Laparoscopic versus open incisional hernia repair: a single-institution analysis of hospital resource utilization for 884 consecutive cases.

BACKGROUND: To analyze hospital resource utilization for laparoscopic vs open incisional hernia repair including the postoperative period. METHODS: Prospectively collected administrative data for incisional hernia repairs were examined. A total of 884 incisional hernia repairs were examined for trends in type of approach over time. Starting October 2001, detailed records were available, and examined for operating room (OR) time, cost data, length of stay (LOS), and 30-day postoperative hospital encounters. RESULTS: Of the total, 469 incisional hernias were approached laparoscopically (53%) and 415 open (47%). Laparoscopic repair had shorter LOS (1 +/- 0.2 days vs 2 +/- 0.6 days), longer OR time (149 +/- 4 min vs 89 +/- 4 min), higher supply costs (2,237 dollars +/- 71 dollars vs 664 dollars +/- 113 dollars), slightly lower total hospital cost (6,396 dollars +/- 477 dollars vs 7,197 dollars +/- 1,819 dollars), and slightly more postoperative hospital encounters (15% vs 13%). Use of laparoscopy increased over time (37% in 2000 vs 68% in 2004). CONCLUSIONS: Laparoscopic incisional hernia repair is becoming increasingly popular, and not at increased cost to the health care system.

[Bone lengthening and soft tissue correction using the Ilizarov technique]. / Knochenverlängerungen und Weichteilkorrekturen mittels der Ilizarov-Technik.

From March 1989 to September 1995 at the Department of Orthopedic Surgery at the Vienna General Hospital 31 limb-lengthenings or corrections of the soft-tissue (contractions of joints, clubfeet) using the Ilizarov method were performed. 15 patients have already finished growth. All complications were analysed according to Paley's classification. The tibia-group (n=13) reached an average lengthening of 3.5 cm (2-5.7 cm) [16% (6-35%) of the initial length] with a healing index of 1.7 mo/cm and a complication rate of 42%. In the femur-group (n=8) a mean lengthening of 5.4 cm (2.5-9.4 cm) [21% (7-34%) of the initial length] could be achieved with a healing rate of 1.3 mo/cm and a complication rate of 40%. The ulna-group (n=3) reached an average lengthening of 2.6 cm (2.2-3.4 cm) [21% (17-24%) or the initial length] with a healing index of 1.4 mo/cm and a complication rate of 17%. The knee contracture group (n=2) was free of complications. The Ilizarov technique has been performed successfully in a high percentage although extensive elongations reported by Ilizarov could not be achieved neither by us nor by other authors. The above-mentioned method has been proved to be efficient and successful to correct deformities of the soft-tissue.

Clinical and sonographic evaluation of the risk of rupture in the Achilles tendon.

Chronic pain in the region of the Achilles tendon is a common problem and often a sign of progressive degeneration of the tendon which may lead to its rupture. We studied the clinical course and sonograms in 36 patients with achillodynia to find a prognostic parameter enabling us to estimate the risk of rupture. The patients were evaluated clinically for swelling and tenderness and by high-resolution real-time sonography. The sonograms were graded according to the tendon thickness as normal (< 6 mm), minimal (6-8 mm), moderate (8-10 mm) to high-grade (> 10 mm) in the sagittal diameter of the transverse section, and alterations of echotexture were described as diffuse, circumscribed, or inhomogenous. At the time of the primary investigation, we found thickening and alterations of the echotexture in 33 of 72 tendons. In 48 tendons we found pain and local or diffuse swelling in the Achilles tendon region (sensitivity 0.58, specificity 0.79). After a follow-up of 48 +/- 8 months, 7 tendons had ruptured spontaneously. Analysis of the sonograms of the patients taken prior to the rupture showed a high-grade thickening in 4 cases, moderate thickening in 2 cases, and a diameter between 6 and 8 nm in one patient. In no case did we find a rupture of a tendon primarily classified as normal. Patients without sonographic changes exhibited a significantly better clinical outcome following conservative treatment. Sonography was found to be a valuable tool for determination of the tendon's thickness and echotexture. In 28% of our patients with thickening, circumscribed lesions of the echotexture, and chronic pain, a spontaneous rupture occurred.

Time course of vinblastine-induced autophagocytosis and changes in the endoplasmic reticulum in murine pancreatic acinar cells: a morphometric and biochemical study.

