Blockade of p-selectin is sufficient to reduce MHC I antibody-elicited monocyte recruitment in vitro and in vivo.

Donor-specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody-mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody-mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc-independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody-mediated rejection, in which B6.RAG1(-/-) recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P-selectin, which supports monocyte binding. In the experimental model, donor-specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL-1-Ig abolished antibody-induced monocyte infiltration in the allograft, but had little effect on antibody-induced endothelial injury. Our data suggest that antagonism of P-selectin may ameliorate accumulation of macrophages in the allograft during antibody-mediated rejection.

Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Oxidative stress and inflammatory response in dermal toxicity of single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNT) represent a novel material with unique electronic and mechanical properties. The extremely small size ( approximately 1 nm diameter) renders their chemical and physical properties unique. A variety of different techniques are available for the production of SWCNT; however, the most common is via the disproportionation of gaseous carbon molecules supported on catalytic iron particles (high-pressure CO conversion, HiPCO). The physical nature of SWCNT may lead to dermal penetration following deposition on exposed skin. This dermal deposition provides a route of exposure which is important to consider when evaluating SWCNT toxicity. The dermal effects of SWCNT are largely unknown. We hypothesize that SWCNT may be toxic to the skin. We further hypothesize that SWCNT toxicity may be dependent upon the metal (particularly iron) content of SWCNT via the metal's ability to interact with the skin, initiate oxidative stress, and induce redox-sensitive transcription factors thereby affecting/leading to inflammation. To test this hypothesis, the effects of SWCNT were assessed both in vitro and in vivo using EpiDerm FT engineered skin, murine epidermal cells (JB6 P+), and immune-competent hairless SKH-1 mice. Engineered skin exposed to SWCNT showed increased epidermal thickness and accumulation and activation of dermal fibroblasts which resulted in increased collagen as well as release of pro-inflammatory cytokines. Exposure of JB6 P+ cells to unpurified SWCNT (30% iron) resulted in the production of ESR detectable hydroxyl radicals and caused a significant dose-dependent activation of AP-1. No significant changes in AP-1 activation were detected when partially purified SWCNT (0.23% iron) were introduced to the cells. However, NFkappaB was activated in a dose-dependent fashion by exposure to both unpurified and partially purified SWCNT. Topical exposure of SKH-1 mice (5 days, with daily doses of 40 microg/mouse, 80 microg/mouse, or 160 microug/mouse) to unpurified SWCNT caused oxidative stress, depletion of glutathione, oxidation of protein thiols and carbonyls, elevated myeloperoxidase activity, an increase of dermal cell numbers, and skin thickening resulting from the accumulation of polymorphonuclear leukocytes (PMNs) and mast cells. Altogether, these data indicated that topical exposure to unpurified SWCNT, induced free radical generation, oxidative stress, and inflammation, thus causing dermal toxicity.

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase.

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.

Complications in stroke patients: a study carried out at the Rehabilitation Medicine Service, Changi General Hospital.

AIM: The aim of this study was to look at the type and frequencies of complications after an acute stroke in an inpatient rehabilitation setting. We also looked at the type of complications which required the transfer of patient care back to the primary referring physician. MATERIALS AND METHODS: A retrospective review of case notes of patients transferred to the rehabilitation team was conducted. The study period was a six-month period from the beginning of January 2001 to the end of June 2001. A list of complications was made. Each pre-determined complication was then defined. The frequency of each complication was then calculated. RESULTS: A total of 140 case notes were reviewed. The overall complication rate was 54.3%. The more common complications, in order, from highest to lowest frequencies, were: constipation (complicating 22.9% of strokes); acute retention of urine (ARU, 20.9%); urinary tract infections (UTI, 14.3%); depression (9.3%); and limb pain (8.6%). Females were more likely to have UTI (p=0.038), ARU (p=0.002) and depression (p=0.018). Patients 65 years and above were more likely to suffer multiple complications although the results did not reach statistical significance (p=0.055). The care for eight patients (5.7% of patients with complications) had to be transferred back to the primary referring team or physician. CONCLUSIONS: Complications post stroke are common. Some patients required transfer of care back to the primary referring physician. A pro-active approach is ideal in all post stroke patients, in order to identify and treat any complications early, thereby, improving outcome and reducing costs.

Outsourcing and benchmarking in a rural public hospital: does economic theory provide the complete answer?

