Declining efficacy of AIDS case list cross-referencing in human immunodeficiency virus look-back: 1981 through 1992

BACKGROUND: The impact of AIDS case list cross-referencing in human immunodeficiency virus look-back was assessed. STUDY DESIGN AND METHODS: Post-1977 blood donations from former donors identified by 11 collaborating health departments as having developed AIDS have been traced at Irwin Memorial Blood Centers since 1983. To assess the changing efficacy of AIDS case list cross-referencing in identifying infected donors and recipients, trends in cases reported through December 1992 were analyzed. RESULTS: Previous donors (n = 638) were identified from 21,917 AIDS case listings, for an overall match rate of 2.9 percent. The rate of detection of previous donors from listings of AIDS cases declined from a peak of 5.3 percent in 1985 to 1.6 percent in 1992. Overall, 86 percent (1824/2122) of donations by persons later reported on AIDS case lists were made prior to January 1983 when risk exclusion measures were initiated. Of the 212 known infected recipients linked to AIDS case list donors, 87 (41%) were previously identified by other look-back approaches. The rate of identification of infected recipients detected exclusively through AIDS case listings declined from a mean of 21 per year from 1984 to 1987 to a mean of 3 per year from 1990 to 1992. No transmissions have been documented from donations prior to 1979. CONCLUSION: These findings demonstrate the declining yield of AIDS case list cross-referencing as a trigger for human immunodeficiency virus look-back. Continued reevaluation of look-back programs is essential so that their various components may be curtailed when justified.     

Granulocyte-macrophage colony-stimulating factor is an endogenous regulator of cell proliferation in juvenile chronic myelogenous leukemia

Juvenile chronic myelogenous leukemia (JCML) is a rare myeloproliferative disorder of early childhood that is clinically and cytogenically distinct from the well-recognized adult type of chronic myeloid leukemia. Unlike the adult disease, growth of hematopoietic progenitors from peripheral blood (PB) occurs in the absence of exogenous stimulus even at low cell densities. This so-called "spontaneous" growth can be abrogated by adherent cell depletion and appears to depend on production of endogenous growth factors. We studied seven children with JCML to determine the nature of endogenous stimulators. With isolated PB mononuclear cells (PBMNCs) and a 3H- thymidine (3H-TdR) incorporation assay, JCML cells were shown to incorporate high levels of 3H-TdR when cultured in the absence of stimulus even at low cell densities. When neutralizing antisera prepared against each of the four known colony-stimulating factors (CSFs), GM-CSF, G-CSF, M-CSF, and interleukin-3 (IL-3), as well as antisera against interleukin-1 (alpha and beta) and tumor necrosis factor (TNF) were added to these cultures, only the antisera against recombinant human GM-CSF (rhGM-CSF) consistently resulted in significant inhibition of cell proliferation, achieving up to 72% inhibition of 3H-TdR incorporation in one case. Monoclonal antibodies (MoAbs) against rhGM-CSF resulted in a similar and highly significant degree of inhibition. A marked inhibitory effect of rhGM-CSF antiserum on "spontaneous" growth of PB CFU-GM derived colonies in semisolid medium was also demonstrated in four of five patients studied (87% to 90% inhibition). Production of growth factors by highly enriched JCML monocytes was variable. When initially studied in five of the seven patients, the monocytes from three of the patients revealed increased release of IL-1-like activities; two patients had levels similar to those of controls. One patient with normal levels when initially studied was later shown to have markedly increased amounts of IL-1-like activities in a second preparation of monocyte-conditioned medium (MCM). High levels of GM-CSF were detected in the initial MCM from one patient, but this may have indirectly reflected elevated IL-1-like activities present in the MCM. IL-3 and M-CSF levels were either low or undetectable in the patients studied as compared with MCM prepared with normal adult monocytes. These results clearly implicate GM-CSF as the primary endogenous regulator of JCML cell proliferation in culture and suggest that this malignant myeloproliferative disease may in part result from paracrine stimulation of marrow progenitor cells by growth factors/cytokines secreted by the malignant monocytes.     

