Identification of Novel Bacteriophages with Therapeutic Potential That Target Enterococcus faecalis.

The Gram-positive opportunistic pathogen Enterococcus faecalis is frequently responsible for nosocomial infections in humans and represents one of the most common bacteria isolated from recalcitrant endodontic (root canal) infections. E. faecalis is intrinsically resistant to several antibiotics routinely used in clinical settings (such as cephalosporins and aminoglycosides) and can acquire resistance to vancomycin (vancomycin-resistant enterococci). The resistance of E. faecalis to several classes of antibiotics and its capacity to form biofilms cause serious therapeutic problems. Here, we report the isolation of several bacteriophages that target E. faecalis strains isolated from the oral cavity of patients suffering root canal infections. All phages isolated were Siphoviridae with similar tail lengths (200 to 250 nm) and icosahedral heads. The genome sequences of three isolated phages were highly conserved with the exception of predicted tail protein genes that diverge in sequence, potentially reflecting the host range. The properties of the phage with the broadest host range (SHEF2) were further characterized. We show that this phage requires interaction with components of the major and variant region enterococcal polysaccharide antigen to engage in lytic infection. Finally, we explored the therapeutic potential of this phage and show that it can eradicate E. faecalis biofilms formed in vitro on a standard polystyrene surface but also on a cross-sectional tooth slice model of endodontic infection. We also show that SHEF2 cleared a lethal infection of zebrafish when applied in the circulation. We therefore propose that the phage described here could be used to treat a broad range of antibiotic-resistant E. faecalis infections.

Improving animal and human health through understanding liver fluke immunology.

Sheep, goats and cattle represent the most numerous and economically important agricultural species worldwide used as sources for milk, fibre and red meat. In addition, in the developing world, these species often represent the sole asset base for small-holder livestock farmers and cattle/buffaloes often provide the majority of draught power for crop production. Production losses caused by helminth diseases of these animals are a major factor in extending the cycle of poverty in developing countries and a major food security issue for developed economies. Fasciola spp. are one of the most important zoonotic diseases with a global economic impact in livestock production systems and a poorly defined but direct effect on human health. Improvements in human and animal health will require a concerted research effort into the development of new accurate and simple diagnostic tests and increased vaccine and drug development against Fasciola infections. Here, the use of definitive natural host breeds with contrasting resistance to Fasciola infections is discussed as a resource to contrast parasite-host interactions and identify parasite immune evasion strategies. Such studies are likely to boost the discovery of new vaccine, drug and diagnostic candidates and provide the foundation for future genetic selection of resistant animals.

Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland.

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH(1) was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.

Nonphosphorylating respiration of mitochondria from brown adipose tissue of rats.

Mitochondria from brown adipose tissue of cold-acclimated rats (6 degrees C) oxidize alpha-ketoglutarate at a rate twice that of controls (26 degrees C). In both groups, however, the phosphorus: oxygen ratio with alpha-ketoglutarate never exceeded unity, and it is essentially zero with either succinate or alpha-glycerophosphate. Adenosine triphosphatase activity of these mitochondria is very low and it is not stimulated by 2,4-dinitrophenol. In addition, both respiration and phosphorylation are unaffected by adenosine diphosphate, 2,4-dinitrophenol, bovine serum albumin, or glutathione. Endogenous respiration of tissue slices is not stimulated by 2-4-dinitrophenol. It is suggested that brown fat mitochondria are not capable of oxidative phosphorylation, but do phosphorylate at the substrate level. Since these findings provide an unusual example of electron transport by means of an energetically nonconservative pathway, their significance to thermogenesis by brown adipose tissue is particularly emphasized.

Iron-manganese nodules from nares abyssal plain: geochemistry and mineralogy.

Three nodules from a core taken north of Puerto Rico are composed chiefly of an x-ray amorphous, hydrated, iron-manganese oxide, with secondary goethite, and minor detrital silicates incorporated during growth of the nodules. No primary manganese mineral is apparent. The nodules are enriched in iron and depleted in manganese relative to Atlantic Ocean averages. The formation of these nodules appears to have been contemporary with sedimentation and related to volcanic activity.

