Dose-response effects of TPI ASM8 in asthmatics after allergen.

BACKGROUND: TPI ASM8 contains two modified antisense oligonucleotides (AON) targeting the beta subunit (ß(c) ) of the IL-3, IL-5, GM-CSF receptors and the chemokine receptor CCR3. A previous study suggested that TPI ASM8 had broader effects than just inhibition of eosinophils in asthmatics. OBJECTIVE: We assessed whether TPI ASM8 caused a dose-dependent attenuation in the inflammatory and physiological changes after inhaled allergen challenge (AIC). METHODS: This single-center, open-label, stepwise-ascending dose study was conducted in fourteen stable, mild allergic asthmatics. Following placebo AIC, subjects underwent AIC after 4 days treatment with 1, 2, and 4 mg BID and finally 8 mg once daily (OD) of TPI ASM8, inhaled via the I-Neb™ nebuliser. Treatments were separated by 2-3-week washout periods. RESULTS: TPI ASM8 was safe and well tolerated at all doses. TPI ASM8 8 mg OD reduced eosinophils in sputum after AIC (by 60.9% at 7 h and 68.4% at 24 h post-AIC, P=0.016 and P=0.007, respectively). Additionally, TPI ASM8 8 mg OD significantly attenuated the early and late airway responses as shown by the reduction in the area under the curve by 45% (P=0.016) and 59%, (P=0.0015), respectively, the increase in eosinophil cationic protein (ECP) by up to 57% (P=0.021), and airway responsiveness to methacholine by more than 1 doubling dose (P=0.012). A dose-response relationship was noted, and efficacy was maintained with once per day administration. CONCLUSIONS: TPI ASM8 attenuated a broad range of inflammatory and physiological changes after AIC, suggesting that CCR3, IL-3, and GM-CSF also are important targets for the management of asthma.

Multitargeted approach using antisense oligonucleotides for the treatment of asthma.

Asthma is characterized by inflammation and hyperresponsiveness related to the accumulation of inflammatory cells, particularly eosinophils, within the airways. We tested the hypothesis that a multitargeted approach is better than a single-targeted approach in a rat model of asthma. We simultaneously delivered oligonucleotides (ODNs) targeting the chemokine receptor CCR3 and the common beta chain subunit of the receptors for IL-3, IL-5, and GM-CSF at the time of ovalbumin challenge in sensitized Brown Norway rats. Fewer eosinophils were detected in bronchoalveolar lavage (BAL) of rats treated with both ODNs as compared to each ODN alone. Moreover, airway responsiveness to LTD(4) was significantly decreased at lower doses in the 2 ODN-treated groups compared to a single ODN. As ODN therapy has raised concerns of toxicity we therefore examined ODNs prepared with modified DNA bases, specifically 2'amino, 2'deoxyadenosine (DAP) in place of adenosine. In vivo, administration of individual DAP-ODN was efficacious in inhibiting airway hyperresponsiveness, whereas delivery of 2 DAP-ODNs (targeting CCR3 and common beta chain) reduced the influx not only of eosinophils but also lymphocytes and macrophages in the lungs of rats as compared to the unmodified ODNs. Blocking multiple inflammatory pathways simultaneously is more effective in preventing eosinophilia and airway hyperresponsiveness than inhibiting either pathway alone. The challenges associated with the development of a product containing two oligonucleotides in humans are discussed.

Medical directors of long-term care facilities: preventing another physician shortage?

