superoxide anion (O(2)). In contrast, exposure of M. tuberculosis-infected cells to D(3) led to significant production of O(2), and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D(3)-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D(3) required prior infection with live M. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47(phox) and p67(phox) to the membrane fraction was observed in cells incubated with live M. tuberculosis and D(3) but not in response to combined treatment with heat-killed M. tuberculosis followed by D(3). Redistribution of p47(phox) and p67(phox) to the membrane fraction in response to live M. tuberculosis and D(3) was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D(3)-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase.

Endogenous brain cytokine mRNA and inflammatory responses to lipopolysaccharide are elevated in the Tg2576 transgenic mouse model of Alzheimer's disease.

Beta-amyloid (Abeta) plaques have been shown to induce inflammatory changes in Alzheimer's disease brains. Cortical, but not cerebellar tissue from 16-month-old Tg2576 (Tg+) mice showed significant increases in interleukin (IL)-1alpha (2.2-fold), IL-1beta (3.4-fold), tumor necrosis factor-alpha (3.9-fold), and monocyte chemoattractant protein-1 (2.5-fold) mRNA levels compared to controls (Tg-). These changes were not apparent in 6-month-old Tg+ mice except for TNF-alpha. mRNA levels of glial fibrillary acidic protein and complement components, C1qA and C3 were also elevated in aged mice. Lipopolysaccharide (LPS) (25 microg/mouse, i.v.) induced a significantly greater production of IL-1beta protein in the cortices and hippocampi of Tg+ vs. Tg- mice at 1, 2, 4, and 6 h. Experiments in 6-month-old mice showed that not only was there less cytokine produced compared to 16-month-old mice, but the exacerbated cytokine response to LPS in Tg+ mice was not apparent. Higher levels of Abeta1-40 were measured in the cortices of 6- and 16-month-old Tg+ mice at 4-6 h after LPS, which returned to baseline after 18 h. We demonstrate that Abeta plaques elicit inflammatory responses in Tg2576 mice that are further exacerbated when challenged by an exogenous inflammatory insult, which may serve to amplify degenerative processes.

Acceleration and increased severity of collagen-induced arthritis in P-selectin mutant mice.

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (-/-) mice to explore the role of P-selectin in the development of joint inflammation. Surprisingly, CIA onset was accelerated and severity was increased in P-selectin mutant mice, compared with wild-type mice (+/+). Increased levels of anti-type II collagen IgG were detected in both nonarthritic and arthritic P-selectin mutant mice from days 14-91. In addition, splenocytes isolated from immunized and nonimmunized P-selectin mutant mice produced significantly less IL-2 and IL-4, but significantly higher levels of IL-10 and IL-5 than splenocytes from wild-type mice. These observations show that P-selectin-mediated leukocyte rolling is not required for the development of murine CIA and that P-selectin expression exerts a controlling effect on the development of Ag-driven inflammatory joint disease, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into lymphoid tissue or inflammatory foci.

Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1.

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.

Reduced susceptibility to collagen-induced arthritis in mice deficient in intercellular adhesion molecule-1.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the firm adhesion of leukocytes to venular endothelium and facilitates leukocyte extravasation from the vasculature into inflamed tissue. In addition, ICAM-1 is an important costimulatory molecule during Ag presentation to lymphocytes. Using mice deficient in ICAM-1, we have investigated the role of this molecule in the development of collagen-induced arthritis. After immunization with type II collagen, 71% of wild-type mice developed arthritis compared with 50% of ICAM-1 heterozygote mutants and 18% of ICAM-1 homozygous mutants. In those ICAM-1 mutants that developed arthritis, the mean day of onset, the mean number of involved paws, and the severity of paw inflammation were not significantly different from those in wild-type mice. The reduced incidence of arthritis in the ICAM-1 homozygous mutant mice was not due to lack of immunity to type II collagen, since these mice developed similar levels of anti-type II collagen IgG compared with wild-type mice and had a positive delayed-type hypersensitivity reaction to type II collagen. The reduction of arthritis in heterozygous as well as homozygous deficient mice indicates that expression of ICAM-1 can be a pivotal variable in the pathogenesis of collagen-induced arthritis in mice. The results suggest that naturally occurring genetic variation in the expression of ICAM-1 or related inflammatory cell adhesion molecules might influence susceptibility to the complex disease of rheumatoid arthritis in humans and support the concept that pharmacologic approaches to chronic reduction in the expression or the function of ICAM-1 may be of therapeutic value.

