Ascorbic acid enhances destruction of Escherichia coli O157:H7 during home-type drying of apple slices.

Destruction of Escherichia coli O157:H7 was evaluated on inoculated apple slices dehydrated at two temperatures with and without application of predrying treatments. Half-ring slices (0.6 cm thick) of peeled and cored Gala apples were inoculated by immersion for 30 min in a four-strain composite inoculum of E. coli O157:H7. The inoculated slices (8.7 to 9.4 log CFU/g) either received no predrying treatment (control), were soaked for 15 min in a 3.4% ascorbic acid solution, or were steam blanched for 3 min at 88 degrees C immediately prior to drying at 57.2 or 62.8 degrees C for up to 6 h. Samples were plated on tryptic soy (TSA) and sorbitol MacConkey (SMAC) agar media for direct enumeration of surviving bacterial populations. Steam blanching changed initial inoculation levels by +0.3 to -0.7 log CFU/g, while immersion in the ascorbic acid solution reduced the inoculation levels by 1.4 to 1.6 log CFU/g. Dehydration of control samples for 6 h reduced mean bacterial populations by 2.9 log CFU/g (TSA or SMAC) at 57.2 degrees C and by 3.3 (SMAC) and 3.5 (TSA) log CFU/g at 62.8 degrees C. Mean decreases from initial inoculum levels for steam-blanched slices after 6 h of drying were 2.1 (SMAC) and 2.0 (TSA) log CFU/g at 57.2 degrees C, and 3.6 (TSA or SMAC) log CFU/g at 62.8 degrees C. In contrast, initial bacterial populations on ascorbic acid-pretreated apple slices declined by 5.0 (SMAC) and 5.1 (TSA) log CFU/g after 3 h of dehydration at 57.2 degrees C, and by 7.3 (SMAC) and 6.9 (TSA) log CFU/g after 3 h at 62.8 degrees C. Reductions on slices treated with ascorbic acid were in the range of 8.0 to 8.3 log CFU/g after 6 h of drying, irrespective of drying temperature or agar medium used. The results of immersing apple slices in a 3.4% ascorbic acid solution for 15 min prior to drying indicate that a predrying treatment enhances the destruction of E. coli O157:H7 on home-dried apple products.

A case-matched molecular comparison of extraovarian versus primary ovarian adenocarcinoma.

BACKGROUND: Extraovarian müllerian adenocarcinoma (EOM) resembles primary ovarian carcinoma (POC) both histologically and clinically, yet little is known regarding the molecular genetic characteristics of this entity. The objective of this study was to compare the expression of three molecular markers of tumor behavior in EOMs and POCs. METHODS: Forty-four patients meeting strict criteria for EOM were identified and matched to POC controls for age, stage, tumor histology and grade, cytoreductive surgery, and survival. Immunohistochemistry was used to determine overexpression of p53 and HER-2/neu. DNA content was evaluated by flow cytometry. Direct DNA sequencing of exons 5-8 of the p53 gene was performed in nine EOM tumors. Statistical comparisons were made using chi-square, Kaplan-Meier, and Mantel-Cox log rank methods. RESULTS: Overexpression of HER-2/neu was demonstrated in 59% (26 of 44) of the EOM group versus 36% overexpression (16 of 44) in the POC controls (P = 0.05). Overexpression of p53 was noted in 48% of the EOM cases, similar to the 59% incidence observed in the control group (P = 0.29). Missense mutations were found in 9 of 9 EOM tumors showing strong p53 nuclear immunostaining. No significant difference in the incidence of aneuploidy was observed when EOM cases were compared with POC controls (65% vs. 63%). High tumor grade was strongly associated with HER-2/neu overexpression in the EOM group (P = 0.002). None of the parameters studied were predictive of prognosis within the EOM and POC groups. CONCLUSIONS: Although overexpression of p53 protein, p53 gene mutations, and abnormal DNA content were similar between EOMs and POCs, EOMs demonstrated almost twice the rate of HER-2/neu overexpression. This result suggests that distinct genetic events may be responsible for malignant transformation in EOMs versus POCs.

Clonal expansions of activated gamma/delta T cells in recent-onset multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease characterized by focal demyelination of the white matter of the brain and spinal cord. Central nervous system damage appears to be mediated by infiltrating T lymphocytes and macrophages, and a central role for autoreactive CD4+ T cells has been proposed. However, the initial immune events that lead to the chronic process of MS remain unidentified. We now present evidence that a subset of T lymphocytes bearing gamma/delta T-cell antigen receptors has been activated in patients with recent-onset disease. Cells recovered from the cerebrospinal fluid of subjects with MS were cultured for short periods of time in medium supplemented with T-cell growth factors. Expansions of V delta 1 and V delta 2 T-cell receptor-bearing lymphocytes were found only in cell populations obtained from subjects with recent-onset disease. Similar populations were not expanded in subjects with chronic MS or other neurological diseases. Junctional region sequencing showed the expanded gamma/delta T cells to be oligoclonal in nature, suggestive of specific stimulation by antigen. These results reveal a fundamental difference in the immunopathogenesis of acute vs. chronic disease and provide additional insight into the autoimmune nature of MS.

