Comparison of the content and subpopulations of CD3 and CD34 positive cells in bone marrow harvests and G-CSF-mobilized peripheral blood leukapheresis products from healthy adult donors

Recombinant human granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem/progenitor cells (PBPC) have replaced bone marrow (BM) harvests for autologous transplantation after myeloablative therapy in cancer patients. G-CSF-mobilized PBPC from healthy donors contain one log excess of T lymphocytes representing a potential risk for graft-versus-host disease (GVHD). However, recent pilot clinical studies of G-CSF-mobilized allogeneic PBPC transplantation have shown rapid haematological recovery and no severe acute GVHD except in a very few cases. Therefore, the risk of inducing severe acute GVHD is not as high as was expected during the pioneering period of allogeneic PBPC transplantation.The present study was performed to address the possible reasons for the rapid haematological recovery and the absence of severe acute GVHD observed after allogeneic PBPC transplantation by comparing the contents and subsets of CD3⁺ and CD34⁺ G-CSF-mobilized PBPC (n = 31) with those of BM (n = 26) allografts from healthy adult donors. The present results revealed that the phenotypic profiles of CD3⁺ and CD34⁺ cells differ between PBPC and BM allografts. The single PBPC leukapheresis product contained 10 times more mononuclear cells, 1.5 times more CD34⁺ cells, 5.5 times more CD3⁺ T lymphocytes, 3 times more CD19⁺ B lymphocytes and 3.8 times more CD14⁺ monocytes than the single BM harvest. Both CD34⁺CD33⁺ myeloid progenitor cells and CD34⁺HLA-DR- long-term reconstituting haemopoietic stem cells were significantly increased in the CD34⁺ G-CSF-mobilized PBPC compared with the CD34⁺ BM cells; median 73.1% and 30.4% vs 60.6% and 5.0%, respectively, P < 0.01.The percentage of CD3⁺ cells coexpressing CD4 (T helper/inducer) was similar in both PBPC and BM allografts, 47.2% and 45.6%, respectively, whereas the percentage of CD3⁺ cells coexpressing CD8 (T suppressor/cytotoxic) was significantly decreased in PBPC compared with BM; 37.0% vs 55.9%, p < 0.01.     

槲寄生化学成分的研究 Ⅶ.槲寄生新甙Ⅶ的分离和结构

自中药槲寄生中分离得到一个新的黄酮甙,经波谱测定及水解试验,确定其结构为鼠李秦素-3-O-β-D-芹糖基(1→2)-[6″-(3-羟基-3-甲基戊二酸半酯)]葡萄糖甙,命名为槲寄生新甙Ⅶ(viscumneoside Ⅶ).     

The LIFESTYLE study: costs and effects of a structured lifestyle program in overweight and obese subfertile women to reduce the need for fertility treatment and improve reproductive outcome. A randomised controlled trial

Background

In the Netherlands, 30% of subfertile women are overweight or obese, and at present there is no agreement on fertility care for them. Data from observational and small intervention studies suggest that reduction of weight will increase the chances of conception, decrease pregnancy complications and improve perinatal outcome, but this has not been confirmed in randomised controlled trials. This study will assess the cost and effects of a six-months structured lifestyle program aiming at weight reduction followed by conventional fertility care (intervention group) as compared to conventional fertility care only (control group) in overweight and obese subfertile women. We hypothesize that the intervention will decrease the need for fertility treatment, diminish overweight-related pregnancy complications, and will improve perinatal outcome.

Methods/Design

Multicenter randomised controlled trial in subfertile women (age 18-39 year) with a body mass index between 29 and 40 kg/m². Exclusion criteria are azoospermia, use of donor semen, severe endometriosis, premature ovarian failure, endocrinopathies or pre-existent hypertensive disorders. In the intervention group the aim is a weight loss of at least 5% to10% in a six-month period, to be achieved by the combination of a diet, increase of physical activity and behavioural modification. After six months, in case no conception has been achieved, these patients will start fertility treatment according to the Dutch fertility guidelines. In the control group treatment will be started according to Dutch fertility guidelines, independently of the patient's weight.

Outcome measures and analysis

The primary outcome measure is a healthy singleton born after at least 37 weeks of gestation after vaginal delivery. Secondary outcome parameters including pregnancy outcome and complications, percentage of women needing fertility treatment, clinical and ongoing pregnancy rates, body weight, quality of life and costs. Data will be analysed according to the intention to treat principle, and cost-effectiveness analysis will be performed to compare the costs and health effects in the intervention and control group.

Discussion

The trial will provide evidence for costs and effects of a lifestyle intervention aiming at weight reduction in overweight and obese subfertile women and will offer guidance to clinicians for the treatment of these patients.

