Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho.

We demonstrate that within the intact and spontaneously active retina, dendritic processes of ganglion cells exhibit rapid and extensive movements during the period of synaptogenesis. Marked restructuring occurs in seconds, but structural changes are relatively balanced across the dendritic arbor, maintaining overall arbor size and complexity over hours. Dendritic motility is regulated by spontaneous glutamatergic transmission. Both the rate and extent of the movements are decreased by antagonists to NMDA and non-NMDA glutamate receptors but are unaffected by tetrodotoxin, a sodium channel blocker. The dendritic movements are actin dependent and are controlled by the Rho family of small GTPases. Transfection of dominant-negative and constitutively active mutants into ganglion cells showed that Rac and Rho exert reciprocal effects on motility. We suggest that the Rho family of small GTPases could integrate activity-dependent and -independent signals from afferents, thereby adjusting target motility and maximizing the chance for initial contact and subsequent synaptogenesis.

Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic beta-cells and other peripheral tissues of mouse and rat.

Glutamic acid decarboxylase (GAD) catalyzes synthesis of the inhibitory neurotransmitter gamma-amino butyric acid. Two homologous forms of GAD encoded by separate genes have been cloned from rat brain, with predicted protein sizes of 67 and 65 kilodaltons. GAD is present outside the brain, and pancreatic islet GAD is believed to be a target of autoimmunity in insulin-dependent diabetes mellitus. However, peripheral expression of the two GAD genes is incompletely characterized. We, therefore, investigated GAD expression in peripheral tissues, including pancreas, of mouse and rat. cDNAs encoding GAD 67 and GAD 65 were cloned from mouse brain and shown to be 95% homologous with the rat sequences. RNase protection assay using specific cRNA probes demonstrated expression of both GAD forms in freshly harvested pancreas and testis. Levels of both GAD mRNAs were greater in rat than mouse pancreas. GAD 67 mRNA was more abundant than GAD 65, and both were localized to islet beta-cells by in situ hybridization. In testis, both GAD mRNAs were localized to spermatocytes. Additionally, GAD 67, but not GAD 65, mRNA was detected in mouse and rat spleen and mouse liver. Thus, both GAD genes are expressed in peripheral tissues, with GAD 67 mRNA being more abundant under physiological conditions. The expression of both GAD 67 and GAD 65 genes specifically in islet beta-cells indicates that both GAD forms are candidate autoantigens in rodent models of insulin-dependent diabetes mellitus.

Cytokines and autoimmune beta cell destruction in NOD mouse fetal pancreas isografts in cyclophosphamide-induced diabetes.

The role of cytokines in a model of cyclophosphamide (CP)-accelerated beta cell destruction in fetal pancreas isografts transplanted into NOD mice was studied. One group of prediabetic NOD mice was injected with CP at a dose of 300 mg/kg i.p. and 7 days later isografts of organ cultured fetal pancreas (FP) were transplanted under the kidney capsule of these and untreated control mice. The mice were killed at several time points post-transplantation and the histological appearance of the host pancreas used to evaluate the disease progress in the grafts since previous studies had shown good correlation between isograft and native pancreas pathology. Intragraft cytokine gene expression was monitored by reverse-transcriptase polymerase chain reaction (RT-PCR) at the same time points and the expression levels between the experimental groups compared to normal kidney tissue. In comparison to isografts from non-CP injected mice, isografts from CP-treated mice showed increased expression of IFN-gamma, TNF-alpha, TNF-beta, IL-5, and eotaxin but no increase in IL-10 expression. The enhanced transcription of these cytokines correlated with massive infiltration of immune cells and ongoing beta cell destruction in the host pancreas of the CP-treated recipients.

Both TH1 and TH2 cytokine mRNAs are expressed in the NOD mouse pancreas in vivo.

