The inability of a bacteriophage to infect Staphylococcus aureus does not prevent it from specifically delivering a photosensitizer to the bacterium enabling its lethal photosensitization.

OBJECTIVES: It has been demonstrated that the efficiency of lethal photosensitization can be improved by covalently binding photosensitizing agents to bacteriophage. In this study we have investigated whether a bacteriophage requires the capacity to infect the bacterium to enhance lethal photosensitization when linked to a photosensitizer. METHODS: Tin (IV) chlorin e6 (SnCe6) was conjugated to bacteriophage Phi11, a transducing phage that can infect Staphylococcus aureus NCTC 8325-4, but not epidemic methicillin-resistant S. aureus (EMRSA)-16. The conjugate and appropriate controls were incubated with these bacteria and either exposed to laser light at 632.8 nm or kept in the dark. RESULTS: The SnCe6/Phi11 conjugate achieved a statistically significant reduction in the number of viable bacteria of both 8325-4 and EMRSA-16 strains by 2.31 log(10) and 2.63 log(10), respectively. The conjugate could not however instigate lethal photosensitization of Escherichia coli. None of the other combinations of controls, such as an equivalent concentration of SnCe6 only, an equivalent titre of bacteriophage only or experiments conducted without laser light, yielded significant reductions in the number of viable bacteria recovered. CONCLUSIONS: The inability of a bacteriophage to infect S. aureus does not prevent it from specifically delivering a photosensitizer to a bacterium enabling its lethal photosensitization.

Peptide growth factors can provoke "fetal" contractile protein gene expression in rat cardiac myocytes.

Cardiac-specific gene expression is intricately regulated in response to developmental, hormonal, and hemodynamic stimuli. To test whether cardiac muscle might be a target for regulation by peptide growth factors, the effect of three growth factors on the actin and myosin gene families was investigated by Northern blot analysis in cultured neonatal rat cardiac myocytes. Transforming growth factor-beta 1 (TGF beta 1, 1 ng/ml) and basic fibroblast growth factor (FGF, 25 ng/ml) elicited changes corresponding to those induced by hemodynamic load. The "fetal" beta-myosin heavy chain (MHC) was up-regulated about four-fold, whereas the "adult" alpha MHC was inhibited greater than 50-60%; expression of alpha-skeletal actin increased approximately two-fold, with little or no change in alpha-cardiac actin. Thus, peptide growth factors alter the program of differentiated gene expression in cardiac myocytes, and are sufficient to provoke fetal contractile protein gene expression, characteristic of pressure-overload hypertrophy. Acidic FGF (25 ng/ml) produced seven- to eightfold reciprocal changes in MHC expression but, unlike either TGF-beta 1 or basic FGF, inhibited both striated alpha-actin genes by 70-90%. Expression of vascular smooth muscle alpha-actin, the earliest alpha-actin induced during cardiac myogenesis, was increased by all three growth factors. Thus, three alpha-actin genes demonstrate distinct responses to acidic vs. basic FGF.

The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load.

Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; however, it is not known whether reactivation of the smooth muscle alpha-actin gene occurs in ventricular hypertrophy. We therefore investigated myocardial expression of the smooth muscle alpha-actin gene after hemodynamic overload. Smooth muscle alpha-actin mRNA was discernible 24 h after coarctation and was persistently expressed for up to 30 d. In hypertrophied hearts, the prevalence of smooth muscle alpha-actin gene induction was 0.909, versus 0.545 for skeletal muscle alpha-actin (P less than 0.05). Ventricular mass after 2 d or more of aortic constriction was more highly correlated with smooth muscle alpha-actin gene activation (r = 0.852; P = 0.0001) than with skeletal muscle alpha-actin (r = 0.532; P = 0.009); P less than 0.0005 for the difference in the correlation coefficients. Thus, smooth muscle alpha-actin is a molecular marker of the presence and extent of pressure-overload hypertrophy, whose correlation with cardiac growth at least equals that of skeletal alpha-actin. Induction of smooth muscle alpha-actin was delayed and sustained after aortic constriction, whereas the nuclear oncogenes c-jun and junB were expressed rapidly and transiently, providing potential dimerization partners for transcriptional control by c-fos.