The effect of high-dose steroids on MRI gadolinium enhancement in acute demyelinating lesions.

Gadolinium (Gd) enhancement of brain lesions by MRI is a marker of active blood-brain barrier damage secondary to an inflammatory process. We studied the effects of high-dose (1,000 mg/d) intravenous (IV) methylprednisolone (Mp) for 4 to 8 days on Gd-enhancing lesions in seven patients with acute demyelinating diseases and compared pretreatment brain MRIs with studies obtained 1 to 4 days after treatment. Five patients had complete suppression, and two had significant suppression of Gd enhancement following treatment. In addition, six of seven patients had Gd-enhancing lesions that explained their clinical signs; in five of six of these patients, suppression of the Gd-enhanced lesions temporally correlated with clinical improvement. Thus, short courses of high-dose IV Mp suppress Gd enhancement in acute demyelinating lesions, and this correlates with clinical improvement.

Immunophenotypic markers in renal cell carcinoma.

UNLABELLED: We studied immunophenotypic and tumor cell markers in renal cell carcinoma (RCC) to determine if there are patterns of expression which may correlate with biologic behavior and response to therapy. Fourteen RCCs from 13 patients were stained by the immunoperoxidase technique using primary antibodies to Leu 4, Leu 14, Leu 2a, Leu 3a and b, lysozyme, dendritic reticulum cell (DRC), S-100, HLA-DR, epithelial membrane antigen (EMA) and beta-2-microglobulin (B2-MG). Staining was correlated with tumor stage, nuclear grade, histologic patterns, degree of cellular infiltrate, and clinical followup. Four RCCs were stage T1, four T2, five T3, and one T4. Most tumors were clear or granular cell type, with a solid or tubular growth pattern. The number of infiltrating lymphocytes and monocytes correlated with tumor grade and stage. Tumor-infiltrating lymphocytes (TILs) were predominantly Leu 3-positive (T-helper phenotype). B-cell markers were negative. Dendritic cells were rare. HLA-DR was present on endothelial cells in 11 tumors and on tumor cells in ten. HLA-DR expression increased with tumor grade. Tumor cells expressed EMA in 12 cases; B2-MG in four cases. Two patients, stages 3 and 4, died at 2 and 6 mo. CONCLUSIONS: (a) T-helper cells and monocytes infiltrate RCCs. Their numbers increase with tumor grade and stage. (b) HLA-DR expression by tumor cells tends to correlate with increasing stage and grade. (c) Dendritic cells are infrequent in RCCs.

The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression.

Posttransplant lymphoproliferative disorders (PTLDs) were diagnosed in 43 patients from the Pittsburgh-Denver series between June 1980 and March 1987. This constitutes a detection rate of 1.7%. Major categories of clinical presentation included a mononucleosislike syndrome, gastrointestinal/abdominal disease, and solid organ disease. The median time of onset in patients initially immunosuppressed with cyclosporine-A (CsA)-containing regimens was 4.4 months after transplant, regardless of tumor clonality. A strong association of PTLD with Epstein-Barr virus (EBV) was observed. A histologic spectrum of lesions from polymorphic to monomorphic was observed. Whereas polymorphic lesions could be either clonal or nonclonal, monomorphic lesions appeared to be clonal in composition. The presence of large atypical cells (atypical immunoblasts) or necrosis did not appreciably worsen the prognosis. Twelve patients had clonal, 13 had nonclonal, and five had both clonal and nonclonal tumors. Clonality was indeterminate in 13 cases. Most patients were treated with a regimen based on reduced immunosuppression and supportive surgery. Almost all nonclonal and about half of the clonal lesions respond to this conservative therapy, indicating that it is an appropriate first line of treatment. This behavior suggests that a spectrum of lesions ranging from infectious mononucleosis to malignant lymphoma constitutes the entity known as PTLD. Some monoclonal tumors can undergo regression, however, apparently in response to host immune control mechanisms. Because of its short latency and strong association with EBV, PTLD is an important model for the study of virus-associated tumor progression in humans.

Fibrolamellar carcinoma of the liver: an immunohistochemical study of nineteen cases and a review of the literature.

Hepatocellular carcinoma (HCC) is a rapidly fatal neoplasm of high worldwide prevalence. Fibromellar carcinoma (FLC), a variant of HCC, lacks the dismal prognosis of "ordinary" HCC (O-HCC) and is characterized by a diagnostic histologic appearance. The current study analyzes the clinical characteristics, immunohistochemistry, and treatment of nineteen cases of FLC. These data, together with a detailed review of the literature, further characterize this unique variant. FLC affects younger patients and lacks the male predominance of O-HCC. Also, FLC lacks specific association with cirrhosis, hepatitis B virus infection, use of oral contraceptives, and alcohol abuse, all of which are implicated in other hepatic tumors. This, along with differences in serum tumor marker prevalence (AFP, B12 binding protein) suggests that its pathogenesis differs from that of O-HCC. Despite these differences, FLC shares a common differentiation with O-HCC. The increased amounts in FLC of stainable alpha-1-antitrypsin, fibrinogen, and C-reactive protein, all of which are acute phase reactants and normal hepatocyte products, implies better differentiation of FLC cells. Finally, the better prognosis of FLC is supported by this study, since only two of the 19 patients died because of tumor. This contrasts with the reported survival of patients with O-HCC, usually measured in weeks. Hepatic transplantation may hold promise for future patients with "surgically unresectable" FLC as procedure-related complications are overcome.

