Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes

We previously characterized a Na+/H+ exchange activity in rat pancreatic zymogen granules [Anderie, I., and Thévenod, F. (1996) J. Membrane Biol, 152, 195-205]. Here we have identified the Na+/H+ exchanger (NHE) isoforms present in zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited zymogen granule Na+/H+ exchange in a concentration dependent manner, maximally to 53 +/- 5% of controls at 100nM. The remaining Na+/H+ exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC50 approximately 25 microM) or benzamil (EC50 approximately 100 microM). Amiloride inhibited weakly suggesting that "amiloride-resistant" and "amiloride-sensitive" NHE are expressed in zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in zymogen granule membranes was confirmed by immunoblots of zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in zymogen granule membranes of rat exocrine pancreas.     

Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.     

Autoimmunity, immunodeficiency and mucosal infections: chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of "molecular mimicry" (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.     

The "Heart Area" of Juan Canalejo Hospital Complex. A new approach to clinical management

The present work describes the process by which the pilot project of clinical management of the Hospital Complex Juan Canalejo, designated as "Heart Area", was implemented. In the first section, the needs and reasons that led to the undertaking of this project are explained. The project's objectives and operative strategies are listed. In the Material and Methods section, three basic aspects of the "Heart Area" are described: selection criteria of the "Area", its structure and function, and its foundation and development. In the Results section, we compare the activity undertaken in the "Area" to the situation present prior to its implementation, in relation to quality and costs. Finally, in the Conclusions, we comment on the important implications that our project can have within the Hospital Complex Juan Canalejo as well as in the health care field in general.     

Compensation for gaze perturbation during inactivation of the caudal fastigial nucleus in the head-unrestrained cat

Muscimol injection in the caudal part of the fastigial nucleus (cFN) leads, in the head-unrestrained cat, to a characteristic dysmetria of saccadic gaze shifts toward visual targets. The goal of the current study was to test whether this pharmacological cFN inactivation impaired the ability to compensate for unexpected perturbations in gaze position during the latency period of the saccadic response. Such perturbations consisted of moving gaze away from the target by a transient electrical microstimulation in the deep layers of the superior colliculus simultaneously with extinction of the visual target. After injection of muscimol in the cFN, targets located in the contralesional hemifield elicited gaze shifts that fell short of the target in both "perturbed" and "unperturbed" trials. The amplitude of the compensatory contraversive gaze shifts in perturbed trials coincided with the predicted amplitude of unperturbed responses starting from the same position. Targets located in the opposite hemifield elicited hypermetric gaze shifts in both trial types, and the error of compensatory responses was not statistically different from that of unperturbed gaze shifts. These results indicate that inactivation of the cFN does not interfere with the ability of the head-unrestrained cat to compensate for ipsiversive or contraversive perturbations in gaze position. Thus the gaze-related feedback signals that are used to compute a reference signal of desired gaze displacement are not impaired by cFN inactivation.     

Subgingival microbial profile of Papillon-Lefèvre patients assessed by DNA-probes

The prevalence of 18 selected bacterial species was assessed by means of "checkerboard" DNA-DNA hybridisation in a group of 12 Saudi-Arabian adolescents with Papillon-Lefèvre syndrome. A total of 36 tooth sites were investigated. The patients exhibited severe periodontal disease with deep pockets. All 12 patients harboured the putative bacterial pathogens P. intermedia, F. nucleatum, P. micros and S. intermedius while T. denticola, B. forsythus, P. nigrescens, E. corrodens, S. noxia and C. rectus were recovered from 11 patients. P. gingivalis was recovered from 9 patients and 18 sites while corresponding figures for A. actinomycetemcomitans were 8 and 19, respectively. A number of the investigated species (B. forsythus, T. denticola, P. intermedia, C rectus) reached high levels (> or =10(6) cells) in more than 1/2 of the patients. On the other hand, bacteria such as A. actinomycetemcomitans and P. gingivalis were infrequently encountered at high levels in these subgingival samples. In conclusion, the analysis failed to demonstrate a PLS-specific profile of the subgingival infection, since the bacterial composition of the sampled sites closely resembled that characterising deep pockets in adult periodontitis patients.     

Screening for colorectal cancer in Catalonia. Results of a population survey

This CT study was designed to assess brain morphology in agoraphobia. 21 patients and 21 normal control subjects matched in age and sex were investigated. Frontal and parietooccipital cortex, temporal cortex, lateral ventricles and third ventricle were evaluated by qualitative assessment on a 3-point scale (normal, questionable, abnormal). Patients showed significant bilateral enlargement of prefrontal cortical cerebrospinal fluid (CSF) spaces (p < .05). The rating "abnormal" was given to none (0%) of the normal controls, but to 6 (28.6%) of the patients in the left hemisphere, and to 4 patients (19%) in the right hemisphere, respectively. No qualitative differences were seen in the temporal cortex, lateral ventricles and third ventricle. These findings support the hypothesis that alterations in brain morphology are involved in the etiology of agoraphobia. The lack of a correlation between CSF enlargement and duration of illness suggests that prefrontal CSF enlargement is a neurobiological vulnerability marker in agoraphobia.     

Dietary factors influence the recovery rates of Helicobacter pylori in a BALB/cA mouse model

We have identified a number of type I and type II keratins in the zebrafish Danio rerio by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot-binding assay and immunoblotting. These keratins range from 56 kDa to 46 kDa in molecular mass and from pH 6.6 to pH 5.2 in isoelectric point. Type II zebrafish keratins exhibit significantly higher molecular masses (56-52 kDa) compared with the type I keratins (50-48 kDa), but the isoelectric points show no significant difference between the two keratin subclasses (type II: pH 6.0-5.5; type I: pH 6.1-5.2). According to their occurrence in various zebrafish tissues, the identified keratins can be classified into "E" (epidermal) and "S" (simple epithelial) proteins. A panel of monoclonal anti-keratin antibodies has been used for immunoblotting of zebrafish cytoskeletal preparations and immunofluorescence microscopy of frozen tissue sections. These antibodies have revealed differential cytoplasmic expression of keratins; this not only includes epithelia, but also a variety of mesenchymally derived cells and tissues. Thus, previously detected fundamental differences in keratin expression patterns between higher vertebrates and a salmonid, the rainbow trout Oncorhynchus mykiss, also apply between vertebrates and the zebrafish, a cyprinid. However, in spite of notable similarities, trout and zebrafish keratins differ from each other in many details. The present data provide a firm basis from which the application of keratins as cell differentiation markers in the well-established genetic model organism, the zebrafish, can be developed.