A Ras-dependent pathway regulates RNA polymerase II phosphorylation in cardiac myocytes: implications for cardiac hypertrophy.

Despite extensive evidence implicating Ras in cardiac muscle hypertrophy, the mechanisms involved are unclear. We previously reported that Ras, through an effector-like function of Ras GTPase-activating protein (GAP) in neonatal cardiac myocytes (M. Abdellatif et al., J. Biol. Chem. 269:15423-15426, 1994; M. Abdellatif and M. D. Schneider, J. Biol. Chem. 272:527-533, 1997), can up-regulate expression from a comprehensive set of promoters, including both cardiac cell-specific and constitutive ones. To investigate the mechanism(s) underlying these earlier findings, we have used recombinant adenoviruses harboring a dominant negative Ras (17N Ras) allele or the N-terminal domain of GAP (nGAP), responsible for the Ras-like effector function. Inhibition of endogenous Ras reduced basal levels of [3H]uridine and [3H]phenylalanine incorporation into total RNA, mRNA, and protein, with parallel changes in apparent cell size. In addition, 17N Ras markedly inhibited phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II), known to regulate transcript elongation, accompanied by down-regulation of its principal kinase, cyclin-dependent kinase 7 (Cdk7). In contrast, nGAP elicited the opposite effects on each of these parameters. Furthermore, cotransfection of constitutively active Ras (12R Ras) with wild-type pol II, rather than a truncated mutant lacking the CTD, demonstrated that Ras activation of transcription was dependent on the pol II CTD. Consistent with a potential role for this pathway in the development of cardiac myocyte hypertrophy, alpha1-adrenergic stimulation similarly enhanced pol II phosphorylation and Cdk7 expression, where both effects were inhibited by dominant negative Ras, while pressure overload hypertrophy led to an increase in both hyperphosphorylated and hypophosphorylated pol II in addition to Cdk7.

High serum lactate as an adjunct in the early prediction of anastomotic leak following oesophagectomy.

BACKGROUND: Anastomotic leak (AL) following oesophagectomy carries a high mortality and morbidity. Early detection and intervention is required for a successful outcome. We have examined the role of a high postoperative serum lactate in predicting which patients are at risk of developing an anastomotic leak(AL). MATERIALS AND METHODS: All patients who underwent transthoracic oesophagectomy over a 3-year period were identified from a prospectively collected database. Medical records were reviewed to identify the highest serum lactate recorded from blood gas analysis over each 24hr post-operative period. Patients who underwent transhiatal and left thoraco-abdominal oesophagectomies were excluded. Patients who developed a chyle leak were excluded. RESULTS: Of a total of 136 oesophagectomies included for analysis, 18 developed an AL (13.2%). Of these patients, 10 underwent thoracoscopic oesophageal mobilization with cervical anastomosis and the rest an Ivor Lewis procedure. Predictive factors for AL included neoadjuvant chemotherapy (15/18 83.3% vs 55/118 46.6% p = 0.0046) and number of positive lymph nodes (mean 4.2 vs control mean 2.3 p = 0.045). Overall net fluid balance was comparable between the 2 groups, although AL patients received slightly more fluid on Day 3. High lactate levels on days 1-3 were associated with an AL. Using a Day 2 lactate of 1.7 mmol/L, the sensitivity of predicting AL was 72% and specificity 88%. The mean lag time using existing diagnostic modalities was 7.9 days. CONCLUSION: A serum lactate of >1.7 mmol/l on day 2 should raise the possibility of a potential AL. Such patients should be selected for more intensive monitoring, optimization and selective gastroscopy.

Investigation of outbreaks of Pneumocystis jirovecii pneumonia in two Scottish renal units.