In NOD mice, autoimmune recognition and destruction of pancreatic islet beta-cells appear to be independently regulated: all mice develop cellular infiltration of the islets (insulitis), but not all develop diabetes. The destructive potential of the insulitis lesion may depend on the balance between the two CD4+ T-cell subsets. TH1 and TH2, that mediate cellular-cytotoxic and humoral responses, respectively. With a semi-quantitative reverse transcriptase-PCR assay, we examined whether the disease process was reflected in the profiles of TH1 (IL-2, IFN-gamma and IL-12) and TH2 (IL-4, IL-6 and IL-10) cytokine mRNAs expressed in pancreata of NOD mice. Pancreata rather than isolated islets were examined to minimize manipulation ex vivo to preserve the expression of cytokine transcripts in vivo. At age 6 weeks, when 70% of mice had insulitis, all cytokine transcripts were detected in most pancreata, and their expression levels corresponded to the degree of insulitis. Similarly, during induction of diabetes with cyclophosphamide all transcripts were detected and levels corresponded with the degree of insulitis. In one-year-old mice without diabetes, all transcripts were detected but levels did not correspond to the degree of insulitis. Thus, in pancreata of NOD mice with different degrees of insulitis, we were unable to demonstrate, at the RNA level, polarisation of cytokine expression into either a TH1 or TH2 profile. This finding does not, however, exclude expression of distinct cytokine transcripts by immuno-inflammatory cells within the islet lesion, which might be revealed by in situ hybridization.

Detection and typing of human papillomavirus using the Vira Type "in situ" kit: comparison with a conventional dot blot technique.

A new commercial kit (Vira Type "in situ", Life Technologies, Inc., Molecular Diagnostics Division, Guithersburg, Maryland, USA) for the detection of human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33 and 35 in routinely processed human anogenital tissue was compared with a conventional dot blot assay for HPV 6, 11, 16 and 18. Both systems use double-stranded genomic DNA probes for the detection of type specific HPV DNA. The probes used on the dot blots were labelled with 32P and visualised autoradiographically. The Vira Type probes were labelled with biotin and visualised using a streptavidin-alkaline phosphatase conjugate with NBT-BCIP substrate. Biopsy specimens from the cervix, vagina, and vulva of 46 women were processed by both methods and compared. The histological diagnoses ranged from benign changes, to dysplasia, and invasive carcinoma. Overall, 50% of biopsy specimens were positive for HPV DNA by dot blot hybridisation; only 39% were positive by Vira Type in situ hybridisation. Three of the specimens positive by the Vira Type "in situ" kit showed no cross hybridisation and were the same HPV type as the dot blot. A further 13 showed hybridisation, but the showed cross hybridisation, but the to the dot blot results. One biopsy specimen was positive for different HPV types by the two tests and one was positive by Vira Type and negative by dot blot. Six biopsy specimens were negative by Vira Type but positive by dot blot. It is concluded that the Vira Type "in situ" kit has a similar specificity but lower sensitivity than the dot blot hybridisation method for the detection of HPV DNA.

Lymphoid aggregates in bone marrow: study of eventual outcome.

The practical importance of finding a morphologically benign lymphoid aggregate in the bone marrow of patients without known lymphoproliferative disease was assessed in 786 consecutive patients who had had 951 iliac crest bone marrow biopsies performed. Of these, 430 patients known to have lymphoproliferative disease at the time of biopsy were excluded. Of 356 patients, 86 (aggregate group) had at least one lymphoid aggregate in their biopsy specimen biopsy specimen (82 morphologically benign, three suspicious, and one malignant). Another 86 patients without aggregates (control group) were matched by age and sex. Both groups were followed up until death, or for a mean of 21.9 and 22.9 months, respectively, to assess their outcome. Eighteen (22%) of the 82 patients with morphologically benign lymphoid aggregates were later proved to have lymphoproliferative disease compared with none of the 86 control patients. Another 12 patients in the aggregate group and seven in the control group were suspected of having a lymphoproliferative disease on clinical grounds, so that altogether 30 (37%) and seven (8%), respectively, developed confirmed or suspected lymphoproliferative disease. In both cases the differences were highly significant (p less than 0.001). It is suggested that lymphoid aggregates in clinical biopsy material may not be a physiological finding and should alert pathologists or haematologists to the possibility of lymphoproliferative disease.

