Neurovirulence of Cryptococcus neoformans determined by time course of capsule accumulation and total volume of capsule in the brain.

Cryptococcal meningitis remains a significant opportunistic infection in HIV-infected individuals worldwide, despite availability of antiretroviral therapies in developed nations. Current therapy with amphotericin B is difficult to administer and only partially effective. Mechanisms of cryptococcal neuropathogenesis are still not clearly defined. In the present study, we used a C57Bl/6 mouse model with intravenous inoculation of three isogenic strains of Cryptococcus neoformans: H99, Cap59, and Pkr1-33. These strains differ in their capsule production and are normocapsular, hypocapsular, and hypercapsular, respectively. We studied the role of capsule in the morbidity and mortality of our host animal. Surprisingly, we found that the hypercapsular strain was least virulent while the strains that produced less capsule were more virulent and had higher concentrations of organism in the brain. These results suggest that neurovirulence is related to total capsule volume and rate of capsule accumulation in the brain, rather than the amount of capsule produced per organism. Therapies which decrease central nervous system dissemination and inhibit replication rates in the brain may be more effective than therapies which target capsule production.

A transmissive laser speckle imaging technique for measuring deep tissue blood flow: an example application in finger joints.

BACKGROUND AND OBJECTIVE: Laser speckle perfusion imaging (LSPI) is a minimally invasive optical measure of relative changes in blood flow, providing real-time, high resolution, two-dimensional maps of vascular structure. Standard LSI imaging uses a light-reflective geometry that limits the measurement to a thin surface layer of 0.2-1 mm. The objective of this study was to test a new LSI instrument geometry with the laser source opposed to the image capture plane (light transmissive). Captured light then travels the entire tissue thickness (10-15 mm), sampling much deeper regions of interest than conventional optical imaging techniques. STUDY DESIGN: Reflective-light (conventional) and transmissive-light LSI modes were used to measure finger joint blood flow during a timed tourniquet occlusion of the brachial artery in volunteer participants. RESULTS: There was greatly increased visibility of vessels underlying the skin in the light-transmissive mode LSI mode. Established LSI algorithms were shown to still work in the light-transmissive mode, despite decorrelation due to finite laser coherence length and the light passing through a tissue thickness of 10-15 mm. CONCLUSION: Transmissive LSI can be used to measure blood flow deep (10-15 mm) into tissues. This could be useful for non-invasive measurements of finger joint synovial blood flow in diagnosing and treating peripheral vascular disorders, such as rheumatoid arthritis.

Endoscopic laser speckle imaging of tissue blood flow: applications in the human knee.

This work represents the first clinical data acquired with the endoscopic laser speckle imaging (eLSPI) system, a new diagnostic tool developed for real-time imaging of tissue blood flow during endoscopic surgical procedures. eLSPI was used to image tissue perfusion in the medial compartment of the knee of five patients requiring arthroscopic knee surgery. The effectiveness of eLSPI as a diagnostic tool was tested by measuring changes in tissue perfusion resultant from tourniquet application, and intra-articular epinephrine. eLSPI produced real-time perfusion video images of tissue blood flow in the knee joint. Tourniquet applications produced consistent decreases in mean perfusion index measurements (29.3% +/- 5.1% in meniscus; 39.5% +/- 8.2% in synovium with an intra patient variability of 6%-9%). A dose-dependent vasoconstrictive response to the administration of intra-articular epinephrine was measured, with maximum dose producing a mean decrease in perfusion of 31.0%-9.3% in meniscus and 41.2%-10.9% in synovium. eLSPI consistently detects decreases in articular tissue blood flow resultant from tourniquet inflation or from the administration of increasing concentrations of epinephrine. These are the first in vivo results indicating physiologic changes in articular tissue as a function of two commonly applied practices in endoscopic joint surgery.

Detection of c-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas.

Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.

Abrogation of p53-induced apoptosis by the hepatitis B virus X gene.

