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PubMed1994

Urea and lactate determined in 1-microL whole-blood samples with a miniaturized thermal biosensor.

A miniaturized flow-injected thermal biosensor was developed for the determination of urea and L-lactate in undiluted blood in 1-microL samples. The sensor employed a small enzyme column constructed of stainless steel tubing and microbead thermistors. Urease and lactate oxidase/catalase were separately immobilized onto controlled-pore glass beads, which, in turn, were charged into the enzyme column. With a flow rate of 70 microL/min, linear analytical ranges from 0.2 to at least 50 mmol/L and 0.2 to 14 mmol/L were obtained for urea and lactate, respectively. The relative standard deviations (CVs) for measurements of analyte in buffer were 0.91% for urea and 1.84% for lactate. For urea in whole blood, the CV for 50 determinations was 4.1%. Contrived samples containing various concentrations of urea and L-lactate in whole blood were determined with this sensor and with a spectrophotometric method. Comparisons of the results gave correlation coefficients of 0.989 and 0.984 for 30 blood urea and 30 blood lactate assays in concentrations ranging from 4 to 20.9 mmol/L and from 1.7 to 12.7 mmol/L, respectively.

Biosensing Techniques↗
PubMed1994

On the postnatal growth of the canalis mandibulae in the miniature pig.

The shape and the growth of the canalis mandibulae in female miniature pigs, MINI-LEWE, were analysed in 11 postnatal age steps. The lower jaw halves were cut frontally in a defined position and the heights and widths of the canalis mandibulae were measured. Changes in the shape of the canalis mandibulae in the frontal saw cuts were observed with regard to the dentition. After the eruption of the deciduous teeth there was no further change in shape. Growth curves of the mandibular canal's measures reached a peak during the eruption of the deciduous teeth and the first molar, decreasing significantly afterwards. A close correlation was observed between the measures of canal height and canal width. On the other hand there was only a slight correlation with the external measures of the mandible, which suggests that there are different growth processes.

Aging↗
PubMed1993

Estrus detection by using vaginal cytologic examination in miniature swine.

Vaginal smears were obtained from four Yucatan miniature swine daily for 69 days and stained with hematologic stain. Epithelial cells were categorized as superficial, large intermediate, small intermediate, or parabasal. Leukocytes were also quantitated. External signs of estrus were recorded, including swelling, discharge, restlessness, or vocalization. Mean age of three of the swine was 147 days at the beginning of the study. The fourth pig was 317 days old. The three younger swine had their first observed estrus at the age of 178 days (range, 167 to 196 days). Mean cycle length was 17 to 21 days. The moving mean leukocyte count (i.e., each value was averaged with the values for the previous day and the following day) always exceeded the epithelial cell count (regardless of type), except during the 3 to 4 days when the pigs exhibited external signs of estrus. Further, epithelial cells were at their peak during estrus, decreasing markedly during diestrus, and increasing again during proestrus. The combined superficial plus large intermediate cell counts were significantly higher during estrus than during diestrus or proestrus. We conclude that daily vaginal smears can be used to determine the stage of estrus in Yucatan pigs.

Animals↗
PubMed1994

Exercise attenuates the anti-lipolytic effect of adenosine in adipocytes isolated from miniature swine.

Four pairs of female and six pairs of male litter-mate Yucatan miniature swine (Sus scrofa) were used in this study which examined the possibility that endurance exercise training reduces the sensitivity of adipocytes to the anti-lipolytic effects of adenosine. One member of each pair was exercise-trained on a treadmill for three months while its litter-mate remained sedentary, after which time over-the-shoulder fat and left brachialis muscle were biopsied. Despite a predominance of type IIB fibres, biopsied muscle of exercised swine had 38% more citrate synthase activity than controls (P < 0.05). The average cell diameters of adipocytes isolated from exercisers were 14% smaller (P < 0.05) than those from controls. Rates of adrenaline-stimulated lipolysis expressed as nmol glycerol released/90 min incubation period per 10,000 cells failed to differ between the two groups; however, when expressed per cm2 surface area, a significant 37% increase was observed. Incubation with 1 microM adrenaline and increasing doses of phenylisopropyladenosine (PIA) caused a rightward shift in the dose-response curve of adipocytes in five of the ten exercisers compared to litter-mate controls. The concentration of PIA causing one-half inhibition of lipolysis was 64% greater in adipocytes from exercisers than controls (4.03 nM vs. 2.49 nM, n = 10, P < 0.05). These data support the hypothesis that endurance exercise-training induces a reduction in adipocyte sensitivity to adenosine, thereby facilitating fatty acid mobilization.

