A gamma-herpesvirus immune evasion gene allows tumor cells in vivo to escape attack by cytotoxic T cells specific for a tumor epitope

DNA vaccines induce CTL attack on target tumor epitopes, but tumor elimination in vivo also requires sufficient effector CTL to enter the site, guided by inflammatory chemokines. Many herpesviruses contain genes for chemokine and chemokine receptor-like proteins to protect infected cells from immune attack. To assess if this evasion strategy could protect tumor cells, we used a model where CTL specific for a single epitope were the only effectors. Following DNA vaccination, CTL eliminated tumor cells from a subcutaneous site. However, introducing a viral gene encoding a secreted broad-spectrum chemokine-binding protein (M3) into tumor cells completely blocked CTL attack. Transduced tumor cells also protected neighboring non-transduced tumor. These findings confirm the importance of chemokines for migration of CTL to a non-lymphoid site. They may have relevance for escape of human virus-associated malignancies, and raise the question of whether analogous molecules might contribute to the failure of CTL to eliminate tumors.     

Acute pulmonary toxicity of inhaledβ-1,3-glucan and endotoxin

The number of inflammatory cells was studied in lung walls and airways after inhalation of endotoxin or -1,3-glucan. In the water unsoluble form, -1,3-glucan caused a delayed response in terms of a decrease in macrophages and lymphocytes in the lung wall, 1 to 7 days after exposure but no invasion of neutrophils into the airways. When solubilized in 0.02 N NaOH, the cell response was the same as that observed after exposure to endotoxin.     

Refined genetic mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.3 and evidence of linkage disequilibrium in European families

Chronic distal spinal muscular atrophy (Chronic DSMA, MIM (*)607088) is a rare autosomal recessive disorder characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of Chronic DSMA gene has been previously mapped to chromosome 11q13 in the 10.3 cM interval defined by loci D11S1889 and D11S1321. By linkage analysis in 12 European Chronic DSMA families, we showed that a disease gene maps to chromosome 11q13.3 (Z(max)=6.66 at theta=0.00 at the DSM4 locus) and suggested that this condition is genetically homogeneous. Recombination events allowed us to reduce the genetic interval to a 2.6 cM region, telomeric to the IGHMBP2 gene, excluding this gene as the disease causing gene in Chronic DSMA. Moreover, partial linkage disequilibrium was found between three rare alleles at loci D11S1369, DSM4 and D11S4184 and the mutant chromosome in European patients. Analysis of the markers at these loci strongly suggests that most Chronic DSMA chromosomes are derived from a single ancestor. Refinement of the Chronic DSMA locus will hopefully allow to test candidate genes and lead to identification of the disease-causing mutations.     

Trisomy 7 mosaicism, maternal uniparental heterodisomy 7 and Hirschsprung's disease in a child with Silver-Russell syndrome

Prenatal trisomy 7 is usually a cell culture artifact in amniocytes with normal diploid karyotype at birth and normal fetal outcome. In the same way, true prenatal trisomy 7 mosaicism usually results in a normal child except when trisomic cells persist after birth or when trisomy rescue leads to maternal uniparental disomy, which is responsible for 5.5-7% of patients with Silver-Russell syndrome (SRS). We report here on the unusual association of SRS and Hirschsprung's disease (HSCR) in a patient with maternal uniparental heterodisomy 7 and trisomy 7 mosaicism in intestine and skin fibroblasts. HSCR may be fortuitous given its frequency, multifactorial inheritance and genetic heterogeneity. However, the presence of the trisomy 7 mosaicism in intestine as well as in skin fibroblasts suggests that SRS and HSCR might possibly be related. Such an association might result from either an increased dosage of a nonimprinted gene due to trisomy 7 mosaicism in skin fibroblasts (leading to SRS) and in intestine (leading to HSCR), or from an overexpression, through genomic imprinting, of maternally expressed imprinted allele(s) in skin fibroblasts and intestine or from a combination of trisomy 7 mosaicism and genomic imprinting. This report suggests that the SRS phenotype observed in maternal uniparental disomy 7 (mUPD(7)) patients might also result from an undetected low level of trisomy 7 mosaicism. In order to validate this hypothesis, we propose to perform a conventional and molecular cytogenetic analysis in different tissues every time mUPD7 is displayed.     

