Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Specific alterations in the expression of α3β1 and α6β4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane

Highly invasive cell subpopulations from a human prostate carcinoma cell line, PC-3, were selected for by allowing the parental PC-3 cells to invade through reconstituted basement membrane, Matrigel. These cells were collected, cultured and then selected further by repeated invasion through the in vitro invasion chamber. The invasive subpopulations (I-PC3 (2) and (3)) were found to be approximately 15-fold more invasive in vitro than the parental cells, had a distinct rounded morphology in culture, and proliferated more rapidly than the parental cells. When injected either subcutaneously or intraperitoneally into immunocompromised SCID mice, the I-PC3 cells were found to form tumors at the primary sites and to be highly invasive and metastatic. In contrast, the parental PC-3 cells formed tumors at the site of inoculation in these mice but failed to invade or metastasize. The I-PC3 cells attached equally as well as PC-3 cells to fibronectin, laminin, collagen type IV and vitronectin, but unlike the parental PC-3 cells these invasive variants failed to spread on any of these substrates. On Matrigel, the PC-3 cells became highly organized, whereas the I-PC3 cells remained rounded, clumped together and penetrated into the Matrigel. Biochemical analysis of the expression of adhesion proteins and integrins demonstrated that whereas the parental cells synthesized and secreted substantial amounts of fibronectin, the I-PC3 cell variants did not secrete any fibronectin. Although both PC-3 and I-PC3 cells expressed equivalent levels of cell surface v 3, 21 and 51 integrins, the expression of the 31 integrin, which is expressed at very high levels on the parental PC-3 cells, was drastically reduced on the invasive I-PC3 cells. This decrease in expression of 3 occurred also at the level of mRNA expression. Finally, whereas the PC-3 cells express 61, in the invasive I-PC3 cells the a6 subunit was associated mostly with the 4 subunit. Since the 64 integrin is analogous to the A9 tumor antigen which is associated with aggressive human squamous cell carcinomas, the apparent overexpression of 64 may also participate in the aggressive behavior of these variant prostate carcinoma cells. Alterations in the expression of the 31 and 64 integrins may thus allow these cells to become more invasive, and lead to an increased propensity for metastasis.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1, IL-1, TNF, IL-2 and Ifn. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1>rIL-1>rTNF>rIfn=BSA (control) following a single sc. injection. However, only rIL-1 and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1 lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These in vivo observations reinforce, the roles of both IL-1 and IL-2 as potent mediators of chronic immunoinflammatory disease.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Establishment of immortalized Schwann cells from Sandhoff mice and corrective effect of recombinant human β-hexosaminidase A on the accumulated GM2 ganglioside

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl--D-glucosamine (-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl--D-glucosamine-6-sulfate (-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells. Incorporation of recombinant human -hexosaminidase isozymes expressed in Chinese hamster ovary cells into the cultured Sandhoff Schwann cells via cation-independent mannose 6-phosphate receptors was found, and the incorporated -hexosaminidase A degraded the accumulated GM2 ganglioside. The established Sandhoff Schwann cell line is useful for investigation and development of therapies for Sandhoff disease.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Inhibition of lung colonization of mouse colon 26 adenocarcinoma by recombinant mouse interferon β through a modification of platelet function

Recombinant murine interferon (MuIFN-) given i.v. efficiently inhibited both pulmonary arrest and formation of lung colonies of NL-17, a highly metastatic variant of mouse colon adenocarcinoma 26. NL-17 was rather resistant to MuIFN- in vitro and was highly resistant to natural killer cells of mice even though they were treated in vivo with MuIFN-. Platelets isolated from MuIFN--treated mice showed reduced aggregating activity induced by NL-17. Since lung colonization by NL-17 is influenced by platelet aggregation, the inhibition of colonization by MuIFN- could be partly mediated through modification of platelet function in vivo. The effect of MuIFN- on platelet function and its subsequent inhibition of lung colony formation give new insights into the action of recombinant MuIFN-.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Is there a role for the tumor cell integrin αIIbβ3 and cytoskeleton in tumor cell-platelet interaction?

In vitro tumor cell-platelet interaction was examined using B16 amelanotic (B16a) melanoma cells. These tumor cells express the _IIb3-type cytoadhesin. Aggregation studies demonstrated that tumor cell surface _IIb3 mediates the recognition of platelets since pretreatment of tumor cells with antibody against _IIb3 prevents platelet-tumor cell interaction as well as platelet activation measured by aggregometry, platelet eicosanoid metabolism and ultrastructural analysis. In B16a cells, disruption of the microfilaments and intermediate filaments inhibits mobility of _IIb3 on the cell surface. Microtubules do not play a role in receptor mobility, because B16a cells do not possess well-defined microtubules in interphase and colchicine does not affect receptor mobility. Disruption of microfilaments or intermediate filaments results in an inhibition of tumor cell-platelet interaction as evidenced by aggregometry studies and ultrastructural analysis. We suggest that platelet interaction with tumor cells begins with _IIb3-mediated receptor recognition followed by not only platelet activation but also microfilament- and vimentin intermediate filament-dependent tumor cell activation.     

Restricted Vβ gene usage of tumour-infiltrating T lymphocytes in primary gastric malignant B-cell lymphoma

Ten cases of primary gastric malignant lymphoma (PGL) were investigated by immunohistochemical and molecular genetic analysis. These cases were diagnosed histopathologically as follicular small cleaved cell type (1 case), diffuse small cleaved cell type (3 cases) and diffuse large cell type (6 cases) based on the WF (Working Formulation) classification. Seven cases classified as small cleaved or diffuse large cell type belong to low (4 cases) or high (3 cases) grade MALT lymphoma according to Isaacson's classification. All PGL belonged to B lineage cells according to immunohistochemical study and immunoglobulin rearrangements. Rearrangements of TCR chain genes were observed in four of the ten cases. The possibility that the TCR rearrangements were caused by tumour-infiltrating T-cells (TILs) was supported by the following observations: the tumours did not show T- and B-cell biphenotype, TCR exhibited functional VDJ rearrangement and V usage pattern was not a neoplastic type. Analysis of the repertoire of the TCR chain in TILs revealed a common usage of V2 in the above four cases, and furthermore, predominant usage of a particular chain composed of V2-D2.1-J2.3 was observed in one of the four cases. These results indicate that the TILs of PGL have a restricted TCR repertoire.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.     

Factors influencing serum neopterin and β2-microglobulin levels in a healthy diverse population

Sera and questionnaire data from a population-based random sample of healthy adults was used to evaluate factors influencing neopterin and 2-microglobulin (2m) values. Both neopterin and 2m levels increased with age and were higher among white than blacks (mean values for whites and blacks: neopterin, 5.06 vs 4.49 nmol/L; 2m, 1.36 vs 1.28 mg/L). Gender differences were noted for 2m but not neopterin values (2m males vs females: 1.37 vs 1.29 mg/L). Neopterin values were lower among current smokers than among nonsmokers (4.32 vs 5.16 nmol/L) and were higher among users of antihistamines (5.46 among users vs 4.65 nmol/L among nonusers). Neopterin and 2m were correlated in this healthy adult population (adjustedr=0.53,P=0.001), yet no other interrelationships with numerous biologic markers except between 2m and serum-soluble interleukin-2 receptor levels (adjustedr=.41,P=0.05) were observed. These findings provide important baseline information to consider before planning or evaluating studies utilizing neopterin or 2m levels.