Tolerance in TCR/Cognate Antigen Double-Transgenic Mice Mediated by Incomplete Thymic Deletion and Peripheral Receptor Downregulation

Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossed with transgenic mice expressing the cognate antigenic protein under the control of the H- 2K^b promoter. Double-transgenic mice show negative selection of thymocytes at the CD4⁺8⁺TCR¹⁰ to CD4⁺8⁺TCR^hi transition stage. A few CD8 T cells, however, escape clonal deletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels of the transgenic receptor and upregulated levels of the CD44 memory marker. Such cells do not proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they can develop low but detectable levels of antigen-specific cytotoxic function after stimulation in vitro in the presence of IL-2.     

Zygomycosis. Report of four cases with formation of chlamydoconidia in tissue

The authors describe spheric to ovoid chlamydoconidia and mucoraceous hyphae in tissues from four patients, two with cutaneous and two with pulmonary zygomycosis. The diagnosis in each case was confirmed by immunofluorescence staining and the presence of characteristic hyphae in tissue. It is important that these conidia be recognized, because they can easily be mistaken for other fungi, nematode ova, or other microorganisms in tissue sections, thereby resulting in the potential for misdiagnosis.     

Pathogenesis of rabies in dogs inoculated with an Ethiopian rabies virus strain. Immunofluorescence,histologic and ultrastructural studies of the central nervous system

Summary Dogs were inoculated with either an Ethiopian or Mexican rabies virus strain. The distribution of viral antigen and lesions were studied by immunofluorescence, histologic and electron microscopic techniques. In all dogs inoculated with the Ethiopian rabies virus strain, tremendous whorls of filamentous fluorescing aggregates were observed throughout the brain; these were not observed in dogs inoculated with the Mexican virus.Lesions consisted of neuronal degeneration and neuronophagia, associated with large inclusion bodies and widespread inflammation in dogs inoculated with the Ethiopian isolate. All observed portions of the brain and spinal cord were affected. In general, lesions were much less severe with the Mexican isolate. Occasional astrocytes were observed to have inclusions in dogs inoculated both with the Ethiopian and Mexican strains.Most neurons examined electronmicroscopically showed signs of infection, varying from a small granular or finely fibrillar viral matrix to numerous matrices accompanied by prolific numbers of virus particles occupying much of the perikaryon. These were found in all dogs inoculated with the Ethiopian strain but were rare with the Mexican isolate. Viral budding occurred from membranes of the rough endoplasmic reticulum, outer lamella of the nuclear envelope, and rarely from the plasma membrane.With 15 FiguresThis work was done while the senior author (Visiting Scientist) was being supported by the Swedish Aid for Research Cooperation with Developing Countries Project 77/89 A 9–49 U-forsk, through the Department of Bacteriology and Epizootology, Biomedicum, P.O. Box 583, S-75123 Uppsala, Sweden.     

Hybridization studies of cultured human pituitary prl and gh producing adenoma cells: Effects of thyrotropin-releasing hormone,somatostatin, and phorbol ester

The effects of the hypothalamic hormones, thyrotropin-releasing hormone (TRH), and somatostatin (SRIH), and of phorbol 12-myristate 13-acetate (PMA) on PRL and GH secretion and messenger RNA (mRNA) levels were analyzed in 10 GH and/or PRL producing adenomas after culturing the tumor cells in the presence of these secretagogues for 7 days. The expression of chromogranin A and B mRNAs was also examined. All four of the clinically diagnosed GH adenomas expressed or secreted both GH and PRL while four of six clinically diagnosed prolactinomas produced or secreted both PRL and GH. Prolactinomas had less than 10% of tumor cells expressing chromogranin A mRNA while more than 40% of the adenoma cells expressed chromogranin B mRNA. TRH stimulated PRL secretion and increased PRL mRNA levels while SRIH decreased GH secretion and mRNA expression in some cases. Unexpectedly, PMA stimulated PRL mRNA levels four- to sevenfold above control levels in two adenomas and generally stimulated chromogranin A and B mRNA expression but not GH mRNA, as determined by Northern hybridization and in situ hybridization analyses. These results indicate that cultured prolactinoma cells express significantly more chromogranin B mRNA than chromogranin A mRNA, and that PMA increases PRL mRNA expression in some prolactinomas, although the effect of PMA on various adenomas reflects the heterogeneity of these tumors with respect to protein kinase C stimulation.     

