Immunomodulation and adenoviral gene transfer to the lungs of nonhuman primates.

Previous data from our laboratory and others have demonstrated a critical role for the CD4+ T lymphocyte in in vivo immune responses to recombinant adenoviral vectors. In rodent models, this subset of T cells is required for T cell proliferation, subsequent cytotoxic T cell generation, and production of anti-adenoviral antibodies by B cells. Both depleting and nondepleting anti-CD4 antibodies can attenuate these immune responses to recombinant adenovirus. On the basis of these data, we hypothesized that a nondepleting CDR-engrafted anti-human CD4 antibody (OKT4A) with cross-reactivity to rhesus macaques would attenuate both T and B cell responses to intrapulmonary administration of recombinant adenovirus and permit prolonged reporter gene expression and permit secondary gene transfer. Juvenile rhesus macaques were treated with PBS or OKT4A antibody (10 mg/kg) daily beginning 1 day prior to and up to 11 days after gene transfer. OKT4A resulted in significant attenuation of lymphocyte recruitment into the lung, lymphocyte-proliferative responses to both adenovirus capsid proteins and transgene protein, and adenovirus-induced interferon-gamma elaboration in whole blood and hilar lymph nodes. However, OKT4A was ineffective in attenuating adenovirus-induced IL-4 production in whole blood or hilar lymph nodes, generating neutralizing anti-adenoviral antibodies, or permitting secondary gene transfer. As all the monkeys in this protocol had baseline-detectable anti-adenoviral antibodies by ELISA that were nonneutralizing, analogous to most patients with cystic fibrosis, we postulate that anti-CD4 did not block the proliferation of memory B cells. Moreover, these data suggest that for transient immunomodulation to be successful, strategies need to focus specifically on B cell activation independent of CD4+ T cell help.

Investigation of capillary electrochromatography with brush-type chiral stationary phases.

Fused-silica capillaries (100 microm ID) were packed with the (3R, 4S)-Whelk-O chiral stationary phase (CSP) bonded on 3.0 microm silica particles. The enantiomers of 41 neutral analytes containing stereogenic centers, axes or planes were examined by packed capillary electrochromatography. More than 30 of these were cleanly resolved, owing to the selectivities and efficiencies afforded by this CSP. High reproducibility with no indication of diminished performance was observed using the same capillary for hundreds of runs (including intermediate change of the buffer system) over a period of several weeks. Acetate, 2-(N-morpholino)ethanesulfonic acid, or phosphate buffers, each modified with either acetonitrile or methanol, were used as mobile phases. The influence of buffer concentration, modifier amount, temperature, applied voltage, and pH on performance of the brush-type CSP was investigated.

RWJ 67657, a potent, orally active inhibitor of p38 mitogen-activated protein kinase.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.

Anti-inflammatory doses of ibuprofen: effect on neutrophils and exercise-induced muscle injury.

The purpose of the study was to determine the effect of anti-inflammatory doses of ibuprofen on neutrophils, neutrophil O2* production, and markers of muscle injury. Males (n=10) performed 2 bouts of one-arm eccentric exercise on opposite arms separated by three weeks. Subjects received 2400 mg x d(-1) of ibuprofen or a placebo 5 d before exercise and during 10 d of recovery. Measurements were made before the treatments, pre-exercise, at 4 h, and at 1, 2, 3, 4 and 10 d post-exercise. Circulating neutrophil counts were similar between the treatments at the sampling points. Neutrophil counts were higher (p<0.05) for ibuprofen and were elevated (p<0.05) at 4h post-exercise relative to pre-exercise in both treatments. Stimulated neutrophil O2* production was lower for ibuprofen relative to placebo at pre-exercise and was increased (p<0.05) at 4 h and 4 d of both treatments. CK activity at 3 d post-exercise was lower (p<0.05) for ibuprofen relative to placebo. Isometric strength, soreness, tenderness, and arm angles were similar between the treatments. In conclusion, anti-inflammatory doses of ibuprofen reduced CK activity but not the neutrophil response or other indirect markers of muscle injury during recovery from eccentric arm exercise.

Interleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signaling pathways and IL-6 production.

