PLT Cells Research Articles

Effects of Nano-Fipronil on Male Rats’ Biochemical, Liver, and Renal Functions

Fipronil has been widely employed for crop protection over the past decade because of its low soil stability and insecticidal potency at modest application rates. The present study was designed to investigate the adverse effects of sub-acute exposure to 10 and 20 mg/kg/b. w. of Nano-Fipronil on biochemical blood profile and histological parameters in male rats according to the permissible limits of acceptable daily intake (ADI). The results hematological parameters indicated that Red blood cells (RBCs) and Hemoglobin(H.b) decreased in all treated groups compared with the control. While PLT and white blood cells (WBCs) was significantly increased in all treated groups compared with the control. The toxicity of effects of nano-fipronil pesticides leads to higher levels of liver function parameters (AST, ALT and ALP), kidney function (Blood urea and Creatinin and Uric acid) and Oxidative stress (MDA, GSH and CAT) results demonstrated highly significant differences (P≤0.05) in all groups treated compared with the control.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

An outbreak of Goatpox virus infection in Wild Red Serow (Capricornis rubidus) in Mizoram, India.

Goatpox virus (GTPV) belongs to the genus Capripoxvirus associated with characteristic clinical lesions in fully susceptible breeds of sheep and goats. To date, there is no report of outbreaks of GTPV infection in any wild animals. This study reports the outbreak of GTPV infection in wild Red Serow (Capricornis rubidus.) in Mizoram, India. A total of 113 wild Serow carcasses were recovered from seven districts of Mizoram between May 2015 to October 2016. A postmortem revealed presumptive pox-like lesions. Clinical specimens (lung, skin, and trachea) were examined for the aetiological agents. GTPV could be isolated in PLT cells and confirmed in PCR assays by targeting RPO30 and P32 genes. The genetic and phylogenetic analysis reveled that over 99.8% sequence identity with GTPV from India and other parts of the world. To the authors' knowledge, this is the first report of GTPV infection in wild ruminants.

Leukopenia Accuracy of the Bloodhound Integrated Hematology System

The Bloodhound Integrated Hematology System (Constitution Medical, Westborough, MA) performs an automated CBC and WBC differential by printing undiluted whole blood onto a microscope slide, staining the slide, then performing image analysis. Data and images from a 600-cell WBC differential, RBC, PLT and other cells are reviewable on a monitor. This study compared the 100-cell WBC differentials of leukopenia …

Association of Hemoglobin Concentration and Cord Blood Cell Characteristics in Healthy Newborn Infants.

