The effect of non-steroidal anti-inflammatory drugs on osteosarcoma cells.

OBJECTIVE: Osteosarcoma (OS), an aggressive malignancy, is the most common primary bone tumor in children. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce pain and inflammation. NSAIDs have shown to be toxic to certain malignancies such as colorectal, breast, and pancreatic cancers, but are not well-studied in OS. The purpose of this study is to assess whether ketorolac induces apoptosis in OS cells, compare this to indomethacin, which has been shown to inhibit OS proliferation, and explore the underlying mechanism. MATERIALS AND METHODS: A rat OS cell line (UMR-108) was exposed to various concentrations of ketorolac and indomethacin. Cell viability, cytotoxicity, apoptosis induction, DNA fragmentation and the expression of apoptosis-related markers were examined by MTT assay, colony formation assay, flow cytometry, agarose gel electrophoresis, and Western blot respectively. RESULTS: The results indicated that ketorolac and indomethacin could induce apoptosis of rat OS cells in a dose- and time-dependent manner. Apoptosis was confirmed by cell morphology and annexin positivity. The molecular data showed that NSAIDs affected expression of Bcl-2, survivin, and Poly (ADP-ribose) polymerase-1 (PARP). CONCLUSIONS: These findings demonstrated that NSAIDs induced apoptosis in rat OS cells in vitro. Further research focusing on the potential cytotoxicity of NSAIDs in vivo is needed.

Latent inhibition in 35-day-old rats is not an "adult" latent inhibition: implications for neurodevelopmental models of schizophrenia.

RATIONALE: Latent inhibition (LI) refers to retarded conditioning to a stimulus as a consequence of its inconsequential preexposure. Amphetamine-induced disruption of LI and its potentiation by antipsychotic drugs (APDs) in the adult rat are well-established models of schizophrenia and antipsychotic drug action, respectively. It is not clear whether LI can be similarly modulated at prepubertal age. OBJECTIVES: In view of the notion that schizophrenia is a neurodevelopmental disorder whose overt expression depends on postpubertal brain maturational processes, we investigated whether several manipulations known to modulate LI in adult rats, including systemic administration of amphetamine and the atypical APD clozapine, are capable of producing the same effects in prepubertal (35-day-old) rats. METHODS: LI was measured in a thirst motivated conditioned emotional response (CER) procedure in which rats received 10 or 40 tone preexposures followed by 2 or 5 tone-footshock pairings. RESULTS: Like in adults, LI was present with 40 preexposures and 2 conditioning trials. In contrast to findings in adults, LI was resistant to disruption by amphetamine at a dose (1 mg/kg) that significantly increased locomotor activity, as well as by reducing the number of preexposures to ten, increasing the number of conditioning trials to five, or changing the context between preexposure and conditioning. Clozapine (5 mg/kg) and the selective 5HT2A antagonist M100907 (0.3 mg/kg) administered in conditioning were without an effect on "persistent" LI with extended conditioning, but were capable of disrupting LI when administered in the preexposure stage, as found in adults. CONCLUSION: The results point to functionality within brain systems regulating LI acquisition but not those regulating LI expression in periadolescent rats, further suggesting that postpubertal maturation of the latter systems may underlie schizophrenia-mimicking LI disruption reported in adult rats following perinatal manipulations and possibly disrupted LI observed in schizophrenia.

Immune responses to therapeutic proteins in humans--clinical significance, assessment and prediction.

There is a large and increasing number of therapeutic proteins approved for clinical use and many more undergoing preclinical studies and clinical trials in humans. Most of them are human or 'humanized' recombinant molecules. Virtually all therapeutic proteins elicit some level of antibody response, which in some cases, can lead to potentially serious side effects. Therefore, immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess immunogenicity of these molecules, appropriate detection, quantitation and characterization of antibody responses are necessary. Immune responses to therapeutic proteins in conventional animal models has not been, except in rare cases, predictive of the response in humans. In recent years there has been a considerable progress in development of computational methods for prediction of epitopes in protein molecules that have the potential to induce an immune response in a recipient. Initial attempts to apply such tools in early development of therapeutic proteins have already been reported. It is expected that computer driven prediction followed by in vitro and/or in vivo testing of any potentially immunogenic epitopes will help in avoiding, or at least minimizing, immune responses to therapeutic proteins.

Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5.

Studies of CD5-deficient mice indicate that the transmembrane glycoprotein CD5 negatively regulates antigen receptor-mediated signals in thymocytes, lymph node T cells and B1a cells. CD5 contains four tyrosine residues in its cytoplasmic domain and is phosphorylated on tyrosine residues following antigen receptor ligation. Recently it has been proposed that CD5 function is dependent on the recruitment of the tyrosine phosphatase SHP-1 to tyrosine-phosphorylated CD5 and subsequent dephosphorylation of signaling molecules. In this study we investigated the requirements for, and sites of, CD5 tyrosine phosphorylation. Using a T cell line deficient in the tyrosine kinase p56(lck) and the same cell line reconstituted with this kinase, we show that p56(lck) expression is required for efficient CD5 tyrosine phosphorylation. Using tyrosine-phosphorylated peptides corresponding to CD5 cytoplasmic sequences we also show that the Src homology 2 (SH2) domain of p56(lck) binds prominently to pY429SQP, with 30-fold less affinity to pY463DLQ and not to pY441PAL. A number of murine CD5 Y --> F and deletion mutants were expressed in Jurkat T cells. The Y441F mutant was tyrosine phosphorylated at levels comparable to wild-type, but the Y429F and Y463F mutants were phosphorylated at lower levels. Two deletion mutants, which contain only one tyrosine residue (Y378) located at the interface of the transmembrane and cytoplasmic domains, were not tyrosine phosphorylated, suggesting that Y378 is not readily available for phosphorylation. Taken together these results suggest that both Y429 and Y463 can recruit p56(lck), and that these residues are the only prominent sites for CD5 tyrosine phosphorylation.

Negative regulation of CD4 lineage development and responses by CD5.

CD5 deficiency results in a hyper-responsive phenotype to Ag receptor stimulation. Here we show that the development and responses of CD4 lineage T cells are regulated by the function of CD5. Thymocytes expressing the I-Ad-restricted DO11.10 TCR undergo abnormal selection without CD5. In H-2d mice, the absence of CD5 causes deletion of double-positive thymocytes, but allows for efficient selection of cells expressing high levels of the DO11.10 clonotype. By contrast, there is enhanced negative selection against the DO11.10 clonotype in the presence of I-Ab. T cell hybridomas and DO11.10 T cells are more responsive to TCR stimulation in the absence of CD5. Such hypersensitivity can be eliminated by expression of wild-type CD5, but not by a form of CD5 that lacks the cytoplasmic tail. Finally, CD5 deficiency partially suppresses the block of CD4 lineage development in CD4-deficient mice. Taken together, the data support a general role for CD5 as a negative regulator of Ag receptor signaling in the development and immune responses of CD4 lineage T cells.

Antiphospholipid antibodies, proteins C and S, and coagulation changes in sickle cell disease.

The significance, interactions, and sources of coagulation abnormalities and their relationship to clinical severity and painful episodes in sickle cell disease are not clear. To evaluate this, we have examined various measures of coagulation in 37 patients with sickle cell disease (20 patients with HbSS disease and 17 patients with HbSC disease). Measurements have included isotypes of antiphospholipid antibodies (IgG, IgM, IgA) to specific phospholipids; proteins C (activity, total antigen) and S (activity, total and free antigen); measures of coagulation activation (prothrombin fragment 1.2, thrombin-antithrombin, fibrinopeptide A, d-dimers); indicators of clinical severity; and studies obtained during steady states and painful episodes. Results in HbSS disease showed that antiphospholipid antibodies were increased, with IgG phosphatidylserine showing the highest and most frequently increased levels (37% of patients). Protein C (activity) and protein S (activity, total, free antigen) were decreased (P<.01), and all measures of coagulation activation were increased (P<.001). In HbSC disease, antiphospholipid antibodies were normal, protein C (activity) and protein S (free antigen) were decreased (P<.001), and all measures of coagulation activation were increased (P<.02). A strong correlation was observed in HbSS disease between IgG-PS and d-dimers. Moderate correlations occurred between protein C activity and thrombin-antithrombin and fibrinopeptide A, between protein S activity and prothrombin fragment 1.2 and d-dimers, and between protein C and protein S activity. In HbSC disease, moderate and fewer correlations occurred. Significant differences between HbSS disease and HbSC disease were observed in aPLs, proteins C and S, and measures of coagulation activation. Measurements during steady states and during painful episodes were not significantly different. We conclude that the antiphospholipid antibody IgG-PS may contribute to coagulation activation in HbSS disease and that IgG-PS, protein C, and protein S relate to each other and jointly to measures of coagulation activation. The increased level of IgG-PS in HbSS disease most likely reflects exposure of the procoagulant phosphatidylserine on the surfaces of red cell-shed vesicles and sickle red cells, which would further affect coagulation activation. The significant differences in coagulation measures between HbSS disease and HbSC disease are consistent with differences in clinical severity between the diseases. The development of painful episodes does not appear to be related to the coagulation changes.

