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Biomedical subjects

M D Fleming

Publications and source records attributed to M D Fleming.

At least 19 recordsLinked to original sources
PubMed1999

The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.

Targeted mutagenesis was used to produce two mutations in the murine hemochromatosis gene (Hfe) locus. The first mutation deletes a large portion of the coding sequence, generating a null allele. The second mutation introduces a missense mutation (C282Y) into the Hfe locus, but otherwise leaves the gene intact. This mutation is identical to the disease-causing mutation in patients with hereditary hemochromatosis. Mice carrying each of the two mutations were bred and analyzed. Homozygosity for either mutation results in postnatal iron loading. The effects of the null mutation are more severe than the effects of the C282Y mutation. Mice heterozygous for either mutation accumulate more iron than normal controls. Interestingly, although liver iron stores are greatly increased, splenic iron is decreased. We conclude that the C282Y mutation does not result in a null allele.

Alleles↗
PubMed1999

Molecular insights into mechanisms of iron transport.

The past 3 years have witnessed extraordinary progress in our understanding of mammalian iron transport and homeostasis. The first transmembrane iron transporter has been found. Mutations in this protein, in two animal models with iron-transport defects, have helped to define the roles of this protein in vivo. The gene defective in patients with hereditary hemochromatosis has been identified, and much has been learned about the structure and function of its gene product. Finally, our ability to make a molecular diagnosis of hereditary hemochromatosis has called attention to new iron-loading disorders, including African iron overload and juvenile hemochromatosis.

Animals↗
PubMed1999

Iron transport across biologic membranes.

Iron is essential for life, but is toxic in excess. Nearly all organisms have therefore developed regulated mechanisms for efficient transport of iron into cells. This paper reviews the current understanding of iron transport, focusing on valuable lessons from studies of yeast iron transport and the discovery of the first mammalian transmembrane iron transporter.

Animals↗
PubMed1998

The G185R mutation disrupts function of the iron transporter Nramp2.

Microcytic anemia (mk) mice and Belgrade (b) rats have severe iron deficiency anemia due to defects in intestinal iron transport and erythroid iron utilization. Both animal mutants carry the same missense mutation in Nramp2, the first mammalian iron transporter to be identified. This mutation, in which glycine 185 is changed to arginine (G185R), occurs within predicted transmembrane domain 4 of the protein. We have performed site-directed mutagenesis of murine Nramp2, focusing on amino acids of transmembrane domain 4 that are highly conserved among Nramp-like proteins. We have expressed each mutant form in transfected cells and examined iron transport function, subcellular localization, and protein amounts. All tested forms of Nramp2 localize to the plasma membrane and to transferrin-containing endosomes. Most transmembrane domain 4 mutations affect the amount of protein detected and consequently show diminished iron transport. The G185R mutation, however, causes near total loss of Nramp2 function that cannot be fully explained by a decreased amount of protein, indicating that G185R disrupts iron transport through an alteration in the function of Nramp2, rather than degradation of the protein.

Amino Acid Substitution↗
PubMed1998

Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport.

The Belgrade (b) rat has an autosomal recessively inherited, microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake and gastrointestinal iron absorption. The b reticulocyte defect appears to be failure of iron transport out of endosomes within the transferrin cycle. Aspects of this phenotype are similar to those reported for the microcytic anemia (mk) mutation in the mouse. Recently, mk has been attributed to a missense mutation in the gene encoding the putative iron transporter protein Nramp2. To investigate the possibility that Nramp2 was also mutated in the b rat, we established linkage of the phenotype to the centromeric portion of rat chromosome 7. This region exhibits synteny to the chromosomal location of Nramp2 in the mouse. A polymorphism within the rat Nramp2 gene cosegregated with the b phenotype. A glycine-to-arginine missense mutation (G185R) was present in the b Nramp2 gene, but not in the normal allele. Strikingly, this amino acid alteration is the same as that seen in the mk mouse. Functional studies of the protein encoded by the b allele of rat Nramp2 demonstrated that the mutation disrupted iron transport. These results confirm the hypothesis that Nramp2 is the protein defective in the Belgrade rat and raise the possibility that the phenotype shared by mk and b animals is unique to the G185R mutation. Furthermore, the phenotypic characteristics of these animals indicate that Nramp2 is essential both for normal intestinal iron absorption and for transport of iron out of the transferrin cycle endosome.