Effect of cumulus cell mass and follicle quality on in-vitro maturation of cynomolgus monkey oocytes

Cynomolgus monkey oocytes were recovered from healthy or atreticfollicles > 1000 µm in diameter at day 8 of gonadotrophinstimulated cycles and cultured in vitro, cumulus enclosed orcumulus free, for 2 days. Germinal vesicle breakdown (GVBD)occurred in 26.7% of healthy cumulus enclosed oocytes (n= 60)compared to 52.6% of atretic cumulus enclosed oocytes (n = 38).The mechanical removal of the cumulus cell mass increased theGVBD rate of the healthy oocytes (56.5%, n = 23) and acceleratedthe passage of the atretic oocytes (n = 23) from metaphase I(4.3%) to metaphase II (47.8%). In the healthy primate follicle,the dktyate stage could be maintained by aninhibitory substancesecreted by the cumulus; foUicular atresia could either decreasethe synthesis of this substance and/or induce the metabolismof a stimulatory substance.     

Histological detection of minimal metastatic involvement in axillary sentinel nodes: a rational basis for a sensitive methodology usable in daily practice.

There is no consensus method for the histological analysis of axillary sentinel nodes (SN). This study aimed to (1) assess the rate of occult metastases in SN using large serial sectioning and immunohistochemistry (IHC), (2) evaluate whether occult metastases were predictive of metastases in the downstream axillary nodes, and (3) specify a methodology of analysis of SN that could be both sensitive and applicable in daily practice. One hundred three patients with breast carcinoma underwent SN biopsy and then axillary dissection. SN free of tumor at standard examination of one section were sectioned at six levels (150-microm intervals) and immunostained for cytokeratin. The number and localization of labeled metastatic cells (occult metastases) were recorded. In 29 of the 103 patients (28%), SN were found to be metastatic after standard examination. The SN of the remaining 74 patients were further analyzed using IHC. Occult metastases were detected in 35 of these patients (47.3%), leading to an overall SN involvement rate of 62% (29+35/103). In 33 of these 35 cases, the plurality and the dispersion of the immunostained cells implied that the screening of only 3 of the 6 levels would have led to the detection of diagnostic positive events. Only one of the 35 patients (2.8%) with occult metastases showed metastatic lymph node in the downstream axilla. In our series of axillary SN, the analysis of one standard histologic section and, when negative, of only three additional sections after IHC revealed >60% of metastasis or occult metastasis. Metastasis detected by standard analysis had a high predictive value of downstream node metastasis, whereas the predictive value of occult metastasis revealed by IHC was poor. The clinical significance of occult metastases in SN needs to be specified by long-term follow-up analysis.     

Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens

Agrobacterium tumefaciens is known to transfer parts of its tumor-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi. We have used this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete Agaricus bisporus. Analysis of transformants shows that the T-DNA integrates at random sites into the host genome and that the selection marker is stable during mitosis and meiosis. The Agrobacterium system allows the transformation of both homokaryons and heterokaryons of A. bisporus. Also, both karyotypes of an heterokaryon can be transformed simultaneously. Furthermore, this is the first report on the transformation of vegetative mycelium of a commercial strain of A. bisporus. Received: 6 June 2000 / Accepted: 10 October 2000     

Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle ( C _t) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Navβ1, Navβ3, Cav1.3, Cav3.1 and Cavα2δ2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cavβ2 and Cavγ4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvβ1, MinK and Cavγ7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.     

Beta2 integrins control the severity of murine Lyme carditis

Infection of C57BL/6 (B6) mice with the Lyme disease spirochete Borrelia burgdorferi can result in development of arthritis and carditis. B. burgdorferi induces expression of beta2/CD18 integrins, adhesion molecules that mediate the firm adhesion of leukocytes to the endothelium necessary for cellular extravasation during inflammation. The important role of beta2/CD18 integrins during extravasation suggests that these molecules play a role in the development of Lyme arthritis and carditis. The dependency of these inflammatory processes on the beta2 integrins was investigated in CD18 hypomorph mice, which express low levels of CD18. The results indicate that CD18 deficiency did not abrogate development of Lyme arthritis or carditis. Moreover, it resulted in increased severity of Lyme carditis. B. burgdorferi-infected CD18 hypomorph mice showed an increased macrophage infiltration of the heart, while they produced lower levels of borreliacidal anti-B. burgdorferi antibodies compared to wild-type mice. In accordance with these results, we demonstrate that dendritic cells from CD18 hypomorph mice secrete higher levels of monocyte/macrophage chemoattractant protein 1 (MCP-1/CCL2) in response to B. burgdorferi. Similarly, we show by real-time PCR that B. burgdorferi-infected hearts from CD18 hypomorph mice express increased levels of MCP-1 RNA compared to wild-type mice. Overall, our results indicate that beta2 integrin deficiency does not abrogate B. burgdorferi-induced inflammation; rather, it results in increased recruitment of macrophages into the B. burgdorferi-infected heart, likely due to the increased expression of MCP-1 in this tissue. Thus, beta2 integrins may play a regulatory role in B. burgdorferi-induced inflammation beyond mediating adhesion of leukocytes to the endothelium.