Inhibition of delayed-type contact hypersensitivity in mice deficient in both E-selectin and P-selectin.

Leukocyte rolling and emigration in response to inflammatory stimuli appears to involve both E-selectin- and P-selectin-dependent adhesion, which suggests that these molecules have overlapping functions. To clarify their relative contributions in chronic inflammation, we examined delayed-type contact hypersensitivity (DTH) responses in P-selectin, E-selectin, and E-/P-selectin-deficient mice. Oxazolone-induced increases in ear thickness and ear weight were equivalent in wild-type mice and in P-selectin and E-selectin mutants, but were significantly reduced in E-/P-selectin mutants. The number and area of microabscesses on the ears of E-/P-deficient mice were decreased by 72% and 93%, and the number of leukocytes invading the subdermal ear tissue was reduced. T cells from E-/P-deficient mice transferred oxazolone reactivity into naive wild-type mice. However, when donor T cells from wild-type mice were transferred into E-/P-selectin-deficient mice, the DTH response was significantly impaired. These results show that leukocyte recruitment into a subacute inflammatory reaction can occur when either P-selectin or E-selectin is present, but is significantly reduced when both selectins are absent. Both P- and E-selectin are likely to play important roles in the development and maintenance of inflammatory diseases.

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.

Role of intercellular adhesion molecule-1 in antigen-induced lung inflammation in brown Norway rats.

We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype. 1A29 treatment produced dose-related increases in circulating 1A29 and blood neutrophils. In the BAL fluid of 1A29-treated animals, significant (P < 0.05) reductions in the numbers of eosinophils and lymphocytes, but not neutrophils or alveolar macrophages, were observed in association with a reduced inflammatory pathology in lung tissue. 1A29 administration reduced the number of detectable ICAM-1 binding sites on T cells in peripheral blood and BAL fluid examined ex vivo by flow cytometry. We conclude that ICAM-1 is critically important for the antigen-specific recruitment of eosinophils and lymphocytes into the lungs.

Type II collagen-induced arthritis in the diabetic-resistant biobreeding rat: inflammatory and histopathological features of joint pathology and effects of antiinflammatory and antirheumatic drugs on this chronic arthritic process.

Diabetic-resistant (DR) BioBreeding (BB) rats developed an erosive hind paw arthritis when immunized with an emulsion of bovine type II collagen (CII) and incomplete Freund's adjuvant. Macroscopic clinical evidence of type II collagen-induced arthritis (CIA) first appeared as periarticular erythema and edema in the hind paws between days 9 and 10 postimmunization with CII. The incidence of CIA was 100% by day 11 in the CII-challenged rats; and CIA severity progressed over a 28-day period with radiographic evaluation revealing focal resorption of bone together with osteophyte formation in the tibiotarsal joint and soft tissue swelling. The histopathology of CIA included an hyperplastic synovium that invaded and eroded articular cartilage at the joint margins, and subchondral bone resorption associated with bone-derived, multinucleated cell-containing granulomatous lesions in the rat hind paw. The corticosteroid, methylprednisolone (medrol), and the nonsteroidal antiinflammatory drug, flurbiprofen (Ansaid), administered at 2 mg/kg (p.o.), suppressed the clinical signs of CIA, and caused 79 to 83% inhibition of hind paw inflammation. However, methylprednisolone, but not flurbiprofen, inhibited the joint pathology in CIA. The antirheumatic drugs, cyclophosphamide (cytoxan, 5 mg/kg, p.o.) and cyclosporin A (CsA, 25 mg/kg, p.o.) suppressed the cartilage erosion in inflamed rat joints, and exerted marked inhibition (89-100%) of hind paw swelling. Methotrexate (0.15 mg/kg, p.o.) treatment reduced hind paw swelling (48%), whereas azathioprine, D-penicillamine (DP) and the oral gold preparation, auranofin, were inactive. Anti-CII antibody titers were completely suppressed by cyclosporin A and cytoxan. Radiographic evidence of protection from bone resorption, osteophyte formation and soft tissue swelling was apparent in the tibiotarsal joints of cytoxan, cyclosporin A, methylprednisolone and methotrexate-treated rat.

NPC 15669-modulated human polymorphonuclear neutrophil functional responsiveness: effects on receptor-coupled signal transduction.