Elevated levels of interleukin-1 beta (IL-1 beta) and IL-6 in serum and increased production of IL-1 beta mRNA in lymph nodes of patients with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome

To evaluate a possible implication of cytokines in the pathogenesis of polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome, we studied five consecutive patients with this condition, of which four had sclerotic bone lesions and four had multicentric Castleman's disease. Interleukin-1 beta (IL-1 beta) and IL- 6 serum levels were determined in these patients (13 serum samples) and in patients with multiple myeloma (5) and Waldenstrom's macroglobulinemia (5). In situ hybridization of the relevant mRNAs was performed on lymph node specimens of two patients with POEMS syndrome who had Castleman's disease. Elevated serum levels of IL-1 beta (13/13 samples), and IL-6 (7/13 samples) were found in patients with POEMS syndrome. In the other patients, serum IL-1 beta was undetectable or slightly increased and IL-6 was elevated in a single patient with Waldenstrom's macroglobulinemia. Abundant IL-1 beta mRNA-producing cells were present in interfollicular spaces in the two tested patients, while IL-6 mRNA-producing cells were rare. We conclude that IL-1 beta and IL-6 serum levels may be chronically elevated in patients with POEMS syndrome, and that lymph node may be one site of IL-1 beta overproduction. These results are in keeping with the hypothesis that cytokines mediate systemic manifestations of POEMS syndrome.     

Uptake of L-carnitine by a human intestinal epithelial cell line, Caco- 2

BACKGROUND & AIMS: The mechanism of intestinal uptake of L-carnitine is controversial. The aim of this study was to clarify the mechanism and regulation of L-carnitine uptake. METHODS: Uptake of [3H]-L-carnitine was measured across the apical membrane of confluent monolayers of Caco- 2 cells. RESULTS: [3H]-L-carnitine uptake was linear and appreciable for up to 7 minutes with minimal metabolic alteration, was temperature- and Na(+)-(but not pH-) dependent, and included a saturable component with an apparent Michaelis constant of 45.5 +/- 6.5 mumol/L and a maximum velocity of 83.5 +/- 5.6 nmol.mg protein-1.5 min-1. Unlabeled L- carnitine and its structurally related analogues significantly (P < 0.01) inhibited [3H]-L-carnitine uptake, whereas unrelated compounds were ineffective. L-Carnitine uptake was also energy-dependent, being significantly (P < 0.01) inhibited by metabolic inhibitors. Our results also suggested that a calmodulin- but not a protein kinase C- or protein kinase A-mediated pathway plays a role in regulating L- carnitine uptake by Caco-2 cells. CONCLUSIONS: L-carnitine uptake by intestinal epithelial cells (Caco-2) involves a carrier-mediated system that is temperature-, Na(+)-, and energy-dependent and seems to be under the regulation of a calmodulin-mediated pathway. (Gastroenterology 1996 Dec;111(6):1534-40)     

Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL- 3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony- forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony- forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.     

Constitutional and Legal Protection for Life Support Limitation in India

Appropriate treatment limitations towards the end of life to reduce unwanted burdens require ethical clarity that is supported by appropriate legislation. The lack of knowledge of enabling legal provisions, physicians feel vulnerable to legal misinterpretation of treatment limiting decisions. In India the lack of societal awareness, inadequate exploration of the gray areas of bio-ethics and unambiguous legal position relating to terminal illness have resulted in poor quality end of life care. Much of the perceived vulnerability by the physician is attributable to insufficient knowledge and understanding of existing constitutional and legal position in India. While we await informed legal and legislative opinion, this paper highlights possible legal liabilities arising from treatment limitation decisions with available defense. It is hoped that such clarity would lead to more confident ethical decisions and improved end of life care for patients.