[Prevalence of cardiometabolic risk in a national sample of Costa Rican youth using the waist-height anthropometric indicator]. / Prevalencia de riesgo cardiometabólico en una muestra nacional de jóvenes costarricenses utilizando el indicador antropométrico cintura-estatura.

Central obesity is known as an indicator of cardiometabolic risk, but better anthropometric measures than Body Mass Index (BMI) are needed to detect it. Waist-to-height ratio (WHtR) is an indicator of central adiposity and a strong predictor of cardiometabolic risk. OBJECTIVE: To determine the pre valence of cardiometabolic risk in a representative sample of Costa Rican children and adolescents. PATIENTS AND METHOD: Cross-sectional and descriptive study carried out with 2,684 students from 64 educational centers in Costa Rica. Validated questionnaires were applied to the students and their weight, height, and waist circumference were determined. Weight and body fat percentage of the students were determined with a Tanita model SC-331 S (without column). Height was measured with a SECA stadiometer model 217. The abdominal circumference was estimated using a tape mea sure. BMI and WHtR were calculated for each participant. The behavior of the indicator WHtR was analyzed with logistic regression models. All procedures were approved by the Ethics Committee of INCIENSA. RESULTS: There was a strong and positive correlation between waist circumference and BMI (r = 0.748, p < 0.001, 56% of shared variance), and a positive and moderate correlation between WHtR and BMI (r = 0.611, p < 0.01, 37% of shared variance). CONCLUSIONS: A third part of the stu dent population of elementary, middle, and high schools (31.8%) is at cardiovascular and metabolic risk.

[Breastfeeding duration, mixed feeding and health risk in Costa Rican children and adolescents]. / Duración de la lactancia materna, alimentación combinada y riesgo para la salud en jóvenes costarricenses.

OBJECTIVE: to analyze the associations between breastfeeding duration and mixed feeding (breast milk and formula) with obesity, cardiometabolic risk (WHtR), body fat, and dietary and physical activity risk behaviors in children and adolescents. SUBJECTS AND METHOD: cross-sectional study carried out with 1,467 students in Costa Rica. An interview was conducted with parents and children to gather information on socioeconomic status, education, dietary and physical activity risk behaviors of the youth, including breastfeeding duration and mixed feeding. Body mass index, waist circumference, and body fat were assessed by bioelectrical impedance analysis and cardiometabolic risk was mea sured with the waist-to-height ratio (WHtR ≥ 0.5) by trained personnel. Descriptive statistics and logistic regression models were applied. RESULTS: the average age was 11.4 ± 2.6 years and 50.9% were male. 55.5% of the population was middle class; 60% were sedentary, and 16% presented obesity. Around 20% were breastfed without the introduction of formula before 6 months, 13% were never breastfed, and more than 60% were breastfed for ≥ 6 months. Those children who were fed only with breast milk or in combination with formula for ≥ 6 months presented a lower percentage of obesity than those who received formula feeding only (60.8 vs 39.2; p < 0.005). Children with cardiometa bolic risk (WHtR ≥ 0.50), unhealthy diet, sedentary lifestyle, and who were fed only with formula are at higher risk of developing obesity (OR = 18.8, 95% CI 13.2-26.0). CONCLUSIONS: these results are consistent with other studies and reinforce the evident protection of breastfeeding against the development of obesity.

[Kawasaki syndrome in a 9-year-old boy as a result of COVID-19]. / Kawasaki-Syndrom bei einem 9-jährigen Jungen infolge von COVID-19.

Since the beginning of 2020 an increase in a Kawasaki-like disease has been noted. The WHO assumes a connection to the COVID-19 pandemic and it is defined as the multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019.A 9-year-old boy attended the pediatric emergency department with persistent fever and then developed a classical Kawasaki syndrome with affection of the left coronary artery. A specific origin of an infection could not be detected. The SARS-CoV-2 PCR was negative. In due course positive SARS-CoV­2 antibodies were detected. The patient was treated with intravenous immunoglobulins, ASS and a glucocorticoid, which led to an improvement in the clinical and echocardiographic state of the patient.

[How can the impact of Health Technology Assessment (HTA) in the Austrian healthcare system be assessed? Design of a conceptual framework]. / Wie kann der Impact von Health Technology Assessment (HTA) im österreichischen Gesundheitssystem ermittelt werden? Die Entwicklung eines Analyseinstruments.

