Biomedical subjects

M S Levy

Publications and source records attributed to M S Levy.

At least 19 recordsLinked to original sources

PubMed2006

Genetic selection for resistance or susceptibility to oral tolerance imparts correlation to both Immunoglobulin E level and mast cell number phenotypes with a profound impact on the atopic potential of the individual.

BACKGROUND: Immunological oral tolerance is being studied with great interest due to its therapeutic potential in allergy and autoimmunity processes, although the cellular and molecular mechanisms linking these different phenomena remain elusive. In the present study, two mouse lines with extreme phenotypes for susceptibility [TS Line] or resistance [TR Line] to oral tolerance and their [TS x TR]F2 segregants were used in order to evaluate the impact of these traits on the atopic potential of the individuals. OBJECTIVE: Demonstrate whether the tr and ts genes, cumulated during 18 generations of bidirectional genetic selection, influence expression of two important immunobiological traits (IgE and mast cell) critical to allergic response. METHODS: Mice with extreme phenotypes for oral tolerance to ovalbumin (OVA), produced by assortative mating (TS and TR Line), and their (TS x TR)F2 segregating were used. Serum IgE levels assayed by ELISA, and mastocytes counted with toluidine blue staining were evaluated in naïve mice. Anaphylaxis was induced by intravenous injection of OVA, intestinal inflammation by oral administration of OVA 7 days after immunization, and pulmonary inflammation by intranasal and nebulization OVA challenges. Specific IgE was dosed by passive cutaneous anaphylaxis. RESULTS: The naïve TS mice have a 20-fold lower serum IgE level and two- to threefold diminished mast cell numbers in mucosal sites, when compared with TR-mice, which were highly susceptible to allergic inflammation and anaphylactic shock. The associations of oral tolerance, serum IgE levels and mast cell numbers in naïve animals were confirmed analysing the simultaneous presence of these traits in individuals of a [TS x TR]F2 -segregating population. CONCLUSION: The results suggest that the complex of genes controlling TS and TR phenotypes play a main role in the regulation of the atopic potential of the individual. The studies of these traits in interline F2 segregants demonstrated a co-segregation of TS and TR phenotypes with IgE responsiveness and mast cell numbers. Thus, the opposite capacity of the genetically modified mice may be involved in co-adaptative mechanisms reflecting a dynamic relation between gene frequencies in a natural population. These correlations give circumstantial evidence to support clinical applications of oral tolerance in allergic and autoimmune diseases.

Administration, Oral↗

PubMed2002

Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption.

There is an increasing interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The use of an integrated unit operation, comprising tangential flow microfiltration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was examined in this work. Experiments were performed using a custom-built tangential flow microfiltration rig (membrane area=12.5 cm(2)). Tangential flow filtration-adsorption of E. coli lysates containing a plasmid product removed most solids (>75%) and decreased chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed, indicating a recovery yield of 100% (transmission equal to 1). Results were similar for E. coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin, and protein levels were also observed. The reproducibility and potential for scale up of this integrated filtration-adsorption operation makes it at attractive option for intermediate- to large-scale pharmaceutical-grade plasmid processing.

Adsorption↗

PubMed2002

The effects of graft width and graft laxity on the outcome of caprine anterior cruciate ligament reconstruction.

We studied how initial graft size and initial graft laxity affected the biomechanics of anterior cruciate ligament (ACL) reconstruction at six months. Sixteen goats had bilateral reconstructions staged eight weeks apart. Autografts 4 and 7 mm wide were taken from the central patellar tendon (PT). Lax grafts were created by adding 4 mm slack to the graft before fixing. We reconstructed each joint using a combination of width and laxity treatments. Both factors were changed for the contralateral joint and all combinations appeared with equal frequency. At six months we measured the joint extension limit, anterior-posterior (AP) translation, and osteoarthritic changes. The grafts were then tested to failure to determine their mechanical properties. After six months the difference in initial treatments had disappeared: there was no difference in graft cross-section due to the different initial widths and there was no difference in joint AP translation due to the initial graft laxity. We did observe that wide grafts were associated with a block to extension, decreased joint AP translation, and increased articular cartilage damage and osteophyte formation. While AP translation was reduced, it was correlated with decreased extension, possibly indicating an increase in scar tissue formation rather than a more functional graft. Neither graft width nor graft laxity produced differences in any graft mechanical properties. This suggests that the use of larger grafts to prevent increased AP translation has undesirable complications. Ultimately, we conclude that neither of these surgical treatments strongly affects the biomechanical result of caprine ACL reconstruction.

