Organic acids and their salts as dipping solutions to control listeria monocytogenes inoculated following processing of sliced pork bologna stored at 4 degrees C in vacuum packages.

Postprocessing contamination of cured meats with Listeria monocytogenes has become a major concern for the meat processing industry and an important food safety issue. This study evaluated aqueous dipping solutions of organic acids (2.5 or 5% lactic or acetic acid) or salts (2.5 or 5% sodium acetate or sodium diacetate, 5 or 10% sodium lactate, 5% potassium sorbate or potassium benzoate) to control L. monocytogenes on sliced, vacuum-packaged bologna stored at 4 degrees C for up to 120 days. Organic acids and salts were applied by immersing (1 min) in each solution inoculated (10(2) to 10(3) CFU/cm2) slices of bologna before vacuum packaging. Growth of L. monocytogenes (PALCAM agar) on inoculated bologna slices without treatment exceeded 7 log CFU/cm2 (P < 0.05) at 20 days of storage. No significant (P > 0.05) increase in L. monocytogenes populations occurred on bologna slices treated with 2.5 or 5% acetic acid, 5% sodium diacetate, or 5% potassium benzoate from day 0 to 120. Products treated with 5% potassium sorbate and 5% lactic acid were stored for 50 and 90 days, respectively, before a significant (P < 0.05) increase in L. monocytogenes occurred. All other treatments permitted growth of the pathogen at earlier days of storage, with sodium lactate (5 or 10%) permitting growth within 20 to 35 days. Extent of bacterial growth on trypticase soy agar plus 0.6% yeast extract (TSAYE) was similar to that on PALCAM, indicating that the major part of total bacteria grown on TSAYE agar plates incubated at 30 degrees C was L. monocytogenes. Further studies are needed to evaluate organic acids and salts as dipping solutions at abusive temperatures of retail storage, to optimize their concentrations in terms of product sensory quality, and to evaluate their effects against various other types of microorganisms and on product shelf life. In addition, technologies for the commercial application of postprocessing antimicrobial solutions in meat plants need to be developed.

Microbial contamination occurring on lamb carcasses processed in the United States.

Lamb carcasses (n = 5,042) were sampled from six major lamb packing facilities in the United States over 3 days during each of two visits (fall or winter, October through February; spring, March through June) in order to develop a microbiological baseline for the incidence (presence or absence) of Salmonella spp. and for populations of Escherichia coli after 24 h of chilling following slaughter. Samples also were analyzed for aerobic plate counts (APC) and total coliform counts (TCC). Additionally, incidence (presence or absence) of Campylobacter jejuni/coli on lamb carcasses (n = 2,226) was, determined during the slaughtering process and in the cooler. All samples were obtained by sponge-sampling the muscle-adipose tissue surface of the flank, breast, and leg of lamb carcasses (100 cm2 per site; 300 cm2 total). Incidence of Salmonella spp. in samples collected from chilled carcasses was 1.5% for both seasons combined, with 1.9% and 1.2% of fall or winter and spring samples being positive, respectively. Mean (log CFU/cm2) APC, TCC, and E. coli counts (ECC) on chilled lamb carcasses across both seasons were 4.42, 1.18, and 0.70, respectively. APC were lower (P < 0.05) in samples collected in the spring versus fall or winter, while TCC were higher in samples collected in the spring. There was no difference (P > 0.05) between ECC from samples collected in the spring versus winter. Only 7 out of 2,226 total samples (0.3%) tested positive for C. jejuni/coli, across all sampling sites. These results should be useful to the lamb industry and regulatory authorities as new regulatory requirements for meat inspection become effective.

A patient acuity and staffing tool for stroke rehabilitation inpatients based on the FIM instrument.

We undertook to develop a tool based on the FIM instrument to predict the number of nursing hours required to care for stroke patients in an acute inpatient rehabilitation program. The initial study to evaluate the feasibility of using the FIM instrument revealed that the total FIM score had a strong inverse relation to the level of care indicated by the Patient Care Index (PCI) at days 1, 5, 7, 10, 15, and 20 of rehabilitation (rs = -.76 to -.87). The results warranted continued investigation of the FIM instrument as a guide for nurse staffing decisions. Based on data from the initial study, five categories of FIM score ranges were designated that demonstrated the most accuracy of placing patients at the correct level of care. Special care considerations unique to institutional settings were identified and incorporated into the tool's final format, as were the calculations to determine the amount of assistance needed. The study reported here was undertaken to evaluate the level of care indicated by the adapted tool, compared with that of the PCI, in a sample of 67 stroke admissions. Spearman correlations revealed a moderate relationship (rs = .49 to .54) between the amount of care determined by the Patient Acuity and Staffing tool and through the PCI at the first, second, and third team meetings. We conclude that the system is an effective, efficient guide for scheduling nurse staffing on the stroke rehabilitation unit.

