Human Bloods Research Articles

Matrix Solid-Phase Dispersion Extraction of Sulfonamides from Blood

Matrix solid-phase dispersion extraction was applied to the extraction of sulfadiazine, sulfamerazine, and sulfamethazine from human and animal bloods. The separation and determination of the analytes were carried out by high-performance liquid chromatography. The effects of the types of the dispersion adsorbents and elution solvents were investigated, and the highest recovery was obtained when diatomaceous earth was used as the dispersion adsorbent, while acetone was used as the elution solvent. Under the optimal conditions, the linear range for determining the sulfonamides in blood samples was 0.020-10.0 µg/mL, and the average recoveries of the three sulfonamides were higher than 87.5%.

The optimal conditions for the estimation of DNA methylation levels using high throughput microarray derived DNA immunoprecipitation (MeDIP)-enrichment in human bloods

Genomic-wide mapping of epigenetic changes is essential for understanding the mechanisms involved in chemical-induced gene regulation. The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is a recently devised method for the extraction of methylated DNA from a sample of interest and used to determine the distribution of DNA-methylation within functional regions (e.g., promoters) or in the entire genome robustly and cost-efficiently. This approach is based on the enrichment of methylated DNA with an antibody that specifically binds to 5-methyl-cytosine (5mC) and can be combined with PCR and microarray. Here we summarize the experimental procedure of MeDIP followed by high throughput microarray and provides a comprehensive summary of quality control strategies.

Molecular Gender Determination of Ancient Human from Malay Peninsular

Problem statement: DNA samples from fourteen modern human bloods (seven males and seven females) and two ancient skeletal samples exc avated from Kalumpang Island and Dungun, Peninsular Malaysia were subjected for molecular ge nders determination using specific primers of human AMELX and AMELY . Approach: A standard multiple PCR mixture with forward primer and either a human X-specific reverse primer for AMELX or a human Y-specific reverse primer for AMELY amplifications were used to assess the presence of these genes in male and female samples. Results: PCR amplification of a modern male sample yielded 329 and 235 bp bands, whereas a modern female sample only 329 bp band. The Kalumpang Island sample produced two positive bands of AMELY and AMELX . After reamplification of the Dungun sample, only an AMELY band was visible. All amplified bands were cloned into TA plasmid and sequenced. BLAST analysis showed that the 329 bp band is AMELX , while the 235 bp product is AMELY . Conclusion/Recommendations: The skeletal remains of both Kalumpang Island and Dungun samples from west and east of Peninsular Malaysia respectively, are males.

Bioinformatics approach to mRNA markers discovery for detection of circulating tumor cells in patients with gastrointestinal cancer

Background: Detection of tumor cells in the blood, or minimal deposits in distant organs as bone marrow, could be important to identify cancer patients at high risk of relapse or disease progression. Quantitative polymerase chain reaction (PCR) amplification of tissue or tumor selective mRNA is the most powerful tool for the detection of this circulating or occult metastatic cells. Our study aims to identify novel gastrointestinal cancer-specific markers for circulating tumor cell detection. Method: Phase I preclinical study was performed by means of computational tools for expression analysis. In silico data were used to identify and prioritize molecular markers highly expressed in gastrointestinal cancers but absent in hematopoietic-derived libraries. Selected genes were evaluated by means of qRT-PCR in gastrointestinal cancer and hematopoietic cell-lines, normal human bone marrows and bloods, tumor tissue, and blood from cancer patients. Results: Novel and known mRNA markers for circulating tumor cell detection in gastrointestinal cancer have been identified. Among all the genes assessed, PKP3, AGR2, S100A16, S100A6, LGALS4, and CLDN3 were selected and assays based on blood qRT-PCR were developed. Reliably qRT-PCR assays for the novel targets plakophilin 3 (PKP3) and anterior gradient-2 (AGR2) to identify blood-borne cells in cancer patients were developed. Conclusions: Novel and known gastrointestinal-specific mRNA markers for circulating tumor cells have been identified through in silico analysis and validated in clinical material. qRT-PCR assay targeted to PKP3 and AGR2 mRNAs might be helpful to detect circulating tumor cells in patients with gastrointestinal cancer.