OBJECTIVE: The long-term care (LTC) sector in Canada is expanding, but little attention has been given to medical human resources in this area. Our objective was to seek LTC medical directors' opinions about medical services in LTC and about strategies for recruitment and retention. DESIGN: Mailed survey. SETTING: Long-term care facilities and nursing homes. PARTICIPANTS: Seven hundred five medical directors of LTC facilities across Canada were identified from the Canadian Healthcare Association database. MAIN OUTCOME MEASURES: Responses to open- and closed-ended questions and to Likert-type scales. RESULTS: The response rate was 55%. The average age of medical directors was 54 years. Most had started work in LTC because of a vacant position, as opposed to self-perceived skills or training. Most (75.3%) reported satisfaction with their role as medical directors, but 82.7% believed that there was a significant shortage of physicians working in LTC, and 42% had seriously considered leaving their positions. Major sources of satisfaction identified were clinical, especially working with older patients and improving care. Important sources of dissatisfaction were remuneration for LTC work, on-call coverage, and excessive paperwork. Directors suggested increases to fee schedules as the main recruitment and retention strategy, and many believed that increasing exposure to LTC during residency would increase recruitment. Development of larger on-call groups for coverage and alternative methods of remuneration were not cited as important factors. Most did not believe that working in a teaching nursing home would increase their satisfaction. Directors did not think the use of nurse practitioners would alleviate concerns about shortages of physicians. CONCLUSION: Medical directors of LTC facilities are aging, and many are considering leaving their work in LTC. Without an increase in the number of physicians willing to work in LTC institutions, the current shortage of LTC physicians could increase in the near future. Medical directors' responses to questions could help guide strategies to recruit and retain physicians. Future areas of research should include the perspectives of physicians who are not medical directors and of family medicine residents.

Size-dependent fine-structure splitting in self-organized InAs/GaAs quantum dots.

A systematic variation of the exciton fine-structure splitting with quantum dot size in single quantum dots grown by metal-organic chemical vapor deposition is observed. The splitting increases from to as much as with quantum dot size. A change of sign is reported for small quantum dots. Model calculations within the framework of eight-band theory and the configuration interaction method were performed. Different sources for the fine-structure splitting are discussed, and piezoelectricity is pinpointed as the only effect reproducing the observed trend.

Cryptogenic epilepsy: an infectious etiology?

PURPOSE: Cryptogenic epilepsy, the group of epilepsy syndromes for which an etiology is unknown, comprises approximately 20% of all epilepsy syndromes. We selected patients in this subgroup of epilepsy and tested them for evidence of Toxoplasma gondii IgG antibodies by the enzyme-linked immunosorbent assay. T. gondii is found in up to 20% of the U.S. population forming dormant brain cysts in the latent bradyzoite form. We investigated the hypothesis that dormant T. gondii infection might be associated with cryptogenic epilepsy. METHODS: We selected patients with cryptogenic epilepsies and tested them for evidence of T. gondii IgG antibodies by the enzyme-linked immunosorbent assay. A control group was also tested for comparison. RESULTS: We have found a statistically-significant elevation of T. gondii antibodies among cryptogenic epilepsy patients as compared to controls [59% increase in optical density (OD), p = 0.013]. This association persisted after adjustment for subjects' gender and age in a multiple logistic regression model; however, it was no longer as statistically significant. CONCLUSIONS: Our results suggest that chronic T. gondii infection with brain cysts may be a cause of cryptogenic epilepsy.

What drugs are our frail elderly patients taking? Do drugs they take or fail to take put them at increased risk of interactions and inappropriate medication use?

OBJECTIVE: To determine whether there were discrepancies between what medications frail elderly outpatients took and what physicians thought they took and whether discrepancies put patients at risk of taking inappropriate drugs and of increasing the potential for drug interactions. DESIGN: Case series. SETTING: Day Hospital Program at St Mary's of the Lake Hospital in Kingston, Ont. PARTICIPANTS: One hundred twenty community-living elderly patients attending the Day Hospital Program in 1998. Three patients and two family physicians declined to participate. MAIN OUTCOME MEASURES: Lists of medications being taken by patients compared with lists of medications in physicians' charts. Category according to explicit criteria that each drug fell into and risk of drug interactions as determined by the Clinidata Drug Interaction Program. RESULTS: Of the 120 patients, 115 had at least one discrepancy between their lists of medications and their physicians' lists. Of the 1390 medications on the lists, 521 (37%) were being taken by patients without their doctors' knowledge, 82 (6%) were not being taken by patients when doctors thought they were, and 133 (10%) were on both patients' and their doctors' lists but with dosages or frequency of administration that were different. More potential drug interactions were identified on patients' lists than on physicians' lists. No increase in risk of inappropriate drug use was identified. CONCLUSION: Family physicians are often unaware of all the medications their patients are actually taking. Medications used by patients without physicians' knowledge increase the likelihood of drug interactions. Family physicians should look at and inquire about all medications, including over-the-counter drugs, their patients are actually taking.