The p53 tumor suppressor gene product is a transcriptional transactivator and a potent apoptotic inducer. The fact that many of the DNA tumor virus oncoproteins bind to p53 and affect these p53 functions indicates that this interaction is an important step in oncogenic transformation. We and others have recently demonstrated that the hepatitis B virus oncoprotein, HBx, can form a complex with p53 and inhibit its DNA consensus sequence binding and transcriptional transactivator activity. Using a microinjection technique, we report here that HBx efficiently blocks p53-mediated apoptosis and describe the results of studies exploring two possible mechanisms of HBx action. First, inhibition of apoptosis may be a consequence of the failure of p53, in the presence of HBx, to upregulate genes, such as p21WAF1, Bax, or Fas, that are involved in the apoptotic pathway. Data consistent with this hypothesis include HBx reduction of p53-mediated p21WAF1 expression. Alternatively, HBx could affect p53 binding to the TFIIH transcription-nucleotide excision repair complex as HBx binds to the COOH terminus of p53 and inhibits its binding to XPB or XPD. Binding of p53 to these constituents of the core TFIIH is a process that may be involved in apoptosis. Because the HBx gene is frequently integrated into the genome of hepatocellular carcinoma cells, inhibition of p53-mediated apoptosis by HBx may provide a clonal selective advantage for hepatocytes expressing this integrated viral gene during the early stages of human liver carcinogenesis.

Effects of p53 mutants on wild-type p53-mediated transactivation are cell type dependent.

The spectrum of p53 mutations differs among human cancer types. We have hypothesized that the p53 mutational spectrum observed in particular tumor types reflects the functional ability of different p53 mutants to modulate wild-type (WT) p53-dependent gene transcription. Missense p53 mutants representing several mutational hotspot codons were cotransfected with WT p53 and analysed for their effects on p53-dependent transactivation of a reporter construct containing a specific p53 binding sequence (PG13-CAT) in human tumor cell lines lacking endogenous p53. Our results show that the ability of p53 mutants to inhibit WT p53-mediated transactivation is cell type dependent. In cell lines derived from a lung adenocarcinoma and a mesothelioma, the transactivation function of WT p53 was strongly inhibited by all p53 mutants examined. However, in cell lines derived from a prostate carcinoma and an osteosarcoma, the mutants examined generally had only minimal dominant negative effects. In cell lines derived from a hepatocellular carcinoma and an ovarian carcinoma, two mutants (248trp and 273his) enhanced WT p53-mediated transactivation of the reporter construct. Additional mutants retained the ability to inhibit WT p53-mediated transactivation in these cell lines. In addition, in a series of four breast tumor cell lines, the p53 mutants examined had similar effects on WT p53 transactivation ability including enhanced transactivation activity in the 273his cotransfectants. The p53 mutants were incapable of transactivating the PG13-CAT reporter in the absence of WT p53 expression. Therefore, the dominant negative effects of p53 mutants on WT p53 function may vary depending on the particular cell type. In addition, mutants with stronger inhibitory capabilities may confer a selective advantage during the tumorigenic process.

A comparison of two laser-based methods for determination of burn scar perfusion: laser Doppler versus laser speckle imaging.

UNLABELLED: Laser Doppler perfusion imaging (LDI) is an established technique for early assessment of burn depth to help determine a course of treatment. Laser speckle perfusion imaging (LSPI) is an alternative laser based, non-invasive perfusion monitoring technique that offers rapid and high resolution images of tissue. We have evaluated the ability of the LSPI instrument in determining and monitoring burn scar perfusion over time and compared it with the LDI instrument as a standard. METHODS: Ten patients with hypertrophic burn scars (time since injury: 1-8 months) were recruited. Burn scars were scanned with both instruments (LSPI and LDI) monthly over a period of 11 months. Clinical grading of the burn scars was assessed on every scan date using the Vancouver burn scar scale. RESULTS: Comparison of the perfusion values determined by each instrument shows a strong positive correlation, r2=0.86 (n=63). Each instrument's output also correlated significantly with the clinical grading of the scar, indicating the expected decrease in perfusion as the clinical condition of the scars improved with time. SIGNIFICANCE: The new LSPI instrument compared favorably with the established LDI instrument, yielding similar results. The considerably faster scan time and higher resolution of the LSPI method provides a distinct clinical advantage, both in terms of patient comfort and for reliably matching perfusion characteristics to their associated anatomical features. The fast temporal response of the LSPI instrument could be used to monitor near real-time responses to mechanical or pharmacological interventions to study dynamic vascular changes to burn damaged tissues.