Multiplex real-time PCR for detection of anaplasma phagocytophilum and Borrelia burgdorferi.

A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >/=4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.

Identification of BMP and Activin Membrane-bound Inhibitor (BAMBI), an Inhibitor of Transforming Growth Factor-β Signaling, as a Target of the β-Catenin Pathway in Colorectal Tumor Cells

The Wnt signaling pathway is activated in most human colorectal tumors. Mutational inactivation in the tumor suppressor adenomatous polyposis coli (APC), as well as activation of beta-catenin, causes the accumulation of beta-catenin, which in turn associates with the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors and activates transcription of their target genes. Here we show that beta-catenin activates transcription of the BMP and activin membrane-bound inhibitor (BAMBI)/NMA gene. The expression level of BAMBI was found to be aberrantly elevated in most colorectal and hepatocellular carcinomas relative to the corresponding non-cancerous tissues. Expression of BAMBI in colorectal tumor cell lines was repressed by a dominant-negative mutant of TCF-4 or by an inhibitor of beta-catenin-TCF interaction, suggesting that beta-catenin is responsible for the aberrant expression of BAMBI in colorectal tumor cells. Furthermore, overexpression of BAMBI inhibited the response of tumor cells to transforming growth factor-beta signaling. These results suggest that beta-catenin interferes with transforming growth factor-beta-mediated growth arrest by inducing the expression of BAMBI, and this may contribute to colorectal and hepatocellular tumorigenesis.

Characteristic expression of aryl hydrocarbon receptor repressor gene in human tissues: Organ-specific distribution and variable induction patterns in mononuclear cells

To investigate the expression of aryl hydrocarbon receptor repressor (AhRR) and related molecules in various tissues and the effects of aromatic hydrocarbons (AHs) on their expression, we developed a reliable technique of quantification of human AhRR as well as aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT) and cytochrome P450 1A1 (CYP1A1) mRNA by real-time TaqMan PCR method. First, we examined the expression of these genes in human adult or fetal tissues. The levels of AhRR expression were extremely high in testis, very high in lung, ovary, spleen and pancreas from adults, whereas those were low in those from fetuses. On the other hand, CYP1A1 expression was extremely high in lung, and AhR and ARNT were ubiquitously expressed in almost all tissues. Second, we compared the expression levels of these genes in mononuclear cells (MNCs) from various sources. Comparison of the basal expression levels of these genes in MNCs demonstrated that MNCs from umbilical cord blood showed higher AhRR or CYP1A1 expression than those from adults. The induction of AhRR or CYP1A1 expression by 3-methylcholanthrene (3-MC) was observed in MNCs from adults but not from umbilical cord blood. Consequently, there existed characteristic differences in the basal levels of AhRR and CYP1A1 expression in MNCs, as well as in their inducibility by 3-MC among MNCs from various types of human bloods. These results will provide basic information for a possible application of AhRR and CYP1A1 measurements to evaluate AH exposure in vivo.

Differentiation of Osteoblast Progenitor Cells from Human Umbilical Cord Blood

Background: Human umbilical cord bloods, which could be taken during the delivery are utilized as a source of hematopoietic stem cells. Also in cord blood, there are several kinds of stem cells such as endothelial and mesenchymal stem cells. Methods: We isolated the mesenchymal stem cells from human umbilical cord bloods and confirmed the differentiation of these cells into osteoblast progenitor cells. The mesenchymal stem cells derived from umbilical cord blood have the ability to differentiate into specific tissue cells, which is one of characteristics of stem cells. These cells were originated from the multipolar shaped cells out of adherent cells of the umbilical cord blood mononuclear cell culture. Results: The mesenchymal stem cells expressed cell surface antigen CD13, CD90, CD102, CD105, α-smooth muscle actin and cytoplasmic antigen vimentine. Having cultrued these cells in bone formation media, we observed the formation of extracellular matrix and the expression of alkaline phosphatase and of mRNA of cbfa-1, ostoecalcin and type I collagen. Conclusion: From these results we concluded that the cells isolated from the umbilical cord blood were mesenchymal stem cells, which we could differentiate into osteoblast when cultured in bone formation media. In short, it is suggested that these cells could be used as a new source of stem cells, which has the probability to alternate the embryonic stem cells. (Immune Network 2002;2(3):166-174)