Functional consequences of costimulation by ICAM-1 on IL-2 gene expression and T cell activation.

LFA-1 is a well-recognized adhesion molecule, but its role in providing costimulatory signals to T cells has remained controversial. We have compared the ability of class II-positive transfectants that do and do not coexpress ICAM-1 (ProAd and ProAd-ICAM) to activate Ag-specific Th1 clones and naive CD4-positive T cells isolated from TCR transgenic mice. Ag presentation by ProAd to Th1 clones can induce calcium-dependent signaling events after engagement of the TCR, as evidenced by the nuclear localization of the transcription factors NF-AT and NF-kappaB. Nevertheless, coexpression of ICAM-1 or B7-1 on ProAd is required to induce detectable levels of IL-2 gene expression in either Th1 clones or naive T cells. In Th1 clones, activation by ProAd-ICAM induces very transient IL-2 mRNA expression that does not result in detectable IL-2 secretion or T cell proliferation. In naive T cells, the duration of IL-2 mRNA expression is longer, allowing for a transient burst of IL-2 protein that is sufficient to drive the cells into the cell cycle. In spite of this initial response, Ag presentation by ProAd-ICAM is a tolerogenic signal to naive T cells, and responding T cells undergo apoptosis 4 to 5 days poststimulation. These data suggest that engagement of LFA-1 can provide sufficient costimulatory signals to induce T cell activation and IL-2 gene expression, but cannot protect against anergy induction or provide for T cell survival.

Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies.

Antigen-specific T cell activation depends initially on the interaction of the T cell receptor (TCR) with peptide/MHC. In addition, a costimulatory signal, mediated by distinct cell surface accessory molecules, is required for complete T cell activation leading to lymphokine production and proliferation. CD28 has been implicated as the major receptor on T cells responsible for delivering the costimulatory signal. Although two distinct ligands for CD28, B7-1 and B7-2, have been identified on antigen-presenting cells (APC), the co-stimulatory role of each molecule during a physiological immune response remains unresolved. In the present study, the relative roles of B7-1 and B7-2 interactions were evaluated in an allogeneic pancreatic islet transplant setting. In isolation, anti-B7-2 mAbs and, to a much lesser degree, anti-B7-1 mAbs suppressed T cell proliferative responses to allogeneic islets or splenic APC in vitro. Maximal inhibition of the allogeneic response was observed using a combination of the anti-B7-1 and anti-B7-2 mAbs. Administration of anti-B7-2 but not anti-B7-1 mAbs prolonged C3H allograft survival in B6 recipients, with a combination of both mAbs significantly prolonging rejection beyond either mAb alone. The immunosuppressive effects of the in vivo mAb treatment were not manifested in in vitro analyses as T cells isolated from suppressed mice responded normally to allogeneic stimuli in terms of both proliferation and lymphokine production. However, combined mAb therapy in vivo selectively delayed CD4+ T lymphocyte infiltration into the graft. These data suggest that both B7-1 and B7-2 costimulatory molecules are active in vivo, although B7-2 plays a clearly dominant role in this allograft model. The mechanism of immune suppression in vivo remains unresolved but may occur at sites distinct from the allograft.

Identification of a unique costimulatory activity for murine T helper 1 T cell clones.