Quantitative localization of Na-K pump sites in the frog sacculus.

The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [3H]ouabain autoradiography. Excised tissue was incubated for 10-30 min (23 degrees C) in micromolar concentrations of high specific activity [3H]ouabain (14-70 muCi ml-1, 5-15 Ci mmol-1), washed for 30 min (4 degrees C), then rapidly frozen (-175 degrees C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K+ (50 mM) in the incubation and (2) low specific activity [3H]ouabain (1 mM, 0.013-0.025 Ci mmol-1) indicated negligible nonspecific binding of the [3H]ouabain. Measurable levels of specific [3H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [3H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [3H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals. Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites micron-3 X 10(3), means +/- S.E.M.): saccule macula, 1.9 +/- 0.2; saccule perimacula, 1.1 +/- 0.1; saccule wall, 2.3 +/- 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (Sv). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and stereological data. The following values were obtained (sites micron-2 +/- 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites micron-2 +/- 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.

Quantitative localization of Na-K pump site in frog inner ear dark cells.

The Na-K pump site distribution within the dark cells of the frog inner ear was examined with quantitative freeze-dry [3H]ouabain autoradiography. Control experiments revealed that ouabain binding was specific. Na-K pumps were located in the basolateral dark cell plasma membrane and were distributed nonuniformly across the epithelial apical-basal axis. The highest concentration (sites per volume) was found over the basal region and the lowest over the apical region. The average pump site concentration for dark cells from four animals was 25 X 10(3) sites/micron3. Stereological analysis of conventionally fixed tissue revealed that the plasma membrane area per volume (Sv) was also nonuniform across the apical-basal axis with the highest surface to volume ratio in the basal region. The average Sv for two dark cell regions was 5.5 micron2/micron3. Combination of autoradiography and stereology revealed that the pump site density (sites per membrane area) was independent of position along the apical-basal axis and was equal to 4500 +/- 25%, a value close to the upper limit as determined by the diameter of the isolated Na-K-ATPase.

Postembryonic growth of the optic tectum in goldfish. II. Modulation of cell proliferation by retinal fiber input.

The proliferation of cells in the germinal zone of the optic tectum of adult goldfish was studied following unilateral optic nerve crush or removal of one eye. Dividing germinal cells were labeled with [3H]thymidine, which was injected at various times (0 to 30 days) following surgery; fish were sacrificed after short (48 hr) survival times. The numbers of labeled nuclei in the tectal germinal zones were compared on the two sides (intact and denervated). We show that permanent removal of optic input (by enucleation) resulted in a sustained depression of [3H]thymidine incorporation in the tectal germinal zone on the denervated compared to the intact side. Temporary denervation (by optic nerve crush) initially had a similar effect; however, upon reinnervation of the tectum by regenerating optic fibers, proliferation was enhanced on the experimental side compared to the intact side. Because cells in the germinal zone are known to produce new tectal cells, neurons as well as glia, in the normal growing adult brain (Raymond, P. A. and S. S. Easter, Jr. (1983) J. Neurosci. 3: 1077-1091), some of the proliferating cells may have been generating neurons. This inference is supported by the observation that in two fish whose right eye had been removed more than 2 years earlier, there were fewer neurons in the denervated tectum than in the intact tectum. Thus, it is likely that the observed decrease in incorporation of [3H]thymidine by cells in the germinal zone of the denervated optic tectum resulted in a slower rate of addition of new tectal cells on the affected side. We conclude that cytogenesis in the germinal zone of the growing optic tectum of adult goldfish is regulated by optic fiber input. This mechanism may be important in matching the rates of growth of retina and tectum in the normal brain of the growing adult fish.

Doubly projecting neurons of the dorsal column nuclei.

In a sample of 1700 neurons recorded from the dorsal column nuclei of the cat, 44 (2.6%) were found to send an axon down the dorsal spinal cord. Fully 70% of these caudally projecting neurons also sent an axon to the ventral thalamus. Nearly all had small cutaneous receptive fields distally on the forelimb and displayed response properties similar to other neurons of the dorsal column nuclei. Most were isolated along the lateral and medial margins of the cuneate nucleus rostral to the obex, and many were excited or inhibited by pericruciate cerebral stimulation. A few clearly were excited monosynaptically from the contralateral cerebral cortex at a latency that required the largest pyramidal tract fibers. These neurons probably comprise an important subset that regulates the flow of sensory information in spinal and brainstem somatic sensory pathways.