Haematologic complications from human babesiosis: a case report.

Formerly known as Nantucket fever, babesiosis is increasing in incidence across the Northeastern United States. Because of its emerging health risk globally, it is important to be aware of its various presenting manifestations. We present the case of a middle-aged man with haemolytic anaemia from Babesia microti infection.

Allelic frequency of a p53 polymorphism in human lung cancer.

p53 is a tumor suppressor gene that is mutated in diverse tumor types. Here we report the frequencies of common polymorphic variants at codon 72 of the p53 gene in germline DNA of lung cancer cases and controls as determined by a polymerase chain reaction strategy. The observed allelic distribution was found to be significantly different between African-Americans and Caucasians in this U.S. population. The frequency of polymorphic variants was similar in lung cancer cases and controls after adjustment for race. However, among lung cancer patients the proline variant at codon 72 was in excess in adenocarcinoma patients by comparison with other histologies.

Evaluation of laser-Doppler perfusion imaging for measurement of blood flow in cortical bone.

Most techniques currently available to measure blood flow in bone are time consuming and require destruction of the tissue, but laser-Doppler technology offers a less invasive method. This study assessed the utility of laser-Doppler perfusion imaging (LDI) to measure perfusion in cortical bone. Twelve mature New Zealand White rabbits were assigned to one of three groups: normal control, constriction (norepinephrine), or dilatation (nitroprusside). The left and right medial tibiae were consecutively scanned at red (634-nm) and near-infrared (810-nm) wavelengths to examine the repeatability of LDI output. The pharmacological intervention groups were injected with the respective drug, and LDI measurements at 810 nm were obtained concurrently with colored microsphere-determined flow in all of the groups. LDI effectively quantified blood flow in cortical bone and detected physiologically induced changes in perfusion. A significant positive correlation was found between microsphere-determined flow and LDI output (r = 0.6, P < 0.05). Repeatability of consecutive LDI measurements was within 5%. The effectiveness of LDI to measure perfusion in bone suggests this method has potential for investigating the role of blood flow in bone metabolism and remodeling.

Dynamic measurement of bone blood perfusion with modified laser Doppler imaging.

Although the mechanisms are not clearly defined, blood flow may play an important role in moderating skeletal adaptation. Most techniques currently available to measure blood flow in bone are time-consuming and require destruction of the tissue, but laser Doppler technology offers a less invasive method. The present study assessed whether laser Doppler perfusion imaging could detect changes in perfusion in cortical bone. By use of modified laser Doppler perfusion imaging with an adjustable, incorporated, near infrared-laser gain photodetection system, perfusion of blood in the mid-diaphyseal tibial cortex of New Zealand White rabbits (n = 5) was measured before, during, and after occlusion of the femoral artery. During occlusion, perfusion decreased 69% compared with control levels; removal of the arterial clip caused flux values to return to near normal. Laser Doppler perfusion imaging provides a two-dimensional image related to blood flow, and the results of this pilot study suggest that it may be an effective technique for imaging in vivo dynamic changes in perfusion in cortical bone.

The c-K-ras gene and human cancer (review).

A significant number of human tumors from diverse histological origins contain c-K-ras oncogenes which have been activated by somatic point mutations resulting in single amino acids substitutions in the encoded p21 ras protein. In addition to these qualitative changes, other genetic alterations leading to increased expression of the c-K-ras gene, especially its mutated form, appear to be important in the activation of its oncogenic potential. These findings support the hypothesis that c-K-ras oncogenes are contributing in a dominant but dose dependent manner to the multistage process of human tumorigenesis. Activated c-K-ras oncogenes have been detected in human tumors at different stages of progression, including premalignant neoplasms. These studies provide evidence for the involvement of somatic mutational activation of this ras gene in the early stages of tumor development in some types of human cancer. We discuss here the structural and functional features of the human c-K-ras gene and the involvement of its activated form in human cancer.

