The axis of interleukin 12 and gamma interferon regulates acute vascular xenogeneic rejection.

Recent advances using transgenic animals or exogenous complement inhibitors have demonstrated prevention of hyperacute rejection of vascularized organs, but not graft loss due to acute vascular rejection. Using various wild-type and cytokine-deficient mice strains, we have examined the mechanisms of acute vascular rejection. C57BL/6 mice deficient in interleukin12 or gamma interferon showed faster acute vascular rejection than did wild-type mice. Furthermore, mice defective in B-cell development showed no acute vascular rejection. These results demonstrate that the axis of interleukin 12 and gamma interferon provides a survival advantage in vascularized xenografts by delaying or preventing acute vascular rejection caused by a B cell-dependent mechanism.

Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery.

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105+/-9 micrometer in length, and had a doubling time of 39+/-2 hours. Spindle-shaped clones (n=3) were larger (267+/-18 micrometer long, P<0.01) and grew slower (doubling time 65+/-4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) alpha-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca(2+) transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM alpha-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.

The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component.

The erythrocytes of the marine polychaete Glycera dibranchiata contain a number of different, single-chain hemoglobins, some of which self-associate into a 'polymeric' fraction. An oligodeoxynucleotide probe was synthesized based on partial amino acid sequences determined by chemical methods, and used to screen a cDNA library constructed from the poly(A+)mRNA of Glycera erythrocytes (Simons, P.C. and Satterlee, J.D. (1989) Biochemistry 28, 8525-8530). The longest positive inserts found were sequenced using the dideoxy nucleotide chain termination method. One complete clone was obtained: clone 5A, 816 bases long, contained 59 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for 147 amino acids and a 3'-untranslated region of 316 bases. The derived amino acid sequence of Glycera globin P1 was in agreement with the partial amino acid sequences obtained by chemical methods. Three additional inserts obtained in the screening were also sequenced: the inferred amino acid sequences proved to be partial globin sequences which were different from each other and from the sequence of P1. Thus, the 'polymeric' fraction of the intracellular hemoglobin of Glycera probably consists of at least four different globin chains much like the 'monomeric' fraction. Comparison of the 'polymeric' sequence with the two known 'monomeric' sequences, M-II and M-IV, shows that they share 54 identical residues. At 74 positions, the identical residues in M-II and M-IV differ from the corresponding residue in P1, including at E-7, where P1 has a distal His, in contrast to Leu in M-II and M-IV. The alignment of Bashford et al. ((1987) J. Mol. Biol. 196, 199-216) and their templates were used to examine the principal differences between the two types of Glycera globin sequences. They appear to consist of uncommon surface amino acid residues at positions C6 (Phe vs. Ala), E10 (Val vs. Lys), E17 (Lys vs. Val), G1 (Arg vs. Lys), G10 (Met vs. Ala) and H5 (Arg vs. Lys). One or more of these residues could be responsible for the self-association exhibited by the 'polymeric' Glycera globins.

Heat shock protein 47 is expressed in fibrous regions of human atheroma and Is regulated by growth factors and oxidized low-density lipoprotein.

BACKGROUND: Heat shock protein 47 (Hsp47) is a stress protein that may act as a chaperone for procollagen. Its involvement in atherosclerosis is unknown. METHODS AND RESULTS: Hsp47 expression in human coronary arteries was assessed by immunostaining. Strong focal expression was evident in atherosclerotic, but not normal, arteries and was prevalent in the collagenous regions. Double immunostaining revealed that all cells expressing type I procollagen also expressed Hsp47. Moreover, parallel regulation of proalpha1(I)collagen and Hsp47 mRNA expression occurred with cultured human smooth muscle cells stimulated with transforming growth factor-beta1 or fibroblast growth factor-2. However, a proportion of Hsp47-expressing cells in plaque did not express type I procollagen, and this pattern could be reproduced in culture. Heat shock and oxidized LDL stimulated the expression of Hsp47 mRNA by smooth muscle cells, without a concomitant rise in proalpha1(I)collagen expression. CONCLUSIONS: These findings identify Hsp47 as a novel constituent of human coronary atheroma. Its localization to the fibrous cap, regulation by growth factors in parallel with type I procollagen, and selective upregulation by stress raise the possibility that Hsp47 is a determinant of plaque stability.

Insensitivity of right ventricular endomyocardial biopsy in the diagnosis of myocarditis.