Comparison of serum mannan, arabinitol, and mannose in experimental disseminated candidiasis

Concentrations of arabinitol, mannose, and mannan in serum have independently been reported to be elevated in patients with invasive candidiasis. These three marker substances were compared in a rabbit model. Twelve rabbits, immunosuppressed with cortisone, were infected intravenously with Candida albicans 3181A. Six uninfected control animals also received cortisone, and four rabbits were neither infected nor immunosuppressed. Blood samples, drawn from 2 days before to 14 days after infection, were assayed for serum mannan by sandwich enzyme immunoassay, antibodies to mannan by indirect enzyme immunoassay, arabinitol and mannose by gas-liquid chromatography, and serum creatinine. Serum mannan, negative before infection, peaked in all infected animals 4 days after infection (mean, 18 ng/ml) and decreased thereafter. Significant increases (2 standard deviations greater than mean in normals) in arabinitol, the arabinitol/creatinine ratio, and mannose were found in 12, 8, and 12 of the infected rabbits, respectively, but also in all 6 uninfected animals receiving cortisone. Only serum mannan was specific in this immunosuppressed rabbit model.     

Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas.

Protein kinase C (PKC) is involved in the differentiation and growth regulation of a variety of tissues including anterior pituitary gland cells. To determine the distribution of PKC in different types of adenomas, PKC activity was analyzed in human pituitary tumors and the effects of hypothalamic hormone stimulation on PKC activity were examined in cultured adenoma cells. Gonadotroph (LH/FSH) and null cell adenomas had significantly higher levels of particulate, soluble, and total PKC activity compared with growth hormone (GH) adenomas (P < 0.05). Chronic stimulation of null cell adenomas with gonadotropin hormone-releasing hormone or of one GH adenoma with GH-releasing hormone for 7 days did not significantly alter total PKC activity in pituitary cells cultured in serum-free medium. Localization of the calcium-dependent PKC isozymes (alpha, beta and gamma) by immunohistochemistry and in situ hybridization revealed predominantly PKC alpha in all adenomas and variable expression of PKC beta and gamma in some tumors. When the calcium-independent PKC isozymes (delta, epsilon, and zeta) were localized by in situ hybridization, normal and neoplastic pituitaries expressed abundant mRNA for PKC epsilon, whereas some tumors and one normal pituitary had a few cells positive for PKC zeta mRNA as evaluated by grain density and the number of cells labeled. These results indicate that there is a variable distribution of PKC mRNA isozymes in human pituitary adenomas and that normal pituitaries and pituitary adenoma cells express the mRNA for both the calcium-dependent and some of the calcium-independent PKC isozymes. Chronic treatment with the hypothalamic gonadotropin hormone-releasing hormone and GH-releasing hormone, which increased LH/FSH and GH secretion, respectively, did not increase PKC activity in cultured adenoma cells. The presence of calcium-dependent and calcium-independent PKC isozymes in normal and neoplastic pituitary cells indicates that PKC probably plays a major role in signal transduction in the human pituitary adenomas examined in this study.     

The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region

We have recently reported isolation of the gene responsible for X- linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22. 3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus , while it seems to be X- specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M. musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.      

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

Mycoplasma pneumoniae attachment: competitive inhibition by mycoplasmal binding component and by sialic acid-containing glycoconjugates.

Attachment of Mycoplasma pneumoniae to human WiDr cell culture monolayers was examined by using radiolabeled M. pneumoniae. The amount of attachment was proportional to the density of the WiDr cells and to the concentration of M. pneumoniae in the assay. Saturation of the monolayers was achieved with 40 micrograms of virulent strain M129 per assay, whereas binding of avirulent strain B176 was 70% less than that of strain M129. A competitive attachment inhibition assay was used to measure specific binding component activity. Attachment was inhibited when WiDr cells were pretreated with unlabeled virulent strain M129, whereas avirulent noncytadsorbing strain B176 did not inhibit attachment as well as the virulent strain. A protein-rich extract prepared from virulent, cytadsorbing strains of M. pneumoniae also inhibited attachment. The amount of inhibition was dependent on the amount of extract used, and units for binding component activity in the extract were calculated from the competitive attachment inhibition assays. The competitive attachment inhibition assay was also used to investigate the nature of the receptor site on the WiDr cells. Attachment was inhibited when the radiolabeled M. pneumoniae suspensions were pretreated with human sialoglycoproteins, such as orosomucoid and ceruloplasmin, and bovine gangliosides. These findings support the present concept that the mammalian receptor site for M. pneumoniae is a sialic acid-containing glycoprotein.