We have examined the ability of several class II-positive tumor cell transfectants to stimulate murine Th1 clones. Most of the transfectants failed to activate the Th1 clones and, in fact, induced Ag-specific anergy. However, we found that one tumor, a UV-induced fibrosarcoma (6130-VAR1), was capable of stimulating both cytokine production and proliferation in Th1 clones. We believe that 6130-VAR1 cells possess a unique costimulatory activity for the following reasons. First, these cells fail to express known costimulatory molecules including B7-1 and B7-2. Second, 6130-VAR1-mediated stimulation of Th1 clones was not blocked by anti-CD28 Fab or by CTLA4Ig, which suggests that members of the B7 family were not up-regulated during the course of stimulation and that activation does not occur via a CD28-dependent pathway. Third, 6130-VAR1 could provide costimulation when presented on a different surface than the class II/peptide ligand for the TCR. This last finding suggested that the activity on these cells was not simply an adhesion molecule that facilitated increased efficiency of T cell:MHC interactions. Finally, like B7-1 transfectants, stimulation by class II-positive 6130-VAR1 cells prevented the induction of anergy in the Th1 clones. Taken together, these results strongly suggest that 6130-VAR1 expresses a unique costimulatory activity (VAM-1) that, like B7-1, can promote T cell activation and prevent anergy induction.

Expression and functional significance of an additional ligand for CTLA-4.

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the T-cell receptor complex in concert with one or more costimulatory molecules. Recent studies have suggested that the B7 molecule, expressed on most antigen presenting cells, functions as a costimulatory molecule through its interaction with CD28 on T cells. Blocking the CD28/B7 interaction with CTLA4Ig inhibits T-cell activation in vitro and induces unresponsiveness. We demonstrate that another molecule(s), termed B7-2, is expressed constitutively on dendritic cells, is differentially regulated on B cells, and costimulates naive T cells responding to alloantigen. B7-2 is up-regulated by lipopolysaccharide in < 6 hr and is maximally expressed on the majority of B cells by 24 hr. In contrast, B7 is detected only on a subset of activated B cells late (48 hr) after stimulation. In addition, Con A directly induces B7-2 but not B7 expression on B cells. Finally, although both anti-B7 monoclonal antibodies and CTLA4Ig blocked T-cell proliferation to antigen-expressing B7 transfectants, only CTLA4Ig had any significant inhibitory effect on T-cell proliferation to antigens expressed on natural antigen presenting cells, such as dendritic cells. Thus, B7 is not the only costimulatory molecule capable of initiating T-cell responses since a second ligand, B7-2, can provide a necessary second signal for T-cell activation.

The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells.

Previous studies demonstrated that a human pre-B acute lymphoblastic leukemia cell line, NALM-6, failed to stimulate a primary MLR, despite expression of class II MHC and adhesion molecules. Here we demonstrate that this is the result of the fact that NALM-6 cells do not express the ligand for CD28, namely B7. NALM-6 transfectants that expressed high levels of B7 gained the capacity to stimulate IL-2 production by class II MHC molecule-specific alloreactive T cells and to costimulate a polyclonal population of purified T cells cultured with immobilized anti-CD3 mAb. In the presence of PMA, NALM-6 cells transfected with B7 polyclonally stimulated T cells in a cyclosporine A-resistant fashion, a property previously attributed only to agonistic anti-CD28 mAb. The gain of these functions could not be explained solely by an increased capacity of the transfectants to form conjugates with T cells, suggesting that the CD28/B7 interaction transduces a costimulatory signal in T cells.

[Bases for a program designed to promote breast-feeding. I. Identification of variables in the immediate post-partum period]. / Bases para la formulacion de un programa de promocion a la lactancia. I. Identificacion de variables en el posparto inmediato

PIP: The variables involved in the duration of breastfeeding were analyzed in a group of 60 mothers in Mexico who were followed from delivery until 6 months postpartum. 2 groups were formed: women without any previous breastfeeding experience (primipara) and women with previous experience (multipara). The analysis was performed by step wise regression, grouping the variables in 2 classification systems. The 1st, which was according to temporal proximity to breastfeeding practice, divided the variables into predisposing prenatal, early, and late neonatal factors. The 2nd, according to the level of organization, used the following categories: social, biological, and behavioral motivation. The results show that both groups of mothers base their breastfeeding on the confidence that they have in their own breastfeeding capacity. For the primiparal, this confidence is expressed by age, the advantages they found in nonhuman milk, their milk production, the difficulty they had in establishing breastfeeding during the 1st days, and their anticipation of future breastfeeding problems. The multiparal base their confidence on their prior experience in addition to the previously mentioned factors. The multivariate regression for the primipara explained 98.41% of the variance. Of the 3 significant variables, early postpartum contact accounted for 74.43%. In the case of multiparal, 66.70% of the variance was explained by variables. Of these, the starting age of supplementation was negatively related and explained 47.14% of the variance.^ieng

A comparison of life events scoring methods as predictors of psychological symptomatology.

