Biomedical subjects

R C Barfield

Publications and source records attributed to R C Barfield.

7 recordsLinked to original sources

PubMed2007

Autologous transplantation of CD133 selected hematopoietic progenitor cells for treatment of relapsed acute lymphoblastic leukemia.

A 21-year-old white male with relapsed acute lymphoblastic leukemia (ALL) developed an invasive Zygomycosis infection 3 weeks after beginning re-induction chemotherapy. Because of the high risk of fatal recurrence of the fungal infection, neither long-term maintenance chemotherapy nor allogeneic hematopoietic stem cell transplant (HSCT) was considered appropriate. Because his ALL blasts expressed CD34 but lacked CD133, he received a CD133 selected autologous graft following high-dose consolidation chemotherapy. The patient survives in remission 19 months after HSCT.

AC133 Antigen↗

PubMed2005

Partial purification and characterization of an aminopeptidase from Eimeria tenella.

Our previous investigation demonstrated the expression in Eimeria tenella sporulated oocysts of an aminopeptidase (AP) with strong homology to AP N. To further understand the role of proteases during development, we investigated the molecular and biochemical properties of E. tenella AP. Greater than 95% AP activity was present in a soluble extract during sporulation of oocysts with highest activity in fully sporulated oocysts. The AP activity was inhibited by the AP inhibitors bestatin and 1,6-phenanthroline, but not by serine protease inhibitors. The AP had specificity for synthetic endopeptidase substrates that contain arginine, alanine, or glycine at the N terminus. Partial purification of the enzyme yielded a major protein band with an Mr of about 106 kDa and an isoelectric point (Ip) of 5.1. Reverse transcription-polymerase chain reaction indicated that the gene for AP is expressed during sporulation, but expression is absent or greatly reduced in the sporozoites and merozoites. On the basis of the deduced gene structure, the predicted Mr is 110 kDa with a pI of 5.59. Database search indicates that the E. tenella AP shares significant homology with the AP from Apicomplexan taxa: Toxoplasma gondii, Cryptosporidium parvum, and Cryptosporidium hominis. Together, these results confirm the presence of a cytosolic AP related to AP N, which is expressed and active during sporulation of E. tenella oocysts.

Amino Acid Sequence↗

PubMed2004

A one-step large-scale method for T- and B-cell depletion of mobilized PBSC for allogeneic transplantation.

BACKGROUND: The presence of T and B cells in allogeneic grafts contributes to GvHD and to EBV-associated lymphoproliferative disease (LPD). Depletion of T and B cells from the graft decreases the risk of these complications. METHODS: T and B cells were depleted from mobilized peripheral stem cells from volunteer donors (n=5) using anti-CD3 and anti-CD19 Abs conjugated to magnetic microbeads, and the CliniMACS device. The function of the stem cells after depletion was evaluated using colony assays and non-obese diabetic (NOD)/SCID repopulating experiments. RESULTS: The mean mononuclear cell (MNC) count prior to T- and B-cell depletion was 2.19x10(10) (range 1.48-3.53). After depletion, the mean percentage of contaminating T cells was 0.02% (range 0.01-0.04%) with a mean log(10) depletion of 3.4 (range 3-3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01-0.4%) with a mean log(10) depletion of 2.2 (range 1.4-3). The mean recovery of CD3- and CD19-negative MNCs after depletion was 70% (range 54-88%) and the mean recovery of CD34(+) stem cells was 69% (range 52-98%). The mean number of natural killer (NK) cells after T- and B-cell depletion was 5.2x10(8) (range 2-10x10(8)). In vitro colony assays and in vivo NOD/SCID repopulation assays showed no negative impact of this method on the function of the hematopoietic stem cells. DISCUSSION: Our results show that the CliniMACS system can be used to efficiently deplete PBSC of T and B cells simultaneously, without adverse effect on the graft.

Animals↗

PubMed2004

A conserved 19-kDa Eimeria tenella antigen is a profilin-like protein.