A feasibility study to investigate post-operative oxygen consumption (POpOC) after colorectal surgery requiring bowel resection.

BACKGROUND: Oxygen consumption after surgery is increased in response to the tissue trauma sustained intra-operatively and the subsequent systemic inflammatory response that ensues. The cardio-respiratory system must match the tissue oxygen and metabolic requirements; otherwise, peri-operative complications may occur. Existing data is several decades old. The primary objective of this feasibility study was to determine the ease of recruiting participants and collecting relevant data to assess the extent and duration of increased oxygen consumption and post-operative complications after major abdominal surgery in contemporaneous times. METHODS: One hundred patients scheduled for elective colorectal surgery requiring a bowel resection were screened to test specific feasibility criteria relating to ease of recruitment, duration of post-operative stay, ease of data collection, and drop-out rates. A calibrated metabolic cart was used to obtain unblinded pre-operative resting oxygen consumption recordings. The metabolic cart was then used to obtain post-operative oxygen consumption readings on days 1 to 5 as long as the participant remained as an inpatient. At the time of the oxygen consumption reading, a Post-Operative Morbidity Survey score (POMS) was calculated. Feasibility outcomes chosen a priori were that at least one participant would be recruited every 2 weeks from the pre-admission colorectal clinic, at least 10% of potential subjects screened would be enrolled, at least 80% of recruited participants would have a minimum post-operative stay of 2 nights, a minimum of 3 consecutive days of oxygen consumption data would be collected for each subject, at least 8 of 9 POMS score domains would be completed per participant per day and the drop-out rate would be no greater than 10%. We deemed that screening 100 patients would be sufficient to test our feasibility outcomes. RESULTS: Twelve participants completed the protocol. All pre-specified feasibility criteria were met. No increase in post-operative oxygen consumption was observed in this feasibility cohort. CONCLUSIONS: The protocol and experiences gained from this feasibility study could be used to plan a larger study to better define changes in post-operative oxygen consumption after major abdominal surgery utilizing current surgical techniques.

Inhibition of a Plasmodium vinckei cysteine proteinase cures murine malaria.

Intraerythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously identified a Plasmodium falciparum trophozoite cysteine proteinase as a putative hemoglobinase and shown that specific inhibitors of this proteinase block the hydrolysis of globin and the development of cultured parasites. We now show that the murine malaria parasite Plasmodium vinckei has an analogous cysteine proteinase with similar biochemical properties to the P. falciparum proteinase, including an acid pH optimum, a preference for the peptide proteolytic substrate benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methylcoumarin, and nonomolar inhibition by seven peptide fluoromethyl ketone proteinase inhibitors. Thus, P. vinckei offers a model system for the in vivo testing of the antimalarial properties of cysteine proteinase inhibitors. One of the proteinase inhibitors studied, morpholine urea (Mu)-Phe-Homophenylalanine (HPhe)-CH2F strongly inhibited the P. vinckei cysteine proteinase in vitro and rapidly blocked parasite cysteine proteinase activity in vivo. When administered four times a day for 4 d to P. vinckei-infected mice, Mu-Phe-HPhe-CH2F elicited long-term cures in 80% of the treated animals. These results show that peptide proteinase inhibitors can be effective antimalarial compounds in vivo and suggest that the P. falciparum cysteine proteinase is a promising target for chemotherapy.

Late left ventricular lead displacement and treatment with a new endovenous active fixation lead.

Left ventricular (LV) lead displacement is an early complication of biventricular pacemakers and leads to loss of capture, diaphragmatic pacing, and symptomatic deterioration, requiring a revision procedure. We report a case of late LV lead displacement following a coughing fit and treatment with a lead with a new principle of active fixation.

Clinical Consequences of Hypertriglyceridemia-Associated Proteinuria in Miniature Schnauzers.