In vivo comparison of scanning technique and wavelength in laser Doppler perfusion imaging: measurement in knee ligaments of adult rabbits.

At present, there are only two laser Doppler perfusion imaging systems (LDIs) manufactured for medical applications: a 'stepwise' and a 'continuous' scanning LDI. The stepwise scanning LDI has previously been investigated and compared with coloured microsphere determined standardised flow. The continuous scanning LDI is investigated and compared with the stepwise scanning LDI for its ability to measure in vivo, hypoaemic, ligament tissue blood flow changes. The continuous scanning system was supplied with two lasers, red and near infrared (NIR), allowing for additional assessment of the effect of wavelength on imaging ligament perfusion. Perfusion images were obtained from surgically exposed rabbit medial collateral ligaments (MCL). Continuous and stepwise LDI scans were compared using correlation and linear regression analysis of image. averages and standard deviations. Using the same method of analysis, LDI measurements using red and NIR lasers indicated a high degree of correlation, at least over the ranges of perfusion assessed, indicating that red and NIR lasers measure similar regions of flow in the rabbit MCL. These experiments confirm that both LDI techniques provide a valid in vivo measure of dynamic changes in connective tissue perfusion and could have significant impact on the understanding and treatment of joint injury and arthritis.

Comparison of laser speckle and laser Doppler perfusion imaging: measurement in human skin and rabbit articular tissue.

Laser Doppler perfusion imaging (LDI) is currently used in a variety of clinical applications, however, LDI instruments produce images of low resolution and have long scan times. A new optical perfusion imager using a laser speckle measurement technique and its use for in vivo blood flow measurements are described. Measurements of human skin and surgically exposed rabbit tissue made using this instrument were compared with a commercial laser Doppler perfusion imaging instrument. Results from blood flow measurements showed that the laser speckle imager measured an 11-67% decrease in blood flow under arterial occlusion. Under similar conditions, the laser Doppler imager measured blood flow decreases of 21-63%. In comparison with LDI, it was observed that the higher temporal resolution of the laser speckle imager was more sensitive to measuring the hyperaemic response immediately following occlusion. This in vivo study demonstrated some of the several advantages laser speckle imaging has over conventional LDI, making the new instrument more versatile in a clinical environment.

Evaluation of laser Doppler imaging to measure blood flow in knee ligaments of adult rabbits.

Laser Doppler imaging (LDI) is investigated as a novel method for in vivo ligament tissue blood flow determination. LDI output signal is obtained from surgically exposed rabbit medial collateral ligaments (MCL). The LDI signal is compared with simultaneously determined, coloured microsphere (CM)-derived standardised MCL blood flow. Correlation of LDI output with the CM flow data and a linear regression of 17 data points in nine rabbits (joint injured to provoke an acute vascular response in the tissues) indicate that LDI provides a reasonable estimate of MCL blood flow, at least over the ranges assessed. If properly calibrated, and given enough tissue-specific data points, LDI may have advantages over conventional, but more invasive, techniques. The potential clinical application of LDI technology to joint injury and arthritis research is discussed.

Factors influencing the appropriateness of restorative dental treatment: an epidemiologic perspective.