Trial registration

Dutch Trial Register NTR1530     

苯乙哌啶合成的改进

目的：改进苯乙哌啶的合成方法,使之更适合工业化生产。方法,以N,N－双（β-氯乙基）对甲苯磺酰胺为原料,利用液－液相转移催化方法制得1－对甲苯磺酰基－4－氰基哌啶,再经水解,酯化,缩合,成盐等步骤,合成了苯乙哌啶。结果：总收率可达51．8％,结论：此合成方法适合工业化生产。     

Hematoxylin and Safranin O Staining of Frozen Sections

Currently the hematoxylin and eosin staining procedure is the most popular among Mohs surgeons for histology. However, safranin O, a cheaper and relatively safer stain which is predominantly used for plant histology, should be considered as it offers similar or improved accuracy in the diagnosis of frozen sections of basal and squamous cell carcinomas.     

Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3' noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.     

Sulfhydryl reducing agents and shape regulation in human erythrocytes

Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced- stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT- induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.     

Association of cell cycle expression of Ia-like antigenic determinations on normal human multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins

An association has been established between human Ia-like antigenic determinants, expression during DNA synthesis on multipotential (CFU- GEMM) and erythroid (BFU-E) progenitor cells, and the regulatory action of acidic isoferritins in vitro. Treatment of human bone marrow cells with monoclonal anti-Ia (NE1-011) plus complement inhibited colony formation of CFU-GEMM) and BFU-E by 50%-70%. Reduction of colonies was similar whether bone marrow cells were exposed to anti-Ia plus complement, high specific tritiated thymidine (3HTdr), or acidic isoferritins. No further decrease was apparent with 3HTdr or acidic isoferritins after Ia-antigen+ CFU-GEMM or BFU-E were removed, or with anti-Ia plus complement or acidic isoferritins after S-phase CFU-GEMM or BFU-E were removed. Anti-Ia, in the absence of complement, had no effect on colony formation but blocked the inhibition of CFU-GEMM and BFU-E by acidic isoferritins. Demonstration of Ia-antigens on BFU-E and inhibition of BFU-E by acidic isoferritins appeared to require the presence of phytohemmagglutinin leukocyte conditioned medium (PHA-LCM) in the culture medium during the 14-day incubation period. these results implicate Ia-antigen+ cells, acidic isoferritins, and PHA-LCM in the regulation of multipotential and erythroid progenitor cells in vitro.     

Concurrent classical Hodgkin lymphoma and plasmablastic lymphoma in a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma treated with fludarabine: a dimorphic presentation of iatrogenic immunodeficiency-associated lymphoproliferative disorder with evidence suggestive of multiclonal transformability of B cells by Epstein-Barr virus

A small fraction of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma develop Epstein-Barr virus–positive B-cell lymphoproliferative disorders. These Epstein-Barr virus–B-cell lymphoproliferative disorders are thought to be related to immune suppression induced by fludarabine/other chemotherapeutic regimens. As in other immunodeficiency-associated lymphoproliferative disorders, these disorders demonstrate a heterogeneous histological spectrum that ranges from polymorphic to monomorphic to classical Hodgkin lymphoma–like lesions. We report a case of concurrent classical Hodgkin lymphoma and plasmablastic lymphoma in a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma treated with fludarabine. Both classical Hodgkin lymphoma and plasmablastic lymphoma were positive for Epstein-Barr virus-encoded RNA, whereas classical Hodgkin lymphoma was also positive for Epstein-Barr virus- latent membrane protein 1, suggesting a different viral latency. Immunoglobulin gene rearrangement studies demonstrated distinct clones in the plasmablastic lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. These findings suggest biclonal secondary lymphomas associated with iatrogenic immunodeficiency. Epstein-Barr virus–B-cell lymphoproliferative disorders in the setting of chronic lymphocytic leukemia/small lymphocytic lymphoma, in particular those arising after chemotherapy, should be separated from true Richter's transformation, and be categorized as (iatrogenic) immunodeficiency-associated lymphoproliferative disorder.     

The 20-Minute Rapid MART-1 Immunostain for Malignant Melanoma Frozen Sections

BACKGROUND Immunohistochemical staining has been used to help detect malignant melanoma on Mohs surgery frozen sections. Previous investigators have developed protocols for reliable MART-1 immunostaining of frozen sections, but these protocols are time-consuming.
OBJECTIVE The objective was to report a rapid 20-minute MART-1 immunostaining protocol for frozen sections.
METHODS The protocol was utilized on 30 melanomas treated with Mohs micrographic surgery.
RESULTS The stain clearly highlighted normal background melanocytes, as well as melanocytic hyperplasia and malignant melanoma.
CONCLUSIONS The 20-minute protocol provides a rapid and reliable method for immunostaining of malignant melanoma. The availability of more rapid immunostaining methods improves efficiency of the Mohs laboratory and significantly reduces patient and physician waiting time.