Do double-stranded RNA receptors play a role in preeclampsia?

Dysregulation of the maternal immune system during pregnancy has been implicated in the development of preeclampsia (PE), however the pathogenetic signals and mechanisms have not been completely elucidated. Here we provide a hypothesis and evidence that dsRNA is a danger signal leading to maternal immune system activation and an "antiviral" immune response that manifests as PE. dsRNA released from necrotic cells and/or from viruses causes excessive activation of dsRNA receptors and PE-like symptoms in animals. Additionally, high expression levels of dsRNA receptors have been identified in human and animal placental tissue as well as trophoblast cells, and these receptors appear to be excessively activated in PE. These key components of the innate immune system that respond to invading pathogens and dead or necrotic tissue likely play a major role in the development of PE.

Marinobufagenin inhibits proliferation and migration of cytotrophoblast and CHO cells.

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na(+)/K(+)-ATPase. Increased plasma levels of MBG have been reported in patients with volume expansion-mediated hypertension and preeclampsia. We have recently demonstrated that MBG impairs both the proliferation and growth factor-induced migration of human first trimester cytotrophoblast (CTB) cells, crucial for proper placental development. However, the intracellular signaling mechanisms regulating the MBG-induced impairment of CTB differentiation, migration and invasion are unknown. The human extravillous CTB cell line SGHPL-4 was utilized for this study. The phosphorylation of MAP kinase protein ERK1/2 was evaluated by Cellular Activation of Signaling ELISA (CASE) in control CTB cells and those treated with MBG. MBG at concentrations of 10 and 100nM inhibited CTB cell proliferation, migration and invasion (60%, 50% and 50%, respectively). MBG also caused a significant decrease in the phosphorylation of ERK1/2. In addition, MBG decreased proliferation, migration, and ERK1/2 activity in another motile cell line, CHO cells. Another sodium pump inhibitor, ouabain, similarly decreased proliferation and ERK1/2 activity in CTB and CHO cells. These data suggest that the changes observed in cell function may be mediated by inhibition of Na(+)/K(+)-ATPase. We demonstrate that the MBG-induced impairment of CTB cell proliferation, migration and invasion is associated with decreased ERK1/2 activity which may be mediated by inhibition of Na(+)/K(+)-ATPase.

Serial exercise performance in children with surgically corrected congenital aortic stenosis.

We examined serial exercise test performance in children with congenital aortic stenosis (AS) treated surgically compared to that of nonoperated children with mild to moderate AS. Maximal treadmill exercise data were assessed in 21 children 5.5 +/- 3.8 years after aortic valve (AO) surgery. Patients had undergone the Ross procedure (n = 6) or previous aortic valvotomy, balloon valvuloplasty, or neonatal aortic valvotomy (n = 15). Follow-up treadmill tests were conducted 3.7 +/- 2.8 years later. Data were compared to those of 19 nonoperated AS patients (mean gradient by echocardiogram <50 mmHg). These patients were exercised 3.6 +/- 3.2 years apart. Endurance time, heart rate, systemic blood pressure, and electrocardiogram were compared as repeated measures between tests and to age- and sex-matched normative data. Postsurgical children with AS had normal endurance times despite low peak heart rates on the initial test, and they maintained endurance over time. Nonoperated children with mean AO gradients <50 mmHg also had normal endurance times on the initial test but increased endurance over 3.6 years. Children with operated and nonoperated AS were able to reach or exceed normal endurance times, which may make it difficult to achieve compliance to imposed activity restrictions in this population.

Detection of varicella zoster virus DNA in some patients with giant cell arteritis.

PURPOSE: The purpose of this study was to determine whether an association exists between giant cell arteritis (GCA) and the presence of varicella-zoster virus (VZV), by using histologic, molecular, immunohistochemical, and ultrastructural analyses of temporal artery biopsy specimens. METHODS: In a randomized masked study, 64 temporal artery biopsy specimens were analyzed by PCR for VZV DNA. The samples included 35 specimens histologically positive and 29 specimens histologically negative for GCA. Immunohistochemical staining for VZV viral antigen IE-63 was performed on seven of the specimens positive for GCA and five negative specimens. Transmission electron microscopy (TEM) was performed on five of the specimens positive for GCA. RESULTS: PCR was positive for VZV DNA in 9 (26%) temporal arteries tested that showed histologic evidence of GCA. The remaining 26 histologically positive temporal arteries and all 29 histologically negative arteries tested gave negative PCR results for VZV DNA. Statistical analysis (z-test) comparing the association of VZV DNA between the specimens that were positive and negative for GCA showed a significant difference (P = 0.010). Immunohistochemical studies were positive in several biopsy specimens within adventitial histiocytes-macrophages, but these results did not correlate with either the presence or absence of VZV DNA or with the histologic evidence of GCA. No viral particles were observed by TEM. CONCLUSIONS: This study showed a significant association of VZV DNA to temporal artery biopsy samples positive for GCA compared with the negative specimens. The results support the hypothesis that VZV may play a role in the pathogenesis of some cases of GCA. However, PCR, immunohistochemical, and electron microscopic findings suggest the virus is present at extremely low quantities, is abortively replicating, or is latent.

Molecular screening of donor corneas for fungi before excision.

