Isolation of Shiga Toxin-Producing Escherichia coli from the Surfaces of Beef Carcasses in Slaughterhouses in Japan.

Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen worldwide. It is necessary to control and prevent STEC contamination on beef carcasses in slaughterhouses because STEC infection is associated with beef consumption. However, the frequencies of STEC contamination of beef carcasses in various slaughterhouses in Japan are not well known. Herein, we investigated the contamination of beef carcasses with STEC in slaughterhouses to assess the potential risks of STEC. In total, 524 gauze samples were collected from the surfaces of beef carcasses at 12 domestic slaughterhouses from November 2020 to February 2023. The samples were measured for aerobic plate counts and tested for pathogenic genes (stx and eae) and major O-serogroups (O26, O45, O103, O111, O121, O145, and O157) by real-time PCR screening. Subsequently, immunomagnetic separation (IMS) was performed on samples positive for stx, eae, and at least one of the seven O-serogroups of STEC. Isolation process without IMS was performed on samples positive for stx, including those subjected to IMS. STEC O157:H7 and stx-positive E. coli other than serotype O157:H7 were isolated from 0.6% and 4.6% of beef carcass surfaces, respectively. Although the STEC O157:H7 isolation rate was low and stx-positive E. coli other than serotype O157:H7 belonged to minor O-serogroups, the results mean a risk of foodborne illness. Furthermore, a moderate correlation was observed between aerobic plate counts and detection rates of stx-positive samples by real-time PCR screening. The STEC O157:H7 isolated facilities showed higher values on aerobic plate counts and detection rates of stx-positive samples than the mean values of total samples. Therefore, these results suggest that it is important to evaluate hygiene treatments against beef carcasses for the reduction of STEC contamination risk, particularly in facilities with high aerobic plate counts.

[Study of surgical procedures for retinal detachment with macular hole].

PURPOSE: To evaluate the surgical outcome of retinal reattachment, the reattachment rate according to the range of detachment, and postoperative visual acuity for macular hole retinal detachment (MHRD). SUBJECTS AND METHODS: Sixty-eight eyes of 67 patients with MHRD were analyzed. The mean follow-up period was 54 months. RESULTS: Retinal reattachment occurred in 42/68 eyes (62%) after initial surgery. The reattachment rates were 6/23 eyes (26%) in the gas tamponade group, 13/19 eyes (68%) in the vitrectomy group, 23/ 26 eyes (88%) in the group that underwent removal of internal limiting membrane as adjunct to vitrectomy (ILM) group. In the additional surgery, the reattachment rates were 5/9 eyes (56%) in the gas tamponade group, 13 eyes (100%) in the vitrectomy group, 1/2 eyes (50%) in the ILM group, and 6 eyes (100%) in the macular prombe buckling group. No significant differences were seen in the detachment extent-related reattachment rate within the same surgery and the postoperative visual acuity between the groups. CONCLUSION: The results show that removal of ILM contributes to successful reattachment in the initial surgery, and that for non-reattachable eyes, macular buckling in the second surgery is the most reliable method.

Rupture and drug release characteristics of multi-reservoir type microspheres with poly(dl-lactide-co-glycolide) and poly(dl-lactide).

For the multi-reservoir type microspheres composed of polylactide (PLA) and poly(dl-lactide-co-glycolide) (PLGA), the influence of inner drug-holding layer/outer layer ratio on drug release profiles was studied. The microspheres were prepared by the O/W type emulsion-solvent evaporation technique, and cisplatin was used as a model drug. The water-uptake and the erosion of each polymer were evaluated to clarify the mechanism of drug release for multi-reservoir type microspheres. The formulations were classified by the influence of the blending ratio on drug-release profiles: the formulations with the drug-release profiles independent of the blending ratio (Typel group) and the formulations with drug-release profiles depending on the blending ratio (type 2 group). The formulations of type 1 group showed the uniform swelling during drug-release test, and provided the drug-release governed by the erosion of the inner drug-holding layer. On the other hand, the formulations of Type2 group showed the rupture of outer layer which was induced by the swelling of inner drug-holding layer, and the microspheres with the low ratio of the PLGA provided the drug-release rate which exceeded the estimate from the erosion profiles. The results of present study revealed that two types of drug-release mechanism exist for multi-reservoir type microspheres.