INTRODUCTION: In Austria research in Health Technology Assessment (HTA) has been conducted since the 1990s. Research in HTA aims at supporting an adequate and efficient use of health care resources in order to sustain a publicly financed and solidary health care system. Ultimately, HTA research should result in better health of the population. Research results should provide independent information for decision makers. For legitimizing further research resources and for prioritizing future HTA research and guaranteeing the value of future research, HTA research needs itself to undergo evaluation. Aim of the study is to design a conceptual framework for evaluating the impact of HTA research in Austria on the basis of the existing literature. METHODS: An already existing review which presents methods and concepts how to evaluate HTA-impact was updated by a systematic research including literature of the years 2004-January 2010. Results were analysed in regard to 4 categories: definition of the term impact, target groups and system levels, operationalisation of indicators and evaluation methods. RESULTS: Overall, 19 publications were included. Referring to the 4 categories, an explanation of impact has to take into account HTAs multidisciplinary setting and needs a context related definition. Target groups, system levels, indicators and methods depend on the impact defined. Studies investigated direct and indirect impact and were focused on different target groups like physicians, nurses and decision makers on the micro-, and meso level, as well as politicians and reimbursement institutions on the macro level. Except for one reference all studies applied already known and mostly qualitative methods for measuring the impact of HTA research. Thus, an appropriate pool of instruments seems to be available. There is a lack of information about validity of applied methods and indicators. By adapting adequate methods and concepts a conceptual framework for the Austrian HTA-Impact evaluation has been designed. CONCLUSIONS: The paper presents an overview of existing methods for the evaluation of the HTA research. This has been used to identify useful approaches for measuring the HTA-impact in Austria. By providing a context sensitive framework for impact evaluation in Austria the Austrian HTA-research contributes to the international trend of impact-evaluation.

Mycoplasma agassizii sp. nov., isolated from the upper respiratory tract of the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus).

Biochemical, serological and molecular genetic studies were performed on seven mycoplasma isolates that were recovered from the upper respiratory tract of clinically ill desert tortoises. The isolates were serologically related to each other but serologically distinct from previously described species. Unique mycoplasma species-specific 16S rRNA nucleotide sequences were found in the proposed type strain. The name Mycoplasma agassizii is proposed for these isolates. The type strain is PS6T (= ATCC 700616T) which caused upper respiratory tract disease (URTD) in experimentally infected tortoises.

Detection of antibodies to a pathogenic mycoplasma in American alligators (Alligator mississippiensis), broad-nosed Caimans (Caiman latirostris), and Siamese crocodiles (Crocodylus siamensis).

An epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995. Mycoplasma alligatoris sp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled for M. alligatoris as the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response to M. alligatoris exposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P < 0.05) beginning 6 weeks after inoculation. The ELISA also detected seroconversion (P < 0.05) in the relatively closely related broad-nosed caiman Caiman latirostris and the relatively distantly related Siamese crocodile Crocodylus siamensis following experimental inoculation with M. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogen M. alligatoris among captive, repatriated, and wild crocodilian species.

Histologic, neurologic, and immunologic effects of methylmercury in captive great egrets.

Captive great egret (Ardea albus) nestlings were maintained as controls or were dosed with methylmercury chloride at low (0.5), and high doses (5 mg/kg, wet weight) in fish. Low dosed birds were given methylmercury at concentrations comparable to current exposure of wild birds in the Everglades (Florida, USA). When compared with controls, low dosed birds had lower packed cell volumes, dingy feathers, increased lymphocytic cuffing in a skin test, increased bone marrow cellularity, decreased bursal wall thickness, decreased thymic lobule size, fewer lymphoid aggregates in lung, increased perivascular edema in lung, and decreased phagocytized carbon in lung. High dosed birds became severely ataxic and had severe hematologic, neurologic, and histologic changes. The most severe lesions were in immune and nervous system tissues. By comparing responses in captive and wild birds, we found that sublethal effects of mercury were detected at lower levels in captive than in wild birds, probably due to the reduced sources of variation characteristic of the highly controlled laboratory study. Conversely, thresholds for more severe changes (death, disease) occurred at lower concentrations in wild birds than in captive birds, probably because wild birds were exposed to multiple stressors. Thus caution should be used in applying lowest observed effect levels between captive and wild studies.

