HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells.

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

Role of controlled cardiac reoxygenation in reducing nitric oxide production and cardiac oxidant damage in cyanotic infantile hearts.

Cardiopulmonary bypass (CPB) is used increasingly to correct cyanotic heart defects during early infancy, but myocardial dysfunction is often seen after surgical repair. This study evaluates whether starting CPB at a conventional, hyperoxic pO2 causes an "unintentional" reoxygenation (ReO2) injury. We subjected 2-wk-old piglets to ventilator hypoxemia (FIO2 approximately 0.06, pO2 approximately 25 mmHg) followed by 5 min of ReO2 on CPB before instituting cardioplegia. CPB was begun in hypoxemic piglets by either abrupt ReO2 at a pO2 of 400 mmHg (standard clinical practice) or by maintaining pO2 approximately 25 mmHg on CPB until controlling ReO2 with blood cardioplegic arrest. The effects of abrupt vs. gradual ReO2 without surgical ischemia (blood cardioplegia) were also compared. Myocardial nitric oxide (NO) production (chemiluminescence measurements of NO2- + NO3-) and conjugated diene (CD) generation (spectrophotometric A233 measurements of lipid extracts) using aortic and coronary sinus blood samples were assessed during cardioplegic induction. 30 min after CPB, left ventricular end-systolic elastance (Ees, catheter conductance method) was used to determine cardiac function. CPB and blood cardioplegic arrest caused no functional or biochemical change in normoxic (control) hearts. Abrupt ReO2 caused a depression of myocardial function (Ees = 25 +/- 5% of control). Functional depression was relatively unaffected by gradual ReO2 without blood cardioplegia (34% recovery of Ees), and abrupt ReO2 immediately before blood cardioplegia caused a 10-fold rise in cardiac NO and CD production, with subsequent depression of myocardial function (Ees 21 +/- 2% of control). In contrast, controlled cardiac ReO2 reduced NO production 94%, CD did not rise, and Ees was 83 +/- 8% of normal. We conclude ReO2 injury is related to increased NO production during abrupt ReO2, nullifies the cardioprotective effects of blood cardioplegia, and that controlled cardiac ReO2 when starting CPB to correct cyanotic heart defects may reduce NO production and improve myocardial status postoperatively.

Indomethacin-associated neutropenia with subsequent Gram-negative sepsis in a preterm infant. Cause or coincidence?

A preterm male infant with a patent ductus arteriosus developed neutropenia during treatment with indomethacin. Afterward, the mother described her own history of indomethacin-associated neutropenia. During the recovery from the neutropenia, the infant became septic with bacteremia caused by Enterobacter cloacae. Although indomethacin-related neutropenia has been described in adults, no case in a neonate has been reported. If neutropenia occurs after indomethacin therapy in a neonate, a familial history of indomethacin-associated neutropenia should be sought and the increased risk of infection should be considered.

Sequence analysis of an immunogenic and neutralizing domain of the human T-cell lymphoma/leukemia virus type I gp46 surface membrane protein among various primate T-cell lymphoma/leukemia virus isolates including those from a patient with both HTLV-I-associated myelopathy and adult T-cell leukemia.

Human T-cell lymphoma/leukemia virus type I (HTLV-I) causes adult T-cell leukemia/lymphoma and HTLV-I-associated myelopathy. Specific regions within the outer envelope proteins of other retroviruses, e.g., human immunodeficiency virus type 1, are highly immunogenic and, because of the selective pressure of the host immune system, quite variable. Mutations in the external envelope protein gene of murine retroviruses and human immunodeficiency virus type 1 influence cellular tropism and disease pathogenesis. By contrast, no disease-specific viral mutations have been identified in HTLV-I-infected patients. However, all isolates studied thus far have originated from leukemic cell lines, peripheral blood mononuclear cells, or cerebrospinal fluid lymphocytes from patients with HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma and, therefore, may not truly reflect tissue-associated variation. The midregion of the HTLV-I gp46 external envelope glycoprotein (amino acids 190-209) induces an antibody response in 90% of infected individuals, and a hexapeptide in this region (amino acids 191-196) elicits antibodies in rabbits which inhibit syncytia formation and infection of target lymphocytes. Because of the above, we expected the neutralizing domain of the gp46 env gene of HTLV-I to possess disease or organ-associated mutations selected by the infected host's immune system. Hence, we amplified, cloned, and sequenced HTLV-I DNA directly from in vivo central nervous system, spleen, and kidney specimens, and a leukemic cell line from a patient (M. J.) with both HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma to discern the possibility of tissue- and/or disease-specific variants. In addition, we sequenced several HTLV-I isolates from different regions of the world, including Papua New Guinea, Bellona, and Liberia, and compared them to other previously published HTLV-I and related retroviral sequences. The 239-base pair sequence corresponding to amino acids 178 to 256 in gp46 displayed minor tissue-specific variation in clones derived from central nervous system tissues from patient M. J., but overall was highly conserved at both the DNA and amino acid levels. Variation was observed in this region among the other HTLV-I, simian T-cell lymphoma virus type I, and HTLV-II isolates in a pattern that was consistent with their known phylogenetic relationship. No consistent disease-related changes were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Superoxide generation by neonatal and adult rabbit alveolar macrophages.

