P. S. Thrane

A synthetic, chemically modified ribozyme eliminates amelogenin, the major translation product in developing mouse enamel in vivo.

Ribozymes are small RNA structures capable of cleaving RNA target molecules in a catalytic fashion. Designed ribozymes can be targeted to specific mRNAs, blocking their expression without affecting normal functions of other genes. Because of their specific and catalytic mode of action ribozymes are ideal agents for therapeutic interventions against malfunctioning or foreign gene products. Here we report successful experiments to ‘knock out’ a major translation product in vivo using synthesized, chemically modified ribozymes. The ribozymes, designed to cleave amelogenin mRNA, were injected close to developing mandibular molar teeth in newborn mice, resulting in a prolonged and specific arrest of amelogenin synthesis not caused by general toxicity. No carriers were required to assist cellular uptake. Amelogenins are highly conserved tissue‐specific proteins that play a central role in mammalian enamel biomineralization. Ultrastructural analyses of in vivo ribozyme‐treated teeth demonstrated their failure to develop normally mineralized enamel. These results demonstrate that synthesized ribozymes can be highly effective in achieving both timed and localized ‘knock‐out’ of important gene products in vivo, and suggest new possibilities for suppression of gene expression for research and therapeutic purposes.

Epithelial expression of HLA, secretory component (poly-Ig receptor), and adhesion molecules in the human alimentary tract.

Epithelial HLA class II is differentially expressed (DR >> DP) only after birth in salivary glands and small intestinal mucosa, in contrast to class I determinants and secretory component (SC) which appear early in gestation. However, there is a brisk postnatal increase in SC expression along with the class II induction, suggesting stimulation by cytokines from activated immune cells. T lymphocytes remain quite scanty in postnatal salivary glands, and the striking SC and class II expression might reflect a synergistic effect of IFN-gamma and TFN-alpha on immature epithelial cells. Enhanced epithelial expression of both SC and class II in salivary glands from sudden infant death victims could be the effect of immunostimulation caused by an infectious agent. Strikingly upregulated SC and epithelial class II expression (DR > DP > DQ) is seen in various inflammatory lesions such as obstructive sialadenitis, Sjögrens syndrome, chronic gastritis, and celiac disease. IFN-gamma and TNF-alpha are most likely involved as the expression patterns can be reproduced with these cytokines in vitro on colonic epithelial cell lines. However, these molecules of the Ig supergene family do not show a selective response in epithelia of inflammatory lesions because increased expression is also seen for lysozyme, lactoferrin and some other proteins. ICAM-1 can be upregulated on epithelial cells by various cytokines in vitro although the situation remains uncertain in mucosal inflammation. The expression pattern in IBD is complicated by dysplastic epithelial changes leading to reduced SC levels which may thus, in turn, jeopardize the poly-Ig transport mechanism. Epithelial class II molecules appear to have antigen-presenting properties, but the immunopathologic role of their increased expression in inflammatory disease in terms of induction of autoimmunity and/or abrogation of oral tolerance is a matter of continuing dispute.

Development of Intestinal Mucosal Immunity in Fetal Life and the First Postnatal Months

ABSTRACT: Nine premature infants who were either stillborn or who died shortly after delivery (gestational age, 24–32 wk), eight full-term infants who died during the first 3 postnatal wk, and four infants who died in the postneo-natal period were studied by immunohistochemistry for duodenal expression of secretory component (SC) and epithelial HLA class I and II determinants and for the presence of IgA-, IgM-, and IgG-producing immunocytes. Only small amounts of SC appeared before the 29th gestational wk, but thereafter it increased rapidly; 1 wk after birth, SC showed an adult distribution pattern. Epithelial class I was expressed throughout the period investigated, whereas class II (HLA-DR) determinants were absent on the duodenal villi until 1 wk after birth. HLA-DP and -DQ were not expressed by the epithelium. No IgA immunocytes were seen before 1 wk after birth, whereas a few IgM- and IgG-producing cells were present throughout the period studied. The intense epithelial HLA-DR expression from the 2nd postnatal wk, along with SC and the appearance of IgA immunocytes, suggests that the intestinal immune system is modulated in response to environmental factors shortly after birth.

Ontogenesis of the secretory immune system and innate defence factors in human parotid glands.