1. The effect of NPC 15669, N-carboxy-L-leucine, N-[(2,7-dimethylfluoren-9-yl)methyl]ester), an inhibitor of human polymorphonuclear neutrophil (PMN) adhesion, on granule exocytosis and the oxidative burst was investigated in PMN activated with receptor-specific pathophysiological stimuli. 2. NPC 15669 caused a concentration-dependent (1-30 microM) inhibition of the extracellular release of azurophil (myeloperoxidase) and specific (vitamin B12-binding protein) granule constitutents from PMN exposed to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4), platelet activating factor (PAF), C5a and interleukin-8 (IL-8). 3. The receptor agonist-triggered PMN oxidative burst, measured as superoxide anion (O2-) production, was suppressed by NPC 15669. 4. Phorbol myristate acetate (PMA)-stimulated degranulation and O2-) production were unaffected by NPC 15669. 5. NPC 15669 (0.1-10 microM) inhibited receptor-triggered inositol 1,4,5-trisphosphate (IP3) production and the IP3-triggered increase in cytosolic-free calcium ([Ca2+]i) in FMLP-activated PMN, but not in cells exposed to the other receptor agonists. 6. NPC 15669 suppressed FMLP but not PMA-stimulated redistribution of protein kinase C (PKC) in PMN. 7. The specific binding of [3H]-FMLP but not [125I]-C5a to PMN was inhibited by NPC 15669. 8. NPC 15669 suppressed O2- production and the rise in [Ca2+]i in PMN treated with the guanine nucleotide-binding protein (G-protein) activators, sodium fluoride (NaF) and mastoparan, respectively. 9. The results show that NPC 15669 inhibits PMN responsiveness to various receptor agonists, and suggest interference with receptor-coupled signal transduction in this inflammatory cell at both the receptor and post-receptor level in a stimulus-specific manner.

FVB/N mice: an inbred strain sensitive to the chemical induction of squamous cell carcinomas in the skin.

The widespread use of FVB/N mice for the establishment of transgenic lines containing active oncogenes suggested the importance of testing the parent FVB/N mice for sensitivity to experimental carcinogenesis. After initiation of mouse skin by a single treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by 20 weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), the skin tumor incidence was compared in FVB/N mice, TPA-sensitive (SENCAR and CD-1) and TPA-resistant mice (BALB/c and C57BL/6). Initiation by 25 micrograms DMBA followed by promotion with a low dose of TPA (2 micrograms/week) induced one or more papillomas in only 25% of FVB/N mice, compared with 100% in SENCAR, 53% in CD-1, 17% in BALB/c and 0% in C57BL/6 mice. At a more effective dose of TPA (5 micrograms/week), FVB/N mice initiated by 5, 25 or 100 micrograms DMBA developed 3.4, 6.9 and 11.8 papillomas per mouse. In contrast, the incidence of squamous cell carcinomas (SCCs) (17-18/30 mice) did not increase with DMBA dose. TPA promotion of non-initiated mice induced only six papillomas, but three progressed to SCCs, a high rate of malignant conversion. Skin tumor induction by 20 weekly treatments with 10 micrograms DMBA produced few papillomas, but 50.0% of the papillomas progressed to carcinomas in FVB/N mice, compared with 9.15% in SENCAR, 37.5% in CD-1, 23.1% in BALB/c and 15.0% in C57CL/6 mice. The first carcinomas appeared after 14 weeks in FVB/N, 24 weeks in SENCAR, 26 weeks in CD-1 and C57BL/6 and 34 weeks in BALB/c mice. Thus, FVB/N mice develop an unusually high incidence of SCCs after treatment with repeated DMBA, DMBA initiation-TPA promotion and even TPA alone.

Reconstitution of glucose uptake and chemotaxis in Pseudomonas aeruginosa glucose transport defective mutants.

Wild-type glucose uptake and glucose chemotaxis activities were restored in glucose transport defective Pseudomonas aeruginosa strains PFB360 and PFB362 after introduction of plasmid pPZ129, containing a 1.1-kilobase DNA fragment that is essential for the expression of the P. aeruginosa periplasmic glucose binding protein. The restoration of glucose uptake and chemotaxis to wild-type levels in these strains was also achieved by reconstitution with cold-shock fluid and purified glucose binding protein isolated from P. aeruginosa PA01 wild-type strain H103 grown in conditions resulting in the induction of the high-affinity glucose transport system. Glucose uptake was determined by whole cell uptake and shock fluid binding of D-[U-14C]glucose, using standard filter binding assays. Positive chemotaxis towards glucose was assessed by capillary assays using 10 mM glucose, the amount required for optimal chemotaxis, and judged by plating capillary contents accumulated after 30 min.