Radiochemotherapy in locally advanced squamous cell carcinomas of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a common disease that develops in the upper aerodigestive epithelium. The most important risk factors are tobacco and alcohol consumption. There is also increasing evidence that human papillomavirus plays an important role in the cause of SCCHN. The complex anatomy, the vital functions of the upper aerodigestive tract and the close proximity to vital structures, explain that the goal of treatment is not only to improve survival outcomes, but also to preserve organ function. Radiotherapy and surgery are the standard modalities of treatment, reflecting the locoregional predominance of SCCHN. Chemotherapy plays an important role in the treatment of patients with locoregionally advanced disease, in conjunction with radiotherapy and surgery. Indeed, standard therapy for resectable locoregionally advanced (stage III or IV) SCCHN cancers consists either of surgery and adjuvant chemoradiotherapy or definitive concomitant chemoradiotherapy, depending upon disease site, stage and resectability of the tumour, or institutional experience. Concomitant chemoradiotherapy has been shown in several randomised trials to improve disease-free and overall survival in the postoperative setting for resected disease with poor prognostic factors. Furthermore, multiple randomised studies and meta-analyses have shown that definitive chemoradiotherapy, as well anti-epidermal growth factor receptor treatment in one randomised study, improved disease-free and overall survival when compared with radiotherapy alone. This overview reviews the most relevant published studies on the multidisciplinary management of SCCHN and discusses future strategies to reduce locoregional failures.

A history of the renewal of the corneal epithelium : a 3D animation

Background: To compare the different schemes that have been proposed during the last thirteen years to explain the renewal of the corneal epithelium. Material and Methods:We analyzed all the data present in the literature to explain the renewal of the corneal epithelium in mammals. According to the schemes proposed in the literature we developed a 3D animation to facilitate the understanding of the different concepts. Results:Three different schemes have been proposed to explain the renewal of the corneal epithelium in mammals during the last thirteen years. 1950-1981: the corneal epithelium was thought being renewed by mitosis of cells located in the basal layer. At this time scientist were not talking about stem cells. 1981-1986 was the period of the "XYZ hypothesis" or the transdifferentiation paradigm. At this time the conjunctival epithelium renewed the corneal epithelium in a centripetal migration. 1986-2008: the limbal stem cell paradigm, there were no stem cells in the corneal epithelium, all the corneal stem cells were located in the limbus and renewed the central cornea after a migration of 6 to 7 mm of transient amplifying cells toward the centre of the cornea. 2008, epithelial stem cells were found in the central cornea in mammals (Nature, Majo et al. November 2008). Discussion:We thought that the renewal of the corneal epithelium was completely defined. According to the last results we published in Nature, the current paradigm will be revisited. The experiments we made were on animals and the final demonstration on human has still to be done. If we find the same results in human, a new paradigm will be define and will change the way we consider ocular surface therapy and reconstruction.

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens.

BACKGROUND: Trichoplax adhaerens is the best-known member of the phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types. RESULTS: We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior, where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide. CONCLUSIONS: Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior.

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa.

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Epithelium-mesenchyme interactions control the activity of peroxisome proliferator-activated receptor beta/delta during hair follicle development.

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

Primary γδ T cell lymphoma of the lung: report of a case with features suggesting derivation from intraepithelial γδ T lymphocytes [Primary gammadelta T cell lymphoma of the lung: report of a case with features suggesting derivation from intraepithelial gammadelta T lymphocytes].