Pneumocystis jirovecii is recognized as an opportunistic pathogen. In recent years, human-to-human transmission of P. jirovecii has been demonstrated. However, outbreaks of P. jirovecii infections are not well defined because the epidemiological setting that facilitates transmission is not fully understood. This article describes two outbreaks of P. jirovecii pneumonia (PCP) in renal transplant patients in the West of Scotland. In total, 25 patients in two geographically contiguous locations were affected. Allele B was identified as the dominant type, along with allele A3. It was not possible to determine the exact reason for clustering of cases, although the outpatient clinic setting featured in one of the outbreaks. The outbreaks ceased with the use of trimethoprim-sulphamethoxazole prophylaxis; the target populations that received prophylaxis were different in the two outbreaks. Infection control teams should be alert to the possibility of outbreaks of PCP.

Chlorpromazine distribution in hamsters and mice bearing transplantable melanoma.

Chlorpromazine (CPZ) distribution was measured in tissues of Syrian golden hamsters bearing Greene melanoma and in BALB/c mice bearing Harding-Passey melanoma. Distribution was evaluated as a function of time (0.5 to 14 days) and as a function of single and multiple doses (up to five) of from 5 to 50 mg CPZ per kg body weight. Routes of administration (i.p., i.v., p.o.) were compared. The physiological behavior of CPZ is of interest as it is used extensively as a tranquilizing drug (Thorazine). Further, since CPZ binds to the pigment melanin, the possibility exists of using CPZ to transport diagnostic or therapeutic agents to melanoma. It was found that, at 2 days postinjection, tumor/tissue concentration ratios exceeded 10 for metabolizing organs, such as liver and 100 for "back-ground" tissues, such as blood and muscle. Absolute concentrations of CPZ in tumor exceeding 100 microgram CPZ per g tumor were obtained with both single and multiple doses. This selective high concentration in tumor would make CPZ an ideal vehicle for the transport of boron to tumor for use in neutron capture therapy via the 10B(n, alpha)7Li reaction.

Melanin content of hamster tissues, human tissues, and various melanomas.

Melanin content (percentage by weight) was determined in both pigmented and nonpigmented tissues of Syrian golden hamsters bearing Greene melanoma. Melanin content was also measured in various other melanoma models (B-16 in C57 mice, Harding-Passey in BALB/c mice, and KHDD in C3H mice) and in nine human melanomas, as well as in selected normal tissues. The purpose was to evaluate the possible efficacy of chlorpromazine, which is known to bind to melanin, as a vehicle for boron transport in neutron capture therapy. Successful therapy would depend upon selective uptake and absolute concentration of borated compounds in tumors; these parameters will in turn depend upon melanin concentration in melanomas and nonpigmented "background" tissues. Hamster whole eyes, hamster melanomas, and other well-pigmented animal melanomas were found to contain 0.3 to 0.8% melanin by weight, whereas human melanomas varied from 0.1 to 0.9% (average, 0.35%). Other tissues, with the exception of skin, were lower in content by a factor of greater than or equal to 30. Melanin pigment was extracted from tissues, and the melanin content was determined spectrophotometrically. Measurements were found to be sensitive to the presence of other proteins. Previous procedures for isolating and quantifying melanin often neglected the importance of removing proteins and other interfering nonmelanic substances.

Determining the effect of social deprivation on the prevalence of healthcare-associated infections in acute hospitals: a multivariate analysis of a linked data set.

BACKGROUND: Healthcare-associated infections (HCAIs) endanger safety by increasing morbidity, mortality, and hospital stay. Studies identifying risk factors for HCAI rarely address the wider determinants of health. However, a well-characterized association exists between increasing social deprivation and poor health outcomes. Therefore it is important to determine whether HCAIs are associated with social deprivation. AIM: To determine the association between social deprivation and the prevalence of HCAI, in all inpatients in an acute hospital in Scotland on a single day across September and October 2011. METHODS: This study linked Scottish data from the 2011 European Point Prevalence Survey of HCAI and Antimicrobial Prescribing to the Scottish Morbidity Record 01, a national dataset with Scottish Index of Multiple Deprivation (SIMD) included. Multivariate logistic regression was used to model HCAI prevalence against SIMD quintile. FINDINGS: No overall association was found between SIMD quintile and prevalence of HCAI in all inpatients. A significant difference was found between HCAI prevalence across SIMD quintile in patients undergoing surgical procedures, with higher prevalence observed with increasing deprivation (P = 0.0071). Variables associated with HCAI prevalence were: intensive care specialty, psychiatric and medical specialties, minimum invasive surgery, and all categories of length of stay. CONCLUSION: This study found a significant difference in HCAI prevalence across SIMD quintile in patients undergoing surgery. To our knowledge this was the first study to examine the overall association between HCAI and SIMD. The findings highlight the broad and comprehensive nature of social deprivation in determining health outcomes.