Determining stroke rehabilitation inpatients' level of nursing care.

The purpose of this study was to evaluate the feasibility of using the Functional Independence Measure (FIM) to predict staffing needs of stroke patients in an acute inpatient rehabilitation program. The Patient Care Index (PCI) was concurrently administered with the FIM on all stroke admissions to a stroke rehabilitation unit over a 3-month period. One hundred fourteen patients 18 years of age or older admitted to the unit with a medical diagnosis of stroke were included in the sample. Total FIM score had a strong inverse relationship to the level of care indicated by the PCI at Days 1, 5, 7, 10, 15, and 20 of rehabilitation (r(s) = -. 76 to -.87). Total FIM score and the need for staff supervision for safety were the two factors predictive of the level of nursing care. The FIM has potential to guide nurse-staffing decisions.

Effects of microgravity on growing cultured skin constructs.

Understanding the cellular, chemical, and physical responses of cells to stimuli is critical to successfully engineering tissue. The effect of culturing a living skin equivalent (LSE) in a submerged microgravity environment was investigated. LSEs were developed by culturing normal human epidermal keratinocyte (NHEK) on a submerged fibroblast and type 1 collagen gel matrix. Once formed, LSEs were brought up to the air/liquid interface, and after 4 days, the cultures were maintained in either (a) a normal air/liquid interface (S), (b) resubmerged in media (R), (c) folded on themselves to enclose the keratinized layer (F/R), or (d) cut into 2-4-mm fragments and suspended in a state of microgravity in a NASA-designed bioreactor (B). All groups were cultured for an average of 3 additional days. LSEs were processed for histologic evaluation. Skin cells were stained for cytokeratin to evaluate function. Images were digitally captured and processed for analysis. Parameters, including epithelial thickness, cellular areas, nuclear number, nuclear area, cytoplasmic area, and stained cytokeratin areas were measured. Removing the air/media interface significantly increased the number of NHEKs present in the skin; microgravity greatly enhanced this effect (p < 0.0001). No significant difference in cellular function as measured by protein expression [stained cytokeratin area (micro(2)) per cell] was found among the groups, though the ratio of nuclear area was significantly increased in all three groups as compared to the S group (p = 0.00227). In the case of the R and F/R groups, this appears due to the loss of the NHEK layer associated with those groups. Additionally, significant nuclear hypertrophy was demonstrated in the B group (p < 0.0001), and cellular hyperplasia was measured in all submerged groups as compared to static (p < 0.0001). Elimination of the air/liquid interface enhanced proliferation of keratinocytes. This effect was further enhanced in the presence of microgravity. No significant effect on cell function was noted with the use of this microgravity environment. We hypothesize that the increased epidermal contact plays a role in this proliferation. Microgravity is also associated with nuclear and cellular hypertrophy over and above that of the submersion methods.

Comparison of sponging and excising as sampling procedures for microbiological analysis of fresh beef-carcass tissue.

Sponging and excising were evaluated as sampling procedures for microbiological analysis of beef-carcass tissue. Brisket tissue portions (10 x 10 cm) were inoculated with 2 ml of an Escherichia coli ATCC 25922 cell suspension (3 x 10(8)CFU/ ml). After 30 min, the portions were sampled by excising (EX) or swabbing (SP) with a sterile sponge and were analyzed for aerobic plate counts on tryptic soy agar and for total coliform counts and E. coli counts on Petrifilm E. coli count plates. Another set of inoculated samples was analyzed after being spray washed, in sequence, with water (6 s, 35 degrees C, 3.4 bar), acetic acid (2%, 6 s, 35 degrees C, 2.1 bar), water (20 s, 42 degrees C, 20.7 bar), and acetic acid (2%, 6 s, 35 degrees C, 2.1 bar). Additional samples were sampled for analysis after chilling at 7 degrees C for 24 h. Bacterial counts recovered were influenced (P < or = 0.05) by procedure of sampling (EX versus SP), time of sampling (0.5 versus 24 h), and by their interactions. Counts recovered 0.5 h after inoculation from unwashed or spray-washed samples were similar between the two sampling procedures (EX and SP). However, counts recovered after 24 h of sample storage were significantly (P < or = 0.05) lower for the SP compared with the EX procedure. The results indicated that as the carcass tissue was stored, recovery of bacteria by SP was less efficient than was recovery by EX.

The effect of leukocyte infiltration on apoptosis in an in vitro thermal injury bioartificial living skin equivalent model.

