Endoluminal Vacuum Therapy for Ivor Lewis Anastomotic Leaks: A Pilot Study in a Swine Model.

Anastomotic leaks are a serious complication associated with Ivor Lewis esophagectomies. Endoluminal negative pressure vacuum devices create a possible treatment alternative to conventional surgical intervention. Ten pigs had an intrathoracic esophageal anastomosis with a 1-cm defect. The experimental group had the device placed intraoperatively across the defect, whereas the control group did not. Once treatment was completed, a contrast fluoroscopic study and necropsy was performed. All control pigs had contrast extravasation on fluoroscopy and contamination on necropsy. The experimental group had no radiologic leak and no contamination on necropsy. The P value for leak is 0.03. This study demonstrated that endoluminal negative pressure vacuum therapy is tolerated in the swine model and is successful in facilitating the healing of anastomotic leaks. Endoluminal negative pressure vacuum therapy has potential clinical benefits, including decreased morbidity and length of hospital stay.

Spontaneous autoimmune gastritis and hypochlorhydria are manifest in the ileitis-prone SAMP1/YitFcs mice.

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4(+) T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.

Retrospective study of Chlamydia trachomatis using the polymerase chain reaction on archival Papanicolaou-stained cytologic smears.

OBJECTIVE: To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique. STUDY DESIGN: A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR. RESULTS: PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted. CONCLUSION: PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.

Comparison of the deduced amino acid sequence of guinea pig adenovirus hexon protein with that of other mastadenoviruses.

PURPOSE: We sought to isolate, clone, and determine the nucleic acid sequence of the guinea pig adenovirus (GPAdV) hexon gene. From this, the amino acid sequence of the cloned portion was deduced and compared with a set of mastadenovirus hexons. METHODS: The DNA isolated from a histologic section of infected guinea pig lung was subjected to high-fidelity amplification, using degenerate primers complementary to a conserved nucleic acid sequence near the 3' end of the hexon gene of mastadenoviruses and a 5' primer from GenBank accession No. X95630 (GPAdV hexon gene partial sequence). The amplified product was cloned, the nucleic acid sequence was determined, and the amino acid sequence was deduced and compared with the hexon amino acid sequences of 25 mastadenoviruses. RESULTS: The cloned fragment comprised 1,603 base pairs (bp) [approximately 50%]) of the hexon. Of the initial 278 nucleic acids of the clone, 276 were identical with GenBank accession No. X95630, and the deduced amino acid sequences of both were identical. The deduced GPAdV hexon amino acid sequence from the clone aligned with structural regions NT, V1, DE1, and FG1 described for human adenovirus types 2 and 5. The GPAdV hexon had < 50% similarity in amino acid sequence, compared with hexons of 25 other mastadenoviruses. Analysis of regional peptide similarities revealed the GPAdV hexon to be more similar to animal mastadenoviruses and human subgroups A, C and F than to other human subgroups. CONCLUSIONS: The cloned portion of the GPAdV hexon contained a sequence nearly identical to that of GenBank accession No. X95630. Compared with the truncated amino acid sequences of human adenovirus types 2 and 5, the deduced GPAdV hexon amino acid sequence was similar in areas structurally conserved, but different in areas associated with type-specific antigenicity.

Diagnostic molecular microbiology in laboratory animal health monitoring and surveillance programs.

Molecular microbiology provides a rapid, reliable, sensitive, and specific means of detecting pathogens in laboratory animals. The author discusses the interpretation of the results of molecular microbiological testing done in conjunction with serologic, bacteriologic, and histopathologic tests, focusing on molecular microbiology as it fits in an overall program of animal health diagnostic profiling.

Evaluation of fentanyl transdermal patches in rabbits: blood concentrations and physiologic response.

