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PubMed1998

Production of soluble single-chain T-cell receptor fragments in Escherichia coli trxB mutants.

Antibodies and T cell receptors (TCR) both belong to the immunoglobulin superfamily whose members are characterised by the possession of one or more immunoglobulin domains. The production of soluble single chain antibody fragments in Escherichia coli has, in recent years, become a routine laboratory procedure. In contrast, the production of T cell receptors in bacteria has remained problematic as the majority of the recombinant protein is insoluble. In this paper we show that single chain TCR produced in E. coli BL21 (DE3) and directed to the periplasm was also insoluble and that this was in part due to the failure of the cell protein processing machinery to cleave the pelB leader sequence. This problem was overcome by expressing the single chain TCR in the cytoplasm of E. coli which carry an inactive thioredoxin reductase gene. This strain allows the formation of disulphide bonds in the cell cytoplasm which we believe encourages the correct folding of the recombinant protein. We have constructed both a human and mouse single chain TCR in these bacteria and demonstrated using BIAcore technology that these molecules have folded in a conformation which allows their recognition by conformational specific ligands. In addition, we have used one of our soluble single chain TCR preparations to isolate a TCR specific Fab molecule from a phage antibody library.

Amino Acid Sequence↗

PubMed2003

Evaluation of herd sampling for Salmonella isolation on midwest and northeast US dairy farms.

Epidemiologic investigations of Salmonella infections in dairy cattle often rely on testing fecal samples from individual animals or samples from other farm sources to determine herd infection status. The objectives of this project were to evaluate the effect of sampling frequency on Salmonella isolation and to compare Salmonella isolation and serogroup classification among sample sources on 12 US dairy farms sampled weekly for 7-8 weeks. Three herds per state were enrolled from Michigan, Minnesota, New York and Wisconsin based upon predefined herd-size criteria. Weekly samples were obtained from cattle, bulk tank milk, milk filters, water and feed sources and environmental sites. Samples were submitted to a central laboratory for isolation of Salmonella using standard laboratory procedures. The herd average number of cattle fecal samples collected ranged from 26 to 58 per week. Salmonella was isolated from 9.3% of 4049 fecal samples collected from cattle and 12.9% of 811 samples from other sources. Serogroup C1 was found in more than half of the samples and multiple serogroups were identified among isolates from the same samples and farms. The percentage of herd visits with at least one Salmonella isolate from cattle fecal samples increased with overall herd prevalence of fecal shedding. Only the three herds with an average fecal shedding prevalence of more than 15% had over 85% of weekly visits with at least one positive fecal sample. The prevalence of fecal shedding from different groups of cattle varied widely among herds showing that herds with infected cattle may be classified incorrectly if only one age group is tested. Testing environmental sample sources was more efficient for identifying infected premises than using individual cattle fecal samples.

Animals↗

PubMedDate not supplied

Serum-ionized calcium in mexican protein-energy malnourished children.

BACKGROUND: Hypocalcemia occurring in children with severe primary protein-energy malnutrition (PEM) has previously been shown to merely reflect a reduction in the protein-bound fraction of serum calcium. On the other hand, ultrafilterable, i.e., nonprotein bound calcium (Ca(U)), remains unaffected. The aim of the present investigation was to test the hypothesis that in infantile PEM, ionized calcium, which represents the major and physiologically most important component of Ca(U), is also kept within a normal concentration, whatever the total serum calcium levels. METHODS: Three milliliters of peripheral venous blood were obtained on admission and periodically afterward from 18 fasting marasmic infants and 6 children with the edematous form of PEM (kwashiorkor). Ionized calcium was determined in 125 microL aliquots of blood serum with ion-selective electrodes. The measurement of related elements and molecules was carried out with customary laboratory procedures. RESULTS: On admission, as well as during the ensuing stages of nutritional recovery, ionized serum calcium exhibited a normal concentration, with the exception of two initial samples. Total calcium, total protein, albumin, and inorganic phosphate concentrations were decreased in a variable degree according to the clinical type, ascending to normal levels during recovery. CONCLUSIONS: Ionized calcium serum concentration remains within normal limits in severe infantile PEM.

Albumins↗

PubMed1986

Diagnosis of canine viral infections.