Abstract 5092 BACKGROUND AND OBJECTIVECord blood (CB), used for hematopoietic stem cell transplantation, is readily available from CB banks, and the banking processes are widely standardized. The data collected in CB banks have been utilized to characterize the contents of CB units and, to some extent, the physiological phenomena of healthy newborn infants. The objective of this study was to evaluate the association of the hemoglobin concentration (Hb), whose measurement is independent of the cell counts, with CB cell characteristics (red blood cells, RBC, hematocrit, Hct, mean platelet volume, MPV, platelets, PLT and white blood cells, WBC). The internal associations of cell counts were also evaluated. MATERIALS AND METHODSThe study comprised 167 consecutive ex utero CB collections accepted for processing. All the infants (52% male) were healthy and delivered at term. Of the deliveries 104 (62%) were by Cesarean section. Perinatal data were obtained from the maternity hospital records. The relative birth weight was expressed as a birth weight z-score to normalize the birth weight for gestational age and gender. The median birth weight was 3784 g (range 2490 – 4975) and the median relative birth weight 0.49 SD (-2.41 – 3.37). The mode of delivery did not significantly influence these values. Umbilical artery (UA) pH samples were drawn after delivery as part of routine hospital care. A hematology analyzer utilizing the electronic resistance detection method coupled with volumetric (cell analysis) and colorimetric (Hb) principles was used for cell counting (Sysmex K1000, Sysmex Corp., Kobe, Japan). All the concentrations were standardized to exclude the effect of the varying blood-to-anticoagulant ratio. (Aroviita et al. Acta Paediatr 2004;93:1323-1329) CD34+ cell analysis was performed with a flow cytometer based on ISHAGE guidelines. Two-sided p values <0.05 were considered statistically significant. The CB banking program was approved by the ethical committees of the institutes. RESULTSThe hematological values and their distributions were within the published ranges. (Nathan and Oski's Hematology of infancy and childhood, 7th ed. 2009) Hb (median 174 g/L, range 130 – 234) correlated with the RBC concentration (4.70 ×1012/L, 3.46 – 6.62; r = 0.96, p < 0.0001), as expected. Interestingly, MPV (8.7 fL, 7.5 – 11.5) correlated with Hb (r = 0.22, p = 0.0043), RBC concentration (r = 0.26, p = 0.0008), and Hct (53.6%, 40.1 – 73.1; r = 0.26, p = 0.0008). The PLT concentration (270 × 109/L, 161 – 607) and plateletcrit (defined as PLT concentration x MPV; 0.24%, 0.15 – 0.48) correlated negatively with Hb (r = -0.31, p < 0.0001 and r = -0.26, p = 0.0008, respectively). Hb also correlated with the WBC concentration (15.1 ×109/L, 5.54 – 39.7; r = 0.23, p = 0.0024). No correlation was observed between UA pH (7.28, 7.04 – 7.4) and the CB cell counts, except for the WBC concentration (r = -0.36, p < 0.0001) and CD34+ cell concentration (43.9 ×106/L, 7.14 - 253; r = -0.37, p < 0.0001). The CB PLT concentration and UA pH differed significantly between vaginal delivery and Cesarean section (p = 0.0053 and p = 0.0002, respectively). CONCLUSIONSValuable information about neonatal hematology can be obtained through careful analysis of perinatal and CB banking data. Hb and the cell characteristics of CB reveal strictly controlled internal associations which seem to reflect the general activity of hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

P589 - T cell receptor gene analysis by cloned PLT cells

P589 - T cell receptor gene analysis by cloned PLT cells

A new HLA-DP (SB) specificity (Cp63) defined in Japanese and Caucasian cell populations by cloned PLT cells

Cp61, Cp62, and Cp63 are HLA-DP (SB) specificities detected by PLT testing using cloned PLT cells derived from Japanese cell donors. It was confirmed, using Japanese and Caucasian cell panels, that Cp61 is identical to DPw5 and Cp62 is identical to DPw4. Cp63, however, was found not to correlate with any known HLA-DP specificities. The gene frequency of Cp63 was found to be 3.6% in Japanese ( N = 42) and 1.9% in Caucasian ( N = 52) cell panels of limited size. No triplet assignment of DPw specificity including Cp63 and found in the Japanese and Caucasian cell populations. The relationship between the Cp63 and HLA antigens demonstrated no significant correlation with HLA-A,B,C,DR, or MT antigens in this study. Linkage between Cp63 and HLA was confirmed from family segregation studies. This is the first report of a new DP specificity termed Cp63.

Identification of four SB antigens by cloned cells. Population studies of Norwegians.

By priming in vitro with allogeneic HLA-DR compatible and also HLA-A,B mostly compatible lymphoid cells, PLT cells resulted in recognizing a group of non D/DR allelic antigens provisionally named K, L, M and N. To improve discrimination these bulk primed typing reagents were cloned and expanded. By typing of previously SB typed lymphoblastoid B cell lines (LCL) the provisional specificities could be identified as SB1, 4, 3 and 2, respectively. Typing of 186 unrelated Norwegians gave the following gene frequences: SB1: 0.05, SB2: 0.16, SB3: 0.13, SB4: 0.42 and SB blank: 0.24. No triplets were found, the calculated gene frequencies fit with Hardy-Weinberg equilibrium, and typing of a B-DR recombinant family confirmed that the SB locus is situated centromeric to B. Associations between SB and A, B, DR antigens in the same material were generally weak, the most significant associations found were between SB1-DR3 and SB4-DR2.