Pathology of upper respiratory tract disease of gopher tortoises in Florida.

Between August 1993 and September 1995, 24 gopher tortoises (Gopherus polyphemus) were received for pathological evaluations from various locations in Florida (USA). All tortoises were examined for clinical signs of upper respiratory tract disease (URTD) including nasal and ocular discharge, palpebral edema, and conjunctivitis. Of the 24 tortoises, 10 had current or previously observed clinical signs of URTD and 14 did not. A blood sample was drawn for detection of anti-mycoplasma antibodies by ELISA, and nasal lavage samples were collected for culture and detection of Mycoplasma agassizii gene sequences by polymerase chain reaction (PCR). Of the 14 clinically healthy tortoises, eight were sero-, culture- and PCR-negative, and six were seropositive for antibodies against M. agassizii. Of those six, five were culture- and/or PCR-positive for M. agassizii, and one was culture- and PCR-negative. Of the 10 ill tortoises, nine were seropositive by the ELISA and one was in the suspect range. Nine of the ill tortoises, including the suspect tortoise, were culture- and/or PCR-positive for M. agassizii, and one was culture- and PCR-negative. For histologic evaluation and discussion, the eight sero-, culture-, and PCR-negative tortoises were designated URTD-negative, and the other 16 were classified as URTD-positive. Histologic evaluation of the upper respiratory tract (URT) indicated the presence of mild to severe inflammatory, hyperplastic, or dysplastic changes in 14 URTD-positive tortoises. Seven of eight URTD-negative tortoises had normal appearing nasal cavities; one had mild inflammatory changes. Transmission electron microscopy revealed an organism consistent with Mycoplasma spp. on the nasal mucosal surface of tortoises with clinical signs and lesions of URTD. Additionally, gram-negative bacteria were isolated more frequently from the nasal cavities of URTD-positive tortoises than URTD-negative tortoises. Because clinical signs of URTD were never observed in six of the URTD-positive tortoises, we also conclude that subclinical URTD can occur in gopher tortoises.

Morbidity and mortality associated with a new mycoplasma species from captive American alligators (Alligator mississippiensis).

Nine of 74 American alligators (Alligator mississippiensis) from a captive Florida herd of 3-4-m-long, 200-350-kg, adult males greater than 30 yr of age died within a 10-day period during 1995. Nonspecific clinical signs included anorexia, lethargy, muscle weakness, paraparesis, bilateral white ocular discharge, and various degrees of periocular, facial, cervical, and limb edema. Pneumonia, pericarditis, and arthritis were found on postmortem evaluation of the spontaneously dead and euthanatized alligators. Rapidly growing mycoplasmas were identified by culture, and mycoplasma nucleotide sequences were identified by polymerase chain reaction testing of fresh lung and synovial fluid from an affected alligator. Culture of banked frozen lung from necropsy specimens and fresh lung and fresh synovial fluid from newly affected alligators confirmed the presence of a new mycoplasma species in seven of eight individuals. Oxytetracycline was administered, but related deaths continued for 6 mo until only 14 of the initial alligators remained. An enzyme-linked immunosorbent assay to detect antibody was developed, and the organism was transmitted experimentally to naive juvenile alligators, although the source of the organism, Mycoplasma sp. (ATCC 700619), has not been identified. The alligator isolate is a novel species in the mycoplasma family because its nucleotide sequence does not match those of over 75 characterized mycoplasma species. Such factors as population density, animal age, and mycoplasmal virulence likely contributed to the course of disease.

Seroepidemiology of upper respiratory tract disease in the desert tortoise in the western Mojave Desert of California.