The time course of the vinblastine(-sulfate; 10 mg/kg body weight, single injection)-induced enlargement and subsequent regression of the autolysosomal compartment was studied by electron microscopic morphometrical and cell biochemical methods in order to gain information concerning some key problems of this major route of intralysosomal degradation of the cell's endogenous macromolecules and structures. Detailed analysis of the dynamics of the total autophagic vacuole (AV) compartment and its different subcompartments (early, advanced, late, and fused AVs), as well as of changes of rough-surfaced endoplasmic reticulum (RER) showed: 1. Pancreatic acinar cells react to vinblastine biphasically, i.e. two expansion phases of the AV compartment, the first in the 0 to 90 min and the second in the 2 to 8 h post-injectional periods, were detected. 2. Fusions of AVs are not inhibited by vinblastine, at least during the second expansion phase when cytoplasmic volume fraction (CVF) of fused AVs steadily increased until the 12th h. Fusion of early, advanced and late AVs or composition of fused complex vacuoles (AVc) are somehow regulated, as the proportion of the three AV stages from the CVF of AVc, was maintained constant throughout the second expansion phase. 3. Stimulation of autophagosome formation and resulting substrate overload seems to be the primary mode of action by which vinblastine causes the enormous expansion of the autolysosomal compartment. 4. Degranulation of the rough-surfaced endoplasmic reticulum (RER) membranes occurs in a biphasic fashion, similarly to the volume and surface changes of the AV compartment, thus supporting our previous hypothesis, that labilization or change of RER may have a role in the formation of autophagosomes. 5. Vinblastine-induced autophagocytosis is a selective process, as mitochondria, Golgi elements and zymogen granules are very much underrepresented, whereas RER is more than twice overrepresented in the volume of early AVs, when compared to their volume fraction in the whole cytoplasm. 6. Immunogold electron microscopy revealed the presence of ubiquitinylated proteins in advanced and late, but not in early AVs.

Related organelles of the endosome-lysosome system contain a different repertoire of ubiquitinated proteins in Sf9 insect cells.

Two components of the endosomal/lysosomal compartment of Sf9 cells, multivesicular bodies (MVB) and light vacuoles with membrane complexes (LVMC) have been isolated and probed for ubiquitin protein conjugates with a specific antibody. Immunogold electron microscopy indicates that whereas ubiquitin-protein conjugates are localised to electron dense areas of MVB they are associated with the membranes of LVMC. Five ubiquitinated polypeptides are revealed in MVB by immunoblotting while numerous ubiquitinated species forming a smear following electrophoresis are present in LVMC. We suggest two possible routes for entry of ubiquitin-protein conjugates into these organelles, via the cell surface and via primary lysosomes.

Extendable tumour endoprostheses for the leg in children.

From 1986 to 1991 we fitted 20 children with endoprostheses after resection of malignant bone tumours of the leg; six have reached skeletal maturity and are the subject of this study. Reconstruction of defects in growing limbs in which the eventual shortening can be predicted requires the use of extendable prostheses. The mean age at operation was 11 years (9.2 to 13.7) and the average follow-up period was 6.3 years (4.3 to 7.6). The diagnosis was osteosarcoma in five patients and Ewing's sarcoma in one. All tumours were Enneking stage-IIB. When seen for follow-up all patients were free from disease. The extendable implants used included the Pafford-Lewis prosthesis and the Kotz Modular Femur Tibia Reconstruction system with a compatible, newly-designed growth module. Telescope-like elongation of the prostheses was performed by insertion of a screwdriver through a small skin incision. Active epiphyseal growth in the adjacent growth plate was preserved by using prosthetic stems with a smooth surface. The mean length gained was 13.15 cm (4.5 to 19.5) requiring 53 planned procedures. Seven revision operations were necessary for complications. Functional evaluation showed excellent and good results in all cases. Stress-shielding at the site of anchorage of the prosthesis was more pronounced than in adults. Implantation of extendable endoprostheses in children provides a reasonable alternative to rotationplasty, but limb salvage requires more operations.

[Results of treatment of primary exclusively pulmonary metastatic Ewing sarcoma. A retrospective analysis of 41 patients]. / Behandlungsergebnisse beim primär ausschliesslich pulmonal metastasierten Ewingsarkom. Eine retrospektive Analyse von 41 Patienten.