Seven scoring methods for the Life Events Survey (LES) were compared to determine which, if any, is superior for prediction of psychological symptomatology as measured by the Brief Symptom Inventory (BSI). Every scoring method tested, except one utilizing an individual's positive ratings of events, was significantly correlated with symptomatology. The method using an individual's negative ratings of events was a significantly better predictor than any other. These findings suggest several conclusions. First, nomothetic methods for weighting life events do not increase a scale's predictive ability beyond that achieved by a frequency count of events. Second, frequency of life events predicts psychological symptomatology only insofar as life events are perceived as negative. That is, positively perceived events do not predict symptomatology. Finally, a life events scale's predictive ability is increased by utilizing the individual's negative perceptions of events.

9-Deazaadenosine--a new potent antitumor agent.

-Deazaadenosine (9-DAA), a novel purine analog, was found to be a potent inhibitor of the growth of nine different human solid tumor cell lines in vitro and of pancreatic carcinoma (DAN) in antithymocyte serum (ATS)-immunosuppressed mice. In culture, IC50 values ranged from 1.1 to 8.5 X 10(-8)M. Ovarian carcinoma (MR) was the only cell line in which the activity of 9-DAA was potentiated (about 10-fold) by pretreatment with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). After incubation of cultured pancreatic DAN cells with 9-DAA (10(-5)M) for 2 hr, a peak appeared in the triphosphate region of HPLC nucleotide profiles that was identified tentatively as 9-deazaATP. Under the same incubation conditions, the incorporation of [3H]uridine into RNA and of [3H]thymidine into DNA was inhibited by 34 and 80% respectively. In vivo studies using ATS-immunosuppressed mice showed that 9-DAA at 0.4 mg/kg/day for 3 consecutive days reduced pancreatic carcinoma (DAN) tumor weights to approximately 50% of untreated controls. The nucleoside transport inhibitor p-nitrobenzyl-6-thioinosine (NBMPR) was shown to selectively protect host tissues from 9-DAA toxicity and, thereby, potentiated the antitumor activity of 9-DAA in vivo at optimal dosages.

The effects of prostacyclin on the coagulation of whole blood.

The effects of prostacyclin (PGI2) on mechanical properties of forming clots were investigated by testing human blood samples on a Thrombelastograph. Concentrations greater than 50 ng/ml (blood) caused a biphasic development of clot stiffness. During the first phase, PGI2 partially inhibited the platelet involvement in coagulation causing initial clot formation at a normal time but with reduced clot stiffness. The second phase occurred after neutralization of PGI2 activity and was characterized by recovery of platelet activity to produce a final clot with normal shear modulus. The duration of the inhibitory effects depended on PGI2 concentration and hematocrit. With a normal hematocrit, a PGI2 concentration of 60 ng/ml caused an inhibition for about 40 min whereas a concentration of 100 ng/ml caused inhibition for about 75 min.

Breast carcinoma: expected results with prevailing therapy.

Computer-stored tumor registry data were scanned to produce the set of patients whose breast carcinoma was either diagnosed or first treated at Evanston Hospital between the years 1973 and 1977, inclusive. Of 560 evaluable patients, 184 were less than or equal to age 50 (LE 50), and 376 were greater than age 50 (GT 50). Comparisons of projected survival show that the survival of all patients is 8% at 5 years and 72% at 7 years. The survival of patients LE 50 is nearly identical to that of the GT 50 age group. Survival of Stage III GT 50 patients, however, is better than Stage III LE 50 (P less than 0.001). Comparison with earlier studies shows an historical trend toward better 5-year survival. Data were generated in this study which allow prediction of prognosis based upon age and stage at diagnosis.