Immunoglobulin‐producing cells and epithelial expression of secretory component (SC), amylase, lysozyme (Ly) and lactoferrin (Lf) were studied by immunohistochemistry to obtain information about the development of mucosal immunity. Tissue specimens were obtained from 20 fetal and 40 postnatal parotid glands, (1) Fetal specimens. Occasional IgM‐ and IgA‐ But no IgD‐, IgG‐ or IgE‐producing cells were seen (ratios, IgM:IgA:IgD:lgG:IgE–4:1:0:0:0). The IgA1 subclass dominated (median 90%, range 50–95%) and these cells were mostly J‐chain‐positive (median 97%, range 94 98%). Only few lgA2‐producing cells were seen (median 10%, range 5–50%) and they were also mostly J‐chain‐positive (median 99%, range 98–100%). Amylase, Ly and Lf were most prominent in early fetal life, while only small amounts of SC were present. (2) Postnatal specimens. Secretory component increased markedly along with a growing number of IgA‐ and IgD‐producing cells (lgA:IgM:IgD:IgG:lgE–4:2:1 : 1:0). The IgA1 subclass remained predominant (median 65%, range 50‐90%) although the proportion of IgA2‐positive cells tended to be raised (median 35%, range 10–50%). Most IgA1 (median 97%, range 67–100%) and IgA2 (median 94%, range 75–100%) cells were J‐chain‐positive. These features probably reflected local activation of the immune system in response to environmental factors. The amount of amylase, Ly and Lf decreased shortly after delivery, perhaps because the cellular stores were emptied by postnatal increase in secretory activity.

A multivariate study of the prognosis of oral squamous cell carcinomas. Are blood group and hemoglobin new prognostic factors

Because blood groups and hemoglobin concentration have been associated with the risk of the development of some cancers, this study evaluated the significance of ABO and Rhesus blood groups and hemoglobin concentration as prognostic factors in oral squamous cell carcinoma (SCC). The authors examined all registered primary SCC of buccal and maxillary alveolar mucosa in the Norwegian population between 1963 and 1972. The biopsy specimens from these patients were reevaluated and borderline cases excluded. The remaining 111 cases were included in the study, and features recorded on first admission were included in the survival analyses. ABO and Rhesus blood groups were found in 99 of these patients. Multivariate survival analysis showed that tumor size, hemoglobin concentration, stage, and Rhesus blood groups were significant prognostic factors, but sex, age, treatment, duration of symptoms, ABO blood groups, and clinical appearance of the tumors were not. The prognostic value of Rhesus blood groups and hemoglobin concentration has not been previously reported for oral SCC.

Development of immune response markers in the trachea in the fetal period and the first year of life

Immunoglobulin (Ig)‐producing cells. T cells (CD3) and epithelial expression of seeretory component (SC) and HLA class II determinants (DR, ‐DP, ‐DQ) were studied by immunohistochemistry in 16 fetal and 15 postnatal specimens from the tracheal wall. Small amounts of secretory component (SC) was present in the traeheal surface and gland epithelium in the fetal period and inereased towards term. A few IgM‐, IgD‐ and IgG‐producing cells were present in some fetal specimens but no IgA‐ and IgE‐produeing cells were found. Only very few CD3 + T‐cells were present in fetal specimens and intraepithelial T‐cells were virtually absent until after birth. Premature infants that lived for 1 week had less SC epithelial expression than mature infants of the same age. The density of CD3 + T‐cells. IgA‐, IgM‐producing cells as well as the epithelial SC expression increased rapidly after birth. Epithelial MHC class II expression was absent in fetal specimens. HLA‐DR was detected on the apical border of the surface epithelium one week after birth and was extensively expressed throughout the remaining postnatal period. E‐pithelial DP and PQ expression were virtually absent during this same period. These features probably reflect local activation of the immune system in response to environmental factors.

Up-regulated epithelial expression of HLA-DR and secretory component in salivary glands: reflection of mucosal immunostimulation in sudden infant death syndrome.