Drug release characteristics of multi-reservoir type microspheres with poly(dl-lactide-co-glycolide) and poly(dl-lactide).

For the multi-reservoir type microspheres composed of poly(dl-lactide-co-glycolide) (PLGA) and poly(dl-lactide) (PLA), the influence of the drug-holding layer and the non-drug-holding layer on drug release profiles was studied. The microspheres with the blend of PLGA and PLA were prepared by the W/O type emulsion-solvent evaporation technique, and cisplatin was used as a model drug. The degree of water uptake and the erosion of each polymer were evaluated to clarify the mechanism of drug release for multi-reservoir type microspheres. The blending of PLA and PLGA provided two types of microspheres in terms of the drug distribution in a microsphere, depending on the ratio of the blend: the microspheres with the drug-holding layer covered by the non-drug layer and the microspheres with the drug on the outer region. The drug release in the early period was governed by the pattern of drug distribution. The drug release rate at a steady state was governed by the erosion of the drug-holding layer. The results of present study indicate that drug release from multi-reservoir type microspheres involves the following process: (a) rapid release of the drug near the surface of microspheres, (b) formation of micropores in the non-drug-holding layer by hydration and erosion, (c) degradation of the drug-holding layer, and (d) diffusion of the drug through micropores.

Anti-tumor effect of intraperitoneal administration of cisplatin-loaded microspheres to human tumor xenografted nude mice.

This study evaluates the anti-tumor effect of cisplatin-loaded microspheres (CDDP-MS) against peritoneal carcinomatosis using human tumor xenografts. The incorporated CDDP was released from CDDP-MS for 3 weeks in vivo as well as in vitro. CDDP-MS at a dose of 35 mg/kg (at maximal tolerable dose (MTD)) showed effective anti-tumor activity (tumor growth inhibition rate (IR)=70.3%) against Li-7 (human liver cancer) xenografts transplanted into the peritoneal cavity. This procedure also resulted in increased life span (ILS (%)=47.2%), whereas CDDP dissolved in saline solution (CDDP-SOL) at a dose of 8 mg/kg (at MTD) was ineffective (IR=15.7%, ILS=2.6%). Likewise, CDDP-MS (35 mg/kg) significantly prolonged the mean survival time (ILS=50.8%) compared with a CDDP-SOL group (8 mg/kg) (ILS=13.1%) in the mice with Li-7 xenografts transplanted into the spleen. Furthermore, CDDP-MS showed markedly effective anti-tumor activity (IR=82.2%) against H-154 (human stomach cancer) xenografts, in which CDDP-SOL was effective (IR=69.5%) at the MTDs. The suppressive effect of CDDP-MS on accumulation of malignant ascites was intimately related to unchanged CDDP concentration in ascites. These results demonstrated that the administration of CDDP-MS resulted in an unchanged CDDP concentration in ascites, and induced a sustained tumor growth inhibition along with a prolonged survival time.

Relation between dissolution profiles and toxicity of cisplatin-loaded microspheres.

The aim of this study is to evaluate and compare the dissolution profiles of cisplatin-loaded microspheres (CDDP-MS) in vitro and in vivo, and to determine the relationship between the dissolution profiles in vitro and systemic toxicity. For this purpose, three types of CDDP-MS that release the CDDP for 1, 2 and 5 weeks without a large amount of initial release in phosphate buffered saline (pH 7.4) were prepared. The dissolution profiles of these formulations in vivo were well correlated with in vitro studies, and resulted in well-controlled plasma platinum concentration. The systemic toxicity of the CDDP-MS and CDDP dissolved in saline (CDDP-SOL) were assessed by intraperitoneal administration in mice. The maximal tolerable dose (MTD) of CDDP-SOL was 13.4 mg/kg, whereas the CDDP-MS of 1, 2 and 5-week types were 34.6, 44.2, 62.6 mg/kg, respectively. The MTD of CDDP increased proportionally when 50% of CDDP had been released from MS in vitro (MTD (mg/kg)=5.22 x T(50(day)) + 13.2, R(2)=0.9935). We demonstrate that the systemic toxicity of CDDP-MS can be predicted by evaluation of the dissolution rate in vitro since in vivo dissolution was correlated with the in vitro.