We compared the abilities of alveolar macrophages (AM) from neonatal and adult rabbits to generate and release superoxide (O2-) after exposure to soluble and particulate stimuli. Basal rates of O2- release, 0.1-0.2 nmol/10(6)AM/10 min, were modestly increased by exposing AM to phorbol myristate acetate, (dihydro)cytochalasin B, or cytochalasin C. Opsonized zymosan was a more effective stimulus, and maximal rates of O2- release were observed when AM were stimulated by a combination of opsonized zymosan and one of the aforementioned cytochalasins. Cytochalasins D and E were each potent activators of O2- production by AM in the absence of additional stimuli. Adult AM released 24.8 +/- 6.0, 20.4 +/- 4.4, and 33.5 +/- 6.4 nmol O2-/10(6)AM/10 min after stimulation by cytochalasin D, cytochalasin E, and dihydrocytochalasin B plus opsonized zymosan, respectively. Following exposure to the same stimuli, AM from 1, 3- and 7-day-old rabbits released O2- at rates that ranged from 45 to 75% of corresponding adult values. The discrepancy between O2- production by neonatal and adult AM was accentuated when comparisons were restricted to AM recovered from bacteriologically sterile respiratory tracts. Our data show the O2- generating capacity of neonatal AM to be substantially less than that of the adult AM. Immaturity of this response could predispose neonates to pulmonary infection.

Seizures in an atelencephalic infant. Is the cortex essential for neonatal seizures?

Clinical and electrographic seizures were recorded in an infant with atelencephaly. Because the infant had no cerebral hemispheres, the ictal discharges were presumed to arise from the disorganized diencephalic derivatives that occupied the entire supratentorial space. The case provided strong support for the concept that, unlike epileptic seizures in older patients, some types of neonatal seizures may originate and propagate exclusively in subcortical structures. This may explain the striking dissociation between the electrographic and behavioral aspects of seizures occasionally observed in newborns, as well as the frequent intractability of such seizures to standard anticonvulsants.

HTLV-I DNA sequences in CNS tissue of a patient with tropical spastic paraparesis and HTLV-I-associated myelopathy.

Human T cell lymphotrophic virus type I (HTLV-I) is the etiologic agent of adult T cell lymphoma/leukemia (ATLL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We studied an HTLV-I-seropositive, white man diagnosed in 1977 with ATLL and 10 years later, 6 months prior to his death, with TSP/HAM. Sections of brain, spinal cord, and visceral tissues were examined histologically, immunohistochemically, by in situ hybridization, and by the polymerase chain reaction (PCR). PCR amplification of a region of the polymerase (pol) gene of HTLV-I from visceral tissue demonstrated the presence of proviral HTLV-I DNA in paraffin-embedded sections from the liver and in DNA extracted from frozen sections of kidney and spleen, but failed to demonstrate viral sequences in paraffin sections of the lung and a lymph node. PCR analysis of CNS tissue demonstrated viral sequences in regions of the brain including frozen samples from cerebellum and cerebral cortex and paraffin sections of the thoracic spinal cord, but failed to detect proviral DNA in sections from a region in the lumbar cord. These results map the distribution of HTLV-I DNA sequences in the CNS of a patient with TSP/HAM for 3 months.

Tracheal asiration and its clinical correlates in the diagnosis of congenital pneumonia.

Tracheal aspirates were obtained from 320 newborns with respiratory distress and one or more perinatal risk factors for infection. Samples were obtained before 8 hours of age, either by direct aspiration or immediately following intubation. Twenty-five infants had bacteria present in the aspirate smear. In each case a pure culture of the organism suspected by smear morphology was grown. The same organism was isolated from blood in 14 of the 25 newborns suspected of having pneumonia. The remaining 11 infants had clinical courses, depressed mature neutrophil counts, and elevated band to total neutrophil ratios consistent with bacterial infection. Twenty-five infants without bacteria in the tracheal aspirate smear were randomly selected as a comparison group. Three of these infants had positive blood cultures, and one of the three also grew the same organism from the tracheal aspirate. The chest radiograph was abnormal in all infants but did not discriminate patients with or without pneumonia. We conclude that examination of the tracheal aspirate obtained within the first 8 hours of age is helpful in the early diagnosis of congenital pneumonia.