Two Human Neonatal IgM Antibodies Encoded by Different Variable-Region Genes Bind the Same Linear Peptide: Evidence for a Stereotyped Repertoire of Epitope Recognition

Two monoclonal IgM Abs have been produced from lymphocytes isolated from two human umbilical cord bloods. These mAbs recognize a conformational epitope present in a CNBr digestion fraction of lactoferrin. Linear epitopes recognized by each mAb were selected from several phage display peptide libraries. In each case, phages displaying a peptide with a motif defined by [WF],G,[EQS],N were recovered. Phages displaying that motif bound equally well to either mAb but did not bind to control IgM. A peptide bearing this motif competed with the phage-displayed peptides for binding to either mAb. The same peptide also competes with a component of the CNBr digestion fraction of lactoferrin for Ab binding in ELISA. The Abs use different families of VH, JH, and VK gene cassettes but use the same JK cassette. All segments are virtually identical to their germline gene counterparts. This work provides further evidence that certain innate specificities are stereotyped among individuals.

Monoclonal antibodies directed against human Rh antigens in tests with the red cells of nonhuman primates

Monoclonal antibodies against Rh related antigens on human red cells often crossreact with the red cells of the highest subhuman primate species. Depending on specificity of antibody, the species tested, and technique used, these reactions can be either species-specific or type specific. In tests with chimpanzee red cells, some of the latter type reactions have specificities related to the R antigen of the R-C-E-F blood group system of chimpanzee; specificities of some others seem to be unrelated to any known chimpanzee blood groups. Monoclonal anti-D reagents that give uniformly positive reactions with human D-positive (common and rare types) red cells, display wide individual differences in tests with chimpanzee blood. This indicates that there are minute structural variations of antibody molecules from one monoclonal anti-D antibodies apparently have no bearing on recognition of the D combining site on the human red cells, but come into play when in contact with chimpanzee rbcs. Some of the monoclonal antibodies directed against Rh and LW molecules are distinguished by unusually strong reactions with the red cells of the Old World monkeys (macaques and baboons), which is in contrast with negative or weak reactions of the same antibodies with the red cells of anthropoid apes and human bloods. One may recall, that polyclonal anti-Rh sera do not react with the blood of rhesus monkeys, the phenomenon that was the source of controversy surrounding the discovery of the rhesus factor of the human blood.

Neutrophil Myeloperoxidase Destruction by Ultraviolet Irradiation

Only a fraction of the UV radiation emitted by the sun reaches the earth; most of the UVB (290-320nm) is eliminated by stratospheric ozone. There is increasing concern, however, that man-made chemicals are damaging this ozone layer. Although the effects of UV on DNA or as a carcinogen are widely known, preleukemia and acute myeloid leukemia (AML) have only rarely been reported in psoriasis patients treated with 8-methoxypsoralen and UV (PUVA). It was therefore of interest to study the effects of UV on the myeloperoxidase (MP) activity of human neutrophils. The peroxidase activity of enriched leukocyte preparations on coverslips was shown cytochemically with a diaminobenzidine medium and cupric nitrate intensification.Control samples (Figs. 1,4,5) of human bloods that were not specifically exposed to UV radiation or light except during routine handling were compared with samples which had been exposed in one of several different ways. One preparation (Fig. 2) was from a psoriasis patient who had received whole-body UVB phototherapy repeatedly.

Rheological behaviors of bovine blood forming artificial rouleaux.