Several factors have combined with an upper respiratory tract disease (URTD) to produce declines on some population numbers of desert tortoises (Gopherus agassizii) in the western USA. This study was designed to determine the seroepidemiology of URTD in a population of wild adult tortoises at the Desert Tortoise Research Natural Area (DTNA) study site in Kern County (California, USA). Prior to initiation of the study, there was a dramatic decline in the number of individuals in this population. At each individual time point, samples were obtained from 12 to 20 tortoises with radiotransmitters during winter, spring, summer, and fall from 1992 through 1995. During the course of the study, 35 animals were sampled at one or more times. Only 10 animals were available for consistent monitoring throughout the 4 yr period. Specific antibody (Ab) levels to Mycoplasma agassizii were determined for individual tortoises by an enzyme-linked immunosorbent assay (ELISA) test. Specific Ab levels were not influenced by the gender of the tortoise. Levels of Ab and distribution of ELISA+, ELISA- and suspect animals were not consistently affected by season within a single year or for a season among the study years. Significantly more tortoises presented with clinical signs in 1992 and 1995. The profile of ELISA+ animals with clinical signs shifted from 5% (1992) to 42% (1995). In 1992, 52% of tortoises lacked clinical signs and were ELISA-. In 1995, this category accounted for only 19% of tortoises. Based on the results of this study, we conclude that URTD was present in this population as evidenced by the presence of ELISA+ individual animals, and that the infectious agent is still present as evidenced by seroconversion of previously ELISA- animals during the course of the study. There is evidence to suggest that animals may remain ELISA+ without showing overt disease, a clinical pattern consistent with the chronic nature of most mycoplasmal infections. Further, there are trends suggesting that the clinical expression of disease may be cyclical. Continued monitoring of this population could provide valuable information concerning the spread of URTD in wild tortoise populations.

Butyrylcholinesterase formulated in liposomes.

Exogenous cholinesterases have the potential to take part in defence against organophosphate toxins, by acting as scavenger systems. Postulating that formulation in liposomes could enhance the toxin-scavenging potential of these enzymes, we have initiated studies of such formulations and are reporting here our first steps, exploring butyrylcholinesterase (BChE) in multilamellar liposomes composed of phosphatidylcholine. We started by developing an essential research tool: a multisample, sensitive and rapid enzyme-activity assay, based on the Ellman reaction, that could be performed directly on liposome-containing samples. Using an ELISA reader equipped to follow time-dependant absorbency changes, 10 min sufficed to assay 96 samples simultaneously. Next, several key properties of liposome-formulated BChE were explored and the major findings were: (i) the encapsulated enzyme was found to retain its activity. (ii) Enzyme activity was found to increase (at constant enzyme concentration) in the presence of the lipid, in a lipid-concentration-dependant manner. Through data analysis it was possible to attribute this effect to changes in k(cat). (iii) Good, reproducible, encapsulation efficiencies (for macromolecules) in the range of 30% were obtained at liposome concentrations of 100 mM lipid. (iv) Free BChE was completely susceptible to proteolysis under conditions mimicking enzymically-hostile biological environments, whereas > or = 60% of the liposome-formulated BChE was protected, found to be inaccessible to the proteolytic enzymes. (v) Short-term exposures of free and liposome-encapsulated BChE to the inhibitor paraoxon, generated significant losses in enzyme activity. Residual activities of both BChE formulations dropped considerably over the paraoxon concentration range of 0.02-0.11 microM, down to 3 and 11% for free and liposome-encapsulated enzyme respectively. These data are a clear indication that the encapsulated BChE was accessible to the inhibitor, indicating that such liposomal formulations have the potential to perform as the desired scavenger systems.

Persistence of maternal antibodies against Mycoplasma agassizii in desert tortoise hatchlings.

OBJECTIVE: To investigate Mycoplasma agassizii-specific maternal antibodies in desert tortoise (Gopherus agassizii) hatchlings. SAMPLE POPULATION: Plasma from 43 captive-reared desert tortoise hatchlings. PROCEDURE: ELISA for M agassizii-specific antibodies was performed. Four hatchlings from 4 clutches of 3 M agassizii-seropositive females with chronic upper respiratory tract disease (URTD) were tested on the day of hatching (set 1), and 20 hatchlings from 4 clutches of 4 M agassizii-seropositive females with URTD and 19 hatchlings from 4 M agassizii-seronegative healthy females were tested at 4, 8, 12, and 29 months old (set 2). Immunoblot analysis was performed to determine immunoglobulin classes in yolk and plasma of hatchlings. To determine infection status of hatchlings, yolk, egg shell membranes (set 1), and nasal lavage fluid (sets 1 and 2) were examined for M agassizii by use of polymerase chain reaction. RESULTS: Yolk and hatchling plasma had significantly lower amounts of specific antibodies than did plasma from adult females. The IgG and IgM antibodies were transferred, but M agassizii-specific antibodies were of the IgG class. Hatchlings were not infected with mycoplasmas. Offspring of sick females had significantly higher specific antibody titers than did offspring of healthy females. Titers were still significantly different in 1-year-old hatchlings. CONCLUSIONS: Desert tortoise females transfer specific IgG and IgM antibodies to their offspring that are still detectable after 1 year. CLINICAL RELEVANCE: Infection with M agassizii may be misdiagnosed in hatchlings with persistent maternal antibodies. Passively acquired antibodies may have a role in pathogenesis of mycoplasma-induced respiratory tract disease and other diseases.