T cell lymphoma of γδ T cell origin is a rare disease that mainly involves extranodal sites and shows aggressive clinical behavior. Here, we report a case of primary γδ T cell lymphoma of the lungs with epitheliotropism in the respiratory epithelium, a feature somewhat reminiscent of what is observed in enteropathy-associated T cell lymphoma. A 63-year-old man presented with chest pain and dyspnea on exertion, weight loss, and general weakness. On a positron emission tomography (PET) scan, multiple hypermetabolic lesions were found in both lungs. Microscopic examination of the wedge lung biopsy revealed nodular infiltration of monomorphic, medium- to large-sized atypical lymphocytes with round nuclei, coarse chromatin, and a variable amount of clear to eosinophilic cytoplasm. Of note, intraepithelial lymphocytosis by atypical lymphoid cells was observed in the respiratory epithelium within and around the nodule. Immunohistochemically, the tumor cells were CD3+, TCRβF1-, TCRγ+, CD5-, CD7+, CD20-, CD79a-, CD30-, CD4-, CD8-, CD10-, BCL6-, CD21-, CD56+, CD57-, and CD138-, and expressed cytotoxic molecules. Epstein-Barr virus (EBV) was not detected by an in situ hybridization assay for EBV-encoded RNA. Interestingly, CD103 was expressed by a subset of tumor cells, especially those infiltrating the epithelium. T cell clonality was detected by multiplex PCR analysis of TRG and TRD gene rearrangements. After 2 months of systemic chemotherapy, PET scan showed regression of the size and metabolic activity of the lesions. This case represents a unique γδ T cell lymphoma of the lungs showing epitheliotropism by CD103+ γδ T cells that is suggestive of tissue-resident γδ T cells as the cell of origin.

Evaluation of the integrity of human corneal epithelium maintained in organ-culture during eye banking using Corneamax®

Purpose: Mediums have been developed to conserve corneal endothelium in organ-culture during eye banking. CorneaMax® is used by 25% of Eye Bank in Europe. Only little is known about conservation of corneal epithelium with this medium during banking. Its preservation could be of interest in clinic to cure corneal disease with stem cells deficiency. Therefore, we wanted to examine the integrity of human corneal epithelium maintained in CorneaMax®. Methods: Human corneas, considered unsuitable for transplantation, were obtained from the Eye Bank in Lausanne. Average post-mortem time was 14 hours. Cornoscleral rings were maintained in organ-culture in Corneamax® at 32°C. Samples were formalin-fixed after period ranging from 0 (D0) to 35 days (D35, N=5 for each time points) and stained with H&E. Proliferation and apoptosis were evaluated by immunostaining with antibody against Ki67 and Caspase3 respectively. Results: Corneas, which were not in organ-cultured (D0), showed different morphology, including intact epithelium with 5 to 7 layers, but also completely denuded basement membrane. In two cases, at D0, the epithelium lost its adherence to the basal lamina of the cornea creating a large epithelial sheet. During the two first days, corneas and limbus area lost totally their epithelium, except for some remaining limbal basal cells. From day 2 to day 10, regeneration of the epithelium took place, starting from the limbal region in direction to the central cornea. From day 10 to day 35, corneal epithelium appeared as an atrophic epithelium, consisting of only two cell layers. Proliferation happened in the whole cornea during the 35 days of organ-culture, as shown by Ki67 positive cells. Apoptosis was rarely detected in the corneal epithelium. Conclusions: Corneas maintained in CorneaMax® showed a complete disappearance of the corneal epithelium during the two first days and a conservation of limbal basal cells in the limbal region. These remaining cells allowed a full regeneration of the tissue, leading to an atrophic epithelium, composed of only two cell layers. This atrophic epithelium could be seen in all the organ-cultured corneas during the 35 days of conservation. This study is a first step to develop medium in organ-culture in order to conserve corneal epithelial cells.

