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.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Quantal release of transmitter is not associated with channel opening on the neuronal membrane.

The traditional view that quantal release of neurotransmitter results from the fusion of transmitter-containing vesicles with the neuronal membrane has been recently challenged. Although various alternative mechanisms have been proposed, a common element among them is the release of cytoplasmic transmitter, which, in one view, could occur through large conductance channels on the presynaptic membrane. Six nerve-muscle cell pairs were examined with a whole-cell patch clamp for the presence of such channels that are associated with the production of miniature end-plate potentials. Examination of the neuronal membrane current during the occurrence of 822 miniature end-plate potentials produced no evidence of large channels. Thus it is unlikely that quantal release is mediated by such channels in the neuromuscular junction.

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.

Propagation of calcium waves between colonic smooth muscle cells in culture.

Intercellular propagation of a diffusible substance through direct cytoplasmic communication between multiple cells could represent an important mechanism for mutual multiple cell signaling between cells in a tissue. The current study was aimed at characterizing the mechanism(s) underlying the intercellular propagation of calcium concentration ([Ca2+]i) transients between colonic smooth muscle cells. Changes in [Ca2+]i in smooth muscle cells from the rabbit distal colon in primary cultures were monitored using videomicroscopy with the fluorescent dye Fura-2. Myocytes responded to light mechanical deformation of the plasma membrane with a localized increase in [Ca2+]i which spread in a wave-like fashion through up to 5 adjacent cells, with little change in wave amplitude. Dye coupling between cells was demonstrated by Lucifer Yellow, and intercellular wave propagation was abolished by octanol, suggesting propagation of Ca2+ waves via gap junctions. Wave propagation was not dependent on extracellular [Ca2+]i suggesting regenerative release of Ca2+ from intracellular stores. Propagation of Ca2+ waves through silent cells suggested a diffusible messenger other than Ca2+. Wave propagation and kinetics were unaffected by ryanodine (50 microM) or caffeine (10 mM), but abolished by depletion of thapsigargin-sensitive Ca2+ stores and by the phospholipase C inhibitor U-73122 (10 microM), implicating inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive stores as the major Ca2+ source for propagated Ca2+ transients. These results indicate that, in a connected complex of colonic smooth muscle cells in culture, multiple cells can monitor the mechanical status of a single cell through diffusion of Ins(1,4,5)P3, Ca2+, or another intercellular messenger.

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.

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro.

The mechanism(s) by which intestinal smooth muscle tension is signaled to extrinsic primary afferent neurons is poorly understood. In order to characterize myocyte-neuron communication, we developed a coculture system using rat dorsal root ganglion (DRG) neurons and myocytes obtained from the circular muscle layer of the rat distal colon. Both cell types maintained their phenotype in culture, as demonstrated by positive immunocytochemical staining for neuron-specific enolase and smooth muscle actin. Myocytes showed mechanosensitivity in the form of increases in [Ca2+]i in response to light mechanical touch of the plasma membrane. This increase in [Ca2+]i was independent of extracellular Ca2+ and passed as a propagated wave from muscle cells into adjacent DRG neurites. The inhibitory effect of octanol on this intercellular propagation suggests propagation of [Ca2+]i gradients via heterologous gap junctions. This preparation may serve a useful model system for the study of the interaction of visceral afferents and their target cells.

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by kappa-opioids.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

Prevention of intraocular pressure rise following intravitreal injection.

The intraocular pressure change produced by an intravitreal injection of ganciclovir, 2 mg in 0.1 ml, was studied in patients with cytomegalovirus retinitis. Using a Tono-pen XL to measure the intraocular pressure (IOP) of four patients (six eyes) we found the mean pressure immediately following injection was 44.5 mm Hg. Measurements taken on separate occasions after a 30 mm Hg decompression of the eye for 15 minutes before the injection showed a mean IOP of only 20.6 mm Hg after the injection. Mercury bag decompression of the eye significantly reduced the rise in IOP following intravitreal injection (difference in the mean IOP rise = 26.4 mm Hg, df = 54, t = 7.67, p < 0.001). Without ocular decompression before injection, all patients complained of temporary loss of vision, and reflux of the injection solution was frequently observed. Use of ocular decompression also reduced the discomfort of the injection. Throughout the full course of treatment by this means there were no adverse effects on the visual acuity. This technique is recommended to those performing intravitreal injections.

Glaucoma Filtering Surgery With Interferon-alpha-2b.

SUMMARY: The effect of subconjunctival interferon-alpha-2b treatment on success rates after glaucoma filtering surgery was compared with that of 5-fluorouracil in a pilot study. Surgery was defined as successful if it reduced the intraocular pressure to less than 21 mm Hg without medication. Initially, doses of 2 x 10 IU of interferon-alpha were given. By 6 months postoperatively, 6 of 16 interferon-treated patients had had to resume medication to control their glaucoma, compared with only 2 of 16 patients treated with 5-fluorouracil (p = 0.08). When the dose of interferon was increased to 1 x 10 IU, 2 of 13 interferon-treated patients had to resume by 6 months (p = 0.14 when compared with the patients receiving the lower dose). Corneal epithelial defects were less common in the 1 x 10 IU interferon-alpha group compared with the 5-fluorouracil group (1 of 13 vs. 11 of 16; p = 0.0002) while other side effects, both local and systemic, were similar between the two groups. This study shows that (a) interferon-alpha-2b, in the doses used, is well-tolerated by the human eye, and (b) interferon-alpha-2b may reduce the risk of failure of glaucoma filtration surgery with fewer corneal complications than are seen with 5-fluorouracil.

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.

Brief sexual counseling during cardiac rehabilitation.

Advances in the understanding and treatment of myocardial infarction have appeared over the past decade. One intervention technique receiving increasing emphasis is the development of multidisciplinary "rehabilitation teams" whose chief aim is to assist individuals in returning to former levels of medical and psychosocial functioning. Within the team approach, the mental health specialist clearly plays a significant role. Counselors can provide support and reassurance in the midst of the medical crisis and help to minimize stress related to rearrangements in family roles and routines. In this instance, brief sex counseling as part of an ongoing rehabilitation program serves to clarify misconceptions, reduce fears, and facilitate return to sexual activities after infarction. It is important to remember, however, that an accurate physiologic evaluation provides the foundation on which to base counseling efforts. Without adequate medical information, no amount of counseling expertise will succeed. Certainly the final decision to resume sexual behavior remains with the individual couple. The counselor's primary task is to emphasize that the myth of total abstinence is not applicable for most cardiac patients. In reality, it is possible and even highly beneficial for patient and spouse to return to their normal sexual relationship.

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.

Effect of a reduction in arterial oxygen content (carbon monoxide) on coronary flow.

Mongrel dogs were chronically instrumented for ventricular pacing, to measure left circumflex coronary flow (CF), left ventricular and arterial pressure, and to obtain blood samples from the left atrium and coronary sinus. Following a 3- to 4-week recovery period, the animals were subjected to a 30% reduction in arterial oxygen saturation by exposure to low levels of carbon monoxide. Three types of experiments were performed: 1) control, 2) pacing at 150 bpm, and 3) double blockade with propranolol and atropine. Reduction in arterial oxygen saturation in the three conditions studied resulted in a significant increase in CF and no change in myocardial oxygen consumption. The relationship between CF and arterial saturation demonstrated that double blockade produced a difference in the magnitude of the CF response. These results indicate a neurogenic mechanism to regulate coronary flow that may aid in maintaining oxygen availability during stressful conditions.

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.