Detection of human papillomavirus DNA and mRNA using synthetic, type-specific oligonucleotide probes.

Type-specific 30'mer-36'mer oligonucleotide probes complementary to mRNA transcribed from the E6 and E7 open reading frames of HPV 6b/11, 16, 18 and 33 were designed using the published nucleotide sequences. As oligonucleotides are easily and relatively cheaply synthesized in large amounts and are free of vector DNA, they were assessed for potential use in routine clinical detection and typing of HPV. Multiple Southern and dot blots of cloned HPV 6b, 11, 16, 18, 31 and 33 DNA, and of DNA extracted from cell lines carrying integrated HPV 16 and 18 genomes were prepared. In addition, Northern and dot blots of RNA extracted from the HPV-containing cell lines HeLa, CaSki and SiHa, were also prepared. All filters were first probed with the oligonucleotide and then with the corresponding full-genomic HPV DNA probe and their relative sensitivities and specificities compared: both probe types were labelled with 32P. The oligonucleotide probes were all as specific as the full-genomic probes for Southern, DNA and RNA dot blot hybridisations. The HPV 16 and 18 oligonucleotide probes detected HPV transcripts of the appropriate sizes in the cell line RNA. For DNA detection, oligonucleotide probes were up to 10 times less sensitive than the full-genomic probes, but for RNA detection, they were more sensitive. The sensitivity for both HPV DNA and RNA detection could be improved by using two type-specific oligonucleotide probes in combination, without reducing the specificity. The ease of preparation and handling of oligonucleotide probes, together with their lack of contaminating vector DNA, suggests that they may have some advantages over full-genomic probes for the clinical detection and typing of HPV.

Cervical cancer--what role for human papillomavirus?

OBJECTIVE: To review the role of human papillomavirus (HPV) as a causative agent of cancer of the cervix. DATA SOURCES, data synthesis, study selection: Medical journals, recently published text books related to cancer of the cervix and HPV and Papillomavirus Reports were examined to review the pathology of cervical cancer and its precursor lesions, its epidemiology in Australia and overseas, methods of detection of HPV (in particular molecular biology techniques used to diagnose HPV) and evidence linking HPV with genital neoplasia. CONCLUSION: While there is compelling evidence strongly linking certain HPV types with genital cancer, a causative role is yet to be proven and the aetiology is most likely multifactorial. Detection and typing of high risk genotypes of HPV in the genital tract as a diagnostic exercise to identify those women most at risk of developing genital neoplasia is not currently recommended.

Multiple cadherin mRNA expression and developmental regulation of a novel cadherin in the developing mouse eye.

Cadherins form a large family of transmembrane glycoproteins whose members include the classical cadherins, the desmosomal cadherins, and the protocadherins. The classical cadherins mediate homophilic cell-cell adhesion and are key regulators of many morphogenetic processes. More than a dozen classical cadherins are expressed in both the developing and the mature central nervous system. Although individual cadherins have been identified in the retina of various species, we wished to determine the range of cadherins expressed at distinct developmental stages in the mouse retina. Using a PCR-based cloning strategy, we detected 10 different classical cadherin mRNAs of both type I and type II subtypes. The most abundant cDNA was that encoding the type II cadherin, Cadherin-11. The other type II cadherins detected were VE- and T2-cadherin and Cadherin-6 and -12. Four type I cadherins, N-, R-, P-, and E-cadherin, were also present. One cadherin cDNA encoded a novel cadherin, called EY-cadherin for cloned from eye. EY-cadherin is most closely related to human Cadherin-14 (93% identical). EY-cadherin mRNA was detected in the adult mouse eye, brain, and testis with a 20-fold increase in expression levels in the embryonic head from E11 to E19 and a 50-fold increase in expression levels in the postnatal eye from PN1 to PN16. Multiple cadherin gene expression is consistent with the hypothesis that different cadherins regulate morphogenetic processes, such as neuronal migration and lamination, and determine the specific interneuronal connections found in the mature retina.

Plasminogen activator in granulocyte-macrophage-CSF transgenic mice.