BACKGROUND: Primary hypertriglyceridemia is a common condition in older Miniature Schnauzers that recently has been associated with proteinuria and underlying glomerular pathology, particularly glomerular lipid thromboemboli. Consequences of glomerular disease can include hypertension, thromboembolic disease, and cardiac disease. The incidence of these sequelae in Miniature Schnauzers with hypertriglyceridemia-associated proteinuria (HTGP) is unknown. OBJECTIVE: To investigate prevalence of hypertension, decreased antithrombin III activity, and cardiac disease in Miniature Schnauzers with and without HTGP. ANIMALS: Thirty-two Miniature Schnauzers ≥7 years old. METHODS: Prospective case-control study. Data collected from dogs included a CBC, biochemistry panel, urinalysis, urine protein-to-creatinine ratio, urine cortisol-to-creatinine ratio, serum total thyroxine concentration, fasting serum triglyceride concentration, indirect blood pressure, antithrombin III activity, and serum cardiac troponin I concentration. Results from dogs with HTGP (serum triglyceride concentration ≥ 100 mg/dL and urine protein-to-creatinine ratio >0.5) were statistically compared to normotriglyceridemic, nonproteinuric dogs. RESULTS: Eighteen of the 32 dogs (56%) had primary hypertriglyceridemia. Of those dogs, 8 of 18 had proteinuria. None of the HTGP dogs were azotemic or hypoalbuminemic. Serum albumin concentration, alkaline phosphatase activity, and cholesterol concentration were significantly increased in dogs with HGTP compared to those without HGTP. No increased risk of hypertension, decreased antithrombin III activity, or cardiac disease was noted. Limited data from 8 dogs with HTGP showed no development of hypoalbuminemia or azotemia over a median follow-up period of 18 months. CONCLUSIONS AND CLINICAL IMPORTANCE: Geriatric Miniature Schnauzers with HGTP may have a good prognosis overall, and are not typically azotemic or hypoalbuminemic.

Association of persistent synthesis of viral DNA with macrophage accessory cell dysfunction induced by avian retrovirus myeloblastosis-associated virus of subgroup B inducing osteopetrosis in chickens.

This investigation concentrates on a regenerative anemia and immunosuppression occurring in the absence of osteopetrosis. Polyclonal activation of T-cells was used as an in vitro test system to study immunosuppression induced by the avian myeloblastosis-associated virus of Subgroup B inducing osteopetrosis [MAV-2(O)]. T-cell unresponsiveness in vitro was attributed to a defect in an accessory cell function of the macrophage. Counterflow centrifugation fractionation followed by mixing experiments indicated that the T-cell population from immunosuppressed chickens responded to mitogen stimulation when added to control macrophage cultures. In addition, lymphocyte fractions from uninfected chickens were unresponsive when added to macrophage cultures isolated from MAV-2(O)-infected chickens. Cultured splenic macrophages isolated from infected chickens contained high levels of both integrated and unintegrated viral DNA and formed syncytia by 21 days in culture. The macrophages remained viable and exhibited mature functional characteristics during mitogen stimulation assays. Therefore, it was speculated that the persistent synthesis of retrovirus DNA might be involved in the inability of infected macrophages to function as accessory cells.

Inhibition of concanavalin A response during osteopetrosis virus infection.

Infection of animals with oncogenic viruses frequently leads to an immunosuppressed state. We have examined immunosuppression induced by an avian osteopetrosis virus, myeloblastosis-associated virus of subgroup B inducing osteopetrosis [MAV-2(O)], and our results suggest that this virus induces immunosuppression by a novel mechanism. Lymphoid cells from osteopetrotic chickens did not respond to a wide dose range of concanavalin A (Con A) over a wide cell density range. Failure to undergo blastogenesis was not due to a lack of Con A-binding sites, since 125I-labeled Con A bound to lymphocytes from infected and uninfected chickens. Infected lymphocytes failed to respond to sodium metaperiodate stimulation, indicating that failure of blastogenesis was not due to a blockage of Con A receptor sites. MAV-2(O) infection of chicks 8 days of age resulted in a transient immunosuppression which appeared 1 to 2 weeks after infection. Cell-mixing experiments showed that MAV-2(O)-induced immunosuppression was not attributable to suppressor cells. In contrast, adherent cells from normal lymphoid preparations restored mitogenicity to lymphocytes from MAV-2(O)-infected animals. Adherent cells were present in the spleen and peripheral blood lymphocytes of MAV-2(O)-infected chickens in numbers comparable to those of the uninfected animal, and both sets of cells contained Fc-dependent phagocytic activity and nonspecific esterase. Peritoneal exudate cells were elicited from osteopetrotic and normal chickens in similar numbers. We conclude that MAV-2(O) induces immunosuppression by interfering with an accessory function of macrophage-like adherent cells.