A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase-polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-L-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.

Actins↗

PubMed2004

Analysis of transcripts expressed by Eimeria tenella oocysts using subtractive hybridization methods.

To characterize the genes expressed by Eimeria tenella oocysts, the sequence of 499 expressed sequence tags (ESTs) was obtained from complementary DNA (cDNAs) enriched for transcripts expressed by unsporulated or sporulated oocysts. Of these, 225 clones were isolated from cDNA of sporulated oocysts and 274 from unsporulated oocysts. A total of 163 unique sequences were found, and the majority of these (64%) represent novel genes with no significant homology to the proteins in GenBank. Approximately half of the unique transcripts generated from sporulated oocysts are also expressed by sporozoites and merozoites, whereas the expression of most (79%) of the transcripts from unsporulated oocysts has not yet been detected at other stages of development. The expression of 4 transcripts obtained from the subtracted cDNAs was confirmed by quantitative reverse transcriptase-polymerase chain reaction. The results confirmed that these transcripts are in fact differentially expressed between sporulated and unsporulated oocysts.

Animals↗

PubMed2003

The effect of dietary betaine on intestinal and plasma levels of betaine in uninfected and coccidia-infected broiler chicks.

Chicks fed betaine supplemented diets and infected with Eimeria acervulina and Eimeria maxima had markedly higher levels of betaine in the duodenum and mid-gut than unsupplemented, infected chicks. Uninfected chicks fed betaine exhibited almost twice the levels of betaine in the gut as infected chicks. Plasma betaine levels were lower in E. maxima-infected chicks than in E. acervulina-or Eimeria tenella-infected chicks. Betaine supplementation reversed the decrease in weight gain in E. maxima- infected chicks but had no effect on the decrease in weight gains in E acervulina- and E. tenella-infected chicks. Coccidia-infected birds on normal diets regularly exhibit increases in plasma NO(2)(+)NO(3). This increase was abolished in E.tenella-infected birds on betaine supplement. Betaine feeding did not alter this effect in E. acervulina- and E. maxima-infected birds. Results indicate that betaine supplementation has a positive effect on gut betaine levels in birds infected with E. acervulina and E. maxima. In all treatment groups, infection lowered the levels of betaine.

Animals↗

PubMed2003

Characterization of a developmentally regulated oocyst protein from Eimeria tenella.

Changes in proteins during sporulation of Eimeria tenella oocysts were investigated. Unsporulated E. tenella oocysts collected from cecal tissue at 7 days postinoculation were sporulated in aerated media at 28 C for 0-48 hr. Gel analysis of soluble protein extracts prepared from oocysts from their respective time points indicated the presence of 2 prominent bands with relative molecular weight (Mr) in the range of 30 kDa and making up 20% of the total protein. These 2 bands, designated as major oocyst proteins (MOPs), were absent or barely detectable by 21 hr of sporulation. MOP bands were weakly reactive with glycoprotein stain but showed no mobility shift on deglycosylation. By gel analysis it was shown that the purified MOPs consisted of 2 bands of Mr 28.7 and 30.1 kDa. However, by matrix-assisted laser deabsorption-time of flight analysis it was shown that masses were about 17% lower. Internal sequence analysis of the 28.7-kDa protein generated 2 peptides of 17 and 14 amino acids in length, consistent with a recently described protein coded by the gam56 gene and expressed in E. maxima gametocytes. Rabbit antibodies made against MOPs were localized to outer portions of sporocysts before excystment and to the apical end of in vitro-derived sporozoites. These same antibodies were found to react with bands of Mr 101 and 65 kDa by Western blot but did not recognize MOPs in soluble or insoluble sporozoite extracts. The data suggest that the MOPs are derived from part of a gametocyte protein similar to that coded by gam56 and are processed during sporulation into sporocyst and sporozoite proteins. Alternatively, the binding of anti-MOP to 101- and 65-kDa proteins may result from alternatively spliced genes as the development of parasite proceeds.

Amino Acid Sequence↗