Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors

Adhesive interactions with the extracellular matrix of the bone marrow (BM) stroma are of critical importance in the regulation of hematopoiesis. In part, these interactions are presumed to play an important role in retaining CD34+ hematopoietic progenitor cells (HPCs) within the BM environment, in close proximity with BM stromal cells and the cytokines they produce. Evidence of a more direct role for cell adhesion in the regulation of hematopoiesis is provided by recent data showing that adhesive interactions can also provide important costimulatory signals. We have previously shown that normal CD34+ HPCs express high levels of fibronectin (Fn) receptors very late antigen-4 (VLA-4) and VLA-5 in a low-affinity state, which do not allow HPCs to strongly adhere on immobilized Fn, and that cytokines such as interleukin-3, granulocyte-monocyte colony-stimulating factor, and stem cell factor transiently activate these receptors, providing HPCs with an adhesive phenotype on Fn. Thus, knowledge of the functional states of adhesion receptors is critical to our understanding of the physiological mechanisms responsible for the regulation of normal hematopoiesis. Herein, we show that combinations of cytokines that synergize to stimulate the proliferation of CD34+ HPCs result in additive stimulation of the adhesion of these cells to Fn. Thus, the activation level of Fn receptors expressed by normal CD34+ HPCs is highly correlated with their proliferative state, suggesting a functional link between these two events. Therefore, we propose a 2- step model with an initial activation of VLA-4 and VLA-5 generated by cytokine receptors that is followed by a secondary signal resulting from Fn binding to VLA-4 and VLA-5, which may cooperate with those generated by cytokine receptors.     

Relationship between minimal residual disease and outcome in adult acute lymphoblastic leukemia

In children with acute lymphoblastic leukemia (ALL), the level of minimal residual disease (MRD) at the end of induction strongly predicts outcome, presumably because it measures both drug sensitivity and the number of leukemic cells requiring elimination. Children with high levels (> 10(-3) leukemic cells per marrow cell) nearly always relapse, whereas those with low levels (<2 x 10(-5)) seldom do. However, the importance of MRD in adult ALL is unclear. We studied 27 patients aged 14 to 74 who were treated with a standard protocol and who attained morphological remission. MRD in the marrow at first remission was quantified by using the polymerase chain reaction (PCR), with the rearranged immunoglobulin heavy chain gene as a molecular marker. Levels of MRD varied from 3 x 10(-1) to <7 x 10(-7). The probability of long-term relapse-free survival was significantly related to the level of MRD and only one of nine patients with MRD >10(- 3) did not relapse. For patients who did relapse, there was an inverse relationship between MRD level and the length of remission. Overall, MRD in adults in whom a translocation had not been identified was significantly higher than in comparably-treated children, suggesting that ALL in adults is more drug-resistant than in children.     

Palliative care management: a Veterans Administration demonstration project

As part of a Veterans Health Administration (VA) commitment to improve end-of-life care the VA Greater Los Angeles Healthcare System (GLA) implemented Pathways of Caring, a 3-year demonstration project targeting patients with inoperable lung cancer and advanced heart failure and chronic lung disease. The program utilized case-finding for early identification of poor-prognosis patients, interdisciplinary palliative assessment, and intensive nurse care coordination to optimize symptom management, continuity and coordination of services across providers and care settings, and support for families. Program evaluation used patient and family surveys as well as reviews of medical records and administrative databases to assess processes and outcomes of care. Despite significant programmatic challenges including organizational instability and evaluation design issues, the program achieved measurable success including high rates of advance care planning, hospice enrollment, and death at home, and low end-of-life hospital and Intensive Care Unit (ICU) use. As a result of its success, the program will be expanded and its care model extended institution-wide.     

Plasma digitalislike activity in essential hypertension or end-stage renal disease

Plasma extracts from 119 subjects showed a digitalislike activity, as evidenced by the ability of these extracts to inhibit ouabain binding to the Na+-K+ pump. High levels of the digitalislike compound were found in 18 of 54 untreated hypertensive subjects, 7 of 21 normotensive subjects with a family history of hypertension, and 10 of 14 patients with end-stage renal failure. Dialysis significantly reduced the activity of this compound. These results suggest 1) that endogenous digitalislike factor is not directly linked to hypertension but rather is related to sodium balance and 2) that it neither originates nor is activated by renal tissue, as it was present in four of six anephric patients.     