Adenosine↗
PubMed1994

Tolerance to discordant xenografts. I. Sharing of human natural antibody determinants on miniature swine bone marrow cells and endothelial cells.

Xenotransplantation could potentially overcome the organ shortage that currently limits the field of transplantation. Because of their breeding characteristics as well as their size and physiologic similarities to humans, we have chosen miniature swine as possible xenograft donors, and are currently attempting to develop a means of using mixed xenogeneic chimerism as an approach to tolerance induction in swine-to-primate species combinations. One major barrier to organ grafting from pig to man is the presence in human serum of preformed natural antibodies (NAb) reacting with antigens expressed on porcine endothelial cells and causing hyperacute rejection. Previous experiments performed in our laboratory have shown that both humoral and cellular tolerance can be induced in a concordant xenogeneic species combination (rat-->mouse) using donor bone marrow infusion following conditioning with a nonmyeloablative regimen. Induction of chimerism in these animals was associated with a marked reduction in the level of IgM natural antibodies that recognize rat bone marrow cells. A similar approach could also lead to humoral and cellular tolerance induction in the swine-->human species combination, permitting transplantation of vascularized organs from the swine donor. To determine the potential of bone marrow transplantation to induce a state of "natural antibody tolerance," it was essential to determine whether or not all human NAb target antigens expressed on swine EC are also expressed on cells derived from swine bone marrow. We have addressed this question by evaluating the ability of various swine bone marrow-derived cells to absorb human IgM and IgG NAb that bind to swine EC. Our results demonstrate that swine bone marrow cells and their progeny can absorb almost all IgM NAb that bind to swine EC, as detected by flow cytometric and ELISA assays. Specificity of absorption was demonstrated, as total serum IgM levels declined only minimally after absorption on swine BMC and to an extent comparable to that observed following absorption with human cells, which did not deplete swine EC-binding NAb. Human IgG binding to swine EC was also completely absorbed by swine BMC. These results suggest that a state of "NAb tolerance" could be induced by successful swine marrow engraftment in man. Furthermore, swine PBL, platelets, and EC were able to absorb most IgM NAb that bound to swine BMC, suggesting that absorption using antigen from any of these tissues might facilitate marrow engraftment, and hence tolerance induction, in this species combination.

Animals↗
PubMed1993

Extensive allelic polymorphism in the CDR2-like region of the miniature swine CD4 molecule.

MHC class II polymorphism is well documented whereas only minimal polymorphism has been reported for the CD4 molecule with which it interacts. We report on the structural basis of an allelic polymorphism of the CD4 molecule in miniature swine. Eleven of 13 nucleotide differences between the 2 alleles cause amino acid replacements. A majority of these replacements are clustered in a region protruding as a loop structure, termed Ig CDR2-like, from the surface of the amino terminal domain. This part of the human CD4 appears to comprise the binding site for the human immunodeficiency virus gp120 protein. The loop structure has also been implicated in the binding of CD4 to MHC class II, but this is currently a matter of some controversy. Our previous results have indicated that the porcine CD4 polymorphism, that we now show is situated in this loop, does not significantly affect the binding to class II. However, because the polymorphism appears to have been selected for and is situated in a very exposed part of the molecule, it is likely to be of functional significance.

Alleles↗
PubMed1993

Hemodynamic parameters of anesthetized pigs: a comparative study of farm piglets and Göttingen and Yucatan miniature swine.

We studied the evolution in time of the main hemodynamic parameters in farm piglets and Göttingen and Yucatan miniature swine anesthetized with droperidol, flunitrazepam, and alpha-chloralose. Measurements included arterial pressure, heart rate, intraventricular pressure, and maximum rate of increase during contraction (dp/dt max). For each parameter and each strain of swine, we determined the mean stability period defined as the longest period of time during which the observed values ranged between their mean value +/- 15% and the mean absolute values during the stability period. In our experimental conditions, the parameters remained constant for 2 to 3.5 hours. Only minor interstrain variations were noticed.

