Reduced folate carrier (RFC) transcripts in human leukemias were measured by a competitive PCR assay. Total RNAs were reverse transcribed and amplified in the presence of competitive templates for ...RFC and beta-actin. RFC transcripts were normalized to transcripts for beta-actin. In a series of K562 sublines, a approximately 30-fold range of RFC transcripts measured by PCR assay closely agreed with results of Northern analysis and varied in proportion to RFC protein on Western blots and 3Hmethotrexate transport. RFC transcripts varied over a 88-fold range in 49 specimens from 48 children with acute lymphoblastic leukemia (ALL). Median RFC transcripts were similar for 15 T-cell and 33 B-precursor ALL samples (RFC/beta-actin = 6.13 x 10(-3) and 7.92 x 10(-3), respectively) and for 41 diagnostic (7.20 x 10(-3)) and 8 relapse (5.58 x 10(-3)) samples. Whereas PCR measurements of RFC transcripts approximated changes in methotrexate transport in B-precursor ALL blasts (n = 10), for T-ALL blasts (n = 12) there was no apparent relationship between these parameters. For hyperdiploid B-precursor blasts (n = 11) with greater than 52 chromosomes and three to five copies of chromosome 21, the median RFC transcript level was approximately 3-fold higher than that for diploid B-precursor blasts. RFC transcripts were also elevated for two of three B-precursor specimens with acquired trisomy 21. Our results suggest that RFC gene expression is far more predictive of methotrexate uptake capacity in B-precursor than T-ALL and that increased copies of chromosome 21 in B-precursor ALL blasts are generally associated with increased RFC transcripts. Hence, the good prognosis for children with hyperdiploid B-precursor ALL treated with antimetabolite-based chemotherapy and the high levels of methotrexate and methotrexate polyglutamates accumulated may, in part, reflect elevated RFC gene expression and capacities for methotrexate transport.
Hemgn (a gene symbol for hemogen in mouse, EDAG in human and RP59 in rat) encodes a nuclear protein that is highly expressed in hematopoietic tissues and acute leukemia. To characterize its ...regulatory mechanisms, we examined the activities of a Hemgn promoter containing 2975 bp of 5' flanking sequence and 196 bp of 5' untranslated region (5' UTR) sequence both in vitro and in vivo: this promoter is preferentially activated in a hematopoietic cell line, not in nonhematopoietic cell lines, and is sufficient to drive the transcription of a lacZ transgene in hematopoietic tissues in transgenic mice. Mutagenesis analyses showed that the 5' UTR including two highly conserved GATA boxes is critical for the promoter activity. GATA1, not GATA2, binds to the GATA binding sites and transactivates the Hemgn promoter in a dose-dependent manner. Furthermore, the expression of human hemogen (EDAG) transcripts were closely correlated with levels of GATA1 transcripts in primary acute myeloid leukemia specimens. This study suggests that the Hemgn promoter contains critical regulatory elements for its transcription in hematopoietic tissues and Hemgn is a direct target of GATA1 in leukemia cells.
IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to ...induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation.
Test whether a martial arts-based therapy, Kids Kicking Cancer (KKC), can reduce pain and emotional distress in children with cancer, other chronic health conditions (e.g., sickle cell), and healthy ...siblings.
This study surveyed children's pain and distress levels immediately before and after a 1-hr in-person KKC class. Eligible participants were enrolled in standard KKC classes, were diagnosed with a chronic health condition (e.g., cancer, sickle cell) or were the sibling of a child diagnosed and were between the ages of 5-17 years (inclusive). Children reported on their pain and distress using Likert-style scales (Coloured Analog Scale and modified FACES scale, respectively). Friedman test was used to test for overall changes in pain and distress, and within subgroups. Age and sex effects were evaluated using Spearman's rank-order correlation. Additional Yes/No questions were administered regarding KKC satisfaction and use of techniques.
Fifty-nine youth (19 cancer patients, 17 non-cancer patients, 23 siblings; 5-17 yrs, 26 females) completed this study. Overall, there was a significant reduction in pain (
= 0.033) and emotional distress (
< 0.001) after a 1-hr class, with 50% and 89% of youth reporting a reduction in pain and distress, respectively. On average, pain levels remained within the mild/moderate range on average (i.e., pre vs. post levels; pre: M = 1.67, post: M = 1.33) and emotional distress went from mild/moderate to none/mild distress, on average (pre: M = 1.92, post: M = 1.08). Youth with higher pre-class pain and distress reported greater reductions (
= 0.001 and
< 0.001, respectively). The reduction in pain appeared to be most pronounced with cancer and non-cancer patients. In contrast, the reduction in distress appeared to be most pronounced among healthy siblings. However, overall, reductions in pain and distress did not significantly differ among subgroups (i.e., cancer patients, non-cancer patients, siblings), and change in pain and distress was not associated with age or sex. Ninety-six percent of youth would recommend KKC to others and 81% reported using KKC techniques (e.g., the Breath Brake
or other martial arts techniques) outside of class, such as at home.
Results support the more widespread application of KKC as a psychosocial intervention for reducing pain and distress in various pediatric populations.
With contributions from the leading experts in the field, Pediatric Plastic and Reconstructive Surgery for Primary Care provides primary care pediatricians and other professionals who care for ...children comprehensive coverage of both common and rare congenital and acquired pediatric conditions.
Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the ...human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.
While chemokines primarily promote chemotaxis, it is apparent that these cytokines also modulate a number of other biologic activities, including adhesion, degranulation, cytotoxicity, and enzyme ...release. We demonstrate here that the beta chemokines, recombinant human macrophage inflammatory protein-1 alpha and -1 beta, RANTES (regulated upon activation, normally T cell expressed and secreted), and macrophage chemotactic peptide-1, are capable of directly costimulating purified human T cell and human T cell clone proliferation and IL-2 production in the presence of anti-CD3 mAb, but not phorbol esters, in vitro. This costimulatory activity was dose and donor dependent and required the presence of free extracellular calcium as well as endogenously produced IL-2. Chemokine treatment of human T cells in vitro increased the level of cell surface CD25 and soluble CD25. In addition, these chemokines enhanced both Ag- and alloantigen-specific T cell and T cell clone proliferation. This activity was further augmented in the presence of the CD28 ligand, B7-1. Neutralization analyses using chemokine-specific Abs revealed that endogenously produced chemokines are important costimulatory mediators in human T cell activation. Together, these results suggest that chemokines not only play an important role in lymphocyte recruitment to inflammatory sites, but also participate in T cell activation.
A deficiency in understanding the steps responsible for colitis is the lack of comprehension for the role chemokines play in mucosal inflammation. IFN-gamma-inducible protein-10 (IP-10) and CXCR3 are ...highly expressed at sites of colitis. Our findings show that IP-10 significantly contributes to the development of Th1 and inflammatory responses. Specifically, IP-10 inhibition in IL-10(-/-) mice attenuates the associated increases in serum and/or local amyloid A, IL-2, IL-6, TNF-alpha, IFN-gamma, IL-1alpha, and IL-1beta with colitis as compared with IL-10(-/-) mice that develop colitis similar to human Crohn's disease. Correspondingly, the rate or intensity of inflammation in IL-10(-/-) mice treated with anti-IP-10 Abs showed improved scoring of inflammation, compared with control IL-10(-/-) mice. This study provides important and novel information regarding IP-10 as a target for the treatment of colitis.
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