Influence of bursectomy on bone growth and anemia induced by avian osteopetrosis viruses.

This study examines the contribution of the bursa of Fabricius to the pathogenic manifestations of two myeloblastosis-associated viruses which primarily cause osteopetrosis [MAV-1(O) and MAV-2(O)]. MAV-2(O) infection of surgically bursectomized 1-month-old chicks resulted in a rapidly fatal anemia whereas infection of untreated chicks of the same age resulted in a transient drop in hematocrit. Surgical bursectomy of embryos before or after embryonal infection with MAV-2(O) did not alter the course of osteopetrosis, indicating that the bursa was not a source of target cells. Bursectomy prolonged the period of susceptibility to MAV-2(O) induced osteopetrosis until one day posthatching; untreated chicks were not susceptible to osteopetrosis induction at that age. MAV-1(O) infection of eight-day-old bursectomized chicks resulted in osteopetrosis in the absence of anemia; untreated eight-day-old chicks infected with MAV-1(O) showed no effects of virus infection. A role for the bursa in MAV-2(O) infection was found in the participation of neutralizing antibodies in the recovery from anemia. A single dose of antiviral antibody was found to prevent the appearance of anemia. The protective effect of antiviral antibody was dose dependent, and antiserum administration had to be initiated within three days after virus in order to be effective. Antiviral antibody against MAV-1(O) did not protect against MAV-2(O)-induced anemia, suggesting subgroup specificity. These results suggest that the bursa does not provide a stem cell which participates in the bone hyperplasia induced by MAV-1(O) and MAV-2(O). Rather, the humoral antibodies provided by cells derived from the bursa may serve to eliminate viremia and limit virus-specific cytopathogenic effects.

Proliferative fibromatosis in avian skeletal muscle caused by cloned recombinant avian leukosis viruses.

Two molecular recombinants (EU-8 and K-3) constructed from ring-necked pheasant virus and UR2AV, the helper virus associated with avian sarcoma virus UR2, caused a high incidence of a hitherto unreported pathological condition in chick skeletal muscle. A disease spectrum was observed in which muscle was infiltrated by proliferating fibroblasts and caused white streaks, white diffuse areas, or well-defined elongated tumors. Fibroblast proliferation was progressive, and the gross presentation depended on the rapidity and extent of proliferation. There was evidence of anaplasia but not frankly malignant disease or metastases. The disease was produced at a high frequency when 10-day-old embryos were infected. Breast muscle (pectoralis major and minor) was affected with variable severity in all birds, thigh muscles were affected occasionally, while other thoracic, external abdominal, and wing muscles were affected very rarely. Progression of proliferative muscle lesions was demonstrated by sequential necropsies and microscopic examination of muscle samples. The disease appeared before 2 weeks of age, and thin white streaks with minimal cellular proliferation were the earliest lesions observed. Elongated, spindle-shaped tumors were the most advanced form of proliferation observed. The advanced lesions showed cellular anaplasia and signs of rapid growth, and appeared most commonly at 6 weeks of age or later. Histologically, mild proliferation correlated with normal appearing fibroblasts producing collagen. Severe proliferation correlated with anaplastic, rapidly dividing cells producing little collagen and a high mitotic index. A decrease in the virus dose resulted in less severe fibromatosis, but at least one chicken infected with a low virus dose showed severe disease. When 10- or 35-day-old hatched chicks were injected in the breast muscle with EU-8 or K-3, a low incidence of lymphoid leukosis was observed, but no fibromatosis resulted during an observation period of 27 weeks. Infectious virus was extracted from the fibromatosis in the breast muscle, and extracted virus caused the same disease when injected in 10-day-old embryos. No transforming activity was observed on normal chick embryo fibroblast monolayers. Cell cultures were established from areas in the breast muscle affected by fibromatosis, and from discrete tumors. These cells did not show any signs of transformation in culture. Supernatants obtained from these cell cultures caused fibromatosis when injected into 10-day-old embryos, but did not transform normal chick embryo fibroblasts.

