Effects of photobiomodulation and caffeine treatment on acute kidney injury in a hypoxic ischemic neonatal rat model.

Hypoxic ischemic encephalopathy (HIE) occurs in 2-5/1000 births, with acute kidney injury (AKI) occurring in 40%. AKI increases morbidity and mortality. Caffeine, an adenosine receptor antagonist, and photobiomodulation (PBM), working on cytochrome c oxidase, are potential treatments for AKI. To examine effects of caffeine and PBM on AKI in rats, Day 7 pups underwent a HIE intervention (Modified Rice-Vannucci model) replicating pathology observed in humans. Caffeine was administered for 3 days and/or PBM for 5 days following HIE. Weights and urine for biomarkers (NGAL, albumin, KIM-1, osteopontin) were collected prior to HIE, daily post intervention and at sacrifice. Both treatments reduced kidney injury seen on electron microscopy, but not when combined. HIE elevated urinary NGAL and albumin on Days 1-3 post-HIE, before returning to control levels. This elevation was significantly reduced by PBM or caffeine. KIM-1 was significantly elevated for 7 days post-HIE and was reduced by both treatments. Osteopontin was not altered by HIE or the treatments. Treatments, individually but not in combination, improved HIE-induced reductions in the enzymatic activity of mitochondrial complexes II-III. PBM and caffeine also improved weight gain. PBM and caffeine reduces AKI diagnosed by urinary biomarkers and confirmed by EM findings.

Intrarenal B Cell Cytokines Promote Transplant Fibrosis and Tubular Atrophy.

Renal transplantation is the optimum treatment for end-stage renal failure. B cells have been identified in chronic allograft damage (CAD) and associated with the development of tertiary lymphoid tissue within the human renal allograft. We performed renal transplantation in mice to model CAD and identified B cells forming tertiary lymphoid tissue with germinal centers. Intra-allograft B220(+) B cells comprised of IgM(high) CD23(-) B cells, IgM(lo) CD23(+) B cells, and IgM(lo) CD23(-) B cells with elevated expression of CD86. Depletion of B cells with anti-CD20 was associated with an improvement in CAD but only when administered after transplantation and not before. Isolated intra-allograft B cells were cultured and shown to synthesize multiple cytokines, the most abundant of these were GRO-α (CXCL1), RANTES (CCL5), IL-6 and MCP-1 (CCL2). Tubular loss was observed with T cell accumulation within the allograft and development of interstitial fibrosis, whilst type III collagen deposition was observed in areas of F4/80(+) macrophages and PDGFR-ß(+) and transgelin(+) fibroblasts, all of which were reduced by B cell depletion. We have shown that intra-allograft B cells are key mediators of CAD. B cells possibly contribute to CAD by intra-allograft secretion of cytokines and chemokines.

Indications for thrombolysis in deep venous thrombosis.

OBJECTIVES: Deep venous thromboses (DVTs) are a significant cause of morbidity and mortality in the general and inpatient population. Current anticoagulation therapy is efficient in reducing thrombus propagation but does not contribute to clot lysis or prevention of post-thrombotic limb syndrome. Catheter directed thrombolysis (CDT) is an alternative method for treating DVTs but there is no consensus regarding indications for its use. DATA SOURCES: PubMed and Cochrane library were searched for all articles on deep vein thrombosis and thrombolysis. REVIEW METHOD: Articles presenting data on DVT thrombolysis, DVT anticoagulation, mechanical thrombectomy, venous stenting and May-Thurner's syndrome were considered for inclusion in the review. RESULTS: CDT reduced clot burden, DVT recurrence and may prevent the formation of post-thrombotic syndrome. Indications for its use include younger individuals with a long life expectancy and few co-morbidities, limb-threatening thromboses and proximal ilio-femoral DVTs. There is a marked lack of randomised controlled trials comparing CDT-related mortality and long term outcomes compared to anticoagulation alone. The effectiveness of combined pharmaco-mechanic thrombectomy, although promising, need to be further investigated, as is the role of caval filters in preventing DVT-associated pulmonary emboli. CONCLUSIONS: These results suggest that the outcome of CDT in DVT management are encouraging in selected patient cohorts, but further evidence is required to establish longer term benefits and cost-effectiveness.