Interleukin (IL)-1 is a proinflammatory cytokine with pleiotropic effects in inflammation. IL-1 binding to its receptor triggers a cascade of signaling events, including activation of the stress-activated mitogen-activated protein (MAP) kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, as well as transcription factor nuclear factor kappaB (NF-kappaB). IL-1 signaling results in cellular responses through induction of inflammatory gene products such as IL-6. One of the earliest events in IL-1 signaling is the rapid interaction of IL-1 receptor-associated kinases, IRAK and IRAK-2, with the receptor complex. The relative roles of IRAK and IRAK-2 in IL-1 signaling pathways and subsequent cellular responses have not been previously determined. To evaluate the importance of IRAK in IL-1 signaling, IRAK-deficient mouse fibroblast cells were prepared and studied. Here we report that IL-1-mediated activation of JNK, p38, and NF-kappaB were all reduced in embryonic fibroblasts deficient in IRAK expression. In addition, IL-6 production in response to IL-1 was also dramatically reduced in IRAK-deficient embryonic fibroblasts and in skin fibroblasts prepared from IRAK-deficient mice. Our results demonstrate that IRAK plays an essential proximal role in coordinating multiple IL-1 signaling pathways for optimal induction of cellular responses.

Potent inhibitors of the MAP kinase p38.

The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines TNF-alpha and IL-1. We have developed a novel series of potent p38 inhibitors that could lead to new methods of treatment for inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.

The effect of preexercise carbohydrate status on resistance exercise performance.

The purpose of this study was to determine the effect of a high vs. a low preexercise carbohydrate (CHO) diet on performance during multiple sets of resistance exercise. Eleven resistance-trained males performed cycle ergometry to deplete quadriceps muscle glycogen stores, followed by 48 hr of a high (HICHO) or a low (LOCHO) CHO diet. Subjects then performed five sets each of squats, leg presses, and knee extensions (resistance = 15 RM) to failure. Blood samples were taken before and during exercise for determination of glucose and lactate (LA). No differences in performance (repetitions x weight lifted) were observed (HICHO = 15,975 +/- 1,381 and LOCHO = 15,723 +/- 1,231 kg). Blood glucose was significantly higher after exercise for HICHO compared to LOCHO (HICHO = 4.8 +/- 0.2 vs. LOCHO = 3.9 +/- 0.2 mmol.L-1). No differences in LA accumulation were observed. The data indicated that preexercise CHO status did not affect resistance exercise performance. Further, the differences in blood glucose and the similarity in LA responses suggest that glycolysis was maintained in the LOCHO condition, and there may have been an increased reliance on blood glucose when preexercise CHO status was low.

Nitroarylhydroxymethylphosphonic acids as inhibitors of CD45.

A series of nitroarylhydroxymethylphosphonic acids was synthesized and evaluated as inhibitors of CD45. It was discovered that both the alpha hydroxy and nitro groups are essential for activity. Potency is enhanced by the addition of a large lipophilic group on the aryl ring adjacent to the phosphonic acid moiety. Kinetics studies have shown that these compounds are competitive inhibitors and thus bind at the active site of this enzyme.

Maximal accumulated oxygen deficit of resistance-trained men.

The primary purpose of the study was to compare maximal accumulated oxygen deficit (MAOD) in resistance-trained (RT), endurance-trained (ET), and untrained men (UT). A secondary purpose was to determine the influence of leg muscle mass (MM) on MAOD by examining the relationship between MM and MAOD and by comparing MAOD expressed relative to MM between the groups. MAOD was determined during 2-4 min of constant-load fatiguing cycling. MM, estimated via anthropometric measurements, was higher (p < .05) for RT (mean +/- SE; 25.5 +/- 3.4 kg) compared to ET (20.3 +/- 3.5) and UT (21.6 +/- 3.4). MAOD in liters O2eq was larger in RT (4.75 +/- 0.3) compared to UT (3.07 +/- 0.3) and ET (3.75 +/- 0.3). A significant positive correlation was observed between MAOD (LO2eq) and MM (kg) for RT only (RT, r = .85; ET, r = .55; UT, r = .20). Based on the correlational and mean MM data, the higher MAOD (LO2eq) in RT relative to ET and UT is predominantly the result of their larger leg muscle mass.

