Functional interleukin-2 receptors are expressed on natural killer-like leukemic cells from a dog with cutaneous lymphoma

We identified a dog with large granular lymphocytic leukemia and cutaneous lymphoma that exhibited constitutive expression of interleukin-2 (IL-2) receptors by the leukemic peripheral blood lymphocytes. The leukemic cells phenotypically resembled natural killer (NK) cells, and their surface IL-2 receptors were functional, as determined by the capacity to bind human recombinant IL-2 with high- affinity resulting in the transduction of proliferation signals and in the development of lymphokine-activated killer cell activity. These cells produced IL-2 spontaneously, and they may have maintained their proliferative state through an IL-2-dependent autocrine growth pathway. Our results indicate that neoplastic lymphocytes of syndromes that involve circulating leukemic cells with dermotropism can originate from NK-like cells. Additionally, the data also suggest that proliferative conditions such as these may be the result of the aberrant production of IL-2. Further, this case illustrates the potential for the use of hematopoietic malignancies in the dog as a suitable animal model for immune targeting of IL-2 receptors as a novel treatment approach for similar malignancies of human beings.     

In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice

We tested whether the in vivo infusion of recombinant, soluble CTLA4 fused with Ig heavy chains, as a surrogate ligand used to block CD28/CTLA4 T-cell costimulation, could prevent efficient T-cell activation and thereby reduce graft-versus-host disease (GVHD). Lethally irradiated B10.BR recipients of major histocompatibility complex disparate C57BL/6 donor grafts received intraperitoneal injections of human CTLA4-Ig (hCTLA4-Ig) or murine CTLA4-Ig (mCTLA4-Ig) in various doses and schedules beginning on day -1 or day 0 of bone marrow transplantation (BMT). In all five experiments, recipients of CTLA4-Ig had a significantly higher actuarial survival rate compared to mice injected with an irrelevant antibody control (L6) or saline alone. Survival rates in recipients of hL6 or PBS were 0% at 29 to 45 days post-BMT. In recipients of CTLA4-Ig, survival rates were as high as 63% mice surviving 3 months post-BMT. However, protection was somewhat variable and recipients of CTLA4-Ig were not GVHD-free by body weight, clinical appearance, and histopathologic examination. There were no significant differences in the survival rates in comparing injection dose, injection duration, or species of CTLA4-Ig (hCTLA4-Ig v mCTLA4- Ig). Splenic and peripheral blood flow cytometry studies of long-term hCTLA4-Ig-injected survivors showed a significant peripheral B-cell and CD4+ T-cell lymphopenia, consistent with GVHD. A kinetic study of splenic reconstitution was performed in mice that received hCTLA4-Ig and showed that mature splenic localized CD8+ T-cell repopulation was not significantly different in recipients of hCTLA4-Ig compared with hL6, despite the significant increase in actuarial survival rate in that experiment. These data suggest that the beneficial effect of hCTLA4-Ig on survival is not mediated by interfering with mature donor- derived T-cell repopulation post-BMT. Neither hCTLA4-Ig nor mCTLA4-Ig interfered with hematopoietic recovery post-BMT. We conclude that CTLA4- Ig (most likely in combination with other agents) may represent an important new modality for GVHD prevention.     

The macrocyclic lactone protein kinase C activator, bryostatin 1, either alone, or in conjunction with recombinant murine granulocyte- macrophage colony-stimulating factor, protects Balb/c and C3H/HeN mice from the lethal in vivo effects of ionizing radiation