Fracture healing in HIV-positive populations.

Highly active anti-retroviral therapy has transformed HIV into a chronic disease with a long-term asymptomatic phase. As a result, emphasis is shifting to other effects of the virus, aside from immunosuppression and mortality. We have reviewed the current evidence for an association between HIV infection and poor fracture healing. The increased prevalence of osteoporosis and fragility fractures in HIV patients is well recognised. The suggestion that this may be purely as a result of highly active anti-retroviral therapy has been largely rejected. Apart from directly impeding cellular function in bone remodelling, HIV infection is known to cause derangement in the levels of those cytokines involved in fracture healing (particularly tumour necrosis factor-alpha) and appears to impair the blood supply of bone. Many other factors complicate this issue, including a reduced body mass index, suboptimal nutrition, the effects of anti-retroviral drugs and the avoidance of operative intervention because of high rates of wound infection. However, there are sound molecular and biochemical hypotheses for a direct relationship between HIV infection and impaired fracture healing, and the rewards for further knowledge in this area are extensive in terms of optimised fracture management, reduced patient morbidity and educated resource allocation. Further investigation in this area is overdue.

Gross and histopathological characteristics of two lipomas and a neurofibrosarcoma detected in aquacultured southern bluefin tuna, Thunnus maccoyii (Castelnau), in South Australia.

Three visible lesions were examined from two specimens of southern bluefin tuna. The lesions were examined grossly and two were identified as lipomas, the third bore similarities to a schwannoma. Histopathology confirmed that two consisted of mature adipocytes consistent with a diagnosis of lipoma. The third lesion consisted of spindle cells in Antoni A and B patterns and was tentatively diagnosed as a malignant schwannoma. Immunohistochemistry identified both S100 and glial fibrillary acid protein expression within the lesion, which, together with the histopathological appearance, is consistent with a diagnosis of neurofibrosarcoma.

A parasite survey of farmed Southern bluefin tuna, Thunnus maccoyii (Castelnau).

Farmed Southern bluefin tuna (SBT) were examined for parasites. Samples of harvest fish, mortalities and some fish showing clinical signs of disease were investigated. Targeted screening was conducted for a scuticociliate, Uronema nigricans, the myxosporean Kudoa sp. and a sanguinicolid digenean, Cardicola forsteri. General parasitological investigation revealed a diverse parasite community of didymozoid digeneans, two species of copepods, a polyopisthocotylean monogenean and larval cestodes. Targeted screening for U. nigricans exposed a low prevalence, most probably due to a lack of sensitivity in the test method. Few of the parasites examined pose a risk to the health of farmed SBT.

Recovery from oxidant-mediated lung injury: response of metallothionein, MIP-2, and MCP-1 to nitrogen dioxide, oxygen, and ozone exposures.

Oxidant-induced lung injury is believed to be mediated by reactive oxygen species. Recovery from oxidant exposure has been associated with pulmonary inflammation. Inflammatory cell accumulation involves the synthesis of chemokines, including neutrophil chemoattractants such as macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractants such as monocyte chemoattractant protein-1 (MCP-1). Antioxidants are the first line of defense of lung cells against inhaled oxidants. Metallothionein (MT) can act as an antioxidant and free-radical scavenger. To better understand the pulmonary response associated with recovery from oxidant-mediated injury, we exposed mice to either 15 ppm nitrogen dioxide for 24 h, >99% oxygen for 72 h, or 1 ppm ozone for 24 h. Mice were examined at the end of exposure or after recovering in room air for 4 or 24 h. Neutrophils were elevated at the end of exposure and remained elevated through the postexposure period, whereas macrophage numbers were decreased at the end of exposure and remained below control levels at 4 and 24 h postexposure. MT, MIP-2, and MCP-1 mRNA levels were elevated at 4 h postexposure; however, after 24 h of recovery only MCP-1 remained elevated. These results indicate that MT, MIP-2, and MCP-1 mRNA levels responded similarly to recovery from nitrogen dioxide, oxygen, and ozone exposure. Monocyte accumulation was delayed as compared to neutrophils and was consistent with the timing of MIP-2 and MCP-1 expression. Peak expression of MT and MIP-2 preceded peak neutrophil accumulation. Consequently, the timing of MT, MIP-2, and MCP-1 expression may be important biological markers in assessing the state of injury and recovery associated with oxidant-mediated injury.