Transmission of human T cell leukemia virus type I to sheep: antibody profile and detection of viral DNA sequences.

Lambs were inoculated intraperitoneally with either 1.8 x 10(7) live peripheral blood cells from an HTLV-I-infected person (five lambs) or with 8 x 10(7) live cells from the HTLV-I-producing cell lines MT-2 (four lambs) or C10 MJ (five lambs). Four control lambs were inoculated with minimal essential medium supplemented with fetal calf serum. The animals were monitored during a period of 24 months. Beginning at 5 to 12 months after inoculation, four of the five lambs inoculated with the fresh HTLV-I-infected peripheral blood cells began to develop detectable levels of antibodies to a recombinant HTLV-I gp21env antigen, as determined by an enzyme-linked immunoassay (ELISA). The anti-gp21 antibodies persisted for the remaining observation period. These antibodies were not detected in the sera from the other sheep. Absorption and blocking experiments demonstrated the specificity of the gp21 reactivity. This reactivity was also confirmed by Western blot (WB). With the exception of the serum of an MT-2-inoculated sheep that formed a weak band with p19 by WB, none of the sera of the four gp21-positive sheep or of the other experimental sheep reacted with other structural or regulatory HTLV-I proteins, as determined by ELISA, WB, and radioimmunoassay. PCR analyses demonstrated the presence of the HTLV-I provirus in peripheral blood leukocytes of the four sheep showing antibodies to gp21env. The remaining sheep were negative. PCR analyses failed to detect BLV sequences in any of the experimental sheep. None of the sheep showed clinical abnormalities during the observation period. The potential value of the sheep model for studying atypical virus-host interactions in infected people is discussed.

Evaluation of HIV type 1 western blot-indeterminate blood donors for the presence of human or bovine retroviruses.

From 1985 through 1990, 1100 of 500,000 human blood donations in Syracuse, New York were repeatedly reactive by ELISA for antibodies to the human immunodeficiency virus type 1 (HIV-1). Nine hundred of the ELISA-reactive samples were confirmed as negative by Western blot (WB), 40 were confirmed as positive, and the remaining 160 sera were indeterminate, reacting mainly with HIV-1 gag gene products. Twenty donors with the most reactive indeterminate WB were selected for follow-up studies. Four of these 20 donors admitted to retroviral risk factors and, interestingly, 12 (60%) had exposure to dairy cattle and drank unpasteurized milk. These 20 donors were analyzed over a 3-year period for the presence of the pathogenic human retroviruses HIV-1, HIV-2, human T cell lymphoma/leukemia virus types I and II (HTLV-I and HTLV-II), as well as bovine immunodeficiency virus (BIV) and leukemia virus (BLV). Retroviral analyses included serology, plasma antigen capture, virus culture, and the polymerase chain reaction. Only one donor seroconverted and was clearly infected with HIV-1. None of the other 19 donor serological reactivities to HIV-1 changed, nor were they positive for any of the above-mentioned retroviruses. Although we cannot ascertain whether these latter 19 HIV-1 WB-indeterminate donors were exposed to human or bovine retroviral proteins, it is unlikely that their HIV-1 seroreactivity was caused by infection with HIV-1, HIV-2, HTLV-I, HTLV-II, BLV, or BIV.

Prototypical HTLV-I/II infection is rare in LGL leukemia.

The etiology of LGL leukemia is not known; however, we recently detected HTLV-II in a patient with LGL leukemia. In this study, we found that sera from 6 of 28 patients with LGL leukemia were positive for HTLV-I/II using a whole virus ELISA; moreover, the ELISA-negative sera were near the positive cut-off value. Therefore, we performed additional studies on these sera using commercially available assays which can confirm and distinguish HTLV-I from HTLV-II infection. Serum from only one patient was confirmed positive using conventional criteria (HTLV-II+). Sera from 25 patients (89%) had indeterminate reactivity on Western blot assays. Of these, sera from 21 (84%) reacted to gag protein p24; 12 (48%) reacted with recombinant env protein p21e, and 10 (40%) reacted with both. We could not detect HTLV-I/II pol or pX gene sequences in these patients using polymerase chain reaction analyses, with the exception of the HTLV-II-infected patient described previously. These data show that most patients with LGL leukemia are not infected with prototypical HTLV-I or HTLV-II. The frequent reactivity of patient sera to HTLV-I/II gag protein p24 and to env protein p21e, however, suggests that a deleted or variant form of HTLV-I/II may be associated with LGL leukemia.