Secondary allergic T cell responses are regulated by dendritic cell-derived thrombospondin-1 in the setting of allergic eye disease.

Allergic eye disease, as in most forms of atopy, ranges in severity among individuals from immediate hypersensitivity to a severe and debilitating chronic disease. Dendritic cells play a key role in stimulating pathogenic T cells in allergen re-exposure, or secondary responses. However, molecular cues by dendritic cells underpinning allergic T cell response levels and the impact that this control has on consequent severity of allergic disease are poorly understood. Here, we show that a deficiency in thrombospondin-1, a matricellular protein known to affect immune function, has subsequent effects on downstream T cell responses during allergy, as revealed in an established mouse model of allergic eye disease. More specifically, we demonstrate that a thrombospondin-1 deficiency specific to dendritic cells leads to heightened secondary T cell responses and consequent clinical disease. Interestingly, whereas thrombospondin-1-deficient dendritic cells augmented activity of allergen-primed T cells, this increase was not recapitulated with naïve T cells in vitro. The role of dendritic cell-derived thrombospondin-1 in regulating secondary allergic T cell responses was confirmed in vivo, as local transfer of thrombospondin-1-sufficient dendritic cells to the ocular mucosa of thrombospondin-1 null hosts prevented the development of augmented secondary T cell responses and heightened allergic eye disease clinical responses. Finally, we demonstrate that topical instillation of thrombospondin-1-derived peptide reduces T cell activity and clinical progression of allergic eye disease. Taken together, this study reveals an important modulatory role of dendritic cell-derived thrombospondin-1 on secondary allergic T cell responses and suggests the possible dysregulation of dendritic cell-derived thrombospondin-1 expression as a factor in allergic eye disease severity.

The role of cysteine residues in the catalytic activity of glycerol-3-phosphate dehydrogenase.

Glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxido-reductase, EC 1.1.1.8) has been shown to be sensitive to inhibition by iodoacetate. The reaction of the enzyme with iodoacetate, which appears to be a simple bimolecular process, is accompanied by a corresponding loss of enzyme activity. In addition to changes in activity, the alkylation reaction was monitored by the incorporation of radioactivity from iodo[2-14C]acetate, by changes in amino acid composition, and by changes in the content of free sulfhydryl groups. It is concluded that there are two cysteine residues in the native dimeric enzyme which are essential for enzymic activity. The rate of inactivation was relatively insensitive to the presence of various compounds with the exception of NADH which markedly inhibited the reaction. Kinetic and binding studies showed that the binding of NADH prevents alkylation and, conversely, alkylation prevents NADH binding. From the pH dependence of the alkylation reaction, the pKa of the essential sulfhydryl groups was calculated to be 8.5 and it is suggested that the binding of coenzyme is independent of the state of ionization of these groups.

Aromatic dental monomers affect the activity of cholesterol esterase.

The dental restorative monomer, BISGMA (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane), and bisphenol A diglycidyl ether (BADGE) increase the velocity of the reaction catalyzed by pancreatic cholesterol esterase (CEase, bovine). The metabolite of these monomers, bisphenol A bis(2,3-dihydroxypropyl) ether, and a common plasticizer, di-2-ethylhexyl phthalate (DEHP), also increase the velocity of CEase-catalyzed ester hydrolysis. BISGMA at concentrations of 1.5-8.0 microM increases the velocity to 126-169% of its value in the absence of BISGMA. Increasing BISGMA above 8 microM caused no further increase in velocity. BADGE at 7-25 microM increases the velocity to 112-205% of its value without BADGE. The metabolite of BISGMA and BADGE at concentrations of 2.0-7.1 microM increases the velocity to 103-113% of its value without metabolite. DEHP at concentrations of 0.52-4.3 microM increases the velocity to 108-187% of its value without DEHP. On the other hand, bisphenol A dimethacrylate is a competitive inhibitor of CEase, with a K(i) of 3.1 microM.