Pulmonary effects induced by ultrafine PTFE particles.

PTFE (polytetrafluoroethylene) fumes consisting of large numbers of ultrafine (uf) particles and low concentrations of gas-phase compounds can cause severe acute lung injury. Our studies were designed to test three hypotheses: (i) uf PTFE fume particles are causally involved in the induction of acute lung injury, (ii) uf PTFE elicit greater pulmonary effects than larger sized PTFE accumulation mode particles, and (iii) preexposure to the uf PTFE fume particles will induce tolerance. We used uf Teflon (PTFE) fumes (count median particle size approximately 16 nm) generated by heating PTFE in a tube furnace to 486 degrees C to evaluate principles of ultrafine particle toxicity. Teflon fumes at ultrafine particle concentrations of 50 microg/m(3) were extremely toxic to rats when inhaled for only 15 min. We found that when generated in argon, the ultrafine Teflon particles alone are not toxic at these exposure conditions; neither were Teflon fume gas-phase constituents when generated in air. Only the combination of both phases when generated in air caused high toxicity, suggesting either the existence of radicals on the surface or a carrier mechanism of the ultrafine particles for adsorbed gas compounds. Aging of the fresh Teflon fumes for 3.5 min led to a predicted coagulation to >100 nm particles which no longer caused toxicity in exposed animals. This result is consistent with a greater toxicity of ultrafine particles compared to accumulation mode particles, although changes in particle surface chemistry during the aging process may have contributed to the diminished toxicity. Furthermore, the pulmonary toxicity of the ultrafine Teflon fumes could be prevented by adapting the animals with short 5-min exposures on 3 days prior to a 15-min exposure. Messages encoding antioxidants and chemokines were increased substantially in nonadapted animals, yet were unaltered in adapted animals. This study shows the importance of preexposure history for the susceptibility to acute ultrafine particle effects.

Amputation neuroma of the great auricular nerve after operations on the parotid gland.

We found an incidence of 6% (5/81) of traumatic neuroma after section of the great auricular nerve during operations on the parotid gland. Excision permits definitive diagnosis, the stump being allowed to retract beneath the belly of the sternomastoid muscle. However, excision is not always indicated, and the diagnosis can be made clinically allowing for a more conservative treatment policy.

A new compound heterozygous mutation of the gonadotropin-releasing hormone receptor (L314X, Q106R) in a woman with complete hypogonadotropic hypogonadism: chronic estrogen administration amplifies the gonadotropin defect.

We describe a woman with complete hypogonadotropic hypogonadism and a new compound heterozygous mutation of the GnRH receptor (GnRHR) gene. A null mutation L314X leading to a partial deletion of the seventh transmembrane domain of the GnRHR is associated with a Q106R mutation previously described. L314X mutant receptor shows neither measurable binding nor inositol phosphate production when transfected in CHO-K1 cells compared to the wild-type receptor. The disease is transmitted as an autosomal recessive trait, as shown by pedigree analysis. Heterozygous patients with GnRHR mutations had normal pubertal development and fertility. The present study shows an absence of LH and FSH response to pulsatile GnRH administration (20 microg/pulse, sc, every 90 min). However, GnRH triggered free alpha-subunit (FAS) pulses of small amplitude, demonstrating partial resistance to pharmacological doses of GnRH. FSH, LH, and FAS concentrations were evaluated under chronic estrogen treatment and repeat administration of GnRH. Not only were plasma FSH, LH, and FAS concentrations decreased, but FAS responsiveness was reduced. This new case emphasizes the implication of the GnRH receptor mutations in the etiology of idiopathic hypogonadotropic hypogonadism. We also have evidence for a direct negative estrogen effect on gonadotropin secretion at the pituitary level, dependent on the GnRHR signaling pathway.