ABSTRACT: Human parotid glands from 55 forensic autopsy subjects, 1–12 mo of age, were examined by immunohistochemistry without knowledge about the cause of death. Various combinations of monoclonal or polyclonal antibody reagents of the following specificities were applied in two-color immunofluorescence analyses: HLA class I or II (DR, DP, or DQ); pan-T cell (CD3); leukocyte common antigen (CD45); and secretory component (poly-Ig receptor). Sudden infant death syndrome victims (n = 17) were shown to have significantly increased numbers of CD45+ stromal leukocytes and intensified epithelial expression of HLA-DR and secretory component as well as increased endothelial expression of both HLA class I and II (DR, DP, and DQ) determinants compared with controls (n = 31) who had died from noninfectious causes. Seven overtly infectious subjects (bronchopneumonia) showed still more up-regulated expression. This result suggested that enhanced stimulation of the local immune system exists in sudden infant death syndrome, with release of certain cytokines that are known to up-regulate epithelial expression of HLA-DR and secretory component.

The Effects of Acidic Fluoride Solutions on Early Enamel Erosion in vivo

Acidic fluoride solutions may reduce dental erosion. The aim of this study was to compare the effect of different acidic fluoride solutions on enamel dissolution using an established in vivo model. When possible 4 anterior teeth (255 teeth in a total of 67 subjects) were isolated and exposed to 0.01 M citric acid. The acid was collected in test tubes before (etch I) and 5 min after (etch II) application of test fluoride preparations. Acidic fluoride solutions (pH range 1.5–2.9), i.e. SnF2, TiF4 and hydrogen fluoride (HF) (all approx. 0.1 M F), HF (0.027, 0.055, 0.082 M F) and neutral NaF solution (0.1 M F) as control were applied to the labial surfaces of the teeth for 1 min (6 ml/min). Enamel dissolution was examined by chemical analysis of calcium content in the citric acid etch solutions using atom absorption spectrometry. The change in calcium concentration (ΔCa) and the percentage of mean calcium reduction were calculated from the difference in calcium loss between etch I and etch II. Statistical analysis was carried out using the Wilcoxon signed rank test and Kruskal-Wallis tests with Dunn’s multiple comparison. Results showed a mean ΔCa of 0.671 mg/l (SD 0.625) for SnF2, and ranged from 0.233 mg/l (SD 0.248) for the weakest HF solution to 0.373 mg/l (SD 0.310) for the strongest HF solution. This represented a 67% reduction in enamel dissolution for SnF2 and a 40–76% reduction for the HF solutions. No reduction was observed for TiF4 or NaF. The types of metal, pH and fluoride concentration are all important for the in vivo effect.

On the genetics of hypodontia and microdontia: synergism or allelism of major genes in a family with six affected members.

Familial severe hypodontia of the permanent dentition is a rare condition. The genetics of this entity remains unclear and several modes of inheritance have been suggested. We report here an increase in the number of congenitally missing teeth after the mating of affected subjects from two unrelated Norwegian families. This condition may be the result of allelic mutations at a single gene locus. Alternatively, incompletely penetrant non-allelic genes may show a synergistic effect as expected for a multifactorial trait with interacting gene products. This and similar kindreds may allow identification of genes involved in growth and differentiation of dental tissues by linkage and haplotype association analysis. Brittle nails, delayed growth of the hair, and delayed teething in the probands support the grouping of these conditions among the ectodermal dysplasias.

Clustering of IgA-Producing Immunocytes Related to HLA-DR-Positive Ducts in Normal and Inflamed Salivary Glands

Ig‐producing immunocytes and epithelial expression of secretory component (SC) and HLA‐DR were evaluated by two‐colour immunofluorescence staining in 10 normal and 20 inflamed salivary glands; the latter included specimens from 10 patients with obstructive sialadenitis and 10 with Sjögrens syndrome (SS), Epithelium adjacent to T‐cell infiltrates showed extensive co‐expression of SC and HLA‐DR, suggesting that leucocyte‐derived cytokines were responsible for this concurrent up‐regulation. Clusters (>2 positive cells) of IgA‐producing cells were spatially related to DR‐positive ducts. The possibility is discussed that DR‐expressing epithelium contributes to local terminal differentiation of IgA‐producing plasma cells. A cytokine‐mediated up‐regulation of SC that simultaneously increases the transport capacity for polymeric IgA would constitute an efficient enhancement of secretory immunity in diseased glandular tissue.