Randomized trial of 3-hour versus 24-hour infusion of high-dose paclitaxel in patients with metastatic or locally advanced breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-26.

PURPOSE: Paclitaxel is an active drug for the treatment of breast cancer; however, the appropriate duration of administration is unknown. We assessed and compared the response rate, event-free survival, survival, and toxicity of paclitaxel 250 mg/m(2) delivered every 3 weeks as a 3-hour or 24-hour infusion. PATIENTS AND METHODS: A total of 563 women with stage IV or IIIB breast cancer were randomized into one of two groups: 279 received 3-hour paclitaxel and 284 received 24-hour paclitaxel. Patients were stratified by age, stage of disease, and prior therapy. RESULTS: A significantly higher rate of tumor response occurred in the first four cycles of therapy in patients who received the 24-hour infusion of paclitaxel (51% v 41%, respectively; P =.025). Tumor response over all cycles was also significantly higher in the group that received 24-hour infusion (54% v 44%, respectively; P =.023). There were no significant differences in event-free survival or survival between the two arms of the study (P =.9 and.8, respectively). No treatment by stage or by age interactions were observed. During the first four cycles of therapy, at least one episode of >/= grade 3 toxicity (excluding nadir hematologic values, alopecia, and weight change) occurred in 45% of patients who received the 3-hour paclitaxel infusion and in 50% of those who received the 24-hour paclitaxel infusion. Febrile neutropenia, >/= grade 3 infection, and >/= grade 3 stomatitis were less frequent, and severe neurosensory toxicity was more frequent in those who received the 3-hour paclitaxel infusion. Ten treatment-related deaths occurred in the first four cycles. Age, stage, and prior chemotherapy did not influence the effect of treatment. CONCLUSION: When administered as a continuous 24-hour infusion, high-dose paclitaxel results in a higher tumor response rate than when administered as a 3-hour infusion but does not significantly improve event-free survival or survival. Paclitaxel as a 24-hour infusion results in increased hematologic toxicity and decreased neurosensory toxicity.

Rapid induction of B-cell lymphomas by avian leukosis virus.

Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene and produce an ALV-myb read-through RNA, which is spliced to produce a truncated Myb protein. The genetic determinants of such recombinant ALVs have been mapped to a 42-nt deletion within the gag gene. This deletion increases splicing efficiency since it is located within a negative regulator of splicing. We propose that the deletion leads to increased production of Myb protein by increasing splicing of an ALV-myb pre-mRNA.

Cell-to-cell and cell-to-matrix interactions mediate chemokine expression: an important component of the inflammatory lesion.

Although many studies have characterized soluble factors that stimulate or inhibit chemokine secretion, in this review we focus on the event of cellular adhesion as a novel mechanism for stimulating chemokine expression. Recent work has demonstrated chemokine expression following cell-to-cell and cell-to-matrix adhesion. The specificity of this finding was demonstrated utilizing various techniques that illustrate that adhesion, and not a soluble stimulus, is in some cases responsible for initiating or augmenting chemokine expression. For example, co-cultures of peripheral blood monocytes and endothelial cells secreted elevated levels of IL-8 and MCP-1 compared with either cell type alone. When co-cultured in transwells, this effect was significantly attenuated. In other experiments, neutralizing monoclonal antibodies to various adhesion molecules inhibited chemokine expression. The effects of adhesion were not limited to leukocytes. Both immune and non-immune cell types were evaluated as potential sources of adhesion-mediated chemokine expression. Not suprisingly, expression of some chemokines was associated with adhesion, whereas others were not, supporting the notion that adhesion differentially signals chemokine secretion during the inflammatory response. We hypothesize that as a recruited leukocyte encounters different adhesion substrates such as endothelial cells, basement membrane, extracellular matrix, and fibroblasts, the expression of chemokines from both the leukocyte and the substrate may be initiated, inhibited, or augmented. Careful characterization of the contribution of adhesion to regulation of chemokine expression will provide insight into the pathogenesis of many human diseases where chemokines have a central role.

TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury.

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.