End-stage renal disease: factors affecting referral decisions by family physicians in Canada, the United States, and Britain.

The objective of this study is to determine how patient age, sex, creatinine level, and comorbidity affect referral decisions for the treatment of end-stage renal disease (ESRD) and whether these decisions are affected by physician characteristics in three countries: Canada, the United States, and Britain. A vignette-based questionnaire was mailed to a random sample of family physicians in Ontario, Canada (1,818 physicians); all family physicians in the state of New York (1,814 physicians); and a sample of general practitioners from the south of England (2,228 physicians) in 1996. Physicians were presented with clinical scenarios involving a patient with varying degrees of renal insufficiency and a complicating comorbidity, including angina, diabetes, cancer, mental illness, or socioeconomic circumstances. They were asked to indicate the likelihood of referral. Half the physicians received a questionnaire describing a male patient, and half, a female patient. Mean creatinine levels at which physicians would refer were 260 micromol/L for British physicians, 297 micromol/L for Canadian physicians, and 340 micromol/L for American physicians. No difference in referral rates was found based on the sex of the patient or physician. Sixty-five percent of American and Canadian physicians would refer regardless of patient age, but only 49% of British physicians would do so. Family physicians in the United States, Canada, and Britain function as gatekeepers for patients with ESRD. They are less likely to refer based on increasing severity of comorbid conditions. They also discriminate based on age, but not sex.

Identification and role of thiols in Toxoplasma gondii egress.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

NK cell-mediated lysis of autologous human oligodendrocytes.

Although considered an autoimmune disease, the mechanisms underlying oligodendrocyte (OL)/myelin injury in multiple sclerosis (MS) remain to be established. We utilized in vitro assays to demonstrate that human OLs, as well as other glial elements (astrocytes, microglia), were susceptible to injury mediated by peripheral blood-derived mononuclear cell preparations (MNCs) enriched for natural killer (NK cells) by depleting CD3(+) +/- CD19(+) cells through use of either magnetic beads or cell sorting. Cytotoxic effects of the NK cell-enriched effectors were dependent on pre-exposure of these cells to IL-2. Furthermore, we found that autologous OLs were as susceptible to injury mediated by IL-2 activated NK cells as were heterologous OLs. In context of the tissue injury that occurs in MS, our results suggest that the inflammatory milieu in MS lesions could provide conditions required for NK cell activation and that such effector cells can bypass the putative protective effects of self-MHC class I molecules that may be expressed on OLs.

Primary care in Bosnia and Herzegovina. Health care and health status in general practice ambulatory care centres.

OBJECTIVE: To assess the health care and health status of patients attending primary care clinics in Bosnia and Herzegovina. DESIGN: Assisted administration patient survey. SETTING: Two ambulatory care clinics (ambulantas) in each of three cities in Bosnia and Herzegovina: Tuzla, Mostar, and Banja Luka. PARTICIPANTS: Patients attending the ambulantas during a 1-week period in March 1999; 885 answered questionnaires. MAIN OUTCOME MEASURES: Each patient listed demographic characteristics and answered questions on satisfaction with health care and with the physical and financial accessibility of health care services and medications. A validated health status questionnaire (EuroQoL), previously used in parts of the former Yugoslavia, was administered. RESULTS: Only 22% of patients were employed; 57% could not pay the nominal fee to see a physician; 71% walked to the clinic; mean distance from patients' homes to the clinics was 2.3 km; 63% could not get the medications prescribed (in 85% of cases because of cost, not availability); 80% to 90% of answers to satisfaction questions suggested high satisfaction with the care patients received from their doctors; 67% of the time patients were referred to a specialist by general practitioners; 33% had problems walking; 17% had problems with self-care; 36% had problems with usual daily activities; 72% had at least some pain or discomfort; and 62% described at least some anxiety or depression. The three cities showed significant differences; patients in Tuzla generally had lower health status and more problems with health care. CONCLUSION: Unemployment and financial considerations reduced health care access in Bosnia and Herzegovina. While only one third of patients had physical difficulties, two thirds had emotional problems or pain. Satisfaction with physicians' care was high.