The effect of zinc on insulin metabolism.

Experiments were designed to study the effect of Zn on in vivo and in vitro insulin metabolism. The in vivo experiments involved pretreating mice with either Zn or Na, followed by ip [125I]iodoinsulin injection. Pretreatment of mice with Zn resulted in an accelerated and increased magnitude of binding of [125I]iodoinsulin to the liver compared to mice pretreated with Na. Results are submitted which support the probability that the changes in the amounts of intact and degraded insulin in circulation with time are related to the binding and degradation of insulin in the liver rather than in the kidney. In vivo ip injected insulin was demonstrated to preferentially bind to the plasma membrane of the liver. Liver plasma membranes isolated from mice pretreated with Zn bound more [125I]iodoinsulin than plasma membranes of Na-pretreated mice. In vitro experiments employing isolated liver plasma membranes demonstrated that added Zn increased the binding and inhibited the degradation of insulin. Evidence is presented that supports the concept that two receptors exist, one at which degradation of [125I]iodoinsulin occurs and another at which degradation does not occur.

Sympathetic stimulation and hypertension in the pyridoxine-deficient adult rat.

Pyridoxal phosphate is the coenzyme of various decarboxylases involved in the formation of monoamine neurotransmitters such as gamma-aminobutyric acid, serotonin, dopamine, and norepinephrine. Adult male Sprague-Dawley rats placed on a pyridoxine-deficient diet for 8 weeks showed significant hypertension compared with pyridoxine-supplemented controls. Hypothalamic contents of pyridoxal phosphate, gamma-aminobutyric acid, and serotonin in the pyridoxine-deficient rats were significantly lower than those in pyridoxine-supplemented controls. Hypertension was associated with sympathetic stimulation. Treatment of pyridoxine-deficient rats with a single dose of pyridoxine (10 mg/kg body weight) reversed the blood pressure to normal levels within 24 hours, with concomitant restorations of hypothalamic serotonin and gamma-aminobutyric acid as well as the return of plasma norepinephrine and epinephrine to normal levels. Also, pyridoxine treatment reversed the hypothalamic hypothyroidism observed in pyridoxine-deficient rats. These results indicate an association between pyridoxine deficiency and sympathetic stimulation leading to hypertension.

Venous endothelial changes in therapeutic arteriovenous fistulae.

Specimens of veins of therapeutic arteriovenous fistulae from five patients were examined by an en face immunohistochemical technique to investigate endothelial morphology and the presence of the vasoactive peptide endothelin-1 (ET-1). These were compared with control segments of long saphenous veins from six patients. Venous endothelium from the arteriovenous shunts was mostly intact even overlying phlebosclerotic plaques. Occasional small areas of denudation, with associated platelets, were present in the depressions of 'jet' lesion however. The endothelial cells were generally elongated and interspersed with foci of polyhedral cells. The control saphenous veins contained elongated endothelial cells without detectable denudation. Image analysis of histological sections of veins from the shunts indicated significantly less intact elastic tissue than control veins but greater mononucleated endothelial cell density in en face preparations. ET-1 staining was considerably stronger in endothelium from the fistulae than in the control saphenous veins and was most intense over the raised crescentic ridges of jet lesions, stenoses and phlebosclerotic plaques. Endothelial mitoses and cells with hyperchromatic nuclei stained more strongly for ET-1 than surrounding cells. These results indicate that the endothelial cells lining veins associated with arteriovenous fistulae are dynamically altered by the increased haemodynamic stresses associated with these shunts. Furthermore ET-1 may act as a localising factor associated with intimal thickening at sites of 'jet' lesions, stenosis and phlebosclerosis.