Molecular mechanisms of follicular associated epithelium differentiation

Les muqueuses respiratoires, genitales et digestives sont continuellement exposées aux antigènes de l?alimentation, à la flore intestinale et aux pathogènes. Cela implique une activité immunologique intense et finement régulée dans ces tissus. On admet que la modulation de ces réponses immunitaires muqueuses s?effectue dans des organes sentinels spécifiques appelés o-MALT (organized mucosal associated lymphoid tissues). Ces processus de modulation et la biologie de ces sites immuno-inducteurs sont peu connus. Ceci est pourtant d?une grande relevance si l?on veut faire un design rationnel de drogues et de vaccins muqueux. Dans l?intestin grèle, ces organes sont composés de follicules multiples et sont appelés plaques de Peyer. Ils sont constitués de follicules enrichis en cellules B comprenant ou non un centre germinatif, de regions interfolliculaires comprenant des cellules T, et d?une région en d ome riche en cellules dendritiques, cellules B naives et cellules T CD4+, surmontée par un epithelium specialisé, le FAE (epithelium associé aux follicules). Le FAE contient des cellules M spécialisées dans le transport de macromolécules et micro-organismes de la lumière intestinale au tissu lymphoide sous-jacent. Ce transport des antigènes est une condition obligatoire pour induire une réponse immunitaire. Les cellules du FAE, outre les cellules M, expriment un programme de différenciation distinct de celui des cellules associées aux villosités. Ceci est characterisé par une baisse des fonctions digestives et de défenses, et l?expression constitutive des chimiokines: CCL20 et CCL25. Le but de l?étude présentée ici est de rechercher les facteurs cellulaires et/ou moléculaire responsables de cette différenciation. Certaines études ont démontré l?importance du contact entre le compartiment mésenchymateux et l?épithelium pour la morphogenèse de ce dernier. En particulier, les molécules de la matrice extracellulaire peuvent activer des gènes clefs qui, à leur tour, vont controler l?adhésion et la differenciation cellulaire. Dans l?intestin, les cellules mésenchymateuses différencient en myofibroblastes qui participent à l?élaboration de la matrice extracellulaire. Dans cette étude, nous avons décrit les différences d?expression de molécules de la matrices sous le FAE et les villosités. Nous avons également montré une absence de myofibroblastes sous le FAE. Suite à plusieurs évidences expérimentales, certains ont proposé une influence des composés présents dans la lumière sur la différenciation et/ou la maturation des plaques de Peyer. La chimiokine CCL20, capable de recruter des cellules initiatrices de la réponse immunitaire, constitue notre seul marqueur positif de FAE. Nous avons pu montrer que la flagelline, un composé du flagelle bactérien, était capable d?induire l?expression de CCL20 in vitro et in vivo. Cet effet n?est pas limité aux cellules du FAE mais est observé sur l?ensemble de l?épithelium intestinal. Molecular mechanisms of FAE differenciation. La signalement induit par la lymphotoxine ß est critique pour l?organogenèse des plaques de Peyer, car des souris déficientes pour cette molécules ou son récepteur n?ont ni plaque de Peyer, ni la plupart des ganglions lymphatiques. Nous avons obtenus plusieurs évidences que la lymphotoxine ß était impliquée dans la régulation du gène CCL20 in vitro et in vivo.<br/><br/>Mucosal surfaces of the respiratory, genital and digestive systems are exposed to food antigens, normal bacterial flora and oral pathogens. This justifies an intense and tuned immunological activity in mucosal tissues. The modulation of immune responses in the mucosa is thought to occur in specific sentinel sites, the organized mucosa associated lymphoid tissues (o-MALT). This immune modulation and the biology of these immune-inductive sites are poorly understood but highly important and relevant in the case of drugs and vaccines design. In the small intestine, these organs (gut associated lymphoid tissue : GALT) consists of single or multiple lymphoid follicles, the so-called Peyer?s patches (PP), with typical B cell-enriched follicles and germinal centers, inter-follicular T cell areas, and a dome region enriched in dendritic cells, naive B cells, and CD4+ T cells under a specialized follicle associated epithelium (FAE). To trigger protective immunity, antigens have to cross the mucosal epithelial barrier. This is achieved by the specialized epithelial M cells of the FAE that are able to take up and transport macromolecules and microorganisms from the environment into the underlying organized lymphoid tissue. The ontogeny of M cells remains controversial: some data are in favor of a distinct cell lineage, while others provide evidence for the conversion of differentiated enterocytes into M cells. In this study we mapped the proliferative, M cells and apoptotic compartments along the FAE. Enterocytes acquire transient M cell features as they leave the crypt and regain enterocyte properties as they move towards the apoptotic compartment at the apex of the FAE, favouring the hypothesis of a plastic phenotype. The follicle-associated epithelium (FAE) is found exclusively over lymphoid follicles in mucosal tissues, including Peyer?s patches. The enterocytes over Peyer?s patches express a distinct phenotype when compared to the villi enterocytes, characterized by the down regulation of digestive and defense functions and the constitutive expression of chemokines, i.e. CCL20 and CCL25. The purpose of this study was to investigate and identify the potential cells and/or molecules instructing FAE differentiation. Contact between the epithelial and the mesenchymal cell compartment is required for gut morphogenesis. Extracellular matrix molecules (ECM) can activate key regulatory genes which in turn control cell adhesion and differentiation. In the gut, mesenchymal cells differentiate into myofibroblats that participate to the elaboration of ECM. We have described a differential expression of extracellular matrix components under the FAE, correlating with the absence of subepithelial myofibroblats. Molecular mechanisms of FAE differenciation. Different studies proposed an influence of the luminal compartment in the differentiation and/or the maturation of PP. CCL20, a chemokine able to recruit cells that initiate adaptive immunity constitutes our first positive FAE molecular marker. We have shown that CCL20 gene expression is inducible in vitro and in vivo in intestinal epithelium by flagellin, a component of bacterial flagella. This effect was not restricted to the FAE. Lymphotoxin ß (LTß) signaling is critical for PPs organogenesis as LT deficient mice as well as LTß-receptor-/- mice lack PPs and most of the lymph nodes (LN). The continuous signaling via LTßR-expressing cells appears necessary for the maintenance throughout the life of PP architecture. We obtained in vitro and in vivo evidence that LTß signalling is involved in CCL20 gene expression.

