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Apoptosis is a genetically controlled and evolutionarily conserved form of active cell death, albeit with an increase in complexity with continuing development. A high conservation at the functional and molecular level has been described between the players of the apoptotic machinery in invertebrates (Caenorhabditis elegans and Drosophila) and mammals. However, fish represent an excellent and advantageous model for the study of vertebrate development and disease, bridging the gap between the C. elegans/Drosophila and mouse/human models. Moreover, contrary to C. elegans and Drosophila, fish can be used for studying the development and function of vertebrate-specific organs and have a fully developed immune system similar to that of mammals. Last but not less important, both the environment and human health will obviously gain by using the knowledge generated through the use of fish models, for developing better prophylactic and therapeutic measures with impact on the aquaculture industry. In the present article, structural and functional data on the most important apoptosis related molecules, namely death-receptor, Bcl-2 and caspase families, and mechanisms are reviewed. The data point to the existence in fish of apoptotic pathways equivalent to those of mammals, making fish useful animal models for studying apoptosis, which may have great applicability for the advance of the knowledge on the role of apoptotic cell death in human apoptosis-related disorders as well as in pharmaceutical design.

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The relevance of fish research has been rising due to the expansion of aquaculture and to the increasing use of fish as replacements for mammals in the study of human physiological and pathological issues. Fish have much smaller genomes compared to mammals, and zebrafish, fugu, medaka and spotted green puffer fish have the sequence of their genomes completed or near completion. Fish have several of the virtues of Drosophila melanogaster and Caenorhabditis elegans for apoptosis research, but offer additional advantages because they are vertebrates and have a developed immune system and apoptotic pathways similar to those of mammals. Many phenotypes in the zebrafish resemble human diseases and this fish has been increasingly used in pharmaceutical design of apoptosis modulating drugs. The roles of microRNAs, bcl-2, p53, insulin-like growth factor-binding protein-3, and cellular apoptosis susceptibility (CAS) and c-Myc genes (involved in the interaction apoptosis/cancer), and Abeta peptides, presenilin enhancer 2, cyclin-dependent kinase 5 and tau (factors with relevant roles in apoptosis-associated human neurodegenerative disorders), have also been successfully investigated in fish models. Results of research with fish that have advanced the knowledge on the participation of apoptosis in viral infections and of apoptosis and secondary necrosis in bacterial infections are also reviewed. It is expectable that the use of fish for research on apoptosis-related issues relevant for human physiology and pathology and for the design of apoptosis-modulating drugs will continue to increase.

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Photobacterium damselae subsp. piscicida is the causative agent of pasteurellosis in wild and farmed marine fish worldwide. Although serologically homogeneous, recent molecular advances have led to the discovery of distinct genetic clades, depending on geographical origin. Further details of the strategies for host colonisation have arisen including information on the role of capsule, susceptibility to oxidative stress, confirmation of intracellular survival in host epithelial cells, and induced apoptosis of host macrophages. This improved understanding has given rise to new ideas and advances in vaccine technologies, which are reviewed in this paper.

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Mycobacteriosis (piscine tuberculosis) has been reported to affect a wide range of freshwater and marine fish species; however, this is the first report describing mycobacterial infections in turbot Scophthalmus maximus. High numbers of granulomas were initially observed in the organs of moribund farmed turbot. Bacteriological analysis of organs with granulomas led to the isolation of Mycobacterium marinum. Further analysis, to determine the prevalence of the infection in the farm and to identify its source, showed the occurrence of a dual infection by M. marinum and M. chelonae. The presence of Nocardia sp. in some of the fish infected with mycobacteria was also detected. The presence of granulomas in internal organs of apparently healthy fish indicated a high prevalence of the disease, a conclusion that was supported by isolating mycobacteria from all fish with or without granulomas. The infection was probably responsible for the mortality observed (approximately 2% mo(-1)), as most of the recently dead fish presented high numbers of granulomas and isolation of mycobacteria was possible from all of the fish. The isolation of M. marinum from the inlet water suggested this as the most plausible source for the infection occurring in the farm.

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The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.