Alleles↗
PubMed1998

Mammalian iron transport: an unexpected link between metal homeostasis and host defense.

Iron deficiency and iron overload disorders are common in clinical practice. Both can result from perturbations in the flux of iron across the absorptive intestinal enterocyte. Until recently iron transport has been poorly understood. In 1997 two independent cloning strategies identified Nramp2 (DCT1) as the first mammalian transmembrane iron transporter. In this review we discuss evidence that Nramp-related proteins play essential roles in metal homeostasis and host defense.

Animals↗
PubMed1998

p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.

We describe the cloning of p63, a gene at chromosome 3q27-29 that bears strong homology to the tumor suppressor p53 and to the related gene, p73. p63 was detected in a variety of human and mouse tissues, including proliferating basal cells of epithelial layers in the epidermis, cervix, urothelium, and prostate. Unlike p53, the p63 gene encodes multiple isotypes with remarkably divergent abilities to transactivate p53 reporter genes and induce apoptosis. Importantly, the predominant p63 isotypes in many epithelial tissues lack an acidic N terminus corresponding to the transactivation domain of p53. We demonstrate that these truncated p63 variants can act as dominant-negative agents toward transactivation by p53 and p63, and we suggest the possibility of physiological interactions among members of the p53 family.

Amino Acid Sequence↗
PubMed1998

Splenic pathology in myelodysplasia: a report of 13 cases with clinical correlation.

Splenomegaly is uncommon in myelodysplasia (MDS) and, although cytopenias may be severe, therapeutic splenectomy is rarely performed. We report the histologic, histochemical, and immunophenotypic findings of nine cases of surgical splenectomy and four postmortem spleens from MDS patients. Four histologic patterns were identified: one dominated by erythrophagocytosis, one characterized by red pulp plasmacytosis, one with extramedullary hematopoiesis as the only salient finding, and one with marked red pulp expansion caused by a monocytic proliferation. Wright-Giemsa and histochemical stains were performed on touch preparations in three cases and played a critical role in the precise subclassification of one MDS patient's hematologic disorder, which ultimately proved to be chronic myelomonocytic leukemia. Splenectomy led to sustained improvement of cytopenias in three cases, but did not eliminate transfusion dependence for the remaining patients. Three splenectomy cases exhibited clustered Leder-negative mononuclear elements: two of these patients experienced disease progression to refractory anemia with excess blasts in transformation or acute myelogenous leukemia during post-splenectomy follow-up, whereas none of the three splenectomy patients without clustered mononuclear elements did. We conclude that splenomegaly in MDS usually reflects the sequelae of dyspoiesis rather than evidence of a proliferative phase, that clustering of Leder-negative large cells may correlate with either a substantial monocytic component or, possibly, increased risk of disease progression, and that the spleen can provide diagnostic as well as prognostic information in MDS patients with splenomegaly.

Adult↗
PubMed1997

Angiosarcoma.

Explore the source record for details and available documents.

Adult↗
PubMed1996

Long-term results of breast conservation therapy for breast cancer.

OBJECTIVE: This study was done to determine the long-term outcome of breast conservation therapy (BCT) for patients with early-stage breast cancer during a period of treatment evolution at a single institution. SUMMARY BACKGROUND DATA: Breast cancer treatment has evolved from extensive surgical extirpation of the breast to treatment options that conserve the breast. Prospective and retrospective studies have confirmed the efficacy of BCT and justify its use for many patients with early breast cancer, but there is no universally accepted consensus as to who benefits from more aggressive application of surgery or radiotherapy in BCT. Prognostic variables for breast cancer and information on factors that contribute to local recurrence help predict BCT results. Continued analysis of BCT still is necessary to improve patient outcome. METHODS: Eighty-five patients treated with BCT (lumpectomy with adjuvant radiation therapy) at the Medical College of Virginia from 1980 to 1990 were identified. Clinicopathologic parameters and treatment details were analyzed for relationship to development of local recurrence, distant metastasis, and survival. Fisher's exact test was used for comparisons. Actuarial survival curves were plotted. The earlier treatment period (1980-1985) was compared with the later treatment period (1985-1990). RESULTS: Median follow-up was 5 years. Actuarial overall survival was 83% at 5 years (69% at 10 years), and 5-year distant metastasis-free survival was 79%. The 5-year actuarial local recurrence rate was 6.6% (crude rate 10.6%, 9/85). Young patients (age < 40 years) were found to be at increased risk for local recurrence (24% < 40 years vs. 6% > or = 40 years, p < 0.05). Tumor margins < or = 3 mm were more frequently found, and lumpectomy site radiation boost was used increasingly from 1986 to 1990. Almost half of all local recurrences occurred after 5 years. CONCLUSIONS: Survival and local recurrence rates were comparable to other series. Young patients were found to be at increased risk for local recurrence. Negative microscopic margins, even when close, can provide low local recurrence rates when adjuvant radiation therapy is administered.