Upper respiratory tract disease in the gopher tortoise is caused by Mycoplasma agassizii.

Upper respiratory tract disease (URTD) has been observed in a number of tortoise species, including the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus). Clinical signs of URTD in gopher tortoises are similar to those in desert tortoises and include serous, mucoid, or purulent discharge from the nares, excessive tearing to purulent ocular discharge, conjunctivitis, and edema of the eyelids and ocular glands. The objectives of the present study were to determine if Mycoplasma agassizii was an etiologic agent of URTD in the gopher tortoise and to determine the clinical course of the experimental infection in a dose-response infection study. Tortoises were inoculated intranasally with 0.5 ml (0.25 ml/nostril) of either sterile SP4 broth (control group; n = 10) or 10(8) color-changing units (CCU) (total dose) of M. agassizii 723 (experimental infection group; n = 9). M. agassizii caused clinical signs compatible with those observed in tortoises with natural infections. Clinical signs of URTD were evident in seven of nine experimentally infected tortoises by 4 weeks postinfection (p.i.) and in eight of nine experimentally infected tortoises by 8 weeks p.i. In the dose-response experiments, tortoises were inoculated intranasally with a low (10(1) CCU; n = 6), medium (10(3) CCU; n = 6), or high (10(5) CCU; n = 5) dose of M. agassizii 723 or with sterile SP4 broth (n = 10). At all time points p.i. in both experiments, M. agassizii could be isolated from the nares of at least 50% of the tortoises. All of the experimentally infected tortoises seroconverted, and levels of antibody were statistically higher in infected animals than in control animals for all time points of >4 weeks p.i. (P < 0.0001). Control tortoises in both experiments did not show clinical signs, did not seroconvert, and did not have detectable M. agassizii by either culture or PCR at any point in the study. Histological lesions were compatible with those observed in tortoises with natural infections. The numbers of M. agassizii 723 did not influence the clinical expression of URTD or the antibody response, suggesting that the strain chosen for these studies was highly virulent. On the basis of the results of the transmission studies, we conclude that M. agassizii is an etiologic agent of URTD in the gopher tortoise.

[Surgery of stomach carcinoma--established and controversial procedures. Retrospective 1-year analysis with discussion of current aspects]. / Magencarcinomchirurgie--Bewährtes und Kontroverses. Retrospektive 10-Jahres-Analyse mit Diskussion aktueller Aspekte.

BACKGROUND: Current problems in gastric cancer surgery concern the extent of gastric resection, the need for abdominal evisceration, the degree of lymphadenectomy, and optimal preoperative tumor staging procedure. PATIENTS AND METHODS: In a restrospective analysis, data on epidemiology, extent of surgery, histopathology, postoperative complications, mortality, and survival in 284 gastric cancer patients were evaluated. RESULTS: Our results are in favor of subtotal gastrectomy performed for all T stages located in the distal or middle third, provided that a tumor-free margin of 5 cm in intestinal type and 10 cm in diffuse Lauren's type tumor can be achieved. Additional organ resections are indicated only if direct tumor invasion has occurred, and should not be part of an extended lymphadenectomy procedure. The degree of lymph node removal should be guided by the primary tumor site. Multimodal therapeutic approaches and high postoperative mortality after exploratory laparotomy justify the use of diagnostic laparoscopy in T3/4 tumors and if diagnostic scans suggest possible tumor spread. CONCLUSION: Even though surgery for gastric cancer is well standardized, a tailored surgical approach to different extent of gastric cancer appears warranted.