In the study reported here, we sought to evaluate transdermal fentanyl patches for their ability to achieve detectable plasma concentrations with minimal adverse effects in New Zealand White rabbits. Fentanyl patches were applied to the dorsum after removing hair either by clipping or by application of a depilatory agent. Blood samples were collected every 12 h for a total of 96 h (24 h after patch removal) for determination of plasma fentanyl concentration. At those times, rabbits were assessed for changes in body temperature, heart rate, respiratory rate, and body weight. In rabbits with clipped hair, where rapid hair re-growth was not a mitigating factor, mean plasma fentanyl concentration reached a mean (+/- SEM) peak of 1.11 +/- 0.32 ng/ml at 24 h, decreased to 0.77 +/- 0.21 ng/ml at 72 h, and was negligible at 96 h. In rabbits with depilated hair, peak concentration was obtained at 12 h (6.7 +/- 0.57 ng/ml) and decreased gradually to 0.27 +/- 0.06 ng/ml at 72 h. In a second group of fentanyl-treated rabbits in which hair started growing back within 24 h, plasma fentanyl concentration was not detectable. Control and fentanyl-treated rabbits with clipped hair had no effect from the experimental manipulations other than slight loss in body weight. In the depilatory group, two rabbits appeared moderately sedated during the initial 12-h period, and had decreased respiratory rate for 24 h. In conclusion, rabbits tolerate the transdermal fentanyl patch well. Hair regrowth in rabbits may present a complicating factor that impedes dermal absorption of fentanyl. The application of a depilatory agent lead to early and rapid absorption of fentanyl causing undue sedation in some rabbits and lack of sustained plasma concentrations for the desired three-day period.

Severe inflammatory defect and reduced viability in CD18 and E-selectin double-mutant mice.

CD18-deficient mice (CD18(-/-) mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E(-/-) mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18(-/-)CD62E(-/-), but not CD18(-/-)CD62P(-/-), mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10-15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E(-/-) mice that were reconstituted with CD18(-/-) bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-alpha-induced model of inflammation. Leukocyte adhesion efficiency in CD18(-/-)CD62E(-/-) mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18(-/-)CD62E(-/-) mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

Latent Pneumocystis carinii infection in commercial rat colonies: comparison of inductive immunosuppressants plus histopathology, PCR, and serology as detection methods.

Histopathologic evaluation combined with a period of immunosuppression has been the standard procedure for detection of Pneumocystis carinii in commercial rat colonies. Variation in induction regimens and in the sensitivity of detection methods may result in underreporting of the presence of P. carinii in breeding colonies or delay its detection. In the present study, methylprednisolone and cyclophosphamide were evaluated for the ability to induce P. carinii infection in rats from an enzootically infected commercial barrier colony. The presence of P. carinii was detected by histopathologic methods and by amplification of a targeted region of the P. carinii thymidylate synthase gene by PCR over the 8-week study period. Sera taken from rats prior to either induction regimen were evaluated for the presence of P. carinii-specific antibodies by the immunoblotting technique. Few significant differences in ability to induce organism burden or in histopathology were observed between the two immunosuppressive regimens. However, a dramatic loss of weight over the study period was observed in rats treated with methylprednisolone but not in rats treated with cyclophosphamide. Although histopathologic changes attributable to P. carinii did not appear before 2 weeks with either immunosuppressant, the presence of the organism in these animals was detected by immunoblotting and PCR. Cyst scores and the intensities of the histopathologic lesions increased during the study period, but the number of rats exhibiting evidence of P. carinii infection did not change after week 3. These results suggest that use of the PCR method on postmortem lung tissue of rats without prior induction regimens or identification of anti-P. carinii antibodies in antemortem serum samples is a sufficiently sensitive method for detection of the presence of a P. carinii carrier state in rodent breeding colonies.

Detection of Pneumocystis carinii in rats by polymerase chain reaction: comparison of lung tissue and bronchoalveolar lavage specimens.

The purpose of this study was to determine and compare the efficiency of detection of Pneumocystis carinii in rats by polymerase chain reaction (PCR) of DNA extracted from two sampling locations: lung tissue and bronchoalveolar lavage. The study involved naturally infected F344 rats that were allotted to groups to intercollate the investigation of several variables, including nonimmunosuppressed rats, rats subjected to a timed induction sequence of 1 to 4 weeks of immunosuppression, and two immunosuppressants: a corticosteroid and cyclophosphamide. The PCR amplified a 357-base pair region contained within the gene encoding the large ribosomal RNA subunit of P. carinii mitochondrial DNA. The identity of the PCR product was confirmed by Southern blot analysis with an oligonucleotide probe. In a comparison of lung bronchoalveolar lavage specimens after immunosuppression, P. carinii was detected by PCR in 100% of lung tissue but in only 87.5% of the lavage specimens. Lung tissue of three animals was test-positive when the corresponding lavage specimen was negative by PCR analysis. The PCR detected P. carinii in both types of specimens from the same two of three nonimmunosuppressed rats. In all there was 88% agreement of PCR results between the two sampling techniques. The difference in diagnostic outcome for the two specimen types was not statistically significant (Fisher's exact test). It was concluded that both specimen types were adequate for PCR detection of P. carinii in rats.

Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.

Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.

Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors.

To evaluate the potential for adenovirus-mediated central nervous system (CNS) gene transfer, the replication deficient recombinant adenovirus vectors Ad.RSV beta gal (coding for beta-galactosidase) and Ad-alpha 1AT (coding for human alpha 1-antitrypsin) were administered to the lateral ventricle of rats. Ad.RSV beta gal transferred beta-galactosidase to ependymal cells lining the ventricles whereas Ad-alpha 1AT mediated alpha 1-antitrypsin secretion into the cerebral spinal fluid for 1 week. These observations, together with beta-galactosidase activity in the globus pallidus and substantia nigra following stereotactic administration of Ad.RSV beta gal to the globus pallidus, suggest that adenovirus vectors will be useful for CNS gene therapy.

Structure of the fibrinogen binding sequence: arginylglycylaspartic acid (RGD).

The crystal structure of a tetrahydrated form of L-arginyl-glycyl-L-aspartic acid (RGD), the consensus sequence for binding of fibrinogen to cell surface receptors, has been determined from diffractometer data. The tripeptide was crystallized in double zwitterionic form via hanging drop vapor diffusion experiments at a pH near 6.5. The orthorhombic unit cell contains four formula units in space group P2(1)2(1)2(1) with lattice parameters a = 4.852(4), b = 11.376(3), c = 34.083(8)A at RT. The structure was solved by direct methods and refined to a final R = 0.067 based upon 1345 observations with I greater than or equal to 2 sigma(I). Peptide bonds both are trans, omega 2 = 174.2(6) degrees and omega 3 = -169.3(6) degrees. The backbone bends at glycine with phi 2 = -85.5(8) degrees. One of the water molecules sits between the arginyl side chain and the C-terminal carboxylate, forming an intramolecular hydrogen bond to the glycyl carboxyl and linking adjacent molecules through two other H-bond interactions. Comparison of the structure to RGD sequences extracted from 3-D protein structures reveals a diversity of conformations for this tripeptide sequence.

Kinesthetic cortical area anterior to primary somatic sensory cortex in the raccoon (Procyon lotor).

Extracellular microelectrode recording of cortical unit activity, with subsequent histological examination, was used to determine the extent, organization, and cytoarchitecture of the zone of muscle afferent projections (kinesthetic cortex) anterior to the primary somatic sensory cortex in anesthetized raccoons. Activity was evoked in response to mechanical stimulation of muscles from which the overlying skin had been dissected away. Most kinesthetic responses were elicited in a contiguous cortical area, which included: the anterior bank of the lateral arm, and the fundus and posterior bank of the medial arm of the medial central sulcus; and the anterior two-thirds of the interfundic rise within the interbrachial sulcus. Some responses were recorded in a separate small area of the anterior bank at the medial end of the lateral central sulcus. Somatotopy was evident with forelimb represented lateral to hindlimb. Proximal limb muscles were represented in the center of the medial central sulcus; distal muscle projections were medial (hindlimb) or lateral (forelimb) in the same sulcus. Most representations were of flexor and extensor muscles of the contralateral carpus and forepaw digits. Activity at a given recording locus in the kinesthetic area could be elicited by both flexor and extensor muscles, which acted about a common joint. Low amplitude units evoked by cutaneous stimulation of the dissected skin were recorded in the kinesthetic area; these were from receptive fields of skin that normally overlay the muscles whose higher-amplitude evoked kinesthetic units were represented in that same recording locus. The kinesthetic zone was anterior to primary somatic sensory cortex, where the outer stripe of Baillarger and granular layer IV become attenuated. In the hindlimb muscle representation area, the additional criterion of area 3a (large pyramidal cells in layer V) was seen. However, no cytoarchitecture could be identified that was consistently associated with the kinesthetic cortex.