Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors.

Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.

BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator.

The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-gamma. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.

Production and purification of novel secreted human proteins.

Our objective during the last year was to produce and purify 50-80 novel, secreted human proteins identified via high throughput cDNA sequencing and computer analysis. We chose the baculovirus expression vector system in order to obtain secreted, correctly folded, bioactive proteins. Recombinant (re-)baculoviruses (BV) were plaque purified, and pulse-labeling was used to verify the synthesis and secretion of the re-proteins. N-terminal microsequencing was performed to simultaneously confirm the identity of the protein(s) as well as the signal peptide (SP) cleavage site(s). Following sequence confirmation, the proteins were purified to homogeneity and functional assays carried out to determine potential therapeutic applications. We identified proteins with antiviral activity, several novel growth factors, proteins influencing the differentiation of specific cell types, novel proteases and protease inhibitors among others. Certain proteins were expressed both in insect cells and in CHO stable cell lines. In the cases analyzed, we found that the same SP cleavage site was utilized in the two expression systems. Significant differences were observed in the carbohydrate moieties attached to the proteins, though no effects on the biological activity due to these differences have been demonstrated. The BV system has served as a viable alternative for the high throughput, high fidelity expression of many novel secreted human genes. To date, more than 75 new genes have been expressed, and the re-proteins purified. This expression system combines many favorable traits including relative speed, moderate cost but perhaps most importantly, the production of biologically active proteins.

Lithium-induced nephrotic syndrome.

The nephrotic syndrome is a rare, idiosyncratic adverse renal effect of lithium that can occur with therapeutic plasma lithium levels. The syndrome is usually reversed by discontinuation of lithium treatment but may require corticosteroids. Renal biopsies reveal fusion of the foot processes of renal epithelial cells, referred to as "minimal change disease." No particular variable identifies individuals at risk for developing the nephrotic syndrome while taking lithium. The authors review the eight published cases in the English-language literature and present the case of an adolescent who developed lithium-induced nephrotic syndrome.

Recombinant human extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro.

Two mRNAs are transcribed from the extracellular matrix protein 1 gene (Ecm1): Ecm1a and an alternatively spliced Ecm1b. We studied Ecm1 mRNA expression and localization during endochondral bone formation and investigated the effect of recombinant human (rh) Ecm1a protein on organ cultures of embryonic mouse metatarsals. Of the two transcripts, Ecm1a mRNA was predominantly expressed in fetal metacarpals from day 16 to 19 after gestation. Ecm1 expression was not found in 16- and 17-day-old metatarsals of which the perichondrium was removed. In situ hybridization and immunohistochemistry demonstrated Ecm1 expression in the connective tissues surrounding the developing bones, but not in the cartilage. Biological effects of rhEcm1a protein on fetal metatarsal cultures were biphasic: at low concentrations, Ecm1a stimulated alkaline phosphatase activity and had no effect on mineralization, whereas at higher concentrations, Ecm1a dose dependently inhibited alkaline phosphatase activity and mineralization. These results suggest that Ecm1a acts as a novel negative regulator of endochondral bone formation.

Extra-nuclear location of histones in activated human peripheral blood lymphocytes and cultured T-cells.

Dextrin-2-sulphate (D2S) is a sulphated polysaccharide which inhibits human immunodeficiency virus type 1 infection of T-cells by binding to the cell surface. During our investigations of the nature of this interaction, a cell membrane fraction was prepared by ultracentrifugation from the T-cell line, HPB-ALL. Separation of membrane proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and analysis for binding proteins using ligand blotting showed that 3H-D2S bound, in a saturable and displaceable manner, to two regions corresponding to molecular weights of 14,000-18,000 and 28,000-32,000. The N-terminal sequences of two of the major protein components in the 14,000-18,000 region were consistent with those of histones H2B and H3. The presence of histone H2B in the cell membrane preparation was confirmed by immunoblotting and enzyme-linked immunosorbent assay using a specific antibody. Histone standards were used to determine the level of each histone in the cell membrane fraction. In addition, the binding of 3H-D2S to purified histone standards was quantified. These results show that all of the binding of 3H-D2S to proteins in the 14,000-18,000 region of the cell membrane preparation can be attributed to the histones present. In contrast to HPB-ALL cells, a cell membrane fraction from freshly isolated human peripheral blood lymphocytes contained very low levels of histones. However, after culture with phytohaemagglutinin for 3 days the cell membrane fraction contained greatly increased levels of histones. To exclude the possibility of contamination of the cell membrane preparation with histones derived from the nucleus, cell membranes were also prepared using an affinity-based method using polyethyleneimine-cellulose. Immunoblotting of adsorbed plasma membranes showed the presence of histone H2B. SDS-polyacrylamide gels stained for protein also indicated that the preparation contained histones H1, H2A, H3 and H4. In further experiments whole cells were used to avoid contamination from nuclear proteins. Lactoperoxidase mediated 125I labelling, a method specific for radiolabelling cell surface proteins, confirmed the presence of histones H2B, H3 and H4 on the surface of HPB-ALL cells. Also, incubation of HPB-ALL cells or phytohaemagglutinin-activated peripheral blood lymphocytes with D2S caused displacement of histones from the cell surface into the supernatant without altering cell viability. In addition, immunocytochemistry of freshly isolated peripheral blood lymphocytes showed that histone H2B was located predominantly in the nucleus. However, in phytohaemagglutinin-activated peripheral blood lymphocytes immunoreactive material was also prominent in the endoplasmic reticulum and on the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Preclinical evaluation of the pharmacokinetics, biodistribution, and elimination of MS-325, a blood pool agent for magnetic resonance imaging.