41 patients presenting with primary metastatic Ewing's sarcoma or malignant peripheral neuroectodermal tumor (PNET) with initial metastases restricted to the lungs and/or pleural space were analysed with respect to clinical manifestation and treatment results retrospectively. All patients were treated according to the protocols CESS 81 and CESS 86 of the German Society of Pediatric Oncology and Hematology (GPOH). The time since diagnosis ranges from 19 to 137 months, with a median of 72 months. Median relapse-free survival time was 21.8 months. 18 patients were female, 23 were male. The majority of primary tumors exceeded 100 ml of volume. Preferred sites were the pelvis with 16 cases, the limbs with 14 cases and the chest wall with 6 cases. The histological specification of the tumor was Ewing's sarcoma in 22 and PNET in 11 patients, in 8 cases no specific distinction was given. As to local therapy of the primary tumor, 12 patients underwent radiotherapy, 11 surgery, and 18 a combination of both. Patients were allocated to one of these three options on an individual basis. Cytostatic drug treatment was given according to the GPOH-CESS 81 and CESS 86 protocols. As calculated by means of the Kaplan-Meier analysis, relapse-free survival was 30% ten years after diagnosis. Surgery or pulmonary irradiation of 12-20 Gy was applied to lung metastases. 12 of 27 patients are in continuous complete remission following this therapeutic approach.

Borderline chondrosarcoma of long and flat bones.

We reviewed histological and clinical findings of six cases of borderline chondrosarcoma and examined the expression of collagen types I, II, III, V, and VI by immunohistochemical analysis of these tumors. Borderline chondrosarcoma is defined as a cartilaginous tumor of bone resembling enchondroma on the basis of histomorphology. Clinically the tumor causes intermittent vague pain unrelated to physical activities. On radiographs borderline chondrosarcoma is characterized by evidence of endosteal erosion. We observed local recurrences in two cases treated by intralesional excision and marginal excision, and one of those cases died of inoperable local tumor recurrence. In our histological analysis based on tissue patterns, there were enchondromatous patterns in five cases, and chondrosarcomatous patterns in four cases. In the second recurrent tumor in one case, a chondrosarcomatous pattern was newly observed, and the recurrent tumor was found to be a low-grade chondrosarcoma cytologically in the other case. In the tumor matrix immunoreactivity for collagen types II and VI was predominant, with collagen types I, III, and V showing heterogeneous expression in some cases. In all cases rimming of tumor lobules with collagen types I and V was absent. Immunoreactivity for collagen type II in the cytoplasm of tumor cells was found in four cases and all three recurrent tumors. Borderline chondrosarcoma, as defined by histology, clinical symptoms and radiological appearance, shows a collagen distribution pattern similar to that of low-grade chondrosarcoma. These findings are in accordance with the clinical outcome of borderline chondrosarcoma which parallels that of low-grade chondrosarcoma. Thus borderline chondrosarcoma may be best treated by wide en-bloc excision rather than curettage.

Comparison of cell surface antigen HBA71 (p30/32MIC2), neuron-specific enolase, and vimentin in the immunohistochemical analysis of Ewing's sarcoma of bone.

The HBA71 antigen is an M(r) 30,000/32,000 cell surface glycoprotein (p30/32MIC2), encoded by the pseudoautosomal MIC2 gene on chromosomes X and Y, that is expressed in Ewing's sarcomas. Immunohistochemical studies demonstrate a striking specificity for HBA71 among neoplasms of diverse histologic types. In the present study, 43 cases of Ewing's sarcoma of bone were tested for HBA71 expression and six additional immunohistochemical markers regularly used in the differential diagnosis of small round-cell tumors of childhood and adolescence (neuron-specific enolase, vimentin, leukocyte common antigen, cytokeratins, muscle-specific actin, desmin). The study design included (a) random selection of Ewing's sarcoma cases from the files of Memorial Hospital beginning in 1968, (b) blind review of the original histopathologic diagnoses of ES, (c) side-by-side immunohistochemical study of recut histologic specimens, and (d) statistical analysis of immunohistochemical findings in view of clinical outcome. Of the seven antigens studied, only HBA71, neuron-specific enolase and vimentin were expressed in a significant proportion of cases. Forty-one of the 43 cases were HBA71+ (95% sensitivity); of these, 21 were neuron-specific enolase+, 29 were vimentin+, and 15 were both neuron-specific enolase+ and vimentin+. One tumor lacked all antigens, and one was vimentin+ only. Comparison of tumor tissues in five patients obtained before and after cytostatic chemotherapy showed no change in HBA71 expression or in the other antigens tested. Product-limit survival analysis (median disease-free survival was 27.3 months for the study cohort) revealed no significance of neuron-specific enolase or vimentin marker status. These results raise doubts about the usefulness of neuron-specific enolase and vimentin immunohistochemistry to distinguish Ewing's sarcoma from other small round-cell tumors of childhood and adolescence or as prognostic indicators in Ewing's sarcoma. The positive identification of Ewing's sarcoma of bone now becomes a reality using HBA71 immunohistochemistry, either as a sole method or in combination with chromosomal breakpoint analysis. This may result in achieving uniform diagnostic criteria for evaluating the biologic, therapeutic, and prognostic aspects of Ewing's sarcoma and related neoplasms.

