Deleterious effects of irradiation and bone marrow transplantation therapy in the genetically anemic an/an mouse

The efficacy and outcome of bone marrow transplantation therapy following lethal irradiation were examined in syngeneic mice that had a hereditary macrocytic anemia (an/an) or were genotypically normal (+/+). Successful RBC and WBC replacement, based on blood cell parameters and donor genetic markers, were observed in all combinations of transplant therapy. Nevertheless, the an/an mice died prematurely several months after treatment, whether they received +/+ or an/an marrow cells. In contrast, the +/+ recipients of either +/+ or an/an marrow cells survived for at least 1 year after transplantation. Premature death of the an/an mice was associated with lymphopenia, anemia, kidney lesions, and severe pathogen-free pneumonitis. On the basis of our results, we hypothesize that the premature deaths of an/an mice are caused by a kind of chronic irradiation damage to which an/an mice are especially susceptible.     

In Vitro free radical production in rat esophageal mucosa induced by nicotine

Oxidative stress induced by nicotine was investigated in the esophageal mucosa of rats. The homogenized mucosa was incubated for 30 min with 50, 100, 200, 400, and 800 ng/mg protein/ml nicotine or with 200 ng/mg protein/ml nicotine for 15, 30, 45, and 60 min. Esophageal mucosa was also incubated for 30 min with 200 ng/mg protein/ml nicotine with or without the scavengers superoxide dismutase (SOD), catalase, SOD + catalase, inactivated SOD, inactivated catalase, or albumin. Incubation with 0.9% NaCl served as control. There was a strong correlation between chemiluminescence and the nicotine dose (r=0.75) or the nicotine incubation time (r=0.77). Thirty-minute incubation of the esophageal mucosa with 200 ng/mg protein/ml nicotine increased chemiluminescence 5.5-fold and lipid peroxidation 3.3-fold. This response was dampened by SOD or catalase and abolished by SOD + catalase. Inactivated enzymes or albumin had no scavenging effect. These results demonstrate that nicotine causes oxidative stress to the esophageal mucosa.     

Expression of specific granule markers on the cell surface of neutrophil cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6- fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12- binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.     

Active immunization against renin in normotensive marmoset.

Primate renins (human and monkey) are very similar. We used pure human renin to immunize marmosets (Callithrix jacchus) and thereby produce a chronic blockade of the renin-angiotensinogen reaction. After a control period of 2 months, five male marmosets, on their usual sodium-poor diet, were immunized against pure human renin by three subcutaneous injections of 30 micrograms each, with complete and then incomplete Freund's adjuvant. Three marmosets were injected with adjuvant only and served as controls. Blood sampling and blood pressure measurements were performed weekly. After the third injection, the five marmosets immunized against renin developed a high titer of renin antibodies (50% binding of 125I-labeled human renin at a dilution of greater than or equal to 1:10,000). The antibodies inhibited the enzymatic activity of both marmoset and human renins. At the same time, systolic blood pressure decreased significantly from 125 +/- 13 mm Hg to 87 +/- 8 mm Hg (mean +/- SD; 1 mm Hg = 133 Pa). Plasma renin enzyme activity was undetectable in three animals. Plasma aldosterone decreased significantly. After 1-4 months with low blood pressure, a normal urinary output, and a normal plasma creatinine, the five marmosets became sick and died within one month. At autopsy an immunological renal disease, characterized by the presence of immunoglobulin and macrophage infiltration colocalized with renin, was found. Granulomatous formations, probably due to Freund's adjuvant, could be seen in the lungs and in the kidney. No immunoglobulin was detectable in extrarenal vessels or in other organs. These experiments demonstrate that, in this primate, a chronic blockade of the renin-angiotensin system can be achieved by active immunization against homologous renin, but this blockade is associated with the development of an autoimmune disease localized in the kidney.     

Myelostimulatory activity of recombinant human interleukin-2 in mice

In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.     