Anesthesia↗
PubMed1993

Microinjection of L-lactate in the preretinal vitreous induces segmental vasodilation in the inner retina of miniature pigs.

PURPOSE: The authors investigated the hypothesis that the retinal vasomotor effect of acute hypoxia is mediated by lactate. METHODS: Retinal vasomotor arteriolar response was measured in the intact eyes of miniature pigs after systemic administration and after local preretinal juxta-arteriolar microinjection of lactate. RESULTS: Injection of L-lactate (physiologically produced lactate) into the systemic circulation decreased the arterial blood pH but did not dilate the retinal arterioles. By contrast, microinjections of L-lactate (0.5 mol/l, pH 2) into the juxta-arteriolar vitreous induced a reversible segmental vasodilation of 32 +/- 4% (standard deviation). This vasodilation did not depend on periarteriolar pH lowering because microinjections of a 0.5 mol/l L-lactate at neutral pH also dilated segmentally the retinal arterioles (37 +/- 5.5%). The effect of lactate was stereospecific because microinjections of the isomer D-lactate (0.5 mol/l, pH 2) did not affect the arteriolar caliber (P = 0.63). Perfusion of the eye with the cyclo-oxygenase inhibitor indomethacin, through cannulization of the sublingual artery, caused a generalized reversible arteriolar vasoconstriction of 51 +/- 9.8% but did not inhibit the segmental vasodilator effect of locally microinjected L-lactate. CONCLUSIONS: It is known that acute hypoxia in the isolated retina causes an increase in lactate production. In the intact eye, there is a retinal vasodilation, which is not inhibited by indomethacin. Hence, it was concluded that retinal, but not blood, lactate is a possible mediator of the acute hypoxia-induced vasodilation.

Animals↗
PubMed1995

Study of immune function in inbred miniature pigs vaccinated and challenged with suid herpesvirus 1.

Specific immune responses of inbred miniature pigs following vaccination and challenge with suid herpesvirus 1 (SHV-1) were determined. Vaccination of swine with SHV-1 elicited both specific neutralizing antibody and lymphoproliferative responses. Moreover, pigs vaccinated with SHV-1 were fully protected against a lethal virus challenge. Pigs vaccinated with a recombinant (r) SHV-1 virus, followed by challenge with a virulent SHV-1, had lower percentages of circulating T- and B-lymphocytes, and showed a significant (P < or = 0.05) reduction in peripheral blood mononuclear cell (PBMC) antibody-dependent cell-cytotoxicity than control (noninfected, SHV-1 sero-negative) animals. From the 5th through the 8th week of postchallenge, rSHV-1 was isolated from 2 of 4 pigs. Presence of r-virus was indicative that PBMC were infectious in vivo. The rSHV-1, with beta-galactosidase activity, was only recovered from ConA- and IL-2-stimulated primary PBMC cocultivated with porcine kidney cells. Control pigs exposed to challenge SHV-1 elicited both specific neutralizing antibody and lympho-proliferative responses followed by subsequent infection. These infected pigs, compared to control pigs, had significantly (P < or = 0.05) lowered percentages of T- and B-lymphocytes, lowered T-cell mitogenic responses, variable PBMC counts, and lowered blood phagocytic cell function. When PBMC from control pigs were cultured and infected with SHV-1, the virus caused a significant (P < or = 0.05) suppression of T-cell proliferation and PBMC mitochondrial dehydrogenase and macrophage activities.

Animals↗
PubMed1995

[Segregation of pigment cell anomalies in Munich miniature swine (MMS) Troll crossed with German Landrace].

In order to study the inheritance of melanocytic lesions in the Munich Miniature Swine (MMS) Troll, we established the F1-, F2- and R1-generations, starting with one melanoma-bearing MMS-Troll boar and four German Landrace sows as founder animals. A total of 168 animals were born, 24 in the F1-, 111 in the F2-, 19 in the B1DL-, and 14 in the B1Troll-generation. Benign lesions with lentigoid melanocytic hyperplasia or nests of hyperplastic melanocytes like in human junctional nevus were seen in 10 (41.7%) F1-, 17 (15.3%) F2-, 2 (10.5%) B1DL-, and 6 B1Troll-animals. Malignant melanomas occurred only in the F2-(4 animals; 3.6%) and in the B1Troll-(1 animal; 7.1%) generation. The observed segreation suggests different modes of inheritance for nevi and melanomas. The segregation of nevi can be explained by a major gene model with additional modification by a polygenic component. For melanoma, a major gene model does not fit the data sufficiently. Therefore, a two-or-three-locus model with doubled or tripled recessive affected animals has to be supposed for the inheritance of melanoma. Influence of SLA-haplotypes could not be observed.