The clinical suspicion of myocarditis relies strongly on endomyocardial biopsy for confirmation, yet the sensitivity of the procedure in this setting has not been clearly defined. Biopsy sensitivity was determined in 14 hearts with histologically proved myocarditis studied ex vivo, including 12 autopsy hearts and 2 native hearts explanted at cardiac transplantation. With use of the Stanford and Cordis bioptomes, endoymocardial biopsy was performed near the apex on the right side of the ventricular septum (four to five samples/bioptome per patient) and repeated in the nonapical portion of the septum from the moderator band to the subpulmonary infundibulum (additional three to five samples/bioptome per patient). In a casewise assessment, 43% to 57% of the endomyocardial samples were diagnostic for myocarditis, as calculated separately for each bioptome in each region of sampling (apical/nonapical). Both apical and nonapical sensitivity improved to 64% when the findings of the two bioptomes were combined (eight to nine samples/patient in each region). By collectively analyzing all available samples for each patient, 11 (79%) of 14 cases could be diagnosed, but this required a mean of 17.2 samples/patient, a number clinically unrealistic. The exclusion of four cases of fungal myocarditis from analysis did not significantly alter the results. In transmural ventricular sections, none of four patients with sudden death had inflammatory disease confined to the conduction system. In conclusion, despite six to eight negative biopsy samples/patient with suspected myocarditis, repeat biopsy may still be warranted.

Chronic relapsing experimental allergic encephalomyelitis in the Lewis rat: studies on immune regulation.

To study immunoregulation of chronic relapsing experimental allergic encephalomyelitis (CR-EAE) in Lewis rats, we adoptively transferred concanavalin A-activated lymph node cells (LNC) or splenocytes, from hind footpad-inoculated donors at the onset (day 11), or recovery (day 16), of the first attack. Popliteal LNC, especially from day 16 donors, provided significant and dose-dependent, but incomplete, protection of recipients from encephalitogenic challenge; maximal mean delay in EAE onset was 10 days later than controls, with subsequent paralysis reduced more than 6-fold. In contrast, particularly from day 11 donors, superficial inguinal LNC recipients developed actively induced disease of normal severity up to 4 days earlier than CR-EAE controls. Furthermore day 11 EAE splenocytes, but not day 16 ones, adoptively transferred disease into 50-88% of naive recipients. In separate studies, we demonstrated unresponsiveness to active induction of disease in all rats re-challenged during stable late remission, as well as in a minority of animals pretreated with antigen in incomplete Freund's adjuvant. These results suggest an organ-dependent and time-dependent balance between effector and suppressor populations in the model.

Syngeneic graft-versus-host disease induced by cyclosporine--a reappraisal.

Cyclosporine-induced graft-versus-host disease has been described in lethally irradiated rats and mice following syngeneic bone marrow reconstitution. To further study this apparently CsA-restricted phenomenon, we followed reported protocols by administering CsA orally at 50 or 100 mg/kg/day to irradiated, syngeneically reconstituted C57B1/6 mice. No clinical evidence of GVHD was observed for more than 8 weeks after CsA discontinuation. Ear biopsies and circulating immunoglobulin levels 2-4 weeks after stopping CsA failed to demonstrate histological or serological evidence of GVHD, respectively, compared with mice allogeneically reconstituted with Balb/c marrow. To further follow a previous report, CsA 50 mg/kg/day orally or 10 mg/kg/day intraperitoneally was given to normal C57B1/6 mice prior to using their spleen or bone marrow cells for reconstitution of lethally irradiated syngeneic mice. Clinical monitoring and histological examination of recipients 2-5 weeks after reconstitution again failed to confirm GVHD. Thus our results were uniformly negative in attempting to reproduce syngeneic GVHD in mice. Existing data on rats and humans are reviewed, showing that syngeneic or autologous GVHD is not CsA-restricted and that the syndrome could be equated to the chronic form of GVHD found in rats and patients after allogeneic bone marrow transplantation.

Evidence for rapid accumulation and persistently disordered architecture of fibrillar collagen in human coronary restenosis lesions.