Immunogenetic analysis of the HLA-D region: Serological and cellular detection of the MB system

The HLA-D region contains three serologically defined systems: DR, MB, and MT. The antigens of MB and MT are strongly associated with groups of DR antigens. This study dealt with the detection of MB by lymphocytotoxicity testing using B cell alloantisera obtained from multiparous women. Selective absorptions of anti-MB and anti-DR sera have demonstrated that MB antigens are distinct and that DR antigens show no crossreactivity. Primed lymphocytes (PLT cells) were generated from mixed leukocyte cultures whereby the stimulator had one incompatible DR-MB combination. Frequently such PLT cells showed specificity not only for DR but also for MB antigens. The latter could be blocked by preincubation of the secondary stimulator with anti-MB serum. Also, it was possible to generate alloreactive lymphocyte clones with specificity for MB. These data suggest that MB antigens may function as lymphocyte-activating determinants. The immunogenetic relationship between DR, MB, and MT suggests a multilocus control of HLA-D region products involved with lymphocyte activation.

Description of a new HLA-linked locus using cloned PLT cells: [formula omitted]. Immunologic Oncology Division, Georgetown University School of Medicine, Washington, D.C. 20007 and the Naval Medical Research Institute, Bethesda, M.D. 20014

Description of a new HLA-linked locus using cloned PLT cells: [formula omitted]. Immunologic Oncology Division, Georgetown University School of Medicine, Washington, D.C. 20007 and the Naval Medical Research Institute, Bethesda, M.D. 20014

Hemizygous HLA mutants of lymphoblastoid cells: A new cell type for histocompatibility testing

HLA variants that have lost expression of multiple cis-linked alleles as determined serologically and enzymatically were analyzed for expression of HLA-D PLT-stimulating (PL) determinants using the primed lymphocyte test. All 19 variants that had lost HLA-DRw3 expression simultaneously had lost expression of the HLA-D region-associated PL specificity. PLT cells made by priming to a variant that was hemizygous for HLA genes resulted in priming to HLA-D PL determinants encoded for by just one haplotype, analogous to priming to an HLA homozygous cell.

A new determinant, defined by PLT, coded for in the HLA region and apparently independent of the HLA-D and DR loci.

A PLT cell was raised between a responder cell which carried the HLA-D and DR specificities Dw3, 8; DRw3, 8 and a stimulator cell which was most likely homozygous for HLA-Dw3, DRw3. The PLT cell appeared to recognize a determinant (PL3A) which was (1) different from the officially recognized HLA-D and DR determinants, (2) was associated with HLA-A1, B8, Dw3 and DRw3, and (3) segregated in three informative families with HLA. Another responder-stimulator combination could be selected to raise PLT cells which recognized the same determinant.

Long-term maintenance of "cloned" human PLT cells in TCGF with LCL cells as a feeder layer.

The long-term maintenance of T cells "cloned" by limiting dilution in TCGF was enhanced by the use of irradiated autologous lymphoblastoid cell line (LCL) cells as well as irradiated LCL cells of the individual to which the T cells were originally primed. It was possible to obtain more than 1 X 10(12) cells from a "clone" seeded at one cell per well. Some of the clones tested express primed LD-typing activity.