Pulmonary chemokine and mutagenic responses in rats after subchronic inhalation of amorphous and crystalline silica.

Chronic inhalation of crystalline silica can produce lung tumors in rats whereas this has not been shown for amorphous silica. At present the mechanisms underlying this rat lung tumor response are unknown, although a significant role for chronic inflammation and cell proliferation has been postulated. To examine the processes that may contribute to the development of rat lung tumors after silica exposure, we characterized the effects of subchronic inhalation of amorphous and crystalline silica in rats. Rats were exposed for 6 h/day, on 5 days/week, for up to 13 weeks to 3 mg/m(3) crystalline or 50 mg/m(3) amorphous silica. The effects on the lung were characterized after 6.5 and 13 weeks of exposure as well as after 3 and 8 months of recovery. Exposure concentrations were selected to induce high pulmonary inflammatory-cell responses by both compounds. Endpoints characterized after silica exposure included mutation in the HPRT gene of isolated alveolar cells in an ex vivo assay, changes in bronchoalveolar lavage fluid markers of cellular and biochemical lung injury and inflammation, expression of mRNA for the chemokine MIP-2, and detection of oxidative DNA damage. Lung burdens of silica were also determined. After 13 weeks of exposure, lavage neutrophils were increased from 0.26% (controls) to 47 and 55% of total lavaged cells for crystalline and amorphous silica, with significantly greater lavage neutrophil numbers after amorphous silica (9.3 x 10(7) PMNs) compared to crystalline silica (6.5 x 10(7) PMNs). Lung burdens were 819 and 882 microg for crystalline and amorphous silica, respectively. BAL fluid levels of LDH as an indicator of cytotoxicity were twice as high for amorphous silica compared to those of crystalline silica, at the end of exposure. All parameters remained increased for crystalline silica and decreased rapidly for amorphous silica in the 8-month recovery period. Increased MIP-2 expression was observed at the end of the exposure period for both amorphous and crystalline silica. After 8 months of recovery, those markers remained elevated in crystalline silica-exposed rats, whereas amorphous silica-exposed rats were not significantly different from controls. A significant increase in HPRT mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to crystalline, but not to amorphous silica. A significant increase in TUNEL staining was detected in macrophages and terminal bronchiolar epithelial cells of amorphous silica-exposed rats at the end of the exposure period; however, crystalline silica produced far less staining. The observation that genotoxic effects in alveolar epithelial cells occurred only after crystalline but not amorphous silica exposure, despite a high degree of inflammatory-cell response after subchronic exposure to both types of silica, suggests that in addition to an inflammatory response, particle biopersistence, solubility, and direct or indirect epithelial cell cytotoxicity may be key factors for the induction of either mutagenic events or target cell death.

Fas-independent apoptosis in T-cell tumours induced by the CD2-myc transgene.

Depending on the cellular context, the Myc oncoprotein is capable of promoting cell proliferation or death by apoptosis. These observations suggest that apoptosis in response to deregulated gene expression may represent a natural brake to tumour development. The pathways by which Myc induces apoptosis are as yet poorly characterised although recent observations on rat fibroblasts over-expressing Myc have demonstrated a requirement for the Fas pathway. To investigate the role of Fas in Myc-induced lymphomagenesis we backcrossed CD2-myc mice onto an lpr background. Rates of tumour development and phenotypic properties, including levels of apoptosis were indistinguishable from CD2-myc controls. Further, tumour cell lines derived from mice expressing a regulatable form of Myc showed inducible apoptosis at similar rates regardless of their lpr genotype. These results show that activation of c-myc and loss of Fas do not collaborate in T lymphoma development and that Myc-induced apoptosis in T-cells occurs by Fas-independent pathways.

Antioxidant and inflammatory response after acute nitrogen dioxide and ozone exposures in C57Bl/6 mice.