[Diagnostic and therapeutic strategies in local recurrence of stomach carcinoma]. / Diagnostische und therapeutische Strategien beim Lokalrezidiv des Magenkarzinoms.

Despite clear margins at the time of resection, 7 to 20% of the patients experience local recurrence of the primary stomach tumor. Intraluminal recurrence is rare but curable in 50% of the cases without distant metastases. Extraluminal recurrent gastric cancer comprises the typical pattern of recurrence and cannot be removed in most of the patients. Predisposing factors that favor the development of recurrent tumors are: higher tumor stages, extended lymph node involvement, tumor grades 3 and 4, diffuse type according to Lauren's classification, and intraoperative perforation of the primary gastric carcinoma.

Treatment of surgically induced acute liver failure by transplantation of conditionally immortalized hepatocytes.

The shortage of human livers available for hepatocyte isolation limits its clinical application. The availability of cloned, conditionally immortalized hepatocytes that could be grown in culture but would lose their transformed phenotype and provide metabolic support upon transplantation would greatly facilitate the treatment of acute liver failure. Toward this goal, we transduced isolated Lewis rat hepatocytes using a replication-defective recombinant retrovirus capable of transferring a gene encoding a thermolabile mutant simian virus 40 T antigen (SV40ts). The cloned, immortalized hepatocytes proliferate at 33 degrees C. At the nonpermissive temperatures (37-39 degrees C), they stop growing and exhibit characteristics of differentiated hepatocytes. These cells did not produce tumors when transplanted in mice with severe combined immunodeficiency disease or in syngeneic rats. To induce acute liver failure, Lewis rats were subjected to 90% hepatectomy (Hpx) and given 5% oral dextrose. All rats that did not undergo hepatocyte transplantation died within 96 hr. Fifty percent of rats that received intrasplenic injection of 10 x 10(6) primary Lewis rat hepatocytes (G2, n=6) or 10 x 10(6) SV40ts-conditionally immortalized (SV40ts-ci) hepatocytes (G3, n=8) 1 day before 90% hepatectomy survived, whereas 80% of rats that received an intraperitoneal injection of 200 x 10(6) primary Lewis rat hepatocytes (G4, n=10) or 200 x 10(6) SV40ts-ci hepatocytes (G5, n=10) on the day of hepatectomy survived. Survival after intraperitoneal injection of a cellular homogenate of 200 x 10(6) primary Lewis rat (G7, n=9) or SV40ts-ci hepatocytes (G8, n=10) on the day of Hpx was 33% and 40%, respectively, whereas survival after intraperitoneal injection of 200 x 10(6) Lewis rat bone marrow cells (G6, n=7) was 29%. Thus, transplanted, conditionally immortalized hepatocytes can be as effective as primary hepatocytes in supporting life during acute liver insufficiency. This work represents the first step in developing an hepatocyte cell line that would partially alleviate the organ-donor shortage and could be of potential clinical value.

Liposome-encapsulated ampicillin: physicochemical and antibacterial properties.

The objectives of this study were to develop high-performance liquid chromatography (HPLC) and antibacterial assays for ampicillin encapsulated in multilamellar liposomes (MLV) and investigate the physicochemical and antibacterial properties of ampicillin-liposome systems. The major findings were fourfold. First, ammonium acetate (0.575%) in methanol:water (450:550; v/v), adjusted to pH 7.2, was suitable as the mobile phase for the HPLC determinations of ampicillin in both aqueous and liposomal systems. This mobile phase also provided (alone or with additional methanol) complete dissolution of liposomal assay samples in a precolumn treatment that made all of the encapsulated drug available for chromatography. Multiple samples were assayed without any technical limitations. Second, the growth-inhibition antibacterial assay developed, which used the USP test organism Micrococcus Luteus and paper disks, was quantitative for both free and liposome-encapsulated ampicillin. Third, the physicochemical properties of encapsulated ampicillin include encapsulation efficiencies of 10 to 50% for liposome concentrations in the range 10-200 mM (lipid), and a single rate constant to sufficiently and quantitatively describe the diffusion of encapsulated ampicillin, with half-lives in the range of 40 h. Fourth, the biological properties include the first direct evidence that encapsulated ampicillin retains full biological activity; that is, liposome-encapsulated ampicillin was active against extracellular bacterial colonies of Micrococcus luteus. Furthermore, encapsulation enhanced ampicillin stability. For example, free ampicillin in an aqueous solution that was stored for 5 weeks at 4 degrees C lost 50% of its initial activity, whereas liposome-encapsulated ampicillin (freed from unencapsulated drug) stored under the same conditions lost only 17% of its initial activity. The findings of this study, provide strong support for ampicillin-liposome formulations as valid dosage forms for this drug that are worthy of further experimental evaluations.