Rhinocerebral mucormycosis in renal transplant recipients: report of three cases and review of the literature

Mucormycosis is an opportunistic infection caused by fungi of the order Mucorales. The commonest clinical form is rhinocerebral mucormycosis, which has been described as characteristically complicating diabetes mellitus and leukemia. Three patients with rhinocerebral mucormycosis complicating renal transplantation are described, and 11 additional cases recorded in the English-language medical literature are reviewed. The mean age of the 14 patients was 36 years, and the ratio of males to females was 1.8:1. Diabetes mellitus was present in only five patients, and polycystic kidney was the most common underlying renal disease. Most kidney grafts were obtained from cadavers. Eight patients had evidence of graft rejection, and the majority had been receiving corticosteroids and azathioprine. The initial manifestations of infections became evident two days to four years after transplantation (median, two months). Facial swelling, tissue necrosis, and cranial nerve involvement were common. Seven of 14 cases occurred in Israel, a finding suggesting the intervention of local factors. Despite antifungal and/or surgical therapy, nine patients died as a consequence of the infection days to months after diagnosis. Although a rare complication, rhinocerebral mucormycosis remains a serious threat to the kidney transplant recipient.     

Hyperammonemia complicating mesenteric vein thrombosis

Hyperammonemic coma developed in a 69-year-old woman with prolonged symptoms of abdominal pain, dysphagia, and fever. At laparotomy for an acute condition within the abdomen, mesenteric vein thrombosis was found and partial intestinal resection was performed. Following surgery, the patient regained consciousness and blood ammonia levels became normal. Hyperammonemia and coma complicating mesenteric vein thrombosis have not yet been described. Venous shunts are suggested as being responsible for this rare complication.     

Structural and immunological characterization of insulin-like growth factor II binding to IM-9 cells

The structural and immunological properties of the insulin-like growth factor II (IGF-II) receptor on IM-9 lymphoblasts were studied using a combination of competitive binding and affinity cross-linking techniques as well as with a panel of polyclonal and monoclonal antireceptor antibodies. Unlike IGF-II binding to the classical type II IGF receptor, [125I] IGF-II binding to IM-9 cells was potently inhibited not only by unlabeled IGF-II, but also by insulin (50% inhibition of binding at 2.5 and 1.5 nM, respectively). Affinity cross-linking of [125I] IGF-II to intact cells, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated that the overwhelming majority of IGF-II binding was to a type I receptor (apparent mol wt, greater than 300,000 unreduced and 135,000 reduced), with minimal binding to a type II receptor (apparent mol wt, 220,000 unreduced and 240,000 reduced). After preincubation with five different antireceptor antibodies, inhibition of [125I] IGF-II binding was comparable to inhibition of [125I]insulin binding. These studies demonstrate that in IM-9 cells, the majority of IGF-II binding is to a type I receptor with high affinity for both insulin and IGF-II. Whether this is an atypical insulin receptor or a unique type I receptor remains to be established.     

Expression of a lactogen-dependent insulin-like growth factor-binding protein in cultured mouse mammary epithelial cells.

The ability of normal mouse mammary epithelial cells (MECs) to express insulin-like growth factor-binding proteins (IGFBPs) was examined. MECs were isolated from day 11 pregnant mice and cultured on floating collagen gels in serum-free basal medium. After 24 h, the medium was replaced with fresh medium with/or without mouse PRL (mPRL), mouse placental lactogen-I (mPL-I), mPL-II, mouse GH (mGH), IGF-I, and IGF-II, either alone or in combinations. The MECs were cultured for an additional 5 days before collection of conditioned medium (CM). The relative amount of IGFBPs present in the CM was determined by Western ligand blotting, and alpha-lactalbumin content was determined with a specific RIA. The CM of the MECs contained two IGFBPs, with approximate mol wt of 29K and 40-45K. The 40-45K IGFBP appears to be the mouse equivalent of IGFBP-3, but the identity of the 29K IGFBP is not presently known. The 29K IGFBP was not N-glycosylated and did not cross-react with antiserum to rodent IGFBP-2 or human IGFBP-1. Basal IGFBP expression was very low, but the addition of mPL-I, or mPL-II stimulated a marked increase in the amount of 29K IGFBP that was released into the CM and a lesser increase in the release of IGFBP-3. This increase in the release of 29K IGFBP was dose dependent, with increases found at concentrations as low as 1 ng/ml lactogen. mGH also stimulated the release of 29K IGFBP, but was less potent than any of the three lactogens. Treatment of MECs with either IGF-I or IGF-II increased the amount of both the 29K IGFBP and IGFBP-3 in the CM, with relative potencies similar to those of the lactogenic hormones. However, when either IGF-I or IGF-II was added together with one of the lactogenic hormones, the release of 29K IGFBP was increased in an additive manner. While the IGFs acted additively with the lactogenic hormones on the expression of 29K IGFBP, they did not stimulate alpha-lactalbumin production by the MECs or act to enhance the effects of the lactogenic hormones in stimulating alpha-lactalbumin production. This study demonstrates that IGFBPs are expressed in normal mouse MECs, and the release of these IGFBPs into the CM is hormonally regulated by both lactogenic hormones and IGFs.     