Nonhuman primate responses to murine and humanized OKT4A.

A nonhuman primate antimurine response (MAMA) has been observed in 17 cynomolgus renal allograft recipients of murine OKT 4A. Neither cyclosporine, nor total-lymphoid irradiation, nor donor bone marrow preparation inhibited this antixenogeneic response. In an attempt to alter the antimurine basis of the response, a humanized chimeric OKT4A (IgG4) containing the entire variable portion of the murine OKT4A and a humanized CDR grafted OKT4A mAb sharing only the Complementarity Determining Region from the murine OKT4A, were administered to 8 cynomolgus allograft recipients. MAMA was detected in each recipient. In contrast to sera from recipients of murine OKT4A, sera from recipients of humanized OKT4A displayed no reactivity to other murine mAbs. MAMA specificity did not assay constant (C) region differences between the murine and humanized mAb; however, C region homology in humans should preclude a human antimouse antibody (HAMA) to the Fc portion of a humanized mAb. Furthermore, cynomolgus recipient serum levels of the humanized OKT4A mAb were maintained (> 1 microgram/ml) for a longer period than following treatment with murine OKT4A (murine < 12 days versus between 12 and 24 days for the humanized). If the HAMA response to humanized mAb in future clinical trials, were to be predictably anti-idiotypic, then the opportunity for treatment with sequential mAbs of differing idiotypes would be retained. Moreover, these current studies also suggest that humanized construction may influence the duration of therapeutic mAb levels. Thus, anti-idiotypic reactivity may not be as consequential to the clinical administration of humanized mAb to allograft recipients.

Familial and sporadic insulin-dependent diabetes: evidence for heterogeneous etiologies?

Heterogeneity within insulin-dependent diabetes mellitus (IDDM) has been hypothesized, but few studies have focused on differences which may exist between familial and sporadic IDDM cases. Presenting characteristics for 330 white, newly diagnosed IDDM cases were evaluated. Familial cases were older (10.2 +/- 5.1 years vs 7.9 +/- 4.2 years, P = 0.010) and had, on average, less severe metabolic disturbances at presentation, as demonstrated by lower mean hemoglobin A1 (12.6 +/- 2.4% vs 14.4 +/- 2.6%, P = 0.001) and mean insulin dose at discharge (0.62 +/- 0.35 U/kg/day vs 0.85 +/- 0.29 U/kg/day, P less than 0.001), and higher mean plasma bicarbonate concentrations (19.3 +/- 3.9 mmol/l vs 15.8 +/- 5.9 mmol/l, P = 0.023) and mean plasma C-peptide levels (0.35 +/- 0.36 pmol/ml vs 0.14 +/- 0.15 pmol/ml, P less than 0.001). Further analyses on a subset of IDDM cases (n = 100) indicated that initial differences in metabolic indices observed at diagnosis were no longer apparent at one-year post-diagnosis. These results suggest that the etiology of familial and sporadic IDDM is similar and that the less severe presentation observed at diagnosis in the familial cases may be due to earlier identification of the disease, reflecting increased parental knowledge of diabetic symptoms and/or frequent testing for diabetes.

T cell adhesion to extracellular matrix molecules secreted by endothelial cells cultured on a substrate of type IV collagen.

T cell emigrating from the bloodstream into lymphoid organs or sites of inflammation in the connective tissue must adhere to, and traverse, the subendothelial basement membrane (BM). The goal of the current investigation was to develop a method to study the adhesion of T cells to endothelial cell (EC)-derived extracellular matrix (ECM) as a model for the interaction of T cells with the subendothelial BM in vivo. To be certain that we were truly measuring T cell adhesion to ECM molecules secreted by the EC, it was necessary to culture the EC on a substrate to which T cells could not attach. Non-tissue culture-treated microtiter plate wells which had been coated with type IV collagen (tIVC), a major constituent of BM in vivo, were found to be suitable for this purpose since EC, but very few T cells, adhered to such wells. After incubating the EC on a substrate of tIVC in non-treated wells for a period of 48 h, the EC were gently removed from their underlying ECM and T cell adhesion to that ECM was examined. Using this system, it was observed that approximately 15-40% of human peripheral blood T cells specifically adhered to ECM molecules produced by the EC. This method should be useful as a model for the interactions of T cells and other leukocytes with the vascular BM in vivo.