OBJECTIVES: An epidemiology analysis was performed to identify patient and dentist factors influencing over- and undertreatment of restorative services in a sample of insured adults. METHODS: At baseline, 681 Washington State employees and their spouses, aged 20 to 34 years and residing in the Olympia or Pullman areas, were interviewed by telephone. Oral assessments were conducted to measure personal characteristics, oral disease, and restoration quality. Adults were followed for two years to measure use of restorative services from dental insurance claims. Each adult's baseline and claims data were linked with provider and practice variables collected from the dentist who provided treatment. RESULTS: For overtreatment, 39 percent of adults received one or more replacement restorations in nondecayed teeth with satisfactory fillings at baseline, while 18 percent of adults had one or more restorations placed in teeth with no decay and fillings. An adult's probability of overtreatment was higher if the adult had more fillings at baseline, or if an adult's dentist was younger, had a busy practice, advertised, charged higher fees, had less continuing education, or had a solo practice. For undertreatment, about 16 percent of adults either received no replacement restorations in teeth with unsatisfactory fillings at baseline, or had decayed teeth at baseline that were not filled or crowned. An adult's probability of undertreatment was higher if an adult had less decayed or more missing surfaces at baseline, or if an adult's dentist believed in sharing information with patients, had a busy practice, or reported not placing fillings when radiographic evidence of new caries was present. CONCLUSIONS: A minority of adults aged 20 to 34 experienced potential over- or undertreatment of restorative services, which are influenced by both patient and dentist factors.

Education in emergency nursing: a new beginning. Part I.

The movement of nurse education to the tertiary sector is all but complete in Australia. Trends are changing as new partnerships are being forged and the focus of education is now responsive to economic and political factors. These factors have resulted in the regionalisation of health care facilities and the de-institutionalisation of health services in Queensland, Australia. Moreover, de-institutionalisation of services coupled with economic rationalism has pre-empted the offering of post-graduate clinical courses by university nursing facilities. This paper addresses the process of developing the content of new courses under the umbrella of the Graduate Diploma/Masters in Emergency Care Nursing offered at Griffith University Gold Coast, Australia. This initiative is the first in Queensland and has revisioned the education of emergency care nursing. This course includes a tapestry of knowledge enhancing, clinical and factual subjects presented by experienced clinicians teaching with academics, recognising the collaborative nature of nursing practice with academics and clinicians working jointly in course development, teaching and clinical placement. Strategies developed collaboratively addressed course content including logistics, time management, the critical selective selection of appropriate content, assessment criteria, registerability within Queensland and other states and clinical competence. Moreover, this process of developing the course utilised the Australian National Registering Authorities' Competencies and Benner's use of the Dreyfus model to guide the knowledge level of the clinician at course completion. In particular this paper will not only address the blending of theory and practice to consolidate the relationship inherent between the clinician and education, but will establish the fact that no gap exists.

Education in emergency nursing: a new beginning. Part 2.

Continuing from the previous issue, this paper discusses the process of developing the content of new courses under the umbrella of the Graduate Diploma/Masters in Emergency Care Nursing offered at Griffith University Gold Coast, Australia. Part 2 of this paper includes information on course structure, implementation, content, assessing methods and competences and the logistics of the course.

The expanded role of acute care nurses: the issue of liability.

For a variety of reasons the activities performed by health care professionals are ever increasing, however the legislative process regulating these specialised workers is not keeping up with the practice realities. While competency statements and credentialling mechanisms are developing, they are not uniformly in place for specialists. Therefore activities completed by these practitioners may be legally controversial. This study documents a variety of tasks completed independently by nurses employed in three acute care hospitals that legally require medical orders and then examines the legal implications of this role extension. Credentialling is one mechanism by which nurses and other health care professionals can ensure they possess the levels of knowledge and skill required to perform the advanced activities required of them and consequently avoid negative legal repercussions.

Neurological impairment in a surviving twin following intrauterine fetal demise of the co-twin: a case study.

It has been established that twin pregnancies are at an increased risk for complications, including the risk of morbidity or mortality for one or both of the infants. Cerebral palsy and other associated neurological deficits also occur at higher rates in twin pregnancies. This report examines two cases of intrauterine demise of one twin with subsequent survival of the co-twin. In both cases, the surviving infant suffered significant neurological sequelae. Impairments observed in these two cases include multicystic encephalomalacia and periventricular leukomalacia as well as the subsequent development of cerebral palsy. This case study explores the predisposing factors, incidence, pathophysiology, consequences, and future research implications of these findings.