Adult↗
PubMed1995

Principles of management of basal and squamous cell carcinoma of the skin.

There are a number of options available to the physician for the primary management of basal cell and squamous cell carcinoma of the skin. The most commonly used treatment modalities are primary resection, radiation therapy, and Mohs' micrographic surgery. Although each of these modalities can be used to treat most skin cancers in a variety of settings, each of these forms of cancer treatment is more appropriate in some settings and less appropriate in others. The role of each mode of therapy is discussed, based on effectiveness for cure, short and long term side effects, and cost of treatment. The appropriate role of the physician treating skin carcinoma is to choose the most effective treatment based on the above criteria and not to make the tumor fit any particular treatment modality.

Basal Cell Carcinoma↗
PubMed1995

Adoptive transfer of bryostatin-activated tumor-sensitized lymphocytes prevents or destroys tumor metastases without expansion in vitro.

Because the requirement for long-term cell culture can make adoptive cellular immunotherapy cumbersome, experiments were designed to determine whether smaller numbers of tumor-sensitized T cells activated briefly with bryostatin 1 and ionomycin (B/I) could be returned immediately to recipient mice without in vitro expansion and still have an anti-tumor effect in vivo. Popliteal tumor-draining lymph nodes (DLNs) from mice bearing progressive MCA-105 and MCA-203 footpad sarcomas were harvested and treated for 18 h with B/I. These cells were then washed and transferred immediately to naive C57B1/6 mice. In some experiments, these mice were irradiated (500 rads) before adoptive transfer and were given interleukin-2 (IL-2, 7,500 IU i.p., b.i.d. for 3 days) after receiving the activated lymphocytes. Recipient mice were challenged with sarcoma cells (4 x 10(5) i.v.) 6 to 32 days after receiving the activated lymphocytes. Mice receiving 10(6) B/I-activated lymphocytes before tumor challenge had significantly fewer metastases than did controls. This protective effect did not require exogenous IL-2 or host irradiation. Using Thy-1 congenic donors, it was shown that B/I-activated T cells expanded in recipients when IL-2 was also given, and these cells were a prominent component (15% of total cells) in the infiltrates found in the lungs of mice 7 days after i.v. tumor challenge. Combining these B/I-"pulsed" cells with cyclophosphamide (CYP) and IL-2 to treat mice with established (3-day) metastases resulted in significant reduction in pulmonary nodules, with complete regression in many of the treated mice, which was rarely seen with CYP alone or with CYP + IL-2. Thus, adoptive transfer of tumor-sensitized, B/I-activated DLN cells confers protection against i.v. tumor challenge, without prior in vitro expansion of the effector cells. Phenotyping studies demonstrate that donor cells activated with B/I do expand in recipient mice after adoptive transfer and can move to sites of tumor. Moreover, these cells can mediate a therapeutic effect on established tumor metastases, when combined with chemotherapy.

Adjuvants, Immunologic↗
PubMed1994

Breast cancer in elderly women.

With the aging of the American population, more older women are being diagnosed with breast cancer. With the possible exception of women under 40 years of age, there is no clear evidence of a biologic or clinical difference between breast cancer in younger and older aged groups. Breast cancer is diagnosed at a more advanced stage in older women. Elderly women with breast cancer frequently are treated with less than standard therapy and are less often included in clinical trials. With the exception of specific comorbid conditions that preclude anesthesia and surgery, older women tolerate breast surgery as well as younger women. The results of good surgical and adjuvant therapy for elderly women are as good as those for younger women. Older women with breast cancer deserve the most effective screening, diagnosis, and surgical treatment available.