RATIONALE AND OBJECTIVES: The authors evaluate MS-325, a new albumin-targeted magnetic resonance imaging (MRI) contrast agent, for its pharmacokinetics, biodistribution, and elimination characteristics in multiple animal species. METHODS: Studies were performed in rats, rabbits, and nonhuman primates at intravenous doses ranging from 0.025 to 0.20 mmol/kg. Concentrations of MS-325 in blood, urine, feces, and organs were determined using gadolinium-153-labeled MS-325 and gamma counting or by using non-labeled MS-325 and inductively coupled plasma atomic emission spectrometry. RESULTS: In rabbits and nonhuman primates, MS-325 is approximately 85% to 95% bound to serum proteins and, as a result, exhibits low volume of distribution (Vd) values, 0.11 to 0.14 L/kg, and a long elimination half-life (Te1/2), 2 to 3 hours. Some dose-dependence in the parameters is apparent in rabbits. MS-325 is eliminated primarily through the renal system in non-human primates. In contrast, the behavior of MS-325 in rats is different, exhibiting increased biliary excretion, a larger Vd value, and a shorter Te1/2. CONCLUSIONS: The pharmacokinetics and elimination profile of MS-325, including vascular retention and renal excretion, are favorable for use in humans as an intravascular contrast agent for MRI.

SAA, an apoprotein of HDL: its structure and function.

The putative precursor of amyloid protein AA appears in serum as an apoprotein (apoSAA) of heavier HDL fractions of lipoproteins found in humans and mice. ApoSAA is found by precipitation with specific anti-AA antibodies to be on a particle that also carries apoA-I and some C-apoproteins. In endotoxin-treated mice a lipoprotein fraction with about 2 moles of apoSAA to 1 mole of apoA-I can be isolated, an apoSAA-carrying subset of HDL being thus suggested. In mice, rapid clearance from plasma of native apoSAA compared to apoA-I suggests a special function for apoSAA. The C-terminal amino acid portion of apoSAA may play a role in this function.

Population pharmacokinetics of methylphenidate in children with attention-deficit hyperactivity disorder.

Sources of individual variation in plasma methylphenidate (MP) concentrations during usual clinical use are not established. This was evaluated in a series of patients receiving clinical treatment with MP. A single plasma MP concentration was determined in each of 273 children and adolescents ages 5 to 18 years (mean: 11.1 years) who were clinically good responders to MP for the treatment of attention-deficit hyperactivity disorder. MP was given on a twice-daily schedule (mean dose: 25 mg/day) in 40% of patients and three times daily (mean dose: 39.3 mg/day) in 60%. A nonlinear regression model was applied to estimate overall population values of MP clearance and elimination half-life (t1/2), assuming a one-component model with first-order absorption and elimination, and further assuming that clearance is linearly related to body weight. The model incorporated each patient's dosage size and schedule, body weight, and time of the plasma sample. Iterated solutions of best fit were: t1/2, 4.5 hours (95% confidence interval [CI]: 3.1-8.1 hours), and apparent clearance, 90.7 ml/min/kg (95% CI: 74.6-106.7 ml/min/kg). The model explained 43% of the overall variance in MP concentrations (r2 = 0.43, p < .001). In a small subsample (N = 16), a second plasma sample was drawn at the same time of day and at the same dose; the correlation between the two concentration values was 0.83. The relatively noninvasive approach used in this study allows the assessment of pharmacokinetic properties of medications under conditions of appropriate clinical use in special populations such as children, adolescents, and the elderly.

Control-related cognitions and depression among inpatient children and adolescents.