INTRODUCTION: The ideology and pronouncements of the Australian Government in introducing 'competitive neutrality' to the public sector has improved efficiency and resource usage. In the health sector, the Human Services Department directed that non-clinical and clinical areas be market tested through benchmarking services against the private sector, with the possibility of outsourcing. These services included car parking, computing, laundry, engineering, cleaning, catering, medical imaging (radiology), pathology, pharmacy, allied health and general practice. Managers, when they choose between outsourcing, and internal servicing and production, would thus ideally base their decision on economic principles. Williamson's transaction cost theory studies the governance mechanisms that can be used to achieve economic efficiency and proposes that the optimal organisation structure is that which minimises transaction costs or the costs of exchange. Williamson proposes that four variables will affect such costs, namely: (i) frequency of exchange; (ii) asset specificity; (iii) environmental uncertainty; and (iv) threat of opportunism. This paper provides evidence from a rural public hospital and examines whether Williamson's transaction cost theory is applicable. METHOD: Case study research operates within the interpretivism paradigm and is used in this research to uncover why the outsourcing decision was made. Such research aims to study real-life experiences by examining the way people think and act and, in contrast to positivism, allows the interviewer to participate to better understand the details and features of the experiences. In the present research, individual interviews were conducted with managers of the hospital and owners and staff of the vendor organisations using semi- and unstructured questions to ascertain the extent of, and processes used in, outsourcing specific functional areas, and areas that were not outsourced. RESULTS: Pathology, radiology, dental technician services and lawn mowing were outsourced while food services was retained internally. The outsourcing of radiology was due to the hospital being unable (or unwilling) to finance new equipment and the problematical relationship between the existing radiologists, and hospital management and staff. Outsourcing resulted in increased staff morale, upgraded capital equipment and improved services. The outsourcing of pathology and dental technical services aimed to increase labour flexibility, thereby decreasing costs. Additional drivers in pathology were the changing nature of the funding arrangements rendering it profitable for the private sector to move into the provision of pathology and the increasing power of the medical scientists' union. The outsourcing of lawn mowing was simply to reduce costs. Food services was not outsourced because there was a lack of evidence that costs could be reduced. In addition, the existing relationships with food services staff were regarded as important because they had previously made immense changes to work practices, reduced staff numbers and decreased costs. CONCLUSION: Transaction costs are important when analysing how managers make the outsourcing decision, but the evidence from this case is that not all transaction costs are included in the decision, and that such costs are more complex than can be included in the type of analysis often undertaken by decision-makers. Taking into account Williamson's variables, the research shows that the outsourcing of services did not comply solely with the levels of transaction frequency or the requirement of asset specificity. In addition, opportunistic behaviour was evident on the part of all parties and was used in some cases as a reason for outsourcing, and in others to sway the decision to the manager's predisposed choice. A variety of arrangements were used to reduce environmental uncertainty, such as the transfer of staff to the contractor and the use of long-term contracts. Indeed the case shows that relationships between the hospital, its staff and the vendor are an important consideration that may not always be factored into an analysis that relies solely on transaction costs.

Acute inflammation and recovery in rats after intratracheal instillation of a 1-->3-beta-glucan (zymosan A).

Although endotoxin is a known potent stimulant of inflammatory responses, the magnitude of pulmonary response following exposure to various organic dusts does not always correlate with endotoxin content of the dusts alone. Other components, such as 1-->3-beta-glucans, derived from the inner cell wall of yeasts and fungi, have been implicated in organic dust toxic syndrome. However, animal studies report conflicting results concerning the inflammatory potency of 1-->3-beta-glucan. In this experiment, the pulmonary reaction of rats to 1-->3-beta-glucan (zymosan A) exposure was assessed. Male Sprague-Dawley rats were exposed via intratracheal instillation (IT) to zymosan A (dose range 0-5 mg/kg body weight). Rats were sacrificed 1-7 d postexposure and the following pulmonary responses were monitored: (1) breathing frequency, (2) differential cell counts of hronchoalveolar lavage (BAL) cells, (3) chemiluminescence (CL) as a measure of alveolar macrophage activation, (4) nitric oxide production by alveolar macrophages, (5) albumin levels, and (6) lactate dehydrogenase (LDH) activity in the first acellular lavage fluid. Upon challenge with zymosan A, rats exhibited a dose-dependent pulmonary response at 1 d post IT that was significantly higher than the control level at a dose of 1-2.5 mg/kg body weight for each of these pulmonary parameters. Post-IT enhancement of breathing frequencies and polymorphonuclear leukocytes (PMN) obtained by BAL both correlated very well with zymosan A concentration (r = .95 and .99, respectively). Elevation of albumin levels and LDH activity of the acellular BAL fluid also correlated (r = .80) with the dose of zymosan. The recovery from a single intratracheal administration of zymosan A (2.5 mg/kg body weight) was monitored over 7 d. PMN and CL showed significant recovery from d 1 level by 3 d postexposure. Breathing frequencies and nitric oxide production showed significant recovery from d 1 level by 4 d postexposure. A good correlation (r2= .8) between recovery of PMN in BAL, CL, or nitric oxide production and the days postexposure was observed.

Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.

Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

Agonist-dependent immobilization of chimeric bombesin/GRP receptors: dependence on c-Src activity and dissociation from internalization.

G-protein-coupled receptors (GPCRs) are membrane proteins that exhibit a decreased mobile fraction compared to a freely mobile plasma membrane protein. Recently, interest has focused on proteins other than heterotrimeric G-proteins that interact with GPCRs as scaffolding structures that affect receptor signal transduction. In order to investigate the physical state of receptors before and after agonist, we used fluorescence recovery after photobleaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to the intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) expressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells at 37 degrees C, addition of GRP (100 nM) caused a rapid decrease in GFP-GRP receptor mobile fraction from 0.8 +/- 0.1 to 0.49 +/- 0.05, which was independent of endocytosis. Concurrently, the remaining mobile GFP-GRPreceptors showed an increase in the diffusion rate with the half-time of fluorescent recovery, tau(1/2) = 46 +/- 7 s for untreated cells, decreasing to tau(1/2) = 30 +/- 6 s for cells treated with GRP. Prior treatment with the Src-specific inhibitor PP-2 (10 microM) blocked GFP-GRP receptor immobilization while treatment with the inactive analog PP-3 (10 microM) did not affect receptor immobilization. These data suggest that agonist-bound GPCR have increased plasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity.

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.