15-deoxy-Delta12,14 prostaglandin J2-induced heme oxygenase-1 in megakaryocytes regulates thrombopoiesis.

BACKGROUND: Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ(2) can exert effects independent of PPARgamma, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. OBJECTIVES: To further investigate the influence of 15d-PGJ(2) on megakaryocytes and to understand whether HO-1 plays a role in platelet production. METHODS: Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ(2) on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. RESULTS AND CONCLUSIONS: 15d-PGJ(2) potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ(2)-induced HO-1 was independent of PPARgamma, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ(2) were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ(2)-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.

The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects.

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of evidence links platelet function and thrombosis to vascular inflammation. peroxisome proliferator-activated receptors (PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARbeta/delta and PPARgamma) were recently identified in platelets. Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons. First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to cardiovascular disease (CVD) progression. Dampening platelet release of proinflammatory mediators, including CD40 ligand (CD40L, CD154), is essential to hinder this cascade. Second, understanding the biologic importance of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options.

Rod pathways in the mammalian retina use connexin 36.

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.

Prevalence of infection in ticks submitted to the human tick test kit program of the U.S. Army Center for Health Promotion and Preventive Medicine.

In 1997, ticks removed from humans and received alive by the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine (USACHPPM) were tested for pathogens by polymerase chain reaction (PCR). Thirty-three of 222 (15%) Amblyomma americanum (L.) DNAs produced amplicons of the expected size of Ehrlichia chaffeensis Anderson, Dawson & Wilson and 26/222 (12%) produced amplicons indicating Borrelia burgdorferi Johnson, Schmid, Hyde, Steigalt & Brenner. Five (2%) appeared to be co-infected with both organisms. Thirteen of 308 (4%) Dermacentor variabilis (Say) were PCR-positive for spotted fever group rickettsiae. Restriction fragment-length polymorphism analysis indicated all were Rickettsia montana. One hundred twenty-seven D. variabilis from Monroe County, WI, were tested for B. burgdorferi and 14 (11%) were positive. Five of 24 (21%) Ixodes scapularis Say were positive for B. burgdorferi and one (2%) was positive for the agent of human granulocytic ehrlichiosis. Different species of ticks transmit different pathogens, and most tick-borne diseases have similar early symptoms, therefore knowing the species and infection status of the tick enhances the physician's ability to consider tick-borne agents as a potential cause of disease and recommend appropriate therapy. Ongoing surveillance of the vector species of human diseases provides an additional estimate of human encounters with infected ticks, and testing ticks removed from humans may increase our knowledge of the vector status of tick species for transmitting tick-borne pathogens.

Excitation--contraction uncoupling by a human central core disease mutation in the ryanodine receptor.

Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca(2+)-induced damage stemming from mutant RyR1 proteins forming "leaky" sarcoplasmic reticulum (SR) Ca(2+) release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164--4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca(2+) release without significantly altering resting cytosolic Ca(2+) levels, SR Ca(2+) content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca(2+) release, again without altering resting cytosolic Ca(2+) levels, SR Ca(2+) content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca(2+) release, rather than the expression of overactive or leaky SR Ca(2+) release channels.

Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) infection in Amblyomma americanum (Acari: Ixodidae) at Aberdeen Proving Ground, Maryland.

Human monocytic ehrlichiosis (HME) is a sometimes fatal, emerging tick-borne disease caused by the bacterium Ehrlichia chaffeensis. It is frequently misdiagnosed because its symptoms mimic those of the flu. Current evidence indicates that Amblyomma americanum (L.), the lone star tick, is the major vector of HME. To determine if E. chaffeensis is present in ticks at Aberdeen Proving Ground, MD, questing A. americanum ticks were collected from 33 sites. Nucleic acid was extracted from 34 adult and 81 nymphal pools. Sequences diagnostic for E. chaffeensis from three different loci (16S rRNA, 120-kDa protein, and a variable-length polymerase chain reaction [PCR] target, or VLPT) were targeted for amplification by the PCR. Fifty-two percent of the collection sites yielded pools infected with E. chaffeensis, confirming the presence and widespread distribution of E. chaffeensis at Aberdeen Proving Ground. Analysis with the both the 120-kDa protein primers and the VLPT primers showed that genetic variance exists. A novel combination of variance for the two loci was detected in two tick pools. The pathogenic implications of genetic variation in E. chaffeensis are as yet unknown.