The pattern of collagen deposition after coronary angioplasty could significantly influence recurrent lesion formation. Traditional histologic assessments of coronary restenosis lesions have not identified abundant collagen fibers in restenotic tissue; however, these methods can suffer from lack of sensitivity and are not quantitative. We analyzed collagen architecture in 40 coronary lesions retrieved from patients by directional atherectomy, by exploiting the birefringent properties of fibrillar collagen. Picrosirius red-stained sections were illuminated with circularly polarized light, and fiber content and thickness were quantified by digital image analysis. Fifteen of 19 restenosis lesions (79%) and 1 of 21 native atherosclerosis lesions (5%) displayed a pattern of reactive intimal modeling, characterized by stellateshaped smooth muscle cells variably oriented in a loose extracellular matrix. There was an apparent paucity of collagen fibers in these regions based on staining with Movat's pentachrome, a traditional connective tissue stain. However, circular polarization light microscopy revealed an extensive distribution of collagen fibers in restenosis tissue, occupying 79.9% +/- 11.8% of the section area. Despite this high collagen content, the restenosis lesions were distinct from de novo atherosclerosis lesions in having a disordered collagen alignment, reduced fiber packing (p < 0.05), and thinner fibers (4.3 +/- 1.7 vs 9.2 +/- 4.3 microns, p < 0.001). Fiber diameter was greater in lesions retrieved between 3 and 17 months after angioplasty than in lesions retrieved between 1 week and 3 months (p < 0.05). However, fiber disorientation was evident in all lesions retrieved after 1 week, with little similarity to that of native plaque. Lesions retrieved within 1 week of angioplasty represented a distinct group with identical collagen features as in de novo atherosclerosis lesions, implying a different mechanism of restenosis in that population. We conclude that human coronary restenosis involves rapid accumulation of collagen fibers, which are persistently disordered. This may be critical in the development of restenosis and could significantly influence therapeutic attempts to control the process.

A prospective randomized controlled trial of initial immunosuppression with ALG versus OKT3 in recipients of cardiac allografts.

Thirty-nine heart transplant recipients were randomized prospectively to receive OKT3 or antilymphoblast globulin (ALG) for 7 days, having otherwise identical protocols (group 1: OKT3, n = 20 patients; group 2: ALG, n = 19 patients). No preoperative immunosuppression was given. The protocol consisted of methylprednisolone, 500 mg intraoperatively, followed by 1 mg/kg/day, intravenously or orally, tapering to 0.2 mg/kg/day at 1 month; oral cyclosporine starting 3 to 5 days after transplantation; selective use of azathioprine, 1 to 4 mg/kg/day; and either OKT3, 5 mg/day for 7 days, or ALG, 15 mg/kg/day for 7 days. Of the 39 patients in the study, 34 are alive 6 months to 2 years after transplantation. The actuarial survival at 2 years for the OKT3 and ALG groups was 92% (+/- 0.07%) and 83% (+/- 0.09%), respectively (not significant [NS]). The time to first rejection for group 1 was 5.6 weeks and for group 2 was 5.3 weeks (NS). The mean number of rejections for group 1 and group 2 was 2.1 episodes per patient and 1.4 per patient, respectively (NS). Three patients in each group were free of rejection at 6 months. The total number of infections at 6 months was 1.05 per patient in group 1, 0.74 per patient in group 2 (NS), with 35% of patients receiving OKT3 and 52% of patients receiving ALG actuarially free of infection by 6 months after surgery (NS). During the first 24 hours after surgery, no significant differences were noted in mean blood pressure, central venous pressure, or Po2 between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced detection of cardiac myocyte damage by polarized light microscopy. Use in a model of coxsackievirus B3-induced myocarditis.

Although accurate detection of cardiac muscle damage is critical in the diagnosis of acute myocarditis or acute cellular rejection in both clinical and experimental settings, the histologic evaluation is frequently uncertain without specialized stains. In a study of adult male A/J mice infected with 2x10(5) plaque-forming units of myocarditic coxsackievirus B3, cardiac muscle injury causing myofibrillar disruption was detected as a loss of muscle birefringence by polarized light microscopy. The technique was corroborated by comparison with Masson's trichrome stain and was helpful for histologic examination especially at the early preinflammatory stages of lesion development or in fringe territories of focal lesions. Polarized light microscopy is thus an available means to enhance the histologic determination of cardiac myocyte damage and has specific advantage in an absence of specialized stains.

Phenotypic analysis of infiltrating cells in human myocarditis. An immunohistochemical study in paraffin-embedded tissue.