Effects of inflammatory cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to endothelial cell monolayers and extracellular matrix proteins.

The accumulation of mononuclear phagocytes at sites of chronic inflammation is dependent on an increase in the rate of extravasation of blood-borne monocytes through the vascular endothelium into the connective tissue. Once the monocytes have emigrated into the connective tissue, they may differentiate into tissue macrophages, presumably following interactions with extracellular matrix proteins. To study these processes, we tested the effects of cytokines and phorbol esters on the adhesion of U937 cells, a human monocyte-like cell line, to cultured endothelial cells (EC) and to matrix proteins. In the absence of cytokines, very few of the U937 cells adhered to EC (5% or less in most experiments). When EC were pretreated for optimal periods of time (4-8 hr) with recombinant interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF alpha), or lymphotoxin (LT; also known as TNF-beta), 35-85% of the U937 cells were able to bind. Interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) did not stimulate U937-EC binding, even though IFN-gamma was shown to increase EC adhesiveness for T lymphocytes. Phorbol esters also greatly stimulated U937-EC adhesion but, in this case, the increase was due to an action on the U937 cells. A monoclonal antibody (MAb), 60.3, against the CD11/CD18 family of leukocyte adhesion molecules partially inhibited the adhesion of untreated and phorbol ester-treated U937 cells to noncytokine-treated EC. However, that MAb had no effect on U937 cell binding to TNF-alpha-treated EC. Thus U937 cells use both CD11/CD18-dependent and -independent mechanisms to adhere to EC. In the absence of stimulating agents, only a small proportion of the U937 cells (2-20%) adhered to fibronectin (FN), and almost none bound to either laminin (LN) or gelatin (denatured type I collagen). In the presence of phorbol esters, a much larger proportion of the U937 cells adhered to FN, with only slight increases in the proportion of cells which bound to LN or gelatin. Additional adhesion assays performed in the presence of a pentapeptide containing the amino acid sequence arg-gly-asp (RGD), which is part of one of the cell-binding domains of FN, demonstrated that the RGD-containing peptide almost totally blocked the phorbol ester-induced adhesion of U937 cells to FN. In contrast, the peptide had no inhibitory effect on the phorbol ester-induced binding of U937 cells to EC.

Organ-specific and non-organ-specific lymphocyte receptors for vascular endothelium.

The recirculation of lymphocytes from blood to lymph and back to blood is necessary for the proper functioning of the immune system as it facilitates interactions between antigen-reactive clones of lymphocytes and antigen-presenting cells. The first step in the emigration of a blood-borne lymphocyte into either a secondary lymphoid organ or an inflammatory lesion is its adherence to vascular endothelial cells (EC) lining unique post-capillary venules known as high endothelial venules (HEV). Several groups have recently cloned and sequenced genes which may encode organ-specific lymphocyte receptors for the EC of such HEV. The extracellular portion of the putative murine lymphocyte homing receptor for peripheral lymph node HEV is composed of an N-terminal lectin-like domain, followed by an epidermal growth factor-like domain, and then two identical repeating domains which are homologous to a number of complement-binding proteins. A hydrophobic transmembrane domain and a cytoplasmic tail complete the structure. A very similar gene structure has been reported for a cytokine-inducible EC surface protein which is involved in neutrophil-EC adhesion in vitro. In marked contrast, the gene for a putative human lymphocyte homing receptor appears to belong to a gene family which encodes cell-surface molecules with receptor activity for extracellular matrix (ECM) proteins. Similarly, the cell-surface molecule which appears to be the murine lymphocyte receptor for Peyer's patch HEV is homologous, if not identical, to the human VLA-4 molecule, another receptor with binding activity for an ECM protein. It has also been demonstrated that lymphocyte function-associated antigen 1 (LFA-1) acts in a non-organ-specific manner to mediate lymphocyte-EC adhesion. Finally, other non-organ-specific lymphocyte adhesion molecules for EC may include CD4 and CD8 (which bind to class II and class I MHC antigens, respectively), and CD2 (which binds to LFA-3).