Histamine immunoreactive axons in the macaque retina.

PURPOSE: The goal of these experiments was to identify the neurotransmitter in centrifugal axons of the macaque retina. METHODS: Macaca mulatta retinas and optic nerves were fixed overnight in carbodiimide and labeled with an antiserum to histamine with the use of an immunofluorescence technique. RESULTS: Several large histamine-immunoreactive axons ran from the optic nerve head to the peripheral retina, where they branched extensively and terminated in the inner plexiform layer, occasionally alongside retinal blood vessels. Other axons that emerged from the optic nerve head ran in the optic fiber layer to the central retina, circled the fovea, and then returned to the optic disc. These may be the source of histamine-immunoreactive axons that have been observed in central visual areas. No labeled cell bodies were present in the retina. Because perikarya in the posterior hypothalamus are the only known source of histamine in the primate central nervous system and because neurons there can be retrogradely labeled from the cut optic nerve, the histamine-immunoreactive axons must have originated there. CONCLUSIONS: Centrifugal axons in the macaque retina are part of the system of axons containing histamine that originate in the hypothalamus and project throughout the brain. Because the activity of these neurons is highest during the morning, histamine might play a role in preparing the retina to operate in daylight. The contacts of histamine-immunoreactive axons with blood vessels suggest that histamine may also play a role in regulating the retinal microvasculature.

Overproduction of SM28GST in a baculovirus expression vector and its use to evaluate the in vivo immune responses of mice vaccinated against Schistosoma mansoni with naked DNA encoding the SM28GST gene.

To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.

Cerebral emboli during cardiac surgery in children.

BACKGROUND: Microemboli occur commonly during cardiac surgery in adults, and, when present, increase the risk of neuropsychological deficits. Their incidence and significance during correction of congenital heart disease is unknown. The authors hypothesized that microemboli would occur before bypass with right-to-left cardiac shunts and would also occur in large numbers when the aortic crossclamp was released in children during repair of congenital heart defects. METHODS: In 25 children studied with carotid artery Doppler, embolic signals were counted and timed in relation to 13 intraoperative events. Patients were classified as either at high risk (obligate right-to-left shunt or uncorrected transposition of the great arteries) or at low risk (net left-to-right shunt or simple obstructive lesions) for paradoxical (venous to arterial) emboli. RESULTS: The median number of emboli detected was 122 (range, 2-2,664). Forty-two percent of all emboli were detected within 3 min of release of the aortic crossclamp. The high-risk group had significantly more emboli (median, 66; range, 0-116) during the time interval before cardiopulmonary bypass than did the low-risk group (median, 8; range, 0-73), with P < 0.01. There was no significant difference between the high- and low-risk groups in the total number of emboli detected. There was no apparent association between number of emboli and gross neurologic deficits. CONCLUSIONS: Microemboli can be detected in the carotid arteries of children undergoing repair of congenital heart disease and are especially prevalent immediately after release of the aortic crossclamp. The role of emboli in causing neurologic injury in children undergoing repair of congenital heart disease remains to be determined.

Intracavity laser absorption measurements at ultrahigh spectral resolution.

Intracavity laser absorption spectroscopy with ultrahigh sensitivity and enhanced spectral resolution is demonstrated. It entails use of an intracavity étalon that selects equally spaced modes of the cavity. These modes are reduced in intensity when they occur at spectral locations where absorption that is due to intracavity species also occurs. We obtained absorption spectra by tuning the étalon in small steps across its free spectral range, recording intracavity spectra at each step, and summing the result. The maximum resolution is determined by the width of the étalon peaks, which was ~0.01 cm(-1). When the finesse of the étalon is increased, the resolution equal to the width of a single laser mode can be achieved. With this technique, spectra at Doppler-free resolution such as that required for studies of high vibrational-overtone transitions of molecules in supersonic jets are possible.

Transesophageal echo probe compression of an aberrant right subclavian artery.

Transesophageal echocardiography (TEE) is frequently used in the operating room to assess surgical repairs in children with congenital heart disease. Complications from the procedure are unusual, but the potential for TEE probe compression of normal and abnormal posterior vascular structures has been recognized. We report a case of TEE probe compression of an aberrant right subclavian artery in a patient undergoing repair of an atrioventricular septal defect.

An effective computer-based tardive dyskinesia monitoring system.