An in vitro bioartificial skin construct (BSC) model was studied to see how inflammatory infiltration affects apoptosis in skin that has been thermally injured. The BSC was used as a target organ. Control BSCs without leukocytes (CON) were burned (BCON) by scalding with phosphate-buffered saline heated to 70 degrees C for 6 seconds, and they were then cooled with room temperature phosphate-buffered saline for 15 seconds. Human alloimmunocytes were added to CON to create rejection cultures (REJ) and to BCON to create burned rejection cultures (BREJ). Slides were stained with hematoxylin and eosin and anti-Lewis antibody. In situ labeling of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Those sections that were immunostained for Lewis Y were analyzed for intensity stain index (ISI = [sigma P0 x I0]/ total tissue area in pixels). TUNEL was quantified with the following equation: no. of total apoptotic cells / total tissue area. Necrosis and blister formation in the epidermal layer were evident in BCON and BREJ. Pyknosis and nuclear fragmentation-indicators of apoptosis-were also present. Phagocytosis of keratinocytes by leukocytes was seen in REJ and BREJ. Immunostaining showed greater expression of Lewis Y antigen, as determined by ISI, in REJ as opposed to CON (58.2+/-2.3 vs. 36.4+/-2.3, respectively, P<.001), but no significant difference was found between BCON and BREJ (55.0+/-5.7 vs. 60.5+/-3.4, respectively) and REJ and BREJ (58.3+/-2.3 vs. 60.5+/-3.4, respectively). TUNEL staining indicated the presence of apoptosis as follows: REJ versus CON (0.0015+/-0.0002 vs. 0.0003+/-0.0001, respectively, P<.001); BREJ versus BCON (0.0031+/-0.0006 vs. 0.0018+/-0.0004, respectively, P<.05); REJ versus BREJ (0.0015+/-0.0002 vs. 0.0031+/-0.0006, respectively, P = .007). The presence of leukocytes and thermal injury induces apoptosis in BSC. The combination of these two variables results in increased apoptosis as determined by TUNEL. These findings suggest that a common pathway for skin injury may include inappropriate regulation of apoptosis exacerbated by a mechanism that includes inflammatory cellular infiltration.

Fas-mediated induction of hepatocyte apoptosis in a neuroblastoma and hepatocyte coculture model.

BACKGROUND: We have previously demonstrated an increase in hepatocyte apoptosis when they are cocultured with neuroblastoma cells. Death receptors in the tumor necrosis factor (TNF) family such as TNFR1 and Fas have been identified as regulators of apoptosis and may be responsible for the altered regulation of apoptosis seen in our coculture model. To evaluate the effects of released factors and remove the potential alterations induced by direct contact, a noncontact coculture system was used to study the interaction between hepatocytes and neuroblastoma cells. METHODS: Human Chang hepatocytes (HC) were plated onto Falcon cell culture inserts with 0.45-micrometer pores in the permeable membrane. Human neuroblastoma cells (NB-IMR-32) were seeded into wells of the Falcon companion plate. After 24 h, inserts containing HC were placed into wells containing NB cells and incubated for 4 days. This provided a coculture environment without actual cellular contact. Immunohistochemical staining for TNFalpha, Fas, and Fas ligand (Fas-L) was performed. Apoptosis was detected via the TUNEL method. Images were analyzed with ImagePro-Plus. Statistical analyses were done with significance determined at P < 0.05. RESULTS: Chang hepatocytes demonstrated a significant increase in the levels of TNF, Fas, and Fas-L when cocultured with neuroblastoma cells (P < 0.005). In addition, the cocultured hepatocytes had a 20-fold increase in the apoptotic rate (P < 0.001). Neuroblastoma cells had no demonstrable level of Fas or TNF when grown alone and in cocultures. Neuroblastoma cells that were grown alone had an elevated level of Fas-L, but this level diminished by 44% when cocultured with hepatocytes (P < 0.001). CONCLUSION: An upregulated TNF/Fas receptor-ligand system may be responsible for increased apoptosis in hepatocytes when cocultured with neuroblastoma. This upregulation may be due to release of neuroblastoma-derived Fas ligand into the media. Tumors may alter the regulation of apoptosis in surrounding tissues via the death receptors.

Induction of apoptosis in a neuroblastoma and hepatocyte coculture model.