Animals↗

PubMed2002

Subject variation in caprine anterior cruciate ligament reconstruction.

We studied the subject and treatment contributions to anterior cruciate ligament (ACL) reconstruction biomechanics by reexaming the results of two bilateral reconstruction studies. Bilateral reconstruction allows a comparison between treatments exposed to the same subject related healing factors. The studies examined the effects of gamma irradiation and the effects of initial graft size and initial graft laxity. In both studies different treatments were applied to contralateral limbs. We found that the subject was the best predictor of outcome, while the surgical treatments had little influence on outcome. There was a large variation between subjects despite similar treatments, and little difference between contralateral limbs despite different surgical treatments. At 26 weeks, the graft cross sectional area and modulus were most strongly influenced (p < 0.002) by the subject. We interpret this as a subject related factor is regulating the quantity and quality of the healing tissue. Potential sources of subject related factors include the subject's pre-operative condition, the activity during the post-operative period, and an intrinsic biologic response. By better understanding the source of subject variation, more successful and consistent ACL reconstructions might be achieved.

Animals↗

PubMed2001

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology.

The invention of novel drugs based on biological macromolecules requires the development of specialized formulation methods. Supercritical fluid technology offers the possibility to produce dry powder formulations suitable for inhalation or needle-free injection. In this article we describe the first application of a process involving supercritical carbon dioxide for the production of plasmid DNA-loaded particles. The technique of solution enhanced dispersion by supercritical fluids (SEDS) is used to coformulate the 6.9 kb plasmid pSV beta with mannitol as excipient. After initial experiments showed a high degradation of the plasmid during powder formation, a systematic investigation of the process revealed pH effects to be crucial for the recovery of intact DNA. The application of high-buffer concentration led to an increase of the recovered supercoiled proportion from 7% to 80%.

Biotechnology↗

PubMed2001

Staged reduction of gastroschisis: a simple method.

Staged reduction of abdominal contents using a silastic sheet has become standard management in gastroschisis where primary closure is not possible. With the introduction of a pre-made Silastic silo coupled to a spring-loaded ring (Ben Tec, Sacramento, CA), the procedure can be done at the bedside. We present a simple technique utilizing a disposable umbilical-cord clamp that makes reduction a fast, one-physician procedure and present a preoperative step that facilitates tension-free closure of the abdominal fascia.

Dimethylpolysiloxanes↗

PubMed2000

Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.

A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.

DNA, Superhelical↗

PubMed2000

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing.

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.

Blotting, Southern↗

PubMed2000

Biochemical engineering approaches to the challenges of producing pure plasmid DNA.

Plasmid-based genes offer promise for a new generation of vaccines and for gene therapy, but the size and character of plasmids pose new challenges to biochemical engineers. By acknowledging these and using bioprocess-design information based on fundamental studies of the system's properties, it will be possible to create efficient and consistent processes for these materials. This review addresses the purity required, the key issue of the sensitivity of the chromosomal DNA contaminant and larger plasmids to hydrodynamic forces, and the impact of this and other characteristics of plasmids on the recovery and purification of DNA for pharmaceutical purposes.

Biotechnology↗

PubMed2000

A conceptualization of the repetition compulsion.

The concept of the repetition compulsion remains an enigma. Its etiology is not fully understood and the purpose it serves continues to be a mystery. Although it is often theorized that the compulsion to repeat may function to facilitate mastery of a past trauma, mastery is rarely achieved. In this article the concept of the repetition compulsion is reviewed and the unanswered questions that continue to exist about this phenomenon are summarized. A way to conceptualize the compulsion to repeat is then offered. The compulsion to repeat as it specifically relates to the attempt to master a previous trauma is reviewed, followed by a examination of the relationship between the compulsion to repeat and reenactments. Finally, how the compulsion to repeat can be viewed as a posttraumatic stress response and the implications of understanding it in this fashion is then examined.

Adaptation, Psychological↗

PubMed1999

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye.

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.

Biotechnology↗

PubMed1999

Evolution of the modified Rossetti fundoplication in children: surgical technique and results.