Endothelin-1 is increased overlying atherosclerotic plaques in human arteries.

The distribution of Endothelin-1 (ET-1), a potent vasoactive peptide, within endothelium of human atherosclerotic arteries was examined using a novel en face immunohistochemical technique. The vast majority of endothelial cells were immunoreactive for ET-1. Staining intensity was increased in areas overlying atherosclerotic plaques, calcified media, fatty streaks and about flow dividers, compared with adjacent regions. Multinucleated 'giant' endothelial cells were more common in regions containing strong ET-1 staining than elsewhere. Clusters of leucocytes (probably monocytes) were frequently observed adhering to the endothelial monolayer but not neighbouring regions of denudation. Occasionally underlying macrophage/foam cells and smooth muscle cells were exposed to the surface and included in the en face (Häutchen) preparation. Smooth muscle cells did not stain for ET-1 while macrophages and the larger foam cells were positive for ET-1. These results support the hypothesis that expression of ET-1, at sites containing atheromatous disease, may be involved in the development of atherosclerosis.

Therapeutic effects of S-35-thiouracil in BALB/c mice carrying Harding-Passey melanoma.

Thiouracil (TU) selectively binds to the pigment melanin during melanogenesis and is rapidly cleared from normal tissues. This compound shows little affinity for pre-formed melanin. BALB/c mice, carrying the subcutaneously transplanted Harding-Passey melanoma, were given i.p. injections of 35S-labeled thiouracil in a range of doses and administration schedules. Injected doses ranged from 1.3 to 10 mCi per mouse with resultant tumor dose rates of 10 to 30 cGy/hr, respectively. At the lower dose rates, growth delay of approximately 1 to 2 weeks was observed in all tumors. At the highest doses used, complete tumor regression (no regrowth) was observed in some cases, with extended growth delays of approximately 6 weeks in the rest. These results illustrate the possible utility of radiolabeled thiouracil as a systemically administered brachytherapy agent for melanoma.

Detection of ocular melanoma with 4-(3-dimethylaminopropylamino)-7-[123I]-iodoquinoline.

Iodine-123-labeled 4-(3-dimethylpropylamino)-7-iodoquinoline was evaluated in nine patients. By using a specially designed dual-pinhole ocular collimator, it was possible to obtain positive images at 2-6 hr for only 70% of the cases with subsequently proven ocular melanomas.

Iodothiouracil as a melanoma localizing agent.

Thiouracil and various derivatives are selectively incorporated into the melanin pigment of melanomas during biosynthesis by serving as false melanin precursors. Using the transplantable Harding-Passey melanoma carried in BALB/c mice, we have extended our previous studies with sulfur-35 (35S) thiouracil. The persistence of high levels of [35S]thiouracil in tumor for periods of up to 2 wk has been demonstrated; during this time the drug content in normal tissues returned to near background levels. The variety of iodine isotopes available makes iodothiouracil a particularly promising melanoma-localizing agent. Tumor uptake and biodistribution of [35S]thiouracil and iodothiouracil (both iodine-127 (127I) and iodine-125 (125I) labeled) have been compared and were found to be essentially the same. The selectivity of [125I]thiouracil for melanoma has been qualitatively demonstrated by autoradiography of whole-body sections and quantitated by analysis of tumor and selected tissues. Iodothiouracil was also shown to localize in remote secondary metastases using a metastatic variant of the Harding-Passey melanoma currently being developed in our laboratory. These studies confirm the melanoma localizing capabilities of an iodinated thiouracil, and therefore the potential of using iodinated thiouracil derivatives for diagnosis and therapy of melanotic melanomas.

Radioactive phosphorus uptake test. An in vitro analysis of choroidal melanoma and ocular tissues.