Studies of controlled reperfusion after ischemia. XXI. Reperfusate composition: superiority of blood cardioplegia over crystalloid cardioplegia in limiting reperfusion damage--importance of endogenous oxygen free radical scavengers in red blood cells.

UNLABELLED: Postischemic damage is caused partially by oxygen free radical-mediated injury. This study will show that (1) crystalloid cardioplegia with room air oxygen is deleterious because it is devoid of free radical scavengers and (2) blood cardioplegia limits damage because it contains endogenous free radical scavengers in red blood cells. METHODS: Thirty-two dogs underwent 2 hours of ligation of the left anterior descending coronary artery followed by 20 minutes of regional blood cardioplegic reperfusion on bypass. Ten dogs received only the blood cardioplegic solution (containing its endogenous free radical scavengers); five received initial blood cardioplegia (5 minutes) with endogenous free radical scavengers (catalase and glutathione peroxidase) blocked by aminotriazole and N-ethylmaleimide, respectively; 12 received initial crystalloid cardioplegic solution oxygenated by room air (oxygen tension = 150 mm Hg); seven without and five with exogenous free radical scavengers (superoxide dismutase, catalase, coenzyme Q10); five received initial deoxygenated crystalloid cardioplegic solution (oxygen tension = 6 mm Hg); and five received deoxygenated crystalloid cardioplegic solution. RESULTS: Blood cardioplegia with endogenous free radical scavengers produced the best recovery of systolic shortening (69% systolic shortening) and resulted in the least histochemical damage (11% triphenyltetrazolium chloride nonstaining). The worst recovery and most damage occurred if blood cardioplegia was preceded by oxygenated crystalloid cardioplegia (3% systolic shortening, 48% triphenyltetrazolium chloride nonstaining; p less than 0.05 versus blood cardioplegia) or if free radical scavengers were blocked in the initial period of blood cardioplegia (3% systolic shortening, 41% triphenyltetrazolium chloride nonstaining; p less than 0.05 versus blood cardioplegia). Conversely, deoxygenation or supplementation of oxygenated crystalloid cardioplegic solution with exogenous free radical scavengers restored 60% systolic shortening (p less than 0.05 versus oxygenated crystalloid cardioplegia) and 54% systolic shortening (p less than 0.05 versus oxygenated crystalloid cardioplegia) and reduced damage to 34% and 21% (both p less than 0.05 versus oxygenated crystalloid cardioplegia). CONCLUSION: Blood cardioplegic solutions containing their own endogenous free radical scavengers are superior to crystalloid cardioplegic solutions, because they limit oxygen-mediated perfusion damage and restore contractile function. Initial crystalloid cardioplegic washout negates the salutary effect of blood cardioplegia. Exogenous free radical scavenger supplementation or deoxygenation of the cardioplegic reperfusate is necessary only if crystalloid cardioplegia is used.

Studies of hypoxemic/reoxygenation injury: without aortic clamping. III. Comparison of the magnitude of damage by hypoxemia/reoxygenation versus ischemia/reperfusion.

The immature heart is more tolerant to ischemia than the adult heart, yet infants with cyanosis show myocardial damage after surgical correction of congenital cardiac defects causing hypoxemia. This study tested the hypothesis that the hypoxemic developing heart is susceptible to oxygen-mediated damage when it is reoxygenated during cardiopulmonary bypass and that this hypoxemic/reoxygenation injury is more severe than ischemic/reperfusion stress. Fifteen Duroc-Yorkshire piglets (2 to 3 weeks old, 3 to 5 kg) underwent 60 minutes of 37 degrees C cardiopulmonary bypass. Five piglets (control) were not made ischemic or hypoxemic. Five underwent 30 minutes of normothermic ischemia (aortic clamping) and 25 minutes of reperfusion before cardiopulmonary bypass was discontinued. Five others underwent 30 minutes of hypoxemia (bypass circuit primed with blood with oxygen tension 20 to 30 mm Hg) and 30 minutes of reoxygenation during cardiopulmonary bypass. Functional (left-ventricular contractility) and biochemical (levels of plasma and tissue conjugated dienes and antioxidant reserve capacity) measurements were made before ischemia/hypoxemia and after reperfusion/reoxygenation. Cardiopulmonary bypass (no ischemia or hypoxemia) caused no changes in left-ventricular function or coronary sinus levels of conjugated dienes. The tolerance to normothermic ischemia was confirmed, inasmuch as left-ventricular function returned to 108% of control values and coronary sinus levels of conjugated dienes did not rise after reperfusion. Conversely, reoxygenation raised plasma levels of conjugated dienes in coronary sinus blood in the hypoxic group 57% compared with end-hypoxic levels (p < 0.05 versus end-hypoxic levels and versus ischemia, by analysis of variance). Antioxidant reserve capacity showed the lowest levels (highest production of malondialdehyde) in the hypoxemic group (51% higher than control values; p < 0.05 by analysis of variance). These biochemical changes were associated with a 62% depression of left-ventricular function after bypass because end-systolic elastance recovered only 38% of control levels (p < 0.05 by analysis of variance). These data confirm the tolerance of the immature heart to ischemia and reperfusion and document a hypoxemic/reoxygenation injury that occurs in immature hearts reoxygenated during bypass. Hypoxemia seems to render the developing heart susceptible to reoxygenation damage that depresses postbypass function and is associated with lipid peroxidation. These findings suggest that starting bypass in cyanotic immature subjects causes an unintended reoxygenation injury that may potentially be counteracted by adding antioxidants to the prime of the extracorporeal circuit.