A purpose of the present study is to make an artificial rouleau of bovine red blood cells which is not capable of rouleau formation under physiological condition. Rheological behaviors of bovine blood forming artificial rouleaux were examined. The modification of cell surface by enzyme trypsin induced rouleau formation, whereas the modification of cell surface by neuraminidase did not cause any aggregate formation. The drastic elevation of the fibrinogen content in bovine red blood cells suspension also brought about the formation of rouleau. The value of dynamic rigidity modulus G' of bovine red blood cells in saline solution containing high concentration of fibrinogen is somewhat smaller than that of trypsin treated bovine red blood cells in plasma. The value of G' of trypsin treated bovine red blood cells in plasma first increased to a maximum value and then decreased with the time. It is supposed that the removal of macro-molecules from the cell surface facilitates the mutual approach of cells and causes the formation of rouleau which seems to be the same as that of human and horse bloods.

Change in the ionic environment of cerebral arteries after subarachnoid hemorrhage. Especially of potassium ion concentration in subarachnoid hematoma and its role in cerebral vasospasm

The possible role of electrolytes in the induction of vasospasm has not been well evaluated. These electrolytes are known to have vasomotor effects, and this study especially investigated potasium ion, which is liberated in the process of blood clot hemolysis. Under pentobarbital anesthesia, the basilar arteries of adult rats were exposed by the transclival approach under the operating microscope. Subarachnoid hemorrhage was induced by puncture on a small branch artery. At 10 minutes, two hours, and 24 hours after subarachnoid bleeding, fresh arterial strips were removed and immediately frozen in liquid nitrogen, cryosectioned, and freeze-dried. Electron probe X-ray microanalysis of the dry cryosections was performed to determine the elemental compositions of the vascular smooth muscle, extravasated erythrocyte, and periarterial space. Cytoplasmic concentrations in normal vascular smooth muscle were-K: 133±5; Cl: 12±3; Na: 22±4 (mmol/kg wet weight±SEM). The potassium concentration increased in the extracellular space to up to 8 times above the baseline 24 hours after subarachnoid bleeding, and the cytoplasmic concentrations of Ca, Na, and Cl also increased. The potassium concentration of the extravasated erythrocyte decreased over 24 hours. The time course of potassium ion liberation from extravasated erythrocytes was also studied. Fresh rat and human arterial bloods were withdrawn in sterile test-tubes and stored at 37°C. Hypertensive intracerebral hematomas were also collected during surgery. With a flamephotometer, the potassium concentrations of the stored blood serum and of the hematoma supernatants were measured chronologically. The potassium ion level almost reached its peak valve within two days, although a gradual increase persisted in rat and human incubated blood. On the other hand, the potassium ion level peak of the intracerebral hematoma was reached around day 3 or 4 post hemorrhage, and the level gradually decreased back to normal in approximately three weeks. Potassium has been known to have potent vasomotor effects. This study shows that the potassium environment is of critical importance in cerebral vasospasm caused by subarachnoid hemorrhage.

Application of the transient flow rheology to the study of abnormal human bloods.

Application of the transient flow rheology to the study of abnormal human bloods.

Rabbit antisera to human cord Tγ cells

T cells with Fc receptors for IgG (Tγ) were isolated by ox cell EA rosetting from 50 human cord bloods. Non-Tγ cells (Tμ and T null) were collected at the same time and frozen live in liquid nitrogen. Antisera produced in rabbits by three or four weekly intravenous injections of cord blood Tγ cells were inactivated at 56°C and extensively absorbed with sheep and ox erythrocytes, B-cell lines, suspensions of fresh human thymus (largely T null), and finally non-Tγ cells from original cord bloods. Absorbed anti-Tγ reagents used as F(ab)′ 2 fragments to avoid inadvertent Fc receptor binding were studied by indirect immunofluorescence with FITC goat anti-rabbit F(ab)′ 2. F(ab)′ 2 anti-Tγ antisera stained 5–14% of T cells and 50–66% Tγ cells. No significant staining was noted with monocytes, macrophages, B cells, or non-Tγ T cells. Absorbed anti-Tγ reagents showed no residual anti-human Ia-like specificity, however, double immunofluorescence staining for human Ia-like antigen positive T cells and cells staining with anti-Tγ reagents showed a 50% overlap. An average of 32.5% Tγ cells showed antigens staining with OKM1, a hybridoma identifying antigens related to monocytes; however, only 18.5% of Tγ cells showed double staining with OKM1 and F(ab)′ 2 anti-Tγ antisera. These findings emphasize possible broad heterogeneity within the human Tγ cell subpopulation.