Cerebral malaria in mice: interleukin-2 treatment induces accumulation of gammadelta T cells in the brain and alters resistant mice to susceptible-like phenotype.

In this study, we report that infection with Plasmodium yoelii 17XL, a lethal strain of rodent malaria, does not result in death in the DBA/2 strain of mice. In contrast to BALB/c mice, DBA/2 mice developed significantly less parasitemia and never manifested symptoms of cerebral malaria (CM) on infection with this parasite. Moreover, the histological changes evident in the brain of susceptible BALB/c were absent in DBA/2 mice. Interestingly, the resistant DBA/2 mice when treated with recombinant interleukin (IL)-2, were found to develop CM symptoms and the infection became fatal by 6 to 8 days after infection. This condition was associated with an augmented interferon-gamma and nitric oxide production. Unexpectedly, IL-10 levels were also elevated in IL-2-treated DBA/2 mice during late stage of infection (at day 6 of infection) whereas the inverse relationship between IL-10 and interferon-gamma or nitric oxide was maintained in the early stage of infection (at day 3 after infection). The level of tumor necrosis factor-alpha production was moderately increased in the late phase of infection in these mice. Histology of brain from IL-2-treated mice demonstrated the presence of parasitized erythrocytes and infiltration of lymphocytes in cerebral vessels, and also displayed some signs of endothelial degeneration. Confocal microscopical studies demonstrated preferential accumulation of gammadelta T cells in the cerebral vessels of IL-2-treated and -infected mice but not in mice treated with IL-2 alone. The cells recruited in the brain were activated because they demonstrated expression of CD25 (IL-2R) and CD54 (intercellular adhesion molecule 1) molecules. Administration of anti-gammadelta mAb prevented development of CM in IL-2-treated mice until day 18 after infection whereas mice treated with control antibody showed CM symptoms by day 6 after infection. The information concerning creating pathological sequelae and death in an otherwise resistant mouse strain provides an interesting focus for the burden of pathological attributes on death in an infectious disease.

HTLV type 1 Tax transduction in microglial cells and astrocytes by lentiviral vectors.

Infection with human T cell leukemia virus type 1 (HTLV-1) can result in the development of HAM/TSP, a nonfatal, chronic inflammatory disease involving neuronal degeneration and demyelination of the central nervous system. Elevated levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 observed in the cerebrospinal fluid of HAM-TSP patients suggest that cytokine dysregulation within the CNS is involved in neuropathogenesis. HTLV-1 infection and enhanced expression of TNF-alpha by microglial cells, astrocytes, and macrophages has been hypothesized to lead to the destruction of myelin and oligodendrocytes in the CNS. Although the association of HTLV-2 infection and development of neurological disease is more tenuous, HTLV-2 has also been found to be associated with peripheral neuropathies. To investigate the roles of HTLV Tax(1) and Tax(2) in the induction of cytokine disregulation in these cell types, we are currently developing gene delivery vectors based on human immunodeficiency virus type-1 (HIV-1) capable of stably coexpressing the HTLV-1 or -2 tax and eGFP reporter genes in primary human cells. Transduction frequencies of up to 50%, as assessed by eGFP expression, can be achieved in human monocyte-derived macrophages and in explanted cultures of human microglia. Preliminary data suggest that Tax(1) expression is sufficient to up-regulate the proinflammatory cytokine profile in explanted human microglial cells. Future experiments will compare and evaluate the effect of tax(1) and tax(2) gene expression on the cellular proinflammatory cytokine expression profile, as well as demonstrate the effects of transducing human fetal astrocytes and PBMC-derived macrophages.

Queen's University alternative funding plan. Effect on patients, staff, and faculty in the Department of Family Medicine.