To define the cellular subpopulations that infiltrate the heart in human myocarditis, formaldehyde-fixed, paraffin-embedded sections from 18 hearts with histologically proved myocarditis were examined immunohistochemically. Disease was classified on routine stains as follows: mixed mononuclear cell (7 cases), granulomatous (3), giant cell (1), rheumatic (2), and fungal (5) myocarditis, respectively. On immunohistochemical examination, T-lymphocyte and macrophage predominance was found in nearly every case, except in fungal myocarditis, in which polymorphonuclear leukocytes and macrophages prevailed. In contrast, B lymphocytes and natural killer cells were conspicuously absent, regardless of histologic classification. Giant cells in giant cell myocarditis and in the Aschoff lesions of rheumatic carditis expressed macrophage, but not myocyte, antigens, suggesting derivation along macrophage lineage. Immunohistochemical data obtainable in paraffin-embedded tissues supplement the study of myocarditis, providing information potentially relevant to immunopathogenesis, natural history, and therapy.

The loop gain of autonomic reflex function in orthostatic hypotension.

The loop gain (G) of the autonomic reflex function in orthostatic stress was assessed in anesthetized dogs subjected to 45 and 90 degrees head-up tilt. We observed the magnitude of orthostatic hypotension before and after 1) sinus denervation and vagotomy (SDVT), or 2) ganglionic blockade (GB) with hexamethonium. The decreases in arterial pressure during the orthostatic stress before and after interruption of the autonomic reflex from either the afferent or efferent limb were defined as E and D, respectively. The loop G of the compensatory system was calculated using closed-loop analysis: G = (D/E) - 1. In the SDVT experiments, the average values of E, D, and G were 18.6 mmHg, 62.6 mmHg, and 2.36, respectively, for 45 degrees tilt; and 31.2 mmHg, 82.7 mmHg, and 1.63, respectively, for 90 degrees tilt. The data provide a quantitative measure of the autonomic reflex function in orthostatic hypotension. Furthermore, we found that the corresponding G values in the SDVT and GB experiments were not significantly different. In each experiment, the G value in 90 degrees tilt was lower than that in 45 degrees tilt. The findings suggest that reflexes from the arterial baroreceptors and cardiopulmonary receptors account for a large part of the autonomic compensation to the orthostatic stress. The whole control system operates in a nonlinear fashion, because the gain value tends to decrease as the degree of tilt is increased.

Closed-loop analysis of the reflex function in orthostatic hypotension.

The arterial baroreceptors and cardiopulmonary receptors are important for the control of blood pressure during orthostatic stress. We assessed the baroreflex loop gain of arterial pressure compensation to orthostatic hypotension in pentobarbital-anesthetized and gallamine-paralyzed dogs subjected to 45 degrees and 90 degrees head-up tilt. The orthostatic hypotension before and after sinus denervation and cervical vagotomy was designated as D' and D, respectively. The loop gain (G) was calculated using closed-loop analysis: G = (D/D')-1. In 10 dogs, the D', D and G were 17.2 +/- 2.2 (mean +/- SE) mmHg, 64.2 +/- 4.4 mmHg and 2.73 +/- 0.26, respectively in 45 degrees tilt; and 30.6 +/- 3.2 mmHg, 80.7 +/- 5.2 mmHg and 1.64 +/- 0.24, respectively in 90 degrees tilt. Atropine sulfate (0.5 mg/kg, i.v.) eliminated the reflex tachycardia, but did not affect the SAP change during the head-up tilt. The effects of cervical vagotomy on the orthostatic hypotension were thus attributed to interruption of the aortic nerves and vagal afferents. The data provide a quantitative measure of the baroreflex function during the orthostatic stress. The loop gain is not a constant function because the value is significantly less in 90 degrees than in 45 degrees tilt (P less than 0.05). The nonlinear control system implies that the baroreflex compensation became less efficient in the face of a greater blood pressure perturbation.

Effect of oral clonidine premedication on perioperative hemodynamic response and postoperative analgesic requirement for patients undergoing laparoscopic cholecystectomy.

BACKGROUND: To investigate the clinical efficacy of oral clonidine premedication in anesthesia and analgesia in patients undergoing laparoscopic cholecystectomy (LC). METHODS: One hundred and ten patients, scheduled for elective laparoscopic cholecystectomy, were recruited for the prospective, randomized, single-blind, comparative study. They were randomly allotted to either of the placebo or clonidine group. Patients of the placebo group (n = 65) were premedicated with oral antacid (alugel hydroxide 300 mg), while those in the clonidine group (n = 45) were premedicated with oral clonidine 150 micrograms prior to anesthesia. The premedication was given 60 to 90 min before the anticipated time of induction of anesthesia. Normocapnia was maintained throughout the perioperative period. Mass spectrometer was used to assess the inspired and expiratory concentrations of isoflurane, the anesthetic used for maintenance of anesthesia. Postoperative pain intensity, sedation scores, adverse events, time to the first dose of postoperative analgesic and cumulative analgesic requirement in 24 hours were recorded. Data were expressed as mean +/- SD. RESULTS: Patients in the clonidine group displayed greater hemodynamic stability perioperatively and the isoflurane requirement was also reduced (30% less). The postoperative analgesic requirement was less (1.5 +/- 1.3 vs. 2.2 +/- 1.3 dose, P < 0.05) and the time for the first dose of analgesic was prolonged (411 +/- 565 vs. 264 +/- 441 min) in comparison with the placebo group but no statistic difference was found. CONCLUSIONS: Oral clonidine premedication helped to provide perioperative hemodynamic stability, spared the use of isoflurane and reduced the requirement of postoperative analgesia so as to smoother the way to recovery in patients undergoing LC.