Rheological hysteresis of blood at low shear rate1

A Couette type servo-controlled microviscometer associated with a low frequency generator modulating the rotational speed of the viscometer outer cylinder has been used to plot the rheological hysteresis curves of blood. It has been shown that the shape of these curves largely depends on the values of the parameters α and tm characterizing the linear variation of the rate of shear with time: γ˙=αt for 0<t<tm and γ˙=α(2tm−t) for tm<t<2tm. Two sets of α and tm have been selected in order to obtain rheograms showing preferentially the viscoelastic or the thixotropic behaviour of blood. This technic is used to compare normal human bloods with bloods submitted to various physical or chemical treatments, or with different pathological bloods. Some interpretations are proposed from considerations on changes in the deformability or aggregability of blood cells.

Appearance of formate in blood after ethanol ingestion

Human and rabbit bloods after ingestion of ethanol were analyzed for carboxylic acids, and were found to contain not only acetate but also formate. The formate level in the rabbit serum was 0.35μmole/ml at 4 hours after introduction of 10ml of 40% ethanol/kg into the stomach. Administration of [1- 14C] ethanol or [2- 14C]ethanol resulted in the presence of radioactivity in serum acetate, but not in serum formate. Pretreatment with tryptophan significantly increased serum formate, and pretreatment with folate suppressed the appearance of formate.

A precise method for the determination of whole blood and plasma sulfhydryl groups

A colorimetric method for the determination of total sulfhydryl content whole blood or plasma is presented. For whole blood, a mixture of fresh blood and 5,5′-dithiobis(2-nitrobenzoic acid) in diluted buffer is separated on a gel column into the yellow anion and the hemoglobin fraction. The plasma-DTNB mixture was read directly, and a correction for turbidity and/or color made after addition of N-ethylmaleimide. Results of a sample of human bloods are presented. The effects of several detergents yield high values of sulfhydryl in whole blood, which we believe to be artifactual. Studies of the stability of stored blood samples indicate that whole blood, but not plasma, can be kept refrigerated for several days without significant loss of sulfhydryl reaction.

A precise method for the determination of whole blood and plasma sulfhydryl groups

A colorimetric method for the determination of total sulfhydryl content whole blood or plasma is presented. For whole blood, a mixture of fresh blood and 5,5′-dithiobis(2-nitrobenzoic acid) in diluted buffer is separated on a gel column into the yellow anion and the hemoglobin fraction. The plasma-DTNB mixture was read directly, and a correction for turbidity and/or color made after addition of N-ethylmaleimide. Results of a sample of human bloods are presented. The effects of several detergents yield high values of sulfhydryl in whole blood, which we believe to be artifactual. Studies of the stability of stored blood samples indicate that whole blood, but not plasma, can be kept refrigerated for several days without significant loss of sulfhydryl reaction.

Red cell pH in human maternal and fetal blood as determined by 5, 5-dimethyl-2, 4-oxazolidine dione

Red cell pH was calculated in human maternal and fetal blood, from plasma pH and the distribution of isotopically labelled 2,5-dimethyl-2,4-oxazoladine dione (DMO). Results show a significant difference, at the same plasma pH, in the red cell pH's of maternal and fetal blood. The difference is in a direction which would reconcile differences observed in oxyhemoglobin dissociation functions of human maternal and fetal whole bloods, and the absence of such differences when maternal and fetal hemoglobin solutions are studied in buffers of the same ionic concentration. The relationship of this observation to the role of DPG and ATP in determining dissociation function is discussed.