Studies of hypoxemic/reoxygenation injury: without aortic clamping. IV. Role of the iron-catalyzed pathway: deferoxamine.

This study tests the hypothesis that an iron chelator, deferoxamine, can reduce oxygen-mediated myocardial injury and avoid myocardial dysfunction after cardiopulmonary bypass by its action on the iron-catalyzed Haber-Weiss pathway. Twenty-one immature 2- to 3-week-old piglets were placed on cardiopulmonary bypass for 120 minutes, and five piglets served as biochemical controls without cardiopulmonary bypass. Five piglets underwent cardiopulmonary bypass without hypoxemia (cardiopulmonary bypass control). Sixteen others became hypoxemic while undergoing cardiopulmonary bypass for 60 minutes by lowering oxygen tension to about 25 mm Hg, followed by reoxygenation at oxygen tension about 400 mm Hg for 60 minutes. Oxygen delivery was maintained during hypoxemia by increasing cardiopulmonary bypass flow and hematocrit level. In seven piglets deferoxamine (50 mg/kg total dose) was given both intravenously just before reoxygenation and by a bolus injection (5 mg/kg) into the cardiopulmonary bypass circuit; nine others were not treated (no therapy). Myocardial function after cardiopulmonary bypass was evaluated form end-systolic elastance (conductance catheter) and Starling curve analysis. Myocardial conjugated diene production and creatine kinase leakage were assessed as biochemical markers of injury, and antioxidant reserve capacity was determined by measuring malondialdehyde in postcardiopulmonary bypass myocardium incubated in the oxidant, t-butylhydroperoxide. Cardiopulmonary bypass without hypoxemia caused no oxidant or functional damage. Conversely, reoxygenation (no therapy) raised myocardial conjugated diene levels and creatine kinase production (conjugated diene: 3.5 +/- 0.7 absorbance 233 nm/min/100 g, creatine kinase: 8.5 +/- 1.5 U/min/100 g; p < 0.05 versus cardiopulmonary bypass control), reduced antioxidant reserve capacity (malondialdehyde: 1115 +/- 60 nmol/g protein at 4 mmol/L t-butylhydroperoxide; p < 0.05 versus control), and produced severe post-bypass dysfunction (end-systolic elastance recovered only 39% +/- 7%, p < 0.05 versus cardiopulmonary bypass control). Deferoxamine avoided conjugated diene production and creatine kinase release and retained normal antioxidant reserve, and functional recovery was complete (95% +/- 11%, p < 0.05 versus no treatment). These findings show that iron-catalyzed oxidants may contribute to a reoxygenation injury and imply that deferoxamine may be used to surgical advantage.

Studies of hypoxemic/reoxygenation injury: without aortic clamping. VI. Counteraction of oxidant damage by exogenous antioxidants: N-(2-mercaptopropionyl)-glycine and catalase.