Lung CD57+ cell density is increased in very severe COPD

Among all inflammatory cells involved in COPD, those with a cytolytic or elastolytic activity are thought to play a key role in the pathogenesis of the disease. However, there is no data about the infiltration of cells expressing the CD57 marker in small airways and parenchyma of COPD patients. In this study, surgical specimens from 43 subjects undergoing lung resection due to lung cancer (9 non-smokers, 18 smokers without COPD and 16 smokers with moderate COPD) and 16 patients undergoing double lung transplantation for very severe COPD were examined. CD57+ cells, neutrophils, macrophages and mast cells infiltrating bronchioles (epithelium, smooth muscle and connective tissue) and parenchymal interstitium were localized and quantified by immunohistochemical analysis. Compared to the other groups, the small airways of very severe COPD patients showed a significantly higher density of CD57+ cells, mainly infiltrated in the connective tissue (p=0.001), and a significantly higher density of neutrophils located characteristically in the epithelium (p=0.037). Also, the density of neutrophils was significantly higher in parenchyma of very severe COPD patients compared with the rest of the groups (p=0.001). Finally, there were significant correlations between the bronchiolar density of CD57+ cells and the FEV1 values (R=-0.43, p=0.022), as well as between the parenchymal density of neutrophils and macroscopic emphysema degree (R=0.43, p=0.048) in COPD groups. These results show that CD57+ cells may be involved in COPD pathogenesis, especially in the most severe stages of the disease.

Cadmium uptake and induction of metallothionein synthesis in a renal epithelial cell line (LLC-PK1).