The pattern of expression of urokinase type plasminogen activator (PA) in granulocyte-macrophage-CSF transgenic mice and their normal littermates was studied using RNAse protection assays and a plasminogen-dependent fibrinolytic assay for PA. Urokinase type PA mRNA was expressed at a high level in transgenic peritoneal cells and at a lower level in transgenic eye tissue and spleen, but not in equivalent tissue from the normal mice. Enzymically active PA was detectable in protein extracts from peritoneal cells taken from transgenic mice of less than 8 wk of age (young mice) but not from normals. Paradoxically, extracts from transgenic mice of more than 12 wk of age (old mice) showed little detectable PA activity despite continuing transcription in some mice of this age. The production of PA by peritoneal cells may be responsible for the spontaneous i.p. bleeding which is a feature of the transgenic mice and production in other tissues may help explain the local pathologic changes.

Transcription and translation of two glutamate decarboxylase genes in the ileum of rat, mouse and guinea pig.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, synthesised from glutamate by glutamate decarboxylase (GAD), in the central nervous system. Two forms of GAD, designated GAD 65 and GAD 67, are encoded by distinct genes and have been demonstrated in the mammalian brain. GABA has been postulated to be synthesised in neurons of the enteric nervous system (ENS), but evidence for its role as an enteric neurotransmitter is equivocal. We therefore aimed to determine whether GAD 65 and GAD 67 messenger RNAs (mRNAs) and proteins were expressed in the ileum of mice, rats and guinea pigs. Using an RNase protection assay, both GAD 65 and GAD 67 mRNAs were detected in the rodent small intestine. Antisera specific for GAD 65 or GAD 67, used in immunoblot analyses, revealed GAD 65-like and GAD 67-like immunoreactivity in rat and guinea pig ileum. Anti-GAD 65 antisera detected a major band of 65 kDa. Anti-GAD 67 antisera detected a major band of 55 kDa, which probably represented a breakdown product, and a minor band of 67 kDa. Analysis of immunoblot extracts of rat and guinea pig ileum revealed more GAD 67-like than GAD 65-like immunoreactivity. GAD enzymatic activity was high in the rat and guinea-pig brain, and low in the whole and dissected ileum. These results demonstrate that both GAD 65 and GAD 67 genes are transcribed and translated in the ileum of three rodent species and lend indirect support to the postulate that GABA is synthesised by neurons of the ENS and intestinal endocrine cells.

Cloning and expression of mouse Cadherin-7, a type-II cadherin isolated from the developing eye.

We report the molecular cloning of Cadherin-7 from the embryonic mouse eye. The deduced amino acid sequence shows it to be a type-II cadherin similar to Xenopus F-cadherin and chick Cadherin-7. The mouse Cadherin-7 gene maps to chromosome 1, outside the conserved linkage group of cadherin genes on chromosome 8. Cadherin-7 is expressed throughout the entire period of neural development and mRNA levels are developmentally regulated in both the embryonic and the postnatal central nervous system (CNS). In adult mice, Cadherin-7 expression is restricted to the CNS, with highest levels in the retina. In the developing eye, Cadherin-7 mRNA is found only in the neural retina. It is expressed by all retinal neuroblasts from E11 onward, but becomes progressively restricted to neurons in the inner neuroblast and developing ganglion cell layers (GCL). In the adult retina it is confined to subpopulations of cells in the GCL and to amacrine cells in the inner part of the inner nuclear layer. This expression pattern suggests a role for Cadherin-7 in mouse retinal development, particularly in the formation and maintenance of the GCL.

Cytokine regulation of glutamate decarboxylase biosynthesis in isolated rat islets of Langerhans.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in beta-cell destruction and immune regulation. A major target of beta-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of beta-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1 beta (1 ng/ml, 24 h), tumour necrosis factor alpha (TNF-alpha; 200 units/ml, 24 h) or interferon gamma (IFN-gamma; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-alpha (1000 units/ml), TNF-beta (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor beta 2 (TGF-beta 2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1 beta, TNF-alpha or IFN-gamma was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1 beta and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1 beta, TNF-alpha or IFN-gamma by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of beta-cell destruction in IDDM.