We have examined the in vivo radioprotective effects of the macrocyclic lactone protein kinase C (PK-C) activator, bryostatin 1, administered either alone or in conjunction with recombinant murine granulocyte- macrophage colony-stimulating factor (rmGM-CSF), in Balb/c and C3H/HeN mice subjected to lethal total body irradiation (TBI). When administered alone on a divided dose schedule (24 hours and 30 minutes before TBI), rmGM-CSF (20 micrograms/kg) was ineffective in increasing survival in either strain. However, in Balb/c mice, bryostatin 1 alone (1 microgram) permitted the long-term survival (60 days) of 70% of the animals following TBI, and 80% when administered in conjunction with rmGM-CSF. Bryostatin 1 administered alone according to this schedule exerted minimal radioprotective effects in C3H/HeN mice, but, when combined with a subeffective dose of rmGM-CSF, allowed 50% of the animals to survive. Treatment of Balb/c mice with bryostatin 1 administered as a single dose 4 hours before TBI resulted in a 20% survival rate, and 45% when administered with rmGM-CSF; corresponding values for the C3H/HeN strain were 60% and 40%, respectively. Lastly, the survival rates of Balb/c mice treated with bryostatin 1 administered as a single dose 4 hours following TBI was 20%, and 25% with rmGM-CSF; corresponding values were 50% and 25% for C3H/HeN mice. These findings indicate that the PK-C activator bryostatin 1 exhibits intrinsic in vivo radioprotective effects in lethally irradiated Balb/c and C3H/HeN mice, and may, under some circumstances, augment the radioprotective capacity of rmGM-CSF. They also underscore the critical role that strain differences and scheduling considerations play in determining the in vivo radioprotective capacity of bryostatin 1, as well as its interactions with rmGM-CSF.     

Nonrandom association of free iron with membranes of sickle and beta- thalassemic erythrocytes

To further define the nature of abnormal iron deposits on the membranes of pathologic red blood cells, we have used sickle cell anemia (HbSS), HbSC, and beta-thalassemic erythrocytes (RBCs) to prepare inside-out membranes (IOM) and insoluble membrane aggregates (AGGs) containing coclustered hemichrome and band 3. Study of IOM from HbSC and thalassemic patients showed that amounts of heme iron and, especially, free iron were much higher in patients who had undergone surgical splenectomy. The membrane AGGs from HbSS and beta-thalassemic RBCs contained much more globin than heme, with this discrepancy being variable from patient to patient. Although these AGGs were enriched (compared with the ghosts from which they were derived) for heme, as expected, less than 10% of total ghost heme was recovered in them. Remarkably, these AGGs also were enriched for nonheme iron, markedly so in some patients. Iron binding studies showed that the association of free iron with these hemichrome/band 3 AGGs is explained by the fact that free iron binds to denatured hemoglobin. These results document that free iron is nonrandomly associated with the membranes of sickle and beta-thalassemic RBCs. Whether this plays a causative role in the premature removal of such cells from the circulation remains to be seen.     

Blood cell dynamics in P-selectin-deficient mice

P-selectin is expressed on the surfaces of activated platelets and endothelium where it mediates binding to leukocytes. P-selectin- deficient mice were shown to exhibit peripheral neutrophilia (Mayadas et al: Cell 74:541, 1993). We now show that this is not caused by changes in bone marrow precursors nor by a lack of neutrophil margination. Both P-selectin-positive and -negative animals displayed similar increases in peripheral blood neutrophil numbers after injection of epinephrine. However, clearance of 51Chromium-labeled neutrophils is delayed in mice deficient for P-selectin, indicating that the neutrophilia is at least in part the result of delayed removal. We detected no obvious alterations in lymphocyte differentiation, distribution, or adhesion to high endothelial venules in peripheral lymph nodes. Through intravital microscopy, we examined the impact of P-selectin deficiency on leukocyte/endothelial interaction beyond the initial stages of inflammation. Four hours after the administration of an inflammatory irritant, leukocyte rolling was observed even in the absence of P-selectin. There were significantly fewer rolling cells relative to wild-type mice, and their velocity was reduced. Moreover, in the peritonitis model, the number of peritoneal macrophages in wild-type mice increased threefold at 48 hours, whereas the macrophages in the mutant mice remained near baseline levels. Thus, whereas P-selectin is known to be involved in early stages of an inflammatory response, our results indicate that it is additionally responsible for leukocyte rolling and macrophage recruitment in more prolonged tissue injury.     

