Canine CD34: cloning of the cDNA and evaluation of an antiserum to recombinant protein

Increasingly, enriched populations of hematopoietic progenitors are used in experimental and clinical transplantation studies. The separation of progenitors is based on the expression of CD34, a marker preferentially expressed on progenitor cells. The dog model has been important for preclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To identify and isolate canine hematopoietic progenitors, we have cloned a cDNA encoding a CD34 homologue from a canine myelomonocytic leukemia cell line, ML2. The CD34 homologue cDNA predicts an amino acid sequence that is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C- terminal end of the extracellular domain, but shows considerable divergence from these sequences at the amino-terminal end of the protein. In Western blotting studies, canine CD34 homologue (caCD34) appears to be a heavily and variably glycosylated protein with a molecular weight of approximately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the expression of caCD34 protein, the extracellular domain of caCD34 was expressed as an Ig fusion protein and used as an immunogen to generate a rabbit polyclonal antiserum. The antiserum reacted against the fusion protein, against vascular endothelium, and with three leukemic cell lines. Approximately 1% of canine bone marrow cells stained brightly with antibodies to caCD34 and this population was 25- to 50-fold enriched for colony- forming units-granulocyte-macrophage as compared to unfractionated marrow mononuclear cells. These findings suggest that the canine CD34 homologue is expressed on bone marrow progenitor cells and, thus, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoiesis and stem cell transplantation.     

Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization

Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.     

Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient's age and/or the conditioning regimen, but it is not associated with an increased risk of relapse

We investigated the chimerism pattern within flow-sorted peripheral blood- or bone marrow-derived cell populations after allogeneic bone marrow transplantation (BMT) for the treatment of leukemia in children. This study was performed to define the identity of persistent host-type cells, to identify prognostic variables for the persistence of host- type hematopoiesis, and to determine the prognostic significance of the chimerism pattern on the duration of the leukemia-free interval, the overall survival, and the leukemia-free survival. The patients received either HLA-identical non-T-cell-depleted (n = 46) or HLA nonidentical T- cell-depleted (n = 7) BMT. In the peripheral blood, the children showed either stable mixed chimerism (SMC; ie, persistent host-type hematopoiesis; n = 14), (transient) mixed T-lymphoid chimerism (MTLC; n = 9), or complete chimerism (CC; n = 30). In the bone marrow, only donor-type cells were found in children with either CC (n = 8) or MTLC (n = 2), and a mixture of donor- and recipient-type cells was found in children with SMC (n = 7). The persistence of host-type hematopoiesis (SMC) was significantly related to a lower age of the recipient, the type of conditioning regimen, a lower total body irradiation dose, T- cell depletion of the bone marrow graft, and the use of cyclosporine A for acute graft-versus-host disease prophylaxis. No significant differences were found between patients with (SMC) or without (CC/MTLC) persistent host-type hematopoiesis with respect to the duration of the leukemia-free interval, the overall survival, or the leukemia-free survival. We conclude that ablation of host-type hematopoiesis is not compulsory for long-term leukemia-free survival after allogeneic BMT for various hematologic malignancies.     

Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.     

Treatment adherence in heart failure patients followed up by nurses in two specialized clinics

Objectives:

to analyze treatment adherence in heart failure (HF) patients followed up by the nursing staff at specialized clinics and its association with patients'' characteristics such as number of previous appointments, family structure, and comorbidities.

Methods:

a cross-sectional study was conducted at two reference clinics for the treatment of HF patients (center 1 and center 2). Data were obtained using a 10-item questionnaire with scores ranging from 0 to 26 points; adherence was considered adequate if the score was ≥ 18 points, or 70% of adherence.

Results:

a total of 340 patients were included. Mean adherence score was 16 (±4) points. Additionally, 124 (36.5%) patients showed an adherence rate ≥ 70%. It was demonstrated that patients who lived with their family had higher adherence scores, that three or more previous nursing appointments was significantly associated with higher adherence (p<0.001), and that hypertension was associated with low adherence (p=0.023).

Conclusions:

treatment adherence was considered satisfactory in less than a half of the patients followed up at the two clinics specialized in HF. Living with the family and attending to a great number of nursing appointments improved adherence, while the presence of hypertension led to worse adherence.     

Prevalence of Brucella spp in humans

Objective:

to determine the seroprevalence of Brucella spp in humans.

Method:

this is an observational study, developed with 455 individuals between 18 and 64 years old, who use the Estratégia de Saúde da Família (Brazil''s family health strategy). The serum samples of volunteers underwent buffered acid antigen tests, such as screening, agar gel immunodiffusion and slow seroagglutination test in tubes and 2-Mercaptoethanol.

Results:

among the samples, 1.98% has responded to buffered-acid antigen, 2.85% to agar gel immunodiffusion test and 1.54% to the slow seroagglutination tests on tubes/2-Mercaptoethanol. The prevalence of Brucella spp was 4.4%, represented by the last two tests.

Conclusion:

the results of this research suggest that the studied population is exposed to Brucella spp infection.     

Mefloquine–Oxazolidine Derivatives: A New Class of Anticancer Agents

A series of 23 racemic mefloquine–oxazolidine derivatives, 4‐[3‐(aryl)hexahydro[1,3]oxazolo[3,4‐a]pyridin‐1‐yl]‐2,8‐bis(trifluoromethyl)quinolines, derived from (R*, S*)‐(±)‐mefloquine and arenealdehydes, have been evaluated for their activity against four cancer cell lines (HCT‐8, OVCAR‐8, HL‐60, and SF‐295). Good cytotoxicities have been determined with IC₅₀ values ranging from 0.59 to 4.79 μg/mL. In general compounds with aryl groups having strong electron‐releasing substituents, such as HO and MeO, or electron‐rich heteroaryl groups, for example imidazol‐2‐y‐l, are active. However, other factors such as steric effects may play a role. As both the active and non‐active conformations of the mefloquine–oxazolidine derivatives are similar, it is concluded that molecular conformations do not play a significant role either. This study is the first to evaluate mefloquine derivatives as antitumor agents. The mefloquine–oxazolidine derivatives are considered to be useful leads for the rational design of new antitumor agents.