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The coding sequence of the sea bass light chain was obtained by sequential anchored PCR on a head kidney cDNA library of a DNP(494)-KLH immunised sea bass. The cDNA sequence obtained codes for a leader peptide of 21aa and a mature IgL chain of 223aa. Both the amino acid sequence comparisons and neighbour-joining trees showed that the IgL chain of sea bass obtained is of the L1/G type. To study the variability of the light chain, additional PCRs on the cDNA library and cDNA from pooled head kidneys were performed. Multiple alignment of unique sequences (N=17) could be performed without introducing gaps, and showed extremely low variability in CDR1, and no variability in CDR2 or CDR3. A possible explanation for this low variability of the IgL1 chain might be the enhanced expression of monospecific anti-DNP antibodies. The isolation and characterisation of partial genomic and cDNA IgL sequences, which showed normal variability, corroborate this explanation.

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Extremely high numbers of antibody secreting cells (ASC) were observed in the gills of sea bass fry immunised at three different age/sizes (initial weight of 0.1, 2 and 5 g) by direct immersion in a Photobacterium damselae spp. piscicida bacterin. The relatively low ASC production in the head kidney and spleen suggests that the systemic compartment was only slightly stimulated upon immersion vaccination. There was no response of corresponding magnitude in the gut as the one observed in the gills. A clear age effect was observed in the ASC response of the different groups, especially visible in the gills. Significantly higher numbers of specific ASC were observed in the gills of the two oldest groups (initial weight of 2 and 5 g) compared with the youngest fish (initial weight of 0.1 g), but the oldest groups were not significantly different from each other. Additionally, a more rapid response was observed with the ageing of the fish, with peak responses in all the organs at day 18, 16 and 8 post-immunisation in the smallest to largest fish, respectively. There was no evidence that direct immersion exposure to P. damselae ssp. piscicida at the earliest stages used in the present study (0.1 g) was tolerogenic. In the context of present knowledge, this study strongly supports the importance of the route of immunisation to locally stimulate ASC and the importance that the gills might have in specific responses.

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Monoclonal antibodies specific to sea bass Ig heavy (WDI 1) and light (WDI 3) chains and T cells (DLT15) were used in an ontogenetic study of sea bass by flow cytometry and immunocytochemistry. The influence of weight and age, as well as season, on B cell development was studied in the fastest and slowest growing offspring from the same spawn (5-305 days post hatch: dph). Additionally, B and T cell development was followed in samples of different offspring (5-137 dph). The results suggest that DLT15 recognises very early (pre-?) T cells as well as mature T cells and that these very early T cells might have their origin in a different compartment and subsequently mature in the thymus. They also appeared much earlier in ontogeny (between 5-12 dph onwards) than pre-B cells having cytoplasmic Ig (from 52 dph onwards). With the monoclonal antibodies used, adult levels of T and B cells were both reached between 137-145 dph, suggesting that sea bass is immunologically mature from at least that age onwards. As in other teleosts, the thymus appears to be the primary organ for T lymphocytes and head kidney the primary organ for B lymphocytes. For sea bass, age seems to be more important in determining B cell maturation than body weight.

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UNLABELLED: Choledochoduodenostomy (CDD) has been reported as a more effective treatment of CBD stones than T-tube drainage but it is regarded as a last resort or obsolete therapeutic method due to fears of higher mobidity, cholangitis, "sump" syndrome and liver dysfunction. We aimed to assess the aforementioned issues analyzing prospectively our experience from 1976 through Dec.92. METHODS: CDD was performed in 89 females and 36 males, aged 60 +/- 8.7 years, 26 during repeat surgery. Duct stones were the indication in 94, Sphincter of oddi (SO) dysfunction in 23 and obstructive pancreatitis nodule in 8. Peroperative liver biopsies were obtained in 44 patients. The "follow-up" schedule (> 2.5 years in 110) included clinical interview and LFT's on an yearly basis. Ultra sound (USG) was obtained every one or two years. ERC was done in 10 symptomatic patients and in 25 others for protocul purposes. Liver biopsies were taken four to nine years post surgery in 11 patients-five at relaparotomy for non-biliary causes and six percutaneously by fine needle. Ductal mucosa biopsy could safely be performed in one patient 10 years after surgery. The long-term results were classified as excellent, good, fair or poor. Poor meant the need for further invasive therapy (resurgery or EST). RESULTS: There were two operative deaths (1.6%). The long-term results (123 survivors) were considered excellent in 89, good in 22, fair in 9 and poor in three. Three patients died from unrelated causes and eight others ceased the "follow-up" evaluation three to five years post surgery. All of them were considered as having excellent or good results. A widely patient anastomosis of approximately 20 mms without mucosal inflammatory changes was documented in every patient assessed via ERC. food "debris" was detected within the distal duct of four patients yet it was easily flushed through the stoma. Normal tissue patterns were observed in all long-term liver biopsies. Likewise the ductal mucosa biopsy failed to reveal any acute or chronic inflammatory changes. CONCLUSIONS: 1) CDD is a highly effective short and long-term treatment of CBD lithiasis.2) It does not lead to bacterial or "chemical" cholangitis, to "sump" syndrome or to hepatic dysfunction, provided a wide anastomosis is accomplished.3) CDD should only be considered as obsolete after extensive, long-term, prospective, randomized assessment of laparoscopic or combined laparoendoscopic approaches have been shown to be as effective as or superior to CDD.