This study tests the hypothesis that antioxidants administered before reoxygenation can reduce oxygen-mediated damage and improve myocardial performance. Of 25 Duroc-Yorkshire piglets (2 to 3 weeks, 3 to 5 kg) five underwent 60 minutes of cardiopulmonary bypass without hypoxemia (control group), and five others underwent 30 minutes of hypoxemia on cardiopulmonary bypass with a circuit primed with oxygen tension about 25 mm Hg blood followed by reoxygenation on cardiopulmonary bypass (no treatment). In vitro studies were performed to obtain the optimal dosage of the antioxidants N-(2-mercaptopropionyl)-glycine and and catalase to be used in subsequent in vivo experimental studies; cardiac homogenates were incubated in 0 to 5 mmol/L concentrations of the oxidant t-butylhydroperoxide and malondialdehyde production was measured. Fifteen piglets were made hypoxemic on cardiopulmonary bypass for 30 minutes, and the antioxidants N-(2-mercaptopropionyl)-glycine at either 30 or 80 mg/kg body weight or N-(2-mercaptopropionyl)-glycine, 30 mg/kg body weight, and catalase, 50,000 U/kg body weight, were added to the cardiopulmonary bypass circuit 15 minutes before reoxygenation. Left ventricular contractility, which was expressed as end-systolic elastance, was measured by conductance catheter before hypoxemia and after reoxygenation. Myocardial antioxidant reserve capacity was determined after reoxygenation by incubating cardiac homogenates in the oxidant t-butylhydroperoxide and measuring subsequent malondialdehyde elution. The in vitro bioassay studies showed a dose-dependent reduction of lipid peroxidation with N-(2-mercaptopropionyl)-glycine, with maximal benefits of a 40% decrease and malondialdehyde elaboration occurring with N-(2-mercaptopropionyl)-glycine and catalase compared with untreated cardiac homogenates. Cardiopulmonary bypass (no hypoxemia) caused no oxidant damage or changes in contractile function after cardiopulmonary bypass. Reoxygenation without treatment raised conjugated diene levels 57%,* lowered antioxidant reserve capacity 51%,* and was associated with only 38%* recovery of contractile function (p < 0.05 vs control). In contrast, treatment with antioxidants avoided lipid peroxidation, maintained antioxidant reserve capacity, and resulted in a dose-dependent improvement in left ventricular contractility with complete recovery occurring in N-(2-mercaptopropionyl)-glycine and catalase-treated piglets (*p < 0.05 vs no treatment). This study confirms the occurrence of hypoxemic/reoxygenation injury in immature hearts placed on cardiopulmonary bypass and shows that biochemical and functional damage can be counteracted by adding antioxidants to the cardiopulmonary bypass priming fluid. Contractile function improved in a dose-dependent manner, and oxygen-mediated damage could be avoided by mercaptopropionyl glycine/catalase treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of hypoxemic/reoxygenation injury: without aortic clamping. VIII. Counteraction of oxidant damage by exogenous glutamate and aspartate.

Previous studies show that (1) hypoxemia depletes immature myocardium of amino acid substrates and their replenishment improves ischemic tolerance, (2) reoxygenation on cardiopulmonary bypass causes oxygen-mediated damage without added ischemia, and (3) this damage may be related to the nitric oxide-L-arginine pathway that is affected by amino acid metabolism. This study tests the hypothesis that priming the cardiopulmonary bypass circuit with glutamate and aspartate limits reoxygenation damage. Of 22 immature Duroc-Yorkshire piglets (< 3 weeks old), five were observed over a 5-hour period (control), and five others underwent 30 minutes of CPB without hypoxemia (cardiopulmonary bypass control). Twelve others became hypoxemic by reducing ventilator inspired oxygen fraction to 6% to 7% (oxygen tension about 25 mm Hg) before reoxygenation on cardiopulmonary bypass for 30 minutes. Of these five were untreated (no treatment), and the cardiopulmonary bypass circuit was primed with 5 mmol/L glutamate and aspartate in seven others (treatment). Left ventricular function before and after bypass was measured by inscribing pressure-volume loops (end-systolic elastance). Myocardial conjugated diene levels were measured to detect lipid peroxidation, and antioxidant reserve capacity was tested by incubating cardiac muscle with the oxidant t-butylhydroperoxide to determine the susceptibility to subsequent oxidant injury. CPB (no hypoxemia) allowed complete functional recovery without changing conjugated dienes and antioxidant reserve capacity, whereas reoxygenation injury developed in untreated hearts. This was characterized by reduced contractility (elastance end-systolic recovered only 37% +/- 8%*), increased conjugated diene levels (1.3 +/- 0.1 vs 0.7 +/- 0.1*), and decreased antioxidant reserve capacity (910 +/- 59 vs 471 +/- 30 malondialdehyde nmol/g protein at 2 mmol/L t-butylhydroperoxide*). In contrast, priming the cardiopulmonary bypass circuit with glutamate and aspartate resulted in significantly better left ventricular functional recovery (75% +/- 8% vs 37% +/- 8%*), minimal conjugated diene production (0.8 +/- 0.1 vs 1.3 +/- 0.1*), and improved antioxidant reserve capacity (726 +/- 27 vs 910 +/- 59 malondialdehyde nmol/g protein*) (*p < 0.05 vs cardiopulmonary bypass control). We conclude that reoxygenation of immature hypoxemic piglets by the initiation of cardiopulmonary bypass causes myocardial dysfunction, lipid peroxidation, and reduced tolerance to oxidant stress, which may increase vulnerability to subsequent ischemia (i.e., aortic crossclamping). These data suggest that supplementing the prime of cardiopulmonary bypass circuit with glutamate and aspartate may reduce these deleterious consequences of reoxygenation.