Successful management of massive blood loss to extremely low hemoglobin in an elderly woman receiving spinal surgery.

Blood transfusion is absolutely indicated in acute anemia when the hemoglobin concentration falls below 6 g/dL. Sometimes it challenges the anesthesiologists if the blood intended for urgent transfusion is not readily or quickly available. In this case report, we describe an 81-year-old lady who accidentally sustained acute anemia after spinal surgery with the hemoglobin concentration falling to 1.4 g/dL. During the long wait for the process of cross-matching tests and delivery of blood from the blood bank in the city remote from the hospital, we could do nothing but administer crystalloid and colloid solutions to maintain the circulatory volume to prevent low cardiac output. Epinephrine was given when systolic blood pressure fell below 70 mmHg. Central venous pressure and arterial blood pressure were monitored to guide all the treatment. Fortunately, patient fully recovered on postoperative day 3 without any adverse events.

Fatal pulmonary embolism in a child undergoing extra-ventricular drainage surgery--a case report.

Thromboembolism is rather common in neurological patients and patients with brain tumor, who are bed-ridden or with partial immobile limb. In serious instances morbidity and mortality are inevitable. We present a case report on a fatal pulmonary embolism in a 2-year-old girl who underwent extra-ventricular drainage procedure under general anesthesia for occipital subdural effusion, a sequela of the former surgery undertaken to remove the choroid plexus papilloma 13 days ago. Sudden cardiac arrest occurred during induction of anesthesia and she finally succumbed in spite of vigorous cardiopulmonary resuscitation. Transthoracic and transesophageal echocardiography performed in the course of resuscitation disclosed thrombi of various sizes scattering in right atrium, the right ventricle, main pulmonary trunk, and the left pulmonary artery. The cause of death was thought to be severe obstruction of right ventricular outflow tract by large thrombi. The etiological factors which possibly led to the thrombosis were discussed, and the methods of diagnosis and treatment were also explored.

Pre-anesthetic oral clonidine is effective to prevent post-spinal shivering.

BACKGROUND: Shivering is a common event during spinal anesthesia. Customarily we just treat it rather than prevent it. This study was designed to evaluate the efficacy of oral clonidine as a premedication to prevent post-spinal shivering. METHODS: One hundred males of ASA physical status I-III, aged above 40, scheduled for elective urological surgery under spinal anesthesia, were included in this study. All participants were randomly divided into the clonidine and control groups. They received either oral clonidine 150 micrograms (n = 48) or placebo (n = 52) 90 min before spinal anesthesia in a double-blind fashion. Spinal blockade was induced with heavy bupivacaine to a dermatomal level near T10. The shivering was graded as: none, no perceptible tension of muscles observed; mild, slight muscle tonus (masseter muscle); moderate, real shivering (proximal muscles); and severe, generalized shivering (whole body). The tympanic membrane temperature was recorded 30 min after spinal anesthesia. Data were expressed as mean +/- standard deviation. Chi-square and Student's t-test were used. A p value less than 0.05 was considered statistically significant. RESULTS: The incidence of post-spinal shivering, which was graded as none, mild, moderate, and severe, showed statistically significant differences (p < 0.05) between clonidine 150 micrograms and placebo (83% vs. 42%, 10% vs. 6%, 10% vs. 19%, 0% vs. 33%, respectively) during the 30 min immediately after spinal anesthesia. The respective mean tympanic temperature in oral clonidine and placebo groups showed no difference (clonidine vs. control = 35.9 +/- 0.8 degrees C vs. 35.9 +/- 0.7 degrees C). CONCLUSIONS: Pre-anesthetic medication with oral clonidine 150 micrograms is effective to prevent post-spinal shivering in patients undergoing elective urological surgery.