OBJECTIVE: To compare the modified Rossetti fundoplication with the classic Nissen. SUMMARY BACKGROUND DATA: The traditional surgical treatment of gastroesophageal reflux in children has been the classic Nissen fundoplication, defined by liver mobilization, crural repair, takedown of short gastric vessels, and floppy wrap. The authors have progressed in our technique of fundoplication and now perform a modified Rossetti fundoplication, defined by liver retraction without mobilization, no crural repair, short gastric vessels left intact, and 2-cm floppy wrap. METHODS: A retrospective chart review was performed on 407 pediatric patients who had open fundoplications (Jan. 13, 1993, to Feb. 25, 1998). Two groups were analyzed: the Nissen group (171 patients) and the Rossetti group (236 patients). Groups were compared for incidence of recurrent reflux, dysphagia, hiatal hernia, need for esophageal dilation, revision of fundoplication, time to discharge, and operative time. RESULTS: Incidence of dysphagia (3.7% vs. 3.3%), postoperative hiatal hernia (1.9% vs. 1.4%), need for esophageal dilation (1.2% vs. 0.5%), and need for fundoplication revision (2.5% vs. 2.3%) were similar between the groups. The mean operative time was significantly decreased in the Rossetti group (65 +/- 25 minutes) versus the Nissen group (73 +/- 33 minutes). Recurrent reflux occurred significantly more often in the Nissen group (11.2%) than in the Rossetti group (5.1 %). CONCLUSION: The modified Rossetti fundoplication has a low complication rate and is the authors' preferred method for the surgical treatment of gastroesophageal reflux in children.

Child, Preschool↗

PubMed1999

Once daily, high dose versus divided, low dose gentamicin for open fractures.

A prospective, randomized study was performed on 75 Gustilo Grades II and III open fractures to determine the efficacy of once daily, high dose aminoglycoside therapy, compared with more conventional dosing, in reducing the infection rate when used in conjunction with an aggressive operative treatment protocol. All patients enrolled in the study were treated with immediate irrigation, debridement, operative stabilization of the fracture, and 1 g of cefazolin every 8 hours. At the time of admission patients were randomized to two groups. Patients in Group I received gentamicin 5 mg/kg divided into twice daily doses, and patients in Group II received gentamicin 6 mg/kg given once daily. All patients were monitored for renal toxicity and observed for radiographic and clinical signs of infection until fracture union. The results of the study revealed no statistically significant difference between once daily, high dose versus divided, low dose gentamicin in infection rates. Thus, daily dosing of gentamicin was found to be safe, effective, and cost efficient in the treatment of open fractures when combined with a cephalosporin and aggressive operative debridement and stabilization.

Adolescent↗

PubMed1998

A helpful way to conceptualize and understand reenactments.

Attempts to understand the purpose and the etiology of reenactments can lead to confusion because reenactments can occur for a variety of reasons. At times, individuals actively reenact past traumas as a way to master them. However, in other cases, reenactments occur inadvertently and result from the psychological vulnerabilities and defensive strategies characteristic of trauma survivors. This article offers a means to conceptualize and understand the many ways in which reenactments can occur. Psychotherapeutic strategies are offered to help individuals integrate past traumas and decrease their chances of becoming involved in destructive reenactments.

Adaptation, Psychological↗

PubMed1997

Nonlymphoid vertebral canal tumors in cats: 11 cases (1987-1995).

OBJECTIVE: To determine outcome of cats with nonlymphoid tumors of the vertebral canal that undergo surgery. DESIGN: Retrospective study. ANIMALS: 11 cats. PROCEDURE: Information obtained from the medical records included signalment, tumor location, gross evaluation of completeness of surgical excision, histologic diagnosis, and survival time. RESULTS: Median age of cats was 12 years: all cats had negative FeLV and feline immunodeficiency virus test results. All cats underwent a hemilaminectomy or dorsal laminectomy. The tumor was located in the thoracic portion of the vertebral column in 6 cats. Six cats had intradural-extramedullary tumors. Tumors included meningioma (n = 5), malignant nerve sheath tumors (2), and meningeal sarcoma, chondrosarcoma, lipoma, and osteosarcoma (1 each). One cat was lost to follow-up, 1 cat with meningioma was alive 1,400 days after surgery, and 1 cat with a nerve sheath tumor was alive 2,190 days after surgery. Median survival time for the other 4 cats with meningioma was 180 days (range, 30 to 600 days). CLINICAL IMPLICATIONS: Cats with nonlymphoid vertebral canal tumors that undergo surgery may have a good prognosis.

Age Factors↗

PubMed1996

Open fracture of the tibia in children.