Studies of hypoxemic/reoxygenation injury: without aortic clamping. IX. Importance of avoiding perioperative hyperoxemia in the setting of previous cyanosis.

This study of an in vivo infantile piglet model of compensated hypoxemia tests the hypothesis that reoxygenation on hyperoxemic cardiopulmonary bypass produces oxygen-mediated myocardial injury that can be limited by normoxemic management of cardiopulmonary bypass and the interval after cardiopulmonary bypass. Twenty-five immature piglets (< 3 weeks old) were placed on 120 minutes of cardiopulmonary bypass and five piglets served as a biochemical control group without cardiopulmonary bypass. Five piglets underwent cardiopulmonary bypass without hypoxemia (cardiopulmonary bypass control). Twenty others became hypoxemic on cardiopulmonary bypass for 60 minutes by lowering oxygen tension to about 25 mm Hg. The study was terminated in five piglets at the end of hypoxemia, whereas 15 others were reoxygenated at an oxygen tension about 400 mm Hg or about 100 mm Hg for 60 minutes. Oxygen delivery was maintained during hypoxemia by increasing cardiopulmonary bypass flow and hematocrit level to avoid metabolic acidosis and lactate production. Myocardial function after cardiopulmonary bypass was evaluated from end-systolic elastance (conductance catheter) and Starling curve analysis. Myocardial conjugated diene production and creatine kinase leakage were assessed as biochemical markers of injury, and antioxidant reserve capacity was determined by measuring malondialdehyde after cardiopulmonary bypass in myocardium incubated in the oxidant, t-butylhydroperoxide. Cardiopulmonary bypass without hypoxemia caused no oxidant or functional damage. Conversely, reoxygenation at an oxygen tension about 400 mm Hg raised myocardial conjugated diene level and creatine kinase production (CD: 3.5 +/- 0.7 A233 nm/min/100 g, creatine kinase: 8.5 +/- 1.5 U/min/100 g, p < 0.05 vs cardiopulmonary bypass control), reduced antioxidant reserve capacity (malondialdehyde: 1115 +/- 60 nmol/g protein at 4.0 mmol t-butylhydroperoxide, p < 0.05 vs control), and produced severe postbypass dysfunction (end-systolic elastance recovered only 39% +/- 7%, p < 0.05 vs cardiopulmonary bypass control). Lowering oxygen tension to about 100 mm Hg during reoxygenation avoided conjugated diene production and creatine kinase release, retained normal antioxidant reserve, and improved functional recovery (80% +/- 11%, p < 0.05 vs oxygen tension about 400 mm Hg). These findings show that conventional hyperoxemic cardiopulmonary bypass causes unintended reoxygenation injury in hypoxemic immature hearts that may contribute to myocardial dysfunction after cardiopulmonary bypass and that normoxemic management may be used to surgical advantage.

Studies of hypoxemic/reoxygenation injury: with aortic clamping. X. Exogenous antioxidants to avoid nullification of the cardioprotective effects of blood cardioplegia.