Adult↗
PubMed1994

Precursor frequency analysis of bryostatin activated lymphocytes.

Bryostatin 1 (Bryo) is a potent activator of protein kinase C. When T cells are stimulated with Bryo and the calcium ionophore ionomycin (Io), they expand rapidly in low-dose IL-2 (20 U/ml). We have shown that Bryo/Io-activated T-cells from murine tumor-draining lymph nodes have striking antigen-specific antitumor efficacy in vivo. To account for the specificity despite using a nonspecific T cell activation method, it was postulated that the Bryo/Io combination might preferentially activate antigen-sensitized T cells. To test this hypothesis, an allogeneic response model was used. C57BL/6 mice were primed by intraperitoneal (ip) injection with DBA/2 mouse splenocytes. After 9 days, the C57BL/6 spleens were harvested and H-2d-specific cytolytic T lymphocyte (CTL) precursor frequency (PF) was determined by limiting dilution analysis (LDA). A portion of the same spleen cells was treated for 18 hr with Bryo/Io and expanded for 7 days in IL-2 (20 U/ml); the LDA was then repeated to analyze PF after expansion. The entire experiment was also done with responder and stimulator strains reversed (DBA/2 mice immunized with C57BL/6 cells). The resulting PF values were [table: see text] Normal spleen cells treated with Bryo/Io exhibited < 10% release vs the same targets at any effector:target ratio, ruling out nonspecific cytolysis after Bryo/Io. T lymphocytes or CD8+ T cells were selected from primed splenocytes and the PF values before and after Bryo/Io were analyzed. These data showed that the increase in PF after expansion could not be attributed to T cell or CD8+ cell enrichment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression.

BACKGROUND: Lymphocytes obtained from tumor-draining lymph nodes (DLN) can have potent in vivo antitumor activity after in vitro activation with bryostatin 1 and ionomycin. However, the presence of visceral metastases in the donor can inhibit the effectiveness of such lymphocytes. In the present study, we tested the ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression in a murine model. METHODS: Mice were injected with MCA-105 sarcoma cells in the footpad alone or in the footpad and the tail vein to establish lung metastases. Cyclophosphamide was given i.p. 1 day before harvesting the draining popliteal lymph nodes. For all donor groups, DLN cells were activated with 5 nM bryostatin 1 and 1 microM ionomycin and cultured for 7 days in 20 U/ml IL-2. Activated DLN cells were then adoptively transferred to syngeneic mice with 3-day lung metastases. RESULTS: The adoptive transfer of DLN cells from mice with footpad tumors only significantly reduced the number of lung metastases compared to untreated mice. However, activated DLN cells obtained from mice with both footpad and lung tumors were significantly less effective. Treatment of similar donor mice with 10 mg/kg cyclophosphamide significantly improved the anti-tumor activity of adoptively transferred cells. This dose of cyclophosphamide did not reduce the number of cells obtained from each lymph node or the expansion of cell numbers in vitro. CONCLUSIONS: These results suggest that the administration of low-dose cyclophosphamide prior to harvesting DLN cells may improve the success of adoptive immunotherapy in cancer patients.

Animals↗
PubMed1992

Effect of helium and oxygen on airflow in a narrowed airway.

A mixture of 80% helium and 20% oxygen has physical properties that increase airflow and decrease resistance in the airway when used as a portion of inspired gas. This study was designed to demonstrate and quantify the effects of a helium-oxygen mixture in a normal airway and when airway resistance is increased. Thirty healthy volunteers were studied breathing room air and the helium-oxygen mixture through a normal airway and an airway that included a resistor. Pulmonary function tests, directed by a registered respiratory therapist, were performed on all subjects using a computerized spirometer. The functional vital capacity, one-second forced expiratory volume, half-second forced expiratory volume, and peak inspiratory flow rate were analyzed. There was a statistically significant increase in 1-second forced expiratory volume using a helium-oxygen mixture in a normal airway. All pulmonary function test scores statistically improved when volunteers inspired helium and oxygen through the restricted airway, demonstrating that helium and oxygen can increase airflow in the presence of an increased airway resistance. This substantiates a role for helium and oxygen in treating conditions associated with decreased airway size and increased airway resistance.

Adult↗