The records of eighty-three children who had had an open fracture of the tibial metaphysis or diaphysis between January 1983 and July, 1993 were studied retrospectively. The average duration of follow-up was fourteen months (range, two to seventy-five months). There were twenty-four grade-I, forty grade-II, thirteen grade-IIIA, six grade-IIIB, and no grade-IIIC fractures, according to the classification scheme of Gustilo et al. Sixty patients (72 per cent) had sustained the fracture when they were struck by an automobile, and forty-eight patients (58 per cent) had other associated major injuries. All fractures were treated with irrigation and débridement, and antibiotics were administered parenterally for a minimum of forty-eight hours. Thirty-two patients were managed with immobilization in a cast only; forty, with transcutaneous fixation with an average of two Steinmann pins followed by immobilization in a cast; nine, with external fixation; one, with open reduction and internal fixation with two screws and two pins; and one, with delayed intramedullary nailing. Fifty-seven wounds were closed primarily (forty-four, over a Penrose drain, and thirteen, without a drain), ten were treated with delayed closure, four were allowed to heal by secondary intention, seven were covered with a soft-tissue flap, and five were treated with skin-grafting (a split-thickness skin graft was used for four, and a split-thickness and a full-thickness skin graft were used for one). The average time to union was fifteen weeks (range, five to sixty-one weeks), with the fracture healing by sixteen weeks in sixty-four patients (77 per cent). Eighteen patients (22 per cent) had delayed union, and only one patient (1 per cent) had non-union. Secondary procedures were necessary to achieve union in only two patients. Two patients had a superficial wound infection, and no patient had osteomyelitis. One patient, who had been managed with external fixation, had a pin-track infection; none of the patients who had had transcutaneous fixation had a pin-track infection. Two patients had a compartment syndrome, and two patients had a transient stretch injury of a nerve (the peroneal nerve in one and the sciatic nerve in the other). Four fractures healed with an angulatory deformity of more than 10 degrees in any plane. Five patients had overgrowth of the limb of one centimeter or more. Physeal arrest did not occur in any patient. We concluded that treatment of unstable open fractures of the tibia in children with débridement and transcutaneous fixation followed by immobilization in a cast leads to good anatomical and functional results. We prefer this technique to external fixation, which is associated with several potential complications. Loose closure of a clean open wound over a Penrose drain is effective and can be safely utilized in selected children.

Adolescent↗

PubMed1995

Fate of the reconstructed carotid artery after extracorporeal membrane oxygenation.

Reconstruction of the right common carotid artery has been shown to be feasible in neonates after extracorporeal membrane oxygenation (ECMO). However, the long-term outcome after carotid artery reconstruction (CAR) remains unknown. The purpose of this study was to evaluate the natural progression of the anastomotic site after CAR. Between February 1990 and June 1993, 201 patients received ECMO. All veno-arterial (VA) ECMO patients (n = 172) were considered candidates for reconstruction unless a significant neurological event (ie, intracranial hemorrhage, stroke) had occurred; the duration of ECMO exceeded 10 days, making carotid mobilization difficult; or the patient's prognosis was deemed poor. Reconstruction was performed by excising the arteriotomy site, followed by primary end-to-end anastomosis. Reconstruction was abandoned and the artery ligated if an intimal flap, arterial thrombosis, or excessive tension was encountered. After reconstruction all patients had early carotid ultrasonography and either head computed tomography (CT) or magnetic resonance imaging (MRI). Subsequent ultrasound examinations were performed at approximately 6-month intervals. Diameter index (DI) (a measure of anastomotic narrowing) was calculated using ultrasound by dividing the anastomotic diameter by the diameter of the carotid artery 5 mm proximal to the anastomosis. Forty-three of 172 VA ECMO patients (25%) had successful reconstruction. Long-term follow-up data were available on 27 patients. These 27 patients had 39 ultrasound examinations, with an average follow-up time of 7.3 months (range, 4 days to 29 months). All carotid arteries were patent. Linear regression analysis showed significant improvement in the DI with time (P = .0001, r2 = .382).(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗

PubMed1994

RecN SOS gene and induced precise excision of Tn10 in Escherichia coli.

A mutant defective in the induced excision of Tn10 was isolated by decreased papillation on MacConkey-galactose plates with mitomycin C. The mutation involved was characterized as recN by genetic mapping and complementation. This mutant, as well as a previously characterized recN mutant (recN262), showed a markedly decreased frequency of excision of Tn10 after treatment with UV or mitomycin C. These observations indicate that recN is involved in the induced excision of Tn10.

DNA Repair↗