The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats.

Subcutaneous implants of a recombinant human form of the bone-inducing protein rhBMP-2 (recombinant human bone morphogenetic protein-2) in rats have resulted in the local induction of endochondral bone formation. To test the osteoinductive activity of rhBMP-2 in an osseous location, we created five-millimeter segmental defects in the femora of forty-five adult male Sprague-Dawley rats. Two doses of lyophilized rhBMP-2 (1.4 or 11.0 micrograms) were implanted in each defect, together with guanidine-hydrochloride extracted demineralized rat-bone matrix as a carrier, and the results were compared with those in rats that had implantation of guanidine-hydrochloride extracted demineralized rat-bone matrix only. The formation and healing of bone were determined by radiographic, histological, and mechanical analysis. Both doses of rhBMP-2 induced formation of endochondral bone in the osseous defects in a dose-related manner. Implantation of 11.0 micrograms of rhBMP-2 yielded significant (p less than 0.05) bone formation, resulting in radiographic, histological, and mechanical evidence of union. Despite new-bone formation in the defects that had received 1.4 micrograms of rhBMP-2, no instances of union were observed.

Dynamics of vinblastine-induced autophagocytosis in murine pancreatic acinar cells: influence of cycloheximide post-treatments.

Accumulation of autophagic vacuoles (AVs) was monitored by electron microscopic morphometry in murine pancreatic acinar cells during the 5-hr period after a single injection of vinblastine (VBL). The expansion of the autophagic compartment (AC) occurred in two waves. AVs accumulated in the first 90 min and regressed in the next hour, but thereafter AC expanded again, and 5 hr following the VBL injection, as much as 5.3% of the cytoplasmic volume was found sequestered into the AC. The high rates of accumulation of AVs indicated that VBL stimulated AV formation (segregation) during both expansion phases. To have a deeper insight into the dynamics of the process segregational inhibitor cycloheximide (CHI) was given 1 and 3 hr after VBL and the subsequent regression of the AC and its subcompartments (i.e., early, advanced, and late AVs) were measured during the next 90 min. We found that regression of AVs was fast in the first expansion and slowed down in the second expansion phase during which only early AVs regressed. CHI proved to be a fast and effective inhibitor of autophagic segregation, whether it was given before, simultaneously, or after the VBL injection. The aforementioned results argue for a dual mode of action of VBL (i.e., a prompt stimulation of segregation and a delayed retardation of AV maturation). The two effects of the alkaloid prevail differently along the time course. A further analysis of the behavior of the AC subcompartments showed that CHI perhaps inhibits segregational step(s) occurring prior to the actual formation of the autolysosomes.

Immunohistochemical analysis of neural cell adhesion molecules. Differential expression in small round cell tumors of childhood and adolescence.