The diagnosis of canine viral infections frequently requires the correlation of clinical, hematologic, pathologic, and laboratory findings. When laboratory diagnostics are utilized, appropriate specimens must be collected. In addition, correct timing of specimen collection and the proper handling and transport of these specimens are essential. Whenever possible, the practitioner should not rely on any one laboratory procedure for making a diagnosis; rather, a combination of techniques should be used. Submission of tissue for histopathology, virus isolation, and antigen detection techniques (FAT), serum for serologic analysis, and feces whenever enteric infections are investigated will maximize the chances of making a successful diagnosis.

Animals↗

PubMed1997

The effect of rings on microbial load of health care workers' hands.

BACKGROUND: The hands of health care workers (HCWs) serve as a major route for transmission of nosocomial infection. Although handwashing is known to reduce cross-transmission of infection, the influence of rings on the efficacy of handwashing and the carriage of bacteria on the hands has not been well established. METHODS: In this study, 50 HCWs with rings were paired by unit with 50 HCWs without rings. Cultures were obtained by use of a timed-friction rinse before and after a timed handwashing. Standard laboratory procedures were followed for identification of the bacteria. RESULT: When colony counts before handwashing are taken into consideration, a significant difference is seen after handwashing between the two groups (R2 = 0.56). The regression model showed that the slope was significantly steeper (p < 0.0014) for the group with rings. This effect is more apparent when the colony count on hands is greater than 1000 colony forming units before handwashing. CONCLUSIONS: A standardized, timed handwashing procedure was effective in decreasing the bioload of HCWs' hands. The effect of rings on the bioload was significant in this study.

Case-Control Studies↗

PubMed1997

Universal precautions: costs for protective equipment.

BACKGROUND: The amount and costs of protective equipment used to implement universal precautions in Thailand have not previously been studied. METHODS: A cross-sectional study was done to determine the frequency of clinical and laboratory procedures requiring universal precautions and the amount of protective equipment needed for each. RESULTS: The study was performed in 24 government hospitals in Thailand in December 1993. Totaling 6549 beds, these hospitals had provided service to 357,391 inpatients and 3,411,122 outpatients during the previous year. The annual number of procedures performed in these hospitals was estimated at 17.5 million, with expenditures for protective equipment of $2.4 million (U.S.) per year. The average overall cost for protective equipment was U.S. $5.37 for one inpatient stay and U.S. $0.15 for one outpatient visit. The projected national expense for these barriers was U.S. $41.5 million per annum. The cost for these barriers after the implementation of universal precautions was 2.5 times the cost before implementation. CONCLUSIONS: Overuse of sterile and examination gloves and gowns and underuse of heavy-duty gloves, masks, aprons, goggles, and boots were discovered during the study. Appropriate use of disposable and reusable universal precautions equipment would free health care dollars for other purposes.

Costs and Cost Analysis↗

PubMed1999

Preparation and properties of totally synthetic immunogens.

Although the induction of antibodies and T cells by synthetic peptides representing defined antigenic determinants is a routine laboratory procedure, their use as vaccines has not yet been generally realised. There are a number of reasons for this and paramount is the limitation of valency which affects not only immunogenicity, but also the coverage of the antigenic universe. This paper is a review of our own work in which we have assembled synthetic peptides into multivalent artificial proteins and then examined their immunological properties.

Amino Acid Sequence↗

PubMed1992

The impact of diagnosis-related groups on the cost of hospitalization for end-stage renal disease patients at Rhode Island Hospital from 1987 to 1990.

The introduction of diagnosis-related groups (DRGs) for the reimbursement of hospital costs in the Medicare population made no provisions to identify end-stage renal disease (ESRD) patients. To assess the impact of the DRGs on ESRD patients, we prospectively evaluated all hospitalizations (901) at the Rhode Island Hospital from July 1987 through April 1990 among a chronic hemodialysis population of 587 patients. The complied data on length of hospital stay, cost per day, and total cost were compared with the accepted allowances as defined by the DRG codes assigned to the admissions. The mean values and comparisons were length of stay, 10.2 +/- 21 days (ESRD) versus 9.7 +/- 3.6 days (DRGs), P = 0.44; cost per day, $1,240 +/- $685 (ESRD) versus $671 +/- $897 (DRGs), P = 0.0001; and total cost, $9,660 +/- $17,170 (ESRD) versus $6,815 +/- $8,872 (DRGs), P = 0.0001, a difference of $2,275 per admission. The increase in daily, and thus total, costs was primarily due to the use of supplies, medications, laboratory procedures, and transfusion services. It appears from our data that the DRGs do not adequately reflect the cost of caring for ESRD patients with the same DRGs as non-ESRD patients because of increased demand for ancillary services. If these observations are confirmed, a reevaluation of the DRG process as it relates to ESRD patients is warranted.