Ozone (O(3)) and nitrogen dioxide (NO(2)) are highly reactive and toxic oxidant pollutants. The objective of this study is to compare chemokine, cytokine, and antioxidant changes elicited by acute exposures of O(3) and NO(2) in a genetically sensitive mouse. Eight-week-old C57Bl/6J mice were exposed to 1 or 2.5 ppm ozone or 15 or 30 ppm NO(2) for 4 or 24 h. Changes in mRNA abundance in lung were assayed by slot blot and ribonuclease protection assay (RPA). Messages encoding metallothionein (Mt), heme oxygenase I (HO-I), and inducible nitric oxide synthase (iNOS) demonstrated increased message abundance after 4 and 24 h of exposure to either O(3) or NO(2). Furthermore, increases in message abundance were of a similar magnitude for O(3) and NO(2). Messages encoding eotaxin, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 were elevated after 4 and 24 h of exposure to 1 ppm ozone. Interleukin-6 was elevated after 4 h of exposure to ozone. After 4 h of 2.5 ppm ozone exposure, increased mRNAs of eotaxin, MIP-1alpha, MIP-2, Mt, HO-I, and iNOS were elevated to a higher magnitude than were detected after 1 ppm ozone. Monocyte chemoattractant protein (MCP-1) was elevated following 15 ppm NO(2) exposure. After 4 h of 30 ppm NO(2) exposure, messages encoding eotaxin, MIP-1alpha, MIP-2, and MCP-1 were elevated to levels similar to those detected after ozone exposure. Our results demonstrate a similar antioxidant and chemokine response during both O(3) and NO(2) exposure. Induction of these messages is associated with the duration and concentration of exposure. These studies suggest that these gases exert toxic action through a similar mechanism.

Newborn mice differ from adult mice in chemokine and cytokine expression to ozone, but not to endotoxin.

Neonatal animals of some mammalian species are more tolerant to several pulmonary oxidative stress-inducing toxicants than adults. Our initial studies during hyperoxic injury demonstrated a rapid chemokine and cytokine response early in the development of injury in newborn mice, whereas adult mice demonstrated little alteration in cytokine abundance until lethality was imminent. Our hypothesis is that altered response between newborn and adult mice is associated with differential cell injury, rather than alterations in the regulation of the inflammatory response. To test this hypothesis we utilized two distinct models of inducing pulmonary toxicity: ozone (O(3)), which causes epithelial cell injury, and endotoxin, which causes pulmonary inflammation independent of direct epithelial cell injury. C57Bl/6J mice (36 h or 8 wk old) were exposed to O(3) at 1 or 2.5 ppm for 4, 20, or 24 h or to a 10-min inhalation of 10 ng endotoxin per mouse (estimated deposited dose) and were examined 2, 6, or 24 h postexposure. Adult mice displayed increased sensitivity to O(3), as demonstrated by increased abundance of mRNAs encoding eotaxin, macrophage inflammatory protein (MIP)-1alpha, MIP-2, interleukin (IL)-6, and metallothionein (Mt). In newborn mice, only Mt was increased after 4 h of exposure. In contrast, newborn and adult mice responded similarly at 2 h post endotoxin exposure, inducing messages encoding tumor necrosis factor (TNF)-alpha, eotaxin, MIP-1alpha, MIP-1beta, MIP-2, interferon inducible protein (IP)-10, and monocyte chemoattractant protein (MCP)-1. Furthermore, interleukin-6 (IL-6) was increased in adults but not newborns. Similar chemokine and cytokine responses of newborn and adult mice in response to an agent not causing epithelial injury (endotoxin) suggest that altered inflammatory control observed between newborn and adult mice following O(3) exposure is secondary to epithelial cell injury.

Chemokine gene expression during Pneumocystis carinii-driven pulmonary inflammation.