PURPOSE: To develop panfungal and Candida albicans species-specific polymerase chain reaction (PCR) assays to screen donor eyes for fungal contamination before corneal excision. METHODS: PCR primers were designed for either the broad-spectrum detection of fungal DNA or the specific detection of C. albicans DNA. Their sequences were based on rDNA regions highly conserved among and specific to fungi and C. albicans, respectively. PCR conditions with the two primer sets were optimized and tested for sensitivity using purified C. albicans genomic DNA and a plasmid containing the relevant region of C. albicans DNA. The specificity of the primer sets was established using higher eukaryotic, fungal, prokaryotic, and viral DNAs as PCR templates. Donor eye swab specimens were collected before corneal excision. DNA was extracted from the specimens and tested by both PCR assays. RESULTS: The lower limit of detection for both primer sets was consistently 10(3) genome equivalents, when using genomic DNA as a template and 10(2) copies of plasmid. The fungal PCR assay amplified DNA from all fungal species tested but did not amplify any of the selected mammalian, bacterial, or viral DNA. The C. albicans PCR detected the C. albicans DNA but was negative for all other DNA substrates, including the other fungal templates. Thirty-five percent of the donor eye samples tested were positive for fungus, and 19% were positive for C. albicans DNA. CONCLUSIONS: The PCR assays allowed the rapid screening of DNA extracted from specimens collected from corneal donors for potential fungal contamination. The assay was highly sensitive and specific for screening corneal surfaces. The results suggest that approximately one-third of donor eyes tested harbor fungi on the ocular surface.

Impaired exercise parameters in pediatric heart transplant recipients: comparison of biatrial and bicaval techniques.

The exercise performance of pediatric heart transplant recipients and the effects of bicaval anastomosis were studied in 19 children using a Bruce protocol. Although all children had decreased exercise capacity and heart rates when compared with normals, the bicaval anastomosis patients had similar endurance and peak heart rates as the standard biatrial group.

Immune transfer protects severely immunosuppressed mice from murine cytomegalovirus retinitis and reduces the viral load in ocular tissue.

Cytomegalovirus (CMV) retinitis is a sight-threatening disease that affects immunosuppressed people and is prevalent in people with AIDS. The purpose of this study was to evaluate murine CMV (MCMV) retinitis in a replenishing model with adoptive immune transfer into severely immunosuppressed animals. Adult BALB/c mice, immunosuppressed with cyclophosphamide, were infected subretinally with 5x102 plaque-forming units of MCMV. Four to six hours later, 3-4x107 donor cells were transferred by intravenous infusion. Eight days after the transfer, the eyes that had received donor cells were studied histologically, titered for infectious virus, and analyzed with polymerase chain reaction (PCR). Adoptive transfer of total MCMV-immune lymph node (LN) cells or enriched LN lymphocytes specifically and significantly protected immunosuppressed mice from retinitis even after the initiation of infection. The transfer resulted in a reduced viral load, as measured by both plaque assay and PCR. This replenishment model will be useful for determining the specific immune parameters of protection from CMV retinitis.

Murine cytomegalovirus DNA in peripheral blood of latently infected mice is detectable only in monocytes and polymorphonuclear leukocytes.

Cytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+ and Mac-1- cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed.

Neuroinvasive properties of herpes simplex virus type 1 glycoprotein variants are controlled by the immune response.

Neuroinvasiveness is a property central to the pathogenesis of herpes simplex virus (HSV) and most isolates demonstrate this property. Exceptions are HSV strains KOS and ANG, for which we have previously shown that the non-neuroinvasive phenotype is referable to single amino acid changes in glycoprotein D for ANG or glycoprotein B for KOS. Because glycoproteins B and D are immunologically significant, the possibility that the phenotype has an immunologic basis was examined. Nonimmunosuppressed mice could not be killed with any dose of these non-neuroinvasive viruses after footpad inoculation, but in cyclophosphamide-suppressed animals, the ratios of plaque-forming units to LD50 decreased by at least four orders of magnitude to levels comparable with that of ANG-path, a neuroinvasive derivative of ANG. KOS and ANG induced a more rapid circulating neutralizing Ab response than did ANG-path, and mice were protected when these agents were co-infected with the neuroinvasive strain. The noninvasive viruses engendered an enhanced mononuclear cell infiltrate in infected spinal ganglia which consisted of increased numbers of CD4+ and CD8+ T cells and an increased production and secretion of IgG. HSV-specific Ab-secreting cells were also observed. In addition, passive transfer of anti-HSV mouse serum protected immunosuppressed mice from lethal HSV challenge. Selective in vivo depletion of T lymphocytes increased the detectable levels of both KOS and ANG viruses in the spinal ganglia at 6 days postinfection, but it did not alter the ratios of plaque-forming units to LD50 or affect the HSV-induced increase in ganglionic IgG. Taken together, these data indicate that in these systems there is an immunologic basis for the control of HSV-1 neuroinvasiveness and that humoral, rather than cell-mediated immunity, is playing the major role.

A procedure for the isolation of specific point mutants of influenza virus.

Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 microgram of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.

Strategies for financing national health insurance: who wins and who loses.

Two sources of funds are available to underwrite the costs of any national health-insurance plan: prepayments (premiums, payroll taxes and income taxes) and out-of-pocket payments (coinsurance and deductibles). The extent to which taxes rather than premiums are used to finance an insurance program will be the major determinant of how large a share of the costs of health care will be borne by higher-income groups. The extent to which coinsurance and deductible provisions are reduced or waived for low-income persons will have a less important, but still substantial, role in determining how the costs of a program are distributed. These financing principles, once understood, provide a basis for the design of health-insurance legislation that will achieve any pattern of income redistribution that may be desired.