Diagnosis-Related Groups↗

PubMed1998

Intraoperative and postoperative kinetics of serum tumor markers in patients with oral carcinoma.

PURPOSE: There are no reliable laboratory procedures to monitor intraoperative tumor antigen dispersal in patients with squamous cell carcinoma of the head and neck. This study evaluated the use of serologic parameters as perioperative indicators of systemic manifestations. PATIENTS AND METHODS: In 28 patients, serial measurements of different tumor markers (squamous cell carcinoma antigen, carcinoembryonic antigen, carbohydrate antigen 19.9, carcinoma antigen 125) were made preoperatively, intraoperatively, and postoperatively at short intervals to determine the influence of tumor ablation on the antigen concentration in the serum. A microparticle enzyme immunoassay was used for the serologic analysis. RESULTS: Squamous cell carcinoma antigen showed elevated serum levels preoperatively, which increased intraoperatively and decreased significantly postoperatively. The serologic examinations in the control group and the other tumor markers showed no correlation with the clinical situation. CONCLUSIONS: The results suggest that the titer of squamous cell carcinoma antigen in serum has a positive correlation with the tumor burden and the operative trauma in the case of surgery. These results support the value of intraoperative and postoperative serum antigen monitoring.

Adult↗

PubMed1979

[Zein of maize grain. Preparation and characterization].

A laboratory procedure for isolation and purification of zein from grains of 4 varieties of Maize was described. The preparations were characterized by their physicochemical properties. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS), native zein (from INRA 260 hybrid) was resolved into 2 major classes with average molecular weights of 45,000 and 22,000. After reduction with mercaptoethanol zein contained only two subunits of 22,000 and 24,000 daltons. Upon starch gel electrophoresis in 6 M urea at pH 3.5, native zein exhibited five major or medium intensity bands and several minor ones. The latter, under reducing conditions, disappeared to reinforce the major bands or to yield some new minor bands. Amino acid analysis revealed a very low content of lysine. The NH2-terminal amino acids were determined to be threonine and phenylalanine with a preponderance of the former. Zeins isolated from the varieties studied appeared tohave the same NH2-terminal residues and similar amino acid compositions with an arginine/histidine ratio ranging from 1.1 to 1.2. They differed in relative importance of components, detected by electrophoresis in the presence of SDS or urea. Changes in zein characteristics with the grain genotype allow one to conclude that the components of molecular weights of 22,000 and 24,000 consist of several subunits differing in charge and amino acid content.

Amino Acid Sequence↗

PubMed2002

Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool.

OBJECTIVE: The aim of this study was to develop a rapid laboratory procedure that is capable of subtyping Fanconi anemia (FA) complementation groups FA-A, FA-C, FA-G, and FA-nonACG patients from a small amount of peripheral blood. MATERIALS AND METHODS: For this test, primary peripheral blood-derived FA T cells were transduced with oncoretroviral vectors that expressed FANCA, FANCC, or FANCG cDNA. We achieved a high efficiency of gene transfer into primary FA T cells by using the fibronectin fragment CH296 during transduction. Transduced cells were analyzed for correction of the characteristic DNA cross-linker hypersensitivity by cell survival or by metaphase analyses. RESULTS: Retroviral vectors containing the cDNA for FA-A, FA-C, and FA-G, the most frequent complementation groups in North America, allowed rapid identification of the defective gene by complementation of primary T cells from 12 FA patients. CONCLUSION: Phenotypic correction of FA T cells using retroviral vectors can be used successfully to determine the FA complementation group immediately after diagnosis of the disease.

3T3 Cells↗

PubMed2001

Diagnostic criteria for multiple sclerosis.