The neural cell adhesion molecule (NCAM) was discovered in a search for cell surface antigens of chicken neurons that contribute to cell adhesion and pattern formation during development. Homologous adhesion molecules have been identified in several species, including humans. In this immunohistochemical study, the authors examine the role of human NCAM in tumor diagnosis. The authors used a monoclonal antibody (MAb), 5.1H11, to examine NCAM immunoreactivity in frozen sections of more than 450 tumors, including more than 80 small round cell tumors (SRCT) of childhood and adolescence (neuroblastomas, Ewing's sarcomas [ES], peripheral neuroepitheliomas [PN], primitive neuroectodermal tumors [PNET], esthesioneuroblastomas, malignant ectomesenchymoma, medulloblastomas, small cell osteosarcomas, mesenchymal chondrosarcomas, embryonal rhabdomyosarcomas, and lymphomas). The authors show that 1) neuroblastomas and primary brain tumors are NCAM+; 2) ES, most PN/PNETs, and melanomas are NCAM-; 3) embryonal rhabdomyosarcomas and various other sarcomas are NCAM+; 4) neuroendocrine tumors are NCAM+; 5) subsets of carcinomas of kidney, ovary, lung and other organs are NCAM+; and 6) lymphoid tumors are NCAM-. Tests with normal fetal and adult tissues indicate that these findings reflect only in part the NCAM phenotypes of corresponding normal tissues. Notably the NCAM- phenotype of ES and PN/PNET is not explained by current histogenetic models for these tumors, which suggest a primitive neuroectodermal origin. Finally the authors show that NCAM expression among SRCT has an inverse relationship with the expression of p30/32MIC2, a cell surface antigen of ES and PN/PNET detected with MAb HBA71. These results suggest that immunohistochemical assays for NCAM and p30/32MIC2 expression may aid in the further characterization of SRCT of childhood and adolescence.

Immunohistochemical analysis of Ewing's sarcoma cell surface antigen p30/32MIC2.

Monoclonal antibody (MAb) HBA71, which was raised against Ewing's sarcoma cells, recognizes a cell-surface glycoprotein, p30/32MIC2, that is encoded by the MIC2 gene in the pseudoautosomal region of human chromosomes X and Y. This immunohistochemical study evaluates the specificity and sensitivity of MAb HBA71 for tumor diagnosis. Frozen and paraffin-embedded tissues of more than 300 tumors of diverse histologic type, including more than 100 small round cell tumors of childhood and adolescence, were tested with this MAb by the avidin-biotin immunoperoxidase procedure. The authors found HBA71 immunoreactivity in 61 of 63 Ewing's sarcomas studied and 9 of 11 primitive neuroectodermal tumors and peripheral neuroepitheliomas. HBA71-negative tumors included neuroblastomas (0 of 24), melanomas (0 of 13), an esthesioneuroblastoma, small cell osteosarcomas (0 of 2), a malignant ectomesenchymoma, desmoplastic SRCT (0 of 5), and medulloblastomas (0 of 5). Heterogeneous expression of HBA71 immunostaining was found in some embryonal rhabdomyosarcomas (3 of 14) and astrocytomas (4 of 7), and in a few neuroendocrine tumors (4 of 26), carcinomas (3 of 94), and lymphomas (6 of 30). Because Ewing's sarcomas are consistently HBA71 positive, the authors searched for antigen-positive normal cells that may represent precursors for these tumors; however, no obvious candidate for the elusive cell of origin for Ewing's sarcoma was identified in the normal fetal tissues tested. Their findings indicate that HBA71 is a highly restricted cell-surface antigen of Ewing's sarcomas and primitive neuroectodermal tumors, and immunohistochemistry employing this antibody may be of value in the differential diagnosis of selected small round cell tumors in childhood and adolescence.

Biochemical and genetic characterization of the HBA71 Ewing's sarcoma cell surface antigen.

Monoclonal antibody HBA71 detects a cell surface antigen of human Ewing's sarcomas and peripheral neuroepitheliomas that distinguishes these tumors from other small round cell tumors of childhood and adolescence. In the present study, we show that monoclonal antibody HBA71 reacts with polypeptides of Mr 32,000 and 30,000 and that the HBA71-coding gene segregates with human chromosomes X and Y in rodent-human hybrids. Therefore, we compared HBA71 to the T-cell leukemia antigen 12E7, which is encoded by the pseudoautosomal region of chromosomes X and Y. We show that monoclonal antibodies HBA71 and 12E7 (a) detect polypeptides of identical size, (b) react with mouse cells transfected with complementary DNA corresponding to the 12E7-coding gene, MIC2, and (c) give similar patterns of reactivity with human tumor cell lines and small round cell tumor tissues. Thus, HBA71 and 12E7 are identical or closely related antigens and the available MIC2 probes will facilitate analysis of the molecular mechanisms that determine differential HBA71 expression in small round cell tumors of childhood and adolescence.

Time course of neutral red-induced autophagy in murine pancreatic acinar cells. A morphometrical and cytochemical study.