In previous studies, children with numerous depressive symptoms have shown two patterns of control-related cognition: (1) low levels of perceived personal competence, and (2) "contingency uncertainty"--confusion regarding the causes of significant events. The generality of these findings was tested for more seriously disturbed children. Three child inpatient samples, from separate psychiatric hospitals, completed the Children's Depression Inventory (CDI) plus measures of control-related beliefs. In all three samples, the findings resembled those of previous studies: CDI scores were significantly related to low perceived competence and to contingency uncertainty; by contrast, CDI scores were only weakly related to perceived noncontingency. The findings suggest that depressive symptoms in children may be (1) more closely linked to "personal helplessness" than to "universal helplessness," and (2) more closely linked to uncertainty about the causes of events than to firm beliefs in noncontingency. The findings carry implications for etiology and treatment of child depression.

Characteristics of children and adolescents in a psychiatric hospital and a corrections facility.

Popular opinion holds that youngsters in corrections programs are delinquents in need of reform, whereas youngsters in psychiatric settings have mental health problems and need therapy. Yet some literature suggests that youth in the two settings may not differ greatly in their mental health status. The authors compared demographic, emotional, and behavioral characteristics of youngsters placed in public psychiatric hospital and corrections settings, and they found few differences. Child Behavior Checklist scores for social competence and total problems were high and quite similar for youngsters in the two settings. White children scored significantly worse than black children on Child Behavior Checklist Internalizing, Externalizing, and total problems in corrections settings but not in the psychiatric hospital. Moreover, race was the only variable that predicted the site in which youth were placed. The findings suggest a need to study (1) the mental health needs of youngsters in nonmental health settings and (2) the procedures by which youth are assigned to service settings.

Whither the state in Yugoslav health care?

As part of their broader sociopolitical system of self-managing socialism, the Yugoslavs have attempted to create a health care system which is simultaneously destaticized, depoliticized, decentralized, democratized and economically and socially efficient. This paper provides a description and evaluation of the evolving self-managed health sector in post-World War II Yugoslavia. I argue that, despite repeated institution restructuring and innovation and some noteworthy accomplishments, the Yugoslavs have fallen short of their health objectives. After presenting alternative explanations for these shortcomings, I propose that they can best be understood in terms of the contradictions the Yugoslavs have encountered in their efforts to simultaneously pursue destatization, depoliticization, decentralization, democratization and social and economic efficiency. I conclude with an ideologically unpopular proposal that some form of continuous and legitimate central state coordination may be necessary to resolve current critical problems in Yugoslav health care (e.g. persistent deficits in health financing, shortages of basic medical supplies, duplication of expensive medical technology, unemployed health workers despite unmet health needs, persistent health inequalities).

Plasma proteins as early biomarkers of exposure to carcinogenic aromatic amines.

Two-dimensional gel electrophoresis (2DG) has been used to study the changes induced in dog plasma polypeptides by the known urinary bladder carcinogens, 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA). Treatment with 3-aminobiphenyl (3-ABP) and 1-naphthylamine (1-NA), both considered to be non-carcinogenic, were used as controls. The purpose of this study was: (1) to determine whether or not changes that occurred in the plasma protein patterns were specific to 4-ABP and/or other related carcinogenic arylamines; (2) to measure the time course in the changes of the major polypeptides during dosing and their resynthesis during a recovery period; and (3) to determine, by microsequencing, the biochemical identity of the affected proteins. The results indicate that only the most potent carcinogen, 4-ABP, had the effect of suppressing the expression of some proteins, while the other aromatic amines caused no discernible change in the 2DG patterns during a 12-week dosing period. The 4-ABP caused dramatic suppression of two sets of proteins. One set of three spots had an apparent molecular weight of 32.5 kDa, and a pI of 5.8-6.0. The major component in this group was identified as the beta-chain of haptoglobin. Expression of this protein decreased markedly during the first 2 weeks of treatment and recovered slowly after dosing stopped. Since haptoglobin functions to bind with free hemoglobin and facilitates its elimination from the blood stream, these results can be rationalized as a consequence of 4-ABP binding to hemoglobin in the erythrocyte, resulting in cell death and hemolysis. The 4-ABP modified hemoglobin then binds to haptoglobin and this tertiary complex is purged from the blood stream, resulting in the disappearance of free haptoglobin. A second set of spots (mol. wt., 65 kDa; pI, 6.5-6.6) disappeared much faster than the haptoglobin, and recovered more quickly. The major protein is about one-fifth the intensity of haptoglobin and appeared to be N-terminally blocked. Internal microsequencing of four fragments obtained from tryptic cleavage of the major spot of this group showed significant similarity to the serum albumin sequence of several species. This spot group is not the major serum albumin spot, however, since the latter is readily identified as the most abundant spot on the plasma map. During the course of this study, several other polypeptides in the 2DG map of dog plasma were identified and are presented here.