LLC-PK1 cells, an established cell line from pig kidney with proximal tubule properties, were cultivated in vitro at confluence on plastic dishes. They were then exposed (apical side) to inorganic cadmium (CdCl2, 5 microM) for periods ranging between 1 to 24 h. Analysis of the cell supernatant after homogenisation and ultracentrifugation indicated that Cd taken up in the first 3 h was bound to cytosolic high molecular weight proteins, but was redistributed to low molecular weight proteins at later stages. Induction of Cd-metallothionein (Cd-Mt) synthesis, as judged from Cd-Mt binding to a specific anti-Cd-Mt antibody and from the rate of 35S-cys incorporation into a specific protein fraction, was apparent 3-6 h after the addition of Cd to the incubation medium.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia

Iron-deficiency anemia is the nutritional deficiency most frequently occurring throughout the world, which manifests as a complex systemic disease involving all cells, affecting enzyme activities and modifying protein synthesis. In view of these considerations, the objective of the present study was to determine the effects of iron-deficiency anemia on disaccharidases and on the epithelial morphokinetics of the jejunal mucosa. Newly weaned male Wistar rats were divided into 4 groups of 10 animals each: C6w received a standard ration containing 36 mg elemental iron per kg ration for 6 weeks; E6w received an iron-poor ration (5-8 mg/kg ration) for 6 weeks; C10w received an iron-rich ration (36 mg/kg ration) for 10 weeks; E10w received an iron-poor ration for 6 weeks and then an iron-rich ration (36 mg/kg) for an additional 4 weeks. Jejunal fragments were used to measure disaccharidase content and to study cell proliferation. The following results were obtained: 1) a significant reduction (P<0.001) of animal weight, hemoglobin (Hb), serum iron and total iron-binding capacity (TIBC) in group E6w as compared to C6w; reversal of the alterations in Hb, serum iron and TIBC with iron repletion (E10w = C10w); animal weights continued to be significantly different in groups E10w and C10w. 2) Sucrase and maltase levels were unchanged; total and specific lactase levels were significantly lower in group E6w and this reduction was reversed by iron repletion (E10w = C10w). 3) The cell proliferation parameters did not differ between groups. On the basis of these results, we conclude that lactase production was influenced by iron deficiency and that this fact was not related to changes in cell population and proliferation in the intestinal mucosa

Cell lineage relationship in the stomach of normal and genetically manipulated mice

The oxyntic mucosa of the mouse stomach is lined with a heterogeneous population of cells that form numerous short pits continuous with long tubular glands. Tritiated thymidine radioautography has made it possible to pinpoint the origin of all cell types and to follow the differentiation/migration of different cell lineages along the pit-gland unit. The proliferating multipotent stem cells functionally anchored in the upper glandular region, the isthmus, give rise to three main lineage precursors: 1) pre-pit cells, which migrate upward to the pit while differentiating into mucus-producing pit cells; 2) pre-neck cells, which migrate downward to the glandular neck while differentiating into mucus-producing neck cells that, by approaching the glandular base, gradually change their phenotype into pepsinogen- and intrinsic factor-producing zymogenic cells; 3) pre-parietal cells, which differentiate into acid-producing parietal cells in the isthmus and then undergo bipolar migration towards the pit and the glandular base. Thus, parietal cells are the only cells that complete their differentiation in the isthmus and then migrate to be scattered throughout the pit-gland unit. To determine whether parietal cells play a role in controlling decisions about cell fate within the pit-gland unit, the gastric epithelium has been examined in transgenic mice expressing the H,K-ATPase ß-subunit-1035 to +24/simian virus 40 large T antigen fusion gene. The blockade in parietal cell differentiation in these mice produces an amplification of lineage precursors, a marked depletion of zymogenic cells and an increase in pit cell census. Ablation of parietal cells in another transgenic mouse model expressing the H,K-ATPase ß-subunit-1035 to +24/diphtheria toxin fragment A fusion gene also produces amplification of lineage precursors, and similar effects on zymogenic and pit cell census. These findings strongly suggest that parietal cells produce regulatory signals that control the cellular differentiation program of both pit and zymogenic cell lineages, and would hopefully improve our ability to identify the cellular pathways leading to malignant transformation

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196°C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.