Relationship between clinical signs of upper respiratory tract disease and antibodies to Mycoplasma agassizii in desert tortoises from Nevada.

Plasma samples collected in 1990 from free-ranging desert tortoises (Gopherus agassizii) with and without clinical signs of upper respiratory tract disease (URTD) from Las Vegas Valley, Clark County, Nevada (USA), were tested by enzyme-linked immunosorbent assay (ELISA) for antibodies to Mycoplasma agassizii, a causative agent of URTD. The relationship between clinical signs and ELISA test results was evaluated. Of the 144 tortoises tested, 45 (31%) had clinical signs while 72 (50%) were seropositive. Presence of clinical signs of URTD was positively related to positive ELISA results (P < 0.0001) regardless of sex or age of the animal. Eighty-four percent of animals with clinical signs tested seropositive. Mucous nasal discharge, the most severe and obvious of the clinical signs, was highly predictive for exposure to M. agassizii based on the ELISA. Ninety-three percent of tortoises with mucous nasal discharge tested seropositive. Serologic testing for M. agassizii antibodies supported clinical signs as useful indicators of URTD, but it also detected potential subclinical infection in 34 (34%) of 99 animals without clinical signs.

Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen.

Hematopoietic chimerism has been used in the laboratory to induce life-long immunologic tolerance to donor antigens. The present study demonstrates that mice transplanted with autologous bone marrow cells retrovirally transduced to express HLA-A2.1 develop a significantly depressed immune response to this antigen while retaining normal reactivity to HLA-B7. Retrovirus-mediated transduction was performed using whole bone marrow-producer cell coculture. This approach did not result in significant gene transfer into hematopoietic progenitor cells. Despite this, the antibody response to HLA-A2.1 in mice reconstituted with genetically modified BMC was completely suppressed three months following bone marrow transplantation. Cell-mediated immunity to HLA-A2.1 was partially suppressed in three-fourths of animals tested three months later, although one animal had a CTL profile similar to that an of HLA-A2.1 transgenic mouse. Complete suppression of the antibody-mediated immune response occurred when only one-third of mice had evidence of the introduced genes in their spleen and one-tenth had the introduced sequences in their circulating WBCs by PCR. In conclusion, engineering of BMC to express donor MHC genes may be an alternative to xenogeneic BMT to induce chimerism and tolerance. More efficient transduction of bone marrow progenitor cells may result in more persistent gene expression and long-lasting transplantation tolerance in recipients of genetically modified bone marrow. Successful application of this technology may also be useful in altering immune responses to other external and self antigens.

Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy.

Transplantation of hepatocytes has been shown to provide metabolic support during liver failure in experimental models. The potential clinical application of hepatocyte transplantation, however, is limited by the need for readily available, well-characterized cells, and a worldwide shortage of donor organs. A clonal hepatocyte cell line that could be grown economically in vitro and would exhibit a differentiated, nontransformed phenotype following transplantation would be an attractive solution to this problem. To test this alternative, primary Lewis rat hepatocytes were conditionally immortalized by retroviral transduction with a thermolabile mutant Simian virus 40 (SV40) large T antigen. The cloned immortalized cells proliferate in culture at 33 degrees C and stop growing at 37 degrees C to 39 degrees C. Transplanted into normal livers, these hepatocytes integrate normally into liver cords. When transplanted into the spleens of portacaval-shunted rats, they protect recipients from hyperammonemia-induced hepatic encephalopathy. The cells engrafted in the spleen exhibit normal morphology, secrete bile, and express albumin messenger RNA. The protection from hyperammonemia is reversed by splenectomy. These studies show that hepatocytes can be conditionally immortalized, expanded in culture, and are capable of providing metabolic support in chronic liver insufficiency. Safeguards that could make these cells clinically useful can be accomplished using currently available technology.