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Malnutrition is a frequently observed complication of total gastrectomy. Does the mode of reconstructing the alimentary tract bear part of the responsibility? We assessed our experience from January 1975 to 1992 to analyze this issue. A series of 64 total gastrectomy patients [40 men, 24 women; aged 59 +/- 11 (SD) years] were considered. Preoperative and periodic follow-up evaluations were prospectively documented: upper gastrointestinal series, endoscopic examination, complete blood count, serum and liver biochemistry profiles, serum proteins, tranferrin, serum iron and calcium, iron-binding capacity, oral glucose tolerance test, ultrasonography or computed tomography, actual and ideal body weight and performance (AJCC/UICC) assessments. Symptoms were classified by means of Cuschieri's scoring system. Esophageal mucosal changes (edema, hyperemia, erosions, ulcerations) were documented on endoscopy. There were 36 of 58 operative survivors who had no evidence of tumor recurrence and were available for long-term evaluation (12-132 months). An RY loop had been constructed in 25 patients, 5 with a Hunt pouch; 9 had an isoperistaltic jejunal interposition (IJI), 4 with a Kock pouch; and 2 had a Braun loop. A 60 to 70 cm long jejunal limb was always utilized. Statistical analyses were obtained by means of the Student t-test and the equality of medians test. Progressive malnutrition was observed in patients with the Braun (omega) loops, both patients displaying persistent esophagitis and dietary restrictions. Both recovered ideal body weight after remedial surgery that transformed the omega loop into an RY loop. Both RY and IJI loops effectively prevented alkaline esophagitis.(ABSTRACT TRUNCATED AT 250 WORDS)

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Progressive malnutrition has been reported as a long-term consequence of total gastrectomy (TG), possibly related to the mode of reconstructing the intestine. In reviewing our personal experience (1975-Sept. 91), we attempted to correlate the reconstructive technique used with the subsequent course of the patient. A consecutive series of 62 TGs (59 adenocarcinomas, 3 lymphomas) in 38 males and 24 females 59 +/- 11 (m Mean +/- SD) years old was reviewed. Preoperative and "follow-up" evaluations, including upper gastrointestinal series and/or endoscopic examination, complete blood count, serum and liver biochemistry profiles, serum iron and plasma transferrin, oral GTT, USG or CT scan, actual and ideal body weight (IBW Life Extension Institute of New York), and "performance status" assessments, were prospectively documented. The follow-up symptoms were classified as per Cuschieri's scoring system. The endoscopic esophageal mucosa assessments were documented as well. Among 56 patients surviving operation, 34 were available, without tumor recurrence, for long-term (12-132 months) evaluation. A Roux-en-Y loop reconstruction had been performed in 23, 5 with a Hunt-Lawrence pouch; an isoperistaltic, esophagoduodenal, jejunal interposition (IR) was performed in 9, 4 with a Kock pouch; and an omega loop reconstruction was performed in 2. A 60-70-cm-long jejunal limb was always utilized.(ABSTRACT TRUNCATED AT 250 WORDS)