Over a hundred years ago, Charcot set down what he considered to be some of the clinical characteristics of multiple sclerosis (MS). His triad was not specific but it was the first attempt to separate this disease from the many others affecting the nervous system. The history of clinical diagnostic criteria demonstrates the evolution from rather tentative classifications of restricted value to the more elaborate 1983 scheme which incorporates some laboratory procedures under the rubric paraclinical tests, considered to be extensions of the neurological examination, as well as a new category based on the presence of specific abnormalities of the cerebrospinal fluid (CSF). It is curious that until then the term definite MS had been avoided except for autopsy-proven cases, perhaps a wise move, since exact diagnosis may require long term observation. All the proposed schemes have been based on the twin principles of dissemination in both time and space. The diagnosis of MS must remain a clinical one, supported but not supplanted by the increasingly popular magnetic resonance imaging, which is non-specific and is frequently overinterpreted by radiologists lacking appropriate clinical information. Reliance on the MRI as the principal if not exclusive basis for the diagnosis leads to error in as many as one third of cases. This assumes a great deal of importance considering that such non-MS patients may be counted in epidemiological surveys and included in therapeutic trials for disease-modifying drugs, or eventually treated with these very expensive drugs with still controversial long term efficacy. Not surprisingly, attempts to develop reliable criteria for the MRI diagnosis of MS have been unsuccessful in view of the lack of specificity of that procedure. Great care should be taken to exclude the presence of extrinsic cervical spine lesions which might impinge on the cord, leading to the formation of plaques, or mimic the course of MS. An MRI of the cervical spine is recommended in all patients suspected of having MS who have symptoms suggestive of spinal cord involvement. The diagnosis of MS is, and will remain, based on clinical criteria which codify the characteristic dissemination in time and space of MS.

Acute Disease↗

PubMed1997

Antiviral susceptibility testing-flow cytometric analysis (AST-FCA) for the detection of cytomegalovirus drug resistance.

Antiviral susceptibility testing-flow cytometric analysis (AST-FCA), an application of flow cytometry in conjunction with cell culture, was developed to identify susceptibility of cytomegalovirus (CMV) isolates to the antiviral drugs ganciclovir (GCV) and foscarnet (PFA). The viral isolates used in this study were obtained from peripheral blood of AIDS patients. Among GCV-susceptible strains, the mean 50% inhibitory concentration (IC50) using AST-FCA was 18 microns (SI50 = 1.4). Among GCV resistant strains, the mean IC50 was 47 microns (SI50 = 3.6). For PFA, the mean IC50 was 80 microns (SI50 = 1.0) for susceptible strains. IC50s for strains resistant to PFA, showed no significant reduction of infectivity at the highest drug concentration (i.e., 200 microns PFA) tested. Additional analyses confirmed the accuracy of AST-FCA by parallel testing using plaque reduction assay. AST-FCA is a novel, nonisotopic, and relatively simple to perform laboratory procedure for the identification of CMV drug-resistant strains.

Antiviral Agents↗

PubMed1996

Idiopathic hypersomnia.

Idiopathic hypersomnia is not as well delineated as narcolepsy and its history is much more recent. There are at least two forms of the disorder: (1) a polysymptomatic form, characterized by excessive daytime sleepiness, nocturnal sleep of abnormally long duration, and signs of sleep drunkenness on awakening, and (2) a monosymptomatic form that manifests only by excessive daytime sleepiness. The most widely used laboratory procedures are nocturnal polysomnographic recording following by an MSLT demonstrating a mean sleep latency of less than 10 minutes. At least in the polysymptomatic form, however, continuous polysomnography on an ad lib protocol deserves to be performed to catch the abnormally long major sleep episode and the long unrefreshing naps. Idiopathic hypersomnia is probably one of the most overdiagnosed sleep disorders. Several other disorders must be excluded before the diagnosis can be considered conclusive. Treatment of idiopathic hypersomnia relies on stimulants, which are frequently less effective and less well tolerated than in narcolepsy.

Adult↗

PubMed1986

When is the vectorcardiogram superior to the scalar electrocardiogram?

The clinical usefulness of the vectorcardiogram is well documented by the numerous reports published in the last 3 decades. It has been found more reliable than the electrocardiogram for the diagnosis of atrial enlargement and right ventricular hypertrophy. It is more sensitive than the electrocardiogram in the recognition of myocardial infarction, especially if the infarction is inferior or if it occurs in the presence of left bundle branch block or left anterior hemiblock. It is helpful in the diagnosis of ventricular pre-excitation and in the localization of the bypass tract. Some repolarization abnormalities are more clearly demonstrated by the vector display. However, some information, such as that on cardiac chamber size and myocardial damage, can also be obtained by other noninvasive tests that are often performed on the same patients. With the increasing awareness of cost-effectiveness of various laboratory procedures in medicine, the vectorcardiogram should no longer be considered a routine cardiac test and should be requested only for a specific clinical purpose. When properly utilized, vectorcardiography should remain a valuable diagnostic as well as teaching tool.