This study tests the hypothesis that reoxygenation of cyanotic immature hearts when starting cardiopulmonary bypass produces an "unintended" reoxygenation injury that (1) nullifies the cardioprotective effects of blood cardioplegia and (2) is avoidable by adding antioxidants N-(2-mercaptopropionyl)-glycine plus catalase to the cardiopulmonary bypass prime. Twenty immature piglets (2 to 3 weeks) underwent 30 minutes of aortic clamping with a blood cardioplegic solution that was hypocalcemic, alkalotic, hyperosmolar, and enriched with glutamate and aspartate during 1 hour of cardiopulmonary bypass. Of these, six piglets did not undergo hypoxemia (blood cardioplegic control) and 14 others remained hypoxemic (oxygen tension about 25 mm Hg) for up to 2 hours by lowering ventilator fraction of inspired oxygen before reoxygenation on cardiopulmonary bypass. The primary solution of the cardiopulmonary bypass circuit was unchanged in eight piglets (no treatment) and supplemented with the antioxidants N-(2-mercaptopropionyl)-glycine (80 mg/kg) and catalase (5 mg/kg) in six others (N-(2-mercaptopropionyl)-glycine and catalase). Myocardial function (end-systolic elastance), lipid peroxidation (myocardial conjugated diene production), and antioxidant reserve capacity were evaluated. Blood cardioplegic arrest produced no biochemical or functional changes in nonhypoxemic control piglets. Reoxygenation caused an approximate 10-fold increase in conjugated production that persisted throughout cardiopulmonary bypass, lowered antioxidant reserve capacity 86% +/- 12%, and produced profound myocardial dysfunction, because end-systolic elastance recovered only 21% +/- 2%. Supplementation of the cardiopulmonary bypass prime with N-(2-mercaptopropionyl)-glycine and catalase reduced lipid peroxidation, restored antioxidant reserve capacity, and allowed near complete functional recovery (80% +/- 8%).** Lipid peroxidation (conjugated diene) production was lower during warm blood cardioplegic reperfusion than during induction in all reoxygenated hearts, which suggests that blood cardioplegia did not injure reoxygenated myocardium. We conclude that reoxygenation of the hypoxemic immature heart causes cardiac functional and antioxidant damage that nullifies the cardioprotective effects of blood cardioplegia that can be avoided by supplementation of the cardiopulmonary bypass prime with antioxidants (*p < 0.05 vs blood cardioplegic control; **p < 0.05 vs reoxygenation).

Studies of hypoxemic/reoxygenation injury: with aortic clamping. XIII. Interaction between oxygen tension and cardioplegic composition in limiting nitric oxide production and oxidant damage.

This study tests the interaction between oxygen tension and cardioplegic composition on nitric oxide production and oxidant damage during reoxygenation of previously cyanotic hearts. Of 35 Duroc-Yorkshire piglets (2 to 3 weeks, 3 to 5 kg), six underwent 30 minutes of blood cardioplegic arrest with hyperoxemic (oxygen tension about 400 mm Hg), hypocalcemic, alkalotic, glutamate/aspartate blood cardioplegic solution during 1 hour of cardiopulmonary bypass without hypoxemia (control). Twenty-nine others were subjected to up to 120 minutes of ventilator hypoxemia (oxygen tension about 25 mm Hg) before reoxygenation on CPB. To simulate routine clinical management, nine piglets underwent uncontrolled cardiac reoxygenation, whereby cardiopulmonary bypass was started at oxygen tension of about 400 mm Hg followed by the aforementioned blood cardioplegic protocol 5 minutes later. All 20 other piglets underwent controlled cardiac reoxygenation, whereby cardiopulmonary bypass was started at the ambient oxygen tension (about 25 mm Hg), and reoxygenation was delayed until blood cardioplegia was given. The blood cardioplegia solution was kept normoxemic (oxygen tension about 100 mm Hg) in 10 piglets and made hyperoxemic (oxygen tension about 400 mm Hg) in 10 others. The cardioplegic composition was also varied so that the cardioplegic solution in each subgroup contained either KCl only (30 mEq/L) or components that theoretically inhibit nitric oxide synthase by including hypocalcemia, alkalosis, and glutamate/aspartate. Function (end-systolic elastance) and myocardial nitric oxide production, conjugated diene production, and antioxidant reserve capacity were measured. Blood cardioplegic arrest without hypoxemia did not cause myocardial nitric oxide or conjugated diene production, reduce antioxidant reserve capacity, or change left ventricular functional recovery. In contrast, uncontrolled cardiac reoxygenation raised nitric oxide and conjugated diene production 19- and 13-fold, respectively (p < 0.05 vs control), reduced antioxidant reserve capacity 40%, and contractility recovered only 21% of control levels. After controlled cardiac reoxygenation at oxygen tension about 400 mm Hg with cardioplegic solution containing KCl only, nitric oxide and conjugated diene production rose 16- and 12-fold, respectively (p < 0.05 vs control), and contractility recovered only 43% +/- 5%. Normoxemic (oxygen tension of about 100 mm Hg) controlled cardiac reoxygenation with the same solution reduced nitric oxide and conjugated diene production 85% and 71%, and contractile recovery rose to 55% +/- 7% (p < 0.05 vs uncontrolled reoxygenation). In comparison, controlled cardiac reoxygenation with an oxygen tension of about 400 mm Hg hypocalcemic, alkalotic, glutamate/aspartate blood cardioplegic solution reduced nitric oxide and conjugated diene production 85% and 62%, respectively, and contractility recovered 63% +/- 4% (p < 0.05 vs KCl only).(ABSTRACT TRUNCATED AT 400 WORDS)