Severe combined immunodeficient (SCID) mice lack functional lymphocytes and therefore develop Pneumocystis carinii pneumonia. However, when infected SCID mice are immunologically reconstituted with congenic spleen cells, a protective inflammatory cascade is initiated. Proinflammatory cytokines are produced, and lymphocytes and macrophages are recruited specifically to alveolar sites of infection. Importantly, uninfected regions of the lung remain free from inflammatory involvement, suggesting that there are specific mechanisms that limit inflammation in the infected lung. Therefore, to determine whether chemokines are involved in targeting the P. carinii-driven inflammatory response, steady-state mRNA levels of several chemokines were measured in the lungs of both reconstituted and nonreconstituted P. carinii-infected SCID mice. Despite significant organism burdens in the lungs of 8- and 10-week-old SCID mice, there was no evidence of elevated chemokine gene expression, which is consistent with the lack of an inflammatory response in these animals. However, when 8-week-old infected SCID mice were immunologically reconstituted, signs of focal pulmonary inflammation were observed, and levels of RANTES, MCP-1, lymphotactin, MIP-1alpha, MIP-1beta, and MIP-2 mRNAs were all significantly elevated. Chemokine mRNA abundance was elevated at day 10 postreconstitution (PR), was maximal at day 12 PR, and returned to baseline by day 22 PR. In situ hybridization demonstrated that during the peak of inflammation, RANTES gene expression was localized to sites of inflammatory cell infiltration and P. carinii infection. Thus, these observations indicate that chemokines play a role in the focal targeting of inflammatory cell recruitment to sites of P. carinii infection after the passive transfer of lymphocytes to the host.

Clara cell secretory protein-deficient mice differ from wild-type mice in inflammatory chemokine expression to oxygen and ozone, but not to endotoxin.

The in vivo function of Clara cell secretory protein (CCSP) is unknown. Biologic and biochemical properties associated with CCSP have led to speculation that it participates in pulmonary inflammatory control. Our earlier studies have demonstrated that CCSP-deficient mice are more sensitive to either hyperoxia or ozone toxicity and show altered oxidant-induced pulmonary proinflammatory responses. In this study we test the hypothesis that altered chemokine responses seen in CCSP-/- mice following oxidant stress are a direct consequence of altered immunoregulation associated with CCSP deficiency. To test this hypothesis we utilized three distinct models of inducing pulmonary toxicity: hyperoxia and ozone (O3), which cause epithelial cell injury, and endotoxin, which causes pulmonary inflammation independent of direct epithelial cell injury. Wild-type (WT) or CCSP-/- strain 129 mice were exposed to O3 at 1.0 ppm for 24 hours, oxygen (O2) > 99% for 68 hours or inhalation of 0.0575 microgram endotoxin per mouse for 10 minutes and examined 6 hours postexposure. Mice displayed increased sensitivity to O3, as demonstrated by increased abundance of mRNAs encoding Eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2, after 4 hours of exposure, whereas WT mice were unaltered from controls. Increased sensitivity to hyperoxia was also observed, as demonstrated by increased abundance of mRNAs encoding Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, and interferon-gamma inducible (IP)-10 after 68 hours of exposure, whereas WT mice were unaltered from controls. In contrast, WT and CCSP-/- mice responded identically 6 hours postinhalation of 0.0575 microgram lipopolysaccharide (LPS) per mouse. PMN response was 63% and 64% in WT and CCSP-/- mice, respectively. Messenger RNAs encoding Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, IP-10, and MCP-1 were increased identically. We conclude that CCSP does not participate in regulation of the endotoxin-elicited pulmonary inflammatory response. Identical inflammatory and chemokine responses of CCSP-/- and WT mice in response to a nonepithelial toxic agent (endotoxin) suggest that altered inflammatory control observed between WT and CCSP-/- mice following O2 and O3 exposure is not the result of altered immunoregulation.

Inflammatory and antioxidant gene expression in C57BL/6J mice after lethal and sublethal ozone exposures.