Evaluation of a QRS scoring system for estimating myocardial infarct size. II. Correlation with quantitative anatomic findings for anterior infarcts

The ability of an independently developed QRS point score to estimate the size of infarcts predominantly within the anterior third of the left ventricle was evaluated by quantitative pathologic-electrocardiographic correlation. The study was limited to 21 patients with a single infarct documented by postmortem examination, for whom an appropriately timed standard 12 lead electrocardiogram was available that did not exhibit signs of left or right ventricular hypertrophy, left or right bundle branch block or anterior or posterior fascicular block. At necropsy the heart was cut into five to seven slices. The location and size of the infarct was quantitated by computer-assisted planimetry of the slices.The electrocardiogram of 19 (90 percent) of the patients exhibited either a Q wave or an R wave of no more than 20 ms in lead V₂. The infarct in the two patients without this electrocardiographic finding was small, occupying 2 and 3 percent of the left ventricle, respectively. The percent infarction of the left ventricle correlated with the QRS point score (r = 0.80). Thus in patients without complicating factors in the electrocardiogram and with a single infarct, the electrocardiogram provides a marker for infarction in the anterior third of the left ventricle and permits estimation of infarct size.     

Acute myocardial infarction with papillary muscle rupture

The subject of this report is a 57-year-old obese, hypertensive woman who had been well until the onset of severe chest pain and hypotension. She had to be defibrillated four times on her way to the hospital. The diagnosis of acute inferior-posterior infarction was made by electrocardiogram (ECG) and there was a markedly elevated serum creatine kinase (CK) (including the MB fraction). The patient had a very low cardiac output and ejection fraction. A lung scan revealed possible pulmonary embolism for which she was anticoagulated. She remained hypotensive and hypoxemic and, on Day 17 of her hospital stay, she had a bout of severe dyspnea. A new systolic murmur was heard and the clinical diagnosis of ruptured papillary muscle was made and confirmed by echocardiography, and later at autopsy. All three coronary arteries were severely atherosclerotic and, in addition, the right coronary artery was completely closed by a thrombus. This case clearly illustrates the major pathological changes in the heart that correlate with the clinical findings in patients with a myocardial infarct that is complicated by left ventricular papillary muscle rupture. The pathophysiological effects of this condition, as illustrated in this case report, include the following: 1. The posterior papillary muscle was almost completely separated from its base, with only a thin strip of muscle intact. The mitral valve thus was insufficient (a “flail valve“); this markedly reduced the ejection fraction of the left ventricle, increased its end-diastolic volume and pressure, produced a damming of blood in the pulmonary circulation, and this resulted in the pulmonary edema seen on the chest x-ray. 2. Cardiac hypertrophy (weight = 561 g) was undoubtedly the result of the increased workload imposed on the left ventricle by the systemic arterial hypertension that this patient had for several years. A high blood pressure that persists after a myocardial infarct predisposes to myocardial rupture, but was probably not a risk factor in her case since it did not persist. 3. The cause of the myocardial infarct was the thrombotic occlusion of the right coronary artery (on top of severe three-vessel coronary atherosclerosis), which resulted in infarction of the right as well as the posterior left ventricle and can partly explain the mistaken clinical impression that she had pulmonary emboli.     

Comparison of infarct size changes with delayed contrast-enhanced magnetic resonance imaging and electrocardiogram QRS scoring during the 6 months after acutely reperfused myocardial infarction

Introduction

Magnetic resonance imaging using the delayed contrast-enhanced (DE-MRI) method can be used for characterizing and quantifying myocardial infarction (MI). Electrocardiogram (ECG) score after the acute phase of MI can be used to estimate the portion of left ventricular myocardium that has infracted. There are no comparison of serial changes on ECG and DE-MRI measuring infarct size.

Aim

The general aim of this study was to describe the acute, healing, and chronic phases of the changes in infarct size estimated by the ECG and DE-MRI. The specific aim was to compare estimates of the Selvester QRS scoring system and DE-MRI to identify the difference between the extent of left ventricle occupied by infarction in the acute and chronic phases.

Methods

In 31 patients (26 men, age 56 ± 9) with reperfused ST-elevation MI (11 anterior, 20 inferior), standard 12-lead ECG and DE-MRI were taken from 1 to 2 days (acute), 1 month (healing), and 6 months (chronic) after the MI. Selvester QRS scoring was used to estimate the infarct size from the ECG.

Results

The correlation values between infarct size measured by DE-MRI and QRS scoring range from 0.33 to 0.43 higher for anterior than inferior infarcts. The infarct size estimated by QRS scoring was larger (about 5% of the left ventricle) than infarct size by DE-MRI acute and 1 month, but at 6 months, there was no difference. In about half of the patients, the QRS score agreed with DE-MRI in change of infarct size from acute to 6 months.

Conclusion

In conclusion, the Selvester QRS scoring system is in half of the patients with reperfused first time MI in good accordance with DE-MRI in identifying a decrease or no change in the extent of left ventricle occupied by infarction in the acute and chronic phases.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.