Organ distribution of cisplatin after intraperitoneal administration of cisplatin-loaded microspheres.

The aim of this study was to clarify the organ distribution of cisplatin (CDDP) after intraperitoneal (i.p.) administration of cisplatin-loaded microspheres (CDDP-MS). The distribution of CDDP to normal organs lying in the peritoneal cavity after i.p. administration of CDDP-MS was assessed by comparing with subcutaneous administration to non-cancerous mice. The organ distribution of CDDP after i.p. administration of CDDP-MS shows that CDDP released from microspheres was distributed to the organs lying in the peritoneal cavity and in the retroperitoneum. These are mainly from the systemic circulation, but are not directly from the organ surface. The distribution of CDDP to tumors was evaluated in sarcoma180 tumor-bearing mice by comparing with a bolus injection. The CDDP-MS delivered CDDP to tumors more effectively than did bolus injection. The distribution of CDDP-MS in the peritoneal cavity was in accord with the tumor distribution. This concordance and sustained exposure of CDDP to the tumors might play a critical role in enhancing the CDDP accumulation in tumors. It is concluded that CDDP-MS have a distinct regional pharmacokinetic advantage for peritoneal carcinomatosis, and that i.p. administration of CDDP-MS is an effective treatment for peritoneal carcinomatosis.

Preparation and properties of alginate lyase modified with poly(ethylene glycol).

Modification of the enzyme alginate lyase (AL) with poly(ethylene glycol) (PEG) was attempted for the degradation and removal of alginate biofilms in infectious diseases. The modification of AL with PEG was attempted with three kinds of N-succinimidyl succinate PEG (SS-PEG), which differed in molecular weight (i.e., 2000, 5000 and 12,000 Da). The conjugation of PEG to free amino groups on AL was confirmed by gel permeation chromatography. Quantification of residual free amino groups revealed that PEG modification progressed further with a higher pH and a larger molar ratio of SS-PEG to AL. The reproducibility of the reaction was fairly good. The enzyme activity decreased with increasing PEG modification but the immunoreactivity toward anti-AL antibodies, as evaluated by an ELISA method, was much more remarkably reduced. The immunoreactivity was more reduced by the conjugated PEG with the larger molecular weight. In the reaction with PEG of molecular weight 12,000 Da, we obtained PEG-modified AL retaining approximately 40% enzyme activity but only 0.5% of the immunoreactivity of native AL.

Identification of transplanted retinal pigment epithelium with a novel chromosomal marker.

PURPOSE: To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. METHODS: Primary pigmented RPE harvested from third trimester fetal pigs were transplanted as microaggregates into the subretinal space of 3 albino rabbits. We then used an in situ probe for a repetitive segment of the porcine chromosome to identify the transplanted RPE. RESULTS: Pigmented cells were visible in the subretinal space 2 weeks after transplantation. Approximately 70% of pigment-containing cells were also labeled with the porcine chromosomal marker. Labeled cells were predominantly flatter in morphology and close to Bruch's membrane whereas unlabeled cells were rounder and further from Bruch's membrane. The outer nuclear layer thickness was normal above the pigmented monolayer but was decreased over areas containing multiple layers of pigmented cells. CONCLUSIONS: Fetal porcine RPE xenografts can be identified with a nuclear marker for a repetitive segment of the porcine chromosome. The presence of pigment within unlabelled cells suggests that pigment is not a robust marker for transplanted RPE.