To promote early recognition and treatment of neuroleptic-induced tardive dyskinesia we used our facility's pharmacy and appointment data bases to develop an automated reminder system that significantly improved physician monitoring of patients receiving antipsychotic drug therapy. The system prompts staff to perform regular examinations for abnormal involuntary movements and to review patients' consent to therapy with antipsychotic medication. The average prevalences in the 15 months after automated reminders began, in a population of over 800 patients, increased from 53% to 85% for an annually completed abnormal involuntary movement scale in medical records and from 38 to 74% for a statement of informed consent. Now, 45 months later, prevalences of both measures approaches 100%. The integrated design of the Department of Veterans Affairs computer system allowed linking pharmacy and appointment scheduling data and facilitated the project. The reminder system effectively promoted rapid, marked, and sustained change in physicians' documentation of antipsychotic drug therapy.

An in vitro comparison of biofilm formation on various biliary stent materials.

Stent occlusion may occur as the result of bacterial adherence and biofilm formation. This occlusion may be prompted by the material or design of the stents. In this study, a bile perfusion model was used to compare the biofilm formation on various stent materials. The copolymer and the wire mesh stents demonstrated less biofilm formation than the traditional stent. The development of new polymers may prolong stent patency.

Mental health services for medical students: perceptions of students, student affairs deans, and mental health providers.

PURPOSE: To examine (1) whether there is any consistency among medical schools in mental health services provided and (2) how these services are perceived by student affairs deans, mental health service providers, and the students themselves. METHOD: Questionnaires were sent in October 1991 to the student affairs dean (or director), the individual responsible for student mental health services, and a student representative in each of the 126 U.S. and Canadian medical schools. Data were sought regarding personnel, individuals served, location, hours, administration, funding, confidentiality, administrative referrals, and respondents' suggestions for improvement. Possible differences among the three groups of respondents were tested by chi-square. RESULTS: Responses were received from 75 student affairs deans, 53 mental health providers, and 30 students. There was much diversity among schools in services provided, especially in the areas of administration and funding. Although perceptions of the three respondent groups were often the same, they differed significantly in a number of areas. Suggestions for improvement of services involved funding, personnel, hours, confidentiality and privacy, specialty services, preventive and support programs, and visibility. The suggestion most frequently made by the students was for increased information and visibility. CONCLUSION: The differences among schools coupled with the differing perceptions within schools indicate a need for a comprehensive consideration of what kinds of mental health services are needed and how they can best be made accessible to a diverse body of students experiencing a variety of academic and personal challenges.

ELISA tests for antibodies in experimental bovine tuberculosis.

It was shown that 10(4) cfu of a field isolate of Mycobacterium bovis caused illness in five experimentally infected calves; one of these died. One of three contact calves also became clinically infected. Considerable variation in the humoral response of the affected animals was demonstrated by ELISAs using purified protein derivative (PPD) and phosphatide antigens. The inoculation of antigens used in the comparative tuberculin skin test significantly enhanced the level of PPD antibodies in the affected animals whereas that of the apparently non-infected contact animals remained unchanged.

Use of azathioprine or 6-mercaptopurine in the treatment of Crohn's disease.

The efficacy and safety of therapy with azathioprine/6-mercaptopurine was studied in 78 patients with Crohn's disease. Mean duration of therapy was 1.6 years; 52 patients were treated greater than or equal to 6 months. All patients were also on other antiinflammatory medications. Evaluations included self-assessment and physician's assessment of well-being, functional capacity, general clinical response, clinical activities indices (National Foundation for Ileitis and Colitis/International Organization for the Study of Inflammatory Bowel Disease and Harvey-Bradshaw), and achievement of specific therapeutic goals. General clinical condition improved in 70% of the patients. Median response time was 3 months. The average Harvey-Bradshaw score decreased 37% with therapy, and a decrease of greater than or equal to 30% occurred in 66% of the subjects. An overall 72% achievement rate for specified therapeutic goals included controlling refractory disease, 73%; corticosteroid "sparing," 76%; and lessening fistulization, 63%. Nine patients got worse despite therapy. Adverse effects requiring discontinuation of therapy occurred in 10%, whereas dosages were briefly lowered for mild side effects in another 10%. This study demonstrates the effectiveness and safety of azathioprine/6-mercaptopurine in the majority of selected patients with chronic, unremitting, or steroid-requiring Crohn's disease.