BACKGROUND: The dysregulation of apoptosis may alter the progression of tumor growth and explain the clinical dichotomy observed in children with neuroblastoma (NB). An overexpression of the bcl-2 proto-oncogene induces resistance to apoptosis and has been observed in unfavorable NB. We hypothesized that alterations in apoptosis may be a result of the interactions between NB and the tissues surrounding it. MATERIALS AND METHODS: Human Chang liver cells (HCL, 10(4) cells/cm2) were plated in two-chamber slides for 3 days. Human NB cells (10(5) cells/cm2) were added to one of the chambers and incubated for 3 more days. Control NB were plated under identical conditions in its own medium and in the HCL medium with growth curves measured. DNA fragmentation was detected via the TUNEL method (TdT-mediated nick end-labeling) and bcl-2 expression was determined by immunostaining. RESULTS: NB growth was unaltered by the change in medium. NB stained mildly positive for bcl-2 when plated alone but became markedly positive in coculture. Histologically, HCL and NB appeared healthy when plated alone, but a halo of apoptotic HCL was seen around NB in the coculture. When plated alone, both NB and HCL demonstrated minimal apoptotic activity as detected via the TUNEL method. In the coculture, a halo of HCL surrounding the NB exhibited markedly increased DNA fragmentation and this intensity diminished in cells distant from the NB. CONCLUSIONS: The regulation of apoptosis was altered in this coculture model of NB and HCL. HCL stimulated NB to overexpress bcl-2 and presumably become resistant to apoptosis. Conversely, NB induced the surrounding HCL to undergo apoptosis. The interaction between the local tissue and NB induced alterations in apoptosis in both cell types and resulted in a survival advantage for NB.

A novel in vitro model for xenorejection and immune mechanisms using bioengineered living skin equivalents.

We hypothesized that an in vitro bioengineered skin (LSE) could be used for the study of xenogeneic inflammatory or immunosuppressive mechanisms. Murine fibroblasts (10(4)/mL) were mixed with type 1 rat-tail collagen to form a matrix (approximately 5 days) on which human keratinocytes (NHEK, 10(5)/75 microL) were seeded. The xeno-LSE was used as a rejection target organ in vitro. Rejection was achieved by the addition of human lymphocytes (10(6)/75 microL). At various intervals of growth, control LSEs without lymphocytes (CON) and rejecting LSEs with immunocytes (REJ) were analyzed. Slides were stained with hematoxylin & eosin and examined under light microscopy. REJ xenocomposite LSEs showed classic histologic signs of rejection. There was separation of the D-E junction with the presence of dyskeratotic and necrotic cells with a concomitant inflammatory infiltrate. The CON LSEs showed essentially normal dermis (collagen fibroblast matrix) and keratinocytes. A significant finding was the separation of the D-E junction in the absence of an inflammatory infiltrate. Previous studies have shown that human keratinocytes can be grown with human fibroblasts that are histologically similar to natural human skin. This discohesive nature of the murine fibroblasts and human keratinocytes may represent an important intercellular interaction associated with xeno-cellular interactions and is worthy of further study. Furthermore, the ability to grow xeno-composite tissues has profound implications in the face of organ donor shortages. They may represent an eventual in vitro replacement of skin and even solid organs.

Interactions between outcomes and tumor response to preoperative cisplatin-sensitized radiotherapy in advanced head and neck cancer. Southern New Jersey Head and Neck Cancer Treatment Group.

BACKGROUND: Whether or not tumor response to chemotherapy-sensitized radiation therapy (CTRT) for head and neck cancer leads to an improved outcome is unknown. METHODS: Forty patients who received preoperative cisplatin plus simultaneous radiotherapy for operable stage III and IV head and neck cancer were reviewed retrospectively regarding clinical demographics, staging, and survival status. RESULTS: Twenty-one (57%) patients had a histologic complete response (HCR) and 16 (43%) had a partial (PR) (9) or clinical complete (7) response (CCR). Tumor response of N1 versus N2-3 nodal disease showed 6 (75%) HCR and 4 (25%). Five-year disease-free survival overall was 82% for HCR versus 38% for PR/CCR (P <0.05). Disease-specific 5-year survival was 100% for HCR versus 27% for PR/CCR (P <0.002). CONCLUSIONS: Histologic complete response to CTRT for head and neck cancer is associated with increased survival and encouraging disease-free status. Response to CTRT is inversely proportional to lymphatic tumor load.

Opening and closing mechanisms of the larynx.

Opening and closing of the larynx are determined by the intrinsic and extrinsic muscles acting on the elastic forces in the tongue, pharynx, larynx, and trachea. The pharynx is opened or closed by two mechanisms: (1) Contractions of the cricothyroid and of the intrinsic muscles of the larynx open and close the vocal cords. (2) The false cords, ventricle, and true cords accordion open or close in a bellows mechanism. We conclude that the posterior cricoarytenoid opens the laryngeal airway. The cricothyroid together with the posterior cricoarytenoid accentuates this opening. The larynx is also opened by the geniohyoid, mylohyoid, sternothyroid, and middle constrictor. The thyrohyoid, cricothyroid, sternohyoid, and inferior constrictor close the laryngeal airway. Abnormalities in the soft tissues of the neck or of the innervation of the larynx, pharynx, and neck muscles may severely interfere with patency of the laryngeal airway. This occurs in such conditions as vocal cord paralysis, sleep apnea, multiple sclerosis, amyotrophic lateral sclerosis, spastic dysphonia, mandibular fractures or hypodevelopment, and cerebrovascular disease.