The knowledge of the time course of the influences of chemicals on autophagy is of great importance in the study of their modes of action and hence provides information relating the mechanism and dynamics of this catabolic process. Neutral red (NR) treatment has long been used to produce an accumulation of autolysosomes in different cell types. In the present study early (AV1), advanced (AV2) and late (AV3), as well as complex (fused) AVs (AVc) were distinguished. In our morphometrical measurements, we found all these AV subcompartments significantly expanded as early as 30 min after the injection of NR (0.4 mg/g b.wt.), i.e. a large number of AVs accumulated in the cells. Since cytoplasmic volume fraction (CVF) of AV increased 3-fold during this early period we conclude that, unlike vinblastine, NR is not a fusion inhibitor. Accumulation of AV1 (3-fold) in the presence of fusions possibly indicates that NR stimulates formation of AVs in this early period, after the accumulation of AVs continued. The maximal CVF of AVs were measured at 4 h, when 7.6% of the cytoplasmic volume was sequestered into the AV compartment, two third of which came from AV3. This finding indicates that NR is probably an inhibitor of intravacuolar degradation. However, the high rate of accumulation of AV2, AV3, and total AVs including a slower but still pronounced accumulation of AV1 cannot be explained solely from inhibition of degradation, but indicates a stimulated segregation (AV formation). Our results therefore argue for a possible coupling of the regulation of autophagic segregation and degradation since vinblastine and possibly some other degradation inhibitors were also found to stimulate AV formation in other studies. Another goal of this study was to follow the time course of changes in distribution of certain lysosomal enzymes after NR treatment. According to our enzyme cytochemical studies, acid phosphatase (AP) of untreated cells is mainly located in large and small lysosomal elements of the Golgi zone, aryl sulfatase B (AS) in trans-Golgi elements including pre-secretory granules and trimetaphosphatase (TP) in basal lysosomes. After NR injection TP seemed to appear first in AV1 whereas AP activity was characteristic of more advanced AVs. AS activity only occasionally appeared in AV3 and exclusively at late times after NR injection.

Time course of vinblastine-induced cellular autophagy in the murine pancreatic acinar cells in vivo. Different regression rates of the autophagic compartment caused by cycloheximide given different times after the vinca alkaloid. A morphometric study.

Autophagy is a three-step process in which parts of cytoplasm are segregated by membranes to form autophagosomes gaining acid hydrolases later, being converted this way into autolysosomes in which lysosomal degradation takes place. The actual size of the autophagic vacuole compartment (AVC) is obviously dependent on the velocity of these main steps. According to our morphometric measurements, a single dose (10 mg/kg b.wt.) of vinblastine (VBL) caused a conspicuous expansion of the AVC in pancreatic acinar cells, occurring in two waves: it expanded in the first 90 min but regressed in the next hour. This was followed by a second expansion monitored until the 5th post-injectional hour. The expansion rates indicate the existence of stimulation of autophagic segregation in both expansion phases. To take a further look, into the dynamics of the process, we blocked segregation by giving cycloheximide (CHI 0.2 mg/g b.wt.) 1 and 3 h after VBL and the subsequent regression of the AVC was followed by morphometry in the next 90 min. At the height of the first wave (1-2 h after VBL) the regression of AVC was not retarded, but rather, degradation rate seemed elevated. When CHI was given 1 h after VBL, 92% of the cytoplasmic volume fraction (CVF) of AVC regressed within the next 30 min. The main factor causing the expansion of AVC might be enhanced segregation in the first wave. Contrarily, at the beginning of the second wave, the turnover of AVs is dramatically slowed down. When CHI was given 3 h after VBL, only 27% of CVF of AVC regressed in the next 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence and inhibition by cycloheximide of apoptosis in vinblastine-treated murine pancreas. A role for autophagy?

While investigating the time course of the influence of autophagy-inducer vinblastine in pancreatic acinar cells, we discovered that about 24 h after the single injection of the drug (10 mg/kg b.wt.) when most of the cells were already recovering from the autophagic wave, dying cells with apoptotic nuclei and peculiar morphology of their cytoplasm appeared in the exocrine pancreas. Apoptotic blebs (ABs) separating from apoptotic cells or already phagocytized by neighbouring cells were also also observed. A large number of autophagic vacuoles (AVs) were seen in these cells termed apoptotic cell (AC). They are most probably derived from cells with morphologically normal nuclei but with unusually high number of AVs in their cytoplasm. We termed these cells highly autophagic cells (HAC). Our morphometric measurements show that the partial volume of both HAC and AC increased to 7.5 and 9.5%, respectively, of total cellular volume in the 25th h after vinblastine treatment. This increase could be inhibited by a treatment at the 24th h with cycloheximide (0.2 mg/g b.wt.) an inhibitor of both translation and autophagic segregation. Thus, synthesis of proteins or an enhanced autophagy may be indispensable step(s) in the apoptotic process in this system.