Animals↗
PubMed1996

Long-term influence of dietary fat (sunflower oil, olive oil, lard and fish oil) in the serum fatty acid composition and in the different lipidic fractions, in miniature swine.

A total of 24 miniature swine (sus scrofa) was fed with four diets of 9% fat content, differing only in the quality of the fat source (sunflower oil, olive oil, lard fat and fish oil) for a 12 months period. After the experimental period, the influence of the dietary fat on the serum fatty acid composition, and the distribution of those fatty acids in the different serum lipidic fractions was studied. The olive oil group had the lowest SI value (total sum of saturated fatty acids) and the highest MUFA value (total sum of monounsaturated fatty acids) in serum. The sunflower group had higher proportions of n6 polyunsaturated fatty acids in serum, when compared with the remaining groups. The lard group and the fish oil group had both the highest values of SI and n3 polyunsaturated fatty acids. The results show that fatty acid composition in serum reflected, but not in all cases, the fatty acid composition of the diet. The highest correspondence was found with sunflower oil and olive oil diets. The existence of interactions between the different fatty acid series was evident, especially with the lard and the fish oil diets, as well as endogenous synthesis mechanisms. In lipidic fractions, higher correspondence with diet was found in EC fraction, but, as well as in serum, the existence of interactions between fatty acid series was evident, and especially activity and biosynthesis of desaturases may have been affected.

Analysis of Variance↗
PubMed1997

Liposomes-coated hydroxyapatite and tricalcium phosphate implanted in the mandibular bony defect of miniature swine.

Hydroxyapatite and tricalcium phosphate have been used as bone implants for some period of time. Now unfortunately, these materials have failed to become the nucleation sites for bone regeneration. We hypothesized that coating hydroxyapatite and tricalcium phosphate with negatively charged liposomes may improve the nucleation process for new bone formation. The present study was designed to test this hypothesis. Experiments were carried out in 15 miniature swines' mandibular angle with artificial bony defects. In each of the swine, the bony defects on one side were implanted with either liposomes coated with hydroxyapatite or liposomes coated with tricalcium phosphate, while the other side served as control. At the end of the third and sixth weeks following the operation, we observed result, took histology and radiographs of the operated area. The results showed that liposomes-coated materials were biocompatible and their clinical endpoint was enhanced. At the end of the third week, the implant material was surrounded by dense connective tissues. At the end of the sixth week, there were new bone formations near the implanted material. In addition, liposomes which were immobilized in agarose gel and implanted in the defects showed new bony bridge formation. These observations suggest that liposomes have the ability in promoting repair of osseous deficiencies.

Alveolar Process↗
PubMed1976

Miniature dark adaptometer using tritium as a light source.

A miniature dark adaptometer to measure monophasic rod function, used with disposable masks, permitted 11 measurements to be taken in 1 1/2 hours. The light source, tritium-phosphoric sulfide gas, had a known brightness decay rate incorporated in the calibration table which is provided with the instrument.

Dark Adaptation↗
PubMed1998

Ligand assays: from electrophoresis to miniaturized microarrays.

The main developments in the "ligand assay" field in which I have been involved are traced. These include the original development of "first generation" competitive assays relying on radiolabeled analyte markers; the development of the first "second generation", noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first "third generation" miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter--applicable both to immunoassay and to DNA/RNA analysis--are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.

Electrophoresis↗
PubMedDate not supplied

Removal torque values of titanium implants in the maxilla of miniature pigs.