The concentration of radioactive phosphorus in uveal melanoma and normal parts of the eye was determined in vitro in 14 eyes. The eyes were enucleated after a positive 32P uptake test. Portions of the melanoma as well as normal choroid, retina, sclera, lens, and vitreous were analyzed. The 32P uptake test had been performed at various intervals after intravenous administration of 32P from 24 to 556 hr. The in vitro uptake of 32P was compared to cell type, tumor volume, time of testing, percent uptake measured clinically, and specific activity. The only positive correlation was between percent uptake measured clinically and 32P concentration (dpm/gm). A higher concentration of phosphorus in melanoma resulted when carrier-free 32P was used. A negative correlation existed between number of hours from injection to clinical measurement of percent uptake, although melanoma to normal choroid ratios did not change from 24 to 72 hr. No correlation was found between uptake and tumor volume. The sample was small; however, we saw no correlation between 32P uptake and degree of malignancy.

Boron neutron capture therapy of anterior chamber melanoma with p-boronophenylalanine.

Boron neutron capture therapy (BNCT) is a form of radiation therapy that requires selective uptake of boron by the tumor and irradiation with thermal neutrons. Phenylalanine is an amino acid precursor of melanin and when boronated (p-boronophenylalanine [BPA]) was found to be selectively taken up by Greene melanoma cells in the anterior chamber of rabbits. This tumor model was irradiated 24 hr after oral administration of BPA and was used for biodistribution studies that compared BPA and sodium pentaborate. Three groups were irradiated: group 1 (11 rabbits) received BPA followed by thermal neutron irradiation, group 2 (9 rabbits) received thermal neutron irradiation only, and group 3 (9 rabbits) served as unirradiated, undrugged control animals. Eight of the 11 tumors in group 1 were treated successfully; all tumors in groups 2 and 3 grew. Histopathologic examination did not reveal vascular or retina damage in group 1. These preliminary experiments confirm that newer boronated compounds, such as BPA, used in BNCT and improved neutron beams can provide selective irradiation of ocular melanomas.

Intraocular radiation blocking.

Iodine-based liquid radiographic contrast agents were placed in normal and tumor-bearing (Greene strain) rabbit eyes to evaluate their ability to block iodine-125 radiation. This experiment required the procedures of tumor implantation, vitrectomy, air-fluid exchange, and 125I plaque and thermoluminescent dosimetry (TLD) chip implantation. The authors quantified the amount of radiation attenuation provided by intraocularly placed contrast agents with in vivo dosimetry. After intraocular insertion of a blocking agent or sham blocker (saline) insertion, episcleral 125I plaques were placed across the eye from episcleral TLD dosimeters. This showed that radiation attenuation occurred after blocker insertion compared with the saline controls. Then computed tomographic imaging techniques were used to describe the relatively rapid transit time of the aqueous-based iohexol compared with the slow transit time of the oil-like iophendylate. Lastly, seven nontumor-bearing eyes were primarily examined for blocking agent-related ocular toxicity. Although it was noted that iophendylate induced intraocular inflammation and retinal degeneration, all iohexol-treated eyes were similar to the control eyes at 7 and 31 days of follow-up. Although our study suggests that intraocular radiopaque materials can be used to shield normal ocular structures during 125I plaque irradiation, a mechanism to keep these materials from exiting the eye must be devised before clinical application.

Ocular anatomy for nuclear medicine.

The small size of the eye has precluded many efforts to image ophthalmologic disease processes. An understanding of those areas where nuclear medicine has been successful in ophthalmology requires a review of the appropriate anatomy. The accessibility of the nasolacrimal system and its pumping action have allowed for a unique physiologic appraisal with nuclear medicine techniques. The "magic bullet" that has eluded the search of oncology has left ophthalmologists without a practical diagnostic modality for ocular cancers. However, by placing detection devices in close proximity to these tumors, some useful information can be obtained. Unfortunately, this requires surgical dissection. New techniques have already been introduced into ophthalmology (fluorophotometry) in diabetic disease of the eye and nuclear medicine may have a considerable impact in this area. Understanding the anatomy of the retinal capillaries and larger vessels is essential to the reading of the pioneering work of Freeman and coworkers.