Mechanism and dynamics of macroautophagy in murine exocrine pancreatic cells. A review of vinblastine-induced changes.

Autophagy is a major pathway of lysosomal degradation of cellular macromolecules. The paper summarizes the results obtained in the studies on macroautophagy using the exocrine pancreatic acinar cell as model system and vinblastine as inducer. Current knowledge about the origin and properties of the limiting membranes of autophagic vacuoles, and the results of quantitative morphological studies into the dynamics and kinetics of vinblastine-induced autophagocytosis, as well as recent achievements in isolation and characterization of subclasses of autophagic vacuoles (autophagosomes and autolysosomes) are reviewed.

Time course of quantitative morphological changes of the autophagic-lysosomal compartment of murine seminal vesicle epithelial cells under the influence of vinblastine.

Changes of the autophagic-lysosomal compartment (ALC) of the murine seminal vesicle epithelial cells were monitored by electron microscopic morphometry during 36 h following a single 10 mg/kg bw dose of vinblastine sulfate (VBL), a widely used tool to cause an accumulation of the autophagic vacuoles (AVs). Three morphologically distinct subcompartments of the ALC, i.e. early autophagic vacuoles (AV1) being presumably prelysosomal autophagosomes, advanced AVs (AV2) containing material under degradation and dense bodies (DB) were defined. The ALC and its subcompartments expanded after VBL in a two-phase reaction. The first subcompartments to react significantly were AV1 and AV2 (at 30 min) followed by DBs with a 30 min delay. The ALC then ceased to grow until the 90th min when a second expansion phase started peaking around 8 h with a cytoplasmic volume fraction 15 times larger than in the untreated control. This second growth was entirely brought about by the expansion of the two AV subcompartments. After 8 h the volume fraction of both AV1 and AV2 decreased to cause a gradual regression of the ALC. AV1, however, already ceased to expand as early as after 6 h, i.e. during the last 2 h of the expansion phase of the ALC. Comparison of this time curve with the one we previously measured in mouse liver shows considerable differences between the two cell types. The growth curves of the AV subcompartments in our experiment along with others' kinetic data obtained in steady state cells not treated with VBL show that segregation (= formation of AV1) is possibly stimulated by VBL in our system.

Isolation of pancreatic autophagic vacuoles induced by vinblastine or neutral red. Separation of autophagosomes and autolysosomes by Percoll density gradient centrifugation.

Our purpose was to elaborate a cell fractionation method for the preparation and purification of macroautophagic vacuoles (AVs) and their subfractions: autophagosomes and autolysosomes. To overcome the difficulties caused in liver and some other cell types by the overlapping buoyant densities and sizes of different subclasses of lysosomes and other subcellular particles, we chose the murine pancreatic acinar cell as experimental system in which enormous numbers of large-sized AVs are readily accumulated upon certain treatments. As we measured by electron microscopic morphometry, cytoplasmic volume fraction of AVs was as small as 0.31% in the untreated cells, while it was elevated to 8.1% 4 h after the injection of 50 mg/kg body weight vinblastine sulfate (a widely used inducer of macroautophagy). From vinblastine-treated pancreas, a 5500g sediment containing AVs and mitochondria (AV-M fraction) was obtained by differential centrifugation. This fraction was resolved in a 50% Percoll gradient (15 min, 92,000g) into three distinct particle populations. Mitochondria were localized near the upper boundary of the gradient at a buoyant density of 1.075 to 1.08, whereas directly under them light AVs (1.085-1.09) were banded. Heavy AVs (1.13-1.14) formed a broad layer near the bottom of the tube. Electron microscopic comparison of the morphology of these fractions and AVs in situ showed that light AVs correspond to AVs in early, whereas heavy AVs to AVs in advanced and late stages of degradation of the segregated material. The activity of lysosomal enzymes were found low in both fractions, being several times higher in the heavy than in the light one.(ABSTRACT TRUNCATED AT 250 WORDS)