The purpose of this study was to compare side-by-side two different titanium screw-type implants in the maxillae of miniature pigs. The test implants had a machined and acid-etched surface (Osseotite) whereas the control implants were sandblasted and acid-etched (SLA). After 4, 8, and 12 weeks of healing, removal torque testing was performed to evaluate the shear strength of the bone-implant interface for both implant types. The results demonstrated significant differences between both implant types (P < .01). Osseotite implants revealed mean removal torque values (RTV) of 62.5 Ncm at 4 weeks, 87.6 Ncm at 8 weeks, and 95.7 Ncm at 12 weeks of healing. In contrast, the SLA implants demonstrated mean RTV of 109.6 Ncm, 196.7 Ncm, and 186.8 Ncm at corresponding healing periods. The mean RTV for SLA implants was 75% to 125% higher than for Osseotite implants up to 3 months of healing.

Analysis of Variance↗
PubMed1998

Distraction osteogenesis of the mandible with a modified intraoral appliance: a pilot study in miniature pigs.

The purpose of this study was to evaluate an intraoral, nontooth-borne, submucosal distraction appliance in the pig mandible from a clinical, radiographic, and histologic perspective. Intraoral distraction appliances were fabricated, tested for strength, and then adapted and rigidly fixated to the mandibles of three growing miniature Yucatan pigs. A bicortical osteotomy was performed on the right side of the mandible, and a buccal and lingual corticotomy on the left. Two amalgam markers were placed on each side of the osteotomy/corticotomy sites as references. Measurements were taken to evaluate the length and vector of the distraction site, and radiographs were obtained perioperatively. After a latency period of 5 days, distraction was commenced and continued for 13 days at a rate of 1 mm/day. Clinical evidence of distraction was apparent in all cases, with resulting Class III skeletal deformities. Distraction progressed further for the corticotomy sides than the osteotomy sides because of distraction device failure. Radiographic examination revealed increased bone density at the periphery of the distracted callus, with less density apparent centrally. Histologic examination showed fibrous connective tissue in the center of the callus, with cartilage and osteoid formation at the periphery. Bone formation was parallel to the vectors of distraction in all cases. This study confirms that the use of a modified intraoral appliance for distraction osteogenesis is feasible. Use of this device overcomes many of the problems associated with extraoral devices and may have clinical applications.

Animals↗
PubMed1999

Peripheral tolerance to class I mismatched renal allografts in miniature swine: donor antigen-activated peripheral blood lymphocytes from tolerant swine inhibit antidonor CTL reactivity.

Studies utilizing partially inbred miniature swine have demonstrated that a short course of cyclosporin A leads to indefinite survival of two haplotype class I mismatched renal allografts. In the present study, we have examined peripheral regulatory mechanisms that may be involved in maintenance of tolerance by coculturing PBL from long-term tolerant animals with naive recipient-matched PBL in cell-mediated lympholysis assays. We show that PBL from tolerant animals, primed in vitro with donor Ag, suppress antidonor CTL reactivity by naive recipient-matched PBL. Suppression was not observed when PBL from naive animals, primed with donor-matched PBL, were cocultured with PBL from a second naive animal, nor did PBL from either tolerant or naive recipient-matched control animals, primed with third-party Ag, suppress the generation of anti-third-party CTL by a second naive animal. The suppression was cell dose-dependent, radiation-sensitive, required cell-to-cell contact not reversed by the provision of exogenous IL-2, and associated with lower levels of IL-2R expression on the suppressive effector group (particularly the CD8 single positive cells) when compared with the control effector group. These data indicate an association between the presence of peripheral regulatory cells demonstrable in vitro and the maintenance of tolerance to renal allografts.

Animals↗
PubMed1998

[Diagnosis of non-palpable testis: value of a new miniaturized laparoscope].

Laparoscopy and magnetic resonance imaging (MRI) are competitive tools in the diagnosis of nonpalpable testis. We investigated 29 boys for this indication with MRI. If MRI failed to locate the testis, laparoscopy was performed with a new, miniaturized set of pediatric instruments (1.9-mm optic). MRI revealed 10 inguinal and 7 abdominal testes. There was no false-positive finding. In 12 boys MRI showed no testis. Four cases were true negative, 8 false negative (32%). In these 8 MRI-negative patients, laparoscopy revealed 7 inguinal and 1 abdominal testis. The optical quality of the mini-telescope was sufficient for a 100% correct diagnosis. Laparoscopy-related complications did not occur. In summary, laparoscopic evaluation is the preferred method in pediatric cases of nonpalpable testes.

Adolescent↗