The Genetics of PLT Response

Abstract PLT response is restricted by the HLA-D region. The present study was undertaken to help define the role of HLA-DRw in PLT restimulation. Haplotype-primed intrafamily PLT cells were made against specificities HLA-Drw1, HLA-DRw3, and HLA-DRw7; each PLT was then restimulated with cells from a 35-member unrelated panel. Restimulation values for each PLT were subjected to bimodal clustering analysis. In addition, blocking experiments were performed with other intrafamily and homozygous typing cell PLT after preincubation with B cell alloantisera. The results show a high correlation (0.881 ≤ r ≤ 1.00) between the HLA-DRw specificity of the priming haplotype and the HLA-DRw specificity of unrelated panel cells that restimulate in PLT. When stimulating cells were absorbed with the corresponding DRw alloantisera or p29,34 heteroantiserum (against B cell specific antigens), PLT restimulation was significantly blocked. However, the PLT cells treated with antisera showed no effect. The results strongly suggest that HLA-DRw is the principal PLT-stimulating determinant.

Detection of HLA-D clusters and segregation studies using primed LD typing.

By testing a group of PLT cells over a panel of unrelated restimulating cells, the PLT's could be grouped into clusters according to their ability to discriminate antigen(s) in unrelated cells. The PLT clusters broadly correlated with the homozygous typing cell-defined HLA-D clusters represented on the panel. The PLTs grouped together clearly segregate with a particular HLA haplotype when tested in both unrelated families not possessing the sensitizing haplotype and in the family with the sensitizing haplotype. No influence of HLA SD antigens could be observed in PLT restimulation in the segregation studies.

Primed LD typing: reagent preparation and definition of the HLA-D-region antigens.

Primed LD typing cells were prepared against single HLA haplotypes within families and used to type a random panel of 48 individuals. PLT cells could be grouped on the basis of highly correlated responses with the panel test cells; the 21 different PLT cells could be used to define 6 different PL antigens. One of these, PL 3, was split by the responses of two of the 21 PLT cells which measured an antigen called PL 3.1.

Lack of restimulation by non-HLA antigens in primed lymphocyte typing.

We have studied the role of non-HLA antigens in restimulation of PLT cells by the following protocols. Responder cells were sensitized to cells from family members differing by one HLA haplotype. The PLT cells were restimulated by family members HLA- or HLA-D-identical to the responder. In none of these cases did we find significant restimulation, indicating that it is unlikely that there is a restimulatory non-HLA locus with even two alleles (p less than 0.05) of equal frequency. Other protocols we have used are consistent with this interpretation. This indicates that antigens determined by genes segregating independently of HLA do not play a major role in the PLT restimulation.

Response of primed LD typing cells to homozygous typing cells.

At least two different methods using cellular responses have been described for defining the determinants of the HLA-D region: typing with HLA-D homozygous cells and primed LD typing. Primed LD typing cells were generated in one-haplotype-different combinations and grouped on the basis of two or more cells appearing to define the same HLA-D-region-determined PL antigen. Such cells were restimulated with homozygous typing cells for several of the presently known HLA DW clusters. A very strong correlation was noted: PLT cells defining the antigen PL1 were restimulated with homozygous typing cells for DW3, those PLT cells defining the antigen PL2 were restimulated by homozygous typing cells for DW2, and those defining PL5 were restimulated by homozygous typing cells for DW1.

Lymphoblastoid cell lines of homozygous typing cells used for sensitization in PLT.

Lymphoblastoid cell lines of homozygous typing cells were used as the sensitizing cells in MLC to prepare PLT cells. Results obtained using such cells against a panel of restimulating cells were compared to those obtained using regular PLT cells in which priming had been accomplished with normal peripheral blood lymphocytes. It appears that lymphoblastoid cell lines can be used for this purpose; the advantages of such an approach are given.

Three HLA-D region antigens defined by primed LD typing.

We have recently described a new method, primed LD typing or PLT, for specific identification of HLA-D antigens. Highly discriminatory PLT cells have been developed which clearly differentiate between cells of individuals that restimulate strongly and those that restimulate weakly. Seven such discriminatory PLT cells have been used to define three antigens called PL1, PL2, and PL3; two more PLT cells may define antigen(s) PL4.