Bone marrow transplantation in patients aged 45 years and older

Increasing age has been reported to be a poor prognostic factor for survival after bone marrow transplantation. We evaluated causes of death and frequency and type of complications after marrow grafting in 24 syngeneic and 39 allogeneic recipients who were 45 to 68 years old at the time of transplant. Most patients were in an advanced stage of hematologic malignancy. Among patients given syngeneic transplants, actuarial disease-free survival at 7 years is 20%. The major causes of death were relapse of leukemia and idiopathic interstitial pneumonia. Among allogeneic recipients, 9 (23%) are currently alive, and actuarial disease-free survival at 7 years is 11%. Cytomegalovirus pneumonia and septicemia were the most frequent causes of death. Patients over 50 years of age had the poorest survival rate (1/13), but many of these were transplanted in an advanced stage of their disease. However, among 12 patients transplanted while in remission or at an early stage of their disease, 5 are surviving 65 to 1,160 days after transplantation, with an actuarial survival rate of 22% at 3 years. This is in contrast to those who received their transplant in relapse: 2 out of 20 patients (10%) became long-term survivors, with a probability of survival of 15% at 3 years. The actuarial incidence of grade II through IV acute graft- v-host disease (GVHD) was 30% for allogeneic recipients 45 to 50 years of age. This was not significantly different from the incidence in younger patients. In patients 51 to 62 years of age, the actuarial incidence of acute GVHD was 79%; however, this group included three partially HLA-mismatched transplants. Ten of 15 patients surviving at least 3 months developed chronic GVHD. These results suggest that marrow transplantation is feasible and should be considered in patients over 45 years, especially if recipients are in good clinical condition and are at an early stage of their disease, such as the chronic phase of chronic myelogenous leukemia and preleukemia. For patients more than 50 years of age, allogeneic marrow grafting cannot presently be considered first-line therapy.     

Case report: Acute hepatitis E infection with coexistent glucose-6-phosphate dehydrogenase deficiency

Hepatitis E virus is one of the leading causes of acute viral hepatitis in India but usually manifests as a mild self-limiting illness. Viral hepatitis in the presence of glucose-6-phosphate dehydrogenase (G6PD) deficiency may be associated with complications such as severe anemia, hemolysis, renal failure, hepatic encephalopathy and even death. The incidence of G6PD deficiency in the general population of northern India is reported to be between 2.2% and 14%. Despite both hepatitis E infection and G6PD deficiency being common, their impact on patient illness has only recently been reported. The present study reports a case of severe hemolysis in a patient with G6PD deficiency and hepatitis E infection. Key Words: Glucose-6-phosphate dehydrogenase, G6PD, Hemolysis, Hepatitis EHepatitis E is an enterically transmitted virus and is one of the most common causes of acute viral hepatitis in India (1). Glucose-6-phosphate dehydrogenase (G6PD) deficiency is found in 2.2% to 14% of the general population in North India (2). The coexistence of viral hepatitis and G6PD deficiency has been reported to be associated with severe jaundice and other complications (3,4). Hepatitis E infection with G6PD deficiency has been associated with more severe illness in only one previous report (5). We report an additional case.     

Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis

Nitric oxide has been implicated as a mediator of inflammatory arthritis, and recent work has shown that pro-inflammatory cytokines stimulate NO production in vitro by activation of the inducible nitric oxide synthase (iNOS) pathway. In order to identify the cellular sources of NO production within the joint, we have used immunohistochemical techniques to study the distribution of iNOS in synovium and cartilage from normal and diseased joints. iNOS was most strongly expressed in the synovial lining layer, subsynovium, vascular smooth muscle and chondrocytes from patients with rheumatoid arthritis (RA). Analysis of serial sections, coupled with double immunofluorescent staining, showed that the CD68+ macrophages in the synovial lining layer and, to a lesser extent, fibroblasts were the predominant source of iNOS within synovium, whereas T cells, B cells and neutrophils were negative. A similar pattern of iNOS staining was seen in osteoarthritis, but fewer cells were iNOS positive and the intensity of staining, particularly in cartilage, was much weaker than in RA. In contrast, no evidence of iNOS was detected in non- inflammatory synovium or in cartilage derived from normal joints (fractured neck of femur). In conclusion, these data support the hypothesis that synovium and cartilage are important sources of increased NO production in patients with inflammatory arthritis. Localization of iNOS at these sites within the inflamed joint raises the possibility that increased local production of NO may contribute to the pathogenesis of inflammatory arthritis by increasing synovial blood flow and by modulating cellular function within synovium and articular cartilage.