OBJECTIVE: To determine the effect of the Queen's University alternative funding plan (AFP) on the Department of Family Medicine in terms of patient, staff, and faculty satisfaction; patient encounter logistics; clinical volume; and academic activity. DESIGN: Before-after study. SETTING: Department of Family Medicine at Queen's University of Kingston, Ont. PARTICIPANTS: Patients, faculty, and staff of the Department of Family Medicine's Family Medicine Centre. INTERVENTIONS: The AFP of Queen's University. MAIN OUTCOME MEASURES: Patient satisfaction, staff and faculty job satisfaction, patient waiting time, time spent with patients, patient volume, number of publications, and amount of research funding obtained by faculty members. These outcomes were measured before the AFP began (time 0), 1 year post-AFP (time 1), and 2.5 years post-AFP (time 2). RESULTS: In some categories patients' satisfaction decreased at time 1, but in all cases it was either unchanged or improved at time 2. Staff and faculty job satisfaction did not change over time. Patients spent less time in the waiting room at time 2 than at time 0. Patient volume dropped about 10% between time 0 and time 2. Publication rate did not change, but external research funding increased significantly during the study period. CONCLUSION: The AFP has improved academic productivity, decreased patient volume by 10%, and improved patient flow during clinics. No negative effects on patient satisfaction or on job satisfaction of staff or faculty are apparent.

Cervicovaginal psammoma bodies in endosalpingiosis. A case report.

BACKGROUND: The presence of psammoma bodies on cervicovaginal smears is a rare finding. These structures have usually been associated with malignant tumors, particularly ovarian carcinoma. However, a review of the literature reveals that up to 50% of these patients will have psammoma bodies in association with benign conditions; thus, this finding does not always correlate with the presence of a malignant tumor. CASE: An asymptomatic, nulliparous woman had psammoma bodies on a cervicovaginal smear. Evaluation, including cervical conization, endometrial sampling, laparoscopy with pelvic washings and ovarian biopsy, revealed cervical intraepithelial neoplasia 1, endosalpingiosis and numerous psammoma bodies in all specimens except from the cervix. Follow-up examinations at three and six months were negative. CONCLUSION: The finding of psammoma bodies in a Pap smear is not always associated with the presence of a pelvic malignancy.

Differential infectivity and division of Toxoplasma gondii in human peripheral blood leukocytes.

When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils or lymphocytes were infected. Although tachyzoites were able to divide in each of these cell types, monocytes and dendritic cells were more permissive to rapid tachyzoite division than neutrophils or lymphocytes.

Attachment ligands of viable Toxoplasma gondii induce soluble immunosuppressive factors in human monocytes.

Previous studies have demonstrated that surface antigen proteins, in particular SAG-1, of Toxoplasma gondii are important to this parasite as attachment ligands for the host cell. An in vitro assay was developed to test whether these ligands and other secretory proteins are involved in the immune response of human cells to toxoplasma. Human monocytes were infected with tachyzoites in the presence of antiparasite antibodies, and their effect on mitogen-induced lymphoproliferation was examined. The presence of antibody to either parasite-excreted proteins (MIC-1 and MIC-2) or surface proteins (SAG-1 and SAG-2) during infection neutralized the marked decrease seen in mitogen-induced lymphoproliferation in the presence of infected monocytes. Conversely, antibodies to other secreted proteins (ROP-1) and cytoplasmic molecules had no effect on parasite-induced, monocyte-mediated downregulation. Fluorescence microscope analysis detected microneme and surface antigen proteins on the monocyte cell surface during infection. These results suggest that microneme and surface antigen proteins trigger monocytes to downregulate mitogen-induced lymphoproliferation.

Sensitized lymphocytes and CD40 ligation augment interleukin-12 production by human dendritic cells in response to Toxoplasma gondii.

Dendritic cells (DC) are potent antigen-presenting cells that can stimulate T cell responses by secreting cytokines. During Toxoplasma gondii infection, host immunity is mediated by interferon-gamma, which is induced by interleukin-12 (IL-12). Whether T. gondii infection would stimulate human DC to produce IL-12 was determined. DC were generated from human peripheral blood mononuclear cells cultured with recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human IL-4. DC secreted high levels of IL-12 in response to lipopolysaccharide but not to either live T. gondii tachyzoites or soluble antigen. However, IL-12 production in response to T. gondii was observed when DC were cocultured in contact with lymphocytes isolated from seropositive donors. Ligation of CD40:CD154 was partially essential for IL-12 secretion. These data demonstrate that signals obtained from contact with sensitized lymphocytes are critical for human DC to secrete IL-12 in response to T. gondii.

Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice.

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8(+) and CD4(+) T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8(+) than CD4(+) T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.