Effect of inflammatory cytokines on human endothelial cell proliferation.

Neovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN-gamma) (10-100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)-stimulated EC proliferation. Recombinant interleukin 1 (rIL-1) and recombinant tumor necrosis factor-alpha (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN-gamma and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by 51Cr release. In contrast, the combination of rIFN-gamma and rIL-1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were 1) the stimulation of proliferation by IFN-gamma at a low concentration and 2) the inhibition by both rIL-1 and rTNF of ECGF-stimulated proliferation.

Interferon-gamma increases susceptibility of murine pancreatic beta cells to lysis by allogeneic cytotoxic T lymphocytes.

The selective loss of insulin-producing pancreatic beta cells which occurs in IDDM has been postulated to result from lysis by beta cell-specific cytotoxic T lymphocytes (CTL). CTL typically recognise antigen in the context of MHC class I molecules, which are normally present at low levels on beta cells. However, hyperexpression of class I antigens on islet cells has been observed in the early stages of beta cell destruction in IDDM. Since interferon-gamma (IFN-gamma) is known to increase class I expression on a number of cell types, we have investigated the responses of murine beta cells to this cytokine under various conditions. Two color immunostaining followed by FACS analysis showed that on average, only 14.9 +/- 3.1% of cultured beta cells were class I positive. However, a majority of beta cells could be induced to express class I after 24 hours of IFN-gamma treatment, and maximal induction (80-90% positive) occurred after 48 hours. Importantly, increased class I expression on beta cells could be achieved with very low concentrations of IFN-gamma (1-10 U/ml). Expression of class II MHC was never detected under any of the conditions employed to up-regulate class I. Interestingly, although islet cells were only moderately susceptible to lysis by allospecific CTL, this susceptibility was markedly enhanced by prior exposure of the islets to IFN-gamma. Taken together, these results suggest that beta cells are extremely susceptible to up-regulation of class I MHC molecules by IFN-gamma, and that this property may render these cells particularly susceptible to lysis by autologous class I-restricted CTL. Since enhanced expression of class I frequently accompanies inflammatory responses and viral infections, this property of beta cells may account in part for their selective destruction in IDDM.

Lymphocyte adhesion to endothelial cells in vitro: models for the study of normal lymphocyte recirculation and lymphocyte emigration into chronic inflammatory lesions.

Lymphocytes preferentially leave the bloodstream to enter secondary lymphoid organs or sites of inflammation by first adhering to, and then migrating through, the walls of specialized postcapillary venules known as high endothelial venules. To study the initial adhesion event between lymphocytes and endothelial cells, two different in vitro assays have been developed. In the first, lymphocytes are incubated on frozen sections of lymphoid organs or other tissues. Under certain conditions, lymphocytes specifically bind to the endothelial cells of high endothelial venules in such sections. The results of monoclonal antibody inhibition studies have suggested that endothelial cells at various lymphoid organs, and at certain inflammatory lesions, may express organ-specific ligands on their surface, which bind to corresponding organ-specific "homing receptors" on the lymphocytes. The second assay measures the adhesion of lymphocytes to confluent monolayers of viable, cultured endothelial cells. Because pretreatment of such cell monolayers with a number of cytokines produced at inflammatory sites stimulates an increase in the adhesiveness of the cells for lymphocytes, it has been suggested that such cytokine-induced increases in endothelial cell adhesiveness may be important in the induction of lymphocyte traffic into such lesions. Unlike the homing receptor type of binding described above, the cytokine-induced increase in lymphocyte-endothelial cell adhesion is presumably non-tissue-specific. Results of monoclonal antibody inhibition studies in this system have suggested that at least two ligand-receptor interactions may be involved. Monoclonal antibodies to the lymphocyte function-associated antigen-1 greatly inhibited the adhesion of T cells to untreated endothelial cells, but had little or no inhibitory effect on T-cell adhesion to cytokine-treated endothelial cells.