Ozone (O3) is a highly reactive and toxic oxidant pollutant. The objective of this study is to compare cytokine, chemokine, and metallothionein (Mt) changes elicited by lethal and sublethal exposure to ozone in a genetically sensitive strain of mice. Eight-week-old C57BL/6J mice were exposed to 0.3 ppm ozone for 0, 24, or 96 hours; 1.0 ppm ozone for 0, 1, 2, or 4 hours; or 2.5 ppm ozone for 0, 2, 4, or 24 hours. After 24 hours of exposure to 0.3 ppm ozone, increases in mRNA abundance were detected for messages encoding eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2. These increases persisted through 96 hours of exposure. At this time point messages encoding lymphotactin (Ltn) and metallothionein were also increased. After 4 hours of 1.0 ppm ozone exposure, increases in mRNA abundance were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, and interleukin (IL)-6. Mt mRNA abundance was increased after 1 hour of exposure and persisted through 4 hours, although the magnitude of the alterations increased. After 2 hours of 2.5 ppm ozone exposure, increases were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, IL-6, and Mt. These increases persisted through 4 hours of exposure. Lung weights of mice exposed to 2.5 ppm ozone for 24 hours were approximately 2 times greater than air-exposed mice. At this dose lethality occurred by 36 hours. Increased mRNAs for eotaxin, MIP-1 alpha, MIP-2, and Mt were to a higher magnitude than were detected after 2 and 4 hours of exposure. Messages encoding IL-12, IL-10, interferon (IFN)-gamma, IL-1 alpha, IL-1 beta, and IL-1Ra were unaltered at all time points and doses examined. Our results demonstrate dose- and time-dependent changes in chemokine, cytokine, and Mt mRNA abundance and that early acute changes may be predictive of subacute and chronic responses to ozone.

Chemokine mRNA alterations in newborn and adult mouse lung during acute hyperoxia.

Chemokines play a major role in the recruitment of inflammatory cells during acute lung injury. Adult and newborn C57BL/6 mice were exposed to > 95% oxygen for up to 72 hours and 7 days, respectively. Chemokine mRNA abundance was evaluated in whole lung RNA by ribonuclease protection assay and in tissue sections by in situ hybridization. Monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and interferon gamma-induced protein (IP)-10 mRNAs were present in whole newborn lung by 4 days of hyperoxia and were markedly elevated by 7 days. Levels of mRNA for MCP-1, MIP-1 alpha, and MIP-2 were elevated to a lesser extent by 72 hours of hyperoxia in adults. MCP-1 mRNA abundance was moderately elevated in scattered areas of perivascular tissue, peribronchiolar tissue, and the alveolar interstitium in 4-day hyperoxic newborns and markedly upregulated diffusely throughout the peripheral airspaces in 7-day hyperoxic newborns. MCP-1 mRNA abundance was limited to scattered perivascular areas and airspaces in 72-hour hyperoxic adults. These differences in the intensity, timing, and distribution of chemokine mRNA abundance between adult and newborn mice may help to explain the marked differences in their susceptibility to oxygen injury.

Clara cell secretory protein deficiency increases oxidant stress response in conducting airways.

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP -/-) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP -/- mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP -/- mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.

Interleukin-1 expression during hyperoxic lung injury in the mouse.

An important component of the pathophysiologic response to hyperoxia (O2) is pulmonary inflammation, although the roles of specific inflammatory mediators during pulmonary O2 toxicity are not completely known. Interleukin-1 (IL-1) is an early inflammatory mediator and is sufficient to elicit many of the responses associated with acute injury. The IL-1 family comprises two bioactive proteins, IL-1alpha and IL-1beta, and their natural antagonist IL-1ra. Here we report studies of IL-1 regulation during hyperoxic lung injury in the adult mouse. When assayed by Northern blot, increases in IL-1beta mRNA were seen after 2 days of hyperoxia. In contrast, IL-1alpha mRNA was barely detectable before 4 days of hyperoxia. To further understand the cellular origin of IL-1beta expression in lungs, in situ hybridization and immunohistochemical analyses were performed. IL-1beta mRNA or protein was not detected in the lungs of unexposed animals. At 3 days, we observed the accumulation of IL-1beta transcripts in pulmonary interstitial macrophages and in a subset of neutrophils, and immunodetectable IL-1beta protein was co-localized in adjacent sections. At 4 days of exposure, IL-1beta transcripts were widespread in lung tissue, but many areas rich in IL-1beta mRNA were devoid of immunodetectable IL-1beta. However, it is not known whether increased synthesis of IL-1beta or the uncoupling of IL-1beta protein and mRNA accumulation has a role in pathophysiology of pulmonary O2 toxicity.