Cardiomegaly↗

PubMed2003

Mycoplasmal mastitis in dairy herds.

Mycoplasmal bovine mastitis is potentially a highly contagious disease that can cause severe economic problems in affected herds. The purchase of replacement heifers and cows are frequently the origin of mycoplasmal mastitis outbreaks in previously Mycoplasma-free herds. Purchased cows and heifers should be quarantined and tested for mycoplasmal mastitis before admission to the regular herd. Detection of Mycoplasma-infected cows by culture of milk is straightforward, although there are problems of sensitivity for its detection in milk samples that are inherent to the nature of the disease and laboratory procedures. After detection of infected cows, the best way to protect the herd is to culture all cows in the herd, cows with clinical mastitis, and all heifers and cows after calving and before entering the milking herd. Control of mycoplasmal mastitis requires test and culling from the herd of Mycoplasma-positive cows if possible. When a large number of cows are infected, strict segregation with adequate management is an option; however, animals in this group should never re-enter the Mycoplama-free herd. The functioning of the milking equipment and milking procedures should be evaluated carefully and any flaws corrected. There is no treatment for mycoplasmal mastitis, and vaccination has not proven to be efficacious to prevent, decrease the incidence, or ameliorate the clinical signs of mycoplasmal mastitis. Waste milk should not be fed to calves without pasteurization. M bovis may cause several other pathologies in animals of different ages on a farm, including pneumonia, arthritis, and ear infections. The survival of mycoplasmas in different farm microenvironments needs to be further investigated for its impact on the epidemiology of the disease.

Animals↗

PubMed2003

Nosocomial pneumonia in immunosuppressed patients.

Nosocomial pneumonia represents a serious challenge for clinicians caring for IC patients. Although there have been advances in prophylactic, preemptive, and therapeutic measures, the implications of an inadequate empirical treatment for survival require a prompt and active attitude. A great diversity of diagnostic and laboratory procedures is currently available. In each case, the clinician must determine the tests that should be performed based on different variables. The proper use of noninvasive and bronchoscopic procedures substantially increases the diagnostic yield causing changes in the empirical treatment in most patients. The authors believe that fiberoptic bronchoscopy must be done early when the pulmonary infiltrates are identified if there is not a rapid (48 hours) and clear response to empiric treatment. This approach allows the establishment of a more specific treatment when the possibilities of full recovery are still high. The potential benefit of treatment modifications for survival in IC patients who require MV and undergo bronchoscopy is most probably minimal, because of the severity and irreversibility of the underlying pulmonary process. It is hoped that the application of molecular tools in diagnosis and the advances in preventive strategies and therapeutic agents will improve the survival of NP in a population of IC patients that is expected to grow over the next years.

Bronchoscopy↗

PubMed2000

Making the diagnosis of fungal infection: when to start treatment.

Fungal infections continue to cause major complications in cancer patients. With the increasing use of aggressive chemotherapy and stem cell transplantation causing profound, prolonged depressed immunity, the risk of invasive mycoses has increased. The prognosis of these infections is poor unless they are diagnosed and treated promptly. The management of opportunistic fungal infections is characterized by a series of unresolved problems, including initial difficulties in obtaining an early diagnosis. Clinical signs indicating a definite diagnosis of fungal infection is frequently absent in cancer patients. There are no distinctive symptoms and fever is the most common and, frequently, the only sign. In only a minority of cases, and usually after recovery from neutropenia, can some clinical features, such as pulmonary pseudomycetoma and hepatosplenic lesions, be suggestive of an invasive mycosis. Thus, laboratory procedures are necessary to reveal or confirm the diagnosis of invasive mycosis and establish disease etiology. Cultures are often negative and positive results need a careful evaluation to determine clinical importance. The significance of fungi isolated from mucosal surfaces and from the respiratory tract in diagnosing invasive mycoses is controversial. The problem in interpreting these microbiological data is the differentiation between fungal infection and colonization. Detection of fungal antigens by molecular methods appears to be promising, but the significance in various clinical settings is still under evaluation. In most cases, the diagnosis depends on a combination of clinical, microbiological, histological and serological results.

Antibodies, Fungal↗