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The occurrence of retained/recurrent calculi after primary CBDE followed by temporary T-tube decompression, have remained at rates varying from 5.4% to 20.9% over the last 10 years in spite of sophisticated pre and intraoperative imaging techniques. It is postulated that a functional obstruction, due to dysmotility of the SO, lies behind most stone-containing ducts. Thus it seems logical to us that a permanent "fenestration" should be the management of most such ducts. We prospectively followed-up, for one to 10 years, two groups of patients submitted to primary CBDE aiming to assess the short and long-term results of two different surgical approaches to duct lithiasis. In one (Group A) 162 CBDE's were performed, out of 680 CHE's (24%), with a "positivity" of 68% and in the other (Group B) 80 CBDE's, out of 438 CHE's (18%), with a "positivity" of 70%. In Group A a T-tube decompression was used in 79(49%) and a definitive drainage in 83(51%) whereas in Group B the T-tube was employed in only 3(4%) and some form of permanent "fenestration" in 77(96%). There were no significant differences between the operative mortality rates, which were 2.5% in Group A (1 death post T-tube, 3 post CDJ) and 1.3% in Group B (1 death post CDD). The long-term results, though, were significantly worse among patients of Group A whose ducts were temporarily decompressed: 10/79 (12.7%) required further aggressive interventional therapy for retained/recurrent stones while only 3.8% (3/80) in Group A and 1.3% (1/76) in Group B required revisional surgery for bilio-digestive anastomotic complications with cholangitis. It is concluded that it is against the long-term efficiency of the approach utilized in Group B that the new laparoscopic techniques should be compared.

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One-stage subtotal colectomy of an acutely obstructed left colon would improve quality of life while shortening the length of hospitalization. Prohibitive mortality rates, however, are ascribed to such an approach. Analyzing the Senior Author's experience we compared the one-stage approach versus the multi-stage resections concerning operative mortality and morbidity rates and the duration of hospitalization. Forty-nine of 291 (17%) large bowel cancers presented acute left-sided obstruction requiring emergency surgery. Colostomy alone was performed in 18 (37%), multi-stage colectomy in 20 (41%, Group A) and one-stage subtotal colectomy in 11 (22%, Group B, all of them after 1979), the years under scrutiny being from 1973 through Sept. 1990. Both groups were comparable in age and sex distribution, TNM staging and ASA classification. Operative mortality and morbidity rates were 10% and 30% in Group A, 9% and 18% in Group B, respectively. The average length of hospitalization was 21.25 days (14-30) in Group A, 9.18 days (7-14) in Group B. Whenever an experienced surgical team is available and in the absence of contra-indications (local factors precluding a swift dissection, hemodynamic instability, gangrenous bowel) a one-stage subtotal colectomy, taking advantage of a better healing ileo-sigmoid or ileo-rectal anastomosis, carries acceptable mortality and morbidity rates while enhancing the quality of life and shortening the length of hospitalization. It should be considered the choice procedure, provided selection requirements and technical demands are met. An evaluation of the Senior Author's team experience (1973-90) in the management of acutely obstructing left colon cancer (49/291 or 17%) provides information on multi-stage resections and one-stage subtotal colectomy (Group A and B) as regards operative mortality (10% in Group A, 9% in Group B) as well as length of hospitalization (21 days in Group A, 9 days in Group B).(ABSTRACT TRUNCATED AT 250 WORDS)

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The experience of the senior author in the management of biliary tract lithiasis and/or associated pathology is analyzed, retrospectively until 1976 and prospectively from then on, in an attempt to ascertain the most efficient manner of handling a duct requiring surgical exploration. Primary surgery was done on 245 patients, while 22 common bile duct reoperations were carried out on 20 patients. In the primary surgery group, 177 simple cholecystectomies were undertaken, and an indication for duct exploration was present in the other 68. Out of 90 common bile duct explorations, 68 primary ones plus 22 reoperations, two operative deaths occurred (one in each group). Fifteen patients had a choledocholithotomy with temporary T-tube decompression, with "normal' postexploratory and predischarge tube cholangiograms. Six of these (40%) required reoperation (recurrent stones in three, residual calculus in two, stenotic papilla in one). Of 70 ducts definitively drained (60 choledochoduodenostomies, nine sphincteroplasties, one Y-loop hepaticojejunostomy) only one (1.4%) of the patients who had a sphincteroplasty has had an episode of jaundice and cholangitis, a highly significant difference (p = 0.001). This experience suggests that a correct biliary fenestration, permanently decompressing the biliary tree, performed during the initial operation will avoid many unnecessary hospital admissions and should, therefore, be seen as the procedure of choice in the overwhelming majority of situations when a pathological common bile duct is encountered.

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