Sensitive colorimetric bioassays for insulin-like growth factor (IGF) stimulation of cell proliferation and glucose consumption: use in studies of IGF analogs.

We have established two in vitro bioassay systems for quantification of insulin-like growth factor (IGF) bioactivity. The first assay was used to quantitate mitogenic activity and the second was used to quantitate metabolic activity. Both assays use BALB/c 3T3 fibroblasts grown under serum-free conditions; detection of bioactivity in assays was performed colorimetrically and did not require the use of radioisotopes. The mitogenic bioassay, which requires 48 h for detection, quantitates changes in cell number and provides an index for determining the mitogenic activity of growth factors. Changes in cell number were measured by the enzymatic reduction of exogenously added MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] to MTT-formazan by mitochondrial enzymes, which was directly correlated to cell number. The metabolic bioassay, which requires 22 h for detection, measures glucose consumption by detecting changes from the initial glucose concentration of conditioned medium after addition of various growth factors. When appropriate standards were established for these bioassays, they demonstrated a high level of reproducibility (coefficients of variation were 0.085-0.096 for the mitogenic bioassay and 0.120-0.191 for the metabolic bioassay). Both assays can be performed in 96-well microtiter plates, without the use of radioisotopes, or the limitations of conventional glucose, amino acid, or thymidine incorporation studies. In initial experiments for assay specificity, epidermal growth factor had no measurable effect in either assay. However, IGF-I, IGF-II, and insulin demonstrated effects on both metabolism and mitogenesis. In the case of the mitogenic bioassay, the maximum mitogenic activation by these growth factors was approximately 180% of control, and these factors demonstrated parallel sigmoidal dose-response curves, ranging from 0.02-2 ng/ml for IGF-I and from 2-200 ng/ml for both IGF-II and insulin. In the metabolic bioassay, in contrast to the mitogenic bioassay, insulin showed a dose-response curve whose shape was different from those of IGF-I and IGF-II. IGF-I and IGF-II stimulated glucose consumption in dose-dependent ranges of 0.02-3 ng/ml and 0.4-40 ng/ml, respectively. However, the dose-response effect of insulin was wider, ranging from 0.1-2000 ng/ml. When these assays were used to measure the bioactivity of IGF analogs, a des(1-3)-IGF-I, which has decreased affinity for IGF binding proteins, demonstrated activity equivalent to IGF-I, a [Leu27]IGF-II, which has markedly diminished affinity for the type 1 IGF receptor, exhibited approximately 0.07% of the potency of IGF-I and 1% of the potency of IGF-II.     

Insulin-like growth factor binding protein expression in the hypothyroid rat is age dependent.

Thyroid hormone is essential for normal growth and development. For certain T4 effects, there is a critical period during ontogeny when normal T4 levels are required, and thyroid replacement after that period cannot correct the changes in hypothyroid animals. We have previously described a prolonged high expression of serum insulin-like growth factor binding protein (IGFBP)-2 during the perinatal period in congenitally hypothyroid rats. To see if this effect was confined only to a certain period during rat ontogeny, we made rats hypothyroid with methimazole treatment either prenatally, or at different postnatal ages from 1 to 14 days of life, and at adult age. Serum IGF-I levels were reduced by approximately 30% in all the 18-day-old hypothyroid animals, and did not correlate with the duration of the hypothyroid state. Serum IGF-I levels in the adult animals were 50% of control levels. At the age of 18 days, control animals had only very low levels of IGFBP-2 demonstrable by western ligand blotting, whereas the congenitally hypothyroid animals had elevated levels. Pups placed on methimazole treatment since the first day of life showed higher IGFBP-2 levels at the age of 18 days, although the change was not as prominent as in the congenitally hypothyroid animals (200% vs. 500% of control levels, respectively). Binding protein changes were approximately 2-fold at the mRNA level. Rats started on methimazole after the first 5 days of life showed normal low levels of IGFBP-2 at the age of 18 days. Abnormal IGBFP-2 expression in congenitally or neonatally hypothyroid animals could be corrected by thyroid hormone replacement, if started during the first week of the life, but not later. In adult hypothyroid animals, there was no induction of IGFBP-2 expression, but the levels of IGFBP-3 and -4 were decreased to 80% and to 30% of control levels, respectively. IGFBP-3 messenger RNA (mRNA) levels were decreased to 50% of control levels but IGFBP-4 mRNA levels were paradoxically increased in the hypothyroid animals. All these changes could be corrected by T4 replacement. In conclusion, there exists a critical period during the perinatal development of the rat, when thyroid hormone is essential for a subsequent normal IGFBP-2 ontogenic pattern. Adult animals show a completely different IGFBP response to hypothyroidism, with a decrease of IGFBP-3 and -4 levels. Thus, the effects of thyroid hormone on IGF-IGFBP axis regulation depend on the developmental stage of the animal.